Cisplatin-induced Autophagy Research Articles

2α, 3α, 24-Thrihydroxyurs-12-en-24-ursolic acid enhances the cytotoxic effect of cisplatin on oral cancer cells by down-regulating autophagy.

This study aimed to investigate the effect and mechanism of 2α, 3α, 24-thrihydroxyurs-12-en-24-ursolic acid (TEOA) alone or in combination with cisplatin on oral cancer. TEOA, a pentacyclic triterpenoid compound isolated from the roots of Actinidia eriantha, has demonstrated antitumor activity in preclinical experiments. However, its role in oral cancer remains poorly understood. Our findings revealed that a low concentration of TEOA did not exhibit significant cytotoxicity against oral squamous cell carcinoma cells. However, when combined with cisplatin, TEOA showed a significant therapeutic effect. The combined treatments resulted in a significant inhibition of proliferation and migration and a significant increase in apoptosis of squamous cell carcinoma cells. Cisplatin exposure increased autophagy levels, which may contribute to chemoresistance. Of note, the presence of TEOA significantly inhibited cisplatin-induced autophagy, leading to improved chemotherapy efficacy. Our findings indicate that a mild low dosage of TEOA may enhance the cytotoxic effect of cisplatin by downregulating autophagy in oral cancer cells.

Research on the role of cellular autophagy in the sensitivity of human tongue cancer cells to radiotherapy and chemotherapy

ObjectiveThis paper aims to investigate the role of cisplatin-induced autophagy in human tongue squamous carcinoma Tca8113 cells. MethodsAfter inhibiting the expression of autophagic proteins with different autophagy inhibitors (3-methyladenine, chloroquine), the sensitivity of human tongue squamous cell carcinoma (Tca8113) cells to killing by gradient concentrations of cisplatin and gradient doses of radiation was detected using a colony formation assay. Further, the changes of autophagy expression in Tca8113 cells that had been treated with cisplatin and radiation were detected using western immunoblot, GFP-LC3 fluorescence and transmission electron microscopy. ResultsThe sensitivity of Tca8113 cells to cisplatin and radiation was significantly increased (P < 0.05) after reducing autophagy expression using different autophagy inhibitors. Meanwhile, the expression of autophagy in the cells was significantly increased by cisplatin and radiation treatment. ConclusionTca8113 cells upregulated autophagy under the effect of either radiation or cisplatin, and the sensitivity of Tca8113 cells to cisplatin and radiation could be improved by inhibiting autophagy using multiple pathways.

TRPM2 protects against cisplatin-induced acute kidney injury and mitochondrial dysfunction via modulating autophagy.

Background: Cisplatin is a widely used anti-tumor agent but its use is frequently limited by nephrotoxicity. Transient receptor potential melastatin 2 (TRPM2) is a non-selective cation channel which is generally viewed as a sensor of oxidative stress, and increasing evidence supports its link with autophagy, a critical process for organelle homeostasis. Methods: Cisplatin-induced cell injury and mitochondrial damage were both assessed in WT and Trpm2-knockout mice and primary cells. RNA sequencing, immunofluorescence staining, immunoblotting and flowcytometry were applied to interpret the mechanism of TRPM2 in cisplatin nephrotoxicity. Results: Knockout of TRPM2 exacerbates renal dysfunction, tubular injury and cell apoptosis in a model of acute kidney injury (AKI) induced by treatment with cisplatin. Cisplatin-caused tubular mitochondrial damage is aggravated in TRPM2-deficient mice and cells and, conversely, alleviated by treatment with Mito-TEMPO, a mitochondrial ROS scavenger. TRPM2 deficiency hinders cisplatin-induced autophagy via blockage of Ca2+ influx and subsequent up-regulation of AKT-mTOR signaling. Consistently, cisplatin-induced tubular mitochondrial damage, cell apoptosis and renal dysfunction in TRPM2-deficient mice are mitigated by treatment with a mTOR inhibitor. Conclusion: Our results suggest that the TRPM2 channel plays a protective role in cisplatin-induced AKI via modulating the Ca2+-AKT-mTOR signaling pathway and autophagy, providing novel insights into the pathogenesis of kidney injury.

