Cisplatin-induced Cytotoxicity Research Articles

Notoginsenoside R1 Attenuates Cisplatin-Induced Ototoxicity by Inducing Heme Oxygenase-1 Expression and Suppressing Oxidative Stress

Cisplatin-induced ototoxicity occurs in approximately half of patients treated with cisplatin, and pediatric patients are more likely to be affected than adults. The oxidative stress elicited by cisplatin is a key contributor to the pathogenesis of ototoxicity. Notoginsenoside R1 (NGR1), the main bioactive compound of Panax notoginseng saponins, has antioxidant and antiapoptotic effects. This study investigated the ability of NGR1 to protect against cisplatin-induced damage in auditory HEI-OC1 cells and neonatal murine cochlear explants. The viability of HEI-OC1 cells treated with NGR1 and cisplatin was greater than that of cells treated with cisplatin alone. The results of Western blots and immunostaining for cleaved caspase-3 revealed that the level of cleaved caspase-3 in the cells treated with cisplatin was repressed by NGR1. NGR1 attenuated cisplatin-induced cytotoxicity in HEI-OC1 cells. Intracellular reactive oxygen species (ROS) were detected with a DCFDA assay and immunostaining for 4-HNE. The result revealed that its expression was induced by cisplatin and was significantly reduced by NGR1. Moreover, NGR1 can promote heme oxygenase-1 (HO-1) expression at both the mRNA and protein levels. ZNPPIX, an HO-1 inhibitor, was administered to cisplatin-treated cells to investigate the role of HO-1 in the protective effect of NGR1. The suppression of HO-1 activity by ZNPPIX markedly abolished the protective effect of NGR1 on cisplatin-treated cells. Therefore, NGR1 protects cells from cisplatin-induced damage by activating HO-1 and its antioxidative activity. In cochlear explants, NGR1 protects cochlear hair cells and attenuates cisplatin-induced ototoxicity by inhibiting ROS generation. In the group treated with cisplatin alone, prominent loss of outer hair cells and severe damage to the structure of the stereociliary bundles of inner and outer hair cells were observed. Compared with the group treated with cisplatin alone, less loss of outer hair cells (p = 0.009) and better preservation of the stereociliary bundles of hair cells were observed in the group treated with cisplatin and NGR1. In conclusion, these findings indicate that NGR1 can protect against cisplatin-induced ototoxicity by inducing HO-1 expression and suppressing oxidative stress.

Novel DNA Repair Inhibitors Targeting XPG to Enhance Cisplatin Therapy in Non-Small Cell Lung Cancer: Insights from In Silico and Cell-Based Studies.

NSCLC is marked by low survival and resistance to platinum-based chemotherapy. The XPG endonuclease has emerged as a promising biomarker for predicting the prognosis of cisplatin-treated patients and its downregulation having been reported to increase cisplatin efficacy. This study presents an integrated strategy for identifying small molecule inhibitors of XPG to improve cisplatin therapy in NSCLC. A structure-based virtual screening approach was adopted, including a structural and physicochemical analysis of the protein, and a library of small molecules with reported inhibitory activities was retrieved. This analysis identified Lys84 as a crucial residue for XPG activity by targeting its interaction with DNA. After molecular docking and virtual screening calculations, 61 small molecules were selected as potential XPG inhibitors, acquired from the ChemBridge database and then validated in H1299 cells, a NSCLC cell line exhibiting the highest ERCC5 expression. The MTS assay was performed as a first screening approach to determine whether these potential inhibitors could enhance cisplatin-induced cytotoxicity. Overall, among the eight compounds identified as the most promising, three of them revealed to significantly increase the impact of cisplatin. The inherent cytotoxicity of these compounds was further investigated in a non-tumoral lung cell line (BEAS-2B cells), which resulted in the identification of two non-cytotoxic candidates to be used in combination with cisplatin in order to improve its efficacy in NSCLC therapy.

Sesamol-mediated targeting of EPHA2 sensitises cervical cancer for cisplatin treatment by regulating mitochondrial dynamics, autophagy, and mitophagy.

