Cisplatin-treated Cells 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.

Integrated global proteomic and phosphoproteomic analysis of cisplatin-induced apoptosis in A549 cells

Protein phosphorylation, a widely occurring and significant post-translational modification, is integral to various biological processes. We previously utilized a protein affinity probe to identify genes damaged by cisplatin, revealing that it inflicts substantial damage on protein kinase and protein phosphatase genes. In this study, we investigated cisplatin-induced alterations in the global proteome and phosphoproteome of A549 cells. Employing Fe-IMAC beads and tyrosine phosphorylation enrichment antibodies, we identified 6944 protein groups and 18,274 phosphorylation sites on 4915 proteins across three biological replicates of both cisplatin-treated A549 cells and control cells. Among these, 730 tyrosine phosphorylation sites were identified—marking the most substantial discovery of such sites in A549 cells following cisplatin treatment. Bioinformatics analysis indicated that the proteins exhibiting significant phosphorylation level changes predominantly involved in RNA processing, modification, transcription, translation, and the spliceosome. This suggests that cisplatin-induced damage to protein kinases and phosphatases may disrupt the normal function of these proteins, consequently impairing DNA replication, RNA translation, and shearing, ultimately culminating in tumor cell death. Moreover, we cross-referenced our proteomic data with our previously obtained cisplatin-damaged genes, observing that the majority of down-regulated proteins derived from cisplatin-induced gene damage. The data are available on ProteomeXchange under the identifier PXD053902.

New application of ombuoside in protecting auditory cells from cisplatin-induced ototoxicity via the apoptosis pathway

Hearing loss is caused by many factors including ototoxic drug-induced hair cell damage. Ombuoside, an antioxidant isolated from Gynostemma pentaphyllum, has been suggested to serve as a new neuroprotective drug. However, the role of ombuoside in protecting inner ear hair cells from ototoxic drug-induced damage has not been investigated. Here, we demonstrated the protective potential of ombuoside in mitigating drug-induced ototoxicity in vivo and in vitro. We used cisplatin, a highly ototoxic anti-tumor drug, to induce hair cell damage. Our results showed that ombuoside significantly increased the survival of cisplatin-treated HEI-OC1 cells. Further mechanism research suggested that ombuoside protects HEI-OCI cells from cisplatin-induced apoptosis by reducing the cisplatin-induced upregulation of apoptosis-promoting proteins Bax, Bak, as well as apoptosis indicator proteins cytochrome C and cleaved-caspase-3, and the downregulation of apoptosis-inhibiting proteins Bcl-2. Ombuoside also protects the cells from the excessive ROS production and mitochondrial membrane depolarization triggered by cisplatin. These results demonstrated the potential for ombuoside in protecting hair cells from cisplatin by suppressing ROS generation and the mitochondrial apoptotic cascade. Ombuoside showed promise in protecting hair cells from cisplatin-induced apoptosis by suppressing ROS generation and the mitochondrial apoptotic cascade. Furthermore, ombuoside co-treatment in mouse cochlear explants and zebrafish lateral neuromasts rescued the decreased number and deformed morphology of hair cells resulting from cisplatin exposure. These findings further validated our conclusions and indicated that ombuoside is a potential protector against hearing loss caused by ototoxicity as a clinical side effect of cisplatin.