YTHDF1 Protects Auditory Hair Cells from Cisplatin-Induced Damage by Activating Autophagy via the Promotion of ATG14 Translation.

N6-methyladenosine (m6A) has been recognized as a common type of post-transcriptional epigenetic modification. m6A modification and YTHDF1, one of its reader proteins, have been documented to play a pivotal role in numerous human diseases via regulating mRNA splicing, translation, stability, and subcellular localization. The chemotherapeutic drug cisplatin (CDP) can damage sensory hair cells (HCs) and result in permanent sensorineural hearing loss. However, whether YTHDF1-mediated modification of mRNA is potentially involved in CDP-induced injury in sensory hair cells was not fully clarified. This study investigated the potential mechanisms for the modification of YTHDF1 in CDP-induced damage in HCs. Here, we discovered that YTHDF1's expression level statistically increased significantly after treating with CDP. Apoptosis and cell death of HCs induced by CDP were exacerbated after the knockdown of YTHDF1, while overexpression of YTHDF1 in HCs alleviated their injury induced by CDP. Moreover, YTHDF1 expression correlated with cisplatin-induced autophagy with statistical significance in HCs; namely, YTHDF1's overexpression enhanced the activation of autophagy, while its deficiency suppressed autophagy and, at the same time, increased the loss of HCs after CDP damage. WB analysis and qRT-PCR results of autophagy-related genes indicated that YTHDF1 promoted the translation of autophagy-related genes ATG14, thus boosting autophagy. Therefore, CDP-induced YTHDF1 expression protected HCs against CDP-induced apoptosis by upregulating the translation of autophagy-related genes ATG14, along with enhancing autophagy. Based on these findings, it can be inferred that YTHDF1 is potentially a target for ameliorating drug-induced HCs damage through m6A modification.

Pantoprazole promotes the sensitivity of cervical cancer cells to cisplatin by inhibiting cisplatin-induced autophagy.

This study aimed to explore the role of pantoprazole (PPZ) in affecting the sensitivity of cervical cancer (CC) cells to cisplatin. HeLa and CaSki cells were exposed to cisplatin and/or PPZ treatment. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony formation, flow cytometry, wound healing, and transwell assays were performed to detect cell viability, proliferation, apoptosis, migration, and invasion of CC cells, respectively. Then, expressions of Beclin-1, LC3, and p62 were measured by western blot. Rapamycin (Rapa), acting as an autophagy activator, was applied to confirm the effect of autophagy on the sensitivity of CC cells to cisplatin. Cisplatin treatment suppressed cell viability and proliferation and accelerated apoptosis of CC cells. Combination of cisplatin and PPZ or PPZ alone significantly inhibited cell viability, proliferation, migration, and invasion, and increased cell apoptosis of CC cells. Cisplatin enhanced expression levels of Beclin1 and LC3II/I, and reduced p62 expression. Combination of cisplatin and PPZ significantly decreased the expression levels of Beclin1 and LC3II/I, but increased p62 expression. The autophagy activator, Rapa, eliminated the inhibitory effects of the combination of cisplatin and PPZ on autophagy, and enhanced cell viability, but inhibited apoptosis of CC cells. PPZ promotes the sensitivity of CC cells to cisplatin by inhibiting cisplatin-induced cell autophagy.

Interferon regulatory factor-1 regulates cisplatin-induced apoptosis and autophagy in A549 lung cancer cells

