DNA repair pathways and cisplatin resistance: an intimate relationship

The main goal of chemotherapeutic drugs is to induce massive cell death in tumors. Cisplatin is an antitumor drug widely used to treat several types of cancer. Despite its remarkable efficiency, most tumors show intrinsic or acquired drug resistance. The primary biological target of cisplatin is genomic DNA, and it causes a plethora of DNA lesions that block transcription and replication. These cisplatin-induced DNA lesions strongly induce cell death if they are not properly repaired or processed. To counteract cisplatin-induced DNA damage, cells use an intricate network of mechanisms, including DNA damage repair and translesion synthesis. In this review, we describe how cisplatin-induced DNA lesions are repaired or tolerated by cells and focus on the pivotal role of DNA repair and tolerance mechanisms in tumor resistance to cisplatin. In fact, several recent clinical findings have correlated the tumor cell status of DNA repair/translesion synthesis with patient response to cisplatin treatment. Furthermore, these mechanisms provide interesting targets for pharmacological modulation that can increase the efficiency of cisplatin chemotherapy

Mechanisms of resistance to chemotherapy in tumors cells.

O câncer é uma das principais causas de morte em todo o mundo e o fator limitante na terapia antitumoral é a resistência a processos terapêuticos. Diversos são os mecanismos de resistência à drogas antitumorais e é fundamental desvendar os mecanismos envolvidos na quimiorresistência para eliminá-lo e ter-se uma melhor eficácia terapêutica. Nesse trabalho investigamos os mecanismos que determinam resistência a cisplatina em células de glioma. Primeiramente, mostramos que a resistência celular a cisplatina era independente de p53, capacidade de reparo de DNA. Utilizando modelo in vitro e in vivo foi demonstrado que os níveis de glutationa (GSH) tinha papel fundamental na resistência a cisplatina e temozolamida (TMZ). A linhagem de glioma resistente a TMZ tinha maior proteção antioxidante, maior expressão do fator de transcrição NRF2, GCLM e GST (envolvidos na geração e utilização de GSH). Utilizando modelo in vitro e in vivo mostramos que BSO (inibidor da síntese de GSH) potencializava o efeito tóxico do TMZ. Assim, a combinação de BSO com cisplatina e TMZ é uma abordagem poderosa para otimizar a citotoxicidade em tumores, sendo uma alternativa excitante para o tratamento de pacientes com glioma ou melanoma.Cancer is one of main cause of death worldwide and the limiting factor is antitumoral therapy resistance. Several mechanisms command drug resistance and fundamental question is how is to unveil the mechanisms involved in it to get a better therapeutic efficacy. We investigated the mechanisms of cisplatin resistance in glioma cells. We demonstrated that cisplatin resistance was p53 independent and DNA repair capacity. It was demonstrated in a in vitro a in vivo model that gluthatione (GSH) is the major resistant factor for cisplatin and temozolomide (TMZ). We observed that the TMZ resistant glioma cell line counted with a high expression of the antioxidant transcription factor NFR2, GCLM and GST. We observed that NFR2 silencing greatly enhanced cell death, high levels of DNA damage upon TMZ treatment. In addition, BSO potentiated TMZ killing in human and murine melanoma using in vitro and in vivo models. Thus, combination of BSO, cisplatin and TMZ is a powerful strategy to optimize tumor killing, thus providing an exciting alternative therapeutic protocol to glioma and melanoma patients

Revealing Temozolomide Resistance Mechanisms via Genome-Wide CRISPR Libraries

Glioblastoma is a severe type of brain tumor with a poor prognosis and few therapy options. Temozolomide (TMZ) is one of these options, however, with limited success, and failure is mainly due to tumor resistance. In this work, genome-wide CRISPR-Cas9 lentiviral screen libraries for gene knockout or activation were transduced in the human glioblastoma cell line, aiming to identify genes that modulate TMZ resistance. The sgRNAs enriched in both libraries in surviving cells after TMZ treatment were identified by next-generation sequencing (NGS). Pathway analyses of gene candidates on knockout screening revealed several enriched pathways, including the mismatch repair and the Sonic Hedgehog pathways. Silencing three genes ranked on the top 10 list (MSH2, PTCH2, and CLCA2) confirm cell protection from TMZ-induced death. In addition, a CRISPR activation library revealed that NRF2 and Wnt pathways are involved in TMZ resistance. Consistently, overexpression of FZD6, CTNNB1, or NRF2 genes significantly increased cell survival upon TMZ treatment. Moreover, NRF2 and related genes detected in this screen presented a robust negative correlation with glioblastoma patient survival rates. Finally, several gene candidates from knockout or activation screening are targetable by inhibitors or small molecules, and some of them have already been used in the clinic