Cervical cancer ranks as the fourth most prevalent cancer among women globally, presenting a significant therapeutic challenge due to its resistance to cisplatin. Ephrin type-A receptor 2 (EPHA2) is prominently overexpressed in cervical cancer and plays a vital role in cisplatin resistance, although the underlying mechanisms remain incompletely elucidated. Mitochondrial dynamics, autophagy, and mitophagy are critical in mediating cisplatin resistance. Sesamol, a phytochemical compound, has exhibited promising anticancer properties. This study aims to investigate the regulatory role of EPHA2 in these pathways underlying cisplatin resistance and to investigate the potential of sesamol in overcoming this resistance and inhibiting cervical cancer progression. In this study, we knocked down EPHA2 in the SiHa cell line and evaluated the resulting changes in molecular markers associated with mitochondrial dynamics, mitophagy, and autophagy. Our results indicated that EPHA2 knockdown (EPHA2-KD) led to enhanced mitochondrial fusion and reduced mitochondrial fission, mitophagy, and autophagy. Furthermore, we investigated the effect of EPHA2-KD and sesamol treatment on sensitising cervical cancer to cisplatin treatment. Our data revealed that EPHA2-KD and sesamol treatment significantly increases cellular sensitivity to cisplatin-induced cytotoxicity. Additionally, we demonstrated that sesamol effectively targets EPHA2, as evidenced by decreased EPHA2 expression levels following sesamol treatment. In summary, targeting EPHA2 through knockdown or sesamol treatment enhances cisplatin sensitivity in cervical cancer by modulating mitochondrial dynamics, autophagy and mitophagy, suggesting promising therapeutic strategies to overcome chemoresistance.

Effect of Ganoderma lucidum on Cisplatin-induced Nephrotoxicity and Gastrointestinal Toxicity.

Cancer remains a major global health challenge, with an estimated 10 million cancer-related deaths in 2020, hindering efforts to extend life expectancy. Cisplatin, an effective platinum-based chemotherapeutic agent used against various malignancies, has numerous side effects. Ganoderma lucidum is a traditional Chinese medicine with extensive historical use and proven biological activity. This study investigated the effects of G. lucidum on cisplatin-induced nephrotoxicity and gastrointestinal toxicity. RAW264.7 cells were treated with cisplatin, G. lucidum, or both. Cytotoxicity and antioxidant capacity were measured. Slc:ICR (ICR) mice were treated with cisplatin, G. lucidum, or both. The survival rate and physiological data were measured. G. lucidum suppressed cisplatin-induced cytotoxicity and apoptosis in RAW264.7 cells. G. lucidum suppressed cisplatin-induced nephrotoxicity and gastrointestinal toxicity via its antioxidant effects in ICR mice. The suppressive mechanism of G. lucidum may be mediated via its antioxidant effects. These findings indicate its potential to reduce the side effects of cisplatin.

Activating transcription factor 6 contributes to cisplatin‑induced ototoxicity via regulating the unfolded proteins response

As a broad-spectrum anticancer drug, cisplatin is widely used in the treatment of tumors in various systems. Unfortunately, several serious side effects of cisplatin limit its clinical application, the most common of which are nephrotoxicity and ototoxicity. Studies have shown that cochlear hair cell degeneration is the main cause of cisplatin-induced hearing loss. However, the mechanism of cisplatin-induced hair cell death remains unclear. The present study aimed to explore the potential role of activating transcription factor 6 (ATF6), an endoplasmic reticulum (ER)-localized protein, on cisplatin-induced ototoxicity in vivo and in vitro. In this study, we observed that cisplatin exposure induced apoptosis of mouse auditory OC-1 cells, accompanied by a significant increase in the expression of ATF6 and C/EBP homologous protein (CHOP). In cell or cochlear culture models, treatment with an ATF6 agonist, an ER homeostasis regulator, significantly ameliorated cisplatin-induced cytotoxicity. Further, our in vivo experiments showed that subcutaneous injection of an ATF6 agonist almost completely prevented outer hair cell loss and significantly alleviated cisplatin-induced auditory brainstem response (ABR) threshold elevation in mice. Collectively, our results revealed the underlying mechanism by which activation of ATF6 significantly improved cisplatin-induced hair cell apoptosis, at least in part by inhibiting apoptosis signal-regulating kinase 1 expression, and demonstrated that pharmacological activation of ATF6-mediated unfolded protein response is a potential treatment for cisplatin-induced ototoxicity.