This study aimed to investigate the expression and function of interferon regulatory factor-1 (IRF-1) in non-small cell lung cancer (NSCLC). IRF-1 expression and its prognostic value were investigated through bioinformatic analysis. The protein expression levels of IRF-1, cleaved caspase 3, and LC3-I/II were analyzed by western blotting. A lentiviral vector was used to overexpress or knockdown IRF-1 in vitro. Mitochondrial membrane potential (MMP) and reactive oxygen species (ROS) were analyzed by JC-1 and DCFH-DA staining, respectively. ATP, SOD, MDA, cell viability, LDH release, and caspase 3 activity were evaluated using commercial kits. Compared to the levels in normal tissues, IRF-1 expression was significantly lower in lung cancer tissues and was a prognostic factor for NSCLC. Cisplatin treatment-induced IRF-1 activation, ROS production, ATP depletion, SOD consumption, and MDA accumulation in A549 lung cancer cells. IRF-1 overexpression promoted mitochondrial depolarization, oxidative stress, and apoptotic cell death and inhibited autophagy in A549 cells, and these effects could be reversed by IRF-1 knockdown. These data suggest that IRF-1 regulates apoptosis, autophagy and oxidative stress, which might be served as a potential target for increasing chemotherapy sensitivity of lung cancer.

TTK inhibition increases cisplatin sensitivity in high-grade serous ovarian carcinoma through the mTOR/autophagy pathway

High-grade serous ovarian cancer (HGSOC) is the most lethal gynecological malignancy. However, the molecular mechanisms underlying HGSOC development, progression, chemotherapy insensitivity and resistance remain unclear. Two independent GEO datasets, including the gene expression profile of primary ovarian carcinoma and normal controls, were analyzed to identify genes related to HGSOC development and progression. A KEGG pathway analysis of the differentially expressed genes (DEGs) revealed that the cell cycle pathway was the most enriched pathway, among which TTK protein kinase (TTK) was the only gene with a clinical-grade inhibitor that has been investigated in a clinical trial but had not been studied in HGSOC. TTK was also upregulated in cisplatin-resistant ovarian cancer cells from two other datasets. TTK is a regulator of spindle assembly checkpoint signaling, playing an important role in cell cycle control and tumorigenesis in various cancers. However, the function and regulatory mechanism of TTK in HGSOC remain to be determined. In this study, we observed TTK upregulation in patients with HGSOC. High TTK expression was related to a poor prognosis. Genetic and pharmacological inhibition of TTK impeded the proliferation of ovarian cancer cells by disturbing cell cycle progression and increasing apoptosis. TTK silencing increased cisplatin sensitivity by activating the mammalian target of rapamycin (mTOR) complex to further suppress cisplatin-induced autophagy in vitro. In addition, the enhanced sensitivity was partially diminished by rapamycin-mediated inhibition of mTOR in TTK knockdown cells. Furthermore, TTK knockdown increased the toxicity of cisplatin in vivo by decreasing autophagy. These findings suggest that the administration of TTK inhibitors in combination with cisplatin may lead to improved response rates to cisplatin in patients with HGSOC presenting high TTK expression. In summary, our study may provide a theoretical foundation for using the combination therapy of cisplatin and TTK inhibitors as a treatment for HGSOC in the future.

Cisplatin-induced hydroxyl radicals mediate pro-survival autophagy in human lung cancer H460 cells

BackgroundAccumulated evidence demonstrates cisplatin, a recommended chemotherapy, modulating pro-survival autophagic response that contributes to treatment failure in lung cancer patients. However, distinct mechanisms involved in cisplatin-induced autophagy in human lung cancer cells are still unclear.ResultsHerein, role of autophagy in cisplatin resistance was indicated by a decreased cell viability and increased apoptosis in lung cancer H460 cells pre-incubated with wortmannin, an autophagy inhibitor, prior to treatment with 50 µM cisplatin for 24 h. The elevated level of hydroxyl radicals detected via flow-cytometry corresponded to autophagic response, as evidenced by the formation of autophagosomes and autolysosomes in cisplatin-treated cells. Interestingly, apoptosis resistance, autophagosome formation, and the alteration of the autophagic markers, LC3-II/LC3-I and p62, as well as autophagy-regulating proteins Atg7 and Atg3, induced by cisplatin was abrogated by pretreatment of H460 cells with deferoxamine, a specific hydroxyl radical scavenger. The modulations in autophagic response were also indicated in the cells treated with hydroxyl radicals generated via Fenton reaction, and likewise inhibited by pretreatment with deferoxamine.ConclusionsIn summary, the possible role of hydroxyl radicals as a key mediator in the autophagic response to cisplatin treatment, which was firstly revealed in this study would benefit for the further development of novel therapies for lung cancer.

