二甲双胍对肺腺癌细胞Nrf2基因表达的调控作用研究

目的探讨Nrf2基因在对顺铂耐药的肺腺癌细胞中的表达及二甲双胍对其调控作用的研究。方法应用细胞增殖实验(CCK-8)和流式细胞仪,检测二甲双胍对肺腺癌A549和A549/DDP细胞增殖抑制率、凋亡率,RealtimePCR检测相关耐药基因表达,Western Blot法检测相关蛋白水平表达。结果 Nrf2、GSTA1、ABCC1等基因在A549、A549/DDP细胞间表达存在显著差异,在A549/DDP细胞中呈高表达;二甲双胍能够抑制A549、A549/DDP细胞Nrf2及下游GSTA1、ABCC1基因的表达,并进一步抑制细胞增殖和诱导A549/DDP细胞凋亡。结论 Nrf2基因及下游基因在肺腺癌细胞不同细胞株表达存在差异,二甲双胍可影响其表达。     

IL-6 signaling contributes to cisplatin resistance in non-small cell lung cancer via the up-regulation of anti-apoptotic and dna repair associated molecules

Cisplatin-based chemotherapy is currently the most effective treatment regimen for non-small cell lung cancer (NSCLC), but eventually tumor resistance develops which limits its success. The potential implication of IL-6 signaling in the cisplatin resistance of NSCLC was explored by testing whether NSCLC cells with different levels of intracellular IL-6 show different responses to the cytotoxic treatment of cisplatin. When the cisplatin cytotoxicity of the IL-6 knocked down human NSCLC cells (A549IL-6si and H157IL-6si) were compared with their corresponding scramble control cells (A549sc and H157sc), higher cisplatin cytotoxicity was found in IL-6 si cells than sc cells. Subcutaneous xenograft mouse models were developed using a pair of A549sc and A549IL-6si cells. When the tumor grew to about 400 mm², mice were treated with cisplatin and tumor regression was monitored. Higher tumor regression was detected in the A549IL-6si xenografts compared to A549sc xenografts following cisplatin treatment. Immunostaining study results from tumor tissues also supported this finding. Expression of anti-apoptotic proteins Bcl-2 and Mcl-1 and DNA repair associated molecules ATM, CHK1, TP73, p53, and ERCC1 were significantly up regulated in cisplatin-treated A549sc and H157sc cells, but no increase was detected in A549IL-6si and H157IL-6si cells. Further inhibitor studies revealed that up regulation of these molecules by IL-6 may be through activation of IL-6 downstream signaling pathways like Akt, MAPK, Stat3, and Erk. These results provide potential for combining cisplatin and inhibitors of IL-6 signaling or its downstream signaling pathway as a future therapeutic approach in preventing development of cisplatin resistant NSCLC tumors.     

PI3K/AKT通路通过Nrf2途径调控PD-L1在非小细胞肺癌中的表达

背景与目的：程序性死亡［蛋白］配体-1（programmed death ligand-1,PD-L1）在肿瘤细胞中的表达对肿瘤逃避机体免疫监视具有重要的意义,其表达通常受MAPK、PI3K-AKT或STAT3等多条通路的影响,但这些通路具体经哪个关键环节尚不明确。拟通过PI3K/AKT信号通路对肺癌细胞A549、H460中PD-L1表达的调控具体机制进行探究。方法：使用shRNA技术选择性地沉默A549、H460细胞中的HEB、HTF4、Nrf2及FOXO3a等基因,构建缺陷细胞株;将目的基因PD-L1的3’UTR区域构建至pGL3-basic载体中,通过luciferase双报告基因体系检测PD-L1转录激活情况,并使用实时荧光定量聚合酶链反应（real-time fluorescence quantitative polymerase chain reaction,RTFQ-PCR）技术验证细胞内PD-L1基因转录的情况;通过蛋白质印迹法（Western blot）检测细胞内PD-L1的总表达情况;免疫细胞与肿瘤细胞共培养,检测Nrf2基因敲除前后,人外周血单个核细胞（peripheral blood mononuclear cells,PBMC）对A549、H460细胞株的杀伤效果。结果：经10μg/mL的insulin刺激后,A549、H460细胞株中蛋白激酶B（AKT）基因磷酸化水平显著增强,伴随着PD-L1基因的转录水平和表达水平显著增强（P<0.001）,在HEB、HTF4及FOXO3a基因敲除的细胞株中,情况相似,而在Nrf2基因敲除的细胞株A549（A549 ^Nrf2- ）和H460（H460 ^Nrf2- ）细胞株中,PD-L1的转录和表达水平则与阴性对照组一样未见明显变化（P>0.05）;活性氧（reactive oxygen species,ROS）诱导剂isoproterenol能提高A549、H460细胞株中PD-L1基因的转录和表达水平,而ROS在被NAC解除后,PD-L1的转录和表达水平显著下调,进一步证明Nrf2对PD-L1基因的转录调控作用;wortmannin能逆转insulin刺激引起的AKT磷酸化水平增加,促进PD-L1基因的转录水平和表达水平下降,表明PD-L1的调控与AKT激活程度相关;在insulin和wortmannin的分别干预下,A549、H460细胞株中的Nrf2磷酸化水平能随着AKT磷酸化水平改变而改变,二者相关系数r分别为0.86和0.93,为强正相关;Nrf2敲除后,A549、H460细胞株与PBMC共培养后凋亡数量显著增加。结论：PI3K/AKT通路对肺癌细胞A549、H460中PD-L1表达具有关键调控作用,该调控是通过磷酸化激活Nrf2而实现的。     