Chemotherapy-induced increase in CD47 expression in epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) remains the most lethal gynecological malignancy with limited treatment options. CD47 is a critical immune checkpoint for tumor immune evasion and has been targeted in various clinical trials. This study aimed to assess the impact of chemotherapy on CD47 expression in EOC in order to determine the potential and optimal timing of CD47-targeted therapy in ovarian cancer. We analyzed the expression of CD47 in ovarian cancer and the effect of chemotherapy on the expression of CD47 in ovarian cancer tissues. Furthermore, we investigated the effect of chemotherapy on the expression of CD47 in ovarian cancer cells. CD47 expression was examined using immunohistochemistry (IHC) of specimens from 78 patients with EOC who underwent neoadjuvant chemotherapy (NACT) and interval debulking surgery (IDS) with paired tissue specimens obtained before and after NACT. A retrospective review of the medical records was conducted to correlate demographic and survival data. Subsequently, we employed reverse transcription quantitative polymerase chain reaction (RT-qPCR) and flow cytometry analysis to examine and compare CD47 expression across human ovarian surface epithelial cell line (HOSE), SKOV3, and ES2 cells and to characterize the changes in CD47 expression in SKOV3 and ES2 cells after cisplatin treatment. CD47 expression was observed in 63 out of 78 (80.8%) cases before NACT. The expression of CD47 was not associated with the clinicopathological characteristics or the prognosis of patients with EOC. After three cancer specimens were excluded due to the absence of cancer cells following NACT, Wilcoxon exact tests of 75 pairs of matched specimens indicated that the expression of CD47 increased after chemotherapy (P=0.006). CD47 was expressed at varying levels in HOSE ovarian epithelial cells and SKOV3 and ES2 ovarian cancer cells. Notably, the CD47 messenger RNA (mRNA) expression of HOSE cells was observed to be lower than that of the SKOV3 and ES2 ovarian cancer cells (P<0.001). Following treatment with cisplatin, RT-qPCR indicated an increase in CD47 mRNA expression in SKOV3 and ES2 cells (P<0.001). Flow cytometry revealed a notable elevation in the cell surface CD47 protein mean fluorescence intensity of cisplatin-treated SKOV3 and ES2 cells (P<0.001). CD47 is highly expressed in ovarian cancer, and NACT increases the expression of CD47, which may contribute to tumor immune evasion. It is possible that platinum-based chemotherapy may result in elevated CD47 mRNA expression and cell surface CD47 protein expression.

Placental mesenchymal stem cell exosome-derived miR-139-5p regulates PTEN gene and influences chemotherapeutic-induced ovarian dysfunction

Objective: To investigate the impact of exosomes and microRNA (miRNA) from placental mesenchymal stem cells on chemotherapy-damaged ovarian granulosa cells. Methods: Various public databases were searched for miRNA targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN) gene. After miRNA transfection into human ovarian granulosa cells, cell growth and expressions of the target miRNA and PTEN were detected. Cisplatin was utilized to induce damage to human ovarian granulosa cells, which were subsequently co-cultured with human placental mesenchymal stem cells and exosomes generated from mesenchymal stem cells, then apoptosis and expressions of PTEN and the target miRNA were detected. Results: After analyzing several databases, miRNA 139-5p (miR-139-5p) was chosen as the target miRNA for this research. Transfection of miR-139-5p mimics into human ovarian granulosa cells elevated miR-139-5p expression level (9 882.080±1 049.130), reduced PTEN protein expression level (0.78±0.11), and increased cell proliferation absorbance (0.85±0.07). Cisplatin treatment severely damaged human ovarian granulosa cells and increased apoptosis, cisplatin-treated cells had a higher apoptosis ratio compared to untreated cells [ (41.9±1.0)% vs (5.0±0.3)%, P<0.001]. In damaged human ovarian granulosa cells, co-cultured with human placental mesenchymal stem cells and exosomes increased miR-139-5p expression levels (1.31±0.04 and 1.20±0.03, respectively) and decreased apoptosis ratios [(20.0±0.4)% and (22.3±1.1)%, respectively]. Conclusion: Placental mesenchymal stem cell-derived exosomes repair damages of cisplatin-induced ovarian granulosa cell and could target PTEN gene through miR-139-5p, which might be a potential option for the treatment of chemotherapy-induced ovarian dysfunction.

The deubiquitinase USP44 enhances cisplatin chemosensitivity through stabilizing STUB1 to promote LRPPRC degradation in neuroblastoma.