Cisplatin induces autophagy-associated apoptosis in human oral squamous cell carcinoma (OSCC) mediated in part through reactive oxygen species

Oral Squamous Cell Carcinoma (OSCC) is the sixth most common cancer worldwide. Chemoresistance is a critical problem in OSCC leading to therapeutic failure and tumour recurrence. Recently, autophagy has acquired an emerging interest in cancer as it has been shown to be frequently activated in tumour cells treated with chemotherapeutics. Whether drug-induced autophagy represents a mechanism that allows cancer cells to survive or a pro-death mechanism associated with apoptosis remains controversial. This study evaluated the cellular response to cisplatin and the role of autophagy in mediating cisplatin resistance in OSCC cells. Our results demonstrated that cisplatin concurrently induced apoptosis and autophagy in OSCC cell lines partially through the ROS/JNK pathway. Moreover, inhibition of cisplatin-induced apoptosis abrogated autophagy, indicating a complex interplay between these pathways. Cisplatin-induced autophagy does not appear to elicit a pro-survival effect in OSCC as early-stage autophagy inhibition, using either a pharmacological inhibitor or knockdown of the key autophagy protein ATG5, did not sensitise cells to cisplatin. Additionally, autophagy did not play a role in acquired resistance to cisplatin in our novel cisplatin-resistant OSSC cell line (SCC-4cisR) obtained by pulsed stepwise exposure of SCC-4 cells to cisplatin (~14-fold change in sensitivity). There was no change in the basal levels of autophagy in the SCC-4cisR cells compared to the SCC-4 cells. Furthermore, a significant increase in cisplatin-induced autophagy was observed only in the SCC-4 cells, but not in the derived SCC-4cisR cells. Collectively, these data indicate that autophagy may not be implicated in acquired cisplatin resistance in OSCC.

PPAR-δ activation reduces cisplatin-induced apoptosis via inhibiting p53/Bax/caspase-3 pathway without modulating autophagy in murine renal proximal tubular cells.

Cisplatin-induced injury of renal proximal tubular cells results basically from increased apoptosis via mitochondrial damage, and is mitigated by appropriate enhancement of autophagy. Peroxisome proliferator-activated receptor-delta (PPAR-δ) reportedly protects against not only mitochondrial damages but also enhances autophagy. Thus, PPAR-δ may protect against cisplatin-induced kidney injury. We examined the protective effects of PPAR-δ activation on cisplatin-induced cellular injury and their detailed mechanisms in a murine renal proximal tubular (mProx) cell line using GW0742, an authentic PPAR-δ activator. Cisplatin-induced cell damages were evaluated by TUNEL assay and immunoblot analyses for p53, 14-3-3, Bax, Bcl2, cytochrome C, and activated caspases. Autophagy status was examined by immunoblot analyses for p62 and LC3. GW0742 suppressed cisplatin-induced apoptosis of mProx cells by reducing the activation of caspase-3 via attenuating the phosphorylation of p53 and 14-3-3, mitochondrial Bax accumulation, cytochrome C release from mitochondria to the cytosol and ensuing cytosolic caspase-9 activation. In contrast, GW0742 did not diminish cisplatin-enhanced activation of caspases-8 or -12 as extrinsic or endothelium reticulum apoptotic pathways, respectively. The inhibitory effect of GW0742 on cisplatin-induced caspase-3 activation was significantly diminished by silencing of the PPAR-δ gene expression. GW0742 itself had no influence on starvation-stimulated or cisplatin-induced autophagy in mProx cells, suggesting that the protective effects were not mediated by autophagy modification. Our results indicate that GW0742 may serve as a candidate agent to mitigate cisplatin nephrotoxicity via inhibiting the mitochondrial apoptotic pathway considerably depending on PPAR-δ, without modulating autophagy.

Inhibition of microRNA-17 enhances cisplatin-induced apoptosis of human tongue squamous carcinoma cell.

