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The potential use of pro-senescence therapies, known as TIS (Therapy-Induced Senescence), for the treatment of colorectal cancer (CRC) generated significant interest since they require lower doses compared to those required for inducing apoptosis. However, the senescent cell cycle-arrested cancer cells are long-lived, and studies have revealed escape mechanisms contributing to tumor recurrence. To deepen our understanding of the survival pathways used by senescent cancer cells, we delved into the potential involvement of the hexosamine biosynthetic pathway (HBP). HBP provides UDP-GlcNAc, the substrate for O-GlcNAc transferase (OGT), which catalyzes O-GlcNAcylation, a post-translational modification implicated in regulating numerous cellular functions and aberrantly elevated in CRC. In this study, we demonstrated, in the p53-proficient colon cancer cell lines HCT116 and LS174T, that TIS induced by low-dose SN38 or etoposide treatment was accompanied with a decrease of GFAT (the rate limiting enzyme of the HBP), OGT and O-GlcNAcase (OGA) expression correlated with a slight reduction in O-GlcNAcylation levels. Further decreasing this level of O-GlcNAcylation by knocking-down GFAT or OGT redirected the cellular response to subtoxic chemotherapy doses from senescence to apoptosis, in correlation with an enhancement of DNA damages. Pharmacological inhibition of OGT with OSMI-4 in HCT116 and LS174T cells and in a patient-derived colon tumoroid model supported these findings. Taken together, these results suggest that combing O-GlcNAcylation inhibitors to low doses of conventional chemotherapeutic drugs could potentially reduce treatment side effects while preserving efficacy. Furthermore, this approach may increase treatment specificity, as CRC cells exhibit higher O-GlcNAcylation levels compared to normal tissues., (© 2024. The Author(s).)

Etoposide is a second-line chemotherapy agent widely used for metastatic colorectal cancer. However, we discovered that etoposide treatment induced greater motility potential in four colorectal cancer cell lines. Therefore, we used microarrays to test the mRNA of these cancer cell lines to investigate the mechanisms of etoposide promoting colorectal cancer metastasis. Differentially expressed genes (DEGs) were identified by comparing the gene expression profiles in samples from etoposide-treated cells and untreated cells in all four colorectal cancer cell lines. Next, these genes went through the Gene Set Enrichment Analysis (GSEA), Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis. Among the top 10 genes including the upregulated and downregulated, eight genes had close interaction according to the STRING database: FAS, HMMR, JUN, LMNB1, MLL3, PLK2, STAG1 and TBL1X. After etoposide treatment, the cell cycle, metabolism-related and senescence signaling pathways in the colorectal cancer cell lines were significantly downregulated, whereas necroptosis and oncogene pathways were significantly upregulated. We suggest that the differentially expressed genes LMNB1 and JUN are potential targets for predicting colorectal cancer metastasis. These results provide clinical guidance in chemotherapy, and offer direction for further research in the mechanism of colorectal cancer metastasis., (© 2024. The Author(s).)

Osteoporosis, a common bone disease in older individuals, involves the progression influenced by N6-methyladenosine (m6A) modification. This study aimed to elucidate the effects of VDAC3 m6A modification on human bone mesenchymal stromal cell (BMSC) senescence and osteogenic differentiation. BMSCs were treated with etoposide to induce senescence. Senescence was assessed by β-galactosidase staining and quantitative real-time PCR (qPCR), and osteogenic differentiation was evaluated using Western blot, alkaline phosphatase, and alizarin red S staining. VDAC3 and ALKBH5 expression were quantified by qPCR, and their interaction was assessed by RNA immunoprecipitation (RIP) and luciferase reporter assay. m6A methylation was analyzed using the Me-RIP assay. VDAC3 expression was significantly decreased in etoposide-treated BMSCs (1.00 ± 0.13 vs. 0.26 ± 0.06). VDAC3 overexpression reduced etoposide-induced senescence and promoted osteogenic differentiation. ALKBH5 overexpression inhibited VDAC3 m6A modification (1.00 ± 0.095 vs. 0.233 ± 0.177) and its stability. ALKBH5 knockdown decreased etoposide-induced senescence and promoted osteogenic differentiation, effects that were reversed by VDAC3 knockdown. YTHDF1 was identified as the m6A methylation reader, and its overexpression inhibited VDAC3 stability. We demonstrated that ALKBH5 inhibited osteogenic differentiation of etoposide-induced senescent cells through the inhibition of VDAC3 m6A modification, and YTHDF1 acted as the m6A methylation reader. These findings provide a novel theoretical basis for the treatment of osteoporosis., (© 2024. The Author(s).)

DNA topoisomerase II (TOP2) is an enzyme that resolves DNA topological problems arising in various nuclear processes, such as transcription. Aclarubicin, a member of the anthracyclines, is known to prevent the association of TOP2 with DNA, inhibiting the early step of TOP2 catalytic reactions. During our research on the subnuclear distribution of human TOP2B, we found that aclarubicin affects the mobility of TOP2B in the nucleus. FRAP analysis demonstrated that aclarubicin decreased the nuclear mobility of EGFP-tagged TOP2B in a concentration-dependent manner. Aclarubicin exerted its inhibitory effects independently of TOP2B enzymatic activities: TOP2B mutants defective for either ATPase or topoisomerase activity also exhibited reduced nuclear mobility in the presence of aclarubicin. Immunofluorescence analysis showed that aclarubicin antagonized the induction of DNA damage by etoposide. Although the prevention of the TOP2-DNA association is generally considered a primary action of aclarubicin in TOP2 inhibition, our findings highlight a previously unanticipated effect of aclarubicin on TOP2B in the cellular environment.

Background: The prognosis of high-risk neuroblastomas (NB) that are resistant to first-line induction chemotherapy is relatively poor. This study explored the mechanism of resistance to first-line chemotherapeutics mediated by TXNDC17 and its potential solutions in NB., Methods: The genetic and clinical data of patients with NB were obtained from the Therapeutically Applicable Research to Generate Effective Treatments dataset. TXNDC17 and BECN1 expressions in NB cells were up- and downregulated by transfection with plasmids and shRNA, respectively. Autophagy-related proteins were detected by western blot. Cell viability was determined using cell proliferation and toxicity experiments. Apoptotic cells were detected using flow cytometry., Results: Overall, 1076 pediatric and adolescent patients with NB were enrolled in this study. The 10-year overall survival (OS) rates and event-free survival (EFS) rates for the patients with a mutation of BECN1 were 37.4 ± 9.1% and 34.5 ± 8.8%, respectively. For patients with a mutation of TXNDC17, the 10-year OS and EFS were 41.4 ± 5.9% and 24.3 ± 5.1%, respectively, which were significantly lower than those in the unaltered group. The overexpression of BECN1 and TXNDC17 reduced NB sensitivity to cisplatin (DDP), etoposide (VP16), and cyclophosphamide (CTX). Autophagy mediated by BECN1 was regulated by TXNDC17, and this process was involved in the resistance to DDP, VP16, and CTX in NB. Suberoylanilide hydroxamic acid (SAHA) can enhance the sensitivity and apoptosis of NB cells to chemotherapeutics by inhibiting TXNDC17, ultimately decreasing autophagy-mediated chemoresistance., Conclusions: Acquired resistance to first-line chemotherapeutics was associated with autophagy mediated by BECN1 and regulated by TXNDC17, which can be reversed by SAHA., (© 2024 The Author(s). Cancer Reports published by Wiley Periodicals LLC.)

Senolytic and senomorphic therapies have gained more and more attention in the last decade. This kind of therapy is based on the killing of cellular senescent cells without harming the "normal" cells. Aging is not a disease. Clinical studies on healthy people will be difficult to conduct. Therefore, one possibility is to draw on the large repertoire of medicinal plants and use their senolytic properties to provide mild anti-aging therapies. Chamomile, goldenrod, reishi , and green tea were tested for their ability to trigger senolysis. Quercetin was used as control substance. Cellular senescence was induced with 25 µM etoposide in human dermal fibroblasts and established for at least 14 days. The plant extracts were tested for their antioxidant potential (DPPH assay) and their polyphenol content. Senolysis was determined by presto blue assay of young and etoposide-induced senescent cells, and SA-β-Gal assays were also performed. The senomorphic properties of the plants were investigated using IL-6 ELISA and qPCR. It turned out that chamomile triggers a kind of cytokine storm and causes the cytokine values in the ELISA and in the qPCR to rise extremely, and other senescence-associated phenotype (SASP) markers were also elevated. Goldenrod and quercetin tend to have a senolytic and senomorphic effect, respectively. Regarding the senolytic and senomorphic properties of herbs, we found that all tested herbs can have a senolytic effect, and a senomorphic effect of quercetin has also been discovered. With regard to the effect of chamomile, however, we can say that seemingly harmless tea products may have harmful effects, especially in combination with chemotherapy, at least in cell culture experiments. Nevertheless, inflammation is a double-bladed mechanism with positive effects, for example, in healing, but also known negative effects.

