Chemotherapeutic resistance in anaplastic astrocytoma cell lines treated with a temozolomide–lomeguatrib combination

The treatment of anaplastic astrocytoma (AA) is controversial. New chemotherapeutic approaches are needed for AA treatment. Temozolomide (TMZ) is one of the chemotherapeutic drugs for the treatment of AA. The cytotoxic effects of TMZ can be removed by the MGMT (O(6)-methylguanine-DNA methyltransferase) enzyme. Then, chemotherapeutic resistance to TMZ occurs. MGMT inhibition by MGMT inactivators (such as lomeguatrib) is an important anticancer therapeutic approach to circumvent TMZ resistance. We aim to investigate the effect of TMZ–lomeguatrib combination on MGMT expression and TMZ sensitivity of SW1783 and GOS-3 AA cell lines. The sensitivity of SW1783 and GOS-3 cell lines to TMZ and to the combination of TMZ and lomeguatrib was determined by a cytotoxicity assay. MGMT methylation was detected by MS-PCR. MGMT and p53 expression were investigated by real-time PCR after drug treatment, and the proportion of apoptotic cells was analyzed by flow cytometry. When the combination of TMZ–lomeguatrib (50 μM) was used in AA cell lines, IC₅₀ values were reduced compared to only using TMZ. MGMT expression was decreased, p53 expression was increased, and the proportion of apoptotic cells was induced in both cell lines. The lomeguatrib–TMZ combination did not have any effect on the cell cycle and caused apoptosis by increasing p53 expression and decreasing MGMT expression. Our study is a pilot study investigating a new therapeutic approach for AA treatment, but further research is needed.     

O(6)-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: Enzyme activity, promoter methylation and immunohistochemistry

O(6)-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O(6)-methylguanine. It also repairs larger adducts on the O(6)-position of guanine, such as O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine and O(6)-chloroethylguanine. These adducts are formed in response to alkylating environmental pollutants, tobacco-specific carcinogens and methylating (procarbazine, dacarbazine, streptozotocine, and temozolomide) as well as chloroethylating (lomustine, nimustine, carmustine, and fotemustine) anticancer drugs. MGMT is therefore a key node in the defense against commonly found carcinogens, and a marker of resistance of normal and cancer cells exposed to alkylating therapeutics. MGMT also likely protects against therapy-related tumor formation caused by these highly mutagenic drugs. Since the amount of MGMT determines the level of repair of toxic DNA alkylation adducts, the MGMT expression level provides important information as to cancer susceptibility and the success of therapy. In this article, we describe the methods employed for detecting MGMT and review the literature with special focus on MGMT activity in normal and neoplastic tissues. The available data show that the expression of MGMT varies greatly in normal tissues and in some cases this has been related to cancer predisposition. MGMT silencing in tumors is mainly regulated epigenetically and in brain tumors this correlates with a better therapeutic response. Conversely, up-regulation of MGMT during cancer treatment limits the therapeutic response. In malignant melanoma, MGMT is not related to the therapeutic response, which is due to other mechanisms of inherent drug resistance. For most cancers, studies that relate MGMT activity to therapeutic outcome following O(6)-alkylating drugs are still lacking.     

Temozolomide Resistance in Glioblastoma Cell Lines: Implication of MGMT,MMR, P-Glycoprotein and CD133 Expression

Background

The use of temozolomide (TMZ) has improved the prognosis for glioblastoma multiforme patients. However, TMZ resistance may be one of the main reasons why treatment fails. Although this resistance has frequently been linked to the expression of O6-methylguanine-DNA methyltransferase (MGMT) it seems that this enzyme is not the only molecular mechanism that may account for the appearance of drug resistance in glioblastoma multiforme patients as the mismatch repair (MMR) complex, P-glycoprotein, and/or the presence of cancer stem cells may also be implicated.

Methods

Four nervous system tumor cell lines were used to analyze the modulation of MGMT expression and MGMT promoter methylation by TMZ treatment. Furthermore, 5-aza-2’-deoxycytidine was used to demethylate the MGMT promoter and O(6)-benzylguanine to block GMT activity. In addition, MMR complex and P-glycoprotein expression were studied before and after TMZ exposure and correlated with MGMT expression. Finally, the effect of TMZ exposure on CD133 expression was analyzed.