Combination treatment with arsenic trioxide and sulindac enhances apoptotic cell death in lung cancer cells via activation of oxidative stress and mitogen-activated protein kinases

Arsenic trioxide (As2O3) has been introduced to the treatment of acute promyelocytic leukemia (APL), and has also been shown to induce apoptosis in a variety of solid tumor cell lines, including non-small cell lung cancer. However, the prohibitively high concentration required for the induction of apoptotic cell death in many solid tumor cells is unacceptable for clinical utilization due to the excessive toxicity associated with this dose. Sulindac is known to enhance the cellular responsiveness of tumors toward chemotherapeutic drugs. Herein, we demonstrated that combination treatment with As2O3 and sulindac resulted in a synergistic augmentation of cytotoxicity in H157 lung cancer cells, which was revealed by apoptotic induction as demonstrated by an increase in the sub-G0/G1 fraction. In addition, combination treatment with As2O3 and sulindac increased reactive oxygen species (ROS) and oxidative stress, as evidenced by the heme oxygenase-1 (HO-1) expression and mitogen-activated protein kinase (MAPK) phosphorylation. MAPK inhibitors blocked the induction of HO-1 by combination treatment. Inhibitors of p38 and JNK partially inhibited the augmented cell death whereas the ERK inhibitor showed poor inhibition. Combination treatment with As2O3 and sulindac induced oxidative DNA damage in a time-dependent fashion, which was evaluated by H2AX phosphorylation along with HO-1 induction.     

BPTF promotes tumor growth and predicts poor prognosis in lung adenocarcinomas

BPTF, a subunit of NURF, is well known to be involved in the development of eukaryotic cell, but little is known about its roles in cancers, especially in non-small-cell lung cancer (NSCLC). Here we showed that BPTF was specifically overexpressed in NSCLC cell lines and lung adenocarcinoma tissues. Knockdown of BPTF by siRNA significantly inhibited cell proliferation, induced cell apoptosis and arrested cell cycle progress from G1 to S phase. We also found that BPTF knockdown downregulated the expression of the phosphorylated Erk1/2, PI3K and Akt proteins and induced the cleavage of caspase-8, caspase-7 and PARP proteins, thereby inhibiting the MAPK and PI3K/AKT signaling and activating apoptotic pathway. BPTF knockdown by siRNA also upregulated the cell cycle inhibitors such as p21 and p18 but inhibited the expression of cyclin D, phospho-Rb and phospho-cdc2 in lung cancer cells. Moreover, BPTF knockdown by its specific shRNA inhibited lung cancer growth in vivo in the xenografts of A549 cells accompanied by the suppression of VEGF, p-Erk and p-Akt expression. Immunohistochemical assay for tumor tissue microarrays of lung tumor tissues showed that BPTF overexpression predicted a poor prognosis in the patients with lung adenocarcinomas. Therefore, our data indicate that BPTF plays an essential role in cell growth and survival by targeting multiply signaling pathways in human lung cancers.     