Dysregulated deubiquitinating enzymes (DUBs) execute as intrinsic oncogenes or tumor suppressors and are involved in chemoresistance in cancers. However, the functions and exact molecular mechanisms remain largely unclear in neuroblastoma. Here, a R2 screening strategy based on the standard deviation values was used to identify the most important DUB, USP44, in neuroblastoma with stage 4. We validated the role of USP44 regulation upon cisplatin treatment in vitro and in vivo experiments, revealing the molecular mechanisms associated with USP44 regulation and cisplatin sensitivity in neuroblastoma. We found that low USP44 expression was associated with an inferior prognosis in neuroblastoma patients. Overexpression of USP44 enhanced neuroblastoma cell sensitivity to cisplatin in vitro and in vivo. Mechanistically, USP44 recruited and stabilized the E3 ubiquitin ligase STUB1 by removing its K48-linked polyubiquitin chains at Lys30, and STUB1 further reinforced the K48-linked polyubiquitination of LRPPRC at Lys453 and promoted its protein degradation, thus enhancing the accumulation of mitochondrial reactive oxygen species (mROS), in turn facilitating neuroblastoma cell apoptosis and cisplatin sensitivity. Additionally, overexpression of LRPPRC reversed the promoting effect of USP44 on cell apoptosis in cisplatin-treated neuroblastoma cells. Our findings demonstrate that the USP44-STUB1-LRPPRC axis plays a pivotal role in neuroblastoma chemoresistance and provides potential targets for neuroblastoma therapy and prognostication.

Dihydroartemisinin restores the immunogenicity and enhances the anticancer immunosurveillance of cisplatin by activating the PERK/eIF2α pathway

BackgroundImmunosurveillance is pivotal in the effectiveness of anticancer therapies and tumor control. The ineffectiveness of cisplatin in activating the immunosurveillance is attributed to its lack of adjuvanticity resulting from its inability to stimulate endoplasmic reticulum stress. Dihydroartemisinin demonstrates the anti-tumor effects through various mechanisms, including the activation of the endoplasmic reticulum stress. This study aimed to develop a novel strategy to enhance the immunogenicity of dying tumor cells by combining cisplatin with dihydroartemisinin, thereby triggering effective anti-tumor immunosurveillance and improving the efficacy of cisplatin in clinical practice.MethodsLewis lung carcinoma (LLC) and CT26 colon cancer cell lines and subcutaneous tumor models were used in this study. The importance of immunosurveillance was validated in both immunocompetent and immunodeficient mouse models. The ability of dihydroartemisinin and cisplatin therapy to induce immunogenic cell death and tumor growth control in vivo was validated by prophylactic tumor vaccination and therapeutic tumor models. The underlying mechanism was elucidated through the pharmaceutical or genetic intervention of the PERK/eIF2α pathway in vitro and in vivo.ResultsDihydroartemisinin enhanced the generation of reactive oxygen species in cisplatin-treated LLC and CT26 cancer cells. The combination treatment of dihydroartemisinin with cisplatin promoted cell death and ensured an optimal release of damage-associated molecular patterns from dying cancer cells, promoting the phagocytosis of dendritic cells. In the tumor vaccination model, we confirmed that dihydroartemisinin plus cisplatin treatment induced immunogenic cell death. Utilizing immunocompetent and immunodeficient mouse models, we further demonstrated that the combination treatment suppressed the tumor growth of CT26 colon cancer and LLC lung cancer, leading to an improved prognosis through the restoration of cytotoxic T lymphocyte responses and reinstatement of anti-cancer immunosurveillance in vivo. Mechanistically, dihydroartemisinin restored the immunogenicity of cisplatin by activating the adjuvanticity of damage-associated molecular patterns, such as calreticulin exposure, through the PERK/eIF2α pathway. Additionally, the inhibition of eIF2α phosphorylation attenuated the anti-tumor efficiency of C + D in vivo.ConclusionsWe highlighted that dihydroartemisinin acts as an immunogenic cell death rescuer for cisplatin, activating anticancer immunosurveillance in a PERK/eIF2α-dependent manner and offering a strategy to enhance the anti-tumor efficacy of cisplatin in clinical practice.

Protective roles of thrombomodulin in cisplatin-induced nephrotoxicity through the inhibition of oxidative and endoplasmic reticulum stress

