TW201737924A - Combination therapy for neuroblastoma using MDA-7/IL-24 with therapeutic agents - Google Patents

Abstract

This invention discloses methods of treating neuroblastoma using Ad.5/3-CTV. In one embodiment, the present invention provides a method of treating neuroblastoma in a subject using Ad.5/3-CTV. In another embodiment, the present invention provides a method of inhibiting the growth of a neuroblastoma tumor using Ad.5/3-CTV. In one embodiment, the present invention provides a method of inhibiting the growth of a neuroblastoma cell using Ad.5/3-CTV. In yet another embodiment, the present invention provides a method of inducing apoptosis or toxic autophagy in a neuroblastoma cell using Ad.5/3-CTV. In one embodiment, the present invention provides a method of maintaining remission of neuroblastoma in a subject which has been treated for neuroblastoma. In one embodiment, the methods described herein further comprise a step of administering to the subject or cell a therapeutic agent selected from the group consisting of poly[IC]-PEI, doxorubicin, and any other therapeutic agents that promote toxic autophagy and/or apoptosis.

Description

Combination therapy with MDA-7/IL-24 and therapeutic neuroblastoma

RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Serial No. 62/315, filed on Jan. The disclosure also refers to a number of publications, the entire disclosure of which is hereby incorporated by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made, at least in part, by the Government of the United States Government and was funded by the following agencies: R01 CA097318 and P30 CA016059 awarded by the National Institutes of Health. The U.S. Government has certain rights in the invention.

FIELD OF THE INVENTION The present invention relates to a method of treating neuroblastoma using a combination of Ad.5/3- CTV and a therapeutic agent that promotes toxic autophagy and/or apoptosis. In one embodiment, the therapeutic agent is polyinosin-polyethylenimine (poly[IC]-PEI), doxorubicin, or any other toxic autophagy and/or apoptosis that promotes toxicity. Pharmacy. In one embodiment, the invention provides a method of inhibiting growth of a neuroblastoma cell, inhibiting growth of a neuroblastoma tumor, and/or inducing apoptosis and/or toxic autophagy of a neuroblastoma cell. In another embodiment, the invention provides a method of maintaining a neuroblastoma remission in a subject after treatment.

BACKGROUND OF THE INVENTION Neuroblastoma is the most frequently occurring extracranial solid tumor in children under five years of age, and one out of every 7,000 children is affected. It is speculated that the development of these tumors is the result of rapid proliferation of neuroblasts during fetal growth (1). Neuroblastoma occurs in the sympathetic nervous system and the adrenal medulla of sympathetic nerve ridge cells (2). It represents both heterogeneous masses both clinically and biologically. When present in infants, neuroblastoma may recurably recur in most cases, whereas in older patients these tumors frequently recur with benign ganglioneuroma (2). At the time of diagnosis, approximately half of the patients were assumed to be at high risk due to distal metastases. Neuroblastoma can be classified by four stages. In stage I and stage II, the disease is limited to the primary lesion. The characteristics of stage III and stage IV are diseases that are beyond the scope of the primary lesion. Once the neuroblastoma reaches advanced stage (III or IV), it continues to expand, even after undergoing strict multimodal therapy (1, 3). Because tumors are often presented in the late stages and lack of surgical options, the patient's prognosis is very poor. Although enhanced multimodal therapy has been used to increase the overall cure rate for a small number of neuroblastomas, these therapies have contributed to significant short-term and long-term toxicity (1, 3, 4). Only about 2% of patients with stage III or IV neuroblastoma remain disease-free after completion of chemotherapy and relapse shortly after chemotherapy, indicating that the long-term effects of these agents can be ignored (5). Drug resistance is thought to be the cause of treatment failure, which emphasizes the need for less toxic and more effective treatment strategies (6). Therefore, a comprehensive understanding of the mechanisms controlling proliferation, differentiation, and cell death can extend the understanding of the molecular pathogenesis of neuroblastoma, so that new biologically-based therapies with reduced toxicity and maximum efficacy can be identified.

Melanoma differentiation-related gene-7/interleukin-24 ( mda -7 /IL -24 ) is a unique member of the IL-10-associated interleukin gene family (7), which does not harm normal cells in a variety of cancers or The broad spectrum of antitumor activity of tissues (8, 9). Mda -7 /IL -24 was originally selected for use in a combination of subtractive hybridization and induction of terminal differentiation of cancer cells (10). Mda -7 /IL -24 is forced to manifest in direct cancer toxicity in cancer cells (autologous phagocytosis via induction of apoptosis or toxicity) (11) and indirect anti-tumor effects (via inhibition of angiogenesis) (8, 12) Promotes anti-tumor immune responses (8), sensitizes cancer cells to radiation and chemotherapy-induced killing (13), and promotes potent 'anti-tumor bystander activity' via autocrine/paracrine secretion (14 )). Mda -7 /IL -24 has shown near-universal in vivo and in vivo anti-tumor properties in almost every cancer background (15, 16), thus successfully entering clinical trials (17, 18). These properties of mda - 7/IL-24 make it a potential candidate gene for the treatment of neuroblastoma, in which adenovirus is administered to a single human neuroblastoma cell line (SH-SY5Y) for both in vitro and in vivo xenogeneic Graft growth produces inhibition (19). To enhance the utility of mda -7 /IL -24 as a cancer gene therapy, a conditional replication competent Ad (20) carrying mda -7 /IL -24 was used. In the cancer terminator virus ( CTV ) (21), adenoviral replication is controlled by a cancer-selective rodent gene, promoter-promoting gene-3 ( PEG -3 ) promoter (22). In order to even further enhance the utility of CTV , adenoviruses have been engineered to more efficiently infect cancer cells, resulting in chimeric CTV against directional modifications (23).

Adenovirus (Ads) uses CAR (Coxsackie-Adenovirus Receptors) to infect normal cells as well as cancer cells, whereas cancer cells express different amounts of CAR on their cell surface. In order to improve the inefficiency of Ad-infected tumor cells, a "trendative modification" method has been developed (23). One of these Ad.5/3 vectors showed equal efficacy compared to wild-type Ad.5, thereby expanding the range of utility of Ad.5/3 in CAR low-expression cells and CAR-high-expression cells (23, twenty four). For this reason, modified Ad.5/3- CTV (Ad.5/3- PEG - E1A- mda -7 ) was used to evaluate therapeutic applications in human neuroblastoma cells.

A previously unrecognized pathway has been described which involves mda -7 /IL -24- mediated induction of caspase-independent induction of apoptosis in neuroblastoma cells.

This pathway involves the regulation of expression of apoptosis-inducing factor (AIF) and translocation into the nucleus of neuroblastoma cells, which is induced by ataxia telangiectasia mutated factor (ATM), subsequent phosphorylation, and histones. The γ-H2AX translocation is mediated by nuclear translocations in the nucleus. These findings provide new evidence for understanding the mechanisms of action of the almost universal cancer suppressor genes and support their use as potential therapies for neuroblastoma.

The present invention demonstrates the existence of a novel cell death pathway in neuroblastoma that is triggered by mda -7 /IL -24 via ATM-mediated H2AX and AIF activation, resulting in caspase-independent apoptosis. This pathway is unique to neuroblastoma cells because the effect is not apparent in human breast cancer or melanoma cells.

To further enhance the therapeutic potential of Ad.5/3- CTV against neuroblastoma, the present invention further develops a combination therapy for neuroblastoma using mda-7/IL-24 and other therapeutic agents, including Polymyosine-polyethyleneimine (poly[IC]-PEI) and doxorubicin. Polyinosine-polycytidine, abbreviated as poly-cytosine, Poly[IC], is a synthetic double-stranded RNA (dsRNA) that directly activates dendritic cells and triggers natural killer (NK) cells to kill tumors. Cells that establish their immune regulation functions (59, 60). It is generally considered an antiviral agent that has been shown to exhibit antiviral activity by mimicking the effects of real viral RNA. The antiviral effect is mainly due to the induction of interferon (IFN) type I and downstream stimulatory genes (60). Poly[IC] has been used as a synthetic dsRNA mimetic to enhance the immune system in an IFN-dependent manner for more than four decades (51). Naked poly[IC] induces cell death in neuroblastoma when used at very high concentrations (52, 53). However, clinical trials using naked poly[IC] have shown poor stability of poly[IC] and interferon (IFN) induction, as well as undetectable antitumor effects (51).

How to deliver polyinosinic acid is the key to defining its function. Cytoplasmic delivery of polyinosinic acid with polyethyleneimine (PEI) and poly-cytosine-PEI (poly[(IC)-PEI) form has a significant effect on cancer cell growth, leading to apoptosis And toxic autophagy, as well as enhanced immunomodulatory activity (61-63). When naked poly[IC] is combined with polyethyleneimine (PEI), which allows cytoplasmic delivery of poly[IC], the poly[IC]-PEI combination induces a therapeutic response that is more effective than naked poly[IC] (51, 50) ). Induction of Apoptosis and Toxicity Autophagy has a common and distinct pathway as the molecular mechanism of the endpoint phenotype in specific cancers. However, in different cancers, depending on the delivery, poly[IC]-PEI promotes changes in gene expression in different clusters to induce apoptosis and a terminal phenotype of toxic autophagy. For example, in melanoma, poly[IC]-PEI is involved in the recruitment of ATG-5, inducing MDA-5 linkage to induce toxic autophagy (61, 62). In breast cancer, poly[IC]-PEI promotes induction of apoptosis via regulation of MDA-5 (62). In pancreatic cancer, poly[IC]-PEI inhibits XIAP and survivin expression and activates immune responses by inducing MDA-5, RIG-I, and NOXA (63). Taking the above studies as an example, a unique molecular mechanism for inducing apoptosis and toxic autophagy has been identified in different cancer types. Indeed, there is some overlap in the mechanisms associated with poly[IC]-PEI-mediated anticancer effects, and the effects of apoptosis/toxic autophagy are, at least in part, dependent on the induction of MDA-5.

Cisplatin and doxorubicin are commonly used as part of a combination therapy regimen for neuroblastoma patients (54-56). Doxorubicin induces different apoptotic pathways, and in certain cancer cells, these pathways include AIF, not MDA-5.

MDA-7/IL-24 is a unique gene that exhibits cancer-selective apoptosis-inducing ability in a broad spectrum of tumors (64). Intracellular delivery of MDA-7/IL-24 can be achieved via a variety of pathways, including viral-mediated gene delivery, administration of recombinant proteins, or direct introduction of expression plasmids. Viral-mediated expression of the mda- 7 /IL -24 gene produces a stable expression that triggers an autocrine/paracrine loop and regulates its own transcription and translation. MDA-7/IL-24 activates a variety of signaling pathways that promote the induction of apoptosis (and toxic autophagy) (64). These pathways involve the expression of stress proteins, the induction of reactive oxygen species, and the switching protection against toxic autophagy. There are currently no reports that direct induction of MDA-7 by MDA-7/IL-24 induces apoptosis. In addition, there is currently no report that any a priori prediction can be made for MDA-7/IL-24 to induce apoptosis via direct induction of NOXA, XIAP. In neuroblastoma, MDA-7/IL-24-mediated apoptosis is unique, at least in our current understanding of how this interleukin induces AIF, ATM, and γ-H2AX in a variety of cancer cells. Promote apoptosis/toxicity in autophagy (67).

