CN116509908A - Application of small molecular compound and pharmaceutical composition thereof - Google Patents

Abstract

The invention belongs to the field of tumor treatment, and in particular relates to a pharmaceutical composition for treating glioblastoma and application of a compound for up-regulating HIF-alpha expression, wherein the composition comprises the following components: oncolytic viruses and compounds for upregulating HIF-alpha expression, wherein the oncolytic virus is a genetically engineered herpes simplex virus or adenovirus. In vitro and in vivo experiments show that the method can obviously induce the death of drug-resistant glioblastoma cell iron by up-regulating the expression of HIF-alpha, inhibit the growth of glioblastoma in tumor-bearing mice, obviously prolong the survival time of the mice, cause no obvious organ toxicity, and can improve the anti-tumor effect by combining the compound for up-regulating the expression of HIF-alpha with oncolytic viruses.

Description

Application of small molecular compound and pharmaceutical composition thereof

Technical Field

The invention relates to the field of tumor treatment, in particular to application of a small molecular compound for up-regulating HIF-alpha expression and a pharmaceutical composition thereof.

Background

Glioblastoma (GBM) is the most common invasive primary brain tumor in adults, accounting for more than 30% of intracranial tumors. Although standard multimodal interventions involve maximal excision, combined with radiotherapy and chemotherapy with the alkylating agent Temozolomide (TMZ), the expected survival of most GBM patients is less than 2 years. It has been found that GBM cells are susceptible to resistance to apoptosis, leading to tumor recurrence and treatment failure, and new therapeutic strategies are needed to improve patient prognosis.

Iron death is a newly discovered form of regulated cell death. Iron death is iron dependent compared to apoptosis, and is characterized by increased free iron accumulation and lipid peroxidation, leading to cell death. It has been found that cancer cells resistant to conventional therapies, including those with strong interstitium and susceptibility to metastasis, are more susceptible to iron death. Activating iron death has been considered an effective anticancer mechanism. GBM cells exhibit higher levels of iron absorption and free iron, which means their inherent susceptibility to iron death. Furthermore, recent studies have demonstrated that promoting iron uptake in GBM can inhibit tumor growth and reduce drug resistance. These findings suggest that targeting iron death in GBM is a potentially viable anticancer strategy that helps optimize post-operative treatment of GBM.

Currently, inhibition of system xc-and GPX4 is the most common method of inducing iron death in cancer cells. However, these methods have not proven suitable for GBM. Sulfadiazine is an oral anti-inflammatory agent that inhibits system xc-and thus induces iron death, but it was found in clinical trials in patients with glioblastoma that it was not only poorly effective, but also may cause serious neurological side effects. Further studies have demonstrated that inhibition of system xc-not only inhibits cysteine influx, but also glutamate efflux, a key molecule involved in seizures and neuropathic pain, and thus inhibition of system xc-can lead to neurological adverse events. Regarding inhibition of GPX4, GPX4 gene knockout is embryonic lethal in mice, and GPX4 has important biological functions in cells, targeting potential adverse side effects associated with GPX4 has been considered as the greatest challenge for its clinical application, and thus clinical application is urgently needed to develop new targets in GBM to induce iron death of GBM.

Disclosure of Invention

In view of the shortcomings of the prior art, it is an object of the present invention to provide a pharmaceutical combination for the treatment of glioblastoma; it is a second object of the present invention to provide the use of a combination of an oncolytic virus and a compound for upregulating HIF- α expression in the preparation of a supplement or medicament for ameliorating or/and treating a tumor; it is a further object of the present invention to provide the use of a compound for upregulating HIF- α expression in the preparation of a supplement or medicament for ameliorating or/and treating a tumor.

To achieve the above object, in a first aspect, the present invention provides a pharmaceutical combination for treating glioblastoma, comprising: oncolytic viruses for use in treating glioblastoma and compounds for upregulating HIF- α expression, wherein the oncolytic viruses are genetically engineered herpes simplex viruses or adenoviruses.

In the above pharmaceutical composition for treating glioblastoma, as a preferred embodiment, the oncolytic virus is an oncolytic type 1 herpes simplex virus, or a recombinant virus obtained by genetic engineering on the basis of the oncolytic type 1 herpes simplex virus, or a recombinant virus obtained by genetic engineering on the basis of an Ad5 oncolytic adenovirus; preferably, the oncolytic virus is a recombinant virus obtained by genetic engineering on the basis of Ad5 oncolytic adenovirus, and the genome of the recombinant virus comprises: an E1A gene expression module, and/or an IL-15 functional gene module; more preferably, the E1A gene expression module comprises: the promoter of the E1A gene is the promoter of the Ki67 gene; E1A gene coding sequence; and a 5' UTR sequence of TGFbeta2, located upstream of the E1A gene coding sequence and downstream of the promoter of the Ki67 gene; the base sequence of the promoter of the Ki67 gene is shown as SEQ ID NO.1 in a sequence table; the base sequence of the 5' UTR of the TGF beta2 is shown as SEQ ID NO.2 in a sequence table; the promoter of the Ki67 gene is linked to the 5' UTR of the TGF beta2 to form a fusion sequence; the IL-15 functional gene module comprises an IL-15 gene coding sequence and an element for expressing the IL-15 gene, wherein the element for expressing the IL-15 gene comprises: the promoter of the IL-15 gene is a CMV promoter; a polyA sequence of SV 40; the base sequence of the IL-15 gene is shown as SEQ ID NO. 3.

In the above pharmaceutical combination for treating glioblastoma, as a preferred embodiment, the glioblastoma is a drug-resistant, preferably temozolomide-resistant glioblastoma; preferably, the glioblastoma is GL261, U87 or LN229 cell line.

Glioblastoma in the present invention can also be primary tumor cells derived from drug-resistant glioblastoma patients.

In the above pharmaceutical combination for treating glioblastoma, as a preferred embodiment, the compound for upregulating HIF- α expression is a prolyl hydroxylase inhibitor; preferably, the compound for up-regulating HIF- α expression is roflumilast FG-4592; more preferably, the HIF- α is HIF-2α and/or HIF-1α; HIF-2α is further preferred.

In the above pharmaceutical combination for treating glioblastoma, as a preferred embodiment, the pharmaceutical combination is in the form of a mixture of oncolytic virus and a compound for upregulating HIF- α expression; preferably, the pharmaceutical combination further comprises a pharmaceutically acceptable carrier; preferably, the dosage form of the pharmaceutical composition is a freeze-dried powder injection, an injection, a tablet, a capsule or a drop.

In the above pharmaceutical combination for treating glioblastoma, as a preferred embodiment, the pharmaceutical combination is in the form of separate packages of an oncolytic virus and a compound for up-regulating HIF-alpha expression.

In a second aspect, the invention provides the use of a combination of an oncolytic virus and a compound for upregulating HIF- α expression in the manufacture of a supplement or medicament for ameliorating or/and treating a tumor, wherein the oncolytic virus is a genetically engineered herpes simplex virus or adenovirus and the tumor is glioblastoma.

In the application of the second aspect of the present invention, as a preferred embodiment, the oncolytic virus is an oncolytic herpes simplex virus type 1, or a recombinant virus obtained by genetic engineering on the basis of the oncolytic herpes simplex virus type 1, or a recombinant virus obtained by genetic engineering on the basis of an Ad5 oncolytic adenovirus; preferably, the oncolytic virus is a recombinant virus obtained by genetic engineering on the basis of Ad5 oncolytic adenovirus, and the genome of the recombinant virus comprises: an E1A gene expression module, and/or an IL-15 functional gene module; more preferably, the E1A gene expression module comprises: the promoter of the E1A gene is the promoter of the Ki67 gene; E1A gene coding sequence; and a 5' UTR sequence of TGFbeta2, located upstream of the E1A gene coding sequence and downstream of the promoter of the Ki67 gene; the base sequence of the promoter of the Ki67 gene is shown as SEQ ID NO.1 in a sequence table; the base sequence of the 5' UTR of the TGF beta2 is shown as SEQ ID NO.2 in a sequence table; the promoter of the Ki67 gene is linked to the 5' UTR of the TGF beta2 to form a fusion sequence; the IL-15 functional gene module comprises an IL-15 gene coding sequence and an element for expressing the IL-15 gene, wherein the element for expressing the IL-15 gene comprises: the promoter of the IL-15 gene is a CMV promoter; a polyA sequence of SV 40; the base sequence of the IL-15 gene is shown as SEQ ID NO. 3;

And/or the glioblastoma is a drug resistant, preferably temozolomide resistant glioblastoma; preferably, the glioblastoma is GL261, U87 or LN229;

and/or the compound for upregulating HIF- α expression is a prolyl hydroxylase inhibitor; preferably, the compound for up-regulating HIF- α expression is roflumilast FG-4592, preferably, HIF- α is HIF-2α and/or HIF-1α; HIF-2α is further preferred.