MicroRNA-17 (miR-17) was reported to promote cell proliferation and migration of various types of cancers. However, the mechanism remains unclear. This present study was designed to explore the potential mechanism. Downregulation of miR-17 in CAL-27 cells was performed by transfecting anti-miR-27 plasmids. Xenograft tumor model was carried out to detect the effect of inhibition of microRNA-17 on tongue squamous carcinoma growth. MiR-17 inhibition promotes cisplatin-induced apoptosis via regulating the expression of apoptotic molecules. MiR-17 inhibition promotes cisplatin-induced autophagy of CAL-27 cells. Mechanically, miR-17 inhibition promotes apoptosis and autophagy through STAT3 signaling pathway. Xenograft tumor model showed that miR-17 inhibition attenuates tongue squamous carcinoma growth and promotes tongue squamous carcinoma cell apoptosis in vivo. MiR-17 inhibition enhances cisplatin-induced apoptosis of human tongue squamous carcinoma cell. Our study supplies the evidence that miR-17 may serve as the potential target for human tongue squamous carcinoma treatment.

Role of autophagy in response of epithelial ovarian cancer cells to cisplatin treatment and cisplatin resistance

Ovarian cancer survival rate is inversely associated with the extent of tumor metastasis. One of the main treatment approaches against ovarian cancer is employment of platinum based therapies, including cisplatin. Majority of ovarian cancer patients develop cisplatin resistance. We aimed to investigate roles for macroautophagy in response of epithelial ovarian cancer cells to cisplatin, including changes in cell motility, as well as in development of cisplatin resistance. Cisplatin treatment induced autophagy in Caov-3 cells in vitro, as well as resulted in increased cell motility. Pharmacologic inhibition of autophagy by wortmannin eliminated the effect of cisplatin on cell motility. We further selected Caov-3 cells with acquired cisplatin resistance and observed elevated baseline expression of autophagy markers in the resistant cells. Our data indicate a role for autophagy in development of cisplatin resistance by the EOC cells, as well as a potential role for cisplatin-induced autophagy in ovarian tumor metastasis.

LncRNA SNHG7 enhances chemoresistance in neuroblastoma through cisplatin-induced autophagy by regulating miR-329-3p/MYO10 axis.

Neuroblastoma (NB) is a type of extracranial solid tumor that usually occurs in children. Drug resistance has become a major obstacle in NB chemotherapy. Long non-coding RNA small nucleolar RNA host gene 7 (SNHG7) is an oncogene in many cancers, including NB. This study aimed to investigate the role of SNHG7 in cisplatin sensitivity of NB and the underlying mechanism. Cell Counting Kit-8 (CCK-8) assay was used to detect cell viability, and the IC50 of cisplatin was calculated. The protein levels of autophagy markers were measured by Western blot assay. The levels of SNHG7, miR-329-3p and myosin X (MYO10) were examined by quantitative real-time polymerase chain reaction (qRT-PCR) or Western blot assay. The interaction among SNHG7, miR-329-3p and MYO10 was confirmed by Dual-Luciferase reporter assay. Cisplatin curbed the viability of NB cells in a dose-dependent manner and facilitated autophagy in NB cells. Silencing of SNHG7 reduced cisplatin resistance and suppressed cisplatin-induced autophagy. SNHG7 was a sponge of miR-329-3p and modulated chemosensitivity and autophagy by regulating miR-329-3p. In addition, SNHG7 upregulated MYO10 by sponging miR-329-3p. MYO10 restored the effect of miR-329-3p on cisplatin sensitivity and autophagy. Moreover, suppression of autophagy blocked SNHG7-induced cisplatin resistance. Depletion of SNHG7 potentiated cisplatin sensitivity through inhibition of autophagy by modulating miR-329-3p/MYO10 axis, providing a new therapeutic approach to overcome cisplatin resistance in NB chemotherapy.