When the agents are used in combination and produce a greater than expected additive effect, it can be said that synergy occurs (this can be measured using the method described in the Chou and Talalay methods used in our study (57)). Synergism may occur when two molecules operate via overlapping molecular mechanisms - the two molecular mechanisms are different but can produce an amplification effect when used in combination. Synergism can be observed when one molecule excites a signaling pathway that is being used by another molecule. Thus, when two molecules a) (at the molecular level) exhibit a similar mode of action, b) act through overlapping paths, or c) promote two different paths, but one path stimulates the action of another compound, There can be synergistic effects between molecules in theory. According to the literature, MDA-7/IL-24 and poly[IC]-PEI present two different pathways in mediating apoptosis or toxic autophagy in different cancer cells. Thus, the synergistic effect of MDA-7/IL-24 and poly(IC)-PEI on neuroblastoma cannot be predicted a priori. According to the proposed mode of action, we assume that Ad.5/3- CTV (encoding mda-7/IL-24) and poly[IC]-PEI have potential additive effects, but instead observe the synergy between the two.

In addition, the synergy of the combination of MDA-7/IL-24 with the second compound is not universal. This can be cited in the experiments of the present invention against neuroblastoma, in which the combination of three compounds with MDA-7IL-24 was tested. MDA-7/IL-24 only synergizes with poly[IC]-PEI and doxorubicin, but does not synergize with cisplatin. The mathematical model (using Chou and Talalay's method) also showed a synergistic effect between the two test compounds (poly[IC]-PEI and doxorubicin). Poly[IC]-PEI is a molecule that has been tested to date with the strongest synergy with MDA-7/IL-24. Thus, the present invention is not only novel in general, but also plays an important role in exhibiting enhanced anti-neuroblastoma activity, which in principle can be used to maximize the clinical outcome of treating patients with neuroblastoma.

In summary, the present invention provides for the targeting of neuroblastoma cells by means of a cancer-selective and conditionally replicating competent toxic adenovirus expressing mda -7 /IL -24 to inhibit growth and induce apoptosis. Novel approach. The present invention further provides novel combination therapies for neuroblastoma using mda -7 /IL -24 and other therapeutic agents that promote toxic autophagy and/or apoptosis, including poly[IC]-PEI, and Adriamycin.

SUMMARY OF THE INVENTION The present invention discloses methods of treating neuroblastoma using a combination of Ad.5/3- CTV and other therapeutic agents that promote toxic autophagy and/or apoptosis.

In one embodiment, the invention provides a method of treating a neuroblastoma in an individual using Ad.5/3- CTV . In another embodiment, the invention provides a method of inhibiting the growth of a neuroblastoma tumor using Ad.5/3- CTV .

In one embodiment, the invention provides a method of inhibiting the growth of neuroblastoma cells using Ad.5/3- CTV . In another embodiment, the invention provides a method of inducing apoptosis in neuroblastoma cells using Ad.5/3- CTV . In another embodiment, the invention provides a method of inducing toxic autophagy in cancer cells using Ad.5/3- CTV .

In one embodiment, the invention provides a method of maintaining neuroblastoma remission in an individual who has been treated with neuroblastoma.

In one embodiment, the methods described herein further comprise the step of administering to the individual or cell a therapeutic agent selected from the group consisting of poly[IC]-PEI or doxorubicin.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention relates to a method of treating neuroblastoma using a combination of Ad.5/3- CTV and other therapeutic agents that promote toxic autophagy and/or apoptosis.

Advanced neuroblastoma (the most common extracranial malignant solid tumor in infants and children's central nervous system) is difficult to treat. Melanoma differentiation-related gene-7/interleukin-24 ( mda -7 /IL -24 ) in preclinical animal models and in phase I clinical trials in patients with advanced cancer (including melanoma and various cancers) Ectopic performance promotes broad-spectrum anti-tumor activity in vitro and in vivo without harming normal cells. Mda -7 /IL -24 regulates cancer cell growth, invasion, metastasis by promoting endoplasmic reticulum (ER) stress and regulating multiple signal transduction pathways to exert cancer-specific toxicity (apoptosis or toxic autophagy) Survival and angiogenesis. To enhance the selective expression of mda -7 /IL -24 and to target anticancer activity, a directional modified cancer terminator virus ( Ad.5/3- CTV ) was constructed , which is selectively in mda -7 /IL -24 replicates in stable cancer cells. Ad.5/3- CTV has been found to induce significant neuroblastoma antiproliferative activity and apoptosis in vitro and in vivo in a caspase 3/9 independent manner in a tumor xenograft model. Ad.5/3- CTV promotes these effects via a unique pathway involving translocation of apoptosis-inducing factor (AIF) to the nucleus. Inhibition of AIF rescued neuroblastoma cells from Ad.5/3- CTV- induced cell death, and pan-Kassin inhibition failed to promote survival. Ad.5/3- CTV- infected neuroblastoma cells enhance ATM phosphorylation, stimulate nuclear translocation and increase γ-H2AX, trigger nuclear translocation and enhance AIF performance. These results were further validated using two ATM small molecule inhibitors that attenuated PARP cleavage by inhibiting γ-H2AX, attenuating PARP cleavage and inhibiting Ad.5/3- CTV- infected neuroblastoma cells AIF changes have occurred. In summary, in the present invention, it has been elucidated that mda -7 /IL -24 induces novel pathways for caspase-independent apoptosis in neuroblastoma cells via AIF, ATM, and Modulation by γ-H2AX is mediated. Novel methods for treating neuroblastoma using mda -7 /IL -24 have also been developed.

Neuroblastoma is a heterogeneous clinical entity that can be a subgroup with a good prognosis and a high probability of spontaneous regression, as well as a subgroup that has undergone invasive therapy but presents a very poor prognosis (1, 25). Given this difficulty and the high recurrence rate of advanced neuroblastoma (stages III and IV) (4, 6), strategies for the treatment of this cancer, especially in advanced stages, need to be prioritized. Currently, a broad-spectrum anti-tumor protein MDA-7/IL-24 delivered using a directional modified chimeric cancer terminator virus (Ad.5/3- CTV ) (23, 43) has been targeted against neuroblastoma cells. Evaluation (11, 12, 16). Ad.5/3- CTV reduces the growth of neuroblastoma cells and increases in vitro apoptosis and reduces tumor growth in vivo, which supports its potential applications in cancer therapy. Mechanistic studies have revealed a new pathway by which mda -7 /IL -24 can promote apoptosis in cancer cells, ie, via induction and translocation of AIF into the nucleus.

Mda -7 /IL -24 depicts strong anti-tumor activity mediated through multiple pathways in a variety of cancers (7, 16). The toxicity mechanism of mda -7 /IL -24 includes ER stress caused by inhibition of anti-apoptotic Bcl-2 family members and apoptosis of tumor cells (26, 44), which was also confirmed in the present invention. MDA-7/IL-24 therapy has previously been shown to increase the production of reactive oxygen species (ROS) in many cancer types (18, 27). The close association of ROS production with early stage apoptosis and mitochondrial dysfunction is well known in the art (28). Early reports indicate that ROS production and mitochondrial release of Cyt C promote cell death (29, 30). This results in increased mitochondrial membrane permeability, decreased transmembrane potential, and AIF activation (45), and ultimately induces caspase-independent apoptosis (31). Ad.5/3- CTV delivery of mda -7 /IL -24 to neuroblastoma cells increased AIF levels. Consistent with previously published experimental data, the results of the present invention indicate that in neuroblastoma cells overexpressing mda -7 /IL -24 , AIF translocates into the nucleus and induces caspase-independent cell death. This result was further confirmed by AIF and a pancacine inhibitor. AIF small molecule inhibitors attenuated PARP cleavage, thereby inhibiting cell death in neuroblastoma cells following Ad.5/3- CTV treatment. In addition, administration of a pancacine inhibitor did not alter mda -7 /IL -24 induced PARP cleavage; thus further confirming that this interleukin induces caspase-independent cell death in neuroblastoma cells.

The response of cells to DNA damage phosphorylates the variant H2AX of the H2A protein family (32). H2AX assists chromatin in promoting DNA repair by providing binding sites to downstream repair factors (33). AIF is a flavoprotein that plays a major role in mediating caspase-independent cell death in the intergranular space of the mitochondria (34, 35). After stimulation with cell death, AIF is cleaved by calpain and cathepsin in the mitochondria (36), possibly released into the cytosol via the mitochondrial permeability transition pore, and translocated into the nucleus where it is passed through the nucleus. A complex formed by H2AX and cyclophilin A to induce chromatin condensation and DNA fragmentation (37). A variety of apoptotic stimuli that have been shown to induce AIF mitochondria to nuclear translocation include DNA damaging agents, hypoxia/ischemia, oxidative stress, and excitatory neurotoxins (such as glutamate) (38). However, the signaling pathways that cause nuclear translocation of AIF cells have not been fully elucidated. In this regard, the effect of Ad.5/3- CTV on key apoptotic proteins, AIF, and poly(ADP-ribose) polymerase-1 (PARP1) was studied, which constitutes a relative in MDA-7/IL-24 therapy. A novel, yet critical, caspase-independent pathway of apoptosis.

H2AX (histone H2A family member) is characterized by a phosphorylated SQE motif in its C-terminal tail (39, 45). It has also been determined that DNA fragmentation induces phosphorylation of H2AX histones at serine 139 (40). Although H2AX is primarily involved in DNA damage repair and DNA packaging, it is also a key regulator of progressive cell death (41, 45). In order to understand the mechanism by which MDA-7/IL-24 causes nuclear translocation of AIF cells, the content of γ-H2AX in cells treated with Ad.5/3- CTV was evaluated, and the degree of activation of H2AX was found to be increased. ATM is known to be a major kinase involved in H2AX phosphorylation and ATM is also known to be one of the first activated kinases in cellular responses to double-strand breaks. After Ad.5/3- CTV treatment, ATM, H2AX, and AIF translocations were found to be activated, and these cell modifications were confirmed by pharmacological inhibition of ATM and AIF. The data obtained from such studies indicate that the important role of ATM activation is to trigger H2AX phosphorylation and AIF activation, thereby causing caspase-independent cell death in Ad.5/3- CTV- treated neuroblastoma cells. Ad.5/3- CTV- infected human breast cancer cells (MDA-MB-231 and ZR-751) and melanoma cells (C8161 and SK-Mel) did not enhance AIF performance compared with neuroblastoma cells (Fig. 15). . The key question of how ATM is accurately activated in MDA-7/IL-24-mediated apoptosis in neuroblastoma cells requires further experimentation. A recent study by Baritaud et al. revealed the significance of ATM and DNA-PK in AIF-mediated caspase-independent necrotic apoptosis in regulating γ- H2AX (45). In particular, it was shown that ATM inhibition prevented H2AX phosphorylation observed after the addition of MNNG and subsequently blocked AIF-mediated cell death. This is consistent with the observations of the present invention in that ATM small molecule inhibitors attenuate Ad.5/3- CTV- induced PARP cleavage and H2AX phosphorylation and inhibit AIF changes in neuroblastoma cells (Fig. 5C). In contrast, AIF small molecule inhibitors reduced ATM phosphorylation and cell death in Ad.5/3- CTV- induced neuroblastoma cells (Fig. 4D and 5D). Taken together, the results of the present invention indicate that ATM is functionally related to AIF in a positive feedback loop where it is modulated in each other in neuroblastoma cells following Ad.5/3-CTV infection (Figure 7). Another recent study reported that treatment with pro-oxidants caused tumor-associated, AIF-dependent apoptosis in ATM-null primary chronic lymphocytic leukemia (CLL) tumors (42). All results indicate that ATM and AIF can function independently or together to induce caspase-independent cell death.