Background: Glioblastoma (GBM) is a highly aggressive tumor with unmet therapeutic needs, which can be explained by extensive intra-tumoral heterogeneity and plasticity. In this study, we aimed to investigate the specific metabolic features of Glioblastoma stem cells (GSC), a rare tumor subpopulation involved in tumor growth and therapy resistance., Methods: We conducted comprehensive analyses of primary patient-derived GBM cultures and GSC-enriched cultures of human GBM cell lines using state-of-the-art molecular, metabolic, and phenotypic studies., Results: We showed that GSC-enriched cultures display distinct glycolytic profiles compared with differentiated tumor cells. Further analysis revealed that GSC relies on pyruvate carboxylase (PC) activity for survival and self-renewal capacity. Interestingly, inhibition of PC led to GSC death, particularly when the glutamine pool was low, and increased differentiation. Finally, while GSC displayed resistance to the chemotherapy drug etoposide, genetic or pharmacological inhibition of PC restored etoposide sensitivity in GSC, both in vitro and in orthotopic murine models., Conclusions: Our findings demonstrate the critical role of PC in GSC metabolism, survival, and escape to etoposide. They also highlight PC as a therapeutic target to overcome therapy resistance in GBM., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Neuro-Oncology.)

Inhibitors of DNA-dependent protein kinase (PRKDC; DNA-PK) sensitize cancers to radiotherapy and DNA-damaging chemotherapies, with candidates in clinical trials. However, the degree to which DNA-PK inhibitors also sensitize normal tissues remains poorly characterized. In this study, we compare tumor growth control and normal tissue sensitization following DNA-PK inhibitors in combination with radiation and etoposide. FaDu tumor xenografts implanted in mice were treated with 10 to 15 Gy irradiation ± 3 to 100 mg/kg AZD7648. A dose-dependent increase in time to tumor volume doubling following AZD7648 was proportional to an increase in toxicity scores of the overlying skin. Similar effects were seen in the intestinal jejunum, tongue, and FaDu tumor xenografts of mice assessed for proliferation rates at 3.5 days after treatment with etoposide or 5 Gy whole body irradiation ± DNA-PK inhibitors AZD7648 or peposertib (M3814). Additional organs were examined for sensitivity to DNA-PK inhibitor activity in ATM-deficient mice, where DNA-PK activity is indicated by surrogate marker γH2AX. Inhibition was observed in the heart, brain, pancreas, thymus, tongue, and salivary glands of ATM-deficient mice treated with the DNA-PK inhibitors relative to radiation alone. Similar reductions are also seen in ATM-deficient FaDu tumor xenografts where both pDNA-PK and γH2AX staining could be performed. DNA-PK inhibitor-mediated sensitization to radiation and DNA-damaging chemotherapy are not only limited to tumor tissues, but also extends to normal tissues sustaining DNA damage. These data are useful for interpretation of the sensitizing effects of DNA damage repair inhibitors, where a therapeutic index showing greater cell-killing effects on cancer cells is crucial for optimal clinical translation., (©2024 American Association for Cancer Research.)

Lung cancer has a high incidence rate and a low cure rate, hence the urgent need for effective treatment methods. Current lung cancer drugs have several drawbacks, including low specificity, poor targeting, drug resistance, and irreversible damage to normal tissues. Therefore, there is a need to develop a safe and effective new drug that can target and kill tumor cells. In this study, we combined nanotechnology and biotechnology to develop a CD133 ligand-modified etoposide-liposome complex (Lipo@ETP-CD133) for targeted therapy of lung cancer. The CD133 ligand targeted lung cancer stem cells, causing the composite material to aggregate at the tumor site, where high levels of ETP liposomes could exert a strong tumor-killing effect. Our research results demonstrated that this nano-drug had efficient targeting and tumor-killing effects, indicating its potential for clinical application., (© 2024 American Institute of Chemical Engineers.)

In response to DNA double-strand breaks (DSBs), the ATM kinase activates NF-κB factors to stimulate gene expression changes that promote survival and allow time for cells to repair damage. In cell lines, ATM can activate NF-κB transcription factors via two independent, convergent mechanisms. One is ATM-mediated phosphorylation of nuclear NF-κB essential modulator (Nemo) protein, which leads to monoubiquitylation and export of Nemo to the cytoplasm where it engages the IκB kinase (IKK) complex to activate NF-κB. Another is DSB-triggered migration of ATM into the cytoplasm, where it promotes monoubiquitylation of Nemo and the resulting IKK-mediated activation of NF-κB. ATM has many other functions in the DSB response beyond activation of NF-κB, and Nemo activates NF-κB downstream of diverse stimuli, including developmental or proinflammatory stimuli such as LPSs. To elucidate the in vivo role of DSB-induced, ATM-dependent changes in expression of NF-κB-responsive genes, we generated mice expressing phosphomutant Nemo protein lacking consensus SQ sites for phosphorylation by ATM or related kinases. We demonstrate that these mice are viable/healthy and fertile and exhibit overall normal B and T lymphocyte development. Moreover, treatment of their B lineage cells with LPS induces normal NF-κB-regulated gene expression changes. Furthermore, in marked contrast to results from a pre-B cell line, primary B lineage cells expressing phosphomutant Nemo treated with the genotoxic drug etoposide induce normal ATM- and Nemo-dependent changes in expression of NF-κB-regulated genes. Our data demonstrate that ATM-dependent phosphorylation of Nemo SQ motifs in vivo is dispensable for DSB-signaled changes in expression of NF-κB-regulated genes., (Copyright © 2024 by The American Association of Immunologists, Inc.)

Small cell lung cancer (SCLC) is a recalcitrant cancer of neuroendocrine (NE) origin. Changes in therapeutic approaches against SCLC have been lacking over the decades. Here, we use preclinical models to identify a new therapeutic vulnerability in SCLC consisting of the targetable Jumonji lysine demethylase (KDM) family. We show that Jumonji demethylase inhibitors block malignant growth and that etoposide-resistant SCLC cell lines are particularly sensitive to Jumonji inhibition. Mechanistically, small molecule-mediated inhibition of Jumonji KDMs activates endoplasmic reticulum (ER) stress genes, upregulates ER stress signaling, and triggers apoptotic cell death. Furthermore, Jumonji inhibitors decrease protein levels of SCLC NE markers INSM1 and Secretogranin-3 and of driver transcription factors ASCL1 and NEUROD1. Genetic knockdown of KDM4A, a Jumonji demethylase highly expressed in SCLC and a known regulator of ER stress genes, induces ER stress response genes, decreases INSM1, Secretogranin-3, and NEUROD1 and inhibits proliferation of SCLC in vitro and in vivo. Lastly, we demonstrate that two different small molecule Jumonji KDM inhibitors (pan-inhibitor JIB-04 and KDM4 inhibitor SD70) block the growth of SCLC tumor xenografts in vivo. Our study highlights the translational potential of Jumonji KDM inhibitors against SCLC, a clinically feasible approach in light of recently opened clinical trials evaluating this drug class, and establishes KDM4A as a relevant target across SCLC subtypes., (© 2024. The Author(s).)

Combination drug therapy represents an effective strategy for treating certain drug-resistant and intractable cancer cases. However, determining the optimal combination of drugs and dosages is challenging due to clonal diversity in patients' tumors and the lack of rapid drug sensitivity evaluation methods. Microfluidic technology offers promising solutions to this issue. In this study, we propose a versatile microfluidic chip platform capable of integrating all processes, including dilution, treatment, and detection, for in vitro drug sensitivity assays. This platform innovatively incorporates several modules, including automated discrete drug logarithmic concentration generation, on-chip cell perfusion culture, and parallel drug treatments of cancer cell models. Moreover, it is compatible with microplate readers or high-content imaging systems for swift detection and automated monitoring, simplifying on-chip drug evaluation. Proof of concept is demonstrated by assessing the in vitro potency of two drugs, cisplatin, and etoposide, against the lung adenocarcinoma A549 cell line, under both single-drug and combination treatment conditions. The findings reveal that, compared to conventional microplate approaches with static cultivation, this on-chip automated perfusion bioassays yield comparable IC 50 values with lower variation and a 50 % reduction in drug preparation time. This versatile dilution-treatment-detection microfluidic platform offers a promising tool for rapid and precise drug assessments, facilitating in vitro drug sensitivity evaluation in personalized cancer chemotherapy., 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 Elsevier B.V. All rights reserved.)

MicroRNA hsa-miR-29 was connected to a number of malignancies. Its target genes are many, among them Mcl-1 that is expressed in three possible isoforms, one of which is anti-apoptotic and another one pro-apoptotic. Ratio of these two isoforms appears to affect cell response to external stimuli. We have demonstrated that miR-29b enhanced etoposide toxicity in HeLa cell line by modulating this ratio of Mcl-1 isoforms. However, it is not known whether the described miR-29 effect is common to various cancer types or even have the opposite effect. This represents a significant problem for possible future applications. In this report, we demonstrate that miR-29b affects toxicity of 60 μM etoposide in cell lines derived from selected malignancies. The mechanism, however, differs among the cell lines tested. Hep G2 cells demonstrated similar effect of miR-29b on etoposide toxicity as was described in HeLa cells, i.e. modulation of Mcl-1 expression. Target protein down-regulated by miR-29b resulting in enhanced etoposide toxicity in Caco-2 cells was, however, Bcl-2 protein. Moreover, H9c2, Hek-293 and ARPE-19 cell lines selected as a representatives of non-malignant cells, showed no effect of miR-29b on etoposide toxicity. Our data suggest that miR-29b could be a common enhancer of etoposide toxicity in malignant cells due to its modulation of Bcl family proteins., (© 2024. The Author(s).)