SERCA2 protects against cisplatin-induced damage of auditory cells: Possible relation with alleviation of ER stress

Aims: SERCA2, one of the P-type pumps encoded by gene ATP2A2, is the only calcium reflux channel of the endoplasmic reticulum (ER) and participates in maintaining calcium homeostasis. The present study was designed to explore SERCA2 expression pattern in auditory hair cells and the possible mechanism underlying the effects of SERCA2 on cisplatin-induced ototoxicity.Main methods: The SERCA2 expression pattern in cochlea hair cells and HEI-OC1 cells was measured by Western blot (WB) and immunofluorescence staining. The apoptosis and its related factors were detected by TUNEL assay and WB. The expression levels of ER stress-related factors, ATF6, PERK, IRE1α, and GRP78, were measured via WB. As for the determination of SERCA2 overexpression and knockdown, plasmids and lentiviral vectors were constructed, respectively.Key findings: We found that SERCA2 was highly expressed in cochlea hair cells and HEI-OC1 cells. Of note, the level of SERCA2 expression in neonatal mice was remarkably higher than that in adult mice. Under the exposure of 30 μM cisplatin, SERCA2 was down-regulated significantly compared with the control group. In addition, cisplatin administration triggered the occurrence of ER stress and apoptosis. Those events were reversed by overexpressing SERCA2. On the contrary, SERCA2 knockdown could aggravate the above processes.Significance: The findings from the present study disclose, for the first time, that SERCA2 is abundantly expressed in cochlea hair cells, and the suppression of SERCA2 caused by cisplatin could trigger ER homeostasis disruption, thereby implying that SERCA2 might be a promising target to prevent cisplatin-induced cytotoxicity of hair cells.

Evodiamine potentiates cisplatin-induced cell death and overcomes cisplatin resistance in non-small-cell lung cancer by targeting SOX9-β-catenin axis.

In recent decades, phytotherapy has remained as a key therapeutic option for the treatment of various cancers. Evodiamine, an excellent phytocompound from Evodia fructus, exerts anticancer activity in several cancers by modulating drug resistance. However, the role of evodiamine in cisplatin-resistant NSCLC cells is not clear till now. Therefore, we have used evodiamine as a chemosensitizer to overcome cisplatin resistance in NSCLC. Here, we looked into SOX9 expression and how it affects the cisplatin sensitivity of cisplatin-resistant NSCLC cells. MTT and clonogenic assays were performed to check the cell proliferation. AO/EtBr and DAPI staining, ROS measurement assay, transfection, Western blot analysis, RT-PCR, Scratch & invasion, and comet assay were done to check the role of evodiamine in cisplatin-resistant NSCLC cells. SOX9 levels were observed to be higher in cisplatin-resistant A549 (A549CR) and NCI-H522 (NCI-H522CR) compared to parental A549 and NCI-H522. It was found that SOX9 promotes cisplatin resistance by regulating β-catenin. Depletion of SOX9 restores cisplatin sensitivity by decreasing cell proliferation and cell migration and inducing apoptosis in A549CR and NCI-H522CR. After evodiamine treatment, it was revealed that evodiamine increases cisplatin-induced cytotoxicity in A549CR and NCI-H522CR cells through increasing intracellular ROS generation. The combination of both drugs also significantly inhibited cell migration by inhibiting epithelial to mesenchymal transition (EMT). Mechanistic investigation revealed that evodiamine resensitizes cisplatin-resistant cells toward cisplatin by decreasing the expression of SOX9 and β-catenin. The combination of evodiamine and cisplatin may be a novel strategy for combating cisplatin resistance in NSCLC.

Long noncoding RNA TUG1 promotes cisplatin resistance in ovarian cancer via upregulation of DNA polymerase eta.

Chemoresistance is a major cause of high mortality and poor survival in patients with ovarian cancer (OVCA). Understanding the mechanisms of chemoresistance is urgently required to develop effective therapeutic approaches to OVCA. Here, we show that expression of the long noncoding RNA, taurine upregulated gene 1 (TUG1), is markedly upregulated in samples from OVCA patients who developed resistance to primary platinum-based therapy. Depletion of TUG1 increased sensitivity to cisplatin in the OVCA cell lines, SKOV3 and KURAMOCHI. Combination therapy of cisplatin with antisense oligonucleotides targeting TUG1 coupled with a drug delivery system effectively relieved the tumor burden in xenograft mouse models. Mechanistically, TUG1 acts as a competing endogenous RNA by downregulating miR-4687-3p and miR-6088, both of which target DNA polymerase eta (POLH), an enzyme required for translesion DNA synthesis. Overexpression of POLH reversed the effect of TUG1 depletion on cisplatin-induced cytotoxicity. Our data suggest that TUG1 upregulation allows OVCA to tolerate DNA damage via upregulation of POLH; this provides a strong rationale for targeting TUG1 to overcome cisplatin resistance in OVCA.

