Cellular senescence (CSEN) is caused by a variety of factors that trigger complex molecular pathways. These include telomere shortening, oncogene activation and replicative stress, as well as DNA damage caused by genotoxic anticancer drugs and endogenous and exogenous genotoxins. Here, we review the induction of CSEN by exogenous genotoxic insults resulting from food and environmental exposures. The available data show that genotoxins/carcinogens in tobacco smoke and smokeless tobacco, in the environment, in food, beverages and life-style products induce CNS. The exposures include N-nitroso compounds, polycyclic aromatic hydrocarbons, heterocyclic aromatic amines, acrylamide, heavy metals, fine dust, mycotoxins, phytotoxins, and phycotoxins. Also, heme in red meat contributes to CSEN as it catalyzes the formation of genotoxic species in the colon. Induction of CSEN by external genotoxins/carcinogens is bound on the DNA damage response pathway (DDR), which relies on activation of the ATM/ATR-CHK2/CHK1-p53-p21 axis and the p53-independent p16/p14 axis, eliciting cyclin-dependent kinase inhibition and permanent cell cycle arrest. Other factors that can be involved are DREAM, MAPK, cGAS/Sting, and NF-κB. The accumulation of non-repaired DNA damage triggering CSEN following external genotoxic exposures may contribute significantly to the amelioration of senescent cells and organ failure with age in humans. Senescent cells drive, via the senescence-associated secretory phenotype (SASP), inflammation that is involved in many diseases, including cancer. Although most of the studies were performed with in vitro cell systems, the consequences of CSEN induction by genotoxic nutritional components and environmental exposures seem to be underestimated. Since CSEN correlates with aging, it is reasonable to conclude that exogenous genotoxic pollutants contribute significantly to the aging process through CSEN induction. In light of these findings, it is deduced that reducing genotoxin exposures and using "rejuvenation" supplements (senotherapeutics) are reasonable strategies to counteract cellular senescence and the aging process.

Therapy-induced senescence (TIS) is a major challenge in cancer therapy as senescent cancer cells provoke local and systemic inflammation and might be the cause of recurrences. Elucidation of pathways leading to TIS is of utmost importance for establishing strategies to counteract this. Previously we have shown that temozolomide (TMZ), an alkylating drug used forefront in glioma therapy, causes majorly cellular senescence, which is triggered by the primary damage O6-methylguanine, activating the mismatch repair dependent ATR/ATM-CHK1/CHK2-p53 damage response pathway. The downstream pathways leading to TIS remained to be explored. Here, we show that TMZ-induced TIS in glioma cells does not require activation of the DREAM complex, but is bound on a G2-specific response. We show that the CDK inhibitor p21CIP1 does not interact with CDK4, but with CDK1 and CDK2 causing abrogation of the B-Myb and FOXM1-signaling pathway and subsequently arrest of cells in the G2-phase. The induced G2-arrest is incomplete as DNA synthesis can be resumed leading to endoreduplications. This process, which is inhibited by the CDK4-blocking drug palbociclib, is preceded by reactivation of the G1/S-specific E2F1-signaling pathway due to lack of functional DREAM activation. These findings provide an explanation for the polyploidization and giant cell phenotype of anticancer drug-induced senescent cells. Incomplete DREAM activation may also explain the observation that downregulation of DNA repair is a transient phenomenon, which goes along with the entrance of cells into the senescent state.