PARP1 is crucial in DNA damage repair, chromatin remodeling, and transcriptional regulation. The principle of synthetic lethality has effectively guided the application of PARP inhibitors in treating tumors carrying BRCA1/2 mutations. Meanwhile, PARP inhibitors have exhibited efficacy in BRCA-proficient patients, further highlighting the necessity for a deeper understanding of PARP1 function and its inhibition in cancer therapy. Here, we unveil PIN2/TRF1-interacting telomerase inhibitor 1 (PINX1) as an uncharacterized PARP1-interacting protein that synergizes with PARP inhibitors upon its depletion across various cancer cell lines. Loss of PINX1 compromises DNA damage repair capacity upon etoposide treatment. The vulnerability of PINX1-deficient cells to etoposide and PARP inhibitors could be effectively restored by introducing either a full-length or a mutant form of PINX1 lacking telomerase inhibitory activity. Mechanistically, PINX1 is recruited to DNA lesions through binding to the ZnF3-BRCT domain of PARP1, facilitating the downstream recruitment of the DNA repair factor XRCC1. In the absence of DNA damage, PINX1 constitutively binds to PARP1, promoting PARP1-chromatin association and transcription of specific DNA damage repair proteins, including XRCC1, and transcriptional regulators, including GLIS3. Collectively, our findings identify PINX1 as a multifaceted partner of PARP1, crucial for safeguarding cells against genotoxic stress and emerging as a potential candidate for targeted tumor therapy., (© 2024. The Author(s).)

Caspase-2 (Casp-2) is an enzyme that regulates the development of apoptosis upon alternative splicing of its mRNA. The long form of Casp-2 (Casp-2L) promotes apoptosis while the short form (Casp-2S) has decreased enzymatic activity and inhibits the development of apoptotic processes. However, very little is known about the mechanism of Casp-2 alternative splicing. Several endonucleases are known to participate in this process. The aim of this study was to determine the role of EndoG in regulation of Casp-2 alternative splicing. Strong correlation between expression levels of EndoG and Casp-2 splice-variants was found in CD4⁺ and CD8⁺ human T lymphocytes. Such correlation increased after incubation of these cells with etoposide. Increased expression of Casp-2S was determined during EndoG over-expression in CD4⁺ T-cells, after EndoG treatment of cell cytoplasm and nuclei and after nuclei incubation with EndoG digested cell RNA. Casp-2 alternative splicing was induced by a 60-mer RNA oligonucleotide in naked nuclei and in cells after transfection. The identified long non-coding RNA of 1016 nucleotides is the precursor of the 60-mer RNA oligonucleotide. Based on the results the following mechanism has been proposed. Casp-2 pre-mRNA is transcribed from the coding DNA strand while long non-coding RNA is transcribed from the template strand of the Casp-2 gene. EndoG digests long non-coding RNA and produces the 60-mer RNA oligonucleotide complementary to the Casp-2 pre-mRNA exon 9 and intron 9 junction place. Interaction of the 60-mer RNA oligonucleotide and Casp-2 pre-mRNA causes alternative splicing.

The emergence of chemoresistance poses a significant challenge to the efficacy of DNA-damaging agents in cancer treatment, in part due to the inherent DNA repair capabilities of cancer cells. The Ku70/80 protein complex (Ku) plays a central role in double-strand breaks (DSBs) repair through the classical non-homologous end joining (c-NHEJ) pathway, and has proven to be one of the most promising drug target for cancer treatment when combined with radiotherapy or chemotherapy. In this study, we conducted a high-throughput screening of small-molecule inhibitors targeting the Ku complex by using a fluorescence polarization-based DNA binding assay. From a library of 11,745 small molecules, UMI-77 was identified as a potent Ku inhibitor, with an IC 50 value of 2.3 μM. Surface plasmon resonance and molecular docking analyses revealed that UMI-77 directly bound the inner side of Ku ring, thereby disrupting Ku binding with DNA. In addition, UMI-77 also displayed potent inhibition against MUS81-EME1, a key player in homologous recombination (HR), demonstrating its potential for blocking both NHEJ- and HR-mediated DSB repair pathways. Further cell-based studies showed that UMI-77 could impair bleomycin-induced DNA damage repair, and significantly sensitized multiple cancer cell lines to the DNA-damaging agents. Finally, in a mouse xenograft tumor model, UMI-77 significantly enhanced the chemotherapeutic efficacy of etoposide with little adverse physiological effects. Our work offers a new avenue to combat chemoresistance in cancer treatment, and suggests that UMI-77 could be further developed as a promising candidate in cancer treatment., 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 Elsevier B.V. All rights reserved.)

Background Objectives: The human blood parasite Leishmania donovani causes visceral leishmaniasis or grayish discoloration of the skin (black fever/kala-azar). Antitumor drugs such as daunorubicin and etoposide can help to treat such diseases. The computational approach is used to find a better interaction of drugs with the active site of the protein and help to design new drugs., Methods: In this study, we have optimized two antitumor drugs, daunorubicin and etoposide. We studied frontier molecular orbitals, electrostatic potential (MEP) maps, and the natural bond order analysis of these anticancer drugs, followed by molecular docking with Leishmania donovani protein., Results: The three-dimensional structure of MapK from Leishmania donovani is LDBPK-331470. Our computational calculations reveal that daunorubicin and etoposide drugs can have an affinity with MapK from Leishmania donovani ., Interpretation Conclusion: Our study predicted that both daunorubicin and etoposide could have a similar affinity with the protein (UvrD) Leishmania donovani ., (Copyright © 2024 Copyright: © 2024 Journal of Vector Borne Diseases.)

Loss of proteostasis and cellular senescence have been previously established as characteristics of aging; however, their interaction in the context of lung aging and potential contributions to aging-associated lung remodeling remains understudied. In this study, we aimed to characterize endoplasmic reticulum (ER) stress response, cellular senescence, and their interaction in relation to extracellular matrix (ECM) production in lung fibroblasts from young (25-45 yr) and old (>60 yr) humans. Fibroblasts from young and old patients without significant preexisting lung disease were exposed to vehicle, MG132, etoposide, or salubrinal. Afterward, cells and cell lysates or supernatants were analyzed for ER stress, cellular senescence, and ECM changes using protein analysis, proliferation assay, and senescence-associated beta-galactosidase (SA-β-Gal) staining. At baseline, fibroblasts from aging individuals showed increased levels of ER stress (ATF6 and PERK), senescence (p21 and McL-1), and ECM marker (COL1A1) compared to those from young individuals. Upon ER stress induction and etoposide exposure, fibroblasts showed an increase in senescence (SA-β-Gal, p21, and Cav-1), ER stress (PERK), and ECM markers (COL1A1 and LUM) compared to vehicle. Additionally, IL-6 and IL-8 levels were increased in the supernatants of MG132- and etoposide-treated fibroblasts, respectively. Finally, the ER stress inhibitor salubrinal decreased the expression of p21 compared to vehicle and MG132 treatments; however, salubrinal inhibited COL1A1 but not p21 expression in MG132-treated fibroblasts. Our study suggests that ER stress response plays an important role in establishment and maintenance of a senescence phenotype in lung fibroblasts and therefore contributes to altered remodeling in the aging lung. NEW & NOTEWORTHY The current study establishes functional links between endoplasmic reticulum (ER) stress and cellular senescence per se in the specific context of aging human lung fibroblasts. Recognizing that the process of aging per se is complex, modulated by the myriad of lifelong and environmental exposures, it is striking to note that chronic ER stress may play a crucial role in the establishment and maintenance of cellular senescence in lung fibroblasts.

Purpose: Small-cell lung cancer (SCLC) is an aggressive disease with a dismal prognosis. The addition of immune checkpoints inhibitors to standard platinum-based chemotherapy in first-line setting achieves a durable benefit only in a patient subgroup. Thus, the identification of predictive biomarkers is an urgent unmet medical need., Experimental Design: Tumor samples from naive extensive-stage (ES) SCLC patients receiving atezolizumab plus carboplatin-etoposide were analyzed by gene expression profiling and two 9-color multiplex immunofluorescence panels, to characterize the immune infiltrate and SCLC subtypes. Associations of tissue biomarkers with time-to-treatment failure (TTF), progression-free survival (PFS) and overall survival (OS), were assessed., Results: 42 patients were included. Higher expression of exhausted CD8-related genes was independently associated with a longer TTF and PFS while increased density of B lymphocytes correlated with longer TTF and OS. Higher percentage of M2-like macrophages close to tumor cells and of CD8+T cells close to CD4+T lymphocytes correlated with increased risk of TF and longer survival, respectively. A lower risk of TF, disease progression and death was associated with a higher density of ASCL1+tumor cells while the expression of POU2F3 correlated with a shorter survival. A composite score combining the expression of exhausted CD8-related genes, B lymphocyte density, ASCL1 tumor expression and quantification of CD163+macrophages close to tumor cells, was able to stratify patients into high-risk and low-risk groups., Conclusions: In conclusion, we identified tissue biomarkers and a combined score that can predict a higher benefit from chemoimmunotherapy in ES-SCLC patients., Competing Interests: Competing interests: GP reports Advisory Boards/Honoraria/Speakers’ fee/Consultant by Amgen, AstraZeneca, BMS, Eli Lilly, Jansenn, MSD, Novartis, Pfizer, Roche, and unconditioned research support by AstraZeneca, Roche, MSD. VG reports personal fees for advisory board membership for AstraZeneca, Daiichi Sankyo, Eisai, Eli Lilly, Exact Sciences, Gilead, Merck Serono, MSD, Novartis, Pfizer, Olema Oncology, Pierre Fabre; personal fees as an invited speaker for AstraZeneca, Daiichi Sankyo, Eli Lilly, Exact Sciences, Gilead, GSK, Novartis, Roche and Zentiva; personal fees for expert testimony for Eli Lilly., (© Author(s) (or their employer(s)) 2024. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

