Trichostatin A sensitizes cisplatin-resistant A549 cells to apoptosis by up-regulating death-associated protein kinase.
ABSTRACT: AIM: To investigate the apoptosis-inducing effect of trichostatin A (TSA) in the human lung adenocarcinoma cisplatin-resistant cell line (A549/CDDP) and to examine whether TSA can enhance sensitivity to cisplatin treatment and the underlying molecular mechanisms of such an enhancement. METHODS: Cell viability was evaluated using the Neutral Red assay. Apoptosis was assessed using Hoechst 33258 staining and flow cytometry analysis. Protein expression was detected by Western blotting. To determine the role of Death-associated protein kinase (DAPK) in TSA-induced apoptosis in the A549/CDDP cell line, cells were transfected with pcDNA3.1(+)-DAPK, which has a higher expression level of DAPK compared to endogenous expression, and DAPK activity was inhibited by both over-expression C-terminal fragment of DAPK which may competitive binding DAPK substrates to inhibit the function of DAPK and RNA interference. RESULTS: TSA induced apoptosis in both A549 cells and A549/CDDP cells. TSA enhanced the sensitivity of A549/CDDP cells to cisplatin, along with concomitant DAPK up-regulation. When DAPK was over-expressed, A549/CDDP cells became sensitive to cisplatin and the cytotoxicity of TSA could be increased. Moreover, the cytotoxicity of TSA could be alleviated by inhibition of DAPK activity by the expression of a recombinant C-terminal fragment of DAPK or RNA interference. CONCLUSION: TSA induced sensitivity to cisplatin treatment in cisplatin-resistant A549 cells. The up-regulation of DAPK is one of the mechanisms mediating sensitization to TSA-induced apoptosis in cisplatin-resistant cells.
Project description:BACKGROUND: The efficacy of cisplatin-based chemotherapy in non-small-cell lung cancer is limited by the acquired drug resistance. Identification the RNAs related to the cisplatin resistance may help to improve clinical response rates. METHODS: Microarray expression profiling of mRNAs, lncRNA and miRNA was undertaken in A549 cells and cisplatin resistant A549/CDDP cells. Differentially expressed mRNAs, lncRNAs and miRNAs, verified by realtime RT-PCR, were subjected to pathway analysis. Expression of NKD2 and β-catenin was assessed by realtime RT-PCR and western blot analysis. The effect of lncRNA AK126698 on cisplatin induced apoptosis was investigated by annexin-V/PI flow cytometry. RESULTS: In total, 1471 mRNAs, 1380 lncRNAs and 25 miRNAs differentially expressed in A549/CDDP and A549 cells. Among them, 8 mRNAs, 8 lncRNAs and 5 miRNAs differentially expressed in gene chip analysis were validated. High-enrichment pathway analysis identified that some classical pathways participated in proliferation, differentiation, avoidance of apoptosis, and drug metabolism were differently expressed in these cells lines. Gene co-expression network identified many genes like FN1, CTSB, EGFR, and NKD2; lncRNAs including BX648420, ENST00000366408, and AK126698; and miRNAs such as miR-26a and let-7i potentially played a key role in cisplatin resistance. Among which, the canonical Wnt pathway was investigated because it was demonstrated to be targeted by both lncRNAs and miRNAs including lncRNA AK126698. Knockdown lncRNA AK126698 not only greatly decreased NKD2 which can negatively regulate Wnt/β-catenin signaling but also increased the accumulation and nuclear translocation of β-catenin, and significantly depressed apoptosis rate induced by cisplatin in A549 cells. CONCLUSION: Cisplatin resistance in non-small-cell lung cancer cells may relate to the changes in noncoding RNAs. Among these, AK126698 appears to confer cisplatin resistance by targeting the Wnt pathway.