Enhancing effect of connexin 32 gene on vinorelbine-induced cytotoxicity in A549 lung adenocarcinoma cells

Purpose Connexin (Cx) genes exert negative growth effects on tumor cells with certain cell specificity, and tumor-suppressive effects of the Cx genes contribute to enhancement of chemotherapeutical agents-induced cytotoxicity in some cancer cells. Since we and others have been reported that Cx32 acts as a tumor suppressor gene in lung adenocarcinomas, this study was undertaken to estimate if the combination of Cx32-dependent tumor-suppressive effect and vinorelbine (VBN), a chemotherapeutic agent which has been utilized for clinical lung adenocarcinoma treatment, could be effective in enhancing the sensitivity of the lung cancer to VBN treatment. Methods We established the A549 cells (a human lung adenocarcinoma cell line) which had stable expression of Cx32 and estimated effect of Cx32 on VBN-induced cytotoxicity in the established cells. Results Cx32 expression in A549 cells significantly potentiated VBN-induced cytotoxicity on the cells due to enhancement of apoptosis induction. The enhancing cytotoxicity in A549 cells by Cx32 mainly depended on a decrease in expression of multi-drug resistance-1 (MDR-1) gene responsible for reduction of VBN accumulation into the cells. We also observed that silencing of Cx32 by siRNA treatment elevated the expression level of MDR-1 mRNA in A549 cells and that inhibition of MDR-1 gene product-dependent function enhanced VBN-induced cytotoxicity in the cells. Conclusion These results suggest that Cx32 contributes to the enhancement of VBN-induced cytotoxicity in A549 cells via the reduction of MDR-1 expression.     

High expression of heme oxygenase-1 is associated with tumor invasiveness and poor clinical outcome in non-small cell lung cancer patients

Background

Heme oxygenase-1 (HO-1), a rate-limiting enzyme in heme catabolism, is known to play a role in the protection of cells against oxidative stress, inflammation, anomalous proliferation and apoptosis. As yet, the role of HO-1 expression in non-small cell lung cancer (NSCLC) development and metastasis remains unclear and insufficient data are available regarding its impact on the prognosis of NSCLC patients.

Methods

Seventy NSCLC patients who underwent surgical resection were included in this HO-1 expression study and, concomitantly, clinical parameters were collected. Two lung adenocarcinoma cell lines (A549 and H441) were used to assess both invasive and migratory parameters in vitro.

Results

NSCLC patients with a high HO-1 expression ratio (tumor tissue/normal tissue) (> 1) exhibited a significantly poorer prognosis and a higher metastatic rate compared to those with a low HO-1 expression ratio (p?<?0.05). The invasive and migratory abilities of A549 and H441 cells significantly increased after exogenous HO-1 over-expression and significantly decreased after siRNA-mediated HO-1 expression silencing. HO-1 up- and down-regulation also positively correlated with the expression of metastasis-associated proteins EGFR, CD147 and MMP-9. In addition, we found that HO-1 expression can be inhibited by PI3K and AKT inhibitors, but not by MAPK inhibitors.

Conclusions

HO-1 is a poor prognostic NSCLC predictor and its over-expression may increase the metastatic potential of NSCLC. Based on our findings and those of others, HO-1 may be considered as a novel NSCLC therapeutic target.     

Simultaneous targeting of ATM and Mcl-1 increases cisplatin sensitivity of cisplatin-resistant non-small cell lung cancer