In vivo antigenotoxic activity of BP-C2 composition (at doses of 60, 80, and 120 mg/kg) based on polyphenolic compounds derived from hydrolyzed lignin was evaluated in mouse germ cells. The BP-C2 composition dose-dependently reduced the aneugenic activity of topoisomerase II inhibitor etoposide in mouse oocytes without affecting the clastogenic activity of the genotoxicant. In mouse testicular cells, the BP-C2 composition reduced the DNA-damaging activity of the pro-oxidant genotoxicant dioxidine, but not etoposide. The cytoprotective activity of BP-C2 composition was revealed in relation to etoposide-induced cytotoxicity., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Intrinsic and extrinsic factors in the tumour microenvironment (TME) contribute to therapeutic resistance. Here we demonstrate that transforming growth factor (TGF)-β1 produced in the TME increased drug resistance of neuroblastoma (NB) cells., Methods: Human NB cell lines were tested in vitro for their sensitivity to Doxorubicin (DOX) and Etoposide (ETOP) in the presence of tumour-associated macrophages (TAM) and mesenchymal stromal cells/cancer-associated fibroblasts (MSC/CAF). These experiments were validated in xenotransplanted and primary tumour samples., Results: Drug resistance was associated with an increased expression of efflux transporter and anti-apoptotic proteins. Upregulation was dependent on activation of nuclear factor (NF)-κB by TGF-β-activated kinase (TAK1) and SMAD2. Resistance was reversed upon pharmacologic and genetic inhibitions of NF-κB, and TAK1/SMAD2. Interleukin-6, leukaemia inhibitory factor and oncostatin M were upregulated by this TGF-β/TAK1/NF-κB/SMAD2 signalling pathway contributing to drug resistance via an autocrine loop activating STAT3. An analysis of xenotransplanted NB tumours revealed an increased presence of phospho (p)-NF-κB in tumours co-injected with MSC/CAF and TAM, and these tumours failed to respond to Etoposide but responded if treated with a TGF-βR1/ALK5 inhibitor. Nuclear p-NF-κB was increased in patient-derived tumours rich in TME cells., Conclusions: The data provides a novel insight into a targetable mechanism of environment-mediated drug resistance., (© 2024. The Author(s).)

Herein, we constructed the branch-shaped SiO 2 /nano GO (nGO)/Fe 3 O 4 /selenium quantum dots (QDs) (SeQDs) nanoparticles (SGF/SeQDs) embodying magnetism, fluorescence, and microwave stimulus response properties to enhance the performance of releasing drugs. The SGF/SeQDs composite was characterized by technologies including powder X-ray diffraction, transmission electron microscopy, infrared spectroscopy, etc. In the nanoparticles, the branch-shaped SiO 2 provides a large specific surface area, nGO as the dielectric loss-style material promotes microwave-absorbing performance, and the Fe 3 O 4 serves as a magnetic targeting agent and microwave absorber. Integrating nGO and Fe 3 O 4 could further strengthen the microwave absorption of the entire composite; selenium features both fluorescence and anticancer effects. The synthesized nanoparticles as carriers exhibited a branch-like mesoporous sphere of ∼260 nm, a specific surface area of 258.57 m 2 g -1 , a saturation magnetization of 24.59 emu g -1 , and good microwave thermal conversion performance that the temperature was elevated from 25 to 70 °C under microwave irradiation. These physical characteristics, including large pore volume (5.30 nm), high specific surface area, and fibrous morphology, are in favor of loading drugs. Meanwhile, the cumulative etoposide (VP16) loading rate of the nanoparticles reached to 21 wt % after 360 min. The noncovalent interaction between the VP16 and SGF/SeQDs was mainly the hydrogen-bonding effect during the loading process. Furthermore, the drug release rates at 180 min were up to 81.46, 61.92, and 56.84 wt % at pH 4, 5, and 7, respectively. At 25, 37, and 50 °C, the rates of drug release reach 25.40, 56.84, and 65.32 wt %, respectively. After microwave stimulation at pH 7, the rate of releasing drug increased distinctly from 56.84 to 71.74 wt % compared to that of nonmicrowave irradiation. Cytotoxicity tests manifested that the carrier had good biocompatibility. Therefore, the nanoparticles are looking forward to paving one platform for further applications in biomedicine and drug delivery systems.

DNA topoisomerase II α (TOP2 α ; 170 kDa, TOP2 α /170) is an essential enzyme for proper chromosome dysjunction by producing transient DNA double-stranded breaks and is an important target for DNA damage-stabilizing anticancer agents, such as etoposide. Therapeutic effects of TOP2 α poisons can be limited due to acquired drug resistance. We previously demonstrated decreased TOP2 α /170 levels in an etoposide-resistant human leukemia K562 subline, designated K/VP.5, accompanied by increased expression of a C-terminal truncated TOP2 α isoform (90 kDa; TOP2 α /90), which heterodimerized with TOP2 α /170 and was a determinant of resistance by exhibiting dominant-negative effects against etoposide activity. Based on 3'-rapid amplification of cDNA ends, we confirmed TOP2 α /90 as the translation product of a TOP2 α mRNA in which a cryptic polyadenylation site (PAS) harbored in intron 19 (I19) was used. In this report, we investigated whether the resultant intronic polyadenylation (IPA) would be attenuated by blocking or mutating the I19 PAS, thereby circumventing acquired drug resistance. An antisense morpholino oligonucleotide was used to hybridize/block the PAS in TOP2 α pre-mRNA in K/VP.5 cells, resulting in decreased TOP2 α /90 mRNA/protein levels in K/VP.5 cells and partially circumventing drug resistance. Subsequently, CRISPR/CRISPR-associated protein 9 with homology-directed repair was used to mutate the cryptic I19 PAS (A ATA AA→A CCC AA) to prevent IPA. Gene-edited clones exhibited increased TOP2 α /170 and decreased TOP2 α /90 mRNA/protein and demonstrated restored sensitivity to etoposide and other TOP2 α -targeted drugs. Together, results indicated that blocking/mutating a cryptic I19 PAS in K/VP.5 cells reduced IPA and restored sensitivity to TOP2 α -targeting drugs. SIGNIFICANCE STATEMENT: The results presented in this study indicate that CRISPR/CRISPR-associated protein 9 gene editing of a cryptic polyadenylation site (PAS) within I19 of the TOP2α gene results in the reversal of acquired resistance to etoposide and other TOP2-targeted drugs. An antisense morpholino oligonucleotide targeting the PAS also partially circumvented resistance., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Introduction: Chemoresistance constitutes a prevalent factor that significantly impacts thesurvival of patients undergoing treatment for smal-cell lung cancer (SCLC). Chemotherapy resistance in SCLC patients is generally classified as primary or acquired resistance, each governedby distinct mechanisms that remain inadequately researched., Methods: In this study, we performed transcriptome screening of peripheral blood plasma obtainedfrom 17 patients before and after receiving combined etoposide and platinum treatment. We firs testimated pseudo-single-cell analysis using xCell and ESTIMATE and identified differentially expressed genes (DEGs), then performed network analysis to discover key hub genes involved in chemotherapy resistance., Results: Our analysis showed a significant increase in class-switched memory B cell scores acrossboth chemotherapy resistance patterns, indicating their potential crucial role in mediatingresistance. Moreover, network analysis identifed PRICKLE3 , TNFSFI0 , ACSLl and EP300 as potential contributors to primary resistance, with SNWl , SENP2 and SMNDCl emerging assignificant factors in acquired resistance, providing valuable insights into chemotherapy resistancein SCLC., Discussion: These findings offer valuable insights for understanding chemotherapy resistance and related gene signatures in SCLC, which could help further biological validation studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Yang, Fan, Yang and Cun.)

RNA-binding proteins are frequently deregulated in cancer and emerge as effectors of the DNA damage response (DDR). The non-POU domain-containing octamer-binding protein NONO/p54 nrb is a multifunctional RNA-binding protein that not only modulates the production and processing of mRNA, but also promotes the repair of DNA double-strand breaks (DSBs). Here, we investigate the impact of Nono deletion in the murine KP ( KRas G12D , Trp53 -/- ) cell-based lung cancer model. We show that the deletion of Nono impairs the response to DNA damage induced by the topoisomerase II inhibitor etoposide or the radiomimetic drug bleomycin. Nono-deficient KP (KPN) cells display hyperactivation of DSB signalling and high levels of DSBs. The defects in the DDR are accompanied by reduced RNA polymerase II promoter occupancy, impaired nascent RNA synthesis, and attenuated induction of the DDR factor growth arrest and DNA damage-inducible beta (Gadd45b). Our data characterise Gadd45b as a putative Nono-dependent effector of the DDR and suggest that Nono mediates a genome-protective crosstalk of the DDR with the RNA metabolism via induction of Gadd45b., (© 2024 Mamontova et al.)

