KR20170055176A - A composition for treating TRAIL-resistant glioblastoma comprising morusin and TRAIL as active ingredients - Google Patents

Abstract

The present invention relates to a composition for treating TRAIL-resistant glioblastoma comprising morusin and TRAIL as active ingredients. TRAIL is regarded as a candidate substance as an anticancer drug by having an extremely excellent effect on selectively killing cancer cells, but most of glioblastoma shows resistance against TRAIL so the TRAIL has limits to be applied to actual clinic application. The composition containing morusin and TRAIL as active ingredients shows an excellent anticancer effect against TRAIL-resistant glioblastoma by the morusin sensitizing glioblastoma cells against TRAIL and both morusin and TRAIL showing a synergetic effect.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for treating TRAIL-resistant glioblastoma comprising moruccine and TRAIL as an active ingredient, and a composition for treating TRAIL-resistant glioblastoma,

The present invention relates to a composition for treating TRAIL-resistant glioblastoma comprising morus and TRAIL as an active ingredient.

Glioblastoma is the most frequent malignant tumor in the brain and central nervous system, accounting for the majority of gliomas in the United States, the second most frequent histologically occurring, and a 5-year survival rate of only 5%. In Korea, glioblastoma is the most common type of brain epithelium, accounting for 15.1% of all brain and CNS origin carcinomas, accounting for 34.4% of all gliomas. However, treatment for gliomas is difficult due to the resistance to therapeutic agents, the repeated occurrence of abnormally activated signaling pathways, the difficulty of delivering therapeutic agents efficiently, and the significant spatial and temporal nonuniformity of individual tumors.

Several pathway pathway proteins and anti-apoptotic proteins such as EGFR, PDGFR, and STAT3 have been implicated in the pathogenesis of glioblastoma resistance to cancer therapy. Epithelial growth factor receptor (EGFR) is an important regulator of cell proliferation, metabolism and survival to environmental stimuli, as one of the tyrosine kinases (RTKs). It is known that amplification and / or mutation of RTK such as EGFR occurs in various cancers including glioblastoma. According to the Cancer Genome Atlas (TCGA), 66% of all primary glioblastoma patients showed RTK overexpression and / or mutation, among which EGFR was most commonly altered. That is, overexpression and / or mutation of EGFR occurred in 50% of all primary glioblastoma patients. In addition, platelet-derived growth factor receptor (PDGFR) overexpression was seen in 13% of all primary glioblastoma patients. EGFR and PDGFR induce their trans-phosphorylation after dimerization, followed by AKT, STAT3 regulates a variety of death-related proteins, anti-apoptosis and apoptosis-promoting proteins. Thus, activation of EGFR and PDGFR Targeting both can be an effective treatment strategy because they can block both cancer cell proliferation and cell survival.

Cell death induction by apoptosis is thought to be a good strategy in cancer treatment. Caspase-dependent apoptosis is typically activated through endogenous or exogenous apoptotic pathways. Endogenous apoptotic pathways are induced by endogenous stresses such as DNA damage, hypoxia, or other cellular stress, and the extrinsic apoptotic pathway is mediated by apoptotic receptors such as the TNFR superfamily.

In humans, a tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, is a truncated cytoplasmic death domain, (DR4 / TRAIL receptor-1 and DR5 / TRAIL receptor-2) and two decoy receptors (DcR1 / TRAIL receptor-3 and DcR2 / TRAIL receptor-4). Therefore, TRAIL can induce selective apoptosis of cancer cells and is attracting attention as a cancer candidate. However, various types of cancer cells including glioblastoma are resistant to TRAIL, and thus there is a limit to practical application in clinical practice.

Previous reports have reported that Morus Rober bark of moraceae, morusin isolated from the alba bark, has various biological activities such as antimicrobial activity, scavenger activity against antioxidant anion radical, and anti-inflammatory activity. Have proven to have. Recently, it has been reported that morus is able to induce apoptosis through inhibition of the NF-κB and STAT3 signaling pathways in human prostate cancer, liver cancer and cervical cancer cells.