Malignant brain cancer, the most severe form is glioblastoma (GBM), has a dismal prognosis, despite maximal resection followed by radio-chemotherapy. First line therapeutics are alkylating drugs, notably the DNA-methylating temozolomide (TMZ), administered concomitantly with radiation. Radio-chemotherapy induces not only apoptosis, but also cellular senescence in GBM cells. Senescent cells change the tumor microenvironment, cause an inflammatory response in the affected area and can be reactivated, contributing to recurrences. To eliminate therapy-induced senescent cells, senotherapeutics have gained attention. Here, we describe the pathways triggered in GBM cells leading to cellular senescence and update drugs and natural compounds acting as senolytics, senomorphics and senopreventics. There is an increasing amount of data showing that temozolomide induces cellular senescence, which is even the main response of GBM cells following treatment. We outline the mechanism of senescence in glioblastoma cells and show that it rests on some unique cellular responses that may explain the low curability and aggressiveness of glioblastoma. Thus, senescent GBM cells are incompletely blocked in G2 following temozolomide treatment and undergo endoreduplications. This is presumably fostered by inactivation of CDKN2A, which is frequently mutated in gliomas. Since cellular senescence is a key event induced by temozolomide and radiation in GBM cells, it is reasonable to conclude that glioma cells cannot be completely eliminated, neither by radiation or chemotherapy alone nor in combination. Based on the data, new treatment options with senopreventics, senolytics and senostatics/senomorphics as important supportive medication during or after radiochemotherapie are discussed.

Background: Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are enzymes that catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate (α-KG), which is essential for many metabolic processes, including some steps in DNA repair. In tumors, notably in gliomas, IDH1 and IDH2 are frequently mutated. The mutation found in different cancers is functionally active, causing, instead of α-KG, the formation of 2-hydroxyglutarate (2-HG), which inhibits α-KG-dependent enzymes. Gliomas harboring mutated IDH1/2 show a better prognosis than IDH1 wild-type (wt) tumors of the same grade, which might result from the inhibition of DNA repair functions. A DNA repair enzyme dependent on α-KG is alkB homolog 2 (ALKBH2), which removes several lesions from DNA. These findings prompted us to investigate the response of glioma cells to artesunate (ART), a plant ingredient with genotoxic and anticancer activity currently used in several trials. Materials and Methods: We used isogenic glioblastoma cell lines that express IDH1 wild-type or, based on a TET-inducible system, the IDH1 mutant (mt) protein, and treated them with increasing doses of artesunate. We also treated glioblastoma cells with 2-HG, generated ALKBH2 knockout cells, and checked their sensitivity to the cytotoxic effects of artesunate. Results: We show that the cell-killing effect of ART is enhanced if the IDH1 mutant (R132H) is expressed in glioblastoma cells. Further, we show that 2-HG imitates the effect of IDH1mt as 2-HG ameliorates the cytotoxicity of ART. Finally, we demonstrate that the knockout of ALKBH2 causes the sensitization of glioblastoma cells to ART. Conclusions: The data indicate that ALKBH2 protects against the anticancer effect of ART, and the mutation of IDH1/2 commonly occurring in low-grade gliomas sensitizes to ART via an ALKBH2-dependent mechanism. The data support the use of ART in the therapy of IDH1/2-mutated cancers both in combination with chemotherapy and adjuvant treatment.

Temozolomide (TMZ) concomitant with radiotherapy is the first-line treatment for glioblastoma. However, treatment resistance is frequently observed in patients. Cellular senescence (CSEN) induced by TMZ has been proposed to be one underlying mechanism resulting in resting cells, causing inflammation and possibly recurrences if senescent cells re-enter the cell cycle after treatment. Inhibition of the K + channels human ether-à-go-go type 1 (Eag1) and human ether-à-go-go-related gene (hERG) has shown promising effects in several tumor types including glioblastoma through growth inhibition and induction of apoptosis. In the present study, we analyzed the impact of hERG/Eag1 inhibition on apoptosis and CSEN on its own and in combination with TMZ in a panel of human glioblastoma cell lines and primary glioblastoma cells. hERG/Eag1 protein expression was determined by Western blotting and immunocytochemistry. Cytotoxicity of astemizole and terfenadine alone or in combination with TMZ was assessed by MTT assays. Apoptotic yields were determined by Annexin V/propidium iodide staining, and CSEN was quantified by determining SA-β-galactosidase levels through flow cytometry. We observed a similar protein expression of hERG and Eag1 in all glioblastoma cell lines and primary glioblastoma cells. Astemizole and terfenadine were cytotoxic in glioblastoma cells at low micromolar concentrations (5–10 µM range) through induction of apoptosis. In combination with TMZ, both drugs synergistically sensitized glioblastoma cells to TMZ-induced apoptosis. Moreover, astemizole reduced significantly the TMZ-induced CSEN level, indicating its impact on CSEN induction. Here, we show for the first time that blocking hERG/Eag1 channels in glioblastoma cells can relief TMZ-induced CSEN and synergistically ameliorates cytotoxicity through the induction of apoptosis.

