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Enhanced Cisplatin Cytotoxicity by RNA Interfering the Excision Repair Cross-complementing Gene 1 in Lung Cancer Cell A549/DDP  [PDF]
Yun GAO,Dan SU,Lisha YING,Wangxia LV
Chinese Journal of Lung Cancer , 2010,
Abstract: Background and objective The excision repair cross-complementing gene 1 (ERCC1), which is important in the repair of cisplatin-DNA adducts, was reported to be related to cisplatin resistance in tumor cells. The aim of this study is to investigate the changes of cisplatin sensitivity by silencing ERCC1 gene in lung cancer cell. Methods The small interfering RNA (siRNA) targeting ERCC1 gene was designed and synthesized, and transfected to lung cancer cell A549/DDP. The mRNA and protein expression levels of ERCC1 were evaluated by RT-PCR and Western blot. The changes of cisplatin sensitivity after RNA interference were examined by methyl thiazolyl assay. Results In A549/DDP cell, the mRNA and protein levels of ERCC1 were decreased and the sensitivity to cisplatin was increased from 12.49 μg/mL to 9.27 μg/mL after transfection. Conclusion The sensitivity to cisplatin of lung cancer cell A549/DDP could be enhanced by RNA interfering ERCC1 gene targeted code 346.
EPS8 Inhibition Increases Cisplatin Sensitivity in Lung Cancer Cells  [PDF]
Lidija K. Gorsic, Amy L. Stark, Heather E. Wheeler, Shan S. Wong, Hae K. Im, M. Eileen Dolan
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0082220
Abstract: Cisplatin, a commonly used chemotherapeutic, is associated with ototoxicity, renal toxicity and neurotoxicity, thus identifying means to increase the therapeutic index of cisplatin may allow for improved outcomes. A SNP (rs4343077) within EPS8, discovered through a genome wide association study of cisplatin-induced cytotoxicity and apoptosis in lymphoblastoid cell lines (LCLs), provided impetus to further study this gene. The purpose of this work was to evaluate the role of EPS8 in cellular susceptibility to cisplatin in cancerous and non-cancerous cells. We used EPS8 RNA interference to determine the effect of decreased EPS8 expression on LCL and A549 lung cancer cell sensitivity to cisplatin. EPS8 knockdown in LCLs resulted in a 7.9% increase in cisplatin-induced survival (P = 1.98×10?7) and an 8.7% decrease in apoptosis (P = 0.004) compared to control. In contrast, reduced EPS8 expression in lung cancer cells resulted in a 20.6% decrease in cisplatin-induced survival (P = 5.08×10?5). We then investigated an EPS8 inhibitor, mithramycin A, as a potential agent to increase the therapeutic index of cisplatin. Mithramycin A decreased EPS8 expression in LCLs resulting in decreased cellular sensitivity to cisplatin as evidenced by lower caspase 3/7 activation following cisplatin treatment (42.7%±6.8% relative to control P = 0.0002). In 5 non-small-cell lung carcinoma (NSCLC) cell lines, mithramycin A also resulted in decreased EPS8 expression. Adding mithramycin to 4 NSCLC cell lines and a bladder cancer cell line, resulted in increased sensitivity to cisplatin that was significantly more pronounced in tumor cell lines than in LCL lines (p<0.0001). An EGFR mutant NSCLC cell line (H1975) showed no significant change in sensitivity to cisplatin with the addition of mithramycin treatment. Therefore, an inhibitor of EPS8, such as mithramycin A, could improve cisplatin treatment by increasing sensitivity of tumor relative to normal cells.