Development of cisplatin-resistance is an obstacle in non-small cell lung cancer (NSCLC) therapeutics. To investigate which molecules are associated with cisplatin-resistance, we analyzed expression profiles of several DNA repair and anti-apoptosis associated molecules in parental (A549P and H157P) and cisplatin-resistant (A549CisR and H157CisR) NSCLC cells. We detected constitutively upregulated nuclear ATM and cytosolic Mcl-1 molcules in cisplatin-resistant cells compared with parental cells. Increased levels of phosphorylated ATM (p-ATM) and its downstream molecules, CHK2, p-CHK2, p-53, and p-p53 were also detected in cisplatin-resistant cells, suggesting an activation of ATM signaling in these cells. Upon inhibition of ATM and Mcl-1 expression/activity using specific inhibitors of ATM and/or Mcl-1, we found significantly enhanced cisplatin-cytotoxicity and increased apoptosis of A549CisR cells after cisplatin treatment. Several A549CisR-derived cell lines, including ATM knocked down (A549CisR-siATM), Mcl-1 knocked down (A549CisR-shMcl1), ATM/Mcl-1 double knocked down (A549CisR-siATM/shMcl1) as well as scramble control (A549CisR-sc), were then developed. Higher cisplatin-cytotoxicity and increased apoptosis were observed in A549CisR-siATM, A549CisR-shMcl1, and A549CisR-siATM/shMcl1 cells compared with A549CisR-sc cells, and the most significant effect was shown in A549CisR-siATM/shMcl1 cells. In in vivo mice studies using subcutaneous xenograft mouse models developed with A549CisR-sc and A549CisR-siATM/shMcl1 cells, significant tumor regression in A549CisR-siATM/shMcl1 cells-derived xenografts was observed after cisplatin injection, but not in A549CisR-sc cells-derived xenografts. Finally, inhibitor studies revealed activation of Erk signaling pathway was most important in upregulation of ATM and Mcl-1 molcules in cisplatin-resistant cells. These studies suggest that simultaneous blocking of ATM/Mcl-1 molcules or downstream Erk signaling may recover the cisplatin-resistance of lung cancer.     

siRNA Silencing EZH2 Reverses Cisplatin-resistance of Human Non-small Cell Lung and Gastric Cancer Cells

Clinical resistance to chemotherapeutic agents is one of the major hindrances in the treatment of humancancers. EHZ2 is involved in drug resistance and is overexpressed in drug-resistant cancer cell lines. In this study,we investigated the effects of EHZ2 on cisplatin -resistance in A549/DDP and AGS/DDP cells. EHZ2 mRNA andprotein were found to be significantly overexpressed in A549/DDP and AGS/DDP cells, compared to parentalcells. EHZ2 siRNA successfully silenced EHZ2 mRNA and protein expression. Proliferation was inhibited anddrug resistance to cisplatin was improved. Flow cytometry showed that silencing of EHZ2 arrested A549/DDPand AGS/DDP cells in the G0/G1 phase, increasing apoptosis, rh-123 fluorescence intensity and caspase-3/8activities. Silencing of EHZ2 also significantly reduced the mRNA and protein expression levels of cyclin D1 andMDR1,while up-regulating p15, p21, p27 and miR-218 in A549/DPP cells. Furthermore, silencing of EHZ2 alsosignificantly increased the expression level of tumor suppressor factor miR-218. We also found down-regulatingEHZ2 expression increased methylation in A549/DDP and AGS/DDP cells. This study demonstrates that drugresistance can be effectively reversed in human cisplatin-resistant lung and gastric cancer cells through deliveryof siRNAs targeting EHZ2.     

Effects of granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor on lung cancer: roles of cyclooxygenase-2

We examined the effects of granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on the lung cancer cell lines PC-9, LA-1 and A549. In addition, we examined if the effects of the cytokines on the cell lines are mediated by activation of cyclooxygenase (COX)-2. The three cell lines did not constitutively produce either G-CSF or GM-CSF. G-CSF did not influence cell growth in the three cell lines, while GM-CSF increased cell growth in the A549 and LA-1 lines. G-CSF and GM-CSF dose-dependently decreased cell death in the three cell lines. RT-PCR demonstrated GM-CSF receptor expression in the three lung cancer cell lines, whereas the G-CSF receptor exists only in the PC-9 line. We suggest that G-CSF might rescue the tumor cells from cytotoxicity due to serum deprivation through cellular pathways independent of the G-CSF receptor. G-CSF and GM-CSF increased cyclooxygenase-2 (COX-2) expression in PC-9 and LA-1 cells whereas they decreased COX-2 expression in A549 cells. The COX-2 inhibitor NS-398 increased cell death in PC-9 and LA-1 cells, whereas it decreased cell death in A549 cells. PC-9 and LA-1 clones transfected with sense G-CSF- or GM-CSF showed an increase in COX-2 expression, while COX-2 expression was decreased in transfected A549 clones. COX-2 expression was increased in anti-sense G-CSF- and GM-CSF-transfected A549 clones. Thus, although COX-2 activation seems to induce different biological behavior depending on the cell type, we propose that G-CSF and GM-CSF might accelerate tumor progression by directly regulating COX-2 expression, independently of an autocrine mechanism.     