ObjectiveIntrahepatic cholangiocarcinoma (iCCA) is the second most common primary liver cancer with limited therapeutic options.KRASmutations are among the most abundant genetic alterations in iCCA associated with poor clinical outcome and treatment response. Recent findings indicate that Poly(ADP-ribose)polymerase1 (PARP-1) is implicated inKRAS-driven cancers, but its exact role in cholangiocarcinogenesis remains undefined.DesignPARP-1inhibition was performed in patient-derived and established iCCA cells using RNAi, CRISPR/Cas9 and pharmacological inhibition inKRAS-mutant, non-mutant cells. In addition,Parp-1knockout mice were combined with iCCA induction by hydrodynamic tail vein injection to evaluate an impact on phenotypic and molecular features ofKras-driven andKras-wildtype iCCA. Clinical implications were confirmed in authentic human iCCA.ResultsPARP-1 was significantly enhanced inKRAS-mutant human iCCA. PARP-1-based interventions preferentially impaired cell viability and tumourigenicity in humanKRAS-mutant cell lines. Consistently, loss ofParp-1provoked distinct phenotype inKras/Tp53-induced versusAkt/Nicd-induced iCCA and abolishedKras-dependent cholangiocarcinogenesis. Transcriptome analyses confirmed preferential impairment of DNA damage response pathways and replicative stress response mediated by CHK1. Consistently, inhibition of CHK1 effectively reversed PARP-1 mediated effects. Finally,Parp-1depletion induced molecular switch ofKRAS-mutant iCCA recapitulating good prognostic human iCCA patients.ConclusionOur findings identify the novel prognostic and therapeutic role ofPARP-1in iCCA patients with activation of oncogenicKRASsignalling.

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.

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.

