Metformin treatment reduces temozolomide resistance of glioblastoma cells.
ABSTRACT: It has been reported that metformin acts synergistically with temozolomide (TMZ) to inhibit proliferation of glioma cells including glioblastoma multiforme (GBM). However, the molecular mechanism underlying how metformin exerts its anti-cancer effects remains elusive. We used a combined experimental and bioinformatics approach to identify genes and complex regulatory/signal transduction networks that are involved in restoring TMZ sensitivity of GBM cells after metformin treatment. First, we established TMZ resistant GBM cell lines and found that the resistant cells regained TMZ sensitivity after metformin treatment. We further identified that metformin down-regulates SOX2 expression in TMZ-resistant glioma cells, reduces neurosphere formation capacity of glioblastoma cells, and inhibits GBM xenograft growth in vivo. Finally, the global gene expression profiling data reveals that multiple pathways are involved in metformin treatment related gene expression changes, including fatty acid metabolism and RNA binding and splicing pathways. Our work provided insight of the mechanisms on potential synergistic effects of TMZ and metformin in the treatment of glioblastoma, which will in turn yield potentially translational value for clinical applications.
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 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 (GADD45A<sup>kd</sup>) enhanced the cell growth arrest and cell death induced by TMZ, even in natural (T98) and adapted (TR-U373) TMZ-resistant cells. Interestingly, GADD45A<sup>kd</sup> decreased the expression of O<sup>6</sup>-methylguanine-DNA methyltransferase (MGMT) in TMZ-resistant cells (T98 and TR-U373). In MGMT-deficient/TMZ-sensitive cells (U87 and U373), GADD45A<sup>kd</sup> 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:Overcoming temozolomide (TMZ) resistance in glioma cancer cells remains a major challenge to the effective treatment of the disease. Increasing TMZ efficacy for patients with glioblastoma (GBM) is urgently needed because TMZ treatment is the standard chemotherapy protocol for adult patients with glioblastoma. O6-methylguanine-DNA-methyltransferase (MGMT) overexpression is associated with TMZ resistance, and low MGMT is a positive response marker for TMZ therapy. Here, we used 3 glioma cell lines (SF767, U373, and LN229), which had different levels of TMZ sensitivity. We found TMZ sensitivity is positively correlated with MGMT expression and multidrug-resistance protein ABC subfamily G member 2 (ABCG2) in these cells. CK2-STAT3 signaling and Hippo-YAP signaling are reported to regulate MGMT expression and ABCG2 expression, respectively. We combined CK2 inhibitor CX-4945 and YAP inhibitor verteporfin with TMZ treatment. We found that CX-4945 but not verteporfin can sensitize TMZ-resistant cells SF767 to TMZ and that CX-4945 and TMZ combinational treatment was effective for glioma treatment in mouse models compared with TMZ alone. IMPLICATIONS: A combination of CK2 inhibitor with TMZ may improve the therapeutic efficiency of TMZ toward GBM with acquired resistance.
Project description:Acquired chemoresistance is a major limiting factor in the clinical treatment of glioblastoma (GBM). However, the mechanism by which GBM acquires therapeutic resistance remains unclear. Here, we aimed to investigate whether METTL3-mediated N6-methyladenosine (m<sup>6</sup>A) modification contributes to the temozolomide (TMZ) resistance in GBM. We demonstrated that METTL3 METTL3-mediated m<sup>6</sup>A modification were significantly elevated in TMZ-resistant GBM cells. Functionally, METTL3 overexpression impaired the TMZ-sensitivity of GBM cells. In contrast, METTL3 silencing or DAA-mediated total methylation inhibition improved the sensitivity of TMZ-resistant GBM cells to TMZ <i>in vitro</i> and <i>in vivo</i>. Furthermore, we found that two critical DNA repair genes (MGMT and APNG) were m<sup>6</sup>A-modified by METTL3, whereas inhibited by METTL3 silencing or DAA-mediated total methylation inhibition, which is crucial for METTL3-improved TMZ resistance in GBM cells. Collectively, METTL3 acts as a critical promoter of TMZ resistance in glioma and extends the current understanding of m<sup>6</sup>A related signaling, thereby providing new insights into the field of glioma treatment.