Project description:Resistance to platinum?based drugs, such as cisplatin (CDDP), has been one of the major factors adversely affecting the clinical prognosis of patients with advanced non?small cell lung cancer (NSCLC). While it has been demonstrated that dysregulation of microRNAs (miRNAs) may contribute to cisplatin resistance in NSCLC, the underlying mechanisms remain largely unclear. In the present study, the effect of exosomal miR?1273a on cisplatin sensitivity of NSCLC was investigated. Microarray analysis was conducted to analyze the miRNA expression profiles in exosomes isolated from A549 cells treated with or without CDDP, and miR?1273a was found to be the most prominently downregulated miRNA in CDDP?treated exosomes. Overexpression of miR?1273a significantly increased the cytotoxicity of CDDP and induced apoptosis in A549 cells. Syndecan binding protein (SDCBP) was predicted to be a direct target of miR?1273a by bioinformatics and was found to be downregulated by miR?1273a in A549 cells. Furthermore, decreased plasma exosomal miR?1273a and increased plasma SDCBP levels were found to be associated with worse therapeutic outcomes of patients with advanced NSCLC receiving platinum?based chemotherapy. These findings suggest that miR?1273a is closely associated with the development of cisplatin resistance and may serve as a potential prognostic biomarker and therapeutic target for NSCLC.
Project description:MicroRNAs (miRNAs) have been identified as important posttranscriptional regulators involved in various biological and pathological processes of cells, but their association with tumor chemoresistance has not been fully understood.We detected miR-27a expression in two lung adenocarcinoma cell lines, A549 and A549/CDDP, and then investigated the effects of miR-27a on the metastasis and the chemosensitivity of cancer cells, using both gain- and loss-of-function studies. The correlation between miR-27a level and chemoresistance was further investigated in clinical lung adenocarcinoma specimens.miR-27a was significantly up-regulated in cisplatin-resistant lung adenocarcinoma A549/CDDP cells compared with parental A549 cells. miR-27a regulates epithelial-mesenchymal transition (EMT) and cisplatin resistance in vitro and modulates response of lung adenocarcinoma cells to cisplatin in vivo. Further studies identified Raf Kinase Inhibitory Protein (RKIP) as a direct and functional target of miR-27a. Small interfering RNA-mediated RKIP knockdown revealed similar effects as that of ectopic miR-27a expression, while overexpression of RKIP attenuated the function of miR-27a in lung adenocarcinoma cells. Increased miR-27a expression was also detected in tumor tissues sampled from lung adenocarcinoma patients treated with cisplatin-based chemotherapy and was proved to be correlated with low expression of RKIP, decreased sensitivity to cisplatin, and poor prognosis.Our results suggest that up-regulation of miR-27a could suppress RKIP expression and in turn contribute to chemoresistance of lung adenocarcinoma cells to cisplatin.
Project description:Osteosarcoma is the most common primary malignant bone tumor. Although cisplatin is the primary chemotherapy used in osteosarcoma treatment, the cisplatin resistance remains a big challenge for improving overall survival. The store-operated calcium (Ca2+) entry (SOCE) and its major mediator Stim1 have been shown to be implicated in a number of pathological processes typical for cancer. In this study, we showed that Stim1 expression was significantly increased in chemo-resistant osteosarcoma tissues compared with chemo-sensitivity tissues. Patients with Sitm1 expression exhibited poorer overall survival than Stim1-negative patients. Moreover, un-regulation of Stim1 expression and SOCE were also observed in cisplatin-resistant MG63/CDDP cells compared with their parental cells. Cisplatin treatment obviously reduced Stim1 expression and SOCE in cisplatin-sensitivity MG63 cells, but had no effects on MG63/CDDP cells. In addition, cisplatin resulted in a more pronounced increase of endoplasmic reticulum (ER) stress in MG63 cells than in their resistant variants, which was evidenced by the activation of molecular markers of ER stress, GRP78, CHOP and ATF4. Knockdown of Stim1 using siRNA remarkably enhanced cisplatin-induced apoptosis and ER stress in MG63/CDDP cells, thereby sensitizing cancer cells to cisplatin. On the other hand, overexpression of Stim1 markedly reversed apoptosis and ER stress following cisplatin treatment. Taken together, these results demonstrate that Stim1 as well as Ca2+ entry contributes cisplatin resistance via inhibition of ER stress-mediated apoptosis, and provide important clues to the mechanisms involved in cisplatin resistance for osteosarcoma treatment. Stim1 represents as a target of cisplatin and blockade of Stim1-mediated Ca2+ entry may be a useful strategy to improve the efficacy of cisplatin to treat osteosarcoma.
