Cucurbitacin I Inducoes Apoptosis And Protective Autophagy In Glioblastoma In Vitro And In Vivo

BackgroudGlioblastoma multiforme (GBM), classified as grade IV astrocytoma by the World Health Organization, is the most aggressive and accounts for54%of all gliomas. Even through a combination of surgery, chemotherapy, and radiotherapy is used, there has been only a minimal improvement in the median survival time of GBM patients (from approximately12to14months) or the5-year survival rate (less than five percent), which points to the critical need to identify and implement therapeutic strategies.A variety of cancers including glioma exhibits aberrant activation of signal transducer and activator of transcription3(STAT3), which plays a pivotal role in malignant transformation and tumor cell survival, and blocking aberrant activation of STAT3results in inhibition of tumor growth and survival and induction of apoptosis with few side effect to normal cells. Thus, abrogation of STAT3activation is considered an effective cancer therapeutic approach.Cucurbitacin Ⅰ (JSI-124), a selective inhibitor of JAK2/STAT3, is a plant natural product and isolated from various plant families and has been used as folk medicines for centuries in China, India, Brazil and Peru. Accumulating evidence shows that cucurbitacin Ⅰ has a potent anticancer effect on a variety of cancer cell types, such as breast cancer, lung cancer, neuroblastoma, melanoma and glioma. However, the exact mechanism of Cucurbitacin Ⅰ is not fully elucidated.Macroautophagy (hereafter called autophagy), known as programmed cell death type Ⅱ, has an important homeostatic role, mediating the removal of dysfunctional or damaged organelles, which are digested and recycled for cellular metabolic needs; consequently, might maintain cancer survival under metabolic stress conditions and mediate resistance to anticancer therapies such as radiation, chemotherapy, and some targeted therapies. Mounting evidence suggests that inhibition of autophagy promotes cancer cell death and potentiates various anticancer therapies, implicating autophagy as a mechanism that enables tumor cells to survive antineoplastic therapy. Treatment of these cells with inhibitors of autophagy, such as Chloroquine, Hydroxychloroquine, BafilomycinAl,monensin etc., or knockdown of essential autophagy genes (Beclin1、 Atg5、Atgl2) resulted in enhanced therapy-induced apoptosis.In this study, we systematically investigated the mechanisms of Cucurbitacin I against GBM including autophagy and apoptosis.ObjectiveTo explore new insights into the molecular mechanisms underlying cucurbitacin I-mediated GBM cells apoptosis and autophay, and provide advantageous insights for development of efficacious therapies for GBM.MethodsIn vitro1. CCK-8assay was performed to assess cell viability in GBM cells treated with different concentrations of cucurbitacin I for24and48h.2. Western blot analysis was utilized to examine the expression from lysates of GBM cells.3. pSELECT-GFP-LC3transient transfection disclosed LC3punctates in GBM cells.4. Imunofluorescence staining of LC3B was used to show expressing LC3aggregation.5. Transmission electron microscopy revealed characteristic autophagosomes in GBM cells6. TUNEL assay was performed to show apoptosis in GBM cells.7. Cell death detection ELISAplus assay was performed to show apoptosis in GBM cells.8. Co-immunoprecipitation was performed to monitor the interaction of Bcl-2with Beclin1/hVps34. 9. Small interfering RNA transfecion was used to knockdown of beclin1.In vivo1. U251cells were used to establish heterotopic model of human glioma in nude mice.2. Average body weight and average tumor volume were performed to examine the side effect and inhibition effect of cucurbitacin Ⅰ and (or) CQ against U251xenografts.3. Immunohistochemical assay was performed to display Ki-67expression.4. TUNEL assay was used to show apoptosis.Results1. Cucurbitacin Ⅰ inhibited the growth of GBM cells in vitro and in vivoCCK-8assay showed that treatment with cucurbitacin Ⅰ resulted in growth inhibition of U251and T98G cells in a dose-dependent manner, but their responses varied. Results in vivo displayed that cucurbitacin Ⅰ markedly inhibited tumor volume and tumor weight as compared with the counterparts treated with DMSO. The average tumor volume of solid tumors in cucurbitacin Ⅰ-treated mice was412mm3(±82), as compared with1286mm3(±251) for control group. Moreover, the average tumor weights at study termination were1,340mg (±260) and418mg (±80) in control and cucurbitacin Ⅰ group, respectively. Furthermore, Ki67immunostaining confirmed a pronounced decrease in tumor cell proliferation.2. Cucurbitacin Ⅰ induced apoptosis in GBM cells and xenograft mice model related to bcI-2family proteinsThe percentage of TUNEL-positive cells remarkably increased in a dose-dependent manner in cucurbitacin Ⅰ-treated U251and T98G cells. Similar to this observation, DNA fragmentation ratio of cucurbitacin Ⅰ-treated groups was predominantly elevated compared with control group in a dose-dependent manner examined by cell death detection ELISAPlus assay. Western blot analysis showed that GBM cells treated with cucurbitacin Ⅰ significantly upregulated Bax and cleaved caspase-3(p17), but decreased anti-apoptotic proteins such as Bcl-2and Bcl-xL in a dose-denpendant manner. In agreement, intraperitoneal injections of cucurbitacin Ⅰ resulted in massive apoptotic cell death, cleaved caspase-3(p17) increase and marked bcl-2and bcl-xL decrease on xenografts sections.3. Cucurbitacin Ⅰ triggered autophagy and activated autophagy-related