In a third aspect, the invention provides the use of a compound for upregulating HIF- α expression in the preparation of a supplement or medicament for ameliorating or/and treating a tumor, the tumor being glioblastoma.

In the use according to the third aspect of the invention, as a preferred embodiment, the compound for upregulating HIF- α expression is a prolyl hydroxylase inhibitor; and/or the glioblastoma is a drug resistant, preferably temozolomide resistant glioblastoma;

preferably, the compound for up-regulating HIF- α expression is roflumilast FG-4592; and/or the glioblastoma is GL261, U87 or LN229;

more preferably, the HIF- α is HIF-2α and/or HIF-1α; HIF-2α is further preferred.

The invention has the following beneficial effects:

the invention uses the roflumilast to activate the HIF channel in the GBM cells, the roflumilast can obviously induce the iron death of the GBM cells so as to inhibit the growth of the GBM cells in vivo and in vitro, especially when the cells show TMZ resistance, the Luo Shasi in vivo can obviously prolong the survival time of the GBM mice with chemotherapy resistance, and obvious organ toxicity is not caused. Further evidence suggests that both HIF-1 and HIF-2 are contributing to iron death induction, with HIF-2 upregulation of lipid gene overexpression being the primary cause of roflumilast leading to excessive lipid peroxidation and ultimately iron death, HIF-2 being a potential therapeutic target for induction of iron death in chemotherapy-resistant GBM cells. The results of the present studies provide new insight into HIF signaling pathways regulating iron death and anti-cancer therapies targeting iron death in GBM. In particular roflumilast is expected to provide a new choice for refractory GBM treatment.

In addition, the roflumilast can also be used as a GBM oncolytic virus treatment synergist, and Luo Shasi can play a role in synergism when being combined with GBM oncolytic virus.

Drawings

FIG. 1, luo Shasi in vitro results of drug resistant GBM cell (GL 261 and U87) death, A.CCK8 cell activity assay for cell growth activity after 48h of each group of drug treated GBM cells, ordinate relative cell activity (Relative cell viability); LDH release assay to detect cell death after 72h of GBM cells treated with each group of drug, with relative LDH release (Relative LDH release) on the ordinate; C. cell growth status after GBM cells were treated for 48h for each group of drugs was observed under an optical microscope (ZEISS Axio Observer A1), scale: 100 μm; D. e, apoptosis staining experiments analyze apoptosis conditions of GBM cells treated by various groups of medicines for 72 hours, wherein Annexin V is a coupled fluorescent dye, PI is a nucleic acid dye, and the ordinate is: percent apoptosis (% cell apoptosis), abscissa: early apoptosis (Early apoptosis), late apoptosis (Late apoptosis) (< 0.05, < P <0.01, < P <0.001, ns no significant difference);

Fig. 2, luo Shasi results of induction of iron death by drug resistant GBM cells (GL 261 and U87) he (RXD), a. Cell growth status after 48h of each group of drug-treated GBM cells was observed under an optical microscope (ZEISS Axio Observer A), scale: 100 μm; CCK8 cell activity experiments to detect cell growth activity of each group of drugs after being treated with GBM cells for 48 hours, and the ordinate is relative cell activity (Relative cell viability); LDH release assay to detect cell death after 72h of GBM cells treated with each group of drug, with relative LDH release (Relative LDH release) on the ordinate; D. iron ion concentration in GBM cells of each treatment group, the ordinate is iron ion concentration (Iron concentration, unit: μmol/gprot); E. the GBM mitochondrial ultrastructure of each group was observed by transmission electron microscopy, scale: (cell: cell) 2. Mu.m, (Mitochondria: mitochondria) 1. Mu.m. P <0.05, < P <0.01, < P < 0.001);

fig. 3, luo Shasi results of lipid peroxidation accumulation and CRT translocation of drug-resistant GBM cells induced by him (RXD), a. Fluorescence microscopy of intracellular Oxidized (Oxidized) and reduced (Non-Oxidized) lipid content of each group of GBM cells after 48h of drug treatment, and overlap (Merge) of both, scale: 100 μm; B. flow cytometry analysis of intracellular lipid peroxidation after 48h of treatment of GMB cells with each group of drugs, wherein PE is (phycoerythrin) fluorescent dye, FITC is fluorescein isothiocyanate, ordinate: LPO positive cell percentage (% LPO positive cells); C. fluorescence microscopy observed expression of GBM cell surface CRT protein after 48h of drug treatment of each group, FITC: fluorescein isothiocyanate staining results, DAPI:4', 6-diamidino-2-phenylindole staining results, mere: overlap of FITC and DAPI staining results, scale: 100 μm; D. flow cytometry analysis of GBM cell surface CRT protein expression after 48h of drug treatment for each group, ordinate: CRT positive cell percentage (% CRT positive cells) (% P <0.05,% P <0.01,% P < 0.001);

Fig. 4, luo Shasi results of in vivo inhibition of drug resistant GBM tumor growth and significant prolongation of survival in mice, a. Results of in vivo imaging technique of mice monitoring tumor growth and quantitative analysis, bar graph ordinate: total fluorescence emission value (Total fluorescence), units; photons/second (p/s, photons/sec/cm) ² R abbreviation), abscissa: days after start of drug injection (Days after drugs injection); B. body weight record of each group after treatment start, ordinate: weight (Weight), unit: g, abscissa: days after start of drug injection (Days after drugs injection); C. survival curves, ordinate of mice of each group: survival (Percent survivinal) (number of mice per group n, n=5) (. P)<0.05，**P<0.01 Abscissa: days of tumor implantation (Days after tumor implantation);

FIG. 5, luo Shasi results of in vivo induction of tumor tissue lipid peroxidation levels and iron levels in RXD; A. histochemical staining analysis of Ki67, 4-HNE, HIF-1α, HIF-2α expression in tumor tissue of each group of mice, scale: 50 μm; B. prussian blue staining detects the trivalent iron content of tumor tissues of mice in each group, scale: 20 μm; C. the ferrous content of the tumor tissue of each group of mice, the ordinate being the ferrous ion concentration (Iron concentration, unit: μmol/gprot) (< 0.001) P;

Figure 6, graph of results of Luo Shasi HE (RXD) treatment without significant visceral toxicity in vivo, a. Mice of each group were stained for Heart (Heart), liver (lever), spleen (Spleen), lung (Lung) and Kidney (Kidney) tissue HE, scale: l00 μm; B. prussian blue staining of liver tissue of each group of mice, scale: 20 μm;

FIG. 7 is a graph of the results of HIF- α overexpression mediated RXD induction of iron death in drug resistant GBM cells, wherein si-NC is a blank control; a. QRT-PCR experiment detects siRNA molecule to interfere with mRNA expression result graphs of HIF-1 alpha and HIF-2 alpha in GBM cells, and the ordinate: relative mRNA expression level (Relative mRNAexpression); b, detecting protein expression result graphs in each group of GBM cells after siRNA molecules interfere HIF-1 alpha and HIF-2 alpha expression by a WB experiment, wherein ACTIN is taken as an internal reference; LDH release assay to detect GBM cell death in each treatment group, with relative LDH release (Relative LDH release) on the ordinate; D. iron ion concentration in GBM cells of each treatment group, the ordinate is iron ion concentration (Iron concentration, unit: μmol/gprot); E. fluorescence microscopy observed the intracellular Oxidized (Oxidized) and reduced (Non-Oxidized) lipid content of each group, as well as the overlap (Merge) of the two, scale: 100 μm; p <0.05, P <0.01, P <0.001, ns have no significant difference);

FIG. 8 is a graph of the results of HIF-2. Alpha. Regulated lipid regulatory genes inducing lipid peroxidation in drug resistant GBM cells. A. Flow cytometry was performed to determine the intracellular oxidized and reduced lipid content of GBM in each treatment group, ordinate: LPO positive cell percentage (% LPO positive cells); qpcr experiments to detect mRNA levels of CHAC1, ACSL4, PTGS2 in GBM cells of each treatment group, ordinate: relative mRNA expression level (Relative mRNAexpression); qpcr experiments to detect mRNA levels of HILPDA and PLIN2 in cells of each treatment group, ordinate: relative mRNA expression level (Relative mRNA expression) (< P <0.05, < P <0.01, < P < 0.001).

FIG. 9A. CCK8 cell Activity assay cell viability inhibition rates after 48h treatment of U251 and U87 cells with different concentrations of TMZ, ordinate: inhibition of cell activity (Cell viability suppression, unit:%); B. apoptosis staining experiments analysis of apoptosis after GBM cells were treated with each group of drugs for 72h, ordinate: percent apoptosis (% cell apoptosis), abscissa: early apoptosis (Early apoptosis), late apoptosis (Late apoptosis) (< 0.05, < P <0.01, < P <0.001, ns no significant difference).