Korean Patent Application No. 10-2014-0003343 (a composition for preventing or treating prostate cancer including morus)

The present invention provides a pharmaceutical composition for treating TRAIL-resistant glioblastoma comprising morus and TRAIL as an active ingredient.

The present invention also provides a pharmaceutical composition for anti-cancer adjuvant therapy for TRAIL-resistant glioblastoma comprising morusin as an active ingredient, wherein the morusin is to provide a composition for sensitizing TRAIL-resistant glioblastoma to TRAIL.

The present invention provides a pharmaceutical composition for treating a TRAIL-resistant glioblastoma comprising morusin and a tumor-necrosis-factor-related apoptosis-inducing ligand (TRAIL) as an active ingredient.

The present invention also provides a pharmaceutical composition for anticancer therapy for TRAIL-resistant glioblastoma comprising morusin as an active ingredient, wherein the morusin provides a composition for sensitizing TRAIL-resistant glioblastoma to TRAIL.

The morusin is a substance isolated from the root shell (mulberry) of mulberry and is represented by the following formula.

The morus sin may be synthesized as long as it possesses the inherent properties of morus and may be used without limitation as long as it is extracted from plants other than the bark.

Glioblastoma is a type of glioma. TRAIL has a potent apoptosis inducing effect selectively on cancer cells, but it has been reported that TRAIL resistance is very high in glioblastoma. Depending on the type of glioblastoma, it exhibits resistance to TRAIL to varying degrees. In the composition of the present invention, morusin has an effect of sensitizing a glioblastoma cell showing a strong resistance to TRAIL, a strong resistance, a moderate resistance and a weak resistance to TRAIL, and strongly synergizes with TRAIL. Therefore, the effect of treating TRAIL resistant glioblastoma is excellent.

The compositions of the present invention may be administered orally and parenterally at the time of clinical administration. Is administered in a conventional manner via, for example, intravenous, intraarterial, intraperitoneal, intramuscular, intraarterial, intraperitoneal, intrasternal, percutaneous, intranasal, inhalation, topical, rectal, can do. Preferably, the compositions of the present invention may be administered orally, intravenously, intratumorally, intradermally, or subcutaneously. However, the route of administration is not limited thereto.

The composition of the present invention may be formulated in a unit-dosage form together with a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers include diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents and surfactants. Specifically, the pharmaceutically acceptable carrier includes lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil and the like.

The unit dosage forms may be administered in a single dose or in divided doses. The unit dosage form may be formulated in the form of oral, granule, tablet, capsule, suspension, emulsion, syrup, aerosol or the like, oral suspension, external preparation, injectable solution or the like according to a conventional method. When the unit dosage form of the present invention is formulated for parenteral use, it may contain injectable esters such as propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and ethyl oleate.

In the composition of the present invention, the dosage of the active ingredient varies depending on the condition and body weight of the patient, the degree of disease, the drug form, the administration route and the period, but may be suitably selected depending on the case. For example, the moruscin in the composition may be administered at a dose of 0.0001 to 500 mg / kg per day, and the TRAIL may be administered at a dose of 0.000001 to 100 mg / kg per day. The above administration may be administered once a day or divided into several times. In addition, the pharmaceutical composition of the present invention may contain 0.0001 to 50% by weight of morus and TRAIL relative to the total weight of the composition.

Although TRAIL is highly selective for cancer cell death, it has attracted attention as an anticancer drug candidate. However, most glioblastomas are resistant to TRAIL and thus have limited clinical application. The composition comprising morusin and TRAIL as an active ingredient of the present invention sensitizes morosine to glioma cells and exhibits an excellent anticancer effect against TRAIL-resistant glioblastoma due to synergistic action between morusin and TRAIL.