In summary, a new cell death pathway has been demonstrated for the first time, triggered by mda -7 /IL -24 , followed by ATM-mediated activation of H2AX and AIF, causing caspase-independent apoptosis (Figure 7 This pathway appears to be unique to neuroblastoma cells because the effect is not apparent in human breast cancer or melanoma cells (Figure 15). Support for this conclusion stems from three experimental evidences: (1) inhibition of AIF by AIF inhibitors reduces MDA-7/IL-24-mediated apoptosis; (2) inhibition of caspase by pan-kas protease inhibitors Blocking MDA-7/IL-24-induced cell death; and (3) inhibiting ATM to alter AIF levels, thereby inhibiting cell death in neuroblastoma cells overexpressing MDA-7/IL-24. Thus, the selective induction of cytolysis of neuroblastoma cells using Ad.5/3- CTV (presenting cancer-specific viral replication and stable MDA-7/IL-24 production and secretion) can be targeted at this invasive cancer. Potentially feasible treatment options.

The invention further provides a combination therapy using Ad.5/3- CTV and other therapeutic agents, including poly[IC]-PEI, doxorubicin, and any other that promotes toxic autophagy and/or apoptosis. Therapeutic agent. This may include radiation, reactive oxygen species inducers, and specific DNA damage chemotherapeutic agents. The results indicate that the combination of Ad.5/3- CTV and poly[IC]-PEI synergistically inhibits the growth of neuroblastoma cells (Figures 16-20) and that growth inhibition may be caused by apoptosis (Figure 21). ). Reports on the combined use of the two agents are not present in the present invention, and the synergistic effects observed herein are significant and unexpected, as Ad.5/3- CTV and poly[IC]-PEI involve different cells. Apoptotic pathway (Ad.5/3- CTV induces apoptosis in neuroblastoma cells via AIF and ATM, whereas polyinosinic acid (poly[I:C]) via XIAP, MDA-5, NOXA and / Or RIG-I induces apoptosis in different cancer cells). For doxorubicin, the results indicate that combination therapy with Ad.5/3- CTV and doxorubicin induces strong additive inhibition of neuroblastoma cell growth (Figures 22-26) and growth inhibition Caused by apoptosis (Figure 27). For cisplatin, it was observed that the combination of Ad.5/3- CTV and cisplatin did not produce any observable synergistic or additive inhibition of neuroblastoma cell growth over the test dose range (Figure 28- 33). Therefore, not all chemotherapeutic agents are capable of providing synergistic effects, and it is well understood by the inventors that combinations and treatments are reported for the first time.

Poly(IC)-PEI allows cytoplasmic delivery of poly[IC]. A unique combination of Ad.5/3-CTV (through viral replication and mda-7/IL-24 expression) with toxic autophagy and the apoptosis inducer poly(IC)-PEI for the treatment of neuroblasts tumor. The mechanisms of action of the two agents are different, but the pathways they induce cause toxic autophagy and apoptosis in neuroblastoma. Although the combination of doxorubicin and mda -7 /IL -24 has been shown to enhance apoptosis induction in colon cancer and liver cancer, it has not been reported that the Ad.5/3-CTV-doxorubicin combination promotes neuroblasts. Tumor apoptosis.

The combination therapy described herein can be used as a primary therapy or as a secondary therapy and has significant clinical value in treating children with neuroblastoma. About 700 children are diagnosed with neuroblastoma each year, and neuroblastoma is considered to be one of the most common solid tumors in early childhood (usually in infants or 1-2 years old). The most effective treatment options include radiation, chemotherapy, and surgery. However, despite these methods, the five-year survival rate for high-risk patients (or stage IV) is only 40-50%. In addition, current treatments may have long-lasting negative effects, including cardiovascular, slower growth, changes in mental function with learning problems, and the potential to produce a second cancer, such as leukemia. Thus, the combination therapies of the invention can significantly reduce the amount of drug and benefit long-term treatment. Combination therapy is also suitable for achieving long-term relief of neuroblastoma. For example, an individual with a neuroblastoma can initially be treated with a combination of Ad.5/3- CTV and poly[IC]-PEI followed by a poly[IC]-PEI alone to maintain remission. In addition, since it is expected that the combination therapy of the present invention does not produce drug resistance, the patient can receive the combination therapy more than once if necessary.

In one embodiment, the present invention provides a method of treating a subject of neuroblastoma, the method comprising the steps of a nucleic acid Ad.5 / 3- CTV administering to the subject an effective amount of the nucleic acid Ad.5 / 3- CTV gland comprising Viral vector Ad.5/3 and nucleic acid expressing MDA-7/IL-24. In one embodiment, the therapeutic agent is selected from the group consisting of poly[IC]-PEI and doxorubicin.

In one embodiment, the Ad.5/3- CTV of the invention comprises an adenoviral vector Ad.5/3 and a nucleic acid molecule of the mda -7 /IL -24 gene as described in Dash (20). In another embodiment, the Ad.5/3- CTV of the invention comprises an adenoviral vector Ad.5/3 and a nucleic acid molecule of the mda -7 /IL -24 gene as described in WO2014093270, the entire disclosure of which The contents are hereby incorporated by reference into this application. In another embodiment, the Ad.5/3- CTV of the invention comprises one or more of the sequences of SEQ ID NO.

In one embodiment, the invention provides a method of inhibiting growth of a neuroblastoma tumor in an individual, the method comprising the step of administering to the individual an effective amount of Ad.5/3- CTV , the Ad.5/3- CTV comprising Adenoviral vector Ad.5/3 and nucleic acid expressing MDA-7/IL-24.

In one embodiment, the invention provides a method of inducing apoptosis or toxic autophagy of a neuroblastoma cell, the method comprising administering to the cell an effective amount of Ad.5/3- CTV or causing the cell to An effective amount of Ad.5/3- CTV contacting, the Ad.5/3- CTV comprises an adenoviral vector Ad.5/3 and a nucleic acid expressing MDA-7/IL-24. In one embodiment, the invention provides a method of inducing apoptosis and toxic autophagy in cancer cells, the method comprising administering to the cell an effective amount of Ad.5/3- CTV or subjecting the cell to an effective amount Ad.5/3- CTV contact step, the Ad.5/3- CTV comprises adenoviral vector Ad.5/3 and nucleic acid expressing MDA-7/IL-24.

In one embodiment, the invention provides a method of inhibiting the growth of a neuroblastoma cell, the method comprising administering to the cell an effective amount of Ad.5/3- CTV or the cell with an effective amount of Ad.5/3 a step of CTV contacting, the Ad.5/3- CTV comprises an adenoviral vector Ad.5/3 and a nucleic acid expressing MDA-7/IL-24.

In one embodiment of the invention, the expression of MDA-7/IL-24 is via AIF, ATM, and modulation of γ-H2AX to induce apoptosis in neuroblastoma cells.

In one embodiment, the methods described herein further comprise the step of administering an effective amount of poly[IC]-PEI or doxorubicin.

In one embodiment, the invention provides a method of treating a neuroblastoma in a subject, the method comprising administering to the individual an effective amount of Ad.5/3- CTV and selected from the group consisting of: poly[IC]-PEI or doxorubicin The molecular steps.

In one embodiment, the invention provides a method of inhibiting growth of a neuroblastoma cell, the method comprising administering to the cell an effective amount of Ad.5/3- CTV and selected from the group consisting of: poly[IC]-PEI or A. Or a step of contacting the cell with an effective amount of Ad.5/3- CTV in contact with a molecule selected from the group consisting of poly[IC]-PEI or doxorubicin. In another embodiment, inhibition of growth of neuroblastoma cells is significantly higher than inhibition by treatment with Ad.5/3- CTV alone or with the molecule.

In one embodiment, the invention provides a neuron cell for an individual who has been treated with Ad.5/3- CTV and poly[IC]-PEI or with Ad.5/3- CTV and poly[IC]-PEI A method of maintaining tumor remission, the method comprising the step of administering to a subject an effective amount of poly[IC]-PEI or doxorubicin to maintain neuroblastoma remission.

In one embodiment, the methods described herein are used as primary or secondary therapies for treating an individual having a neuroblastoma.

In one embodiment, the poly[IC]-PEI is applied at a concentration ranging from 0.05 μg/ml to 5 μg/ml. In another embodiment, the concentration of poly[IC]-PEI is in the range of 0.2 to 2.5 μg/ml. In yet another embodiment, the concentration of poly[IC]-PEI is in the range of 1 ng/ml to 100 μg/ml. In one embodiment, the concentration of poly[IC]-PEI ranges from 1 ng to 100 μg per dose. In yet another embodiment, the concentration of poly[IC]-PEI ranges from 1 ng to 100 μg per kilogram of body weight.

In one embodiment, the doxorubicin is administered at a concentration ranging from 1 to 20 μM. In another embodiment, the doxorubicin is administered at a concentration ranging from 5 to 10 μM. In another embodiment, the concentration of doxorubicin ranges from 0.1 mg to 20 mg per kg of body weight. In yet another embodiment, the concentration of doxorubicin ranges from 1 mg to 500 mg per dose.

In one embodiment, Ad.5/3- CTV is administered concurrently with poly[IC]-PEI or doxorubicin. In another embodiment, the poly[IC]-PEI or doxorubicin is administered 12-96 hours after Ad.5/3- CTV administration. In another embodiment, the poly[IC]-PEI or doxorubicin is administered 24-96 hours after Ad.5/3- CTV administration.

In one embodiment of the methods described herein, the dose of Ad.5/3- CTV is in the range of from 1 to 500 plaque-forming units (pfu). In another embodiment, the dosage of Ad.5/3- CTV is in the range of 6.25 pfu to 50 pfu. In another embodiment, the dosage of Ad.5/3- CTV is 5, 10, 25, 30, 40, 50 or 75 pfu. In another embodiment of the methods described herein, the dose of Ad.5/3- CTV is in the range of 1 x 10 ² to 1 x 10 ¹⁵ pfu per ml or dose. In another embodiment, the dose of Ad.5/3- CTV is in the range of 1 x 10 ⁴ to 1 x 10 ⁶ pfu per ml or dose. In one embodiment, Ad.5 / 3- CTV dose within 1x10 ⁶ to 1x10 ¹⁰ pfu per kilogram of body weight range. In another embodiment, the dosage Ad.5 / 3- CTV is lower than the LD ₅₀ (50% killing of individual Ad.5 / 3- CTV minimum dose).