Attenuation of Nicotine Effects on A549 Lung Cancer Cells by Synthetic α7 nAChR Antagonists APS7-2 and APS8-2.

Nicotine binds to nicotinic acetylcholine receptors (nAChRs) that are overexpressed in different cancer cells, promoting tumor growth and resistance to chemotherapy. In this study, we aimed to investigate the potential of APS7-2 and APS8-2, synthetic analogs of a marine sponge toxin, to inhibit nicotine-mediated effects on A549 human lung cancer cells. Our electrophysiological measurements confirmed that APS7-2 and APS8-2 act as α7 nAChR antagonists. APS8-2 showed no cytotoxicity in A549 cells, while APS7-2 showed concentration-dependent cytotoxicity in A549 cells. The different cytotoxic responses of APS7-2 and APS8-2 emphasize the importance of the chemical structure in determining their cytotoxicity on cancer cells. Nicotine-mediated effects include increased cell viability and proliferation, elevated intracellular calcium levels, and reduced cisplatin-induced cytotoxicity and reactive oxygen species production (ROS) in A549 cells. These effects of nicotine were effectively attenuated by APS8-2, whereas APS7-2 was less effective. Our results suggest that APS8-2 is a promising new therapeutic agent in the chemotherapy of lung cancer.

Abstract 7599: Evaluate the treatment efficacy of magnolol combined with cisplatin in non-small cell lung cancer

Abstract Lung cancer is one of the five most common cancers in the world. Statistics indicate that approximately 80% to 90% of lung cancer cases fall under the classification of non-small cell lung cancer (NSCLC). For several decades, non-small cell lung cancer (NSCLC) has often been treated with chemotherapy drugs. However, lung cancer has high incidence and recurrence rates, mainly due to drug resistance and side effects during chemotherapy and radiotherapy. This is the hinder problem in the treatment of NSCLC, which highlight the necessity of developing new drugs or new treatments for NSCLC. Cisplatin is a chemotherapy drug that can treat about 50% of solid tumors, but the cisplatin-induced cytoprotective autophagy is the resistance to clinical treatment. It is urgent to elucidate the underlying molecular mechanisms regulating cisplatin resistance to improve its therapeutic value in lung cancer. Magnolol is a phenolic component isolated from the roots and bark of Magnolia officinalis and is used in traditional Chinese medicine. With a wide range of pharmacological activities, various studies have focused on the anti-cancer properties of magnolol in recent years. The effect of the drug is to keep cells in the G2/M phase, induce mitochondrial dysfunction and ROS production, affect the supply of cellular energy, and then lead to cell apoptosis. In this study, we investigated the ability of magnolol enhance cisplatin-induced cytotoxicity, showing dose dependence in NSCLC cell by MTT assay. We utilized the immunofluorescence staining and western blotting assay to confirm the downgrade expression of LC3B and the upregulate expression of p62 that define magnolol is able to suppress cisplatin-induced cytoprotective autophagy in NSCLC. We also detected the extrinsic/intrinsic apoptosis induction ability of magnolol combined with cisplatin in NSCLC. In the result, the cisplatin combined with magnolol have more significantly triggered the loss of mitochondrial membrane potential, and activate more protein cleaved caspase-3, -8 and -9 than monotherapy. We also used western blotting assay to confirm the expression of apoptosis inhibitory protein Bcl-2 and pro-apoptotic protein Bax to measure the degree of apoptosis. The migration and invasion effects of magnolol and cisplatin were identified by transwell invasion and wound healing on NSCLC. The magnolol combined with cisplatin extended the mouse survival rate and can inhibit tumor progression. These results suggest that combinational therapy of cisplatin and magnolol could be a promising treatment strategy for NSCLC. The potential regulation of magnolol on NSCLC is associated with triggering cisplatin-induced apoptosis and diminishing cisplatin-induce cytoprotective autophagy. Citation Format: Rong-Er Lu, Bo-Xun Chen, Fei-Ting Hsu, I-Tsang Chiang. Evaluate the treatment efficacy of magnolol combined with cisplatin in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7599.