FIG. 10, luo Shasi results of synergistic inhibition of drug resistant GBM cell growth by He (RXD) in combination with Oncolytic Virus (OV), A-B.CCK8 cell activity assay to measure cell growth activity after 72h of GBM cells treated with different RXD concentration gradients, ordinate is cell activity (% relative to solvent treated group); C-D.CCK8 cell Activity assay cell growth Activity after 72h gradient treatment of GBM cells with different oncolytic viruses MOI, the ordinate is cell Activity (cell viability%) relative to control treatment group; e, detecting the cell growth activity of each treatment group GBM cell after 72 hours of treatment by using a CCK8 cell activity experiment, wherein the ordinate is relative cell activity (Relative cell viability); cck8 cell activity assay to examine the growth inhibition of GBM cells in different combination mode groups after the same time relative to OV alone, with the ordinate representing inhibition of cell activity (cell viability suppression%), the results of the o3+r3 group and the o3+r2 group being relative to the inhibition of cell activity in the O3 group (3 days of OV alone), the results of the o4+r2 group being relative to the inhibition of cell activity in the O4 group (4 days of OV alone), the inhibition of cell activity= (1-combination OD/OD) 100%; gcck 8 cell activity assay to examine the growth inhibition of GBM cells in different combination mode groups after the same time period relative to RXD alone, with the ordinate representing inhibition of cell activity (cell viability suppression%), with the results for the r3+o3 and r3+o2 groups being relative to the inhibition of cell activity in the R3 group (RXD alone for 3 days), and with the results for the r4+o2 group being relative to the inhibition of cell activity in the R4 group (RXD alone for 4 days), with inhibition of cell activity= (1-combination OD/alone) 100%; ldh release assay to detect cell death after 96h of GBM cells in each treatment group, relative cell death on the ordinate (Relative cell death); ldh release assay to examine the proportion of GBM cells in different combination mode groups relative to GBM cells after the same time of OV treatment alone, the ordinate indicates relative cell death (Relative cell death), the results of the o4+r4 group, the o4+r3 group, the o4+r2 group being relative cell death relative to the O4 group (4 days of OV treatment alone), relative cell death = OD value of combination treatment group/OD value of treatment alone; ldh release assay to examine the proportion of GBM cells in different combination mode groups after the same time relative to RXD alone, with relative cell death (Relative cell death) on the ordinate, with the results for the r4+o4, r4+o3, r4+o2 groups being relative cell death relative to the R4 group (RXD alone for 4 days), relative cell death = combined OD/OD alone; * P <0.05.

Detailed Description

Hypoxia is one of the fundamental characteristics of GBMs, hypoxia response is regulated by Hypoxia Inducible Factors (HIFs), which are heterodimers consisting of an alpha subunit (HIF-1. Alpha., HIF-2. Alpha. Or HIF-3. Alpha.) and a beta subunit (HIF-beta.). HIF-1 a and HIF-2 a are considered to be major regulators of hypoxia and are involved in promoting malignant progression of a variety of tumors. However, the role of HIF-1α in GBM is currently controversial, and HIF-1α is significantly positively correlated with IDH1/2 mutations. HIF-2α is thought to function similarly to HIF-1α, but is less reported in GBM.

Luo Shasi he (also known as FG-4592), a chemical small molecule HIF stabilizer, which prevents HIF-alpha from being degraded by inhibiting Prolyl Hydroxylase (PHD) which has hydroxylation effect on HIF-alpha, is currently a first-line oral medicine for clinical treatment of renal anemia. In the present invention, the inventors used roflumilast to activate HIF pathway in GBM cells, and found that roflumilast can significantly induce iron death in GBM cells to inhibit GBM cell growth in vivo and in vitro, particularly when the cells exhibit chemotherapy resistance. Luo Shasi he significantly prolonged the survival of chemotherapy-resistant GBM mice in vivo without causing significant organ toxicity. In addition, it was further demonstrated that both HIF-1 a and HIF-2 a activation are involved in the induction of iron death by roflumilast, wherein HIF-2 a upregulation of lipid gene overexpression is the major cause of roflumilast leading to excessive lipid peroxidation and ultimately iron death. In view of the foregoing, the present invention provides new insight into HIF signaling pathways regulating iron death and anti-cancer therapies targeting iron death in GBM.

In order to make the technical content of the present invention more clearly understood, the following detailed description of the technical solution of the present invention will be given with reference to the accompanying drawings and test examples. It should be understood that the detailed description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the invention.

At least 3 replicates were performed for each experiment. Statistical analysis was performed using student's t-test. All data are expressed as mean ± standard deviation. GraphPad Prism 7.0 was used to prepare all charts and perform statistical analysis. P <0.05 was considered significant. Asterisks are used to indicate significance in the figures: * P <0.05; * P <0.01; * P <0.001; NS, has no meaning.

Conditions and procedures not noted in the examples below are generally carried out in accordance with conventional conditions and procedures, and may be carried out with reference to those described in the molecular cloning Experimental guidelines, by Sambrook et al, or in accordance with the experimental conditions and instructions recommended by the vendor. The chemical reagents not illustrated are conventional commercial products.

The materials adopted in the embodiment of the invention are as follows:

1. cell lines

The cell lines GL261, U87 and U251 employed in the present invention are derived from: cell center of Chinese medical science center.

2. Oncolytic viruses

In the invention, the oncolytic virus is genetically engineered herpes simplex virus or adenovirus, and the oncolytic virus can be oncolytic type 1 herpes simplex virus, or recombinant virus obtained by carrying out genetic engineering on the basis of the oncolytic type 1 herpes simplex virus, or recombinant virus obtained by carrying out genetic engineering on the basis of Ad5 oncolytic adenovirus. The oncolytic virus used in the invention is a recombinant virus obtained by carrying out genetic engineering on the basis of Ad5 oncolytic adenovirus, and is specifically obtained by carrying out transformation according to the method of example 2 in the patent publication No. CN 112961840B.

The sequence of the Ki67 gene promoter is (SEQ ID NO. 1):

the sequences of the 5' UTRs are all (SEQ ID NO. 2):

GCCCCTCCCG TCAGTTCGCC AGCTGCCAGC CCCGGGACCT TTTCATCTCT TCCCTTTTGG CCGGAGGAGC CGAGTTCAGA TCCGCCACTC CGCACCCGAG ACTGACACAC TGAACTCCAC TTCCTCCTCT TAAATTTATT TCTACTTAAT AGCCACTCGT CTCTTTTTTT CCCCATCTCA TTGCTCCAAG AATTTTTTTC TTCTTACTCG CCAAAGTCAG GGTTCCCTCT GCCCGTCCCG TATTAATATT TCCACTTTTG GAACTACTGG CCTTTTCTTT TTAAAGGAAT TCAAGCAGGA TACGTTTTTC TGTTGGGCAT TGACTAGATT GTTTGCAAAA GTTTCGCATC AAAAACAACA ACAACAAAAA ACCAAACAAC TCTCCTTGAT CTATACTTTG AGAATTGTTG ATTTCTTTTT TTTATTCTGA CTTTTAAAAA CAACTTTTTT TTCCACTTTT TTAAAAA

IL15 base sequence (Gene ID: 3600) (SEQ ID NO. 3) is as follows:

ATGAGAATTT CGAAACCACA TTTGAGAAGT ATTTCCATCC AGTGCTACTT GTGTTTACTT CTAAACAGTC ATTTTCTAAC TGAAGCTGGC ATTCATGTCT TCATTTTGGG CTGTTTCAGT GCAGGGCTTC CTAAAACAGA AGCCAACTGG GTGAATGTAA TAAGTGATTT GAAAAAAATT GAAGATCTTA TTCAATCTAT GCATATTGAT GCTACTTTAT ATACGGAAAG TGATGTTCAC CCCAGTTGCA AAGTAACAGC AATGAAGTGC TTTCTCTTGG AGTTACAAGT TATTTCACTT GAGTCCGGAG ATGCAAGTAT TCATGATACA GTAGAAAATC TGATCATCCT AGCAAACAAC AGTTTGTCTT CTAATGGGAA TGTAACAGAA TCTGGATGCA AAGAATGTGA GGAACTGGAG GAAAAAAATA TTAAAGAATT TTTGCAGAGT TTTGTACATA TTGTCCAAAT GTTCATCAAC ACTTCTTGA

example 1

The following test methods were used: drug concentration refers to its final concentration in the system.