In one embodiment of the methods described herein, the dose of Ad.5/3- CTV is in the range of 0.1 international units (IU) to 100 IU. In another embodiment, the dose of Ad.5/3- CTV is in the range of 2.5 IU to 50 IU. In another embodiment, the dosage of Ad.5/3- CTV is 5, 7.5, 10, 12.5 or 25 IU. In another embodiment of the methods described herein, the dose of Ad.5/3- CTV is in the range of ¹ x 10 ¹ to 1 x 10 ⁶ per ml or dose. In yet another embodiment, the dose of Ad.5/3- CTV is in the range of ¹ x 10 ¹ to 1 x 10 ⁶ by weight. In another embodiment, the dosage Ad.5 / 3- CTV is lower than the LD ₅₀ (50% killing of individual Ad.5 / 3- CTV minimum dose).

In one embodiment of the methods described herein, the neuroblastoma cells are selected from the group consisting of SK-N-AS, SK-N-SH, and NB1691.

In one embodiment, the invention provides a combination of Ad.5/3- CTV and a medicament for use in a method of treating cancer in a subject, wherein the Ad.5/3- CTV synergizes with the agent to induce cancer cells Growth inhibition, toxic autophagy, and apoptosis. In one embodiment, the Ad.5/3- CTV comprises an adenoviral vector Ad.5/3 and a nucleic acid that exhibits MDA-7/IL-24. In another embodiment, the agent is selected from the group consisting of poly[IC]-PEI, doxorubicin, and other agents capable of promoting toxic autophagy and/or apoptosis.

In one embodiment of this combination, the expression of MDA-7/IL-24 is via AIF, ATM, and modulation of γ-H2AX to induce apoptosis in cancer cells.

In one embodiment of this combination, the Ad.5/3- CTV is administered concurrently with the agent. In yet another embodiment, the agent is administered 12-96 hours after administration of Ad.5/3- CTV .

In one embodiment of the combination, the cancer is a neuroblastoma. In yet another embodiment, the cancer is melanoma, glioblastoma, prostate cancer, breast cancer, colorectal cancer, lung cancer, or pancreatic cancer.

In one embodiment of the combination, the agent is poly[IC]-PEI. In yet another embodiment, the agent is doxorubicin.

In one embodiment of the combination, the agent induces one or more of MDA-5, NOXA, and RIG-I.

In one embodiment, the invention provides a method of treating cancer in an individual, the method comprising administering to the individual an effective amount of Ad.5/3- CTV and an agent, wherein the Ad.5/3- CTV synergizes with the agent Role to induce cancer cell growth inhibition and apoptosis. In one embodiment, Ad.5/3- CTV comprises adenoviral vector Ad.5/3 and a nucleic acid that expresses MDA-7/IL-24. In another embodiment, the agent is selected from the group consisting of poly[IC]-PEI, doxorubicin, and other agents that promote toxic autophagy and/or apoptosis. In one embodiment of the method, the expression of MDA-7/IL-24 is via AIF, ATM, and modulation of γ-H2AX to induce apoptosis in cancer cells.

In one embodiment of the method, the Ad.5/3- CTV is administered concurrently with the agent. In another embodiment, the agent is administered 12-96 hours after Ad.5/3- CTV administration.

In one embodiment of the method, the cancer is a neuroblastoma. In yet another embodiment, the cancer is melanoma, glioblastoma, prostate cancer, breast cancer, colorectal cancer, lung cancer, or pancreatic cancer.

In one embodiment of the method, the agent is poly[IC]-PEI. In another embodiment, the agent is doxorubicin. In one embodiment of the method, the agent induces one or more of MDA-5, NOXA, and RIG-I.

The invention will be more fully understood with reference to the following examples. However, it will be readily understood by those skilled in the art that the examples are provided for illustrative purposes only and are not intended to limit the scope of the invention, which is defined by the scope of the appended claims.

In this application, it should be noted that the term "comprising" is synonymous with "including", "containing" or "characterized" and is intended to be inclusive or open-ended and does not exclude other elements or method steps that are not described. Results showed that mda -7 /IL -24 CTV inhibited proliferation of neuroblastoma cells in vitro

In order to examine the overexpression of mda -7 /IL -24 on in vitro and in vivo growth of neuroblastoma tumors, genetic methods were applied to the targeted modified cancer terminator virus (Ad.5/3- CTV ) ( 23, 26), that is, an ectopically expressed conditional replication adenovirus of mda -7 /IL -24 . Adenovirus entry is associated with cell surface receptors, so the three cell lines of neuroblastoma SK-N-AS, NB1691 and SK-N-SH used in this study (for Ad.5), desmoglein And the status of CD46 (for Ad.3) receptors was determined. The CAR profile was variable, with SK-N-AS having similarly lower amounts in NB1691 cells and higher amounts in SK-N-SH cells (Figures 8A and 8B). The CD46 expression was similar in all three cell lines and the desmoglein content was similar to that of CAR, with the highest performance in SK-N-SH cells. When the transgene expression (MDA-7/IL-24 and E1A) was monitored after Ad.5/3- CTV infection, all three neuroblasts were compared to mock and Ad.5/3-Null infected cells. The tumor cell lines all showed the expected transgenic genes (Fig. 1A). A dose-dependent increase in MDA-7/IL-24 protein content in cells infected with Ad.5/3- CTV was found compared to the control group. To confirm that the increase in MDA-7/IL-24 protein content represents up - regulation of mda - 7 /IL -24 mRNA transcription, mRNA transcript levels in cells treated with Ad.5/3- CTV were assessed. Neuroblastoma cells treated with Ad.5/3- CTV showed mda -7 /IL -24 mRNA compared to cells treated with mock, Ad.5/3-null or Ad.5/3- E1A The content increased significantly (data not shown). Next, MTT assay was used to assess the effect of Ad.5/3- CTV on the growth of neuroblastoma cells. Although Ad.5/3- E1A inhibits cell proliferation to a certain extent via its oncolytic activity, the use of Ad.5/3- CTV forcing mda -7 /IL -24 is the most potent cell compared to other therapies. The dose-dependent decrease in proliferation (Fig. 1B). Ad .5 /3 - CTV induces cell death in neuroblastoma cells

Ectopic manifestations of mda -7 /IL -24 induce apoptosis in a variety of cancer types (16). Excessive expression of mda -7 /IL -24 in neuroblastoma cell lines resulted in an increase in TUNEL-positive cells compared to cells treated with mock, Ad.5/3-Null or Ad.5/3- E1A treatments (Fig. 2A). Further proof of induction of apoptosis was confirmed by FACS analysis, in which the sub-G1 population (DNA content) in cells infected with Ad.5/3- CTV was increased. The DNA frequency distribution histogram (where the sub-G1 region corresponds to apoptotic cells) indicates that Ad.5/3- CTV increases the number of apoptotic cells of SK-N-AS to 40-50%, making SK-N- The number of apoptotic cells in SH increased to 35-50% and the apoptotic cells of NB1691 cells increased to 40-50%, compared with 5% of the control group infected with Ad.5/3-Null or The cells infected with Ad.5/3- E1A were approximately 20% (Fig. 2B). According to Western blot analysis, it is apparent that PARP cleavage is mainly present in neuroblastoma cells infected with Ad.5/3- CTV (Fig. 2C). After infection of neuroblastoma cells with Ad.5/3- CTV , the expression of mda -7 /IL -24 downstream molecules and signals involved in cell death was also analyzed. In many cancers, mda -7 /IL -24 is known to enhance pro-apoptotic genes and attenuate anti-apoptotic genes in the Bcl-2 protein family (44). After Ad.5/3- CTV infection, similar expression characteristics were found in neuroblastoma cells, ie, BAX and P-JNK (pro-apoptotic proteins) were enhanced and BCL-2 and BCL-xL (anti-cells) The expression of apoptotic proteins was reduced (Figure 9). Notably, these changes were only apparent after Ad.5/3- CTV infection of neuroblastoma cells, but not after Ad.5/3-null or Ad.5/3- E1A infection of neuroblastoma cells obvious. Mda -7 /IL -24 induces caspase-independent apoptosis in neuroblastoma cells in vitro

Although it has been well established that mda -7 /IL-24 has tumor suppressor and apoptosis-promoting properties for a broad spectrum of human cancer cells, the molecular mechanism by which mda -7 /IL -24 induces apoptosis is very divergent and depends on the tumor type. And involves different paths (44). Caspase-dependent apoptosis is a common pattern of progressive cell death (46), so this experiment was designed to determine whether mda -7 /IL -24 induces cell death in neuroblastoma whether it is caspase-dependent. Treatment of neuroblastoma cells with Ad.5/3-CTV did not induce activation of caspase-3 or caspase-9 by Western blot analysis or by luminescence-based assays (Figures 3A and 3B) ). To determine potential pathways involved in exogenous apoptosis, the performance of caspase 8 was monitored after Ad.5/3- CTV infection and no activation was found in these cell lines (Figure 10). In another validation study, neuroblastoma cells were treated with z-vad fmk (a pancacine inhibitor) prior to Ad.5/3- CTV infection and incubated for an additional 48 hours. The PARP cleavage in the cells was then analyzed by Western blotting, demonstrating the presence of similar PARP cleavage with or without treatment with the pancacine inhibitor prior to Ad.5/3- CTV infection (Fig. 3C). This result further supports the presence of a caspase-independent cell death mechanism following MDA-7/IL-24 ectopic expression in neuroblastoma cells. Mda -7 /IL -24 mediates apoptosis in neuroblastoma, involving AIF activation and nuclear translocation

The tumor suppressor p53 plays an important role in suppressing tumorigenesis by inducing genomic stability, cell cycle arrest or apoptosis (47). Apoptosis-inducing factor (AIF) is a mitochondrial protein that causes apoptosis when translocated to the nucleus, causing cell death mainly through induced chromosome condensation and DNA fragmentation in a caspase-independent context. Dead (48). P53 is a defined regulator of AIF in caspase-independent cell death and AIF promotes p53-mediated cell death (49). After Ad.5/3- CTV infection, p53 content was moderately increased in a dose-dependent manner compared to mock- or Ad.5/3-Null-infected cells (Figure 11). AIF content in Ad.5/3- CTV- infected neuroblastoma cells increased in a dose-dependent manner (Fig. 4A), indicating that AIF potentially involves caspase-independent cell death.

Previous studies have shown that BCL-2 protein promotes the insertion of BAK or BAX into the mitochondrial membrane to form functional oligomers, thereby depolarizing the mitochondrial inner membrane and subsequent AIF nuclear translocation, promoting caspase-independent Apoptosis (50). To determine if mda- 7 /IL -24 exerts a similar effect on nuclear translocation of AIF cells, immunostaining and cell fractionation methods were used. AIF showed a granular pattern in the mitochondria of untreated control cells, whereas after treatment with Ad.5/3- CTV , AIF was detected in the nucleus (Fig. 4B). This result was confirmed by cell fractionation, which clearly showed that after Ad.5/3- CTV infection, AIF accumulated in the nucleus lysate and the content in the cytoplasm fraction was simultaneously decreased (Fig. 4C).