Chloroquine enhances cisplatin-induced apoptosis of nasopharyngeal carcinoma cells by inhibiting autophagy via upregulating miR129

To investigate the role of miR129 in mediating the effect of chloroquine to enhance cisplatin- induced apoptosis in nasopharyngeal carcinoma cells (HNE1). MTT assay was used to detect the viability of HNE1 cells treated with different concentrations of cisplatin. Colony formation of HNE1 cells treated with cisplatin and chloroquine, alone or in combination, was observed using crystal violet staining. BALB/C unde mice were inoculated with HNE1 cells and randomly divided into 4 groups with 6 mice in each group. The mice received intraperitoneal injections of cisplatin and chloroquine, alone or in combination once every 3 days for 4 consecutive weeks, and the tumor growth was observed in each group. The expression of miR129 in HNE1 cells treated with chloroquine, cisplatin, or both was detected with qPCR. The effects of miR129 suppression with a miR129 inhibitor on the expressions of autophagy related proteins p62, LC3B, Beclin1 and the drug-resistant related protein P-glycoprotein (P-gp) were examined using Western blotting in HNE1 cells treated with chloroquine, cisplatin, or both; the changes in cell apoptosis were detected Annexin V/PI double staining. Chloroquine combined with cisplatin significantly inhibited HNE1 cell proliferation in vitro and the growth of HNE1 cell-derived tumor in nude mice as compared with cisplatin alone (P < 0.01). In cultured HNE1 cells, inhibition of the expression of miR129 significantly promoted autophagy and up-regulated P-gp expression (P < 0.01); Chloroquine obviously inhibited cisplatin-induced autophagy and up-regulated the expression of miR129 in HNE1 cells (P < 0.01). Transfection of the cells with the miR129 inhibitor abolished the inhibitory effect of chloroquine on cisplatin-induced autophagy, and significantly increased the cell survival rate (P < 0.05) and lower the cell apoptotic rate (P < 0.01) after combined treatment with chloroquine and cisplatin. Chloroquine enhances the pro-apoptotic effect of cisplatin by up-regulating miR129 to inhibit autophagy and drug resistance in HNE1 cells.

Curcumin attenuates cognitive impairment by enhancing autophagy in chemotherapy

Cisplatin, a commonly used chemotherapy drug, can increase the survival rate of cancer patients. However, it often causes various side effects, including neuronal deficit-induced cognitive impairment. Considering that curcumin is effective in neuronal protection, the action of curcumin on cognitive improvement was evaluated in cisplatin-treated C57BL/6 mice in the present study. Our results first showed that curcumin restored impaired cognitive behaviors. Consistent with this, neurogenesis and synaptogenesis were improved by curcumin. In addition, cisplatin-induced dysfunction of apoptosis-related proteins was partly reversed by curcumin. Moreover, cisplatin-induced autophagy was enhanced by curcumin. Our results also indicated that cisplatin induced autophagy through the endoplasmic reticulum (ER) stress-mediated ATF4-Akt-mTOR signaling pathway. Curcumin activated AMPK-JNK signaling, which mediated both mTOR inhibition and Bcl-2 upregulation and in turn enhanced autophagy and suppressed apoptosis, respectively. In contrast, pretreatment with the autophagy inhibitor 3-methyladenine (3-MA) completely abolished the effects of curcumin on cognitive improvement and improved neurogenesis, synaptogenesis and autophagy. Our results show that cognitive improvement induced by curcumin during chemotherapy is mediated by the enhancement of hippocampal autophagy.

Ambra1 induces autophagy and desensitizes human prostate cancer cells to cisplatin.

Prostate cancer (PCa), the second most mortal cancer from developed countries, presents a high level of chemoresistance. There is emerging evidence underscores the critical role of autophagy in the onset, progression, and chemoresistance of PCa. In the present study, we investigated the possible role of a novel autophagy regulator, activating molecule in beclin1-regulated autophagy1 (Ambra1), a novel ATG gene in the sensitivity or PCa cells to cisplatin. We explored the regulation by the Ambra1 manipulation on the induction of apoptosis and autophagy in human PCa DU145 cells in the presence of cisplatin, via up- or down-regulating Ambra1 expression. In addition, we examined the colony forming of DU145 cells post cisplatin treatment and Ambra1 manipulation. Our results demonstrated that the Ambra1 up-regulation reduced, whereas Ambra1 knockdown increased the cisplatin-induced apoptosis, caspase 3 cleavage, and poly ADP-ribose polymerase (PARP) cleavage. Interestingly, we also found significant autophagy induction in the cisplatin-treated DU145 cells, with increased autophagic vesicles, up-regulated autophagy-related markers. However, the cisplatin-induced autophagy was up-regulated by the Ambra1 overexpression or was down-regulated by the Ambra1 knockdown. In addition, the colony forming was also positively regulated by Ambra1 in DU145 cells post cisplatin treatment. In conclusion, Ambra1 negatively regulates the cisplatin-induced apoptosis and the cisplatin-mediated growth reduction in DU145 cells, in association with the Ambra1-mediated autophagy promotion. It implies that Ambra1-mediated autophagy might be an important mechanism underlining the sensitivity reduction of PCa cells.

