Role of Metformin and AKT Axis Modulation in the Reversion of Hypoxia Induced TMZ-Resistance in Glioma Cells.
ABSTRACT: Hypoxia is a key driver of tumor adaptation promoting tumor progression and resistance to therapy. Hypoxia related pathways might represent attractive targets for the treatment of Glioblastoma Multiforme (GBM), that up to date is characterized by a poor prognosis. Primary aim of this study was to investigate the role of hypoxia and hypoxia-related modifications in the effect of temozolomide (TMZ) given alone or in association with the antidiabetic agent Metformin (MET) or the PI3K/mTOR blocker, BEZ235. The study was conducted in the TMZ responsive U251 and resistant T98 GBM cells. Our results showed that during hypoxia, TMZ plus MET reduced viability of U251 cells affecting also CD133 and CD90 expressing cells. This effect was associated with a reduction of HIF-1? activity, VEGF release and AKT activation. In T98 TMZ-resistant cells, TMZ plus MET exerted similar effects on HIF-1?. However, in this cell line, TMZ plus MET failed to reduce CD133 positive cells and AKT phosphorylation. Nevertheless, the administration of the dual PI3K/mTOR inhibitor BEZ235 potentiated the effect of TMZ plus MET on cell viability, inducing a pro-apoptotic phenotype during hypoxic condition also in T98 cells, suggesting the block of the PI3K/AKT/mTOR pathway as a complementary target to further overcome GBM resistance during hypoxia. In conclusion, we proposed TMZ plus MET as suitable treatment to revert TMZ-resistance also during hypoxia, an effect potentiated by the inhibition of PI3K/mTOR axis.
Project description:<h4>Background</h4>Despite aggressive treatment with radiation therapy and concurrent adjuvant temozolomide (TMZ), glioblastoma multiform (GBM) still has a dismal prognosis. We aimed to identify strategies to improve the therapeutic outcome of combined radiotherapy and TMZ in GBM by targeting pro-survival signaling from the epidermal growth factor receptor (EGFR).<h4>Methods</h4>Glioma cell lines U251, T98G were used. Colony formation, DNA damage repair, mode of cell death, invasion, migration and vasculogenic mimicry as well as protein expression were determined.<h4>Results</h4>U251 cells showing a low level of methyl guanine transferase (MGMT) were highly responsive to the radiosensitizing effect of TMZ compared to T98G cells having a high level of MGMT. Treatment with a dual inhibitor of Class I PI3K/mTOR, PI103; a HSP90 inhibitor, 17-DMAG; or a HDAC inhibitor, LBH589, further increased the cytotoxic effect of radiation therapy plus TMZ in U251 cells than in T98G cells. However, treatment with a mTOR inhibitor, rapamycin, did not discernibly potentiate the radiosensitizing effect of TMZ in either cell line. The mechanism of enhanced radiosensitizing effects of TMZ was multifactorial, involving impaired DNA damage repair, induction of autophagy or apoptosis, and reversion of EMT (epithelial-mesenchymal transition).<h4>Conclusions</h4>Our results suggest possible strategies for counteracting the pro-survival signaling from EGFR to improve the therapeutic outcome of combined radiotherapy and TMZ for high-grade gliomas.
Project description:Although several antipsychotic drugs have been shown to possess anticancer activities, haloperidol, a "first-generation" antipsychotic drug, has not been extensively evaluated for potential antineoplastic properties. The aim of this study was to investigate the antitumoral effects of haloperidol in glioblastoma (GBM) U87, U251 and T98 cell lines, and the effects of combined treatment with temozolomide (TMZ) and/or radiotherapy, using 4 Gy of irradiation. The viability and proliferation of the cells were evaluated with trypan blue exclusion assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis, using the annexin-propidium iodide (PI), and cell cycle, cluster of differentiation (CD) expression and caspase-8 activation were measured using flow cytometry. Treatment with haloperidol significantly reduced cell viability in U87, U251 and T98 GBM cell lines. Haloperidol induced apoptosis in a dose-dependent manner, inhibited cell migration and produced an alteration in the expression of CD24/CD44. The additional effect of haloperidol, combined with temozolomide and radiation therapy, increased tumor cell death. Haloperidol was observed to induce apoptosis and to increase caspase-8 activation. In conclusion, haloperidol may represent an innovative strategy for the treatment of GBM and further studies are warranted in glioma xenograft models and other malignancies.