(1) Luo Shasi he inhibits GBM cell activity in vitro and induces chemotherapy-resistant GBM cell death

The test method comprises the following steps:

CCK8 cell activity assay:

using the CCK-8 kit (Cell Counting Kit-8, japan Comdech) and according to the kit instructions: tumor cells cultured by adherence, after trypsin digestion, were inoculated in 96-well plates for detection. Cells were inoculated 2000 cells/well, while control (DMSO equal to the volume of RXD), luo Shasi he RXD (U87: 100. Mu.M; GL261: 200. Mu.M), temozolomide TMZ (200. Mu.M) and RXD+TMZ (U87: 100. Mu.M RXD+200. Mu.M TMZ; GL261: 200. Mu.M RXD+200. Mu.M TMZ) were added respectively to treat cells for 48 hours, and 6 multiplex wells were set in each group. The enzyme-labeled instrument detects OD450, performs statistical analysis by using Graphpad, and draws a growth curve.

LDH cell death assay:

lactate dehydrogenase toxicity assay kit (bi yun tian) was used and performed according to kit instructions: tumor cells cultured by adherence are inoculated in 96-well plates for detection after trypsin digestion, and 6 compound wells are arranged in each group. Cells were inoculated 2000 cells/well, while cells were treated with control (DMSO equal to the volume of RXD), luo Shasi he RXD (U87: 100. Mu.M; GL261: 200. Mu.M), temozolomide TMZ (200. Mu.M) and RXD+TMZ (U87: 100. Mu.M RXD+200. Mu.M TMZ; GL261: 200. Mu.M RXD+200. Mu.M TMZ) were added, respectively, for 72 hours. After that, the mixture was centrifuged in a centrifuge for 400 g.times.5 minutes, 120. Mu.l of the supernatant from each well was added to a new well plate, and 60. Mu.l of LDH detection working solution was added thereto and mixed for 30 minutes in a dark place. The microplate reader detects the OD490 and performs statistical analysis using Graphpad.

Flow apoptosis assay:

the Annexin V-FITC/PI apoptosis kit (next holothurian) was used and performed according to the kit instructions: cells in good condition are inoculated on a 12-hole culture plate, and the number of inoculated cells is 1 multiplied by 10 ⁵ Cells were treated with control (DMSO equal to RXD in volume), luo Shasi HeRXD (U87: 100. Mu.M; GL261: 200. Mu.M; U251: 200. Mu.M), temozolomide TMZ (200. Mu.M) and RXD+TMZ (U87: 100. Mu.M RXD+200. Mu.M TMZ; GL261: 200. Mu.M RXD+200. Mu.M TMZ; U251: 200. Mu.M RXD+200. Mu.M TMZ) for 72 hours, respectively after cell attachment, and culturing was continued. Cells were collected after pancreatin digestion, centrifuged at 300g,4℃for 5 minutes, and the cells were washed 2 times with pre-chilled PBS, each 300g, and centrifuged at 4℃for 5 minutes. The supernatant was aspirated and the cells were resuspended with 100. Mu.l Binding Buffer. Adding 5 μl of Annexin V-FITC staining solution and 10 μl of PI staining solution, and mixingAfter that, incubation was carried out at room temperature for 10 to 15 minutes in the absence of light. The cells were diluted with 400. Mu.l Binding Buffer, and the samples were placed on ice and detected by flow cytometry over 1 hour.

Test results: the mouse glioblastoma cell line GL261 and the human glioblastoma cell line U87 were treated with DMSO (Control), luo Shasi, his RXD (RXD), temozolomide TMZ (TMZ), and rxd+tmz (rxd+tmz), respectively, in four treatment groups, control (DMSO), RXD, TMZ, and rxd+tmz, respectively. It can be seen under the light microscope that RXD and rxd+tmz treated groups had lower cell densities and rounded cell morphologies (fig. 1. C), suggesting GBM cell death occurred;

CCK8 cell activity experiments examined cell growth activity RXD and rxd+tmz treated groups significantly inhibited GBM cell growth (fig. 1. A), wherein RXD treated groups GL261 and U87 had 0.497 and 0.507 fold, respectively, of the control groups and rxd+tmz treated groups GL261 and U87 had 0.658 and 0.700 fold, respectively, of the TMZ treated groups. Meanwhile, LDH release was significantly increased in the LDH cell death experiments for RXD and rxd+tmz treated groups (fig. 1. B), where LDH release was 2.836-fold and 2.041-fold for RXD treated groups GL261 and U87 cells, respectively, compared to control groups, and LDH release was 2.337-fold and 1.356-fold for rxd+tmz treated groups GL261 and U87 cells, respectively, indicating that RXD was effective in inducing GBM cell death. Flow apoptosis analysis found that RXD treated groups increased predominantly the late stage apoptosis proportion compared to TMZ treated groups, and rxd+tmz treated groups did not exhibit synergy of apoptosis induction (fig. 1. D-E), indicating that RXD induced GBM cell death was present in a pathway other than apoptosis. Meanwhile, the experimental results also show that the GL261 and U87 two groups of cells obviously resist apoptosis induction of TMZ and have stronger chemotherapy resistance.

Another human glioblastoma cell line, U251, exhibited a higher sensitivity to TMZ chemotherapy than U87 cells (figure 9.A); flow apoptosis detection found that the proportion of apoptosis in the TMZ treated group in U251 was higher than that in the RXD treated group compared to GL261 and U87 cells, especially RXD did not affect early apoptosis of U251 and RXD combined with TMZ treatment did not show synergy as well (figure 9.B). This result suggests that RXD-induced cell death effects are more pronounced in cells with strong drug resistance.

(2) Luo Shasi he induced chemotherapy-resistant GBM cell iron death

The test method comprises the following steps:

iron ion concentration detection:

serum iron assay kit (built in south kyo) was used and performed according to kit instructions: cells in good condition were inoculated into 6-well culture plates, and the number of cell inoculations was 2×10 ⁵ The experiment is carried out by adopting two cell lines of U87 and GL261, and a control group, an Erastin group, a RXD group, a RXD+Fer-1 group and a RXD+GSH group are respectively arranged, wherein the treatment method of each group is as follows:

control group: each cell was treated with DMSO in equal volume to RXD for 48h, respectively;

erastin group: each cell was treated with 10 μm Erastin for 48h, respectively;

RXD group: RXD was added to U87 cells at a final concentration of 100. Mu.M; RXD was added to GL261 cells at a concentration of 200. Mu.M;

RXD+Fer-1 group: firstly, adding 1 mu M ferrostatin-1 into different cell systems for pretreatment for 2 hours, and then adding RXD for treatment for 48 hours, wherein the final concentration of RXD added into a U87 cell system is 100 mu M, and the final concentration of RXD added into a GL261 cell system is 200 mu M;

rxd+gsh group: firstly, respectively adding 500 mu M GSH into different cell systems for pretreatment for 2 hours, and then adding RXD for treatment for 48 hours, wherein the final concentration of RXD added into a U87 cell system is 100 mu M, and the final concentration of RXD added into a GL261 cell system is 200 mu M; after the treatment, the cells were collected: washing cells with ice PBS for 3 times, adding appropriate amount of RIPA lysate containing 1/100 protease inhibitor, scraping cells, collecting cells, performing ice lysis for 30 min, centrifuging 12000g at 4deg.C for 30 min, collecting supernatant, and discarding precipitate. Each of the measurement tubes was charged with 0.5ml of the sample to be measured and 1.5ml of the iron color developer, and the blank tube and the standard tube were charged with equal amounts of distilled water and 2mg/L of the iron standard solution, respectively, and 1.5ml of the iron color developer. After mixing the tubes, boiling water bath or metal bath at 100deg.C for 5 min, cooling, centrifuging 3500 rpm, centrifuging for 10 min, and measuring absorbance of each tube with enzyme-labeled instrument at 520 nm. The iron concentration was calculated by comparing the absorbance with the absorbance of the standard, the protein concentration of each tube sample was measured by BCA method, and the cellular iron concentration was calculated by protein concentration normalization.

Transmission Electron Microscope (TEM) imaging:

mitochondrial morphology analysis by TEM imaging, the specific steps include: well-grown cells were treated with control (DMSO equal to RXD in volume), erastin (10. Mu.M) and RXD (U87: 100. Mu.M; GL261: 200. Mu.M), respectively, for 48h. Cell pellet was collected by pancreatin digestion, 2.5% glutaraldehyde +2% paraformaldehyde at 4 ℃ for 2 hours, washed three times with PBS buffer for 10 minutes each. 1% osmium acid cells were fixed at 4℃for 2 hours. Then washing three times by double distilled water, and dehydrating by alcohol with gradient of 10 minutes each time: 50% alcohol for 10 minutes, 70% alcohol for 10 minutes, 90% alcohol for 10 minutes, and finally repeating 2 times 100% alcohol for 15 minutes. Replacement: propylene oxide 2 times, 15 minutes each time, propylene oxide: resin = 1:1 displacement of room temperature for 1 hour, propylene oxide: resin = 1:3 displacement room temperature for 1 hour pure resin wet room temperature for 2 hours. Pure resin embedding (EPON 812) polymerization: 35℃for 16 hours, 45℃for 8 hours, 55℃for 14 hours, 60℃for 48 hours. And (5) performing semi-thin section after finishing the trimming, performing azure-meran staining, and positioning under an optical microscope. And then ultrathin sections: uranyl acetate-lead citrate staining was observed under a transmission electron microscope (H7650, hitachi, japan).