FIG. 1 is a graph showing the viability of human glioblastoma cell line treated with TRAIL for 24 hours.
FIG. 2A shows the viability of human glioblastoma cell line treated with TRAIL for 72 hours. FIG. 2B is a diagram showing cell viability after treatment of morcellin with human glioblastoma cell line and normal astrocytic cells. FIG. FIG. 2C shows cell viability in human glioblastoma cell line when morusin and TRAIL were co-treated. FIG. FIG. 2D is an analysis of morus and TRAIL by calculating the combination index (CI) when morus and TRAIL are co-treated with human glioblastoma cell line.
FIG. 3A shows Western blot analysis of cleaved PARP, cleaved caspase 3, cleaved caspase 8 and cleaved caspase 9, which are apoptosis-related proteins, when human glioblastoma cell line is co-treated with morusin and TRAIL. FIG. 3B is a graph showing the number of apoptotic cells in a human glioblastoma cell line by co-treatment with morusin and TRAIL and by flow cytometry.
FIG. 4A is a graph showing the expression of DR4, DR5, DcR1, and DcR2 by western blotting in human glioblastoma cell line treated with morusin at 0, 12.5, or 25 μM. FIG. 4B shows Western blot analysis of DR4, DR5, DcR1, and DcR2 expression at 0, 3, 6, 9, and 18 hours after treatment of human glioblastoma cell line with 25 μM of moruscin. FIG. 4C shows morphogenesis of human glioblastoma cell line and confirmation of expression of DR4 and DR5 by flow cytometry. FIG. FIG. 4D shows morphogenesis of human glioblastoma cell line and confirmation of DR5 mRNA expression by RT-qPCR. FIG. 4E shows transient transfection of siRNA against DR5 mRNA into human glioblastoma cell line and cell viability after co-treatment with TRAIL and morin.
FIG. 5A is a graph showing Western blotting of the expression of c-IAP1, XIAP, and Survivin, which are apoptosis-related proteins, in human glioblastoma cell lines treated with morusin at 0, 12.5, or 25 μM. FIG. 5B shows Western blot analysis of the expression of c-IAP1, XIAP, and Survivin at 0, 3, 6, 9, and 18 hours after morucine treatment on human glioblastoma cell lines. FIG. 5C shows Western blot analysis of XIAP mRNA and Survivin mRNA expression in human glioblastoma cell lines treated with morusin at 0, 12.5, or 25 μM and RT-qPCR. Figure 5D shows that human glioblastoma cell lines were treated with moruccine and treated with XIAP mRNA, Survivin < RTI ID = 0.0 > mRNA expression was confirmed by Western blotting.
FIG. 6A is a diagram showing Western blot analysis of human glioblastoma cell line treated with 0 or 25 μM of moruscin and expression of PDGFR and EGFR. FIG. 6B shows Western blot analysis of phospho-ERK, ERK, phospho-STAT3, STAT3, phospho-EGFR and EGFR in human glioblastoma cell line treated with 0, 12.5 or 25 μM of moruscin. FIG. 6C shows Western blot analysis of expression of phospho-ERK, ERK, phospho-STAT3, STAT3, phospho-EGFR and EGFR at 3, 6, 9 and 18 hours after treatment with 25 μM morcellin in human glioblastoma cell line .
The upper graph of FIG. 7 is taken from FIG. 2B, and the lower diagram shows the process of morphine (a compound having a chemical formula at the center of the figure) sensitizing human glioblastoma cells to TRAIL.

Embodiments are provided to facilitate understanding of the present invention. The following examples are provided to further understand the present invention, and the present invention is not limited thereto.

The cell lines, reagents and antibodies used in the following examples were prepared as follows. Human glioblastoma cell lines U251MG and LN18 were obtained from Cancer Res. 2011, 71 , 6514-6523 and J. Pharmacol. Sci. 2013, 121 , 192-199. U87MG, T98G and U373MG cell lines were purchased from Korean Cell Line Bank (KCLB). U138MG was obtained from Dr. Jae - Soo Kim, Catholic University Medical School in Seoul, Korea. U251MG and U138MG were cultured in DMEM supplemented with 10% FBS and 1% antibiotic / antifungal agent. U87MG and T98G were cultured in MEM and U373MG was cultured in RPMI supplemented with 10% FBS and 1% antibiotic / antifungal agent. All cells were cultured in a wet incubator at 37 ° C with 5% CO ₂ , and the viability of the cultured cells was monitored by a LUNA-FL automated cell counter (Logos Biosystems, Kyonggi-do, Korea).

