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Background and Purpose: Standard of care for glioblastomas includes radio-chemotherapy with the monoalkylating compound temozolomide. Temozolomide induces primarily senescence, inefficiently killing glioblastoma cells. Recurrences are inevitable. Although recurrences presumably arise from cells evading/escaping TMZ-induced senescence, becoming resistant, they are often again treated with TMZ. As an alternative treatment, irinotecan could be used. Our aim was to examine to what extent and conditions the topoisomerase I inhibitor irinotecan induces senescence and to analyze the underlying mechanism., Results: Multiple glioblastoma lines with different genetic signatures for p53, p21 CIP1 , p16 INK4A , p14 ARF , and PTEN were used. By means of LN229 glioblastoma clones which escaped from temozolomide-induced senescence, thus, being potentially recurrence-forming, we show that this escape is accompanied by increased p21 CIP1 protein levels in temozolomide-unexposed senescence-evading clones and inability of temozolomide to induce p21 CIP1 . In contrast, irinotecan was still able to induce p21 CIP1 and could elevate senescence and cell death. In combination with the senolytic drug BV6, irinotecan-induced senescence was significantly reduced. Differential response clusters were also observed in paired samples of newly diagnosed and recurrent patients' tumors. This can partially explain a significantly prolonged progression-free time until surgery for recurrence in patients additionally treated with irinotecan after temozolomide consolidation and upon the first onset of recurrence., Conclusions: p21 CIP1 is essentially involved in induction and maintenance of irinotecan-induced senescence. Neither p16 INK4A , p14 ARF , nor PTEN contribute to senescence, if p21 CIP1 cannot be induced. Based on the positive results of the irinotecan/BV6 treatment, combatting recurrent glioblastomas by targeting senescence cell antiapoptotic pathways (SCAPs) should be considered., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background/aim: Fisetin is a yellow-coloring flavonoid that can be found in a wide variety of plants, vegetables, and fruits, such as strawberries, apples, and grapes. It has been shown to have biological activity by targeting different pathways regulating survival and death and to bear antioxidant and anti-inflammatory activity. Fisetin was shown to be cytotoxic on different cancer cell lines and has the ability to kill therapy-induced senescent cancer cells. The aim of the study was to investigate the DNA damaging and cytotoxic potential of fisetin and its ability to enhance the killing effect of temozolomide on glioblastoma cells., Materials and Methods: We used LN229 glioblastoma cells and measured survival and apoptosis by flow cytometry, DNA strand breaks by the alkaline comet and γH2AX assay, and the DNA damage response by western blot analysis., Results: Fisetin was cytotoxic on glioblastoma cells, inducing apoptosis. In the dose range of 40-80 μM it also induced DNA damage, as measured by the alkaline comet and γH2AX assay, and triggered DNA damage response, as revealed by p53 activation. Furthermore, fisetin enhanced the genotoxic effect of methyl methanesulfonate, presumably due to inhibition of DNA repair processes. When administered together with temozolomide, the first-line therapeutic for glioblastoma, it enhanced cell death, reduced the yield of senescent cells following treatment and exhibited senolytic activity on glioblastoma cells., Conclusion: Data show that high-dose fisetin has a genotoxic potential and suggest that, harnessing the cytotoxic and senolytic activity of the flavonoid, it may enhance the effect of anticancer drugs and eliminate therapy-induced senescent cells. Therefore, it may be useful for adjuvant cancer therapy, including glioblastoma, which is worth to be studied in clinical trials., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

A first-line therapeutic for high-grade glioma, notably glioblastoma (GBM), is the DNA methylating drug temozolomide (TMZ). Previously, we showed that TMZ induces not only apoptosis and autophagy, but also cellular senescence (CSEN). We presented the hypothesis that GBM cells may escape from CSEN, giving rise to recurrent tumors. Furthermore, the inflammatory phenotype associated with CSEN may attenuate chemotherapy and drive tumor progression. Therefore, treatments that specifically target senescent cells, i.e., senolytic drugs, may lead to a better outcome of GBM therapy by preventing recurrences and tumor inflammation. Here, we tested Bcl-2 targeting drugs including ABT-737, ABT-263 (navitoclax), several natural substances such as artesunate, fisetin and curcumin as well as lomustine (CCNU) and ionizing radiation (IR) for their senolytic capacity in GBM cells. Additionally, several proteins involved in the DNA damage response (DDR), ATM, ATR, Chk1/2, p53, p21, NF-kB, Rad51, PARP, IAPs and autophagy, a pathway involved in CSEN induction, were tested for their impact in maintaining CSEN. Treatment of GBM cells with a low dose of TMZ for 8-10 days resulted in >80% CSEN, confirming CSEN to be the major trait induced by TMZ. To identify senolytics, we treated the senescent population with the compounds of interest and found that ABT-737, navitoclax, chloroquine, ATMi, ATRi, BV-6, PX-866 and the natural compounds fisetin and artesunate exhibit senolytic activity, inducing death in senescent cells more efficiently than in proliferating cells. Curcumin showed the opposite effect. No specific effect on CSEN cells was observed by inhibition of Chk1/Chk2, p21, NF-kB, Rad51 and PARP. We conclude that these factors neither play a critical role in maintaining TMZ-induced CSEN nor can their inhibitors be considered as senolytics. Since IR and CCNU did not exhibit senolytic activity, radio- and chemotherapy with alkylating drugs is not designed to eliminate TMZ-induced senescent cancer cells., Competing Interests: The authors declare no conflict of interest.

To clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.

The DNA-methylating drug temozolomide, which induces cell death through apoptosis, is used for the treatment of malignant glioma. Here, we investigate the mechanisms underlying the ability of temozolomide to induce senescence in glioblastoma cells. Temozolomide-induced senescence was triggered by the specific DNA lesion O 6 -methylguanine (O 6 MeG) and characterized by arrest of cells in the G 2 -M phase. Inhibitor experiments revealed that temozolomide-induced senescence was initiated by damage recognition through the MRN complex, activation of the ATR/CHK1 axis of the DNA damage response pathway, and mediated by degradation of CDC25c. Temozolomide-induced senescence required functional p53 and was dependent on sustained p21 induction. p53-deficient cells, not expressing p21, failed to induce senescence, but were still able to induce a G 2 -M arrest. p14 and p16, targets of p53, were silenced in our cell system and did not seem to play a role in temozolomide-induced senescence. In addition to p21, the NF-κB pathway was required for senescence, which was accompanied by induction of the senescence-associated secretory phenotype. Upon temozolomide exposure, we found a strong repression of the mismatch repair proteins MSH2, MSH6, and EXO1 as well as the homologous recombination protein RAD51, which was downregulated by disruption of the E2F1/DP1 complex. Repression of these repair factors was not observed in G 2 -M arrested p53-deficient cells and, therefore, it seems to represent a specific trait of temozolomide-induced senescence. SIGNIFICANCE: These findings reveal a mechanism by which the anticancer drug temozolomide induces senescence and downregulation of DNA repair pathways in glioma cells., (©2018 American Association for Cancer Research.)

Integrins have been suggested as possible targets in anticancer therapy. Here we show that knockdown of integrins αVβ3, αVβ5, α3β1 and α4β1 and pharmacological inhibition using a cyclo-RGD integrin αVβ3/αVβ5 antagonist sensitized multiple high-grade glioma cell lines to temozolomide (TMZ)-induced cytotoxicity. The greatest effect was observed in LN229 cells upon integrin β3 silencing, which led to inhibition of the FAK/Src/Akt/NFκB signaling pathway and increased formation of γH2AX foci. The integrin β3 knockdown led to the proteasomal degradation of Rad51, reduction of Rad51 foci and reduced repair of TMZ-induced DNA double-strand breaks by impairing homologous recombination efficiency. The down-regulation of β3 in Rad51 knockdown (LN229-Rad51kd) cells neither further sensitized them to TMZ nor increased the number of γH2AX foci, confirming causality between β3 silencing and Rad51 reduction. RIP1 was found cleaved and IκBα significantly less degraded in β3-silenced/TMZ-exposed cells, indicating inactivation of NFκB signaling. The anti-apoptotic proteins Bcl-xL, survivin and XIAP were proteasomally degraded and caspase-3/-2 cleaved. Increased H2AX phosphorylation, caspase-3 cleavage, reduced Rad51 and RIP1 expression, as well as sustained IκBα expression were also observed in mouse glioma xenografts treated with the cyclo-RGD inhibitor and TMZ, confirming the molecular mechanism in vivo. Our data indicates that β3 silencing in glioma cells represents a promising strategy to sensitize high-grade gliomas to TMZ therapy.

Background: The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) causes resistance of cancer cells to alkylating agents and, therefore, is a well-established predictive marker for high-grade gliomas that are routinely treated with alkylating drugs. Since MGMT is highly epigenetically regulated, the MGMT promoter methylation status is taken as an indicator of MGMT silencing, predicting the outcome of glioma therapy. MGMT promoter methylation is usually determined by methylation specific PCR (MSP), which is a labor intensive and error-prone method often used semi-quantitatively. Searching for alternatives, we used closed-tube high resolution melt (HRM) analysis, which is a quantitative method, and compared it with MSP and pyrosequencing regarding its predictive value., Results: We analyzed glioblastoma cell lines with known MGMT activity and formalin-fixed samples from IDH1 wild-type high-grade glioma patients (WHO grade III/IV) treated with radiation and temozolomide by HRM, MSP, and pyrosequencing. The data were compared as to progression-free survival (PFS) and overall survival (OS) of patients exhibiting the methylated and unmethylated MGMT status. A promoter methylation cut-off level relevant for PFS and OS was determined. In a multivariate Cox regression model, methylation of MGMT promoter of high-grade gliomas analyzed by HRM, but not MSP, was found to be an independent predictive marker for OS. Univariate Kaplan-Meier analyses revealed for PFS and OS a significant and better discrimination between methylated and unmethylated tumors when quantitative HRM was used instead of MSP., Conclusions: Compared to MSP and pyrosequencing, the HRM method is simple, cost effective, highly accurate and fast. HRM is at least equivalent to pyrosequencing in quantifying the methylation level. It is superior in predicting PFS and OS of high-grade glioma patients compared to MSP and, therefore, can be recommended being used routinely for determination of the MGMT status of gliomas.