Lipid peroxidation assay:

cell lipid peroxidation content was measured using BODIPY 581/591C11 fluorescent probe (Thermo) and according to the reagent instructions: well-grown cells were inoculated into 12-well cell culture plates at a rate of 1X 10 ⁵ Holes, the following group treatment is carried out after the adhesion:

control group: each cell system was treated with DMSO in the same volume as RXD for 48h, respectively;

erastin group: each cell system was treated with 10 μm Erastin for 48h, respectively;

RXD group: each cell line was treated with RXD for 48h, wherein U87: 100. Mu.M RXD, GL261: 200. Mu.M RXD, respectively;

rxd+gsh group: each cell system was pretreated with 500 μm GSH for 2h and then RXD for 48h, where U87:100 μM RXD, GL261:200 μM RXD;

a working solution of BODIPY 581/591C11 fluorescent probe was prepared at a final concentration of 10. Mu.M using a serum-free medium, the medium of the cells was changed to the working solution, and the cells were incubated in an incubator at 37℃for 30 minutes. The cells were washed with PBS buffer, the fluorescence of the cells was observed under a fluorescence microscope (ZEISS Axio Imager M2) and the images were recorded by photographing.

Lipid peroxidation flow cytometry was further performed: cells were treated as above, after which cells were collected by trypsinization, centrifuged at 1000 rpm for 3 minutes in a centrifuge, washed with PBS buffer, centrifuged again for 3 minutes (1000 rpm centrifugation), the supernatant was aspirated to retain pellet, and 300. Mu.l of PBS was resuspended and detected by flow cytometry (Biosciences Accuri C6).

Calreticulin (CRT) detection:

well-grown cells were inoculated into 12-well cell culture plates at a rate of 1X 10 ⁵ Holes, the following group treatment is carried out after the adhesion:

control group: each cell system was treated with DMSO in the same volume as RXD for 48h, respectively;

erastin group: each cell system was treated with 10 μm Erastin for 48h, respectively;

RXD group: each cell line was treated with RXD for 48h, wherein U87: 100. Mu.M RXD, GL261: 200. Mu.M RXD, respectively;

rxd+gsh group: each cell system was pretreated with 500 μm GSH for 2h and then RXD for 48h, where U87:100 μM RXD, GL261:200 μM RXD;

cells were then washed with PBS solution, 4% paraformaldehyde fixed for 20 minutes, followed by a PBS solution rinse for 5 minutes each for 3 times. The blocking solution was blocked for 60 minutes and the PBS solution was rinsed out for a total of 3 times for 10 minutes each. anti-CRT antibodies (# 62304, CST) were then diluted 1:50, cells were incubated and at 4℃overnight. The next day the cells were washed with PBS for 10 minutes each time, 3 total times. And then DAPI cell nuclear staining and anti-fluorescence quenching tablet sealing. The cells were observed for fluorescence under a fluorescence microscope (ZEISS Axio Imager M2) and recorded images were photographed.

Further, calreticulin flow cytometry: cell treatment as above, trypsin digested cells, complete medium stopped digestion after cell collection, centrifuge centrifugation for 3 minutes (1000 rpm), after which PBS buffer washed cells, centrifuge again for 1000 minutes centrifugation for 3 minutes, supernatant retention pellet was aspirated. 1:50 dilutions of anti-CRT antibody (FITC fluorescent label) cells were incubated for 30 min at 4 ℃. The cells were washed with PBS buffer, centrifuged for 3 min at 1000 rpm for 2 times, the supernatant was aspirated to retain pellet, and the cells were resuspended in 300 μl PBS and detected on a flow cytometer (Biosciences Accuri C6).

Test results: GL261 and U87 cells were pretreated with iron death inducer Erastin (10. Mu.M) as a positive control with iron death inhibitor ferrostatin-1 (Fer-1, 1. Mu.M) and GSH (500. Mu.M) for 2 hours, and then RXD (U87: 100. Mu.M; GL261: 200. Mu.M) was added thereto for 48 hours, and it was examined whether RXD-induced cell death had iron death characteristics, i.e., increased intracellular iron ions, increased lipid peroxidation, marked mitochondrial change and immunogenic cell death characteristics. Five treatment groups were set up in total, namely a control group Control, erastin treatment group, a RXD treatment group, a RXD+Fer-1 treatment group and a RXD+GSH treatment group.

Under the mirror, erastin and RXD treatment groups were observed to decrease cell density and shape rounding, and the Fer-1 pretreatment (RXD+Fer-1) and GSH pretreatment (RXD+GSH) blocked this change (FIG. 2. A); in both CCK8 cell activity experiments (fig. 2. B) and LDH release cell death experiments (fig. 2. C), RXD was significantly induced GBM cell death similar to the Erastin treated group and could be blocked by Fer-1 and GSH pretreatment. Further iron ion concentration detection (fig. 2.D) and mitochondrial morphology analysis (fig. 2. E) showed that RXD significantly induced increases in GBM intracellular iron ions while causing cell mitochondrial atrophy and mitochondrial membrane density increase, consistent with iron death characteristics. Likewise, lipid peroxidation analysis showed that RXD and Erastin were similar, induced a significant increase in GBM peroxidized lipids, and could be blocked by iron death inhibitors, conforming to the iron death profile (fig. 3. A-B). One of the immunogenic cell death markers is the eversion of Calreticulin (CRT) from the inside of the cell to the surface of the cell, and both immunofluorescent staining and flow cytometry analysis showed that RXD treatment resulted in a significant increase in GBM cell surface CRT protein, with RXD-induced cell death belonging to immunogenic death and being inhibited by pretreatment (fig. 3. C-D). Thus, the results of the study demonstrate that RXD induces iron death in drug resistant GBM cells.

(3) Luo Shasi in vivo inhibition of chemotherapy drug-resistant glioma growth and remarkable prolongation of survival time of tumor-bearing mice

The test method comprises the following steps:

establishing an intracranial in-situ model of a GL261-C57BL/6 mouse: intracranial GBM modeling using 6 week old C57BL/6 male mice: pre-operation animal surgical room disinfection, the mice are continuously anesthetized by isoflurane gas, the heads are fixed on a stereotactic instrument, and the skin of the heads is disinfected by amoliodine II; cutting the skin with the length of 0.5cm longitudinally in the middle of the top of the head to expose the skull, and applying hydrogen peroxide to the skull until the structures such as bregma, sagittal suture, coronal suture and the like are exposed; the locator was located 2.5mm to the right of the sagittal suture, 1.0mm anterior to the coronal suture, 2.5mm below the cortex, and 5. Mu.l of cell suspension (containing 1.5X10) ⁵ GL261-Luc cells) vertically enter 4.0mm along a bone hole by a microinjector, retreat by 1.0mm, and slowly and uniformly push the inner core within 20 min; pushing the injection needle for 15min, slowly pulling the needle, closing bone holes by bone wax, suturing the scalp and sterilizing; after natural recovery, the animals are fed into an SPF-class animal house plastic film isolator. After 7 days of inoculation, the mice are subjected to in vivo imaging scanning and detection of the tumor formation condition, and the mice which are not tumor-formed (in vivo imaging is non-fluorescent) and have overlarge tumor formation volume (fluorescence value is overlarge) are removed, so that the establishment of the intracranial in-situ GBM model of the mice is completed.

GL261-C57BL/6 intracranial in situ model mice were randomly divided into four groups (n=10 mice per group): control group (ddH with 5% DMSO in the same volume as RXD-treated group) ₂ O, intraperitoneal injection), RXD treatment group (RXD: 10mg/kg/day X7 days, drug solvent: 5% DMSO+40% PEG300+5% Tween 80+50% ddH ₂ O, intraperitoneal injection), TMZ treatment group (TMZ: 35.7mg/kg×7 days, drug solvent: 5% DMSO+30% PEG300+65% ddH ₂ O, intraperitoneal injection), RXD in combination with TMZ treatment group (rxd+tmz, where RXD:10mg/kg/day X7 days, i.p., TMZ:35.7mg/kg X7 days, i.p. injection, 3-4 hours apart from two injections in one mouse). The medicines are injected in groups after the tumor is planted for 7 days, the medicine injection is continued for 7 days,mice were monitored for tumor growth and quantitatively analyzed on days 0, 7, 14, and 21 after tumor inoculation, while mice weights were recorded on the day of drug injection and every three days after drug injection.