The Role of Chaperone-Mediated Autophagy in Cell Cycle Control and Its Implications in Cancer

The cell cycle involves a network of proteins that modulate the sequence and timing of proliferation events. Unregulated proliferation is the most fundamental hallmark of cancer; thus, changes in cell cycle control are at the heart of malignant transformation processes. Several cellular processes can interfere with the cell cycle, including autophagy, the catabolic pathway involved in degradation of intracellular constituents in lysosomes. According to the mechanism used to deliver cargo to the lysosome, autophagy can be classified as macroautophagy (MA), microautophagy (MI), or chaperone-mediated autophagy (CMA). Distinct from other autophagy types, CMA substrates are selectively recognized by a cytosolic chaperone, one-by-one, and then addressed for degradation in lysosomes. The function of MA in cell cycle control, and its influence in cancer progression, are already well-established. However, regulation of the cell cycle by CMA, in the context of tumorigenesis, has not been fully addressed. This review aims to present and debate the molecular mechanisms by which CMA can interfere in the cell cycle, in the context of cancer. Thus, cell cycle modulators, such as MYC, hypoxia-inducible factor-1 subunit alpha (HIF-1α), and checkpoint kinase 1 (CHK1), regulated by CMA activity will be discussed. Finally, the review will focus on how CMA dysfunction may impact the cell cycle, and as consequence promote tumorigenesis

ATR/Chk1 pathway is activated by oxidative stress in response to UVA light in human Xeroderma Pigmentosum Variant cells.

The crucial role of DNA polymerase eta in protecting against sunlight-induced tumors is evidenced in Xeroderma Pigmentosum Variant (XP-V) patients, who carry mutations in this protein and present increased frequency of skin cancer. XPV cellular phenotypes may be aggravated if proteins of DNA damage response (DDR) pathway are blocked, as widely demonstrated by experiments with UVC light and caffeine. However, little is known about the participation of DDR in XP-V cells exposed to UVA light, the wavelengths patients are mostly exposed. Here, we demonstrate the participation of ATR kinase in protecting XP-V cells after receiving low UVA doses using a specific inhibitor, with a remarkable increase in sensitivity and cH2AX signaling. Corroborating ATR participation in UVA-DDR, a significant increase in Chk1 protein phosphorylation, as well as S-phase cell cycle arrest, is also observed. Moreover, the participation of oxidative stress is supported by the antioxidant action of N-acetylcysteine (NAC), which significantly protects XP-V cells from UVA light, even in the presence of the ATR inhibitor. These findings indicate that the ATR/Chk1 pathway is activated to control UVA-induced oxidatively generated DNA damage and emphasizes the role of ATR kinase as a mediator of genomic stability in pol eta defective cells

Ferroptosis Modulation: Potential Therapeutic Target for Glioblastoma Treatment

Glioblastoma multiforme is a lethal disease and represents the most common and severe type of glioma. Drug resistance and the evasion of cell death are the main characteristics of its malignancy, leading to a high percentage of disease recurrence and the patients’ low survival rate. Exploiting the modulation of cell death mechanisms could be an important strategy to prevent tumor development and reverse the high mortality and morbidity rates in glioblastoma patients. Ferroptosis is a recently described type of cell death, which is characterized by iron accumulation, high levels of polyunsaturated fatty acid (PUFA)-containing phospholipids, and deficiency in lipid peroxidation repair. Several studies have demonstrated that ferroptosis has a potential role in cancer treatment and could be a promising approach for glioblastoma patients. Thus, here, we present an overview of the mechanisms of the iron-dependent cell death and summarize the current findings of ferroptosis modulation on glioblastoma including its non-canonical pathway. Moreover, we focused on new ferroptosis-inducing compounds for glioma treatment, and we highlight the key ferroptosis-related genes to glioma prognosis, which could be further explored. Thereby, understanding how to trigger ferroptosis in glioblastoma may provide promising pharmacological targets and indicate new therapeutic approaches to increase the survival of glioblastoma patients

Glutathione depletion sensitizes cisplatin- and temozolomide-resistant glioma cells in vitro and in vivo.