FTO attenuates the cytotoxicity of cisplatin in KGN granulosa cell-like tumour cells by regulating the Hippo/YAP1 signalling pathway.

Premature ovarian failure (POF) is a devastating condition for women under 40 years old. Chemotherapy, especially the use of cisplatin, has been demonstrated to promote the apoptosis of granulosa cells in primary and secondary follicles, leading to POF. Our previous studies demonstrated that fat mass- and obesity-associated (FTO) plays an essential role in protecting granulosa cells from cisplatin-induced cytotoxicity. Various studies have suggested that the Hippo/YAP signalling pathway plays a significant role in regulating cell apoptosis and proliferation. Additionally, YAP1 is the main downstream target of the Hippo signalling pathway and is negatively regulated by the Hippo signalling pathway. However, whether the Hippo/YAP signalling pathway is involved in the protective effect of FTO on granulosa cells has not been determined. In this study, we found that after cisplatin treatment, the apoptosis of granulosa cells increased in a concentration-dependent manner, accompanied by the downregulation of FTO and YAP1. Furthermore, overexpression of FTO decreased cisplatin-induced granulosa cell apoptosis, inhibited the Hippo/YAP kinase cascade-induced phosphorylation of YAP1, and promoted the entry of YAP1 into the nucleus. The downstream targets of YAP1 (CTGF, CYR61, and ANKRD1) were also increased. Si-RNA-mediated downregulation of FTO promoted cisplatin-induced granulosa cell apoptosis, activated the Hippo/YAP kinase cascade, and inhibited the YAP1 entry into the nucleus. These effects were completely reversed by the small molecule inhibitor of YAP1-verteporfin (VP). Taken together, these data suggested that FTO-YAP1 plays a positive role in regulating the proliferation of injured granulosa cells induced by cisplatin.

Inhibition of PI3K/AKT signaling using BKM120 reduced the proliferation and migration potentials of colorectal cancer cells and enhanced cisplatin-induced cytotoxicity.

Although extensive efforts have been made to improve the treatment of colorectal cancer (CRC) patients, the prognosis for these patients remains poor. A wide range of anti-cancer agents has been applied to ameliorate the clinical management of CRC patients; however, drug resistance develops in nearly all patients. Based on the prominent role of PI3K/AKT signaling in the development of CRC and current interest in the application of PI3K inhibitors, we aimed to disclose the exact mechanism underlying the efficacy of BKM120, a well-known pan-class I PI3K inhibitor, in CRC-derived SW480 cells. The effects of BKM120 on SW480 cells were studied using MTT assay, cell cycle assay, Annexin V/PI apoptosis tests, and scratch assay. In the next step, qRT-PCR was used to investigate the underlying molecular mechanisms by which the PI3K inhibitor could suppress the survival of SW480 cells. The results of the MTT assay showed that BKM120 could decrease the metabolic activity of SW480 cells in a concentration and time-dependent manner. Investigating the exact mechanism of BKM120 showed that this PI3K inhibitor induces its anti-survival effects through a G2/M cell cycle arrest and apoptosis-mediated cell death. Moreover, the scratch assay demonstrated that PI3K inhibition led to the inhibition of cancer invasion and inhibition of PI3K/AKT signaling remarkably sensitized SW480 cells to Cisplatin. Based on our results, inhibition of PI3K/AKT signaling can be a promising approach, either as a single modality or in combination with Cisplatin. However, further clinical studies should be performed to improve our understanding.

Protection from cisplatin-induced hearing loss with lentiviral vector-mediated ectopic expression of the anti-apoptotic protein BCL-XL

Cisplatin is a highly effective chemotherapeutic agent, but it can cause sensorineural hearing loss (SNHL) in patients. Cisplatin-induced ototoxicity is closely related to the accumulation of reactive oxygen species (ROS) and subsequent death of hair cells (HCs) and spiral ganglion neurons (SGNs). Despite various strategies to combat ototoxicity, only one therapeutic agent has thus far been clinically approved. Therefore, we have developed a gene therapy concept to protect cochlear cells from cisplatin-induced toxicity. Self-inactivating lentiviral (LV) vectors were used to ectopically express various antioxidant enzymes or anti-apoptotic proteins to enhance the cellular ROS scavenging or prevent apoptosis in affected cell types. In direct comparison, anti-apoptotic proteins mediated a stronger reduction in cytotoxicity than antioxidant enzymes. Importantly, overexpression of the most promising candidate, Bcl-xl, achieved an up to 2.5-fold reduction in cisplatin-induced cytotoxicity in HEI-OC1 cells, phoenix auditory neurons, and primary SGN cultures. BCL-XL protected against cisplatin-mediated tissue destruction in cochlear explants. Strikingly, invivo application of the LV BCL-XL vector improved hearing and increased HC survival in cisplatin-treated mice. In conclusion, we have established a preclinical gene therapy approach to protect mice from cisplatin-induced ototoxicity that has the potential to be translated to clinical use in cancer patients.