Results

Our results showed two clearly differentiated groups of tumor cells characterized by low (A172 and LN229) and high (SF268 and SK-N-SH) basal MGMT expression. Interestingly, cell lines with no MGMT expression and low TMZ IC₅₀ showed a high MMR complex expression, whereas cell lines with high MGMT expression and high TMZ IC₅₀ did not express the MMR complex. In addition, modulation of MGMT expression in A172 and LN229 cell lines was accompanied by a significant increase in the TMZ IC₅₀, whereas no differences were observed in SF268 and SK-N-SH cell lines. In contrast, P-glycoprotein and CD133 was found to be unrelated to TMZ resistance in these cell lines.

Conclusions

These results may be relevant in understanding the phenomenon of TMZ resistance, especially in glioblastoma multiforme patients laking MGMT expression, and may also aid in the design of new therapeutic strategies to improve the efficacy of TMZ in glioblastoma multiforme patients.     

O6-methylguanine DNA methyltransferase and p53 status predict temozolomide sensitivity in human malignant glioma cells

Temozolomide (TMZ) is a methylating agent which prolongs survival when administered during and after radiotherapy in the first-line treatment of glioblastoma and which also has significant activity in recurrent disease. O6-methylguanine DNA methyltransferase (MGMT) is a DNA repair enzyme attributed a role in cancer cell resistance to O6-alkylating agent-based chemotherapy. Using a panel of 12 human glioma cell lines, we here defined the sensitivity to TMZ in acute cytotoxicity and clonogenic survival assays in relation to MGMT, mismatch repair and p53 status and its modulation by dexamethasone, irradiation and BCL-X(L). We found that the levels of MGMT expression were a major predictor of TMZ sensitivity in human glioma cells. MGMT activity and clonogenic survival after TMZ exposure are highly correlated (p < 0.0001, r2 = 0.92). In contrast, clonogenic survival after TMZ exposure does not correlate with the expression levels of the mismatch repair proteins mutS homologue 2, mutS homologue 6 or post-meiotic segregation increased 2. The MGMT inhibitor O6-benzylguanine sensitizes MGMT-positive glioma cells to TMZ whereas MGMT gene transfer into MGMT-negative cells confers protection. The antiapoptotic BCL-X(L) protein attenuates TMZ cytotoxicity in MGMT-negative LNT-229 but not in MGMT-positive LN-18 cells. Neither ionizing radiation (4 Gy) nor clinically relevant concentrations of dexamethasone modulate MGMT activity or TMZ sensitivity. Abrogation of p53 wild-type function strongly attenuates TMZ cytotoxicity. Conversely, p53 mimetic agents designed to stabilize the wild-type conformation of p53 sensitize glioma cells for TMZ cytotoxicity. Collectively, these results suggest that the determination of MGMT expression and p53 status will help to identify glioma patients who will or will not respond to TMZ.     

A novel approach to overcome temozolomide resistance in glioma and melanoma: Inactivation of MGMT by gene therapy

Malignant glioma is the most common primary brain tumor. Malignant melanoma is the most malignant of skin tumor. The two malignancies are poorly responsive to conventional treatment regimens such as chemotherapy. Temozolomide (TMZ) is a DNA-alkylating agent used for the treatment of glioma, astrocytoma, and melanoma. Resistance to alkylating agents such as TMZ correlates with increased expression of DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT). Several studies in animal models have demonstrated that decreasing MGMT level with gene therapy could overcome TMZ resistance and enhance tumor cell death. In the present review, we provide an overview of recent advances in this field.     

Enhanced Anti-Tumor Effect of Zoledronic Acid Combined with Temozolomide against Human Malignant Glioma Cell Expressing O6-Methylguanine DNA Methyltransferase