Cisplatin is an effective chemotherapeutic agent widely used for the treatment of various solid tumors. However, cisplatin has an important limitation in its use; currently, there is no method to ameliorate cisplatin-induced acute kidney injury (AKI). Thrombomodulin (TM) is well known not only for its role as a cofactor in the clinically important natural anticoagulation pathway but also for its anti-inflammatory properties. Here, we investigated the effects of TM in cisplatin-induced AKI. In mice intraperitoneally injected with 15 mg/kg cisplatin, TM (10 mg/kg) or PBS was administered intravenously at 24 h after cisplatin injection. TM significantly attenuated cisplatin-induced nephrotoxicity with the suppressed elevation of blood urea nitrogen and serum creatinine, and reduced histological damages. Actually, TM treatment significantly alleviated oxidative stress-induced apoptosis by reducing reactive oxygen species (ROS) levels in cisplatin-treated renal proximal tubular epithelial cells (RPTECs) in vitro. Furthermore, TM clarified cisplatin-induced apoptosis by reducing caspase-3 levels. In addition, TM attenuated the endoplasmic reticulum (ER) stress signaling pathway in both renal tissues and RPTECs to protect the kidneys from cisplatin-induced AKI. These findings suggest that TM is a potential protectant against cisplatin-induced nephrotoxicity through suppressing ROS generation and ER stress in response to cisplatin.

The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin.

Toxicity and the emergence of resistance are the main challenges in cancer treatment. The optimal dose of cisplatin, one of the most widely used chemotherapeutic anticancer drugs, is currently being widely debated. Furthermore, the dose-dependent molecular mechanisms of its action are poorly understood. To assess the role of protein kinase JNK (cJun N-terminal kinase) signaling in lung cancer treatment, we combined small-molecule JNK inhibitors and cisplatin. Wild-type p53 (tumor suppressor transcription factor TP53) and mutated RAS-bearing lung adenocarcinoma cell line A549 was used as a model in our studies. Here, we demonstrate cisplatin concentration-dependent opposing roles of JNK in killing cancer cells: a cell-protective role at low cisplatin concentrations and an apoptosis-promoting (or neutral) role at high concentrations. Time- and dose-dependent activation of pro-survival protein kinase AKT and TP53 was shown, with similar activation dynamics in cells exposed to different (low and high) cisplatin concentrations. Selective inhibition of AKT and activation of TP53 (expression and phosphorylation) led to a decrease in cell survival, indicating their involvement in cisplatin-induced cell death regulation. The activation levels of TP53 and AKT in cisplatin-treated A549 cells after cotreatment with the JNK inhibitor SP600125 correlated with their role in regulating cell death. TP53 and AKT were proposed as signaling proteins mediating the outcome of JNK inhibition in A549 cells exposed to different concentrations of cisplatin. Our findings suggest that a combination of stress kinase JNK inhibition and low-dose cisplatin, together with manipulation of drug-induced signaling, could be considered as a promising treatment strategy for certain lung cancers.

Pristimerin enhances cisplatin-induced apoptosis in nasopharyngeal carcinoma cells via ROS-mediated deactivation of the PI3K/AKT signaling pathway

To explore the effect of pristimerin combined with cisplatin on proliferation and apoptosis of nasopharyngeal carcinoma cells. CCK-8 assay was used to examine the survival rate of HNE-1 and CNE-2Z cells following treatment for 24 h with different concentrations of pristimerin, cisplatin or their combination. The changes in colony formation ability, apoptosis, and intracellular reactive oxygen species (ROS) levels of the treated cells were analyzed using colony formation assay and flow cytometry. Western blotting was performed to detect the changes in protein expressions in the cells. The effects of pre-treatment with NAC on proliferation, apoptosis, and PI3K/AKT signaling pathway were observed in pristimerin- and/or cisplatin-treated cells. Both pristimerin and cisplatin significantly lowered the survival rate of HNE-1 and CNE-2Z cells (P < 0.05). Compared with pristimerin or cisplatin alone, their combination more strongly inhibited survival and colony formation ability of the cells, increased cell apoptosis rate and intracellular ROS levels, upregulated the protein expressions of Bax, cleaved caspase-3, and cleaved PARP, and downregulated the protein expressions of Bcl-2, Mcl-1, PARP and p-PI3K and p-AKT (P < 0.05). NAC pretreatment significantly attenuated proliferation inhibition and apoptosis-promoting effects of pristimerin combined with cisplatin, and partially restored the downregulated protein expressions of p-PI3K and p-AKT in HNE-1 and CNE-2Z cells with the combined treatment (P < 0.05). Pristimerin can enhance cisplatin-induced proliferation inhibition and apoptosis in nasopharyngeal carcinoma cells, the mechanism of which may involve ROS-mediated deactivation of the PI3K/AKT signaling pathway.