Upregulation of microRNA-451 increases cisplatin sensitivity of non-small cell lung cancer cell line (A549)
Hai-Bo Bian, Xuan Pan, Jin-Song Yang, Zhao-Xia Wang, Wei De
Journal of Experimental & Clinical Cancer Research , 2011, DOI: 10.1186/1756-9966-30-20
Abstract: Quantitative RT-PCR assay was performed to detect the expression of miR-451 in 10 pairs of NSCLC and noncancerous tissue samples. pcDNA-GW/EmGFP-miR-451 was stably transfected into NSCLC cell line (A549). Then, the effects of miR-451 upregulation on growth, colony formation and apoptosis of A549 cells were investigated. Finally, the effects of miR-451 upregulation on in vitro and in vivo sensitivity of A549 cells of DDP were also determined.The level of miR-451 expression in NSCLC tissues was significantly higher than that in corresponding noncancerous tissues. Ectopic overexpression of miR-451 could significantly inhibit growth and induce apoptosis of A549 cells. Moreover, ectopic overexpression of miR-451 could sensitize A549 cells to DDP possibly by increasing DDP-induced apoptosis which might be associated with the inactivation of Akt signaling pathway.This study demonstrated for the first time that combination of DDP application with miR-451 upregulation might be a potential strategy for the treatment of human NSCLC.NSCLC accounts for the majority of lung cancer cases and chemotherapy has been the mainstay of treatments of lung cancers [1]. Up to date, DDP still remains the most widely used first-line chemotherapeutic agent for NSCLC treatment. However, continuous infusion or multiple administration of DDP often cause severe side effects, including myelosuppression, asthenia, and gastrointestinal disorders, as well as long-term cardiac, renal, and neurological consequences [2]. Therefore, improving the sensitivity to drug doses strategies is still a challenge for chemotherapy efficacy. Novel therapeutic modalities combining genetic and chemotherapeutic approaches will play important roles in the fight against cancer in future.MicroRNAs (miRNAs) are small, endogenous non-coding RNAs that have been identified as post-transcriptional regulators of gene expression. MiRNAs exert their functions through imperfect base-pairing with the 3'-untranslated region (3'-UTR)
Ku80 is highly expressed in lung adenocarcinoma and promotes cisplatin resistance  [cached]
Ma Qingshan,Li Ping,Xu Minyu,Yin Jinzhi
Journal of Experimental & Clinical Cancer Research , 2012, DOI: 10.1186/1756-9966-31-99
Abstract: Background Ku80 is crucially implicated in DNA repair, apoptosis, and chemoresistance. In this study, we aimed to assess the expression of Ku80 in clinical lung adenocarcinoma specimens, and investigate its role in the regulation of cisplatin sensitivity in cisplatin resistant human lung adenocarcinoma cells A549/DDP. Methods Tumor specimens and medical records of 106 patients with operable lung adenocarcinoma were obtained from 1998 to 2003. Ku80 mRNA and protein levels of the tumor samples, cultured human lung adenocarcinoma cells A549 cells and their cisplatin resistant variant A549/DDP cells were examined by reverse transcription PCR and western blot analysis. Ku80-specific siRNA or control scramble siRNA was transfected into A549/DDP cells, then cell sensitivity to cisplatin was examined by 3-(4,5-dimethylthia-zol-2-yl)-2,5-diphenyltetrazolium bromide assay and apoptosis was assessed by flow cytometric analysis. In addition, the levels of cleaved caspase-3 and cleaved PARP in the treated cells were detected by western blot analysis. Results Total 83.3% (20/24) cisplatin-resistant tumors had high Ku80 expression, while 8.3% (4/48) cisplatin-sensitive tumors had high Ku80 expression (p < 0.01). Univariate analysis indicated that overall survival and progression-free survival were significantly better in lung adenocarcinoma patients with low vs. high Ku80 expression level (p < 0.01). Ku80 mRNA and protein expression levels were significantly increased in A549/DDP cells compared to parental A549 cells. siRNA mediated knockdown of Ku80 resensitized A549/DDP cells to cisplatin-induced apoptosis. Conclusions Ku80 expression level could predict the outcome and the sensitivity to cisplatin-based chemotherapy in patients with lung adenocarcima. Ku80-siRNA could be utilized as a therapeutic strategy to resensitize nonresponders to cisplatin.
SIRT1 Influences the Sensitivity of A549 Non-small Cell Lung Cancer Cell Line to ?Cisplatin via Modulating the Noxa Expression  [PDF]
Bin CAO, Xiaofeng HE, Wengong WANG, Minke SHI
- , 2016, DOI: : 10.3779/j.issn.1009-3419.2016.02.01
Abstract: Background and objective The resistance of non-small cell lung cancer cells to cisplant is a common clinical phenomenon which could induce a poor therapeutic effect and should be difficult problem to be solved. SIRT1 and Noxa expression are associated with the chemotherapy for tumors. The present study focused on how SIRT1 expression influence the senstivity of non-small cell lung cancer cells and dissected the potential mechanism involved with Noxa. Methods The difference of SIRT1 and Noxa expression between A549 cells and A549/DDP cells was detected by real-time quantitative PCR (qRT-PCR) and Western blot. SIRT1 targeted siRNA was uesed to inhibit the SIRT1 expression in A549/DDP, after transfection, Cell Titer Blue assay, flow cytometry were performed to analyze the cell viability, cell cycle and cell apoptosis in order to reveal the effect of inhibition of SIRT1 on sensitivity of A549/DDP cells to cisplant. Moreover, the expression changes of Noxa in A549/DDP cells after siRNA treatment were detected by qRT-PCR and Western blot. Results There was a significant difference in senstivity to cisplant between A549 and A549/DDP cells. Compared with A549 cells, the A549/DDP cells showed a higher SIRT1 expression and lower Noxa expression. After transfected with SIRT1 targeted siRNA, the cell viability decreased accompanied with a increasing apoptosis rate, meanwhile, higher percent of G2/M phase was detected after the 4 μg/mL cisplant treatment. Further more, inhibition of SIRT1 could induce the Noxa expression in A549/DDP cells. Conclusion Higher SIRT1 expression may induce resistance to cisplant in A549 cells. SIRT1 inhibition may improve the sensitivity of A549/DDP cells to cisplantin though modulating the Noxa expression.