The AIF inhibitor N-phenyl maleimide was used to confirm Ad.5/3- CTV- induced, AIF-mediated cell death in neuroblastoma cells. Treatment with AIF inhibitor at a concentration of 50 μM/L for 1 hour followed by treatment with Ad.5/3- CTV for 48 hours reduced cell death as monitored by FACS analysis (data not shown). This phenomenon was further confirmed by Western blot analysis for PARP cleavage. Treatment with an AIF inhibitor followed by infection with Ad.5/3- CTV caused a reduction in PARP cleavage (Fig. 4D). Collectively, these results indicate that Ad.5/3- CTV induces caspase-independent AIF-mediated apoptosis in neuroblastoma cells. ATM -γ- H2AX axis mediates AIF- induced cell death in neuroblastoma cells

The mechanisms regulating AIF induction as well as nuclear translocation and causing caspase-independent apoptosis are not fully understood. Previous studies have shown that γ-H2AX plays a key role in AIF-mediated necrotic apoptosis in MEF (45). To test this hypothesis, the effect of Ad.5/3- CTV on the expression and activation of H2AX in SK-N-AS and NB1691 neuroblastoma cells was examined. Ad.5/3- CTV infection increased H2AX content and H2AX phosphorylation (γ-H2AX) compared to cells infected with control, Ad.5/3-Null or Ad.5/3- E1A (Fig. 5A) And Figure 12). To further decipher the molecular mechanism of H2AX phosphorylation, ATM content and activation in Ad.5/3- CTV treated SK-N-AS, SK-N-SH, and NB1691 neuroblastoma cells were evaluated. ATM content and ATM phosphorylation were increased compared to cells treated with control, Ad.5/3-Null or Ad.5/3- E1A (Fig. 5B and Fig. 13). Taken together, these results indicate that AIF-mediated caspase-independent apoptosis is required for ATM-induced histone H2AX phosphorylation in Ad.5/3- CTV- treated neuroblastoma cells.

To further demonstrate that ATM is an upstream regulator of H2AX and AIF-mediated cell death in Ad.5/3- CTV- treated neuroblastoma cells, a small molecule ATM inhibitor KU-60019 that blocks ATM kinase is used. And KU-55933. ATM phosphorylates a variety of proteins at specific locations, including H2AX at S139 (γ-H2AX). Neuroblastoma cells were treated overnight with KU-60019 (3 μM) or KU-55933 (5 μM, Figure 14) prior to Ad.5/3- CTV infection. Treatment with KU60019 followed by Ad.5/3- CTV infection inhibited ATM phosphorylation, thereby inhibiting γ-H2AX in SK-N-AS and NB1691 cells (Fig. 5C). This also caused a decrease in PARP cleavage, reflecting a decrease in apoptosis, as confirmed by TUNEL analysis (Fig. 5D and Fig. 14). Taken together, these results indicate that ATM acts as an upstream regulator of H2AX phosphorylation, resulting in AIF-mediated cell death in neuroblastoma cells following Ad.5/3- CTV treatment. Mda - 7 / IL - 24 inhibits tumor growth in neuroblastoma in vivo

To directly assess the effect of mda -7 /IL -24 delivered by Ad.5/3- CTV on tumor growth in vivo, NB1691 cells were subcutaneously implanted on both sides of nude mice. Tumors on the left abdomen were stimulated with an intratumoral injection of Ad.5/3-Null, Ad.5/3- E1A or Ad.5/3- CTV (mda -7 /IL -24 transduced virus ) . Tumor growth in mice was monitored by measuring the tumor every other day with a vernier caliper. There was a significant decrease in tumor volume in mice treated with Ad.5/3- CTV compared to mice treated with Ad.5/3-Null or Ad.5/3- E1A (Fig. 6A). Although Ad.5/3- E1A reduced the growth of the tumor on the left side of the injection to a certain extent, it was apparent that the uninjected right side was observed only in the animals treated with Ad.5/3- CTV on the left side of the tumor. Tumors have "bystander activity" (Figure 6). These results confirm previously published data in which MDA-7/IL-24 exhibits strong anti-tumor "bystander activity". To determine whether MDA-7/IL-24 caused AIF-mediated apoptosis in vivo, immunoassays were performed on MDA-7/IL-24, pATM, γ-H2AX, and AIF in tumor sections. Apoptotic content was determined by TUNEL analysis. Consistent with the in vitro observations of the present invention, tumor sections from mice treated with Ad.5/3-CTV showed increased staining for MDA-7/IL-24, pATM, γ-H2AX, and AIF (Fig. 6C). In addition, the apoptosis index of tumor cells quantified by the number of TUNEL staining positive cells was increased in the case of Ad.5/3- CTV treatment (Fig. 6B). Ad .5 / 3 -CTV neuroblastoma (NB) a combination of poly [IC] -PEI the synergistic induction of cell growth inhibition

NB cell lines (SK-N-AS and SK-N-SH) were infected with Ad.5/3 E1A (12.5 IU) or Ad.5/3- CTV (12.5 IU) and cultured for 24 hours. These cells were then retreated with various doses of poly[IC]-PEI for 24 hours. MTT assay was used to determine cell proliferation and plot (Figures 16-19). The use of Ad.5/3- CTV alone caused 30-40% growth inhibition in both test cell lines. The use of poly[IC]-PEI alone caused 20-40% inhibition of cell growth. Interestingly, the combination of different concentrations of poly[IC]-PEI and fixed low concentrations of Ad.5/3-CTV produced strong synergy in cell proliferation with a combination index (CI) of (0.15-0.40). Inhibition (Figure 20). The CI system was determined using the Chou and Talalay methods (57). The inhibition of cell growth by Ad.5/3- CTV in combination with poly[IC]-PEI may be caused by induction of apoptosis, which may be increased from the PARP lysis phase in the combination treatment group compared to the single treatment group. Proof (Figure 21). Ad .5 / 3 -CTV combination of doxorubicin and neuroblastoma (NB) cell growth was induced by adding a strong inhibition

NB cell lines (SK-N-AS and SK-N-SH) were infected with Ad.5/3 E1A (12.5 IU) or Ad.5/3-CTV (12.5 IU) and cultured for 24 hours. These cells were then treated with various doses of doxorubicin for an additional 24 hours. MTT assay was used to determine cell proliferation and plot (Figures 22-25). Infection with Ad.5/3- CTV alone caused 30-40% growth inhibition in both test cell lines. Doxorubicin alone causes 40-50% inhibition of cell growth. Interestingly, the combination of different concentrations of doxorubicin and fixed low concentrations of Ad.5/3- CTV has a strong additive/synergy-inhibiting effect on cell proliferation with a CI index of (0.52-0.59). (Figure 26). The combination index was determined using the Chou and Talalay methods (57). The inhibition of cell growth by Ad.5/3- CTV in combination with doxorubicin may be caused by induction of apoptosis, as evidenced by the increase in PARP cleavage in the combination treatment group compared to the single treatment group (Fig. 27). The combination of Ad .5 /3 -CTV and cisplatin does not enhance the therapeutic efficacy of either agent alone on neuroblastoma (NB ) cells

NB cell lines (SK-N-AS and SK-N-SH) were infected with Ad.5/3 E1A (12.5 IU) or Ad.5/3- CTV (12.5 IU) and cultured for 24 hours. These cells were then retreated with various doses of cisplatin for 24 hours. MTT assay was used to determine cell proliferation and plot (Figures 28-31). Infection with Ad.5/3- CTV alone caused 30-40% growth inhibition in both test cell lines. The use of cisplatin alone caused 10-30% inhibition of cell growth. However, the combination of different concentrations of cisplatin with a fixed low concentration of Ad.5/3- CTV does not enhance the therapeutic efficacy of either agent alone. In addition, the CI determined using Chou and the Talalay method (57) indicates that this CI is antagonistic in the case of CI values ranging from 0.9 to 1.4 (Fig. 32). Furthermore, the Western blot analysis (apoptosis-inducing marker) for PARP cleavage shown in Figure 33 does not indicate any further increase in PARP cleavage when Ad.5/3-CTV is added to cisplatin.

In summary, the above study proposes a novel viral-chemical combinatorial approach that can provide enhanced therapeutic options for early and advanced neuroblastoma. The immunostimulatory properties of mda -7 /IL -24 indicate that this combination method can even enhance the activity by enhancing the immune response against tumor cells when used in vivo (12, 59). Materials and methods cells and reagents

Human neuroblastoma cell lines SK-N-AS and SK-N-SH were obtained from ATCC (Manassas, VA) and NB1691 cells were obtained from Dr. Alan Houghton of St. Jude Children's Research Hospital (Memphis, TN). The NB1691 cell line was identified using the "CellCheck" service provided by the Research Animal Diagnostic Laboratory and compared to the initial STR map generated by the collaborator (IDEXX BioResearch). After recovery, the cumulative culture duration of the cells (SK-N-SH and SK-N-AS) was less than 6 months. Early passage cells were used in all experiments. All cell lines were tested for mycoplasma contamination frequently using the Sigma Mycobacterium detection kit. SK-N-AS cells were cultured in DMEM containing non-essential amino acids, SK-N-SH cells were cultured with RPMI 1640 and NB1691 cells were supplemented with 10% fetal bovine serum (FBS), 50 units/ml penicillin and Incubate with 50 μg/mL streptomycin (Life Technologies Inc., Frederick, MD) in DMEM (Invitrogen, Carlsbad, CA). The cells were incubated at 37 ° C in a humidified 5% CO 2 atmosphere. Antibodies specific for the following antibodies were used in this study: AIF (#4642), ATM (#2873), pATM (ser 1981, #13050), BCL-2 (#2876), BAX (2772), PARP (#9542) , Caspase 3 (#9662), Caspase 8 (#9746), H2AX (#7631), and γ-H2AX (ser 139, #9718) (Cell signaling Technology, Boston, MA); MDA-7/IL -24 (#K101, GenHunter, Nashville, TN), HRP-conjugated secondary antibody (Dako, Carpinteria, CA), β-actin (#NB600-501, Novus Biologicals, Inc., Littleton, CO). The other materials used in this study were Transcriptor's first cDNA synthesis kit, in situ cell death detection kit, luciferin (#11684795910, Roche Applied Science, Indianapolis, IN), MTT cell growth assay kit (# CT02, Millipore Corporation, Billerica, MA), KU60019 (#SML1416) and KU-55933 (#SML-1109) (Sigma, St Louis, MO). Transfection with plastid

All transfection experiments were performed using FuGene HD transfection reagent according to the manufacturer's protocol (Roche, Indianapolis, IN). Briefly, plastid/siRNA was mixed with FuGene HD reagent (1:3 ratio) in 500 μL of serum-free medium and left for 30 minutes to form a complex. The complex was then added to a 100 mm dish with 2.5 mL serum-free medium (2 μg plastid per ml medium). After 6 hours of transfection, complete medium was added and the cells were cultured for a further 24 hours (1). Cell proliferation assay (MTT analysis )

Determination of cells using a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay as a measure of mitochondrial metabolic activity Growth rate, as described earlier (8). Cells were treated with mock, Ad.5/3-Null, Ad.5/3- E1A or the indicated dose of Ad.5/3- CTV and incubated at 37 °C. After 0-96 hours, the MTT reagent was added and the cells were incubated for 4 hours at 37 °C. After the medium was removed, the formazan crystals were dissolved in DMSO, and the absorbance at 550 nm was read using a micro-quantitative disc spectrometer and the results are graphically represented. Terminal deoxynucleotidyl transferase-mediated gap labeling (TUNEL ) analysis