Transposable elements alter gene expression and may impact response to cisplatin therapy in ovarian cancer.

Cisplatin is widely employed for cancer treatment; therefore, understanding resistance to this drug is critical for therapeutic practice. While studies have delved into differential gene expression in the context of cisplatin resistance, findings remain somewhat scant. We performed a comprehensive investigation of transposable elements (TEs) expression and their impact in host genes in two cisplatin-treated ovarian cancer cell lines. RNA-seq, ATAC-seq, and in-depth bioinformatics analysis were used to compare cisplatin-sensitive and -resistant ovarian cancer cell lines. Our results reveal that cisplatin therapy alters not only the expression of protein-coding genes, but also key TEs, including LINE1, Alu, and endogenous retroviruses, in both cisplatin-sensitive and -resistant cell lines. By co-expressing with downstream genes or by creating chimeric transcripts with host genes at their insertion sites, these TEs seem to control the expression of protein-coding genes, including tumor-related genes. Our model uncovers TEs influencing the expression of cancer genes and cancer pathways. Collectively, our findings indicate that TE alterations associated with cisplatin treatment occur in critical cancer genes and cellular pathways synergically. This research highlights the importance of considering the entire spectrum of transcribed elements in the genome, especially TE expression, for a complete understanding of complex models like cancer response to treatment.

Transfected SARS-CoV-2 spike DNA for mammalian cell expression inhibits p53 activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2 proteins in cancer cells and increases cancer cell viability after chemotherapy exposure.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and COVID-19 infection has led to worsened outcomes for patients with cancer. SARS-CoV-2 spike protein mediates host cell infection and cell-cell fusion that causes stabilization of tumor suppressor p53 protein. In-silico analysis previously suggested that SARS-CoV-2 spike interacts with p53 directly but this putative interaction has not been demonstrated in cells. We examined the interaction between SARS-CoV-2 spike, p53 and MDM2 (E3 ligase, which mediates p53 degradation) in cancer cells using an immunoprecipitation assay. We observed that SARS-CoV-2 spike protein interrupts p53-MDM2 protein interaction but did not detect SARS-CoV-2 spike bound with p53 protein in the cancer cells. We further observed that SARS-CoV-2 spike suppresses p53 transcriptional activity in cancer cells including after nutlin exposure of wild-type p53-, spike-expressing tumor cells and inhibits chemotherapy-induced p53 gene activation of p21(WAF1), TRAIL Death Receptor DR5 and MDM2. The suppressive effect of SARS-CoV-2 spike on p53-dependent gene activation provides a potential molecular mechanism by which SARS-CoV-2 infection may impact tumorigenesis, tumor progression and chemotherapy sensitivity. In fact, cisplatin-treated tumor cells expressing spike were found to have increased cell viability as compared to control cells. Further observations on γ-H2AX expression in spike-expressing cells treated with cisplatin may indicate altered DNA damage sensing in the DNA damage response pathway. The preliminary observations reported here warrant further studies to unravel the impact of SARS-CoV-2 and its various encoded proteins including spike on pathways of tumorigenesis and response to cancer therapeutics. More efforts should be directed at studying the effects of the SARS-CoV-2 spike and other viral proteins on host DNA damage sensing, response and repair mechanisms. A goal would be to understand the structural basis for maximal anti-viral immunity while minimizing suppression of host defenses including the p53 DNA damage response and tumor suppression pathway. Such directions are relevant and important including not only in the context of viral infection and mRNA vaccines in general but also for patients with cancer who may be receiving cytotoxic or other cancer treatments.

Oleanolic Acid Acetate Alleviates Cisplatin-Induced Nephrotoxicity via Inhibition of Apoptosis and Necroptosis In Vitro and In Vivo.