Myeloperoxidase (MPO) is found almost exclusively in granulocytes and immature myeloid cells. It plays a key role in the innate immune system, catalysing the formation of reactive oxygen species that are important in anti-microbial action, but MPO also oxidatively transforms the topoisomerase II (TOP2) poison etoposide to chemical forms that have elevated DNA damaging properties. TOP2 poisons such as etoposide are widely used anti-cancer drugs, but they are linked to cases of secondary acute myeloid leukaemias through a mechanism that involves DNA damage and presumably erroneous repair leading to leukaemogenic chromosome translocations. This leads to the possibility that myeloperoxidase inhibitors could reduce the rate of therapy-related leukaemia by protecting haematopoietic cells from TOP2 poison-mediated genotoxic damage while preserving the anti-cancer efficacy of the treatment. We show here that myeloperoxidase inhibition reduces etoposide-induced TOP2B-DNA covalent complexes and resulting DNA double-strand break formation in primary ex vivo expanded CD34 + progenitor cells and unfractionated bone marrow mononuclear cells. Since MPO inhibitors are currently being developed as anti-inflammatory agents this raises the possibility that repurposing of these potential new drugs could provide a means of suppressing secondary acute myeloid leukaemias associated with therapies containing TOP2 poisons., (© 2024 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Upregulation of PRAME (preferentially expressed antigen of melanoma) has been implicated in the progression of a variety of cancers, including melanoma. The tumor suppressor p53 is a transcriptional regulator that mediates cell cycle arrest and apoptosis in response to stress signals. Here, we report that PRAME is a novel repressive target of p53. This was supported by analysis of melanoma cell lines carrying wild-type p53 and human melanoma databases. mRNA expression of PRAME was downregulated by p53 overexpression and activation using DNA-damaging agents, but upregulated by p53 depletion. We identified a p53-responsive element (p53RE) in the promoter region of PRAME. Luciferase and ChIP assays showed that p53 represses the transcriptional activity of the PRAME promoter and is recruited to the p53RE together with HDAC1 upon etoposide treatment. The functional significance of p53 activationmediated PRAME downregulation was demonstrated by measuring colony formation and p27 expression in melanoma cells. These data suggest that p53 activation, which leads to PRAME downregulation, could be a therapeutic strategy in melanoma cells. [BMB Reports 2024; 57(6): 299-304].

Rationale: Synergic reprogramming of metabolic dominates neuroblastoma (NB) progression. It is of great clinical implications to develop an individualized risk prognostication approach with stratification-guided therapeutic options for NB based on elucidating molecular mechanisms of metabolic reprogramming. Methods: With a machine learning-based multi-step program, the synergic mechanisms of metabolic reprogramming-driven malignant progression of NB were elucidated at single-cell and metabolite flux dimensions. Subsequently, a promising metabolic reprogramming-associated prognostic signature (MPS) and individualized therapeutic approaches based on MPS-stratification were developed and further validated independently using pre-clinical models. Results: MPS-identified MPS-I NB showed significantly higher activity of metabolic reprogramming than MPS-II counterparts. MPS demonstrated improved accuracy compared to current clinical characteristics [AUC: 0.915 vs. 0.657 ( MYCN ), 0.713 (INSS-stage), and 0.808 (INRG-stratification)] in predicting prognosis. AZD7762 and etoposide were identified as potent therapeutics against MPS-I and II NB, respectively. Subsequent biological tests revealed AZD7762 substantially inhibited growth, migration, and invasion of MPS-I NB cells, more effectively than that of MPS-II cells. Conversely, etoposide had better therapeutic effects on MPS-II NB cells. More encouragingly, AZD7762 and etoposide significantly inhibited in-vivo subcutaneous tumorigenesis, proliferation, and pulmonary metastasis in MPS-I and MPS-II samples, respectively; thereby prolonging survival of tumor-bearing mice. Mechanistically, AZD7762 and etoposide-induced apoptosis of the MPS-I and MPS-II cells, respectively, through mitochondria-dependent pathways; and MPS-I NB resisted etoposide-induced apoptosis by addiction of glutamate metabolism and acetyl coenzyme A. MPS-I NB progression was fueled by multiple metabolic reprogramming-driven factors including multidrug resistance, immunosuppressive and tumor-promoting inflammatory microenvironments. Immunologically, MPS-I NB suppressed immune cells via MIF and THBS signaling pathways. Metabolically, the malignant proliferation of MPS-I NB cells was remarkably supported by reprogrammed glutamate metabolism, tricarboxylic acid cycle, urea cycle, etc. Furthermore, MPS-I NB cells manifested a distinct tumor-promoting developmental lineage and self-communication patterns, as evidenced by enhanced oncogenic signaling pathways activated with development and self-communications. Conclusions: This study provides deep insights into the molecular mechanisms underlying metabolic reprogramming-mediated malignant progression of NB. It also sheds light on developing targeted medications guided by the novel precise risk prognostication approaches, which could contribute to a significantly improved therapeutic strategy for NB., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Research on the relationships between oligoelements (OE) and the development of cancer or its prevention is a field that is gaining increasing relevance. The aim was to evaluate OE and their interactions with oncology treatments (cytarabine or etoposide) to determine the effects of this combination on biogenic amines and oxidative stress biomarkers in the brain regions of young Wistar rats. Dopamine (DA), 5-Hydroxyindoleacetic acid (5-Hiaa), Glutathione (Gsh), Tiobarbituric acid reactive substances (TBARS) and Ca +2 , Mg +2 ATPase enzyme activity were measured in brain regions tissues using spectrophometric and fluorometric methods previously validated. The combination of oligoelements and cytarabine increased dopamine in the striatum but decreased it in cerebellum/medulla-oblongata, whereas the combination of oligoelements and etoposide reduced lipid peroxidation. These results suggest that supplementation with oligoelements modifies the effects of cytarabine and etoposide by redox pathways, and may become promising therapeutic targets in patients with cancer., (© 2024. The Author(s).)

Although the selective and effective clearance of senescent cancer cells can improve cancer treatment, their development is confronted by many challenges. As part of efforts designed to overcome these problems, prodrugs, whose design is based on senescence-associated β-galactosidase (SA-β-gal), have been developed to selectively eliminate senescent cells. However, chemotherapies relying on targeted molecular inhibitors as senolytic drugs can induce drug resistance. In the current investigation, we devised a new strategy for selective degradation of target proteins in senescent cancer cells that utilizes a prodrug composed of the SA-β-gal substrate galactose (galacto) and the proteolysis-targeting chimeras (PROTACs) as senolytic agents. Prodrugs Gal-ARV-771 and Gal-MS99 were found to display senolytic indexes higher than those of ARV-771 and MS99. Significantly, results of in vivo studies utilizing a human lung A549 xenograft mouse model demonstrated that concomitant treatment with etoposide and Gal-ARV-771 leads to a significant inhibition of tumor growth without eliciting significant toxicity.

Triple-negative breast cancer (TNBC) remains the most lethal subtype of breast cancer, characterized by poor response rates to current chemotherapies and a lack of additional effective treatment options. While approximately 30% of patients respond well to anthracycline- and taxane-based standard-of-care chemotherapy regimens, the majority of patients experience limited improvements in clinical outcomes, highlighting the critical need for strategies to enhance the effectiveness of anthracycline/taxane-based chemotherapy in TNBC. In this study, we report on the potential of a DNA-PK inhibitor, peposertib, to improve the effectiveness of topoisomerase II (TOPO II) inhibitors, particularly anthracyclines, in TNBC. Our in vitro studies demonstrate the synergistic antiproliferative activity of peposertib in combination with doxorubicin, epirubicin and etoposide in multiple TNBC cell lines. Downstream analysis revealed the induction of ATM-dependent compensatory signaling and p53 pathway activation under combination treatment. These in vitro findings were substantiated by pronounced anti-tumor effects observed in mice bearing subcutaneously implanted tumors. We established a well-tolerated preclinical treatment regimen combining peposertib with pegylated liposomal doxorubicin (PLD) and demonstrated strong anti-tumor efficacy in cell-line-derived and patient-derived TNBC xenograft models in vivo. Taken together, our findings provide evidence that co-treatment with peposertib has the potential to enhance the efficacy of anthracycline/TOPO II-based chemotherapies, and it provides a promising strategy to improve treatment outcomes for TNBC patients.