LebensmittelchemieVolume 78, Issue S1 p. S1-070-S1-070 Abstract Auswirkungen von DNA-Reparaturfaktoren auf die Schädigung von Darmzellen und Entzündungen, ausgelöst durch diätisches Häm-Eisen Simon Wittmann, Simon Wittmann RPTU Kaiserslautern-Landau, Abteilung für Lebensmittelchemie und Toxikologie, Fachbereich Chemie, 67663 Kaiserslautern, DeutschlandSearch for more papers by this authorNina Seiwert, Nina Seiwert RPTU Kaiserslautern-Landau, Abteilung für Lebensmittelchemie und Toxikologie, Fachbereich Chemie, 67663 Kaiserslautern, Deutschland Justus-Liebig-Universität Gießen, Rudolf-Buchheim-Institut für Pharmakologie, 35392 Gießen, DeutschlandSearch for more papers by this authorPhilipp Demuth, Philipp Demuth RPTU Kaiserslautern-Landau, Abteilung für Lebensmittelchemie und Toxikologie, Fachbereich Chemie, 67663 Kaiserslautern, DeutschlandSearch for more papers by this authorDaniel Heylmann, Daniel Heylmann Justus-Liebig-Universität Gießen, Rudolf-Buchheim-Institut für Pharmakologie, 35392 Gießen, DeutschlandSearch for more papers by this authorBernd Kaina, Bernd Kaina Universitätsklinikum Mainz, Institut für Toxikologie, 55131 Mainz, DeutschlandSearch for more papers by this authorSebastian Försch, Sebastian Försch Universitätsklinikum Mainz, Institut für Pathologie, 55131 Mainz, DeutschlandSearch for more papers by this authorJörg Fahrer, Jörg Fahrer RPTU Kaiserslautern-Landau, Abteilung für Lebensmittelchemie und Toxikologie, Fachbereich Chemie, 67663 Kaiserslautern, Deutschland Justus-Liebig-Universität Gießen, Rudolf-Buchheim-Institut für Pharmakologie, 35392 Gießen, DeutschlandSearch for more papers by this author Simon Wittmann, Simon Wittmann RPTU Kaiserslautern-Landau, Abteilung für Lebensmittelchemie und Toxikologie, Fachbereich Chemie, 67663 Kaiserslautern, DeutschlandSearch for more papers by this authorNina Seiwert, Nina Seiwert RPTU Kaiserslautern-Landau, Abteilung für Lebensmittelchemie und Toxikologie, Fachbereich Chemie, 67663 Kaiserslautern, Deutschland Justus-Liebig-Universität Gießen, Rudolf-Buchheim-Institut für Pharmakologie, 35392 Gießen, DeutschlandSearch for more papers by this authorPhilipp Demuth, Philipp Demuth RPTU Kaiserslautern-Landau, Abteilung für Lebensmittelchemie und Toxikologie, Fachbereich Chemie, 67663 Kaiserslautern, DeutschlandSearch for more papers by this authorDaniel Heylmann, Daniel Heylmann Justus-Liebig-Universität Gießen, Rudolf-Buchheim-Institut für Pharmakologie, 35392 Gießen, DeutschlandSearch for more papers by this authorBernd Kaina, Bernd Kaina Universitätsklinikum Mainz, Institut für Toxikologie, 55131 Mainz, DeutschlandSearch for more papers by this authorSebastian Försch, Sebastian Försch Universitätsklinikum Mainz, Institut für Pathologie, 55131 Mainz, DeutschlandSearch for more papers by this authorJörg Fahrer, Jörg Fahrer RPTU Kaiserslautern-Landau, Abteilung für Lebensmittelchemie und Toxikologie, Fachbereich Chemie, 67663 Kaiserslautern, Deutschland Justus-Liebig-Universität Gießen, Rudolf-Buchheim-Institut für Pharmakologie, 35392 Gießen, DeutschlandSearch for more papers by this author First published: 01 March 2024 https://doi.org/10.1002/lemi.202452053AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onEmailFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume78, IssueS1Supplement: Abstracts der Vorträge der Regionalverbände und die der Posterflashtalks der AG JLCMarch/April 2024Pages S1-070-S1-070 RelatedInformation

Most solid metastatic cancers are resistant to chemotherapy. However, metastatic testicular germ cell tumors (TGCT) are cured in over 80% of patients using cisplatin-based combination therapy. Published data suggest that TGCTs are sensitive to cisplatin due to limited DNA repair and presumably also to a propensity to undergo apoptosis. To further investigate this aspect, cisplatin-induced activation of apoptotic pathways was investigated in cisplatin-sensitive testis tumor cells (TTC) and compared to cisplatin-resistant bladder cancer cells. Apoptosis induction was investigated using flow cytometry, caspase activation and PARP-1 cleavage. Immunoblotting and RT-PCR were applied to investigate pro- and anti-apoptotic proteins. Transfections were performed to target p53- and Fas/FasL-mediated apoptotic signaling. Immunoblotting experiments revealed p53 to be induced in TTC, but not bladder cancer cells following cisplatin. Higher levels of pro-apoptotic Bax and Noxa were observed in TTC, anti-apoptotic Bcl-2 was solely expressed in bladder cancer cells. Cisplatin led to translocation of Bax to the mitochondrial membrane in TTC, resulting in cytochrome C release. Cisplatin increased the expression of FasR mRNA and FasL protein in all tumor cell lines. Targeting the apoptotic pathway via siRNA-mediated knockdown of p53 and FAS reduced death receptor-mediated apoptosis and increased cisplatin resistance in TTC, indicating the involvement of FAS-mediated apoptosis in the cisplatin TTC response. In conclusion, both the death receptor and the mitochondrial apoptotic pathway become strongly activated in TTC following cisplatin treatment, explaining, together with attenuated DNA repair, their unique sensitivity toward platinum-based anticancer drugs.