The results show that: the TMZ single drug has no obvious treatment effect, and further proves the chemotherapy resistance of GL261 cells; RXD treatment and rxd+tmz combination treatment significantly inhibited tumor growth (fig. 4. A); RXD treatment (40 days) and rxd+tmz combination treatment (37 days) significantly extended tumor-bearing mice survival compared to control (30 days) and TMZ treated groups (26 days) (fig. 4. C); thus, RXD is able to significantly inhibit drug resistant GBM tumor growth in vivo.

Body weight recordings found that RXD-treated mice had significantly reduced body weight compared to controls, and the combination treatment (rxd+tmz) had significantly reduced body weight compared to the TMZ single drug treatment (fig. 4. B), indicating that TMZ treatment may have stronger chemotherapeutic side effects, no significant adverse effects with RXD treatment, and that the combination treatment with RXD and TMZ may reduce the chemotherapeutic side effects of TMZ.

(4) Luo Shasi he induced chemotherapy-resistant GBM iron death in vivo and did not cause significant visceral toxicity

The test method comprises the following steps:

in the experiment of Luo Shasi in vivo of example 1 (3) for inhibiting the growth of chemotherapy-resistant glioma and remarkably prolonging the survival time of tumor-bearing mice, the control group, the RXD treatment group, the TMZ treatment group and the RXD combined TMZ treatment group were taken for carrying out drug toxicity analysis. On day 15 after tumor inoculation, 3-4 mice per group were left with tumor tissue; experimental observations endpoint each group of mice was left with liver, lung, spleen, kidney and heart specimens for relevant histochemical analysis.

HE staining

Dewaxing and hydrating: 2 times of slicing by xylene solution, 10 minutes each time, 2 times of slicing by absolute ethanol solution, 5 minutes each time, 5 minutes of slicing by 95% ethanol, 5 minutes of slicing by 80% ethanol, 5 minutes of slicing by 70% ethanol, and 5 minutes of slicing by tap water; (2) dyeing: soaking the slice in hematoxylin staining solution (Zhonghua gold bridge), soaking the slice in 1% ethanol hydrochloric acid solution for 2 seconds after 1-3 minutes, washing with tap water, observing the color depth under a lens from time to time, soaking the slice in eosin staining solution (Zhonghua gold bridge) for 5 minutes after the cell nuclei are stained satisfactorily, washing with tap water, and observing the color effect under a lens; (3) and (3) dehydration and transparency: soaking the slices in 75% ethanol for 15 seconds, soaking the slices in 85% ethanol for 15 seconds, soaking the slices in 95% ethanol for 5 minutes, soaking the slices in absolute ethanol for 10 minutes, soaking the slices in xylene for 10 minutes, and soaking fresh xylene for 10 minutes again; (4) sealing and microscopic examination: the slide was removed from the xylene, neutral gum was added dropwise, the coverslip was gently placed to avoid air bubbles, air dried at room temperature, and then the tissue staining was observed under an optical microscope and the image was recorded by photographing.

Immunohistochemical staining

(1) Conventional dewaxing to water, washing with PBS buffer solution for 2 times for 5 minutes, heating with strong fire in a microwave oven for 5 minutes, soaking slices in citrate buffer solution after boiling, putting into the microwave oven again, boiling again for 10 minutes, closing the microwave oven, and naturally cooling at room temperature; (2) the PBS buffer was washed 2 times, each time for 5 minutes, an appropriate amount of endogenous peroxidase blocking agent was added dropwise, and the mixture was incubated at room temperature for 10 minutes, after which the PBS buffer was washed 3 minutes each time for a total of 3 times. Sealing for 30 minutes by using sheep serum sealing liquid, horizontally placing the slices into a light-resistant wet box, diluting the primary antibodies, and respectively obtaining the working liquid concentrations of the primary antibodies: ki67 (1:50, sc-15402, santa Cruz), 4-HNE (1:25, ab48506, abcam), HIF-1α (1:100, #36169S, CST), HIF-2α (1:200, ab109616, abcam) were incubated overnight at 4 ℃; (3) rewarming at normal temperature for 45 minutes, and flushing with PBS buffer solution for 3 minutes each time, for a total of 3 times. Reaction enhancement solution was added dropwise and incubated at room temperature for 20 minutes, with a 3 minute wash of PBS buffer for a total of 3 times. Dripping the enhanced enzyme-labeled goat anti-mouse/rabbit IgG polymer, and incubating for 20 minutes at room temperature; the PBS buffer was washed 3 minutes each time for a total of 3 times; (4) preparing fresh DAB color development liquid, using in a dark place, immediately observing the antigen dyeing degree after dripping, and putting the slice into water for about 1 minute to terminate color development; (5) conventional hematoxylin is used for dying nuclei, differentiating, washing, dehydrating and transparentizing, sealing with center gum, observing tissue dying condition under an optical microscope, photographing and recording images.

Prussian Lan Tie dyeing

Prussian blue staining kit (Beijing Soy Bao) was used and performed according to the kit instructions: (1) conventionally dewaxing to water, washing slices with distilled water for 1 minute; (2) immersing the slices in Prussian blue dyeing working solution for 15-30 minutes, and fully washing with distilled water for 2-5 minutes; (3) immersing the slices in the nuclear solid red staining solution for 5-10 minutes, and then flushing for 15 seconds by tap water; (4) conventional dehydration is transparent, the center gum is sealed, the tissue staining is observed under an optical microscope, and the image is photographed and recorded.

Test results:

further study the effect of RXD treatment at the tissue level, histochemical staining and Prussian blue iron staining of mouse tumor tissue (fig. 5. A-B), analysis of RXD effect on HIF pathway activation in GBM (HIF-1 a, HIF-2 a), as well as on tumor proliferation activity (Ki 67) and lipid peroxidation by-products (4-HNE), and extraction of tissue iron to detect ferrous ion concentration (fig. 5. C). The results show that: RXD treatment significantly reduced tumor proliferation activity (reduced Ki 67), induced tumor HIF-alpha overexpression, especially HIF-2 alpha increase significantly, and simultaneously, RXD treatment significantly increased lipid peroxidation level (increased 4-HNE) and tissue iron content, indicating that RXD treatment effectively induced drug resistant GBM iron death to inhibit tumor growth.

In addition, heart, liver, spleen, lung and kidney of the model mice were collected to prepare organ specimens for HE staining morphology analysis (fig. 6. A), and liver of the mice was stained with prussian blue iron (fig. 6. B), which showed no significant pathological changes, suggesting that RXD treatment did not have significant visceral toxicity, and that it may have good tolerance as an antitumor drug.

(5) HIF-alpha mediated roflumilast to regulate chemotherapy-resistant GBM cell iron death

The test method comprises the following steps:

siRNA transfection:

the cells are inoculated on a culture plate and cultured until reaching 40-50% confluence, and fresh culture medium is replaced. siRNA premix was prepared following Lipofectamine 3000 transfection kit (Invitrogen) protocol. siRNA premix was mixed at 1:1 proportion into diluted Lipofectamine 3000 reagent, incubating for 10-15min at room temperature. The prepared siRNA-lipid complex is added into a cell culture medium, and the cell culture medium is incubated for 48 hours at 37 ℃ for subsequent experiments. The siRNA sequences used for transfection were:

SEQ ID NO.4:5’-GCCGCUCAAUUUAUGAAUATT-3’(siRNAHIF-1α-homo)；

SEQ ID NO.5:5’-GCUGAUUUGUGAACCCAUUTT-3’(siRNAHif-1α-mouse)；

SEQ ID NO.6:5’-GGUGGAGCUAACAGGACAUTT-3’(siRNAHIF-2α-homo)；

SEQ ID NO.7:5’-CCGACCAGCAAAUGGAUAATT-3’(siRNAHif-2α-mouse).

siRNA Hif-1. Alpha. -mouse and siRNA Hif-2. Alpha. -mouse were used for transfection of GL261, and siRNA HIF-1. Alpha. -homoo and siRNAHIF-2. Alpha. -homoo were used for transfection of U87. The experimental section below simplifies the siRNA Hif-1. Alpha. -mouse and siRNA HIF-1. Alpha. -homoo to si-HIF-1. Alpha., and the siRNAHif-2. Alpha. -mouse and siRNAHIF-2. Alpha. -homoo to si-HIF-2. Alpha.).