Neuroblastoma cells and mocks, Ad.5/3-Null, Ad.5/3- E1A or Ad.5/ were evaluated using the TUNEL enzyme reagent following the manufacturer's instructions and as previously described (50). Induction of apoptosis in xenograft tumor tissue sections of 3- CTV- treated mice. Briefly, 5×10 ³ neuroblastoma cells were cultured and treated with the mimic, Ad.5/3-Null, Ad.5/3- E1A or Ad.5/3- CTV for 72 hours. Immobilized in 4% paraformaldehyde in PBS for 1 hour at room temperature (RT) and permeabilized on ice with 0.1% Triton-X 100 formulated with 0.1% sodium citrate formulated in PBS. 2 minutes (for cells) or 10 minutes (for tissue sections). The samples were incubated for 1 hour in the TUNEL reaction mixture in a humidified atmosphere at 37 ° C in the dark. Images were captured and counted using an Olympus Research Fluorescence Microscope attached to a CCD camera. Positively stained apoptotic cells in each of the tumor tissues from 3 animals were counted from 5 microscope fields. Western ink point method

Western blot analysis was performed as previously described (50). Briefly, neuroblastoma cells treated with mock, Ad.5/3-Null, Ad.5/3- E1A or Ad.5/3- CTV were dissolved in radioimmunoprecipitation assay (RIPA) to dissolve In the buffer, the lysis buffer contained 1 mM sodium orthovanadate, 0.5 mM PMSF, 10 μg/mL aprotinin, and 10 μg/mL anti-plasmin peptide. An equal amount of total protein fraction in the lysate was resolved by SDS-PAGE and transferred to a PVDF membrane. The dots were blocked with 5% skim milk powder / 5% BSA and probed overnight with primary antibody followed by HRP-conjugated secondary antibody. The chemiluminescent signal is detected using an ECL system. All blots were re-examined with beta-actin antibody to confirm equal loading. In vivo study

To directly assess the effect of the mimic, Ad.5/3-Null, Ad.5/3- E1A or Ad.5/3- CTV on tumor growth in vivo, 5×10 ⁶ NB1691 cells were subcutaneously implanted into 4 Two flank of 6 week old athymic nude mice. Seven days after tumor cell implantation, when the tumor reached a palpable size, the left abdomen tumor was injected with 8 intratumoral injections, Ad.5/3-Null, Ad.5/3- E1A or Ad.5. /3- CTV stimulation for three weeks (3 injections per 2 weeks and 2 injections in the last week). Tumor growth in mice was monitored every other day by measuring the size of each flank tumor by caliper until the experiment was completed. Each treatment group has two groups of animals. One group was sacrificed on the 2nd day after the last dose was administered (1 mouse per group) and the other group (N=5) was followed until the tumor control group reached its point of need to be sacrificed (according to the IACUC protocol). After the experiment was completed, the tumor was fixed and sections were used for immunohistochemical analysis. Statistical analysis

All data were presented as mean ± standard deviation (SD) of at least three independent experiments (at least three times each). Multiple comparisons were made using a combination of single factor variance analysis (ANOVA) and Tukey post hoc testing. Statistical differences were presented at a probability level of p < 0.05, p < 0.01, and p < 0.001. Ad .5 /3 - Combination study of CTV and other therapeutic agents

Neuroblastoma cell lines (SK-N-AS and SK-N-SH) were infected with Ad.5/3 E1A (12.5 IU) or Ad.5/3- CTV (12.5 IU) and cultured for 24 hours. These cells were then retreated with various doses of poly[IC]-PEI, doxorubicin or cisplatin for 24 hours. Cell proliferation was determined and plotted using MTT assay. The combination index (CI) of various combination treatments was determined using the Chou and Talalay methods (57). Western blot analysis was performed to compare PARP cleavage between the combination treatment group and the single treatment group. In addition to poly[IC]-PEI and doxorubicin studied in the present invention, other therapeutic agents known to affect neuroblastoma cells can be used in combination with Ad.5/3- CTV . 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Enhanced delivery of mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) improves therapeutic efficacy in low CAR prostate cancer cells. Cancer gene therapy 2010;17( 7): 447-56. 21. Sarkar D, Su ZZ, Vozhilla N, Park ES, Gupta P, Fisher PB. Dual cancer-specific targeting strategy cures primary and distant breast carcinomas in nude mice. Proceedings of the National Academy of Sciences Of the United States of America 2005;102(39):14034-9. 22. Su ZZ, Sarkar D, Emdad L, Duigou GJ, Young CS, Ware J, et al. Targeting gene expression selectively in cancer cells by using the Progress-elevated gene-3 promoter. Proceedings of the National Academy of Sciences of the United States of America 2005;102(4):1059-64. 23. Aza b BM, Dash R, Das SK, Bhutia SK, Sarkar S, Shen XN, et al. Enhanced prostate cancer gene transfer and therapy using a novel serotype chimera cancer terminator virus (Ad.5/3-CTV). Journal of cellular physiology 2014;229(1):34-43. 24. 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Figure 1A and Figure 1B show the inhibitory effect of Ad.5/3- CTV on the growth of neuroblastoma cells. In Figure 1A , neuroblastoma cells were infected with Ad.5/3-Null (25 pfu), Ad.5/3- E1A (25 pfu) or Ad.5/3- CTV (12.5 or 25 pfu). The E1A and MDA-7/IL-24 proteins in the cell lysate were evaluated by Western blotting using an antibody at specific hours. In Figure IB , neuroblastoma cells were plated in quadruplicate in 96-well plates and infected with virus as above for the indicated time. MTT assays were used to measure cell growth and expressed as relative proliferation rates (compared to control cells). *, p < 0.01 vs. control.

2A- 2C show that Ad.5/3- CTV induces apoptosis in neuroblastoma cells. In Figure 2A , the neuroblastoma cell line was cultured in 8-well chamber slides with 25 pfu of Ad.5/3-Null or Ad.5/3- E1A or the indicated dose of Ad.5/3- CTV Handle for 72 hours. Cells were fixed and subjected to TUNEL analysis. Data are presented as TUNEL positive cells in a microscope field (compared to untreated control cells). In Figure 2B , neuroblastoma cells were infected for 72 hours as described above and collected and subjected to FACS analysis, where the DNA content was stained with propidium iodide and the data was presented from three independent experiments, presented graphically. Column chart: The average of three replicates. Bar graph: SD, *, p < 0.001 vs. control group. In Figure 2C , neuroblastoma cells were treated as described above for 72 hours. Cells were harvested and Western blot analysis was performed on PARP using specific antibodies and β-actin as internal references.

Figures 3A- 3C show that Ad.5/3- CTV induces caspase-independent cell death in neuroblastoma cells. In Figure 3A , neuroblastoma cells were infected with 25 pfu of Ad.5/3-Null or Ad.5/3- E1A or the indicated dose of Ad.5/3- CTV for 72 hours. Cells were harvested and Western blot analysis was performed against caspase-3 and caspase-9 using specific antibodies and β-actin as internal references. Staurosporine acts as a positive control for caspase 3 activation. In Figure 3B , neuroblastoma cells were treated as above for 72 hours, collected and assayed for caspase-3 activation according to the manufacturer's protocol. Staurosporine served as a positive control. The results represent three independent experiments, displayed graphically. Column chart: average of three replicates; bar chart, SD. (C) Neuroblastoma cells were pretreated with 20 μM Z-VAD-FMK and infected for 72 hours as described above. Cells were harvested and Western blot analysis was performed on PARP using specific antibodies and β-actin as internal references. The results represent three independent experiments.

FIG. 4A -4D shows Ad.5 / 3- CTV promote neuroblastoma cells cell death mediated by AIF. In Figure 4A , neuroblastoma cells were infected with 25 pfu of Ad.5/3-Null or Ad.5/3- E1A or the indicated dose of Ad.5/3- CTV for 72 hours. Cells were harvested and Western blot analysis was performed on AIF using specific antibodies and β-actin as internal references. The results represent three independent experiments. In Figure 4B , neuroblastoma cells were cultured in 8-well chamber slides and treated as described above in Figure 4A for 72 hours. These cells were then subjected to immunofluorescence analysis of AIF using anti-AIF antibodies and Alexa Flour-594 secondary antibody (red fluorescence). The nuclei were stained with DAPI (blue fluorescence). Fluorescent cells were visually inspected and photographed from 10 different fields and representative images were drawn. In Figure 4C , Western blot analysis was used to determine the subcellular distribution of AIF. Neuroblastoma cells were infected with 25 pfu of Ad.5/3- E1A or the indicated dose of Ad.5/3- CTV for 72 hours. The cytoplasm and nuclear fraction were isolated and AIF was examined by Western blotting using specific antibodies. HDAC3 acts as an internal reference for nuclear extracts and β-actin acts as an internal reference for cytoplasmic extracts. In Figure 4D , neuroblastoma cells were pretreated with AIF inhibitor and infected with 25 pfu of Ad.5/3- E1A or the indicated dose of Ad.5/3- CTV for 48 hours. Cells were harvested and Western blot analysis was performed for AIF and PARP using specific antibodies and β-actin as internal references. The results represent three independent experiments.

Figures 5A- 5E show that Ad.5/3- CTV requires ATM and gamma-H2AX phosphorylation to induce AIF-mediated cell death. Neuroblastoma cells were infected with 25 pfu of Ad.5/3-Null or Ad.5/3- E1A or the indicated dose of Ad.5/3- CTV for 72 hours. In Figure 5A , cells were harvested and specific antibodies were used and β-actin was used as an internal reference, and Western blotting was performed for γ-H2AX and H2AX. In Figure 5B , Western blotting was performed using specific antibodies and β-actin as internal references to determine pATM and ATM protein content. In Figure 5C , neuroblastoma cells were either untreated or treated overnight with KU-60019 (3 μM) and infected with 25 pfu Ad.5/3- E1A or the indicated dose of Ad.5/3- CTV for 48 hours. Cells were harvested and Western blotting was performed against MDA-7/IL-24, pATM, γ-H2AX, AIF, and PARP using specific antibodies and β-actin as internal references. The results represent three independent experiments. In Figure 5D , neuroblastoma cells were pretreated with AIF inhibitor and infected with 25 pfu of Ad.5/3- E1A or the indicated dose of Ad.5/3- CTV for 48 hours. Cells were harvested and Western blot analysis was performed on pATM using specific antibodies and β-actin as internal references. The results represent three independent experiments. In Figure 5E , neuroblastoma cells were either untreated or treated overnight with KU-60019 (3 μM) and with 25 pfu of Ad.5/3-Null or Ad.5/3- E1A or the indicated dose of Ad. 5/3- CTV infection for 48 hours. Cells were fixed and subjected to TUNEL analysis. TUNEL positive cells were counted and presented graphically according to TUNEL positive cells in each microscope field, bar graph: average number of TUNEL positive cells per 5 different microscope fields; bar graph, SD, *, p <0.001 vs. control; @, p<0.01 vs. the corresponding dose of Ad.5/3- CTV alone.