Supplementary Figure Legend

Figure S1: HIPK2 does not affect TMZ-induced senescence. Figure S2: HIPK2 does not contribute to CCNU-induced apoptosis. Figure S3: Expression of HIPK2 in LN-229 cells following treatment with TMZ in the absence and presence of the ATM inhibitor KU-60019 (AstraZeneca), the ATR inhibitor VE821 (Selleckchem) and both.

The genotoxic methylating agents temozolomide (TMZ) and procarbazine and the chloroethylating nitrosourea lomustine (CCNU) are part of the standard repertoire in the therapy of malignant gliomas (CNS WHO grade 3 and 4). This review describes the mechanisms of their cytotoxicity and cytostatic activity through apoptosis, necroptosis, drug-induced senescence, and autophagy, interaction of critical damage with radiation-induced lesions, mechanisms of glioblastoma resistance to alkylating agents, including the alkyltransferase MGMT, mismatch repair, DNA double-strand break repair and DNA damage responses, as well as IDH-1 and PARP-1. Cyclin-dependent kinase inhibitors such as regorafenib, synthetic lethality using PARP inhibitors, and alternative therapies including tumor-treating fields (TTF) and CUSP9v3 are discussed in the context of alkylating drug therapy and overcoming glioblastoma chemoresistance. Recent studies have revealed that senescence is the main trait induced by TMZ in glioblastoma cells, exhibiting hereupon the senescence-associated secretory phenotype (SASP). Strategies to eradicate therapy-induced senescence by means of senolytics as well as attenuating SASP by senomorphics are receiving increasing attention, with therapeutic implications to be discussed.

p14 and p16 status of the used glioblastoma cell lines

Class I HDACs protect melanoma cells in vivo. (A) Effect of HDAC inhibition on growth inhibition of A375 melanoma xenografts following TMZ. Tumor volume is shown as a function of time. See materials and methods for detailed treatment conditions. Data are represented as mean {plus minus} SEM. *p<0.05. (B) Effect of treatment on the weight of the mice carrying A375 melanoma xenografts as a function of time.

Effects of CCNU and HRi on transcription in glioblastoma cells

Cell death in p53 deficient cells, efficiency of p21 knockdown and expression of CDK1/CyklinB1

HDAC1, 2 and 3 expression in melanomas. (A) Microarray expression data of HDAC1, HDAC2 and HDAC3 from 45 primary melanoma,18 benign skin nevi, and 7 normal skin tissue specimens. (B) Western blot analysis of HDAC1, HDAC2, HDAC3 and HDAC8 protein levels in the melanoma cell lines D05, G361, SK-Mel187, Mel537, A2058 and A375 compared to the normal (non-cancerous) cells PBLCs, VH10tert and Hema-LP. β-actin served as loading control. Relative expression (R.E.) compared to the lowest protein level is indicated. (C) Relative HDAC protein level in melanoma cells (D05, G361, Mel187, Mel537, A2058 and A375) compared to non-cancerous (PBLCs, VH10tert and Hema-LP) cells as determined by Western blot analysis. (D) Western blot analysis of acetylated-H3 in Hema-LP, PBLCs and VH10tert cells following VPA treatment (1mM for 168 h). H3 was used as loading control. **p<0.005, ***p<0.0001.

Legends for Table S1, Table S2, Figore S1, Figure S2 and Figure S3

Expression and phosphorylation of E2F1 and DP1