Primary primary astrocytes were isolated from the cortex of P1 and P4 ICR (Institute of Cancer Research) mice (DBL Co., Ltd., Chungbuk province, Korea) and maintained in astrocytic culture medium (10% heat- Inactivated FBS and high glucose DMEM supplemented with 1% penicillin / streptomycin). All applicable international and institutional guidelines for animals were followed, and studies using primary brain cells isolated from mice were approved by Kyung Hee University ethics committee (KHUASP (SE) -14-056).

Recombinant human TRAIL / Apo2L was purchased from ATGen (South Korea, Kyunggi Province, Seongnam). Morusin (BP0961, purity (HPLC, 270 nm) ≥ 98%) was purchased from Biopurify Phytochemicals Ltd. (China, Sichuan, Chengdu) and dissolved in DMSO.

Antibodies used for Western blotting were purchased from the following companies. Cleaved caspase 3, cleaved caspase 8, cleaved caspase 9, XIAP, ERK, phospho-ERK, phospho-EGFR, EGFR, PDGFR, STAT3 and phospho-STAT3 from Cell Signaling Technology (Danvers, MA, USA) Antibodies were purchased and DR4, DR5, cIAP1 and Survivin antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and antibodies against DcR1 and DcR2 were purchased from Sigma-Aldrich for β-actin. Other secondary antibodies HRP-conjugated anti-mouse IgG and anti-rabbit IgG (1: 5000; Cell Signaling Biotechnology) were used for immunoblotting.

Example  1. Cells from human glioblastoma cells To survival  TRAIL FOR Morphine  Effect of Combination Therapy

Treatment of TRAIL with 0.01, 0.1, 1, 10, 100, and 1000 ng / ml of TRAIL was performed for 24 hours to human glioblastoma cell lines LN18, U87MG, U373MG, T98G, U251MG and U138MG And cell viability was observed by MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) analysis. Specifically, each cell (3-5 × 10 ³ cells / well) was inoculated into a 96-well plate, treated with TRAIL at various concentrations for 24 hours, and cultured for 24 hours. Then, the medium was removed, and 1 mg / mL MTT Was added to each well. Cells were then incubated for 2 hours at 37 ° C and an equal volume of MTT lysis buffer was added to each well followed by overnight incubation at 37 ° C. The cell viability was then monitored by measuring the OD at 570 nm using a microplate reader (Tecan Austria GmbH, Grodig, Austria). As a result, it was confirmed that all cell lines maintained cell viability of 50% or more and showed resistance to TRAIL (FIG. 1).

In order to more accurately confirm the degree of TRAIL resistance, TRAIL was treated with 0.01, 0.1, 1, 10, 100, and 1000 ng / ml of each of the above glioblastoma cell lines for 72 hours and MTT analysis was performed in the same manner as described above, Sex was observed. As a result, the cell survival rate of U251MG and U138MG was the highest, and the cell lines showed very strong resistance to TRAIL, U87MG and U373MG showed strong resistance, and T98G and LN18 showed moderate resistance 2A).

In order to confirm the effect of treatment with morusin alone, 0, 5, 10, 20, and 30 μM of moruscin was treated for 24 hours in each of the glioblastoma cell line and normal primary astrocytic cells, MTT assays were performed to observe cell viability. As shown in Figure 2B, it is to reduce the cell viability of the anvil god all glioblastoma cell line, but represented by the IC ₅₀ value of about 25-40 μM sikijineun cells were not significantly reduce viability. On the other hand, moruscin did not decrease the viability of normal primary astrocytes.