Synergistic antitumor activity of TRAIL combined with chemotherapeutic agents in A549 cell lines in vitro and in vivo

Purpose To investigate the synergistic cytotoxicity of TRAIL in combination with chemotherapeutic agents in A549 cell lines, we systematically evaluated the cytotoxicity of TRAIL alone and TRAIL in combination with cisplatin, paclitaxel (Taxol) or actinomycin D in A549 cell lines in vitro and in vivo, and whether the sensitivity was correlated with the expression level of TRAIL receptors.Methods We investigated the cytotoxicity of TRAIL alone and the synergistic antitumor effects of TRAIL in combination with chemotherapeutic agents in A549 cells by crystal violet staining and FACS in vitro. The expression levels of DR4, DR5, DcR1 and DcR2 were measured in TRAIL-treated and chemotherapeutic agent-treated A549 cells by Western blotting. The growth inhibition of tumors was evaluated in terms of incidence, volume and weight in a A549-implanted nude mice model.Results Chemotherapeutic agents cisplatin (5.56 g/ml), Taxol (10 and 30 g/ml) or actinomycin D (9.26, 83.3 and 750 ng/ml) augmented the cytotoxicity of TRAIL in A549 cell lines within a range of concentrations of TRAIL (1.98–160 ng/ml) in vitro. The expression levels of DR4 and DR5 were not significantly different and the expression of DcR2 was slightly downregulated, but the expression of DcR1 was not detected in non-treated, TRAIL-treated and chemotherapeutic agent-treated A549 cells. The rates of tumor inhibition following treatment with TRAIL alone (15 mg/kg per day, daily for 10 days) and TRAIL/cisplatin (15 mg/kg per day TRAIL, daily for 10 days; 1.5 mg/kg per day cisplatin, daily for 10 days with 7-day intervals) were 28.3% and 76.8% by tumor weight (P<0.05 for TRAIL alone versus control, P<0.05 for TRAIL/cisplatin versus cisplatin alone and TRAIL alone) on day 65 in vivo.Conclusion TRAIL in combination with chemotherapeutic agents cisplatin, Taxol or actinomycin D exerted synergistic antitumor effects in A549 cell lines in vitro and TRAIL/cisplatin demonstrated synergistic antitumor effects in vivo. The expression levels of TRAIL receptors suggested that the synergistic effects of TRAIL in combination with chemotherapeutic agents are not at the receptor level in A549 cell lines.Abbreviations TNF Tumor necrosis factor - TRAIL TNF-related apoptosis-inducing ligand - NSCLC Non-small cell lung cancer - DR Death receptor - DcR Decoy receptor - IB Inclusion body - FACS Fluorescence activated cell sorter - PI Propidium iodide - IR Rate of inhibition - PMSF Phenyl methyl sulfonyl fluoride     

非小细胞肺癌中PAX6表达及其对肿瘤细胞增殖与侵袭作用机制研究

背景与目的：转录因子PAX6主要表达于胚胎期,不同肿瘤中PAX6呈现高表达,通过不同的信号通路发挥肿瘤抑制或促进作用。检测PAX6在非小细胞肺癌(non-small cell lung cancer,NSCLC)中的表达,通过RNAi下调其表达,研究PAX6对肿瘤细胞侵袭和增殖力以及cyclin E、p38表达水平的影响。方法：应用蛋白印记检测86例NSCLC肿瘤组织及癌旁配对组织以及2例细胞系中PAX6的表达情况。应用PAX6特异性siRNA序列,转染NSCLC细胞系A549,MTT法、Transwell小室、划痕实验检测转染前后细胞增殖、侵袭和迁移能力的对比。蛋白质印记法(Western blot)检测转染前后cyclin E及p38表达情况。结果：对比癌旁组织及正常人支气管上皮细胞16HBE,PAX6在NSCLC组织及A549细胞系中显著高表达。选取高效siRNA序列转染细胞系后,PAX6表达的下调抑制了肿瘤细胞的增殖及集落形成,G1期细胞比例增加,细胞侵袭及迁移力均下降。PAX6基因敲低细胞cyclin E及p38活性受到抑制,表达下调。结论：PAX6通过调控MAPK通路及cyclin E的表达加速肿瘤细胞的增殖、侵袭及迁移,是潜在的NSCLC诊疗靶点。     

siRNA-mediated MDM2 inhibition sensitizes human lung cancer A549 cells to radiation