Project description:Glioblastoma multiforme (GBM) is one of the most malignant human intracranial tumors. Temozolomide (TMZ) is the primary alkylating agent for GBM patients. However, many GBM patients are resistant to TMZ. Therefore, patients with GBM urgently need more effective therapeutic options. 20(S)-ginsenoside-Rg3 (20(S)-Rg3) is a natural chemical with anti-tumor effects, but at present there is little understanding of its functional mechanism. Several research reports have demonstrated that O<sup>6</sup> -methylguanine DNA-methyltransferase (MGMT) repairs damaged DNA and contributes to TMZ resistance in gliomas. In addition, recent studies have shown that MGMT gene expression could be regulated by the Wnt/β-catenin pathway. However, whether 20(S)-Rg3 inhibits MGMT expression and augments chemosensitivity to Temozolomide (TMZ) in glioma cells remains unclear. In this study, we explored the modulating effects of 20(S)-Rg3 on MGMT. We used glioma cell lines, primary cell strain (including T98G, U118 and GBM-XX; all of them are MGMT-positive glioma cell lines) and xenograft glioma models to examine whether 20(S)-Rg3 increased the sensitivity to TMZ and to reveal the underlying mechanisms. We found that the MGMT expression was effectively downregulated by 20(S)-Rg3 via the Wnt/β-catenin pathway in glioma cell lines, and TMZ resistance was significantly reversed by 20(S)-Rg3. Meanwhile, 20(S)-Rg3 shows no obvious cytotoxicity at its effective dose and is well tolerated in vivo. In addition, we found that 20(S)-Rg3 significantly restrains the epithelial-mesenchymal transition (EMT) progression of glioma cells. Taken together, these results indicate that 20(S)-Rg3 may be a novel agent to use in treatment of GBM, especially in TMZ-resistant GBM with high MGMT expression.
Project description:About 95% of Glioblastoma (GBM) patients experience tumor relapse as a consequence of resistance to the first-line standard chemotherapy using temozolomide (TMZ). Recent studies reported consistently elevated expression levels of carbonic anhydrase CA2 in recurrent glioblastoma and temozolomide-resistant glioblastoma stem-like cells (GSCs). Here we show that CA2 is preferentially expressed in GSCs and upregulated by TMZ treatment. When expressed in GBM cell lines, CA2 exerts significant metabolic changes reflected by enhanced oxygen consumption and increased extracellular acidification causing higher rates of cell invasion. Notably, GBM cells expressing CA2 respond to combined treatment with TMZ and brinzolamide (BRZ), a non-toxic and potent CA2 inhibitor. Interestingly, brinzolamide was more effective than the pan-CA inhibitor Acetazolamide (ACZ) to sensitize naïve GSCs and TMZ-resistant GSCs to TMZ induced cell death. Mechanistically, we demonstrated that the combined treatment of GBM stem cells with TMZ and BRZ caused autophagy of GBM cell lines and GSCs, reflected by enhanced LC3 cleavage (LC3-II) and p62 reduction. Our findings illustrate the potential of CA2 as a chemo-sensitizing drug target in recurrent GBM and propose a combined treatment of TMZ with CA2 inhibitor to tackle GBM chemoresistance and recurrence.
Project description:Objective: Temozolomide (TMZ) is commonly used for glioblastoma multiforme (GBM) chemotherapy. However, drug resistance limits its therapeutic effect in GBM treatment. RNA-binding proteins (RBPs) have vital roles in posttranscriptional events. While disturbance of RBP-RNA network activity is potentially associated with cancer development, the precise mechanisms are not fully known. The SNRPG gene, encoding small nuclear ribonucleoprotein polypeptide G, was recently found to be related to cancer incidence, but its exact function has yet to be elucidated. Methods: SNRPG knockdown was achieved via short hairpin RNAs. Gene expression profiling and Western blot analyses were used to identify potential glioma cell growth signaling pathways affected by SNRPG. Xenograft tumors were examined to determine the carcinogenic effects of SNRPG on glioma tissues. Results: The SNRPG-mediated inhibitory effect on glioma cells might be due to the targeted prevention of Myc and p53. In addition, the effects of SNRPG loss on p53 levels and cell cycle progression were found to be Myc-dependent. Furthermore, SNRPG was increased in TMZ-resistant GBM cells, and downregulation of SNRPG potentially sensitized resistant cells to TMZ, suggesting that SNRPG deficiency decreases the chemoresistance of GBM cells to TMZ via the p53 signaling pathway. Our data confirmed that SNRPG suppression sensitizes GBM cells to TMZ by targeting Myc via the p53 signaling cascade. Conclusions: These results indicated that SNRPG is a probable molecular target of GBM and suggested that suppressing SNRPG in resistant GBM cells might be a substantially beneficial method for overcoming essential drug resistance.