When new antitumor therapy drugs are discovered, it is essential to address new target molecules from the point of view of chemical structure and to carry out efficient and systematic evaluation. In the case of natural products and derived compounds, it is of special importance to investigate chemomodulation to further explore antitumoral pharmacological activities. In this work, the compound podophyllic aldehyde, a cyclolignan derived from the chemomodulation of the natural product podophyllotoxin, has been evaluated for its viability, influence on the cell cycle, and effects on intracellular signaling. We used functional proteomics characterization for the evaluation. Compared with the FDA-approved drug etoposide (another podophyllotoxin derivative), we found interesting results regarding the cytotoxicity of podophyllic aldehyde. In addition, we were able to observe the effect of mitotic arrest in the treated cells. The use of podophyllic aldehyde resulted in increased cytotoxicity in solid tumor cell lines, compared to etoposide, and blocked the cycle more successfully than etoposide. High-throughput analysis of the deregulated proteins revealed a selective antimitotic mechanism of action of podophyllic aldehyde in the HT-29 cell line, in contrast with other solid and hematological tumor lines. Also, the apoptotic profile of podophyllic aldehyde was deciphered. The cell death mechanism is activated independently of the cell cycle profile. The results of these targeted analyses have also shown a significant response to the signaling of kinases, key proteins involved in signaling cascades for cell proliferation or metastasis. Thanks to this comprehensive analysis of podophyllic aldehyde, remarkable cytotoxic, antimitotic, and other antitumoral features have been discovered that will repurpose this compound for further chemical transformations and antitumoral analysis.

Protein tyrosine phosphatase receptor type E (PTPRE) is a member of the "classical" protein tyrosine phosphatase subfamily and regulates a variety of cellular processes in a tissue-specific manner by antagonizing the function of protein tyrosine kinases. PTPRE plays a tumorigenic role in different human cancer cells, but its role in retinoblastoma (RB), the most common malignant eye cancer in children, remains to be elucidated. Etoposide-resistant RB cell lines and RB patients display significant higher PTPRE expression levels compared to chemosensitive counterparts and the healthy human retina, respectively. PTPRE promotor methylation analyses revealed that PTPRE expression in RB is not regulated via this mechanism. Lentiviral PTPRE knockdown (KD) induced a significant decrease in growth kinetics, cell viability, and anchorage-independent growth of etoposide-resistant Y79 and WERI RB cells. Caspase-dependent apoptosis rates were significantly increased and a re-sensitization for etoposide could be observed after PTPRE depletion. In vivo chicken chorioallantoic membrane (CAM) assays revealed decreased tumor formation capacity as well as reduced tumor size and weight following PTPRE KD. Expression levels of miR631 were significantly downregulated in etoposide-resistant RB cells and patients. Transient miR631 overexpression resulted in significantly decreased PTPRE levels and concomitantly decreased proliferation and increased apoptosis levels in etoposide-resistant RB cells. These impacts mirror PTPRE KD effects, indicating a regulation of PTPRE via this miR. Additionally, PTPRE KD led to altered phosphorylation of protein kinase SGK3 and-dependent on the cell line-AKT and ERK1/2, suggesting potential PTPRE downstream signaling pathways. In summary, these results indicate an oncogenic role of PTPRE in chemoresistant retinoblastoma., Competing Interests: The authors declare no conflicts of interest.

DNA topoisomerase II β (TOP2 β /180; 180 kDa) is a nuclear enzyme that regulates DNA topology by generation of short-lived DNA double-strand breaks, primarily during transcription. TOP2 β /180 can be a target for DNA damage-stabilizing anticancer drugs, whose efficacy is often limited by chemoresistance. Our laboratory previously demonstrated reduced levels of TOP2 β /180 (and the paralog TOP2 α /170) in an acquired etoposide-resistant human leukemia (K562) clonal cell line, K/VP.5, in part due to overexpression of microRNA-9-3p/5p impacting post-transcriptional events. To evaluate the effect on drug sensitivity upon reduction/elimination of TOP2 β /180, a premature stop codon was generated at the TOP2 β /180 gene exon 19/intron 19 boundary (A G A A //GTAA→A T A G //GTAA) in parental K562 cells (which contain four TOP2 β /180 alleles) by CRISPR/Cas9 editing with homology-directed repair to disrupt production of full-length TOP2 β /180. Gene-edited clones were identified and verified by quantitative polymerase chain reaction and Sanger sequencing, respectively. Characterization of TOP2 β /180 gene-edited clones, with one or all four TOP2 β /180 alleles mutated, revealed partial or complete loss of TOP2 β mRNA/protein, respectively. The loss of TOP2 β /180 protein correlated with decreased (2-{4-[(7-chloro-2-quinoxalinyl)oxy]phenoxy}propionic acid)-induced DNA damage and partial resistance in growth inhibition assays. Partial resistance to mitoxantrone was also noted in the gene-edited clone with all four TOP2 β /180 alleles modified. No cross-resistance to etoposide or mAMSA was noted in the gene-edited clones. Results demonstrated the role of TOP2 β /180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents. SIGNIFICANCE STATEMENT: Data indicated that CRISPR/Cas9 editing of the exon 19/intron 19 boundary in the TOP2β/180 gene to introduce a premature stop codon resulted in partial to complete disruption of TOP2β/180 expression in human leukemia (K562) cells depending on the number of edited alleles. Edited clones were partially resistant to mitoxantrone and XK469, while lacking resistance to etoposide and mAMSA. Results demonstrated the import of TOP2β/180 in drug sensitivity/resistance in K562 cells and revealed differential paralog activity of TOP2-targeted agents., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

RNA-binding proteins emerge as effectors of the DNA damage response (DDR). The multifunctional non-POU domain-containing octamer-binding protein NONO/p54nrb marks nuclear paraspeckles in unperturbed cells, but also undergoes re-localization to the nucleolus upon induction of DNA double-strand breaks (DSBs). However, NONO nucleolar re-localization is poorly understood. Here we show that the topoisomerase II inhibitor etoposide stimulates the production of RNA polymerase II-dependent, DNA damage-inducible antisense intergenic non-coding RNA (asincRNA) in human cancer cells. Such transcripts originate from distinct nucleolar intergenic spacer regions and form DNA-RNA hybrids to tether NONO to the nucleolus in an RNA recognition motif 1 domain-dependent manner. NONO occupancy at protein-coding gene promoters is reduced by etoposide, which attenuates pre-mRNA synthesis, enhances NONO binding to pre-mRNA transcripts and is accompanied by nucleolar detention of a subset of such transcripts. The depletion or mutation of NONO interferes with detention and prolongs DSB signalling. Together, we describe a nucleolar DDR pathway that shields NONO and aberrant transcripts from DSBs to promote DNA repair., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Herein, we envisioned the design and synthesis of novel pyrazolopyrimidines (confirmed by elemental analysis, 1 H and 13 C NMR, and mass spectra) as multitarget-directed drug candidates acting as EGFR/TOPO II inhibitors, DNA intercalators, and apoptosis inducers. The target diphenyl-tethered pyrazolopyrimidines were synthesized starting from the reaction of phenyl hydrazine and ethoxymethylenemalononitrile to give aminopyrazole-carbonitrile 2. The latter hydrolysis with NaOH and subsequent reaction with 4-chlorobenzaldhyde afforded the corresponding pyrazolo[3,4-d]pyrimidin-4-ol 4. Chlorination of 4 with POCl 3 and sequential reaction with different amines afforded the target compounds in good yields (up to 73 %). The growth inhibition % of the new derivatives (6a-m) was investigated against different cancer and normal cells and the IC 50 values of the most promising candidates were estimated for HNO97, MDA-MB-468, FaDu, and HeLa cancer cells. The frontier derivatives (6a, 6i, 6k, 6l, and 6m) were pursued for their EGFR inhibitory activity. Compound 6l decreased EGFR protein concentration by a 6.10-fold change, compared to imatinib as a reference standard. On the other side, compounds (6a, 6i, 6k, 6l, and 6m) underwent topoisomerase II (TOPO II) inhibitory assay. In particular, compounds 6a and 6l exhibited IC 50 s of 17.89 and 19.39 μM, respectively, surpassing etoposide with IC 50 of 20.82 μM. Besides, the DNA fragmentation images described the great potential of both candidates 6a and 6l in inducing DNA degradation at lower concentrations compared to etoposide and doxorubicin. Moreover, compound 6l, with the most promising EGFR/TOPO II inhibition and DNA intercalation, was selected for further investigation for its apoptosis induction ability by measuring caspases 3, 7, 8, and 9, Bax, p53, MMP2, MMP9, and BCL-2 proteins. Additionally, molecular docking was used to explain the SAR results based on the differences in the molecular features of the investigated congeners and the target receptors' topology., 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 Elsevier Inc. All rights reserved.)

Introduction and Objective: Including additional compounds that disturb the energy metabolism of cancer cells in advanced cancer therapy regimens may be an approach to overcome the problem of drug resistance and the therapeutic effectiveness of classic chemotherapeutics. One of the compounds that decouple oxidative phosphorylation, and thus alter the activity of energy-producing pathways, is 2,4-DNP (2,4- dinitrophenol)., Objective: The aim of the study was to assess the ability of the 2,4-DNP to sensitize prostate cancer cells to the action of cisplatin and etoposide, or to intensify their action., Material and Methods: The research was carried out on three prostate cancer cell lines (LNCaP, PC-3, DU-145. To assess the effect of cisplatin or etoposide with 2,4-DNP on prostate cancer cells, MTT assay, analysis of the cell cycle and apoptosis detection was performed. Oxidative stress was investigated by CellRox fluorescence staining and expression of genes related to antioxidant defence. In addition, analysis was conducted of the expression of genes related to cell cycle inhibition, transporters associated with multi-drug resistance and DNA repair., Results: The study showed that the simultaneous incubation of 2,4-DNP with cisplatin or etoposide enhances the cytotoxic effect of the chemotherapeutic agent only in LNCaP cells (oxidative phenotype)., Conclusions: The enhanced cytotoxic effect of chemotherapeutics by 2,4-DNP may be the result of disturbed redox balance, reduced ability of cells to repair DNA, and the oxidative metabolic phenotype of prostate cancer cells.