FIG 6A -6C shows that intratumoral injection Ad.5 / 3- CTV-induced cell death mediated by AIF and inhibits human xenografts neuroblastoma tumor growth. NB1691 human neuroblastoma cells were subcutaneously implanted into the bilateral abdomen of nude mice and the left tumor was treated with 8 intratumoral injections, including the mimetic (solvent), Ad.5/, as described in the materials and methods below. 3- E1A or Ad.5/3- CTV . A total of 6 study animals in each group. Once the tumor of the control animal reached the maximum allowable limit, the collected tumor was fixed in the formalin and embedded in paraffin. In Figure 6A , tumor volumes of the left abdomen and right abdomen were quantified and the results presented graphically. The line represents the mean of all tumor volumes for each group at the indicated time points; bar graph, SD, *, p < 0.05 vs. control; **, p < 0.001 vs. control. In Figure 6B , the formalin-fixed, paraffin-embedded tissue sections were stained for H&E and TUNEL according to standard methods; representative images of the designated treatment groups are depicted. In Figure 6C , immunohistochemical analysis of MDA-7/IL-24, AIF, γ-H2AX, and pATM in tumor sections was performed as described in Materials and Methods below. Representative sections are shown.

Figure 7 shows that ATM and γ-H2AX phosphorylation induce AIF-mediated cell death in Ad.5/3- CTV- treated neuroblastoma cells. Ad.5/3- CTV induces cell death in neuroblastoma cells.

Figures 8A- 8B illustrate the situation in which neuroblastoma cells express adenoviral receptors. In Figure 8A , neuroblastoma cells were cultured to 60-70% confluence and cells were harvested and stained for CAR, CD46, and desmoglein surface receptors using FACS analysis. The results are presented as a percentage of positive cells in the total cell population. The bar chart represents 3 independent experiments; the bar chart represents SD. In Figure 8B , cells were harvested at 60-70% confluence and Western blotting was performed on CAR, CD46, and desmoglein using specific antibodies.

Figure 9 shows that Ad.5/3- CTV induces pro-apoptotic molecules and down-regulates anti-apoptotic molecules. Neuroblastoma cells were infected with Ad.5/3-Null (25 pfu), Ad.5/3- E1A (25 pfu) or Ad.5/3- CTV (12.5 or 25) for 72 hours, and cells were harvested and used. Specific antibodies and β-actin served as internal references, and Western blot analysis was performed for pJNK, JNK, BCL-2, BAX, and BCL-xL. The results represent three independent experiments.

Figure 10 shows that Ad.5/3- CTV induces caspase-independent cell death in neuroblastoma cells. Neuroblastoma cells were infected with Ad.5/3-Null (50 pfu), Ad.5/3- E1A (50 pfu) or Ad.5/3- CTV (25 or 50 pfu) for 72 hours. Cell lysates were prepared and Western blot analysis was performed on caspase-8 using specific antibodies. --actin acts as an internal reference.

Figure 11 shows that Ad.5/3- CTV increases p53 levels in neuroblastoma cells. Neuroblastoma cells were infected with Ad.5/3-Null (25 pfu), Ad.5/3- E1A (25 pfu) or Ad.5/3- CTV (12.5 pfu, 25 pfu or 50 pfu) for 72 hours. . Cells were harvested and Western blotting was performed on p53 using specific antibodies and β-actin as internal references. The results represent three independent experiments.

Figure 12 depicts nuclear translocation of gamma-H2AX in Ad.5/3- CTV treated neuroblastoma cells. Neuroblastoma cells were cultured in 8-well chamber slides with Ad.5/3-Null (25 pfu), Ad.5/3- E1A (25 pfu) or Ad.5/3- CTV (25 or 50) Pfu) was treated for 72 hours. These cells were then subjected to immunofluorescence analysis of the γ-H2AX distribution using an anti-γ-H2AX antibody and an Alexa Fluor-594 secondary antibody (red fluorescence). The nuclei were stained with DAPI (blue fluorescence). Fluorescent cells were visually visualized and photographed from 10 different fields and representative images are depicted in this figure.

Figure 13 shows that Ad.5/3- CTV- infected neuroblastoma cells promote nuclear translocation of pATM. Neuroblastoma cells were cultured in 8-well chamber slides with Ad.5/3-Null (25 pfu), Ad.5/3- E1A (25 pfu) or Ad.5/3- CTV (25 or 50) Pfu) was treated for 72 hours. These cells were then subjected to immunofluorescence analysis of pATM distribution using anti-pATM antibodies and Alexa Fluor-594 secondary antibody (red fluorescence). The nuclei were stained with DAPI (blue fluorescence). Fluorescent cells were visually visualized and photographed from 10 different fields and representative images are depicted in this figure.

Figure 14 shows that ATM inhibitors rescue neuroblastoma cells from Ad.5/3- CTV- induced cell death. Neuroblastoma cells were either untreated or treated overnight with KU-55933 (5 μM) and used Ad.5/3-Null (25 pfu), Ad.5/3- E1A (25 pfu) or Ad.5/3 - CTV (25 or 50 pfu) was infected for 48 hours, cells were fixed and subjected to TUNEL analysis. TUNEL positive cells under each microscope field were counted and presented graphically according to TUNEL positive cells, bar graph: mean number of TUNEL positive cells per 5 different microscope fields; bar graph, SD. *, p < 0.001 vs. control; @, p < 0.01 vs. the corresponding dose of Ad.5/3- CTV alone.

Figure 15 depicts the effect of Ad.5/3- CTV on breast cancer and MDA-7/IL-24 and AIF expression in melanoma cells. Breast cancer (MDA-MB-231 and ZR-751) or melanoma (C8161 and SK-Mel) cells were untreated (control) or with Ad.5/3- E1A (25 pfu) or Ad.5/3- CTV (12.5 or 25 pfu) infection for 72 hours and assessment of MDA-7/IL-24 or AIF expression in cell lysates by Western blot analysis. --actin acts as an internal reference.

Figure 16 depicts the absorbance measured under different processing conditions in an MTT assay. SK-N-AS cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU) for 24 hours and then further treated with the indicated dose of poly[IC]-PEI for 24 hours. . The MTT analysis was then performed according to standard methods.

Figure 17 depicts the % cell proliferation under different treatment conditions. SK-N-AS cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU) for 24 hours and then further treated with the indicated dose of poly[IC]-PEI for 24 hours. . The MTT analysis was then performed according to standard methods. Use 100% as a control value and calculate other values accordingly.

Figure 18 depicts the absorbance measured under different processing conditions in an MTT assay. SK-N-SH cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU) for 24 hours and then further treated with the indicated dose of poly[IC]-PEI for 24 hours. . The MTT analysis was then performed according to standard methods.

Figure 19 depicts % cell proliferation under different treatment conditions. SK-N-SH cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, the cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU) for 24 hours and then further treated with the indicated dose of poly[IC]-PEI. hour. The MTT analysis was then performed according to standard methods. Use 100% as a control value and calculate other values accordingly.

Figure 20 depicts the combination index of Ad.5/3 CTV-poly[IC]-PEI.

Figure 21 depicts Western blot analysis for Ad.5/3 CTV-poly[IC]-PEI. About ² ×10 ⁶ neuroblastoma cells (SK-N-AS or SK-N-SH) cells were cultured for 24 hours. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU) for 24 hours and then further with poly[IC]-PEI (0.5 ug/ml). Handle for 24 hours. Cells were then harvested, the proteins isolated were lysed and used for Western blot analysis.

Figure 22 depicts the absorbance measured under different processing conditions in an MTT assay. SK-N-AS cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU), cultured for 24 hours and then further treated with the indicated dose of doxorubicin for 24 hours. The MTT analysis was then performed according to standard methods.

Figure 23 depicts % cell proliferation under different treatment conditions. SK-N-AS cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU), cultured for 24 hours and then further treated with the indicated dose of doxorubicin for 24 hours. The MTT analysis was then performed according to standard methods. Use 100% as a control value and calculate other values accordingly.

Figure 24 depicts the absorbance measured under different processing conditions in an MTT assay. SK-N-SH cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU), cultured for 24 hours and then further treated with the indicated dose of doxorubicin for 24 hours. The MTT analysis was then performed according to standard methods.

Figure 25 depicts the % cell proliferation under different treatment conditions. SK-N-SH cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU), cultured for 24 hours and then further treated with the indicated dose of doxorubicin for 24 hours. The MTT analysis was then performed according to standard methods. Use 100% as a control value and calculate other values accordingly.

Figure 26 depicts the combination index of Ad.5/3 CTV-Doxorubicin.

Figure 27 depicts Western blot analysis for Ad.5/3 CTV-Doxorubicin. About ² ×10 ⁶ neuroblastoma cells (SK-N-AS or SK-N-SH) were cultured for 24 hours. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU), cultured for 24 hours and then further treated with doxorubicin (5 uM) for 24 hours. Cells were then harvested, the proteins isolated were lysed and used for Western blot analysis.

Figure 28 depicts the absorbance measured under different processing conditions in an MTT assay. SK-N-AS cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU), cultured for 24 hours and then further treated with the indicated dose of cisplatin for 24 hours. The MTT analysis was then performed according to standard methods.

Figure 29 depicts % cell proliferation under different treatment conditions. SK-N-AS cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU), cultured for 24 hours and then further treated with the indicated dose of cisplatin for 24 hours. The MTT analysis was then performed according to standard methods. Use 100% as a control value and calculate other values accordingly.

Figure 30 depicts the absorbance measured under different processing conditions in an MTT assay. SK-N-SH cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU), cultured for 24 hours and then further treated with the indicated dose of cisplatin for 24 hours. The MTT analysis was then performed according to standard methods.

Figure 31 depicts % cell proliferation under different treatment conditions. SK-N-SH cells were cultured for 24 hours (60-70% confluence), then the cells were trypsinized and approximately 5,000 cells were plated in each well of a 96-well plate. MTT analysis was performed for each treatment using 8 wells. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU), cultured for 24 hours and then further treated with the indicated dose of cisplatin for 24 hours. The MTT analysis was then performed according to standard methods. Use 100% as a control value and calculate other values accordingly.

Figure 32 depicts the combination index of Ad.5/3 CTV-cisplatin.

Figure 33 depicts Western blot analysis for Ad.5/3 CTV-cisplatin. About ² ×10 ⁶ neuroblastoma cells (SK-N-AS or SK-N-SH) were cultured for 24 hours. Once the cells were attached to the wells, cells were treated with Ad.5/3 E1A (12.5 IU) or Ad.5/3 CTV (12.5 IU), cultured for 24 hours and then further treated with cisplatin (7.5 ug) for 24 hours. Cells were then harvested, the proteins isolated were lysed and used for Western blot analysis.