Malignant glioma is a severe type of brain tumor with a poor prognosis and few options for therapy. The main chemotherapy protocol for this type of tumor is based on temozolomide (TMZ), albeit with limited success. Cisplatin is widely used to treat several types of tumor and, in association with TMZ, is also used to treat recurrent glioma. However, several mechanisms of cellular resistance to cisplatin restrict therapy efficiency. In that sense, enhanced DNA repair, high glutathione levels and functional p53 have a critical role on cisplatin resistance. In this work, we explored several mechanisms of cisplatin resistance in human glioma. We showed that cellular survival was independent of the p53 status of those cells. In addition, in a host-cell reactivation assay using cisplatin-treated plasmid, we did not detect any difference in DNA repair capacity. We demonstrated that cisplatin-treated U138MG cells suffered fewer DNA double-strand breaks and DNA platination. Interestingly, the resistant cells carried higher levels of intracellular glutathione. Thus, preincubation with the glutathione inhibitor buthionine sulfoximine (BSO) induced massive cell death, whereas N-acetyl cysteine, a precursor of glutathione synthesis, improved the resistance to cisplatin treatment. In addition, BSO sensitized glioma cells to TMZ alone or in combination with cisplatin. Furthermore, using an in vivo model the combination of BSO, cisplatin and TMZ activated the caspase 3–7 apoptotic pathway. Remarkably, the combined treatment did not lead to severe side effects, while causing a huge impact on tumor progression. In fact, we noted a remarkable threefold increase in survival rate compared with other treatment regimens. Thus, the intracellular glutathione concentration is a potential molecular marker for cisplatin resistance in glioma, and the use of glutathione inhibitors, such as BSO, in association with cisplatin and TMZ seems a promising approach for the therapy of such devastating tumors

Chloroquine - induced glioma cells death is associated with mitochondrial membrane potential loss, but not oxidative stress.

Chloroquine (CQ), a quinolone derivative widely used to treat and prevent malaria, has been shown to exert a potent adjuvant effect when combined with conventional glioblastoma therapy. Despite inducing lysosome destabilization and activating p53 in human glioma cells, the mechanisms under lying cell death induced by this drug are poorly under stood. Here, we analyzed inatime – anddose – dependent manner, the effects of CQ up on mitochondria integrity, autophagy regulation and redox processes in four human glioma cell lines that differin their resistance to this drug. NAC – containing media protected cells against CQ-induced loss of mitochondrial membrane potential (MMP), autophagyic vacuoles (LC3II) accumulation and loss of cell viability induced by CQ. However, we noticed that part of this protection was due to media acidification in NAC preparations, alerting for problems in experimental procedures using NAC. The results indicate that although CQ induces accumulation of LC3II, mitochondria, and oxidative stress, neither of these events is clearly correlated to cell death induced by this drug. The only event elicited in all cell lines at equitoxic doses of CQ was the loss of MMP, indicating that mitochondrial stability is important for cells resistance to this drug. Finally, the data indicate that higher steady-state MMP values can predict cell resistance to CQ treatment

XPD/ERCC2 mutations interfere in cellular responses to oxidative stress.

Nucleotide excision repair (NER) is a conserved, flexible mechanism responsible for the removal of bulky, helix-distorting DNA lesions, like ultraviolet damage or cisplatin adducts, but its role in the repair of lesions generated by oxidative stress is still not clear. The helicase XPD/ERCC2, one of the two helicases of the transcription complex IIH, together with XPB, participates both in NER and in RNA pol II-driven transcription. In this work, we investigated the responses of distinct XPDmutated cell lines to the oxidative stress generated by photoactivated methylene blue (MB) and KBrO3 treatments. The studied cells are derived from patients with XPD mutations but expressing different clinical phenotypes, including xeroderma pigmentosum (XP), XP and Cockayne syndrome (XP-D/CS) and trichothiodystrophy (TTD). We show by different approaches that all XPD-mutated cell lines tested were sensitive to oxidative stress, with those from TTD patients being the most sensitive. Host cell reactivation (HCR) assays showed that XP-D/CS and TTD cells have severely impaired repair capacity of oxidised lesions in plasmid DNA, and alkaline comet assays demonstrated the induction of significantly higher amounts of DNA strand breaks after treatment with photoactivated MB in these cells compared to wild-type cells. All XPD-mutated cells presented strong S/G2 arrest and persistent ?-H2AX staining after photoactivated MB treatment. Taken together, these results indicate that XPD participates in the repair of lesions induced by the redox process, and that XPD mutations lead to differences in the response to oxidatively induced damage