Development of effective strategies to manage the inevitable acquired resistance to osimertinib, a third-generation EGFR inhibitor for the treatment of EGFR-mutant (EGFRm) non-small cell lung cancer (NSCLC), is urgently needed. This study reports that DNA topoisomerase II (Topo II) inhibitors, doxorubicin and etoposide, synergistically decreased cell survival, with enhanced induction of DNA damage and apoptosis in osimertinib-resistant cells; suppressed the growth of osimertinib-resistant tumors; and delayed the emergence of osimertinib-acquired resistance. Mechanistically, osimertinib decreased Topo IIα levels in EGFRm NSCLC cells by facilitating FBXW7-mediated proteasomal degradation, resulting in induction of DNA damage; these effects were lost in osimertinib-resistant cell lines that possess elevated levels of Topo IIα. Increased Topo IIα levels were also detected in the majority of tissue samples from patients with NSCLC after relapse from EGFR tyrosine kinase inhibitor treatment. Enforced expression of an ectopic TOP2A gene in sensitive EGFRm NSCLC cells conferred resistance to osimertinib, whereas knockdown of TOP2A in osimertinib-resistant cell lines restored their susceptibility to osimertinib-induced DNA damage and apoptosis. Together, these results reveal an essential role of Topo IIα inhibition in mediating the therapeutic efficacy of osimertinib against EGFRm NSCLC, providing scientific rationale for targeting Topo II to manage acquired resistance to osimertinib.

Therapy-related leukemia carries a poor prognosis, and leukemia after chemotherapy is a growing risk in clinic, whose mechanism is still not well understood. Ikaros transcription factor is an important regulator in hematopoietic cells development and differentiation. In the absence of Ikaros, lymphoid cell differentiation is blocked at an extremely early stage, and myeloid cell differentiation is also significantly affected. In this work, we showed that chemotherapeutic drug etoposide reduced the protein levels of several isoforms of Ikaros including IK1, IK2 and IK4, but not IK6 or IK7, by accelerating protein degradation, in leukemic cells. To investigate the molecular mechanism of Ikaros degradation induced by etoposide, immunoprecipitation coupled with LC-MS/MS analysis was conducted to identify changes in protein interaction with Ikaros before and after etoposide treatment, which uncovered KCTD5 protein. Our further study demonstrates that KCTD5 is the key stabilizing factor of Ikaros and chemotherapeutic drug etoposide induces Ikaros protein degradation through decreasing the interaction of Ikaros with KCTD5. These results suggest that etoposide may induce leukemic transformation by downregulating Ikaros via KCTD5, and our work may provide insights to attenuate the negative impact of chemotherapy on hematopoiesis., (© 2024 Walter de Gruyter GmbH, Berlin/Boston.)

Circulating endothelial cells (CEC) are arising as biomarkers for vascular diseases. However, whether they can be utilized as markers of endothelial cell (EC) senescence in vivo remains unknown. Here, we present a protocol to isolate circulating endothelial cells for a characterization of their senescent signature. Further, we characterize different models of EC senescence induction in vitro and show similar patterns of senescence being upregulated in CECs of aged patients as compared to young volunteers. Replication-(ageing), etoposide-(DNA damage) and angiotensin II-(ROS) induced senescence models showed the expected cell morphology and proliferation-reduction effects. Expression of senescence-associated secretory phenotype markers was specifically upregulated in replication-induced EC senescence. All models showed reduced telomere lengths and induction of the INK4a/ARF locus. Additional p14ARF-p21 pathway activation was observed in replication- and etoposide-induced EC senescence. Next, we established a combined magnetic activated- and fluorescence activated cell sorting (MACS-FACS) based protocol for CEC isolation. Interestingly, CECs isolated from aged volunteers showed similar senescence marker patterns as replication- and etoposide-induced senescence models. Here, we provide first proof of senescence in human blood derived circulating endothelial cells. These results hint towards an exciting future of using CECs as mirror cells for in vivo endothelial cell senescence, of particular interest in the context of endothelial dysfunction and cardiovascular diseases., (© 2024. The Author(s).)

Type II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction cycle. Curiously, most type II topoisomerases (topos II, IV and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase IIβ (hTOP2β) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss elevates the enzyme's propensity for DNA damage. The unstructured C-terminal domains (CTDs) of hTOP2β strongly potentiate strand passage activity in ATPase-less enzymes, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleavage in vitro, as well as in vivo. By contrast, aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are present. Our findings are consistent with the proposal that type II topoisomerases acquired ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Hematopoietic stem cells (HSC) from cord blood can be applied as an alternative to bone marrow in transplantation to treat hematological diseases. Umbilical cord blood (UCB) consists of cycling and non-cycling CD34+/CD45low cells needed for long-term and short-term engraftment. After sorting and subsequent in vitro culture, quiescent HSCs enter the cell cycle. This enables the analysis of HSCs in 2 different cell cycle stages and the comparison of their responses to different genotoxic noxae. To analyze different mechanisms of DNA damage induction in cells, 2 different genotoxins were compared: etoposide, a topoisomerase II inhibitor that targets mitosis in the S/G2-phase of the cell cycle and the alkylating nitrosamine N-Nitroso-N-methylurea (MNU), which leads to the formation of methyl DNA adducts resulting in DNA double breaks during DNA replication and persistent mutations. Cycling cells recovered after treatment even with higher concentrations of etoposide (1.5µM/ 5µM/10µM), while sorted cells treated with MNU (0.1mM/0.3mM/0.5mM/1mM/3Mm/ 5mM) recovered after treatment with the lower MNU concentrations whereas high MNU concentrations resulted in apoptosis activation. Quiescent cells were not affected by etoposide treatment showing no damage upon entry into the cell cycle. Treatment with MNU, similarly to the cycling cells, resulted in a dose-dependent cell death. In conclusion, we found that depending on the genotoxic trigger and the cycling status, CD34+cells have distinct responses to DNA damage. Cycling cells employ both DDR and apoptosis mechanisms to prevent damage accumulation. Quiescent cells predominantly undergo apoptosis upon damage, but their cell cycle status protects them from certain genotoxic insults., (© The Author(s) 2023. Published by Oxford University Press.)

As part of our efforts geared towards developing mechanism-based cancer sensitizing agents, we have previously synthesized and characterized novel deazaflavin analogs as potent tyrosyl DNA phosphodiesterase 2 (TDP2) inhibitors for combination treatments with topoisomerase II (TOP2) poisons. Interestingly, the sensitizing effect of a few analogs toward TOP2 poison etoposide (ETP) was associated with a significant increase in intracellular drug accumulation, which could be an alternative mechanism to boost the clinical efficacy of ETP in cancer chemotherapies. Hence, we evaluated more deazaflavin TDP2 inhibitors for their impact on drug retention in cancer cells. We found that all but one tested TDP2 inhibitors substantially increased the ETP retention in DT40 cells. Particularly, we identified an exceptionally potent analog, ZW-1226, which at 3 nM increased the intracellular ETP by 13-fold. Significantly, ZW-1226 also stimulated cellular accumulation of two other anticancer drugs, TOP2 poison teniposide and antifolate pemetrexed, and produced an effect more pronounced than those of ABC transporter inhibitors verapamil and elacridar in human leukemic CCRF-CEM cells toward ETP. Lastly, ZW-1226 potentiated the action of ETP in the sensitive human CCRF-CEM cells and a few resistant non-small-cell lung cancer (NSCLC) cells, including H460 and H838 cells. Collectively, the results of this study strongly suggest that deazaflavin analog ZW-1226 could be an effective cancer sensitizing agent which warrants further investigation., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interest., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

In childhood, retinoblastoma (RB) is the most common primary tumor in the eye. Long term therapeutic management with etoposide of this life-threatening condition may have diminishing effectiveness since RB cells can develop cytostatic resistance to this drug. To determine whether changes in receptor-mediated control of Ca 2+ signaling are associated with resistance development, fluorescence calcium imaging, semi-quantitative RT-qPCR analyses, and trypan blue dye exclusion staining patterns are compared in WERI-ETOR (etoposide-insensitive) and WERI-Rb1 (etoposide-sensitive) cells. The cannabinoid receptor agonist 1 (CNR1) WIN55,212-2 (40 µM), or the transient receptor potential melastatin 8 (TRPM8) agonist icilin (40 µM) elicit similar large Ca 2+ transients in both cell line types. On the other hand, NGF (100 ng/mL) induces larger rises in WERI-ETOR cells than in WERI-Rb1 cells, and its lethality is larger in WERI-Rb1 cells than in WERI-ETOR cells. NGF and WIN55,212-2 induced additive Ca 2+ transients in both cell types. However, following pretreatment with both NGF and WIN55,212-2, TRPM8 gene expression declines and icilin-induced Ca 2+ transients are completely blocked only in WERI-ETOR cells. Furthermore, CNR1 gene expression levels are larger in WERI-ETOR cells than those in WERI-Rb1 cells. Therefore, the development of etoposide insensitivity may be associated with rises in CNR1 gene expression, which in turn suppress TRPM8 gene expression through crosstalk.

