Inhibition of DNA repair for sensitizing resistant glioma cells to temozolomide (original) (raw)
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Biomedicines
Temozolomide, a DNA methylating drug, is currently being used first-line in glioblastoma therapy. Although the mode of action of this so-called SN1 alkylating agent is well described, including the types of induced DNA damage triggering the DNA damage response and survival and death pathways, some researchers expressed doubt that data mostly obtained by in vitro models can be translated into the in vivo situation. In experimental settings, high doses of the agent are often used, which are likely to activate responses triggered by base N-alkylations instead of O6-methylguanine (O6MeG), which is the primary cytotoxic lesion induced by low doses of temozolomide and other methylating drugs in O6-methylguanine-DNA methyltransferase (MGMT) repair incompetent cells. However, numerous studies provided compelling evidence that O6MeG is not only a mutagenic, but also a powerful toxic lesion inducing DNA double-strand breaks, apoptosis, autophagy and cellular senescence. MGMT, repairing the le...
Background The current standard of care for Glioblastoma Multiforme (GBM) consists of fractionated focal irradiation with concomitant temozolomide (TMZ) chemotherapy. A promising strategy to increase the efficacy of TMZ is through interference with the DNA damage repair machinery, by poly(ADP-ribose) polymerase protein inhibition(PARPi). The objective of the present study was to investigate the therapeutic benefit of combination therapy in patientderived glioma stem-like cells (GSC). Methods Combination therapy feasibility was tested on established GBM cell lines U373 and T98. We developed an in vitro drug-screening assay based on GSC cultures derived from a panel of primary patient tissue samples (n = 20) to evaluate the effect of PARPi (ABT-888) monotherapy and combination therapy with TMZ. Therapeutic effect was assessed by viability, double stranded breaks, apoptosis and autophagy assays and longitudinal microscopic cell monitoring was performed. O-6-methylguanine-DNA methyltransferase (MGMT) status was determined by methylation assay and protein expression by western blots. Results PARPi monotherapy was found to decrease viability by more than 25% in 4 of the 20 GSCs (20%) at 10μM. TMZ monotherapy at 50μM and 100μM was effective in 12 and 14 of the 20 GSCs, respectively. TMZ resistance to 100μM was found in 7 of 8 MGMT protein positive cultures. Potentiation of TMZ therapy through PARPi was found in 90% (n = 20) of GSCs, of which 6 were initially resistant and 7 were sensitive to TMZ monotherapy. Increased induction of double stranded breaks and apoptosis were noted in responsive GSCs. There was a trend noted, albeit statistically insignificant, of increased autophagy both in western blots and accumulation of autophagosomes. Conclusion PARPi mediated potentiation of TMZ is independent of TMZ sensitivity and can override MGMT(-) mediated resistance when administered simultaneously. Response to combination therapy was associated with increased double strand breaks induction, and coincided by increased apoptosis and autophagy. PARPi addition potentiates TMZ treatment in primary GSCs. PARPi could potentially enhance the therapeutic efficacy of the standard of care in GBM.
Effect of temozolomide on the U-118 glioma cell line
Oncology Letters, 2011
are the most lethal subtype of astrocytomas, with a mean patient survival rate of 12 months after diagnosis. The gold standard treatment of GBM, which includes surgery followed by the combination of radiotherapy and chemotherapy with temozolomide (TMZ), increases the survival rate to 14.6 months. The success of TMZ appears to be limited by the occurrence of chemoresistance that allows glioma cells to escape from death signaling pathways. However, the mechanism of TMZ action is yet to be clarified although some controversial results have been reported. Therefore, our aim was to evaluate the occurrence of apoptosis and autophagy in glioma cells treated with TMZ and to correlate TMZ action with the survival pathways Pi3K/ Akt and ERK1/2 MAP kinase. Cell proliferation was evaluated by incorporation of bromodeoxyuridine. Apoptosis was studied by flow cytometry as well as by fluorescence confocal microscopy in order to evaluate the sub G0/G1 percentage of cells and chromatin condensation. The expression of the autophagy-associated protein, LC3, as well as Akt and ERK1/2 was performed by Western blotting. In TMZ-treated GBM cells the expression of LC3, the autophagy-associated protein was increased and only a reduced percentage of cells underwent apoptosis. In addition, we showed that the phosphorylation status of Pi3K/Akt and ERK1/2 MAP kinase was maintained during the treatment with TMZ, suggesting that glioma cells escape from TMZ-induced cell death due to these signaling pathways. The chemoresistance of U-118 cells to TMZ was partially eradicated when cells were simultaneously treated with specific inhibitors of Pi3K/Akt and ERK1/2 MAP kinase signaling pathways and TMZ. Therefore, we hypothesized that in order to induce glioma cell death it is essential to evaluate the activation of the survival pathways and establish a combined therapy using TMZ and inhibitors of those signaling pathways.