gene beclin1in GBM cellsTransmission electron microscopy revealed abundant characteristic autophagosomes in GBM cells treated with cucurbitacin Ⅰ, while scarce autophagosomes in control cells. We determined the induction of autophagy by localizing an autophagosome-specific protein LC3by GFP-LC3trasient transfection, abundant autophagosomes in a dose-dependented manner in cucurbitacin Ⅰ-treated cells were observed. We found similar results through imunofluorescence staining of LC3B. Western blot analysis revealed that LC3-Ⅱ and Beclin1expression were more pronounced with the dose of cucurbitacin Ⅰ increased. Although increased LC3-Ⅱ levels were detected in bafilomycin A1-treated cells due to inhibition of lysosomal degradation of LC3-Ⅱ, LC3-Ⅱ levels were even higher in the cucurbitacin I-treated cells. In addition, in the absence of bafilomycin A1, expression of p62protein was decreased in cucurbitacin Ⅰ-treated cells, suggesting that autophagy was activated and the p62protein was degraded via autophagy. The p62level was obviously elevated in cells treated with bafilomycin A1and cucurbitacin Ⅰ, indicating autophagy was blocked by bafilomycin A1and p62was accumulated in GBM cells. Furthermore, massive LC3B accumulation was noted on tumor sections in cucurbitacin Ⅰ-treated xenografts.4. Constitutive activation of AMPK/mTOR/p70S6K pathway was involved in cucurbitacin Ⅰ-induced autophagy in GBM cellsWestern blot analysis showed that treatment with cucurbitacin Ⅰ for48h down-regulated p-mTOR expression in a dose-dependent manner, but failed to note any alteration of p-AKT. Cucurbitacin Ⅰ-mediated phosphorylation of AMPK was more pronounced with the dose of cucurbitacin Ⅰ increased. In addition, the decreased phosphorylation of p70S6K, a downstream target of mTOR, was observed dose-dependently in GBM cells.5. Cucurbitacin Ⅰ suppressed JAK2/STAT3/HIF-1α signaling in GBM cellsWestern blot analysis showed that cucurbitacin Ⅰ inhibited the JAK2/STAT3cascade and resulted in HIF-1α downregulation in a dose-dependent manner.6. Downregulation of HIF-1α played pivotal roles in cucurbitacin Ⅰ-induced autophagy in GBM cellsTransmission electron microscopy and GFP-LC3transient transfection revealed that in the presence of FG-4497, U251cells showed a significant decrease in the numbers of autophagosomes and the percentage of cells with GFP-LC3dots after treatment with cucurbitacin Ⅰ, which indicated that overexpression of HIF-la might prevent autophagy occurrence after cucurbitacin Ⅰ treatment. Western blot analysis showed that U251cells overexpressing HIF-1α induced by FG-4497showed a significant decrease in the level of conversion LC3B-II to LC3B-I. In addition, we found that cucurbitacin I decreased the level of bcl-2protein that occurred along with the induction of autophagy in U251cells, and that overexpression of HIF-1α prevented cucurbitacin Ⅰ-induced downregulation of bcl-2and autophagy. Compared with the results in siRNA controls, knockdown of beclin1prevented the increase in the level of LC3-II by cucurbitacin Ⅰ. Similar results were observed after pretreating the cells with3-MA. Co-immunoprecipitation was performed to monitor the interaction of Bcl-2with Beclin1/hVps34. We found that under basal conditions Bcl-2and Beclin1/hVps34co-immunoprecipitated with each other in U251cells whereas the interaction markedly decreased in cucurbitacin Ⅰ-treated cells.7. Inhibition of autophagy enhanced cucurbitacin I-induced apoptosis in GBM cells CCK8assay showed that CQ significantly enhanced cucurbitacin I-induced suppression of GBM cells. Aligned with this observation, cucurbitacin Ⅰ-induced apoptotic cell death was augmented in the presence of CQ, which was demonstrated by cell death detection ELISAPIus assayand TUNEL assay. A genetic approach was applied to block the formation of autophagosomes by knocking down expression of beclin1through RNA interference. CCK-8assay was utilized to determine cell viability and cell death detection ELISAPlus assay was performed to examine the apoptosis level in GBM cells. Our data demonstrated that silencing expression of beclin1markedly enhanced cucurbitacin I-induced inhibition of GBM cells growth and promoted the apoptotic cell death.8. CQ enhanced cucurbitacin I-induced tumor growth inhibition in xenograft tumor modelNo major side effects were noted throughout the study. Average tumor volumes at the end of the study were control:616mm3(±130), CQ:580mm3(±107), cucurbitacin I:346mm3(±79), and combination:220mm3(±62). While no statistically significant difference was found between the CQ and control arms (P=0.25), the differences in tumor volume between cucurbitacin I and control, combination and control, and combination and cucurbitacin I arms were significant (P<0.05). Furthermore, combination-treated tumors exhibited a significant (P<0.01) lower average tumor weight at study termination than control. Moreover, there was no effect on body weight of mice. Finally, a pronounced decrease in tumor cell proliferation (Ki67) and increase in apoptosis (TUNEL) were noted in combination-treated xenografts.Conclusion1. Cucurbitacin I significantly inhibited the growth of GBM cells in vitro and in vivo.2. Cucurbitacin I induced apoptosis in GBM cells related to bcl-2family proteins. 3. Constitutive activation of AMPK/mTOR/p70S6K pathway was involved in cucurbitacin I-induced autophagy in GBM cells.4. Cucurbitacin I downregulated HIF-1α and Bcl-2expression and inhibited Beclin1/hVps34complex interaction.5. Inhibition of autophagy markedly enhanced cucurbitacin I-induced apoptosis in GBM in vitro and in vivo.