Determination of Medicago orbicularis Antioxidant, Antihemolytic, and Anti-Cancerous Activities and Its Augmentation of Cisplatin-Induced Cytotoxicity in A549 Lung Cancer Cells

The anti-lung cancer properties of the plant Medicago orbicularis have not been explored yet. Therefore, we identified its phytochemical composition and investigated the antioxidant, anti-hemolytic, and anti-cancerous properties of extracts of this plant in A549 human lung adenocarcinoma cells. The results show that all parts of M. orbicularis (stems, leaves, and fruits) exhibit remarkable hemolytic activities and modest antioxidant capacity. In addition, all extracts showed a dose-dependent anti-cancerous cytotoxic activity against A549 cells, with fruit extracts being the most potent. This cytotoxic effect could be related, at least partly, to the induction of apoptosis, where M. orbicularis fruit extracts reduced the ratio of anti-apoptotic BCL-2/pro-apoptotic BAX, thereby promoting cellular death. Furthermore, the use of M. orbicularis, in combination with a conventional chemotherapeutic agent, cisplatin, was assessed. Indeed, the combination of cisplatin and M. orbicularis fruit extracts was more cytotoxic and induced more aggregation of A549 cells than either treatment alone. GC-MS analysis and total polyphenol and flavonoid content determination indicated that M. orbicularis is rich in compounds that have anti-cancerous effects. We propose M. orbicularis as a potential source of anti-cancerous agents to manage the progression of lung cancer and its resistance to therapy.

Unveiling Novel ERCC1-XPF Complex Inhibitors: Bridging the Gap from In Silico Exploration to Experimental Design.

Modifications in DNA repair pathways are recognized as prognostic markers and potential therapeutic targets in various cancers, including non-small cell lung cancer (NSCLC). Overexpression of ERCC1 correlates with poorer prognosis and response to platinum-based chemotherapy. As a result, there is a pressing need to discover new inhibitors of the ERCC1-XPF complex that can potentiate the efficacy of cisplatin in NSCLC. In this study, we developed a structure-based virtual screening strategy targeting the inhibition of ERCC1 and XPF interaction. Analysis of crystal structures and a library of small molecules known to act against the complex highlighted the pivotal role of Phe293 (ERCC1) in maintaining complex stability. This residue was chosen as the primary binding site for virtual screening. Using an optimized docking protocol, we screened compounds from various databases, ultimately identifying more than one hundred potential inhibitors. Their capability to amplify cisplatin-induced cytotoxicity was assessed in NSCLC H1299 cells, which exhibited the highest ERCC1 expression of all the cell lines tested. Of these, 22 compounds emerged as promising enhancers of cisplatin efficacy. Our results underscore the value of pinpointing crucial molecular characteristics in the pursuit of novel modulators of the ERCC1-XPF interaction, which could be combined with cisplatin to treat NSCLC more effectively.