Cisplatin is a widely used anti-cancer drug for treating solid tumors, but it is associated with severe side effects, including nephrotoxicity. Various studies have suggested that the nephrotoxicity of cisplatin could be overcome; nonetheless, an effective adjuvant drug has not yet been established. Oleanolic acid acetate (OAA), a triterpenoid isolated from Vigna angularis, is commonly used to treat inflammatory and allergic diseases. This study aimed to investigate the protective effects of OAA against cisplatin-induced apoptosis and necroptosis using TCMK-1 cells and a mouse model. In cisplatin-treated TCMK-1 cells, OAA treatment significantly reduced Bax and cleaved-caspase3 expression, whereas it increased Bcl-2 expression. Moreover, in a cisplatin-induced kidney injury mouse model, OAA treatment alleviated weight loss in the body and major organs and also relieved cisplatin-induced nephrotoxicity symptoms. RNA sequencing analysis of kidney tissues identified lipocalin-2 as the most upregulated gene by cisplatin. Additionally, necroptosis-related genes such as receptor-interacting protein kinase (RIPK) and mixed lineage kinase domain-like (MLKL) were identified. In an in vitro study, the phosphorylation of RIPKs and MLKL was reduced by OAA pretreatment in both cisplatin-treated cells and cells boosted via co-treatment with z-VAD-FMK. In conclusion, OAA could protect the kidney from cisplatin-induced nephrotoxicity and may serve as an anti-cancer adjuvant.

A novel GSK3β inhibitor 5n attenuates acute kidney injury

Acute kidney injury (AKI) is a clinical syndrome with high morbidity and mortality caused by various factor. The specific strategies for AKI are still lacking. GSK3β is widely expressed in the kidneys. In acute models of injury, GSK3β promotes the systemic inflammatory response, increases the proinflammatory release of cytokines, induces apoptosis, and alters cell proliferation. We screened a series of 3-(4-pyridyl)-5-(4-sulfamido-phenyl)-1,2,4-oxadiazole derivatives which are recognized as new GSK3β inhibitors, and found that 5n had the least toxicity and the best cell protection. We then tested the anti-inflammatory and reno-protective effect of 5n in cisplatin-treated tubular epithelial cells. 5n had anti-inflammation effect indicated by phosphor–NF–κB detection. Finally, we found that 5n ameliorated renal injury and inflammation in cisplatin-induced AKI mouse model. Silencing GSK3β inhibited cell injury and inflammation induced by cisplatin. We found that GSK3β interacted with PP2Ac to modulate the activity of NF-κB. In conclusion, 5n, the novel GSK3β inhibitor, protects against AKI via PP2Ac-dependent mechanisms which may provide a potential strategy for the treatment of AKI in clinic.

Abstract 2034: The NAE1 inhibitor MLN4924 sensitizes both BRCA1 wild type and mutant triple negative breast cancers to cisplatin by reducing the nucleotide excision repair protein XPC level

Abstract Background: Triple-negative breast cancer (TNBC) shows poor survival and prognosis, high recurrence, and poor response to conventional therapeutics. To develop novel therapeutics for TNBC, we explored the efficacy of the neddylation inhibitor, MLN4924. We showed that MLN4924 displayed increased sensitivity against TNBC compared to non-TNBC subtypes. Mechanistically, MLN4924 induced extensive re-replication and DNA damage leading to cell death. Furthermore, MLN4924 improved cisplatin efficacy by elevating the DNA damage levels. To demonstrate the mechanisms of MLN4924+cisplatin-mediated enhanced cell death, we examined the XPC level, a key factor in the nucleotide excision repair (NER) pathway, playing a central role in cisplatin damage repair. Neddylation of CUL4A promotes XPC ubiquitination which influences XPC binding to the DNA damage sites for NER. Methods: We examined the XPC protein level by Western blot assay and RNA level by Real Time PCR using both BRCA1-wild type (MDA-MB-231) and BRCA1-mutated (MDA-MB-436) cell lines and mouse xenograft tumor samples. We evaluated if the proteosome inhibitor MG132 stabilizes XPC ubiquitination and protein levels by treating these cells with MG132 for various times. We also overexpressed XPC in these cell lines and examined the effect on survival upon MLN4924 and MLN4924+cisplatin treatments. Results: Our results demonstrate that MLN4924 reduces XPC ubiquitination upon shorter drug treatment but reduces the protein level upon longer drug treatment in both BRCA1-wild type and BRCA1-mutated cell lines. MG132 treatment did not stabilize the XPC ubiquitination level upon shorter drug treatment but stabilized the protein level upon longer drug treatment. We also observed a reduction in the XPC protein level in tumor samples which correlated with the increase in the DNA damage level. Surprisingly, XPC overexpression improved the cell survival in both MLN4924- and MLN4924+cisplatin-treated BRCA1-wild type and mutated TNBC cells. Conclusion: We hypothesize that a reduction in the XPC ubiquitination and protein levels plays a role in cisplatin-induced DNA damage repair reducing NER efficiency and consequently increasing the DNA damage upon MLN4924+cisplatin treatment. Since overexpression of XPC results in improved survival of both MLN4924-and MLN4924+cisplatin-treated cells, we anticipate that XPC not only regulates cisplatin damage repair through NER but may have a broader role in MLN4924 sensitization beyond the NER pathway. Importantly, MLN4924 treatment does not affect the XPC mRNA level emphasizing that neddylation influences the XPC protein level. In summary, our research uncovers novel insights into the role of XPC in both MLN4924 and MLN4924+cisplatin sensitization, shedding light on potential avenues for innovative therapeutic strategies for TNBC patients. Citation Format: Shrilekha Misra, Mateusz Opyrchal, Alo Ray. The NAE1 inhibitor MLN4924 sensitizes both BRCA1 wild type and mutant triple negative breast cancers to cisplatin by reducing the nucleotide excision repair protein XPC level [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 2034.