<110> Virginia Commonwealth University <120> Combination therapy with MDA-7/IL-24 and neuroblastoma for therapeutic agents <130> 2207-A-TW<150> US 62/315,126<151> 2016 -03-30<160> 3 <170> PatentIn version 3.5<210> 1<211> 961<212> DNA<213> Artificial sequence<220><223> Description of artificial sequence: synthetic polynucleotide<400> 1catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 120gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 180gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 300agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta gggagatctc 360agagaggaga gagaaagaga aagagaatgg gacagcatgt gactgcctga tgaagttggc 420gtgcttgctc aaaagttctg cgagattgac ggctctctgg atttgagcca aggacacgcc 480tgggaagcca cggtgacctc Acaaggcccg gaatctccgc gagaatttca gtgttgtttt 540cctctctcca cctttctcag ggacttccga aactccgcct ctccggtgac gtcagcatag 600cgctgcgtcg gtacccacga g tggccatcg attcgacgtg tatttatacc cggtgagttc 660ctcaagaggc cactcttgag tgccagcgag tagagttttc tcctccgagc cgctccgaca 720ccgggactga aaatgagaca tattatctgc cacggaggtg ttattaccga agaaatggcc 780gccagtcttt tggaccagct gatcgaagag gtactggctg ataatcttcc acctcctagc 840cattttgaac cacctaccct tcacgaactg tatgatttag acgtgacggc ccccgaagat 900cccaacgagg aggcggtttc gcagattttt cccgactctg taatgttggc ggtgcaggaa 960g 961 <210> 2 <211> 2156 <212> DNA <213 > artificial sequence <220> <223> Description of artificial sequence: synthetic polynucleotide <400> 2ccacatggtg gcagatgctg cattcgaaaa cgtttgaatt gataattatt atcatttgcg 60ggtcctttcc ggcgatccgc cttgttacgg ggcggcgacc tcgcgggttt tcgctattta 120tgaaaatttt ccggtttaag gcgtttccgt tcttcttcgt cataacttaa tgtttttatt 180taaaataccc tctgaaaaga aaggaaacga caggatcttc tagacccggg agcggccgct 240gtcgacattg attattgact agttattaat agtaatcaat Tacggggtca ttagttcata 300gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 360ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 420ggactttcca ttgacg tcaa tgggtggagt atttacggta aactgcccac ttggcagtac 480atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 540cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 600tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 660agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 720tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 780aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc tggctaacta 840gagaacccac tgcttactgg cttatccaaa ttaatacgac tcactatagg gagacccaag 900ctggctagcg tttaaactta agcttggtac cgagctcgga tccactagta acggccgcca 960gtgtgctgga actcggctta caagacatga ctgtgatgag gagctgcttt cgccaattta 1020acaccaagaa gaattgaggc tgcttgggag gaaggccagg aggaacacga gactgagaga 1080tgaattttca acagaggctg caaagcctgt ggactttagc cagacccttc tgccctcctt 1140tgctggcgac agcctctcaa atgcagatgg ttgtgctccc ttgcctgggt tttaccctgc 1200ttctctggag ccaggtatca ggggcccagg gccaagaatt ccactttggg ccctgccaag 1260tgaagggggt tgttccccag aaactgtggg aagccttctg gg ctgtgaaa gacactatgc 1320aagctcagga taacatcacg agtgcccggc tgctgcagca ggaggttctg cagaacgtct 1380cggatgctga gagctgttac cttgtccaca ccctgctgga gttctacttg aaaactgttt 1440tcaaaaacta ccacaataga acagttgaag tcaggactct gaagtcattc tctactctgg 1500ccaacaactt tgttctcatc gtgtcacaac tgcaacccag tcaagaaaat gagatgtttt 1560ccatcagaga cagtgcacac aggcggttcc tgctattccg gagagcattt aaacagttgg 1620acgtagaagc agctctgacc aaagcccttg gggaagtgga cattcttctg acctggatgc 1680agaaactcta caagctctga atgtctagac caggacctcc ctccccctgg cactggtttg 1740ttccctgtgt catttcaaac agtctaagcc gaattctgca gatatccatc acactggcgg 1800ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt gccttctagt 1860tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact 1920cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat 1980tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc 2040aggcatgctg gggatgcggt gggctctatg gcttcgcggc cgcaatcact agtgaattcg 2100cggccgcctg caggtcggat ccgaattcga tatcactagt ggtacccacc cagtgg 2156 <210> 3<211> 2356<212> DNA<213> Artificial sequence <220><223> Description of artificial sequence: synthetic polynucleotide<400> 3tggaatgtca gtttcctcct gttcctgtcc atccgcaccc actatcttca tgttgttgca 60gatgaagcgc gcaagaccgt ctgaagatac cttcaacccc gtgtatccat atgacacgga 120aaccggtcct ccaactgtgc cttttcttac tcctcccttt gtatccccca atgggtttca 180agagagtccc cctggggtac tctctttgcg cctatccgaa cctctagtta cctccaatgg 240catgcttgcg ctcaaaatgg gcaacggcct ctctctggac gaggccggca accttacctc 300ccaaaatgta accactgtga gcccacctct caaaaaaacc aagtcaaaca taaacctgga 360aatatctgca cccctcacag ttacctcaga agccctaact gtggctgccg ccgcacctct 420aatggtcgcg ggcaacacac tcaccatgca atcacaggcc ccgctaaccg tgcacgactc 480caaacttagc attgccaccc aaggacccct cacagtgtca gaaggaaagc tagccctgca 540aacatcaggc cccctcacca ccaccgatag cagtaccctt actatcactg cctcaccccc 600tctaactact gccactggta gcttgggcat tgacttgaaa Gagcccattt atacacaaaa 660tggaaaacta ggactaaagt acggggctcc tttgcatgta acagacgacc taaacacttt 720gaccgtagca actggtccag gtgtgactat taataatact tccttgcaaa ctaaagttac 78 0tggagccttg ggctttgatt cacaaggcaa tatgcaactt aatgtagcag gaggactaag 840gattgattct caaaacagac gccttatact tgatgttagt tatccgtttg atgctcaaaa 900ccaactaaat ctaagactag gacagggccc tctttttata aactcagccc acaacttgga 960tattaactac aacaaaggcc tttacttgtt tacagcttca aacaattcca aaaagcttga 1020ggttaaccta agcactgcca aggggttgat gtttgacgct acagccatag ccattaatgc 1080aggagatggg cttgaatttg gttcacctaa tgcaccaaac acaaatcccc tcaaaacaaa 1140aattggccat ggcctagaat ttgattcaaa caaggctatg gttcctaaac taggaactgg 1200ccttagtttt gacagcacag gtgccattac agtaggaaac aaaaataatg ataagctaac 1260cctatggaca gctccaaaac cagaagccaa ctgcataatt gaatacggga aacaaaaccc 1320agatagcaaa ctaactttaa tccttgtaaa aaatggagga attgttaatg gatatgtaac 1380gctaatggga gcctcagact acgttaacac cttatttaaa aacaaaaatg tctccattaa 1440tgtagaacta tactttgatg ccactggtca tatattacca gactcatctt ctcttaaaac 1500agatctagaa ctaaaataca agcaaaccgc tgactttagt gcaagaggtt ttatgccaag 1560tactacagcg tatccatttg tccttcctaa tgcgggaaca cataatgaaa attatatttt 1620tggtcaatgc tactacaaag caagcgatgg tgcccttttt ccgttggaag ttactgttat 1680gcttaataaa acacggtttc 2100ctgtcgagcc aaacgctcat cgcctgccag atagtcgcac atcctatgtt atgacttttt tattggtcct 1740tgaatgctgg tctagctcca gaaactactc aggcaaccct cataacctcc ccatttacct 1800tttcctatat tagagaagat gactaataaa ctctaaagaa tcgtttgtgt tatgtttcaa 1860cgtgtttatt tttcaattgc agaaaatttc aagtcatttt tcattcagta gtatagcccc 1920accaccacat agcttataca gatcaccgta ccttaatcaa actcacagaa ccctagtatt 1980caacctgcca cctccctccc aacacacaga gtacacagtc ctttctcccc ggctggcctt 2040aaaaagcatc atatcatggg taacagacat attcttaggt gttatattcc cagtgatatt aataaactcc Ccgggcagct cacttaagtt 2160catgtcgctg tccagctgct gagccacagg ctgctgtcca acttgcggtt gcttaacggg 2220cggcgaagga gaagtccacg cctacatggg ggtagagtca taatcgtgca tcaggatagg 2280ggtggtgctg cagcagcgcg cgaataaact gcttgcggcc gcggctccgt cctgcaggaa 2340tacaacatgg cagtgg 2356

(no)

Claims (22)

5/3- CTV in combination with a medicament for use in a method of treating cancer in an individual, wherein the Ad.5/3- CTV synergizes with the agent to induce cancer cell growth inhibition, toxic autophagy, and cell wilting Die. The combination of claim 1, wherein the Ad.5/3- CTV comprises an adenoviral vector Ad.5/3 and a nucleic acid that exhibits MDA-7/IL-24. The combination of claim 1, wherein the agent is selected from the group consisting of polyinosinic acid-PEI (poly[IC]-PEI), doxorubicin, and others capable of promoting toxic autophagy and/or apoptosis. Pharmacy. A combination of claim 2, wherein MDA-7/IL-24 is expressed in said cancer cells to modulate AIF, ATM, and γ-H2AX to induce apoptosis. A combination of claim 1, wherein the Ad.5/3- CTV is administered concurrently with the pharmaceutical system. A combination of claim 1 wherein the agent is administered within 12-96 hours after administration of Ad.5/3- CTV . The combination of any one of claims 1 to 6, wherein the cancer is a neuroblastoma. The combination of any one of claims 1 to 6, wherein the cancer is melanoma, glioblastoma, prostate cancer, breast cancer, colorectal cancer, lung cancer or pancreatic cancer. A combination of claim 7, wherein the agent is poly[IC]-PEI. A combination of claim 7, wherein the agent is doxorubicin. A combination of claim 3, wherein the agent induces one or more of MDA-5, NOXA, and RIG-I. A method of treating cancer in an individual comprising administering to the individual an effective amount of Ad.5/3- CTV and an agent, wherein the Ad.5/3- CTV and the agent produce synergy to induce cancer cell growth inhibition and apoptosis effect. The method of claim 12, wherein the Ad.5/3- CTV comprises an adenoviral vector Ad.5/3 and a nucleic acid that exhibits MDA-7/IL-24. The method of claim 12, wherein the agent is selected from the group consisting of poly[IC]-PEI, doxorubicin, and other agents capable of promoting toxic autophagy and/or apoptosis. The method of claim 13, wherein the expression of MDA-7/IL-24 in the cancer cells modulates AIF, ATM, and γ-H2AX to induce apoptosis. The method of claim 12, wherein the Ad.5/3- CTV is administered concurrently with the pharmaceutical system. The method of claim 12, wherein the agent is administered 12-96 hours after administration of Ad.5/3- CTV . The method of any one of claims 12 to 17, wherein the cancer is a neuroblastoma. The method of any one of claims 12 to 17, wherein the cancer is melanoma, glioblastoma, prostate cancer, breast cancer, colorectal cancer, lung cancer or pancreatic cancer. The method of claim 18, wherein the agent is poly[IC]-PEI. The method of claim 18, wherein the agent is doxorubicin. The method of claim 14, wherein the agent induces one or more of MDA-5, NOXA, and RIG-I.