SNHG16 promotes osteosarcoma progression and enhances cisplatin resistance by sponging miR-16 to upregulate ATG4B expression

BackgroundHuman osteosarcoma is the most common primary cancer of the bone. Multiple mechanisms underlying cell growth, apoptosis, bone development, and drug resistance are important in the development of osteosarcoma therapy, which remains to be fully studied. MethodsWe collected thirty-paired tumor tissues and the adjacent normal ones from osteosarcoma patients. Two osteosarcoma cell lines (SAOS2, U2OS) were used for in vitro experiments. RT-qPCR and Western blot were used for gene expression detection. We applied starBase to predict the potential binding sites. Then, the luciferase reporter assay was used to confirm the potential direct interaction. Besides, we applied CCK-8, EdU assay, and flow cytometric assays to detect cell growth and apoptosis rate. We used wound healing and transwell assays to determine cell migration and invasion abilities. Additionally, we constructed a cisplatin-resistant osteosarcoma cell line to study the potential impact of the regulatory axis on drug resistance. ResultsSmall nucleolar RNA host gene 16 (SNHG16) and autophagy-related 4B (ATG4B) were significantly upregulated in osteosarcoma tissues than the normal ones, and the higher expression level of SNHG16 predicted a poor prognosis in osteosarcoma patients. By contrast, the expression level of miR-16 was markedly lower in tumor tissues and was negatively correlated with SNHG16 (p < 0.001). SNHG16 was shown to promote cell growth, migration, and invasion, while miR-16 reversed this impact. Meanwhile, overexpression of ATG4B significantly promoted the development of osteosarcoma cells attenuated by SNHG16 knockdown or miR-16 mimics. Specifically, overexpression of ATG4B promoted cisplatin-induced autophagy and inhibited cell apoptosis rate, which enhanced the cisplatin resistance in osteosarcoma cell lines. ConclusionsOverall, our findings showed the importance of the regulatory axis of SNHG16/miR-16/ATG4B underlying osteosarcoma progression and chemoresistance to cisplatin. This research would benefit the therapy development in the treatment of osteosarcoma.

Abstract 4278: A comparison of drug sensitivity in isogenic tumor cell lines confirms that cytoprotective autophagy confers intrinsic resistance to cisplatin

Abstract Although autophagy inhibition often increases tumor cell sensitivity to chemotherapy and radiation, these observations do not provide definitive proof that autophagy is actually an intrinsic mechanism of resistance since virtually all tumor cells undergo autophagy in response to therapy. To directly address whether autophagy induction can confer chemoresistance, the current studies utilized a paired set of isogenic cell lines in which (cisplatin-induced) autophagy was either cytoprotective (crp53 cells where p53 was knocked out) or nonprotective (parental p53 wt cells) in function, but where the overall extent of autophagy was essentially identical. Cisplatin sensitivity was significantly lower in the crp53 cells where cisplatin-induced autophagy was protective compared to the parental p53 wt cells where cisplatin-induced autophagy was nonprotective, in support of the premise that cytoprotective autophagy could confer a relative degree of intrinsic resistance to chemotherapy. Accordingly, we can draw the following conclusions: (i) radiation and chemotherapy can promote both protective and nonprotective autophagy in tumor cells; (ii) although p53 can act as an autophagic switch, it cannot be reliably predicted whether autophagy will be protective or nonprotective based solely on p53 function; and (iii) even in the case(s) where autophagy is cytoprotective, it cannot be assumed that autophagy will confer drug or radiation resistance, but only that autophagy inhibition may, in select cases, increase drug or radiation sensitivity. These findings highlight the complexity of efforts to exploit autophagy inhibition as a therapeutic strategy to enhance tumor response to chemotherapy and radiation. Citation Format: Jingwen Xu, Nipa Patel, Tareq Saleh, Yingliang Wu, Santiago Lima, David A. Gewirtz. A comparison of drug sensitivity in isogenic tumor cell lines confirms that cytoprotective autophagy confers intrinsic resistance to cisplatin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4278.