RNA Interference Targeting GRP75 Decreases Cisplatin Resistance in Human Lung Adenocarcinoma Cell  [PDF]
Sien SHI,Zefeng HE,Jianchun CAI,Jiangfeng QIU
Chinese Journal of Lung Cancer , 2011,
Abstract: Background and objective GRP75, a member of HSPs which is overexpressed in some resistant cancer cells, is a molecular chaperone mainly located in mitochondrial membrane. The aim of this study is to investigate the role of GRP75 on the resistant mechanisms of cancer cells by downregulating GRP75 expreesion via RNAi approach. Methods Cisplatin-resistant cell A549/CDDP was established from their parental human lung adenocarcinoma cell line A549 by combining gradually increasing concentrations of cisplatin with high dosage impact. The shRNA for GRP75 was transfected into A549 and A549/CDDP cells by lentivirus. Western blot and methyl thiazolyl tetrazolium (MTT) assay were applied to detect the influence of silencing GRP75 expression on sensitivity of the cells to cisplatin. Results The infection rate of six groups were all over 90%. After infection, the level of expression of GRP75 in both A549 and A549/CDDP were down-regulated (P < 0.05); the level of expression of p53 in A549/CDDP was up-regulated (P < 0.05) and the level of expression of bcl-2 of A549/CDDP was down-regulated (P < 0.05). The resistance index of A549/CDDP before and after infection were 21.52 and 4.14 respectively. Conclusion Cisplatin resistance of lung cancer cells is associated with overexpression of GRP75 gene, which could regulate the expressions of p53 and bcl-2.
PPAR-γ Silencing Inhibits the Apoptosis of A549 Cells by Upregulating Bcl-2  [cached]
Jingyu YANG
Chinese Journal of Lung Cancer , 2013, DOI: 10.3779/j.issn.1009-3419.2013.03.02
Abstract: Background and objective Drug resistance is the one of primary causes of death in patients with lung cancer, PPAR-γ could induce the apoptosis and reverse drug resistance. The aim of this study is to investigate the expression of PPAR-γ on cisplatin sensitivity and apoptosis response of human lung cancer cell line A549. Methods Reconstruction of PPAR-γ silencing A549 cells (A549/PPAR-γ(-)) by siRNA. MTT assay was employed to determine the effect of cisplatin on the proliferation of A549/PPAR-γ(-), flow cytometry to determine the effect of cisplatin on the cell apoptosis, Western blot to determine the change of phosphorylation of Akt, caspase-3 and expression of bcl-2/bax. Finally, RT-PCR was employed to determine the transcriptional level of bcl-2. Results Two PPAR-γ silencing A549 cell clones were established successfully, and the expression of PPAR-γ was downregulated significantly as confirmed by RT-PCR and Western blot. After PPAR-γ silencing, the resistance of these two A549 clones to cisplatin was increased by 1.29-fold and 1.60-fold respectively. Flow cytometry showed that the apoptosis rate was decreased, and Western Blot showed that the phosphorylation of Akt and expression of bcl-2/bax were upregulated, caspase-3 was downregulated. Finally, RT-PCR showed that the transcriptional level of bcl-2 was upregulated as well. Conclusion Downregulation of PPAR-γ in A549 cells led to increase of cisplatin resistance. One of the mechanisms was upregulatin of phosphorylation of Akt and expression of bcl-2, which inhibited the apoptosis of cells. The downregulation of PPAR-γ is a possible mechanism that leads to the clinical drug resistance of cancer.