Project description:Treatment of glioblastoma (GBM) remains a challenge using conventional chemotherapy, such as temozolomide (TMZ), and is often ineffective as a result of drug resistance. We have assessed a novel nitrone-based agent, OKN-007, and found it to be effective in decreasing tumor volumes and increasing survival in orthotopic GBM xenografts by decreasing cell proliferation and angiogenesis and increasing apoptosis. In this study, we assessed combining OKN-007 with TMZ in vivo in a human G55 GBM orthotopic xenograft model and in vitro in TMZ-resistant and TMZ-sensitive human GBM cell lines. For the in vivo studies, magnetic resonance imaging was used to assess tumor growth and vascular alterations. Percent animal survival was also determined. For the in vitro studies, cell growth, IC50 values, RNA-seq, RT-PCR, and ELISA were used to assess growth inhibition, possible mechanism-of actions (MOAs) associated with combined OKN-007?+?TMZ versus TMZ alone, and gene and protein expression levels, respectively. Microarray analysis of OKN-007-treated rat F98 glioma tumors was also carried out to determine possible MOAs of OKN-007 in glioma-bearing animals either treated or not treated with OKN-007. OKN-007 seems to elicit its effect on GBM tumors via inhibition of tumorigenic TGF-?1, which affects the extracellular matrix. When combined with TMZ, OKN-007 significantly increases percent survival, decreases tumor volumes, and normalizes tumor blood vasculature in vivo compared to untreated tumors and seems to affect TMZ-resistant GBM cells possibly via IDO-1, SUMO2, and PFN1 in vitro. Combined OKN-007?+?TMZ may be a potentially potent treatment strategy for GBM patients.
Project description:Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.
Project description:Glioblastoma (GBM) is a malignant, primary brain tumor, highly resistant to conventional therapies. Temozolomide (TMZ) is a first line therapeutic agent in GBM patients, however, survival of such patients is poor. High level of DNA repair protein, O<sup>6</sup>-methylguanine-DNA-methyltransferase (MGMT) and occurrence of glioma stem-like cells contribute to GBM resistance to the drug. Here, we explored a possibility of epigenetic reprograming of glioma cells to increase sensitivity to TMZ and restore apoptosis competence. We combined TMZ treatment with BIX01294, an inhibitor of histone methyltransferase G9a, known to be involved in cancerogenesis. Two treatment combinations were tested: BIX01294 was administered to human LN18 and U251 glioma cell cultures 48 h before TMZ or 48 h after TMZ treatment. Despite their different status of the <i>MGMT</i> gene promoter, there was no correlation with the response to TMZ. The analyses of cell viability, appearance of apoptotic alterations in morphology of cells and nuclei, and markers of apoptosis, such as levels of cleaved caspase 3, caspase 7 and PARP, revealed that both pre-treatment and post-treatment with BIX01294 sensitize glioma cells to TMZ. The additive effect was stronger in LN18 cells. Moreover, BIX01294 enhanced the cytotoxic effect of TMZ on glioma stem-like cells, although it was not associated with modulation of the pluripotency markers (<i>NANOG, SOX2, CD133</i>) expression or methylation of <i>NANOG</i> and <i>SOX2</i> gene promoters. Accordingly, knockdown of methyltransferase G9a augments TMZ-induced cell death in LN18 cells. We found the significant increases of the LC3-II levels in LN18 cells treated with BIX01294 alone and with drug combination that suggests involvement of autophagy in enhancement of anti-tumor effect of TMZ. Treatment with BIX01294 did not affect methylation of the <i>MGMT</i> gene promoter. Altogether, our results suggest that G9a is a potential therapeutic target in malignant glioma and the treatment with the G9a inhibitor reprograms glioma cells and glioma stem-like cells to increase sensitivity to TMZ and restore apoptosis competence.