Macrophages constitute a major part of tumor microenvironment, and most of existing data demonstrate their ruling role in the development of anti-drug resistance of cancer cell. One of the most powerful protection system is based on heat shock proteins whose synthesis is triggered by activated Heat Shock Factor-1 (HSF1); the inhibition of the HSF1 with CL-43 sensitized A549 lung cancer cells to the anti-cancer effect of etoposide. Notably, analyzing A549 tumor xenografts in mice we observed nest-like pattern of co-localization of A549 cells demonstrating enhanced expression of HSF1 with macrophages, and decided to check whether the above arrangement has a functional value for both cell types. It was found that the incubation of A549 or DLD1 colon cancer cells with either human monocytes or THP1 monocyte-like cells activated HSF1 and increased resistance to etoposide. Importantly, the same effect was shown when primary cultures of colon tumors were incubated with THP1 cells or with human monocytes. To prove that HSF1 is implicated in enhanced resistance caused by monocytic cells, we generated an A549 cell subline devoid of HSF1 which did not respond to incubation with THP1 cells. The pharmacological inhibition of HSF1 with CL-43 also abolished the effect of THP1 cells on primary tumor cells, highlighting a new target of tumor-associated macrophages in a cell proteostasis mechanism., (© 2024. The Author(s).)

The nucleolus serves a multifaceted role encompassing not only rRNA transcription and ribosome synthesis, but also the intricate orchestration of cell cycle regulation and the modulation of cellular senescence. G-patch domain containing 4 (GPATCH4) stands as one among the nucleolar proteins; however, its functional significances remain still unclear. In order to elucidate the functions of GPATCH4, we examined the effects of its dysfunction on cellular proliferation, alterations in nucleolar architecture, apoptotic events, and cellular senescence. Through experimentation conducted on cultured neuroblastoma SH-SY5Y cells, the reduction of GPATCH4 caused inhibition of cellular proliferation, concurrently fostering escalated apoptotic susceptibilities upon exposure to high-dose etoposide. In the realm of nucleolar morphology comparisons, a discernible decline was noted in the count of nucleoli per nucleus, concomitant with a significant expansion in the area occupied by individual nucleoli. Upon induction of senescence prompted by low-dose etoposide, GPATCH4 knockdown resulted in decreased cell viability and increased expression of senescence-associated markers, namely senescence-associated β-galactosidase (SA-β-GAL) and p16. Furthermore, GPATCH4 dysfunction elicited alterations in the gene expression profile of the ribosomal system. In sum, our findings showed that GPATCH4 is a pivotal nucleolar protein that regulates nucleolar morphology and is correlated with cell viability., 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 © 2023 Elsevier Inc. All rights reserved.)

Impaired spermatogenesis and male infertility are common consequences of chemotherapy drugs used in patients with testicular cancer. The present study investigated the effects of sodium alginate (NaAL) on testicular toxicity caused by bleomycin, etoposide, and cisplatin (BEP). Rats in group 1 received normal saline, while groups 2 and 3 were treated with 25 and 50 mg/kg of NaAL, respectively. Group 4 was treated with a 21-day cycle of BEP (0.5 mg/kg bleomycin, 5 mg/kg etoposide, and 1 mg/kg cisplatin), and groups 5 and 6 received BEP regimen plus 25 and 50 mg/kg of NaAL, respectively. Then, sperm parameters, testosterone levels, testicular histopathology and stereological parameters, testicular levels of malondialdehyde (MDA), nitric oxide (NO), and total antioxidant capacity (TAC), and the expression of apoptosis-associated genes including Bcl2, Bax, Caspase3, p53, and TNF-α were evaluated. Our findings revealed that NaAL improved sperm parameters, testosterone levels, histopathology, and stereology parameters in BEP-administrated rats. NaAL also improved testis antioxidant status by enhancing TAC and ameliorating MDA and NO. Further, modifications to the expression of Bcl2, Bax, Caspase3, p53, and TNF-α suggested that NaAL alleviated BEP-induced apoptosis and inflammation. Collectively, NaAL protects rats' testes against BEP-evoked toxicity damage through the modulation of nitro-oxidative stress, apoptosis, and inflammation., (© 2024. The Author(s).)

Nanofibrous scaffolds have emerged as promising candidates for localized drug delivery systems in the treatment of cutaneous cancers. In this study, we prepared an electrospun nanofibrous scaffold incorporating 5-fluorouracil (5-FU) and etoposide (ETP) for chemotherapy targeting melanoma cutaneous cancer. The scaffold was composed of polyvinyl alcohol (PVA) and chitosan (CS), prepared via the electrospinning process and loaded with the chemotherapeutic agents. We conducted relevant physicochemical characterizations, assessed cytotoxicity, and evaluated apoptosis against melanoma A375 cells. The prepared 5-FU/ETP co-loaded PVA/CS scaffold exhibited nanofibers (NFs) with an average diameter of 321 ± 61 nm, defect-free and homogenous morphology. FTIR spectroscopy confirmed successful incorporation of chemotherapeutics into the scaffold. Additionally, the scaffold demonstrated a hydrophilic surface, proper mechanical strength, high porosity, and efficient liquid absorption capacity. Notably, sustained and controlled drug release was observed from the nanofibrous scaffold. Furthermore, the scaffold significantly increased cytotoxicity (95%) and apoptosis (74%) in A375 melanoma cells. Consequently, the prepared 5-FU/ETP co-loaded PVA/CS nanofibrous scaffold holds promise as a valuable system for localized eradication of cutaneous melanoma tumors and mitigation of adverse drug reactions associated with chemotherapy., Competing Interests: Declaration of conflicting interestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Cellular senescence is a dynamic biological process triggered by sublethal cell damage and driven by specific changes in gene expression programs. We recently identified ANKRD1 (ankyrin repeat domain 1) as a protein strongly elevated after triggering senescence in fibroblasts. Here, we set out to investigate the mechanisms driving the elevated production of ANKRD1 in the early stages of senescence. Our results indicated that the rise in ANKRD1 levels after triggering senescence using etoposide (Eto) was the result of moderate increases in transcription and translation, and robust mRNA stabilization. Antisense oligomer (ASO) pulldown followed by mass spectrometry revealed a specific interaction of the RNA-binding protein RBMS1 with ANKRD1 mRNA that was confirmed by ribonucleoprotein immunoprecipitation analysis. RBMS1 abundance decreased in the nucleus and increased in the cytoplasm during Eto-induced senescence; in agreement with the hypothesis that RBMS1 may participate in post-transcriptional stabilization of ANKRD1 mRNA, silencing RBMS1 reduced, while overexpressing RBMS1 enhanced ANKRD1 mRNA half-life after Eto treatment. A segment proximal to the ANKRD1 coding region was identified as binding RBMS1 and conferring RBMS1-dependent increased expression of a heterologous reporter. We propose that RBMS1 increases expression of ANKRD1 during the early stages of senescence by stabilizing ANKRD1 mRNA.

Introduction: Antibiotic-resistant bacterial infections, such as Pseudomonas aeruginosa and Staphylococcus aureus, are prevalent in lung cancer patients, resulting in poor clinical outcomes and high mortality. Etoposide (ETO) is an FDA-approved chemotherapy drug that kills cancer cells by damaging DNA through oxidative stress. However, it is unclear if ETO can cause unintentional side effects on tumor-associated microbial pathogens, such as inducing antibiotic resistance., Objectives: We aimed to show that prolonged ETO treatment could unintendedly confer fluoroquinolone antibiotic resistance to P. aeruginosa, and evaluate the effect of tumor-associated P. aeruginosa on tumor progression., Methods: We employed experimental evolution assay to treat P. aeruginosa with prolonged ETO exposure, evaluated the ciprofloxacin resistance, and elucidated the gene mutations by DNA sequencing. We also established a lung tumor-P. aeruginosa bacterial model to study the role of ETO-evolved intra-tumoral bacteria in tumor progression using immunostaining and confocal microscopy., Results: ETO could generate oxidative stress and lead to gene mutations in P. aeruginosa, especially the gyrase (gyrA) gene, resulting in acquired fluoroquinolone resistance. We further demonstrated using a microfluidic-based lung tumor-P. aeruginosa coculture model that bacteria can evolve ciprofloxacin (CIP) resistance in a tumor microenvironment. Moreover, ETO-induced CIP-resistant (EICR) mutants could form multicellular biofilms which protected tumor cells from ETO killing and enabled tumor progression., Conclusion: Overall, our preclinical proof-of-concept provides insights into how anti-cancer chemotherapy could inadvertently allow tumor-associated bacteria to acquire antibiotic resistance mutations and shed new light on the development of novel anti-cancer treatments based on anti-bacterial strategies., 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 © 2023. Production and hosting by Elsevier B.V.)