Temozolomide (TMZ), a DNA methylating agent, is widely used in the adjuvant treatment of malignant gliomas. O⁶-methylguanine-DNA methyltranferase (MGMT), a DNA repair enzyme, is frequently discussed as the main factor that limits the efficacy of TMZ. Zoledronic acid (ZOL), which is clinically applied to treat cancer-induced bone diseases, appears to possess direct anti-tumor activity through apoptosis induction by inhibiting mevalonate pathway and prenylation of intracellular small G proteins. In this study, we evaluated whether ZOL can be effectively used as an adjuvant to TMZ in human malignant glioma cells that express MGMT. Malignant glioma cell lines, in which the expression of MGMT was detected, did not exhibit growth inhibition by TMZ even at a longer exposure. However, combination experiment of TMZ plus ZOL revealed that a supra-additive effect resulted in a significant decrease in cell growth. In combined TMZ/ZOL treatment, an increased apoptotic rate was apparent and significant activation of caspase-3 and cleavage of poly-(ADP-ribose) polymerase were observed compared with each single drug exposure. There were decreased amounts of Ras-GTP, MAPK and Akt phosphorylation and MGMT expression in the ZOL-treated cells. Subcutanous xenograft models showed significant decrease of tumor growth with combined TMZ/ZOL treatment. These results suggest that ZOL efficaciously inhibits activity of Ras in malignant glioma cells and potentiates TMZ-mediated cytotoxicity, inducing growth inhibition and apoptosis of malignant glioma cells that express MGMT and resistant to TMZ. Based on this work, combination of TMZ with ZOL might be a potential therapy in malignant gliomas that receive less therapeutic effects of TMZ due to cell resistance.     

Inhibition of Casein Kinase II by CX-4945, But Not Yes-associated protein (YAP) by Verteporfin,Enhances the Antitumor Efficacy of Temozolomide in Glioblastoma

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.ImplicationsA combination of CK2 inhibitor with TMZ may improve the therapeutic efficiency of TMZ toward GBM with acquired resistance.     

O-Methylguanine-DNA methyltransferase (MGMT) in normal tissues and tumors: Enzyme activity,promoter methylation and immunohistochemistry

O⁶-Methylguanine-DNA methyltransferase (MGMT) is a suicide enzyme that repairs the pre-mutagenic, pre-carcinogenic and pre-toxic DNA damage O⁶-methylguanine. It also repairs larger adducts on the O⁶-position of guanine, such as O(6)-[4-oxo-4-(3-pyridyl)butyl]guanine and O⁶-chloroethylguanine. These adducts are formed in response to alkylating environmental pollutants, tobacco-specific carcinogens and methylating (procarbazine, dacarbazine, streptozotocine, and temozolomide) as well as chloroethylating (lomustine, nimustine, carmustine, and fotemustine) anticancer drugs. MGMT is therefore a key node in the defense against commonly found carcinogens, and a marker of resistance of normal and cancer cells exposed to alkylating therapeutics. MGMT also likely protects against therapy-related tumor formation caused by these highly mutagenic drugs. Since the amount of MGMT determines the level of repair of toxic DNA alkylation adducts, the MGMT expression level provides important information as to cancer susceptibility and the success of therapy. In this article, we describe the methods employed for detecting MGMT and review the literature with special focus on MGMT activity in normal and neoplastic tissues. The available data show that the expression of MGMT varies greatly in normal tissues and in some cases this has been related to cancer predisposition. MGMT silencing in tumors is mainly regulated epigenetically and in brain tumors this correlates with a better therapeutic response. Conversely, up-regulation of MGMT during cancer treatment limits the therapeutic response. In malignant melanoma, MGMT is not related to the therapeutic response, which is due to other mechanisms of inherent drug resistance. For most cancers, studies that relate MGMT activity to therapeutic outcome following O⁶-alkylating drugs are still lacking.     

O6-methylguanine DNA methyltransferase as a promising target for the treatment of temozolomide-resistant gliomas

Temozolomide (TMZ) is an alkylating agent currently used as first-line therapy for gliomas treatment due to its DNA-damaging effect. However, drug resistance occurs, preventing multi-cycle use of this chemotherapeutic agent. One of the major mechanisms of cancer drug resistance is enhanced activity of a DNA repair enzyme, O⁶-methylguanine-DNA-methyltransferase (MGMT), which counteracts chemotherapy-induced DNA alkylation and is a key component of chemoresistance. MGMT repairs TMZ-induced DNA lesions, O⁶-meG, by transferring the alkyl group from guanine to a cysteine residue. This review provides an overview of recent advances in the field, with particular emphasis on the inhibitors of MGMT and underlying mechanisms. Literature search was performed through PubMed and all relevant articles were reviewed, with particular attention to MGMT, its role in TMZ-resistant gliomas, effects of MGMT inhibitors and the underlying mechanisms. Several strategies are currently being pursued to improve the therapeutic efficacy of TMZ via inhibition of MGMT to reduce chemoresistance and improve overall survival. MGMT may be a promising target for the treatment of TMZ-resistant gliomas.     