Project description:Glioblastoma multiforme (GBM) is one of the most aggressive cancers. Despite recent advances in multimodal therapies, high-grade glioma remains fatal. Temozolomide (TMZ) is an alkylating agent used worldwide for the clinical treatment of GBM; however, the innate and acquired resistance of GBM limits its application. Here, we found that TMZ inhibited the proliferation and induced the G2/M arrest of GBM cells. Therefore, we performed microarrays to identify the cell cycle- and apoptosis-related genes affected by TMZ. Notably, GADD45A was found to be up-regulated by TMZ in both cell cycle and apoptosis arrays. Furthermore, GADD45A knockdown (GADD45Akd) enhanced the cell growth arrest and cell death induced by TMZ, even in natural (T98) and adapted (TR-U373) TMZ-resistant cells. Interestingly, GADD45Akd decreased the expression of O6-methylguanine-DNA methyltransferase (MGMT) in TMZ-resistant cells (T98 and TR-U373). In MGMT-deficient/TMZ-sensitive cells (U87 and U373), GADD45Akd decreased TMZ-induced TP53 expression. Thus, in this study, we investigated the genes influenced by TMZ that were important in GBM therapy, and revealed that GADD45A plays a protective role against TMZ treatment which may through TP53-dependent and MGMT-dependent pathway in TMZ-sensitive and TMZ-resistant GBM, respectively. This protective role of GADD45A against TMZ treatment may provide a new therapeutic strategy for GBM treatment.
Project description:Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with poor survival. Cytoreduction in association with radiotherapy and temozolomide (TMZ) is the standard therapy, but response is heterogeneous and life expectancy is limited. The combined use of chemotherapeutic agents with drugs targeting cell metabolism is becoming an interesting therapeutic option for cancer treatment. Here, we found that metformin (MET) enhances TMZ effect on TMZ-sensitive cell line (U251) and overcomes TMZ-resistance in T98G GBM cell line. In particular, combined-treatment modulated apoptosis by increasing Bax/Bcl-2 ratio, and reduced Reactive Oxygen Species (ROS) production. We also observed that MET associated with TMZ was able to reduce the expression of glioma stem cells (GSC) marker CD90 particularly in T98G cells but not that of CD133. In vivo experiments showed that combined treatment with TMZ and MET significantly slowed down growth of TMZ-resistant tumors but did not affect overall survival of TMZ-sensitive tumor bearing mice. In conclusion, our results showed that metformin is able to enhance TMZ effect in TMZ-resistant cell line suggesting its potential use in TMZ refractory GBM patients. However, the lack of effect on a GBM malignancy marker like CD133 requires further evaluation since it might influence response duration.