KRAS is a molecular determinant of platinum responsiveness in glioblastoma

BackgroundKRAS is the undisputed champion of oncogenes, and despite its prominent role in oncogenesis as mutated gene, KRAS mutation appears infrequent in gliomas. Nevertheless, gliomas are considered KRAS-driven cancers due to its essential role in mouse malignant gliomagenesis. Glioblastoma is the most lethal primary brain tumor, often associated with disturbed RAS signaling. For newly diagnosed GBM, the current standard therapy is alkylating agent chemotherapy combined with radiotherapy. Cisplatin is one of the most effective anticancer drugs and is used as a first-line treatment for a wide spectrum of solid tumors (including medulloblastoma and neuroblastoma) and many studies are currently focused on new delivery modalities of effective cisplatin in glioblastoma. Its mechanism of action is mainly based on DNA damage, inducing the formation of DNA adducts, triggering a series of signal-transduction pathways, leading to cell-cycle arrest, DNA repair and apoptosis.MethodsLong-term cultures of human glioblastoma, U87MG and U251MG, were either treated with cis-diamminedichloroplatinum (cisplatin, CDDP) and/or MEK-inhibitor PD98059. Cytotoxic responses were assessed by cell viability (MTT), protein expression (Western Blot), cell cycle (PI staining) and apoptosis (TUNEL) assays. Further, gain-of-function experiments were performed with cells over-expressing mutated hypervariable region (HVR) KRASG12V plasmids.ResultsHere, we studied platinum-based chemosensitivity of long-term cultures of human glioblastoma from the perspective of KRAS expression, by using CDDP and MEK-inhibitor. Endogenous high KRAS expression was assessed at transcriptional (qPCR) and translational levels (WB) in a panel of primary and long-term glioblastoma cultures. Firstly, we measured immediate cellular adjustment through direct regulation of protein concentration of K-Ras4B in response to cisplatin treatment. We found increased endogenous protein abundance and involvement of the effector pathway RAF/MEK/ERK mitogen-activated protein kinase (MAPK) cascade. Moreover, as many MEK inhibitors are currently being clinically evaluated for the treatment of high-grade glioma, so we concomitantly tested the effect of the potent and selective non-ATP-competitive MEK1/2 inhibitor (PD98059) on cisplatin-induced chemosensitivity in these cells. Cell-cycle phase distribution was examined using flow cytometry showing a significant cell-cycle arrest in both cultures at different percentage, which is modulated by MEK inhibition. Cisplatin-induced cytotoxicity increased sub-G1 percentage and modulates G2/M checkpoint regulators cyclins D1 and A. Moreover, ectopic expression of a constitutively active KRASG12V rescued CDDP-induced apoptosis and different HVR point mutations (particularly Ala 185) reverted this phenotype.ConclusionThese findings warrant further studies of clinical applications of MEK1/2 inhibitors and KRAS as ‘actionable target’ of cisplatin-based chemotherapy for glioblastoma.

Sodium thiosulfate does not protect ovarian reserve from cisplatin-induced gonadotoxicity†.

Cisplatin, a platinum-containing alkylating agent, is used in the treatment of various tumors owing to its potent antitumor activity. However, it causes permanent and adverse effects, particularly hearing loss and depletion of ovarian reserve. Until recently, there were no clinically available protective agents to mitigate the adverse side effects of cisplatin-induced cytotoxicity. In 2022, sodium thiosulfate (STS) was approved by the Food and Drug Administration for mitigating hearing loss in children and adolescents undergoing cisplatin treatment. Consequently, our investigation aimed to determine if STS could protect ovarian reserve against cisplatin-induced gonadotoxicity. In an ex vivo culture, the cisplatin-only group exhibited a loss of primordial follicles, while post-STS administration after cisplatin exposure effectively protected primordial follicles. However, when post-STS was administrated either 6 or 4h after cisplatin exposure, it did not confer protection against cisplatin-induced gonadotoxicity in postnatal day 7 or adolescent mouse models. Immunofluorescence assays using γH2AX and cPARP revealed that oocytes within primordial follicles exhibited DNA damage after cisplatin exposure, irrespective of post-STS administration. This underscores the rapid and heightened sensitivity of oocytes to gonadotoxicity. In addition, oocytes demonstrated an increased expression of pCHK2 rather than pERK, suggesting that the pathway leading to oocyte death differs from the pathway observed in the inner ear cell death following cisplatin exposure. These results imply that while the administration of STS after cisplatin is highly beneficial in preventing hearing loss, it does not confer a protective effect on the ovaries in mouse models.

Effects of Nutraceuticals on Cisplatin-Induced Cytotoxicity in HEI-OC1 Cells.