Project description:As an inflammatory factor, IL-25 has been studied in variouscancers, but it is rarely reported in cancer chemotherapy resistance. Major vault protein (MVP), as a gene associated with lung multidrug resistance, is associated with multiple chemotherapy resistances of lung cancer. However, the relationship between IL-25 and MVP in lung cancer cells has not been studied. In this study, we found that both IL-25 and MVP were elevated expressed in cisplatin-resistant lung adenocarcinoma cell line (A549/CDDP). Silencing of IL-25 resulted in down-regulation of MVP expression and reduced cisplatin tolerance of A549/CDDP cells. Overexpression of IL-25 resulted in increase of MVP expression and the cisplatin tolerance in A549 cells. In addition, we found that the extracellular IL-25 could stimulate the expression of MVP and activate the NF-κB signaling pathway. Further, animal models also confirmed that IL-25 reduced the sensitivity of xenografts to chemotherapy. Taken together, we believe that the up-regulation of IL-25 induces MVP expression contributing to chemotherapy resistances of lung cancer cells. Our findings suggest that interference the expression of IL-25 might be potential treatment strategies for the clinical reversing the chemotherapy resistance.
Project description:Chemoresistance is a serious issue in the therapy of many cancers, but the molecular mechanism is little understood. The mRNA level of occludin (OCLN), a tight junctional protein, was increased in the cisplatin (CDDP), doxorubicin (DXR), 7-ethyl-10-hydroxy-camptothecin, or gemcitabine-resistant human lung adenocarcinoma A549 cells. Here, we investigated the regulatory mechanism and pathophysiological role of OCLN. OCLN was mainly localized at tight junctions in A549 and CDDP-resistant A549 (A549/CDDP) cells. The level of p-Akt in A549/CDDP cells was higher than that in A549 cells, and the mRNA and protein levels of OCLN were suppressed by a phosphoinositide 3-kinase (PI3K)/Akt pathway inhibitor, LY-294002, suggesting that a PI3K/Akt pathway is involved in the elevation of OCLN expression. The overexpression of OCLN in A549 cells decreased paracellular permeability to DXR. Cytotoxicity to CDDP was unaffected by OCLN-overexpression in 2D culture model. In 3D culture model, the spheroid size, hypoxic level, and cell viability were significantly elevated by CDDP resistance, but not by OCLN-overexpression. The accumulation inside the spheroids and toxicity of DXR were correlated with OCLN expression. Our data suggest that OCLN is not directly involved in the chemoresistance, but it enhances chemoresistance mediated by suppression of accumulation of anticancer drugs inside the spheroids.
Project description:Cisplatin resistance is a major obstacle in the treatment of NSCLC, and its mechanism has not been fully elucidated. The objectives of the study were to determine the role of miR-378 in the sensitivity of lung adenocarcinoma cells to cisplatin (cDDP) and its working mechanism. With TargetScan and luciferase assay, miR-378 was found to directly target sCLU. miR-378 and sCLU were regulated in A549/cDDP and Anip973/cDDP cells to investigate the effect of miR-378 on the sensitivity and apoptotic effects of cDDP. The effect of miR-378 upregulation on tumor growth was analyzed in a nude mouse xenograft model. The correlation between miR-378 and chemoresistance was tested in patient samples. We found that upregulation of miR-378 in A549/cDDP and Anip973/cDDP cells significantly down-regulated sCLU expression, and sensitized these cells to cDDP. miR-378 overexpression inhibited tumor growth and sCLU expression in a xenograft animal model. Analysis of human lung adenocarcinoma tissues revealed that the cDDP sensitive group expressed higher levels of miR-378 and lower levels of sCLU. miR-378 and sCLU were negatively correlated. To conclude, we identified sCLU as a novel miR-378 target, and we showed that targeting sCLU via miR-378 may help disable the chemoresistance against cisplatin in lung adenocarcinoma cells.