Project description:Glioblastoma (GBM) is the most lethal primary brain cancer that lacks effective molecular targeted therapies. PI3K/AKT/mTOR signaling pathway is activated in 90% of all Glioblastoma Multiforme (GBM) tumors. To gain insight into the impact of the PI3K Pathway on GBM metabolism, we treated U87MG GBM cells with 50nM NVP-BEZ235 (PI3K and mTOR a dual inhibitor) for four days and identified differentially expressed genes with RNA-seq analysis. Overall design: U87MG cells were treated with 50nM NVP-BEZ235 for 4 days and analyzed by RNA seq
Project description:Despite multimodal treatment, glioblastoma (GBM) therapy with temozolomide (TMZ) remains inefficient due to chemoresistance. Matrix metalloproteinase (MMP) and a disintegrin and metalloprotease (ADAM), increased in GBM, could contribute to chemoresistance and TMZ-induced recurrence of glioblastoma.TMZ inducibility of metalloproteases was determined in GBM cell lines, primary GBM cells, and tissues from GBM and recurrent GBM. TMZ sensitivity and invasiveness of GBM cells were assessed in the presence of the metalloprotease inhibitors batimastat (BB-94) and marimastat (BB-2516). Metalloprotease-dependent effects of TMZ on mitochondria and pAkt/phosphatidylinositol-3 kinase (PI3K) and phosphorylated extracellular signal-regulated kinase 1/2 (pERK1/2) pathways were analyzed by fluorescence activated cell sorting, morphometry, and immunoblotting. Invasiveness of GBM cells was determined by Matrigel invasion assays. Potential metalloprotease substrates were identified by proteomics and tested for invasion using blocking antibodies.TMZ induces expression of MMP-1, -9, -14, and ADAM8 in GBM cells and in recurrent GBM tissues. BB-94, but not BB-2516 (ADAM8-sparing) increased TMZ sensitivity of TMZ-resistant and -nonresistant GBM cells with different O(6)-methylguanine-DNA methyltransferase states, suggesting that ADAM8 mediates chemoresistance, which was confirmed by ADAM8 knockdown, ADAM8 overexpression, or pharmacological inhibition of ADAM8. Levels of pAkt and pERK1/2 were increased in GBM cells and correlated with ADAM8 expression, cell survival, and invasiveness. Soluble hepatocyte growth factor (HGF) R/c-met and CD44 were identified as metalloprotease substrates in TMZ-treated GBM cells. Blocking of HGF R/c-met prevented TMZ-induced invasiveness.ADAM8 causes TMZ resistance in GBM cells by enhancing pAkt/PI3K, pERK1/2, and cleavage of CD44 and HGF R/c-met. Specific ADAM8 inhibition can optimize TMZ chemotherapy of GBM in order to prevent formation of recurrent GBM in patients.
Project description:Here we investigate the effects of the novel transforming growth factor-? receptor I (TGF-?RI) serine/threonine kinase inhibitor LY2109761 on glioblastoma when combined with the present clinical standard combination regimen radiotherapy and temozolomide (TMZ). Human GBM U87 (methylated MGMT promoter), T98 (unmethylated MGMT promoter), and endothelial cells (HUVECs) were treated with combinations of LY2109761, TMZ, and radiation. We found that LY2109761 reduced clonogenic survival of U87 and T98 cells and further enhanced the radiation-induced anticlonogenicity. In addition, LY2109761 had antimigratory and antiangiogenic effects in Matrigel migration and tube formation assays. In vivo, in human xenograft tumors growing subcutaneously on BALB/c nu/nu mice, LY2109761 delayed tumor growth alone and in combination with fractionated radiation and TMZ. Interestingly, as expected, the methylated U87 model was more sensitive to TMZ than the unmethylated T98 model in all experiments, whereas the opposite was found for LY2109761. Moreover, with respect to tumor angiogenesis, while LY2109761 decreased the glioblastoma proliferation index (Ki-67) and the microvessel density (CD31 count), the relative pericyte coverage (?-SMA/CD31 ratio) increased in particular after triple therapy, suggesting a vascular normalization effect induced by LY2109761. This normalization could be attributed in part to a decrease in the Ang-2/Ang-1 messenger RNA ratio. LY2109761 also reduced tumor blood perfusion as quantified by noninvasive dynamic contrast-enhanced magnetic resonance imaging. Together, the data indicate that the addition of a TGF-?RI kinase inhibitor to the present clinical standard (radiation plus TMZ) has the potential to improve clinical outcome in human glioblastoma, especially in patients with unmethylated MGMT promoter status.