Chemoresistance to temozolomide in human glioma cell line U251 is associated with increased activity of O6-methylguanine-DNA methyltransferase and can be overcome by metronomic temozolomide regimen

Temozolomide (TMZ) is a novel cytotoxic alkylating agent for chemotherapy of malignant gliomas. However, intrinsic or acquired resistance to TMZ often defines poor efficacy of chemotherapy in malignant gliomas. A growing number of studies indicate that expression of O ⁶-methylguanine-DNA methyltransferase (MGMT) is one of the principal mechanisms responsible for this chemoresistance. In the present study, we evaluated the relationship between expression of MGMT and resistance to TMZ. We generated a TMZ-resistant cell line, U251/TR, by stepwise (8 months) exposure of parental U251 cells to TMZ. The resistance to TMZ was quantified using SRB assay. MGMT expression was evaluated at mRNA (RT-PCR) and protein (Western blot) levels. U251/TR cells showed increased (~ sevenfold) resistance to TMZ. The MGMT expression (both mRNA and protein) was significantly (P < 0.01) increased in U251/TR cells compared with parental U251 cells. Further, MGMT expression fluctuated during exposure of U251/TR cells to TMZ. The resistance of U251/TR cells to TMZ could be overcome by application of elevated doses of TMZ when MGMT expression was at the lowest level. In conclusion, our results demonstrate that the primary mechanism responsible for resistance of U251/TR cells to TMZ is associated with increased expression of MGMT. Resistance of malignant gliomas to TMZ can be overcome by synchronizing metronomic TMZ regimen with MGMT expression.     

BMP2 sensitizes glioblastoma stem-like cells to Temozolomide by affecting HIF-1�� stability and MGMT expression

Glioblastoma multiforme (GBM) is the most common brain tumour, characterized by a central and partially necrotic (i.e., hypoxic) core enriched in cancer stem cells (CSCs). We previously showed that the most hypoxic and immature (i.e., CSCs) GBM cells were resistant to Temozolomide (TMZ) in vitro, owing to a particularly high expression of O6-methylguanine-DNA-methyltransferase (MGMT), the most important factor associated to therapy resistance in GBM. Bone morphogenetic proteins (BMPs), and in particular BMP2, are known to promote differentiation and growth inhibition in GBM cells. For this reason, we investigated whether a BMP2-based treatment would increase TMZ response in hypoxic drug-resistant GBM-derived cells. Here we show that BMP2 induced strong differentiation of GBM stem-like cells and subsequent addition of TMZ caused dramatic increase of apoptosis. Importantly, we correlated these effects to a BMP2-induced downregulation of both hypoxia-inducible factor-1α (HIF-1α) and MGMT. We report here a novel mechanism involving the HIF-1α-dependent regulation of MGMT, highlighting the existence of a HIF-1α/MGMT axis supporting GBM resistance to therapy. As confirmed from this evidence, over-stabilization of HIF-1α in TMZ-sensitive GBM cells abolished their responsiveness to it. In conclusion, we describe a HIF-1α-dependent regulation of MGMT and suggest that BMP2, by down-modulating the HIF-1α/MGMT axis, should increase GBM responsiveness to chemotherapy, thus opening the way to the development of future strategies for GBM treatment.     

DNA repair protein O6-methylguanine-DNA methyltransferase in testis and testicular tumors as determined by a novel nonradioactive assay

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT, alkyltransferase) is an important suicide enzyme involved in defense against O6-alkylating endogenous metabolites and environmental carcinogens. It also plays a pivotal role in primary and acquired resistance of tumors to alkylating anticancer drugs targeting the O6-position of guanine (i.e., methylating and chloroethylating agents). MGMT can thus be considered a crucial biomarker for individual susceptibility to alkylating carcinogens and tumor drug resistance. This implies a need for a fast and convenient method for determination of MGMT. Routinely, MGMT is being quantified by radioactive assays which are relatively laborious. Here we report a nonradioactive MGMT enzyme-linked immunosorbent assay (ELISA) for quantification of MGMT in cell and tissue homogenates. We compared the MGMT-ELISA with the standard radioactive assay and found it to be as sensitive but less time consuming. Therefore, it represents an alternative for the quantification of MGMT in cell and tissue homogenates. We applied the assay for determining MGMT in normal and tumor tissue of testes. In both normal and tumor tissue MGMT was quite variable, ranging from zero to 1300 fmol/mg protein. In various tumor samples MGMT was lower than MGMT in the normal tissue from the same patient or was even not detectable. The MGMT-ELISA might become a useful tool for MGMT determination in clinical routine and health control.     