In order to confirm the effect of co-treatment of moruccin and TRAIL, 0, 5, 10, or 20 μM of morusin was treated alone (control), or TRAIL (100 ng / mL) and 0, 5, 10, or 20 μM of moruscin were co-treated for 24 hours and MTT assay was performed in the same manner as above to analyze cell viability. As a result, cell viability was further reduced in both of the above four cell lines when TRAIL and morusin were treated together with morusin alone (Fig. 2C). In addition, fractions of surviving cells were examined using CalcuSyn software (Biosoft, MO, USA) to determine the fraction affected (Fa) between TRAIL and morin, and the Combination Index (CI) We analyzed the synergy of TRAIL and moruscin. As a result, the CI value was found to be less than 0.8 at all Fa points, and it was found that TRAIL and morushine were strongly synergistic.

Example  2. In human glioblastoma cells To apoptosis  TRAIL FOR Morphine  Effect of Combination Therapy

TRAIL is known to kill cancer cells through a mechanism that induces apoptosis of cancer cells. Therefore, in order to confirm whether the decrease in cell viability shown in Example 1 was caused by an apoptosis mechanism, the following experiment confirmed the effect of the combination treatment of TRAIL and moruscin on the expression of apoptosis-related proteins in human glioblastoma cells.

U87MG cell line and T98G cell line were prepared and treated with TRAIL (50 ng / mL) alone, morusin (5 μM) alone, or TRAIL (25 ng / mL) and morusin Respectively. Cell lysates were obtained and incubated with cleaved PARP, cleaved caspase 3, cleaved caspase 8, an antibody against cleaved caspase 9, and secondary antibody HRP-conjugated anti-mouse IgG and anti-rabbit IgG (1: 5000; Biotechnology).

As a result, when the combination of TRAIL (25 ng / mL) and morusin (2.5 μM) was treated, the cleaved caspase 3 , 8 and 9, and cleaved PARP concentrations were significantly increased (Fig. 3A). The results show that the combination of TRAIL and morusin induces apoptosis of human glioma cells. In addition, cleaved caspase 8 is a protein associated with the exogenous apoptotic pathway, and cleaved caspase 9 is a protein associated with the endogenous apoptotic pathway. Thus, the combination of TRAIL and moruscin induces both apoptotic and exogenous apoptotic pathways leading to potent apoptosis inducing effects It implies.

U87MG and T98G cell lines were also prepared and treated for 24 hours with a combination of TRAIL (50 ng / mL) and morusin (20 μM). Were stained with Annexin V-FITC and PI (propidium iodide) kit (Bio Vision Technology Inc., Golden, CO, USA). Flow cytometry was then performed using a FACS Calibur flow cell counter. As a result, it was confirmed that the number of apoptotic cells increased when the combination of TRAIL and morusin was treated more than that of TRAIL alone or morushine alone (FIG. 3B).

Example  3. Expression of death receptor and decoy receptor in human glioblastoma cells Morphine  effect

Downregulation of death receptors (DR4 and DR5) and / or upregulation of decoy receptors (DcR1 and DcR2) are known to be important mechanisms by which tumor cells acquire resistance to TRAIL. Therefore, the following experiment was conducted to confirm the effect of moruccin treatment on the expression of the receptors in human glioma cells.

U87MG, U373MG, U251MG, and LN18 cell lines were first prepared and treated with 0, 12.5, 25 μM of moruccin for 24 hours to confirm the effect of moruccin in a dose-dependent manner. DR4, DR5, DcR1 and DcR2 were detected by Western blotting. The results are shown in FIG. 4A.

U87MG cell line and U373MG cell line were also prepared and treated with 25 μM morcellin, and after 0, 3, 6, 9, and 18 hours, the cells were treated with DR4, DR5, DcR1, and DcR2. The results are shown in Figure 4B.

In Figures 4A and 4B, moruscin strongly increased the expression of DR5 in a dose- and time-dependent manner in glioma cells, while it appeared to have little effect on other death receptors (DR4, DcR1 and DcR2).