The outcome of cancer therapy strongly depends on the complex network of cell signaling pathways, including transcription factor activation following drug exposure. Here we assessed whether and how the MAP kinase (MAPK) cascade and its downstream target, the transcription factor AP-1, influence the sensitivity of malignant glioma cells to the anticancer drugs temozolomide (TMZ) and nimustine (ACNU). Both drugs induce apoptosis in glioma cells at late times following treatment. Activation of the MAPK cascade precedes apoptosis, as shown by phosphorylation of Jun kinase (JNK) and c-Jun, a main component of AP-1. Pharmacological inhibition and siRNA mediated knockdown of JNK and c-Jun reduced the level of apoptosis in LN-229 glioma cells treated with TMZ or ACNU. Analyzing the underlying molecular mechanism, we identified the pro-apoptotic gene BIM as a critical target of AP-1, which is upregulated following TMZ and ACNU. Importantly, shRNA mediated downregulation of BIM in the malignant glioma cell lines LN-229 and U87MG led to an attenuated cleavage of caspase-9 and, consequently, reduced the level of apoptosis following TMZ and ACNU treatment. Overall, we identified JNK/c-Jun activation and BIM induction as a late pro-apoptotic response of glioma cells treated with alkylating anticancer drugs.

The DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) is a key player in tumor cell resistance. Promoter methylation, MGMT activity and immunohistochemistry are used for determining the MGMT status. However, it is unclear whether MGMT promoter methylation correlates with MGMT activity and whether MGMT promoter methylation of the pretreatment tumor predicts the MGMT status of recurrences. To address these questions, we determined MGMT activity promoter methylation and immunoreactivity in pretreatment and recurrent glioblastomas (GB, WHO Grade IV), and in astrocytomas (WHO Grade III). We show that GB that were promoter methylated display a range of 0-62 fmol/mg MGMT and tumors that were nonmethylated 0-423 fmol/mg protein. For astrocytomas, promoter-methylated samples displayed 0-28 fmol/mg and, nonmethylated samples, 23-107 fmol/mg. No correlation was found between the intensity of promoter methylation and MGMT activity. Given a threshold level of 30 fmol/mg of protein, we found a correlation between promoter methylation and no/low MGMT activity in 82.4% of the tumors. This high correlation level was only observed when tumors were excluded showing a hemimethylated promoter (20%). Therefore, classification of hemimethylated tumors remains questionable. Further, we show that 39.1% of pretreatment GB and 5.3% of recurrences were promoter methylated, which is in line with the observed increase of MGMT activity in recurrences. Although individual exceptions were found, the data show an overall correlation between promoter methylation and lack/low MGMT activity in GB and astrocytomas. We also show that promoter methylation assay is superior over immunohistochemistry in determining the MGMT status defined by a given MGMT activity level.

Glioblastoma multiforme is the most severe form of brain cancer. First line therapy includes the methylating agent temozolomide and/or the chloroethylating nitrosoureas [1-(2-chloroethyl)-1-nitrosourea; CNU] nimustine [1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea; ACNU], carmustine [1,3-bis(2-chloroethyl)-1-nitrosourea; BCNU], or lomustine [1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; CCNU]. The mechanism of cell death after CNU treatment is largely unknown. Here we show that ACNU and BCNU induce apoptosis in U87MG [p53 wild-type (p53wt)] and U138MG [p53 mutant (p53mt)] glioma cells. However, contrary to what we observed previously for temozolomide, chloroethylating drugs are more toxic for p53-mutated glioma cells and induce both apoptosis and necrosis. Inactivation of p53 by pifithrin-alpha or siRNA down-regulation sensitized p53wt but not p53mt glioma cells to ACNU and BCNU. ACNU and BCNU provoke the formation of DNA double-strand breaks (DSB) in glioma cells that precede the onset of apoptosis and necrosis. Although these DSBs are repaired in p53wt cells, they accumulate in p53mt cells. Therefore, functional p53 seems to stimulate the repair of CNU-induced cross-links and/or DSBs generated from CNU-induced lesions. Expression analysis revealed an up-regulation of xpc and ddb2 mRNA in response to ACNU in U87MG but not U138MG cells, indicating p53 regulates a pathway that involves these DNA repair proteins. ACNU-induced apoptosis in p53wt glioma cells is executed via both the extrinsic and intrinsic apoptotic pathway, whereas in p53mt glioma cells, the mitochondrial pathway becomes activated. The data suggest that p53 has opposing effects in gliomas treated with methylating or chloroethylating agents and, therefore, the p53 status should be taken into account when deciding which therapeutic drug to use.