Project description:Background:Although cisplatin is an effective chemotherapeutic drug that is commonly used for non-small-cell lung cancer (NSCLC) treatment, the drug resistance usually occurs during the long-term use of it. It is urgent to develop strategies to reduce the resistance of NSCLC cells to cisplatin. Methods:Cisplatin-resistant NSCLC cell lines (PC9/R and A549/R) were acquired through long-term exposure of PC9 and A549 cells to cisplatin. QRT-PCR analysis was performed to compare the expression of miR-140 between routine NSCLC cells and cisplatin-resistant NSCLC cells. CCK-8 assay was used to evaluate the effect of miR-140 on the sensitivity of PC9/R and A549/R to cisplatin. Western blot assay and luciferase reporter assay were used to confirm the regulation of miR-140 on SIRT1. Western blot and flow cytometry analysis were performed to evaluate the effect of miR-140 on the apoptosis pathway induced by cisplatin. Results:PC9/R and A549/R exhibited obviously lower sensitivity compared to their parental PC9 and A549 cells, respectively. Furthermore, PC9/R and A549/R cells expressed significantly lower levels of miR-140 compared to their parental PC9 and A549 cells, respectively. However, transfection with miR-140 mimics significantly resensitized the PC9/R and A549/R to cisplatin-induced cytotoxicity. In the mechanism research, we confirmed that SIRT1 was overexpressed and was targeted by miR-140 in PC9/R and A549/R. Furthermore, overexpression of SIRT1 was responsible for the resistance to cisplatin in PC9/R and A549/R cells. Transfection with miR-140 was able to inhibit the expression of SIRT1 and thus inhibited the SIRT1/ROS/JNK pathway. As a result, the PC9/R and A549/R cells restored the sensitivity to cisplatin-induced apoptosis. Conclusion:MiR-140 resensitizes cisplatin-resistant NSCLC cells to cisplatin treatment through the SIRT1/ROS/JNK pathway.
Project description:Introduction:Lung adenocarcinoma (LUAD), which is associated with high morbidity and mortality, is prone to cisplatin resistance, resulting in poor patient prognosis. Long non-coding RNAs (lncRNAs) have complex biological functions in a variety of tumors. Elucidating the underlying molecular mechanisms between lncRNA and cisplatin resistance in LUAD is expected to enable identification of new targets for drug development. Methods:Cell proliferation was measured by CCK-8 assay and cell apoptosis was detected using flow cytometry analysis. Luciferase reporter assay was conducted to determine the interaction between lncRNA and MicroRNA. Gene expression was evaluated by Real-Time Quantitative Reverse Transcription Polymerase Chain Reaction and Western blot analysis. Results:Long non-coding RNA activated by TGF-? (lncRNA-ATB) was shown to be significantly up-regulated in A549 cells resistant to cisplatin/cis-dichlorodiammineplatinum (II) (cis-DDP) (A549/CDDP cells), compared with corresponding levels in parental A549 cells. Overexpression of lncRNA-ATB significantly elevated cisplatin resistance in LUAD cell lines (A549 and H1975 cells), and this was associated with activation of apoptosis-related genes. Conversely, silencing of lncRNA-ATB decreased cisplatin resistance in LUAD cells. Mechanistically, lncRNA-ATB increased expression of ?-catenin by directly binding to MicroRNA-200a (miR-200a), thereby promoting cell survival and cisplatin resistance. Transfection with a miR-200a mimic or treatment with the ?-catenin downstream pathway inhibitor IWR-1 could reverse the phenotypes induced by lncRNA-ATB overexpression. Conclusion:In summary, this study revealed that lncRNA-ATB is dramatically up-regulated in cisplatin-resistant LUAD cell lines, and that lncRNA-ATB facilitates cell survival by targeting the miR-200a/?-catenin pathway in these cells.
Project description:Therapy against nasopharyngeal carcinoma (NPC) is hurdled by chemoresistance. Recent studies found that microRNA (miRNA) are important regulators of cancer resistance. In this study, we aimed to explore the role and mechanism of miR-26b in regulating NPC cisplatin (CDDP) resistance. Real-time PCR was used to evaluate miR-26b levels in CDDP-resistant and CDDP-sensitive NPC cells, as well as human NPC tissues. MiR-26b was ectopically overexpressed in CDDP-resistant cells, followed by monitoring changes in cell viability and apoptosis. Interaction between JAG1 and miR-26b was characterized by dual-luciferase reporter assay. Furthermore, we investigated whether ectopic JAG1 expression reversed CDDP sensitivity induced by miR-26b overexpression. The effect of FOXD3 down-regulation on miR-26b was also evaluated. Our results indicate that miR-26b was lower in the CDDP-resistant NPC cells, human NPC tissue, particularly in secondary metastases. Ectopic expression of miR-26b sensitized NPC cells to CDDP. JAG1 is a target of miR-26b, and its expression is inversely correlated with miR-26b. Overexpression of JAG1 reversed the CDDP sensitivity induced by miR-26b overexpression. FOXD3 expression was also down-regulated in CDDP-resistant NPC. FOXD3 promoted miR-26b expression and down-regulation of FOXD3 suppressed miR-26b expression. Down-regulation of miR-26b is closely correlated with the CDDP resistance in NPC.