Cisplatin is a chemotherapeutic drug for the treatment of several solid tumors, whose use is limited by its nephrotoxicity, neurotoxicity, ototoxicity, and development of resistance. The toxicity is caused by DNA cross-linking, increase in reactive oxygen species and/or depletion of cell antioxidant defenses. The aim of the work was to study the effect of antioxidant compounds (Lisosan G, Taurisolo®) or hydrogen sulfide (H2S)-releasing compounds (erucin) in the auditory HEI-OC1 cell line treated with cisplatin. Cell viability was determined using the MTT assay. Caspase and sphingomyelinase activities were measured by fluorometric and colorimetric methods, respectively. Expression of transcription factors, apoptosis hallmarks and genes codifying for antioxidant response proteins were measured by Western blot and/or RT-qPCR. Lisosan G, Taurisolo® and erucin did not show protective effects. Sodium hydrosulfide (NaHS), a donor of H2S, increased the viability of cisplatin-treated cells and the transcription of heme oxygenase 1, superoxide dismutase 2, NAD(P)H quinone dehydrogenase type 1 and the catalytic subunit of glutamate-cysteine ligase and decreased reactive oxygen species (ROS), the Bax/Bcl2 ratio, caspase-3, caspase-8 and acid sphingomyelinase activity. Therefore, NaHS might counteract the cytotoxic effect of cisplatin by increasing the antioxidant response and by reducing ROS levels and caspase and acid sphingomyelinase activity.

Novel 3-hydroxypyridin-4(1H)-One derivatives as ferroptosis inhibitors with iron-chelating and reactive oxygen species scavenging activities and therapeutic effect in cisplatin-induced cytotoxicity

Recent advances in understanding the role of iron and ROS in cell death suggest new therapeutic avenues to treat organ damage including acute kidney injury (AKI). Inhibiting ferroptosis was expected to have great potential for the treatment of this disease. Ferroptosis is characterized by iron-dependent lipid peroxidation and currently, a majority of reported ferroptosis inhibitors belong to either radical-trapping antioxidants or iron chelators. However, clinically used iron chelators such as deferoxamine and deferiprone have limited efficacy against ferroptosis (generally with EC50 > 100 μM), despite their proven safety. Herein, we present the rational design of novel ferroptosis inhibitors by incorporating the naturally occurring cinnamic acid scaffold and the 3-hydroxypyridin-4(1H)-one iron-chelating pharmacophore. Through ABTS˙+ radical-scavenging assay, oxygen radical absorbance capacity (ORAC) measurement, Fe3+ affinity evaluation, and anti-erastin-induced HT22 cell ferroptosis assays, we identified compound 9c as the most prospective ferroptosis inhibitor (ABTS˙+, IC50 = 4.35 ± 0.05 μM; ORCA = 23.79 ± 0.56 TE; pFe3+ = 18.59; EC50 = 14.89 ± 0.08 μM, respectively). Notably, 9c dose-dependently alleviated cell death in cisplatin-induced AKI model. Our results provide insight into the development of new ferroptosis inhibitors through rational incorporation of pharmacophores from existing ferroptosis inhibitors, and compound 9c could be a promising lead compound worth further investigation.

The identification of BCL-XL and MCL-1 as key anti-apoptotic proteins in medulloblastoma that mediate distinct roles in chemotherapy resistance

Medulloblastoma is the most common malignant paediatric brain tumour, representing 20% of all paediatric intercranial tumours. Current aggressive treatment protocols and the use of radiation therapy in particular are associated with high levels of toxicity and significant adverse effects, and long-term sequelae can be severe. Therefore, improving chemotherapy efficacy could reduce the current reliance on radiation therapy. Here, we demonstrated that systems-level analysis of basal apoptosis protein expression and their signalling interactions can differentiate between medulloblastoma cell lines that undergo apoptosis in response to chemotherapy, and those that do not. Combining computational predictions with experimental BH3 profiling, we identified a therapeutically-exploitable dependence of medulloblastoma cells on BCL-XL, and experimentally validated that BCL-XL targeting, and not targeting of BCL-2 or MCL-1, can potentiate cisplatin-induced cytotoxicity in medulloblastoma cell lines with low sensitivity to cisplatin treatment. Finally, we identified MCL-1 as an anti-apoptotic mediator whose targeting is required for BCL-XL inhibitor-induced apoptosis. Collectively, our study identifies that BCL-XL and MCL-1 are the key anti-apoptotic proteins in medulloblastoma, which mediate distinct protective roles. While BCL-XL has a first-line role in protecting cells from apoptosis basally, MCL-1 represents a second line of defence that compensates for BCL-XL upon its inhibition. We provide rationale for the further evaluation of BCL-XL and MCL-1 inhibitors in the treatment of medulloblastoma, and together with current efforts to improve the cancer-specificity of BCL-2 family inhibitors, these novel treatment strategies have the potential to improve the future clinical management of medulloblastoma.