U87MG, U373MG and U251MG cell lines were prepared and treated with 25 μM of moruccine for 24 hours to specifically bind to DR4, DR5, or IgG control, to clearly confirm the effect of morusin on DR4 and DR5 Stained with FITC-conjugated antibodies, and flow cytometry was performed. As a result, it was confirmed that moruscine increases the expression of DR5 but does not increase the expression of DR4 (Fig. 4C).

U87MG cell line was prepared and treated with 25 μM of moruscin, and then total RNA was extracted with TRI Reagent® solution (Ambion, Waltham, MA, USA) to observe the effect of moruccin on DR5 expression at the mRNA level They were separated according to the manufacturer's instructions. Total RNA (1 μg) was reverse transcribed with cDNA using the PrimeScript ™ 1st strand cDNA Synthesis Kit (Takara Bio Biomedical Inc., Seoul, Korea) according to the manufacturer's instructions and amplified with RT-qPCR. Amplification of cDNA was monitored using the Sensi FAST SYBR No-ROX kit (Bioline, Taunton, Mass., USA) on a LightCycler instrument (Roche Applied Sciences, Indianapolis, IN, USA) according to the manufacturer's protocol. RT-qPCR was performed using the specific primers of Table 1 below. GAPDH was used as an internal control.

[Table 1]

Mean ± SD is shown in the graph of Figure 4D (*, p <0.05; **, p <0.01; and ***, p <0.001 for mock control). In FIG. 4D, the DR5 mRNA level increases significantly with time.

From the above results, we confirmed that moruscine increases the expression of DR5 at the mRNA level, thereby increasing the resistance of human glioma cells to TRAIL. Therefore, it was confirmed by the following experiment whether DR5 is knocked down using siRNA for DR5 mRNA to suppress the effect of moruccin.

U87MG cells were prepared in 6-well plates, and DR5 -specific siRNA (Mbiotech, Korea, Kyunggi Province, Hanam) or a proven scrambled control siRNA (control) was transiently transfected using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). The sequence of DR5 siRNA is shown in Table 2 below.

[Table 2]

Forty-eight hours after transfection, the cells were co-treated with TRAIL (50 ng / mL) and morusin (20 μM) for 24 hours. After that, cell viability was confirmed by MTT analysis. Mean ± SD is shown in the graph of Figure 4E (*, p <0.05; **, p <0.01; and ***, p <0.001 for mock control). In the graph of Figure 4E, the leftmost bar is the mock control without any treatment, the middle bar is the result from cells treated with TRAIL and morusin (T + M) after scrambled control, and the rightmost bar Is the result of TRAIL and morusin treated cells after treatment with DR5 siRNA. DR5 siRNA treatment showed a 66% reduction in cell death by TRAIL and morin-synuclein treatment compared to the scrambled control treatment.

Taken together, these results indicate that moruscin sensitizes human glioma cells to TRAIL by increasing the expression of DR5.

Example  4. In human glioblastoma cells, anti- Apoptosis  Effect of Morusin on the Expression of Protein

Upregulation of anti-apoptotic proteins is another important mechanism by which follicular cells acquire resistance to TRAIL. Therefore, the following experiment was conducted to confirm the effect of moruccin treatment on the expression of anti-apoptotic proteins in human glioma cells.

U87MG and U373MG cells were treated with moruccine at 0, 12.5, 25 μM for 24 h and incubated with apoptosis-related proteins c-IAP1, XIAP , Western blotting was performed using an antibody against Survivin. The results are shown in Figure 5A.

U87MG and U373MG cells were treated with 25 μM of moruccine and incubated with c-IAP1, XIAP (5 μM) at 0, 3, 6, 9, and 18 hours to observe the effect of moruccin on the expression of proteins involved in apoptosis in a time- , Western blotting was performed using an antibody against Survivin. The results are shown in Figure 5B.