Effects of apoptosis protein XIAP inhibitor on the apoptosis and sensitivity of chemotherapy in A549 cell  [cached]
Fuhui CHEN,Hongyan QU,Guangjie SUI
Chinese Journal of Lung Cancer , 2008,
Abstract: Background and objective X-linked inhibitor of apoptosis protein (XIAP) is a newly discovered inhibitor of apoptosis protein which prevents apoptosis by inhibiting the activation of caspase. After down-regulating XIAP gene expression in A549 cells, a non-small cell lung cancer (NSCLC) cell lines, we investigated the role of XIAP specific siRNA in apoptosis and chemotherapy sensitivity. Methods The mRNA levels of XIAP gene in A549 cells were assessed using a semi-quantitative reverse transcriptase-PCR (RT-PCR). The expression vector of XIAP small interfering RNA (XIAP siRNA)was constructed and transfected into A549 cells. The transfection was proved effective by the fluorescence microscope. Cell proliferation and cell killing rate after chemotherapeutics treatment were investigated by MTT assay. The rate of apoptosis was detected by flow cytometry assay. Results XIAP siRNA construction was proved successful by enzyme digestion and DNA sequencing. The transfection efficiency in A549 cells from positive transfection group and negative transfection group had no differences. Compared to those in cell from control group, the level of XIAP mRNA expression was significantly decreased, the inhibition activity of Cisplatin was significantly higher in cells from positive transfection group. Proliferation of cells from positive transfection group was significantly inhibited after 24, 48, 72, 96 hours . The rates cell killing and apoptosis in cells from positive transfection group caused by Cisplatin were significantly higher compared to those cells from control group. Conclusion The increased expression of XIAP in NSCLC can inhibit the apoptosis of NSCLC cells and result in NSCLC chemotherapy drug resistance. XIAP siRNA could inhibit the NSCLC cell growth specifically, down-regulation of XIAP gene expression promote apoptosis and increase the chemotherapy sensitivity of NSCLC. XIAP siRNA sequence might become a therapeutic target of NSCLC.
The Long Noncoding RNA HOTAIR Contributes to Cisplatin Resistance of Human Lung Adenocarcinoma Cells via downregualtion of p21WAF1/CIP1 Expression  [PDF]
Zhili Liu, Ming Sun, Kaihua Lu, Jing Liu, Meiling Zhang, Weiqin Wu, Wei De, Zhaoxia Wang, Rui Wang
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0077293
Abstract: HOTAIR, a long intervening non-coding RNA (lincRNA), associates with the Polycomb Repressive Complex 2 (PRC2) and is reported to reprogram chromatin organization and promote tumor progression. However, little is known about the roles of this gene in the development of chemoresistance phenotype of lung adenocarcinoma (LAD). Thus, we investigated the involvement of HOTAIR in the resistance of LAD cells to cisplatin. In this study, we show that?HOTAIR?expression was significantly upregulated in cisplatin-resistant A549/DDP cells compared with in parental A549 cells. Knockdown of?HOTAIR by RNA interference could resensitize the responses of A549/DDP cells to cisplatin both in vitro and in vivo. In contrast, overexpression of HOTAIR could decrease the sensitivity of A549 and SPC-A1 cells to cisplatin. We also found that the siRNA/HOTAIR1-mediated chemosensivity enhancement was associated with inhibition of cell proliferation, induction of G0/G1 cell-cycle arrest and apoptosis enhancement through regulation of p21WAF1/CIP1 (p21) expression. Also, pcDNA/p21or siRNA/p21 could mimic the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on the sensitivity of LAD cells to cisplatin. Importantly, siRNA/p21 or pcDNA/p21 could partially rescue the effects of siRNA/HOTAIR1 or pcDNA/HOTAIR on both p21 expression and cisplatin sensitivity in LAD cells. Further, HOTAIR was observed to be significantly downregulated in cisplatin-responding LAD tissues, and its expression was inversely correlated with p21 mRNA expression. Taken together, our findings suggest that upregulation of HOTAIR contributes to the cisplatin resistance of LAD cells, at least in part, through the regulation of p21 expression.
Conditional Replication Adenovirus Sensitizes A549 Cancer Cell to Cisplatin  [PDF]
Yanan Liu, Yinghui Huang
Engineering (ENG) , 2012, DOI: 10.4236/eng.2012.410B043

Objective: The aim of the study was to improve the therapeutic effect for lung cancer using a synergetic strategy of adenovirus-based gene therapy combined with chemotherapy. Methods: A conditional replication adenovirus(CRAd) was employed to treat the A549 lung cancer cells and cisplatin-resisted A549(A549-DDP) cells. In vitro MTS / PMS assay were used to evaluate the cell viability. PCR were used to detect expression of Coxsackie receptor (CAR) and Multidrug resistance(MDR) gene. The in vivo anti-tumor effect of CRAd and cisplatin was evaluated using a subcutaneous mouse model. Results: The CRAd sensitizes A549 cancer cells to cisplatin. The mechanism of enhanced cell growth inhibition is associated with the increased CAR and MDR expression. Conclusion: Our approach is better than the conventional gene therapy and chemotherapy strategy.

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