The strategy for enhancing temozolomide against malignant glioma
Frontiers in Oncology, 2012
A combined therapy of the alkylating agent temozolomide (TMZ) and radiotherapy is standard treatment, and it improves the survival of patients with newly diagnosed glioblastoma (GBM). The DNA repair enzyme O 6 -methylguanine-DNA methyltransferase (MGMT) removes the most cytotoxic lesions generated by TMZ, O 6 -methylguanine, establishing MGMT as one of the most important DNA repair mechanisms of TMZ-induced DNA damage. Thus, the expression of MGMT, its activity, and its promoter methylation status are associated with the response of GBM to TMZ, confirming that MGMT promotes clinical resistance to TMZ. Previous studies have shown that a variety of drugs such as interferon-β (IFN-β), levetiracetam (LEV), resveratrol, and valproic acid (VAP) increased the sensitivity of TMZ through MGMT-dependent or MGMT-independent mechanisms. In this review, we describe drugs and promising molecules that influence the responsiveness of GBM to TMZ and discuss their putative mechanism of action. In MGMT-positive GBMs, drugs that modulate MGMT activity could enhance the therapeutic activity of TMZ. Thus, administration of these drugs as an adjunct to TMZ chemotherapy may have clinical applications in patients with malignant gliomas to improve the outcome.
Frontiers in oncology, 2012
Alkylating agents have long played a central role in the adjuvant therapy of glioblastoma (GBM). More recently, inclusion of temozolomide (TMZ), an orally administered methylating agent with low systemic toxicity, during and after radiotherapy has markedly improved survival. Extensive in vitro and in vivo evidence has shown that TMZ-induced O(6)-methylguanine (O(6)-meG) mediates GBM cell killing. Moreover, low or absent expression of O(6)-methylguanine-DNA methyltransferase (MGMT), the sole human repair protein that removes O(6)-meG from DNA, is frequently associated with longer survival in GBMs treated with TMZ, promoting interest in developing inhibitors of MGMT to counter resistance. However, the clinical efficacy of TMZ is unlikely to be due solely to O(6)-meG, as the agent produces approximately a dozen additional DNA adducts, including cytotoxic N3-methyladenine (3-meA) and abasic sites. Repair of 3-meA and abasic sites, both of which are produced in greater abundance than O(6...
Oncogene, 2007
Methylating drugs such as temozolomide (TMZ) are widely used in the treatment of brain tumours (malignant gliomas). The mechanism of TMZ-induced glioma cell death is unknown. Here, we show that malignant glioma cells undergo apoptosis following treatment with the methylating agents N-methyl-N 0 -nitro-N-nitrosoguanidine (MNNG) and TMZ. Cell death determined by colony formation and apoptosis following methylation is greatly stimulated by p53. Transfection experiments with O 6methylguanine-DNA methyltransferase (MGMT) and depletion of MGMT by O 6 -benzylguanine showed that, in gliomas, the apoptotic signal originates from O 6 -methylguanine (O 6 MeG) and that repair of O 6 MeG by MGMT prevents apoptosis. We further demonstrate that O 6 MeGtriggered apoptosis requires Fas/CD95/Apo-1 receptor activation in p53 non-mutated glioma cells, whereas in p53 mutated gliomas the same DNA lesion triggers the mitochondrial apoptotic pathway. This occurs less effectively via Bcl-2 degradation and caspase-9, -2, -7 and -3 activation. O 6 MeG-triggered apoptosis in gliomas is a late response (occurring >120 h after treatment) that requires extensive cell proliferation. Stimulation of cell cycle progression by the Pasteurella multocida toxin promoted apoptosis whereas serum starvation attenuated it. O 6 MeGinduced apoptosis in glioma cells was preceded by the formation of DNA double-strand breaks (DSBs), as measured by cH2AX formation. Glioma cells mutated in DNA-PK cs , which is involved in non-homologous endjoining, were more sensitive to TMZ-induced apoptosis, supporting the involvement of DSBs as a downstream apoptosis triggering lesion. Overall, the data demonstrate that cell death induced by TMZ in gliomas is due to apoptosis and that determinants of sensitivity of gliomas to TMZ are MGMT, p53, proliferation rate and DSB repair.
Multifaceted resistance of gliomas to temozolomide
Clinical cancer …, 2002
Purpose and Experimental Design: The contributions of O 6-methylguanine-DNA-methyltransferase (MGMT), p53 status, mismatch repair, and apoptotic response to the resistance of glial tumors to temozolomide (TMZ) were tested using seven established human glial tumor cell lines in culture and xenografts in athymic mice. Results: Resistance to TMZ was only marginally dependent on MGMT activity, because subtoxic doses of TMZ easily eliminated MGMT reserves for at least 18 h after treatment. Resistance to TMZ varied most notably with the p53 status of the tumor. Tumors with wild-type (wt) p53 and a functional p53 response to DNA damage (SWB40 and SWB61) were most sensitive. The p21-related cell cycle arrest was intimately linked to TMZ toxicity because tumors with wt p53 but lacking a robust increase in p21 protein level (D-54) were resistant to TMZ. In contrast, tumors with a dysfunctional p53 cycle and a weak cell cycle response to DNA damage (SWB39 and SWB77) were extremely unresponsive to treatment even with the aid of MGMT inactivators. Notable exceptions to the above were observed with the p53 mutated tumors SWB33 and SWB95, which were arrested by TMZ in G 1-S and consequently underwent apoptosis despite their failure to express p21. Conclusions: By testing a limited number of glial tumors in cell culture and also as xenografts, we have shown that mobilization of the p53 in response to TMZ damage is likely to induce a cell cycle arrest and apoptosis in glial tumors. Additional pathways linking cell cycle arrest and apoptosis contribute to the efficacy of TMZ against p53 mutated glial tumors. The unusual resistance of tumors, of which the cell cycle was not arrested in response to TMZ treatment, was associated with allelic losses during regrowth of treated tumors. Nevertheless such resistance was not related to dysfunctional mismatch repair.