U in SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 and SEQ ID No.7 of the sequence Listing is denoted by T due to the restriction of the software format of the sequence Listing.

si-RNA interference efficiency and specificity detection:

and extracting cellular RNA and total protein respectively 48h and 72h after siRNA transfection, and respectively carrying out RT-qPCR and WB experiments to detect the interference efficiency and the specificity of si-HIF-1 alpha and si-HIF-2 alpha. Total cellular RNA was extracted using TRIzol reagent (Invitrogen) with reference to the conditions described in molecular cloning experiments guidelines, compiled by Sambrook et al; cDNA was obtained using a reverse transcription kit (Promega A3500); qPCR was performed using qPCR kit SYBR premix Ex Taq (Takara) and qPCR instrument (Quantum studio5, ABI), and the primer sequences used for the experiments were as follows:

extracting total cell proteins with RIPA lysate (Biyun-Tian) containing 1/100 PMSF; the WB experiment used anti-HIF-1α (1:1000, #36169S, CST), anti-HIF-2α (1:1000, abcat 616, abcam) and anti- β -actin (1:5000, #4970S, CST) for detection of the relative protein expression levels.

Lipid peroxidation probe staining experiments:

detection of cellular lipid peroxidation content using BODIPY 581/591C11 fluorescent probe (Thermo): inoculating well-grown cells into 6-well cell culture plate with an inoculation number of 1×10 ⁵ And (3) culturing the cells until reaching 40-50% confluence, carrying out transfection of si-NC, si-HIF-1 alpha and si-HIF-2 alpha, changing the liquid 48h after transfection, and respectively adding medicines with the same volume as RXD (control) and RXD (U87: 100 mu M; GL261:200 mu M) for 48h. Cell staining was performed using BODIPY581/591C11 fluorescent probe working solution at a final concentration of 10. Mu.M, and the cell peroxidation levels of each group were analyzed by fluorescence microscopy (ZEISS Axio Imager M2) and lipid peroxidation flow cytometry, respectively, as before.

Test results: the siRNA molecules are used for respectively interfering the expression of HIF-1 alpha and HIF-2 alpha in drug-resistant GL261 and U87 cells, so that the expression quantity is reduced, and the influence of the knockdown of the expression of the HIF-1 alpha and the expression of the HIF-2 alpha on the death process of RXD induced iron is analyzed. Designing si-HIF-1 alpha and si-HIF-2 alpha molecules, and verifying the interference efficiency and the specificity by using RT-qPCR and WB experiments, and simultaneously verifying that RXD is used as a HIF stabilizer to induce the overexpression of the HIF-1 alpha and the HIF-2 alpha of GBM cells (FIG. 7. A-B);

LDH release experiments and cell iron ion concentration detection show that knocking down the expression of HIF-1 alpha and HIF-2 alpha can obviously reduce RXD-induced cell death and intracellular iron ion concentration increase (FIG. 7. C-D), and the expression of the HIF-alpha is mediated by RXD to induce drug resistant GBM cell iron death.

Analysis of lipid peroxidation probe staining experiments showed (fig. 7.E-fig. 8. A), HIF-2α knockdown alone significantly blocked RXD-induced lipid peroxidation accumulation, suggesting that HIF-2α activation was achieved primarily by regulating Luo Shasi he-induced lipid peroxidation.

(6) HIF-2 alpha activation-mediated lipid gene overexpression mediates Luo Shasi he induction of lipid peroxidation of chemotherapy-resistant GBM

The test method comprises the following steps:

qRT-PCR and Western blot:

inoculating well-grown cells into 6-well cell culture plate with an inoculation number of 1×10 ⁵ And (3) culturing the cells until reaching 40-50% confluence, carrying out transfection of si-NC, si-HIF-1 alpha and si-HIF-2 alpha, changing the liquid 48h after transfection, and respectively adding medicines with the same volume as RXD (control) and RXD (U87: 100 mu M; GL261:200 mu M) for 48h. Then, by referring to the conditions described in the molecular cloning laboratory Manual, which is compiled by Sambrook et al, total cell proteins are extracted using RIPA lysate (Biyun-Tian) containing 1/100 PMSF; the WB experiment uses anti-GPX4 (1:1000, sc-166570,Santa Cruz), anti-FTH1 (1:1000, #3998S, CST) and anti-beta-actin (1:5000, #4970S, CST) to detect the relative protein expression; extracting total RNA of cells using TRIzol reagent (Invitrogen); cDNA was obtained using a reverse transcription kit (Promega A3500); qPCR detection was performed using qPCR kit SYBR premix Ex Taq (Takara) and qPCR instrument (Quantum studio5, ABI).

The primer sequences used for qPCR experiments were:

test results: to further analyze the mechanism by which HIF- α regulates GBM cell iron death, expression of the common iron death marker proteins GPX4 and FTH1 was examined by WB experiments (fig. 7. B), and it was found that knocking down expression of both HIF-1α and HIF-2α did not affect GPX4 and FTH1 expression, indicating that HIF- α activation-induced iron death did not involve regulation of GPX4 and FTH 1. The overexpression of CHAC129, ACSL431 and PTGS2 genes is also an important marker of iron death, and qRT-PCR experiments show that RXD can significantly induce the overexpression of CHAC129, ACSL431 and PTGS2 in GBM, and that both HIF-1α and HIF-2α knockdown can block the induction (FIG. 8. B), and again show that both HIF-1α and HIF-2α are involved in the process of RXD inducing iron death of drug resistant GBM cells.

Meanwhile, considering the effect of HIF-2α on lipid peroxidation, we also examined the expression of lipid regulatory genes HILPDA and PLIN2 (fig. 8.C), and the results indicate that RXD can significantly up-regulate the expression of HILPDA and PLIN2 in GBM, but only the knockdown of HIF-2α can significantly block the overexpression of lipid genes. Therefore, HIF-2 a activation up-regulates lipid gene overexpression may be the primary cause of RXD-induced excessive lipid peroxidation in GBM.

In conclusion, the invention discovers for the first time that Luo Shasi he activates HIF-alpha to induce chemotherapy-resistant GBM cells to die, thereby obviously inhibiting the growth of GBM cells in vitro and in vivo, having no obvious visceral toxicity and providing a new idea for improving the current state of GBM treatment.

Example 2 test of sensitivity to GBM in combination with FG-4592 and oncolytic Virus

Combination therapy:

oncolytic viruses: adenovirus-based oncolytic virus TS-2021 (with Ki67 promoter control System and loaded with IL-15 functional Gene) was constructed as described in patent CN112961840B, example 2, for Ad5-Ki67-UTR/IL-15 adenovirus

Chemical small molecules: FG-4592 (trade name: roxadustat Luo Shasi he)

The test method comprises the following steps: the synergistic effect of FG-4592 (Luo Shasi, heRXD) and Oncolytic Virus (OV) TS-2021 combination on GBM (U87, GL 261) was tested using the CCK8 cell activity assay and LDH cell death assay methods of example 1, with the cell processing time in the specific assay being as described below.

Different concentration gradients of RXD and OV (RXD: 50. Mu.M, 100. Mu.M, 200. Mu.M, 400. Mu.M; OV: 10) were set ⁰ MOI，10 ¹ MOI，10 ² MOI，10 ³ MOI) is respectively treated with GBM cells (U87, GL 261), meanwhile, each concentration is provided with a control, each RXD concentration control is added with an equivalent solvent DMSO, each OV concentration control is added with an equivalent solvent PBS, and the IC50 corresponding to the GBM cells (U87, GL 261) is respectively determined through CCK8 cell activity experiments _RXD And IC50 _OV ；

Cell treatments were divided into five groups: control group (Control group, DMSO and PBS added in the same amount as the combination treatment group), luo Shasi other treatment group (RXD group, treatment concentration IC 50) _RXD ) Oncolytic virus treatment group (OV group, treatment concentration IC 50) _OV ) Combination treatment group (RXD+OV group) with treatment concentration of IC50 _RXD +IC50 _OV ) And half-dose combination treatment group (1/2 RXD+1/2OV, treatment concentration 1/2IC 50) _RXD +1/2IC50 _OV ) CCK8 cell Activity experiments were performed after 72h of cell treatment, respectivelyAnd performing LDH cell death experiments after 96 hours of treatment, and detecting the cell activity and cell death of each group of treated cells.

With IC50 _RXD And IC50 _OV The effect concentration of (2) is treated with RXD and OV respectively, and different combination effect modes are set, and the effect of different combination modes on the combination effect is further detected through CCK8 cell activity experiment and LDH cell death experiment. The combined action modes are as follows: RXD treatment for 3 days in combination with OV treatment for 3 days (r3+o3 group or o3+r3 group), RXD treatment for 4 days in combination with OV treatment for 4 days (r4+o4 group or o4+r4 group), RXD treatment for 3 days in combination with OV treatment for 2 days (OV is added 24h after RXD treatment, r3+o2 group), RXD treatment for 4 days in combination with OV treatment for 2 days (OV is added 48h after RXD treatment, OV is added R4+o2 group), RXD treatment for 4 days in combination with OV treatment for 3 days (OV is added 24h after RXD is added to OV, r4+o3 group), OV treatment for 3 days in combination with RXD treatment for 2 days (OV is added 48h after RXD is added to o4+r2 group), and OV treatment for 4 days in combination with RXD (OV is added 24h after RXD is added to RXD, o4+r3 group).