Simple Summary: Temozolomide (TMZ) is the first-line drug in the treatment of glioblastoma. Due to the formation of the DNA damage O6-methylguanine, it is toxic to cancer cells, resulting in the induction of apoptosis. However, the damage also induces cellular senescence. Here, we show that not apoptosis, but cellular senescence is the main response triggered by TMZ-induced DNA damage. We analyzed the senescent cells in detail and show that they are characterized by a high level of non-repaired DNA double-strand breaks that trigger the DNA damage response. The primary damage, O6-methylguanine, is the upstream trigger, but it is not required for maintaining the senescent state. Cells which acquired temozolomide resistance also became resistant to the induction of senescence. Comparing specimens from primary and recurrent glioblastoma, we show that recurrences contain a higher proportion of senescent cells than the primary tumor, indicating that induction of senescence also occurs upon treatment in vivo, which likely plays a role in therapy resistance. First-line drug in the treatment of glioblastoma, the most severe brain cancer, is temozolomide (TMZ), a DNA-methylating agent that induces the critical damage O6-methylguanine (O6MeG). This lesion is cytotoxic through the generation of mismatch repair-mediated DNA double-strand breaks (DSBs), which trigger apoptotic pathways. Previously, we showed that O6MeG also induces cellular senescence (CSEN). Here, we show that TMZ-induced CSEN is a late response which has similar kinetics to apoptosis, but at a fourfold higher level. CSEN cells show a high amount of DSBs, which are located outside of telomeres, a high level of ROS and oxidized DNA damage (8-oxo-guanine), and sustained activation of the DNA damage response and histone methylation. Despite the presence of DSBs, CSEN cells are capable of repairing radiation-induced DSBs. Glioblastoma cells that acquired resistance to TMZ became simultaneously resistant to TMZ-induced CSEN. Using a Tet-On glioblastoma cell system, we show that upregulation of MGMT immediately after TMZ completely abrogated apoptosis and CSEN, while induction of MGMT long-term (>72 h) after TMZ did not reduce apoptosis and CSEN. Furthermore, upregulation of MGMT in the senescent cell population had no impact on the survival of senescent cells, indicating that O6MeG is required for induction, but not for maintenance of the senescent state. We further show that, in recurrent GBM specimens, a significantly higher level of DSBs and CSEN-associated histone H3K27me3 was observed than in the corresponding primary tumors. Overall, the data indicate that CSEN is a key node induced in GBM following chemotherapy. [ABSTRACT FROM AUTHOR]

Abstract: Therapy of malignant glioma relies on treatment with the O6‐methylating agent temozolomide (TMZ) concomitant with ionizing radiation followed by adjuvant TMZ. For the treatment of recurrences, DNA chloroethylating drugs are also used. The main killing lesion induced by these drugs is O6‐alkylguanine. Since this damage is repaired by O6‐methylguanine‐DNA methyltransferase (MGMT), the repair enzyme represents a most important factor of drug resistance, limiting the therapy of malignant high‐grade gliomas. Although MGMT has been shown to be transcriptionally up‐regulated in rodents following genotoxic stress, it is still unclear whether human MGMT is subject to up‐regulation. Here, we addressed the question whether MGMT in glioma cells is enhanced following alkylating drugs or ionizing radiation, using promoter assays. We also checked the response of glioma cell lines to dexamethasone. In a series of experiments, we found no evidence that the human MGMT promoter is significantly up‐regulated following treatment with TMZ, the chloroethylating agent nimustine or radiation. It was activated, however, by dexamethasone. Using deletion constructs, we further show that the basal level of MGMT is mainly determined by the transcription factor SP1. The high amount of SP1 sites in the MGMT promoter likely prevents transcriptional up‐regulation following genotoxic stress by neutralizing inducible signals. The regulation of MGMT by miRNAs plays only a minor role, as shown by DICER knockdown experiments. Since high dose dexamethasone concomitant with temozolomide is frequently used in glioblastoma therapy, induction of the MGMT gene through glucocorticoids in MGMT promoter unmethylated cases might cause further elevation of drug resistance, while radiation and alkylating drugs seem not to induce MGMT at transcriptional level. [ABSTRACT FROM AUTHOR]