Multifaceted Resistance of Gliomas to Temozolomide1
2002
Purpose and Experimental Design: The contributions of O 6-methylguanine-DNA-methyltransferase (MGMT), p53 status, mismatch repair, and apoptotic response to the resistance of glial tumors to temozolomide (TMZ) were tested using seven established human glial tumor cell lines in culture and xenografts in athymic mice. Results: Resistance to TMZ was only marginally dependent on MGMT activity, because subtoxic doses of TMZ easily eliminated MGMT reserves for at least 18 h after treatment. Resistance to TMZ varied most notably with the p53 status of the tumor. Tumors with wild-type (wt) p53 and a functional p53 response to DNA damage (SWB40 and SWB61) were most sensitive. The p21-related cell cycle arrest was intimately linked to TMZ toxicity because tumors with wt p53 but lacking a robust increase in p21 protein level (D-54) were resistant to TMZ. In contrast, tumors with a dysfunctional p53 cycle and a weak cell cycle response to DNA damage (SWB39 and SWB77) were extremely unresponsive to treatment even with the aid of MGMT inactivators. Notable exceptions to the above were observed with the p53 mutated tumors SWB33 and SWB95, which were arrested by TMZ in G 1-S and consequently underwent apoptosis despite their failure to express p21. Conclusions: By testing a limited number of glial tumors in cell culture and also as xenografts, we have shown that mobilization of the p53 in response to TMZ damage is likely to induce a cell cycle arrest and apoptosis in glial tumors. Additional pathways linking cell cycle arrest and apoptosis contribute to the efficacy of TMZ against p53 mutated glial tumors. The unusual resistance of tumors, of which the cell cycle was not arrested in response to TMZ treatment, was associated with allelic losses during regrowth of treated tumors. Nevertheless such resistance was not related to dysfunctional mismatch repair.
Glia, 2002
Temozolomide (TZM) is a novel methylating agent currently under investigation for treatment of recurrent high-grade gliomas. Although TZM generates a wide spectrum of methyl adducts, its cytotoxicity has been attributed to mismatch repair (MR)-mediated processing of O6-methylguanine:T mispairs. N3-methyladenine and N7-methylguanine adducts are promptly repaired by the base excision repair system, unless a poly(ADP-ribose) polymerase (PARP) inhibitor is combined to TZM. In this case, the repair process of N-methylpurines cannot be completed and the deriving DNA strand breaks contribute to cytotoxicity. In this study, we investigated the influence on cell growth and cell cycle of treatment with TZM + PARP inhibitor in glioma cells characterized by different susceptibility to TZM. The results indicated that PARP inhibitor increases growth inhibition induced by TZM in either p53–wild-type or p53-mutant glioblastoma cells, as early as 24 h after drug exposure. The enhancing effect exerted by PARP inhibitor was particularly evident in glioma cells characterized by a defective expression of MR, since these cells are tolerant to O6-methylguanine damage and show low sensitivity to TZM. In O6-alkylguanine-DNA alkyltransferase (OGAT)-deficient and MR-proficient tumor cells bearing wild-type p53, the drug combination markedly reduced cell accumulation in the G2/M phase of cell cycle and induction of the G2 checkpoint regulator Chk1 kinase. In short-term cultures of glioma cells derived from surgical specimens, PARP inhibitor enhanced chemosensitivity to TZM and this effect was especially evident in OGAT-proficient tumors. Thus, a pharmacological strategy based on the interruption of N-methylpurine repair might represent a novel strategy to restore or increase glioma sensitivity to TZM. GLIA 40:44–54, 2002. © 2002 Wiley-Liss, Inc.
Biomedicines
The current standard first-line treatment for adult patients with newly diagnosed glioblastoma includes concurrent radiotherapy and daily oral temozolomide (TMZ), followed by adjuvant TMZ. As a prodrug, TMZ undergoes spontaneous hydrolysis generating a methylating agent. O6-methylguanine is considered the most preponderant toxic damage mechanism at therapeutically relevant TMZ doses, whereas MGMT, which encodes the O6-methylguanine-DNA methyltransferase DNA repair enzyme, is the most relevant resistance mechanism. Speculations on clinically relevant TMZ concentrations, cytotoxic and cytostatic effects of TMZ, and resistance mechanisms exist in the literature. Here, we raise the following principal issues: What are the clinically relevant TMZ concentrations in glioma patients, and which TMZ-induced molecular lesion(s) and corresponding resistance mechanism(s) are important for TMZ therapeutic effects at clinically relevant concentrations? According to clinical data from patients with...