Project description:The outlook for patients with advanced renal cell cancer (RCC) has been improved by targeted agents including inhibitors of the PI3 kinase (PI3K)-AKT-mTOR axis, although treatment resistance is a major problem. Here, we aimed to understand how RCC cells acquire resistance to PI3K-mTOR inhibition. We used the RCC4 cell line to generate a model of in vitro resistance by continuous culture in PI3K-mTOR kinase inhibitor NVP-BEZ235 (BEZ235, Dactolisib). Resistant cells were cross-resistant to mTOR inhibitor AZD2014. Sensitivity was regained after 4 months drug withdrawal, and resistance was partially suppressed by HDAC inhibition, supporting an epigenetic mechanism. BEZ235-resistant cells up-regulated and/or activated numerous proteins including MET, ABL, Notch, IGF-1R, INSR and MEK/ERK. However, resistance was not reversed by inhibiting or depleting these pathways, suggesting that many induced changes were passengers not drivers of resistance. BEZ235 blocked phosphorylation of mTOR targets S6 and 4E-BP1 in parental cells, but 4E-BP1 remained phosphorylated in resistant cells, suggesting BEZ235-refractory mTORC1 activity. Consistent with this, resistant cells over-expressed mTORC1 component RAPTOR at the mRNA and protein level. Furthermore, BEZ235 resistance was suppressed by RAPTOR depletion, or allosteric mTORC1 inhibitor rapamycin. These data reveal that RAPTOR up-regulation contributes to PI3K-mTOR inhibitor resistance, and suggest that RAPTOR expression should be included in the pharmacodynamic assessment of mTOR kinase inhibitor trials.
Project description:Drug resistance to temozolomide (TMZ) contributes to the majority of tumor recurrence and treatment failure in patients with glioblastoma multiforme (GBM). Autophagy has been reported to play a role in chemoresistance in various types of cancer, including GBM. The anticancer effect of statins is arousing great research interests and has been demonstrated to modulate autophagic function. In this study, we investigated the combinational effects of lovastatin and TMZ on treating U87 and U251 GBM cell lines. Cytotoxicity was measured by MTT and colony formation assays; apoptosis was measured by flow cytometry; the cellular autophagic function was detected by the EGFP-mRFP-LC3 reporter and western blot assay. The results showed that lovastatin might enhance the cytotoxicity of TMZ, increase the TMZ-induced cellular apoptosis, and impair the autophagic flux in GBM cells. Lovastatin triggered autophagy initiation possibly by inhibiting the Akt/mTOR signaling pathway. Moreover, lovastatin might impair the autophagosome-lysosome fusion machinery by suppressing LAMP2 and dynein. These results suggested that lovastatin could enhance the chemotherapy efficacy of TMZ in treating GBM cells. The mechanism may be associated with impaired autophagic flux and thereby the enhancement of cellular apoptosis. Combining TMZ with lovastatin could be a promising strategy for GBM treatment.
Project description:The PI3K/Akt pathway is activated in many cancers; therefore, we investigated NVP-BEZ235, a dual PI3K/mTOR inhibitor. BEZ235 was more potent than either the mTOR inhibitor rapamycin or the PI3K inhibitor LY294002 in blocking HIF-1? induction. BEZ235 decreases protein translation, and 7-methyl GTP chromatography showed that the drug induced robust recruitment of 4E-BP1 to eIF4E and a near absence of binding of eIF4G. BEZ235 also decreased expression of other proteins known to be regulated by eIF4E including cyclin B1 and D1 and vascular endothelial growth factor (VEGF). BEZ235 also decreased the level of eIF4G but not eIF4E. As HIF-1? has been associated with adaptation to hypoxic stress, we examined the effect of the drug on cell survival in low pO 2. BEZ235 increased killing of cells under hypoxia, measured by short-term (MTT) and long-term (clonogenic) assays. To understand the underlying mechanism, we examined BEZ235's effect on the expression of factors associated with cell survival. Under normoxia, Akt Ser473 phosphorylation decreased within an hour of BEZ235 treatment, but then increased by 24 h. In contrast, under hypoxia, BEZ235 caused prolonged suppression of Akt Ser473 phosphorylation. Furthermore, there was greater PARP cleavage in hypoxic cells than in normoxic cells, consistent with increased apoptosis. BEZ235 increased autophagy as measured by LC3-I to LC3-II conversion under both normoxic and hypoxic conditions, but our data indicate that this is actually a pro-survival mechanism. In conclusion, we have found that BEZ235 blocks HIF-1? induction by decreasing protein translation and increases cell killing under hypoxia, likely by increasing apoptosis.