MGMT hypermethylation: a prognostic foe, a predictive friend

Alkylation of DNA at the O(6)-position of guanine is one of the most critical events leading to mutation, cancer, and cell death. O(6)-alkylguanine-DNA alkyltransferase (AGT), also known as O(6)-methylguanine-DNA methyltransferase (MGMT), is the DNA repair protein responsible for removing alkylation adducts from the O(6)-position of guanine in DNA. The promoter CpG island hypermethylation-associated gene silencing of MGMT is associated with a wide spectrum of human tumors. This epigenetic inactivation of MGMT has two main consequences in human cancer. First, it uncovers a new mutator pathway that causes the accumulation of G-to-A transition mutations that can affect genes required for genomic stability. Second, there is a strong and significant positive correlation between MGMT promoter hypermethylation and increased tumor sensitivity to alkylating drugs. These findings underline the importance of MGMT promoter hypermethylation in basic and translational cancer research.     

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma

Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.     

替莫唑胺治疗胶质母细胞瘤的耐药性产生机制研究进展

胶质母细胞瘤作为胶质瘤中恶性程度最高的原发性脑部肿瘤,具有治愈率低、复发率高、呈浸润性生长等特点,在不使用化疗药物的情况下,患者中位生存期仅为12.1个月。胶质母细胞瘤患者的标准治疗方法以手术切除为主,放化疗为辅,其中替莫唑胺（temozolomide,TMZ）作为一种新型的口服烷化剂,是目前用于胶质瘤化学治疗的一线药物。但经过替莫唑胺治疗后,患者中位生存期仅提高了2个月,主要原因为胶质母细胞瘤可对TMZ产生耐药性。胶质母细胞瘤对TMZ产生的耐药机制主要为DNA修复机制,其包括了O⁶?甲基鸟嘌呤DNA甲基转移酶（O⁶?methyl guanine DNA methyltransferase,MGMT）对药物作用位点进行的直接修复、错配修复（mismatch repair,MMR）及碱基切除修复（base excision repair,BER）,这些修复机制可修复TMZ引起的DNA损伤,从而降低肿瘤细胞对TMZ敏感性。通过对近年来胶质母细胞瘤的TMZ耐药机制的研究进展进行介绍,旨在为发展新的治疗手段提供理论基础。     

O(6)-Methylguanine-DNA methyltransferase in glioma therapy: Promise and problems

Gliomas are the most frequent adult primary brain tumor, and are invariably fatal. The most common diagnosis glioblastoma multiforme (GBM) afflicts 12,500 new patients in the U.S. annually, and has a median survival of approximately one year when treated with the current standard of care. Alkylating agents have long been central in the chemotherapy of GBM and other gliomas. The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT), the principal human activity that removes cytotoxic O(6)-alkylguanine adducts from DNA, promotes resistance to anti-glioma alkylators, including temozolomide and BCNU, in GBM cell lines and xenografts. Moreover, MGMT expression assessed by immunohistochemistry, biochemical activity or promoter CpG methylation status is associated with the response of GBM to alkylator-based therapies, providing evidence that MGMT promotes clinical resistance to alkylating agents. These observations suggest a role for MGMT in directing adjuvant therapy of GBM and other gliomas. Promoter methylation status is the most clinically tractable measure of MGMT, and there is considerable enthusiasm for exploring its utility as a marker to assign therapy to individual patients. Here, we provide an overview of the biochemical, genetic and biological characteristics of MGMT as they relate to glioma therapy. We consider current methods to assess MGMT expression and discuss their utility as predictors of treatment response. Particular emphasis is given to promoter methylation status and the methodological and conceptual impediments that limit its use to direct treatment. We conclude by considering approaches that may improve the utility of MGMT methylation status in planning optimal therapies tailored to individual patients.