Test results: determination of the treatment concentration IC50 of U87 in combination with the results of the preliminary experiments (FIG. 1) and the results of the concentration gradient cell activity experiments (FIGS. 10. A-D) _RXD 100 mu M, IC50 _OV IC50 for treatment concentration of 1000MOI, GL261 _RXD 200 mu M, IC50 _OV 600MOI, and gradient experiments revealed that GL261 cells were more sensitive to oncolytic viruses and U87 was more sensitive to RXD;

CCK8 cell activity experiments examined cell growth activity in each treatment group, the combined treatment groups (1/2 rxd+1/2OV group and rxd+ov group) had stronger growth inhibition than the treatment groups alone (RXD group and OV group): among GL261 cells, RXD group, OV group, 1/2RXD+1/2OV group and RXD+OV group are 0.92.+ -. 0.02 times, 0.50.+ -. 0.02 times, 0.37.+ -. 0.02 times and 0.30.+ -. 0.03 times, respectively, of Control group; among U87 cells, RXD group, OV group, 1/2RXD+1/2OV group and RXD+OV group were 0.45.+ -. 0.03-fold, 0.77.+ -. 0.01-fold, 0.45.+ -. 0.04-fold and 0.33.+ -. 0.05-fold, respectively, of the Control group (FIG. 10. E); experimental results show that the combination of FG-4592 and oncolytic virus for the treatment of GBM has a synergistic effect. Also, there was no significant difference in synergy efficiency for the different combination treatments (fig. 10. F-G) compared to either the OV alone treated group of the same days (fig. 10. F) or RXD alone treated group of the same days (fig. 10. G).

LDH release experiments examined cell death in each treatment group, the combination treatment groups (1/2 rxd+1/2OV group and rxd+ov group) each had a higher proportion of cell death than the treatment groups alone (RXD group and OV group). Among GL261 cells, RXD group, OV group, 1/2RXD+1/2OV group and RXD+OV group were 1.01.+ -. 0.11-fold, 0.91.+ -. 0.03-fold, 1.45.+ -. 0.15-fold and 1.61.+ -. 0.41-fold, respectively, of Control group; among U87 cells, RXD group, OV group, 1/2RXD+1/2OV group and RXD+OV group were 2.26.+ -. 0.12 times, 2.56.+ -. 0.26 times, 3.49.+ -. 0.20 times and 3.55.+ -. 0.38 times, respectively, as compared to Control group (FIG. 10. H); experimental results show that the combination of FG-4592 and oncolytic virus for the treatment of GBM has a synergistic effect. In addition, there was no significant difference in synergy efficiency for the different combination treatments (fig. 10. I-J) compared to either the ovd alone treated group (fig. 10. I) or RXD alone treated group (fig. 10. J).

It will be appreciated by those skilled in the art that the present invention can be carried out in other embodiments without departing from the spirit or essential characteristics thereof. Accordingly, the above disclosed embodiments are illustrative in all respects, and not exclusive. All changes that come within the scope of the invention or equivalents thereto are intended to be embraced therein.

Claims (10)

1. A pharmaceutical combination for treating glioblastoma comprising: oncolytic viruses for use in treating glioblastoma and compounds for upregulating HIF- α expression, wherein the oncolytic viruses are genetically engineered herpes simplex viruses or adenoviruses.

2. The pharmaceutical combination according to claim 1, wherein the oncolytic virus is an oncolytic type 1 herpes simplex virus, or a recombinant virus genetically engineered on the basis of the oncolytic type 1 herpes simplex virus, or a recombinant virus genetically engineered on the basis of an Ad5 oncolytic adenovirus; preferably, the oncolytic virus is a recombinant virus obtained by genetic engineering on the basis of Ad5 oncolytic adenovirus, and the genome of the recombinant virus comprises: an E1A gene expression module, and/or an IL-15 functional gene module; more preferably, the E1A gene expression module comprises: the promoter of the E1A gene is the promoter of the Ki67 gene; E1A gene coding sequence; and a 5' UTR sequence of TGFbeta2, located upstream of the E1A gene coding sequence and downstream of the promoter of the Ki67 gene; the base sequence of the promoter of the Ki67 gene is shown as SEQ ID NO.1 in a sequence table; the base sequence of the 5' UTR of the TGF beta2 is shown as SEQ ID NO.2 in a sequence table; the promoter of the Ki67 gene is linked to the 5' UTR of the TGF beta2 to form a fusion sequence; the IL-15 functional gene module comprises an IL-15 gene coding sequence and an element for expressing the IL-15 gene, wherein the element for expressing the IL-15 gene comprises: the promoter of the IL-15 gene is a CMV promoter; a polyA sequence of SV 40; the base sequence of the IL-15 gene is shown as SEQ ID NO. 3.

3. The pharmaceutical combination according to claim 1 or 2, wherein the glioblastoma is a drug resistant, preferably temozolomide resistant glioblastoma; preferably, the glioblastoma is GL261, U87 or LN229 cell line.

4. The pharmaceutical combination of any one of claims 1-3, wherein the compound for up-regulating HIF-a expression is a prolyl hydroxylase inhibitor; preferably, the compound for up-regulating HIF- α expression is roflumilast FG-4592; more preferably, the HIF- α is HIF-2α and/or HIF-1α; HIF-2α is further preferred.

5. The pharmaceutical combination according to any one of claims 1-4, wherein the pharmaceutical combination is in the form of a mixture of an oncolytic virus and a compound for upregulating HIF- α expression; preferably, the pharmaceutical combination further comprises a pharmaceutically acceptable carrier; preferably, the dosage form of the pharmaceutical composition is a freeze-dried powder injection, an injection, a tablet, a capsule or a drop.

6. The pharmaceutical combination of any one of claims 1-4, wherein the pharmaceutical combination is in the form of separate packages for oncolytic virus and a compound for upregulating HIF-a expression.

7. Use of a combination of an oncolytic virus and a compound for upregulating HIF- α expression in the manufacture of a supplement or medicament for ameliorating or/and treating a tumor, wherein the oncolytic virus is a genetically engineered herpes simplex virus or adenovirus and the tumor is glioblastoma.

8. The use according to claim 7, wherein,

the oncolytic virus is oncolytic type 1 herpes simplex virus, or recombinant virus obtained by carrying out genetic engineering on the basis of the oncolytic type 1 herpes simplex virus, or recombinant virus obtained by carrying out genetic engineering on the basis of Ad5 oncolytic adenovirus; preferably, the oncolytic virus is a recombinant virus obtained by genetic engineering on the basis of Ad5 oncolytic adenovirus, and the genome of the recombinant virus comprises: an E1A gene expression module, and/or an IL-15 functional gene module; more preferably, the E1A gene expression module comprises: the promoter of the E1A gene is the promoter of the Ki67 gene; E1A gene coding sequence; and a 5' UTR sequence of TGFbeta2, located upstream of the E1A gene coding sequence and downstream of the promoter of the Ki67 gene; the base sequence of the promoter of the Ki67 gene is shown as SEQ ID NO.1 in a sequence table; the base sequence of the 5' UTR of the TGF beta2 is shown as SEQ ID NO.2 in a sequence table; the promoter of the Ki67 gene is linked to the 5' UTR of the TGF beta2 to form a fusion sequence; the IL-15 functional gene module comprises an IL-15 gene coding sequence and an element for expressing the IL-15 gene, wherein the element for expressing the IL-15 gene comprises: the promoter of the IL-15 gene is a CMV promoter; a polyA sequence of SV 40; the base sequence of the IL-15 gene is shown as SEQ ID NO. 3; and/or

The glioblastoma is a glioblastoma with drug resistance, preferably temozolomide drug resistance; preferably, the glioblastoma is GL261, U87 or LN229; and/or

The compound for up-regulating HIF-alpha expression is a prolyl hydroxylase inhibitor; preferably, the compound for up-regulating HIF- α expression is roflumilast FG-4592, preferably, HIF- α is HIF-2α and/or HIF-1α; HIF-2α is further preferred.

9. Use of a compound for up-regulating HIF-a expression in the manufacture of a supplement or medicament for ameliorating or/and treating a tumor, wherein the tumor is glioblastoma.

10. The use of claim 9, wherein the compound for upregulating HIF-a expression is a prolyl hydroxylase inhibitor; and/or the glioblastoma is a drug resistant, preferably temozolomide resistant glioblastoma;

preferably, the compound for up-regulating HIF- α expression is roflumilast FG-4592; and/or the glioblastoma is GL261, U87 or LN229;

more preferably, the HIF- α is HIF-2α and/or HIF-1α; HIF-2α is further preferred.