Downregulation of BAG3 attenuates cisplatin resistance by inhibiting autophagy in human epithelial ovarian cancer cells.

Epithelial ovarian cancer (EOC) is one of the most common malignant gynecological tumors. Interval cytoreductive surgery and cisplatin-based chemotherapy are the standard treatments. However, acquired resistance to cisplatin presents a major challenge for improving the overall survival and prognosis of patients. Recent evidence indicates that cytoprotective functions of autophagy in cancer cells is a potential mechanism for chemoresistance. The present study aimed to investigate the mechanisms responsible for cisplatin resistance in EOC cell lines. The results revealed that cisplatin activated autophagy, measured by an increase in the expression of LC3-II by western blot analysis, protecting ovarian cancer cells from cisplatin toxicity. The present study also identified Bcl-2-associated athanogene 3 (BAG3) as a novel autophagy regulator that serves a role in cisplatin resistance. Treatment with cisplatin was observed to enhance BAG3 expression in parental and cisplatin-resistant ovarian cancer cell lines, and the downregulation of BAG3 blocked cisplatin-induced autophagy, thereby increasing cisplatin sensitivity in the EOC cell lines. In conclusion, BAG3 attenuates cisplatin resistance by inhibiting autophagy, suggesting that downregulation of BAG3 may be a useful therapeutic strategy to overcome cisplatin resistance by preventing cytoprotective autophagy in EOC.

PDIA6 modulates apoptosis and autophagy of non-small cell lung cancer cells via the MAP4K1/JNK signaling pathway.

BackgroundNon-small cell lung cancer (NSCLC) is the most common type of lung cancer with a poor prognosis. We previously found that protein disulfide isomerase family 6 (PDIA6) is upregulated in lung squamous cell carcinoma (LSCC). This study aimed to elucidate the clinical relevance, biological functions, and molecular mechanisms of PDIA6 in NSCLC.MethodsThe expression of PDIA6 in NSCLC was assessed using the TCGA database, western blotting, and immunohistochemistry. Correlations of PDIA6 expression with clinicopathological and survival features were evaluated. The functions of PDIA6 in regulating NSCLC cell growth, apoptosis, and autophagy were investigated using gain-and loss-of-function strategies in vitro or in vivo. The underlying molecular mechanisms of PDIA6 function were examined by human phospho-kinase array and co-immunoprecipitation.FindingsPDIA6 expression was upregulated in NSCLC compared with adjacent normal tissues, and the higher PDIA6 expression was correlated with poor prognosis. PDIA6 knockdown decreased NSCLC cell proliferation and increased cisplatin-induced intrinsic apoptosis, while PDIA6 overexpression had the opposite effects. In addition, PDIA6 regulated cisplatin-induced autophagy, and this contributed to PDIA6-mediated apoptosis in NSCLC cells. Mechanistically, PDIA6 reduced the phosphorylation levels of JNK and c-Jun. Moreover, PDIA6 interacted with MAP4K1 and inhibited its phosphorylation, ultimately inhibiting the JNK/c-Jun signaling pathway.InterpretationPDIA6 is overexpressed in NSCLC and inhibits cisplatin-induced NSCLC cell apoptosis and autophagy via the MAP4K1/JNK/c-Jun signaling pathway, suggesting that PDIA6 may serve as a biomarker and therapeutic target for NSCLC patients.FundNational Natural Science Foundation of China and Institutions of higher learning of innovation team from Liaoning province.