Accumulation of Temozolomide-Induced Apoptosis, Senescence and DNA Damage by Metronomic Dose Schedule: A Proof-of-Principle Study with Glioblastoma Cells

Beltzig, Lea; Stratenwerth, Björn; Kaina, Bernd

doi:10.3390/cancers13246287

Cited by 12 publications

(8 citation statements)

References 37 publications

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“…Following oral 200 mg/m 2 TMZ, the plasma peak level was 72 µM and the concentration in the cerebrospinal fluid was 9.9 µM [50]. It is important to note that TMZ is administered daily, and, in a respective experimental setting, repeated low daily doses gave rise to significant accumulation of DSBs, apoptosis, and CSEN [51]. Therefore, we consider that the results obtained in a clinically relevant dose range are therapeutically relevant.…”

Section: Discussionmentioning

confidence: 98%

Senescence Is the Main Trait Induced by Temozolomide in Glioblastoma Cells

Beltzig

Schwarzenbach

Leukel

et al. 2022

Cancers

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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.

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Section: Discussionmentioning

confidence: 98%

Senescence Is the Main Trait Induced by Temozolomide in Glioblastoma Cells

Beltzig

Schwarzenbach

Leukel

et al. 2022

Cancers

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“…Apoptosis and senescence were analyzed by flow cytometry as described previously [ 16 ]. In brief, for apoptosis measurement, cells including the supernatant were collected by trypsinization, resuspended in annexin-binding buffer containing 2.5 µL annexin-FITC (Miltenyi Biotec, Bergisch-Gladbach, Germany) and incubated for 15 min at RT.…”

Section: Methodsmentioning

confidence: 99%

Abrogation of Cellular Senescence Induced by Temozolomide in Glioblastoma Cells: Search for Senolytics

Beltzig

Christmann

Kaina

2022

Cells

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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.

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“…Even at these low dose levels, TMZ is effective, as indicated by clinical studies ( 4 , 5 ). Moreover, at least in O 6 -methylguanine-DNA methyltransferase (MGMT)-deficient tumors, repeated TMZ treatments very likely lead to an accumulation of critical DNA damages that trigger cell death, which is supported by our studies on glioblastoma cells in vitro ( 6 ). The concurrent administration of TMZ with IR results in a prolongation of overall survival (OS) and progression free survival (PFS) by a few months ( 1 ).…”

Section: Introductionmentioning

confidence: 60%

Temozolomide – Just a Radiosensitizer?

2022

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Radiation concomitant with the DNA methylating drug temozolomide (TMZ) is the gold standard in the treatment of glioblastoma. In this adjuvant setting, TMZ is regarded to be a radiation sensitizer. However, similar to ionising radiation, TMZ induces DNA double-strand breaks and is itself a potent trigger of apoptosis, cellular senescence and autophagy, suggesting that radiation and TMZ act independently. Although cell culture experiments yielded heterogeneous results, some data indicate that the cytotoxic effect of radiation was only enhanced when TMZ was given before radiation treatment. Based on the molecular mechanism of action of TMZ, the importance of specific TMZ and radiation-induced DNA lesions, their repair as well as their interactions, possible scenarios for an additive or synergistic effect of TMZ and radiation are discussed, and suggestions for an optimal timing of radio-chemical treatments are proposed.

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Accumulation of Temozolomide-Induced Apoptosis, Senescence and DNA Damage by Metronomic Dose Schedule: A Proof-of-Principle Study with Glioblastoma Cells

Cited by 12 publications

References 37 publications

Senescence Is the Main Trait Induced by Temozolomide in Glioblastoma Cells

Senescence Is the Main Trait Induced by Temozolomide in Glioblastoma Cells

Abrogation of Cellular Senescence Induced by Temozolomide in Glioblastoma Cells: Search for Senolytics

Temozolomide – Just a Radiosensitizer?

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