5A and 5B show that morusin decreases expression of XIAP and Survivin in a dose-dependent and time-dependent manner in human glioma cells, but does not decrease the expression of c-IAP1.

The effect of morus on the expression of XIAP and Survivin was observed at the mRNA level. In order to confirm the dose-dependent effect, U87MG cell line was treated with 0, 12.5, 25 μM of moruccin for 24 h and mRNA expression of XIAP and Survivin was observed with RT-qPCR. RT-qPCR was performed using the specific primers shown in Table 3 below. On the other hand, GAPDH was used as an internal control (FIG. 5C).

[Table 3]

In order to confirm the time-dependent effect, the U87MG cell line was treated with 25 μM of moruccine and the mRNA expression of XIAP and Survivin was observed by RT-qPCR at 0, 6 and 18 hours (FIG. 5D).

Mean ± SD is shown in the graphs of Figures 5C and 5D (*, p <0.05; **, p <0.01; and ***, p <0.001 for mock control). From Figures 5C and 5D it can be seen that morusin decreases mRNA expression of XIAP and Survivin in a dose-dependent and time-dependent manner.

From the above results, it can be seen that morusin partially sensitizes human glioma cells to TRAIL by decreasing anti-apoptotic protein expression at the mRNA level.

Example  5. In human glioblastoma cells EGFR / PDGFR - STAT3  For the signaling pathway Morphine  effect

The EGFR / PDGFR-STAT3 signaling pathway plays an important role in glioma progression. Therefore, the following experiment was conducted to confirm the effect of moruscin on the EGFR / PDGFR-STAT3 signaling pathway.

LN18, U87MG, T98G, U251MG and U373MG cells were treated with 0 or 25 μM of moruccin for 24 hours. Western blots were then performed using antibodies against PDGFR and EGFR. As a result, moruscin significantly inhibited the expression of PDGFR and EGFR (Fig. 6A).

In addition, the effects of morushine on the expression of phospho-ERK, ERK, phospho-STAT3, STAT3, phospho-EGFR and EGFR were confirmed in a dose-dependent and time-dependent manner.

First, U87MG and U373MG cells were treated with 0, 12.5 or 25 μM of moruccin for 24 hours. Cell lysates were obtained and western blots were performed using antibodies against phospho-ERK, ERK, phospho-STAT3, STAT3, phospho-EGFR, and EGFR. The results are shown in Figure 6B.

U87MG and U373MG cells were treated with 25 μM morcellin and cell lysates were obtained after 0, 3, 6, 9 and 18 hours, and phospho-ERK, ERK, phospho-STAT3, STAT3, phospho-EGFR, Expression of EGFR was confirmed. The results are shown in Figure 6C.

6B and 6C, the concentration of phospho-EGFR and phospho-STAT3 significantly decreased, indicating that moruscine reduced the phosphorylation of EGFR and its downstream target protein, STAT3, in a dose-dependent and time-dependent manner. On the other hand, there was no significant change in the concentration of phospho-ERK.

These results suggest that moruscine inhibits EGFR / PDGFR-STAT3 pathway by inhibiting EGFR phosphorylation and down-regulating PDGFR expression.

Claims (6)

A pharmaceutical composition for the treatment of TRAIL-resistant glioblastoma comprising morusin and TRAIL (tumor-necrosis-factor-related apoptosis-inducing ligand) as an active ingredient. 2. The composition of claim 1, wherein the composition is administered orally or parenterally. 2. The composition of claim 1, wherein the composition is formulated in a unit-dosage form together with a pharmaceutically acceptable carrier. 4. The composition of claim 3 wherein the unit dosage form is formulated as any one selected from the group consisting of capsules, granules, powders, tablets, solutions and suspensions. A pharmaceutical composition for anticancer assistance for TRAIL-resistant glioblastoma, comprising morusin as an active ingredient, wherein said morusin sensitizes TRAIL-resistant glioblastoma to TRAIL. 6. The composition of claim 5, wherein the composition is administered orally or parenterally.

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