Mass spectrometry-based proteomic analysis to characterize cisplatin induced early signaling events in head and neck squamous cell carcinoma

ABSTRACT Cisplatin is the commonly used chemotherapeutic drug in treatment of various cancers. However, development of resistance towards cisplatin results in tumor recurrence. Here, we aim to understand the mechanisms of action of cisplatin and emergence of resistance to cisplatin using mass spectrometry-based proteomic approach. A panel of head and neck squamous cell carcinoma (HNSCC) cell lines were treated with cisplatin at respective IC50 for 24 h and label-free mass spectrometry analysis was carried out. Proteomic analysis of A253, FaDu, Det562 and CAL27 cell lines upon cisplatin treatment resulted in the identification of 5,060, 4,816, 4,537 and 4,142 proteins, respectively. Bioinformatics analysis of differentially regulated proteins revealed proteins implicated in DNA damage bypass pathway, translation and mRNA splicing to be enriched. Further, proteins associated with cisplatin resistance exhibited alterations following short-term cisplatin exposure. Among these, class III tubullin protein (TUBB3) was found to be upregulated in cisplatin-treated cells compared to untreated cells. Western blot analysis confirmed the elevated expression of TUBB3 in cells treated with cisplatin for 24 h, and also in cisplatin resistant HNSCC cell lines. This study delineates the early signaling events that enable HNSCC cells to counteract the cytotoxic effects of cisplatin and facilitate the development of resistance.

Cisplatin and Starvation Differently Sensitize Autophagy in Renal Carcinoma: A Potential Therapeutic Pathway to Target Variegated Drugs Resistant Cancerous Cells.

Cisplatin, a powerful chemotherapy medication, has long been a cornerstone in the fight against cancer due to chemotherapeutic failure. The mechanism of cisplatin resistance/failure is a multifaceted and complex issue that consists mainly of apoptosis inhibition through autophagy sensitization. Currently, researchers are exploring ways to regulate autophagy in order to tip the balance in favor of effective chemotherapy. Based on this notion, the current study primarily identifies the differentially expressed genes (DEGs) in cisplatin-treated autophagic ACHN cells through the Illumina Hi-seq platform. A protein-protein interaction network was constructed using the STRING database and KEGG. GO classifiers were implicated to identify genes and their participating biological pathways. ClueGO, David, and MCODE detected ontological enrichment and sub-networking. The network topology was further examined using 12 different algorithms to identify top-ranked hub genes through the Cytoscape plugin Cytohubba to identify potential targets, which established profound drug efficacy under an autophagic environment. Considerable upregulation of genes related to autophagy and apoptosis suggests that autophagy boosts cisplatin efficacy in malignant ACHN cells with minimal harm to normal HEK-293 growth. Furthermore, the determination of cellular viability and apoptosis by AnnexinV/FITC-PI assay corroborates with in silico data, indicating the reliability of the bioinformatics method followed by qRT-PCR. Altogether, our data provide a clear molecular insight into drug efficacy under starved conditions to improve chemotherapy and will likely prompt more clinical trials on this aspect.

Identification of TACSTD2 as novel therapeutic targets for cisplatin-induced acute kidney injury by multi-omics data integration.

Cisplatin-induced acute kidney injury (CP-AKI) is a common complication in cancer patients. Although ferroptosis is believed to contribute to the progression of CP-AKI, its mechanisms remain incompletely understood. In this study, after initially processed individual omics datasets, we integrated multi-omics data to construct a ferroptosis network in the kidney, resulting in the identification of the key driver TACSTD2. In vitro and in vivo results showed that TACSTD2 was notably upregulated in cisplatin-treated kidneys and BUMPT cells. Overexpression of TACSTD2 accelerated ferroptosis, while its gene disruption decelerated ferroptosis, likely mediated by its potential downstream targets HMGB1, IRF6, and LCN2. Drug prediction and molecular docking were further used to propose that drugs targeting TACSTD2 may have therapeutic potential in CP-AKI, such as parthenolide, progesterone, premarin, estradiol and rosiglitazone. Our findings suggest a significant association between ferroptosis and the development of CP-AKI, with TACSTD2 playing a crucial role in modulating ferroptosis, which provides novel perspectives on the pathogenesis and treatment of CP-AKI.

Determination of apoptotic effects of newly synthesized avobenzone Sm(III) complex in non-small cell lung cancer

In this study, we aimed to investigate the apoptotic effects of the lanthanide complex TBK-23, which was used in biological imaging due to its magnetic and spectroscopic properties. The study utilized HTB-54 and BEAS-2B cell lines. The cells were subjected to TBK-23 and cisplatin at active doses for a duration of 24 hours. Subsequently, flow cytometry was employed to assess the quantity of apoptotic cells, while quantitative RT-PCR was used to measure the expression levels of BAX, BCL-2, and BCL-xL. While cisplatin at 31 µM increased the proapoptotic BAX expression level in HTB-54 and BEAS-2B cells, TBK-23 increased BAX in cancer cells at the same concentration, but there was no observable effect in healthy cells. TBK-23 was found to have higher inhibitory effect on the expression of the antiapoptotic BCL-2 gene in cancer cells compared to that of cisplatin-treated cells. Consequently, it was determined that TBK-23, a novel metal-based compound, exhibited apoptotic effects similar to that of cisplatin and could be a potential chemotherapeutic agent.

Impact of Conventional and Potential New Metal-Based Drugs on Lipid Metabolism in Osteosarcoma MG-63 Cells.

This work investigated the mechanisms of action of conventional drugs, cisplatin and oxaliplatin, and the potentially less deleterious drug Pd2Spermine (Spm) and its Pt(II) analog, against osteosarcoma MG-63 cells, using nuclear-magnetic-resonance metabolomics of the cellular lipidome. The Pt(II) chelates induced different responses, namely regarding polyunsaturated-fatty-acids (increased upon cisplatin), suggesting that cisplatin-treated cells have higher membrane fluidity/permeability, thus facilitating cell entry and justifying higher cytotoxicity. Both conventional drugs significantly increased triglyceride levels, while Pt2Spm maintained control levels; this may reflect enhanced apoptotic behavior for conventional drugs, but not for Pt2Spm. Compared to Pt2Spm, the more cytotoxic Pd2Spm (IC50 comparable to cisplatin) induced a distinct phospholipids profile, possibly reflecting enhanced de novo biosynthesis to modulate membrane fluidity and drug-accessibility to cells, similarly to cisplatin. However, Pd2Spm differed from cisplatin in that cells had equivalent (low) levels of triglycerides as Pt2Spm, suggesting the absence/low extent of apoptosis. Our results suggest that Pd2Spm acts on MG-63 cells mainly through adaptation of cell membrane fluidity, whereas cisplatin seems to couple a similar effect with typical signs of apoptosis. These results were discussed in articulation with reported polar metabolome adaptations, building on the insight of these drugs' mechanisms, and particularly of Pd2Spm as a possible cisplatin substitute.