MDM2 (murine double minute 2) is well-documented to play a key role in radiation response and tumor radiosensitivity, thus offering an attractive clinic drug target to enhance tumor sensitivity to anti-cancer radiotherapy. In this study, we designed and tested two siRNA fragments against human MDM2 in non-small cell human lung cancer A549 cells. Transfection of mammalian expression vector pUR/U6 containing either MDM2 siRNA1 or siRNA2 fragment was shown to reduce MDM2 mRNA levels by 72% and 31%, respectively. Western blotting detected a similar inhibition of MDM2 protein levels in cells transfected with MDM2 siRNA1. A549 cells transfected with the expression vector for siRNA1 significantly decreased cell proliferation and rendered cells more sensitive to radiation. The basal apoptotic and necrotic cells, 1% and 2%, respectively, detected among A549 cells were increased to 2.6% and 14.4% after gamma-irradiation with 5 Gy. Transfection of MDM2 siRNA1 induced 30.1% apoptosis and 12.7% necrosis while combined treatment of siRNA1 and 5-Gy radiation increased apoptosis and necrosis to 45.9% and 15.2%, respectively. These data provide the first evidence that specific siRNA fragment (MDM2 siRNA1) targeting human MDM2 mRNA is able to enhance lung cancer radiosensitivity.     

siRNA沉默STMN1基因抑制肺癌细胞增殖与侵袭的机制探讨

目的:观察下调肺癌细胞中STMN1基因的表达对癌细胞增殖、侵袭及PI3K/AKT信号通路的影响。方法:以人正常肺细胞MRC-5作为对照,通过Western bloting检测人肺癌A549、SPC-A1和H322细胞中STMN1的蛋白表达;STMN1的siRNA转染A549细胞为实验组,另设空白组和阴性组,转染48 h后,Western bloting检测转染效果;CCK8法及Transwell小室分别检测细胞的增殖及侵袭能力;Western bloting检测增殖细胞核抗原（PCNA）、基质金属蛋白酶2（MMP-2）、磷脂酰肌醇3-激酶（PI3K）、磷酸化的丝氨酸苏氨酸激酶（p-AKT）的蛋白表达。结果:STMN1在肺癌A549、SPC-A1和H322细胞中的蛋白表达均显著高于在MRC-5细胞中的表达（P<0.05）,选择A549细胞为研究对象。转染STMN1 siRNA的A549细胞STMN1的蛋白表达显著低于空白组（P<0.05）。与空白组比较,实验组细胞增殖及侵袭能力均显著降低,PCNA、MMP-2、PI3K、p-AKT蛋白表达均显著降低（P<0.05）。结论:siRNA沉默STMN1的表达通过下调PI3K/AKT信号通路降低肺癌细胞增殖及侵袭能力。     

Heme oxygenase-1: a molecular brake on hepatocellular carcinoma cell migration

Hepatocellular carcinoma (HCC) is a fatal disease with great public health impact worldwide. Heme oxygenase (HO)-1 has recently been reported as an important player in tumor angiogenesis and metastasis. However, the role of HO-1 in liver cancer metastasis is unclear. In this study, we explored genetic differences and downstream signal transduction pathways of HO-1 in liver cancer cell lines. HO-1 wild-type and mutant cell lines were generated from human liver cancer cell line HepG2. The overexpression of wild-type HO-1 decreased the migration of HepG2 cells. In contrast, the overexpression of mutant HO-1G143H increased the migration of the cancer cells. Interleukin (IL)-6 is one of the major downstream molecules that mediated this process because IL-6 expression and migration are suppressed by HO-1 and increased when HO-1 is knocked down by shRNA. In addition, we demonstrated carbon monoxide (CO) and p38MAPK are the cofactors in this signal pathway. In vivo animal model demonstrated HO-1 inhibited the tumor growth. In conclusion, in vitro and in vivo data show HO-1 inhibits the human HCC cells migration and tumor growth by suppressing the expression of IL-6. The heme degradation product CO is a cofactor in this process and inhibits p38MAPK phosphorylation.     

Emodin enhances cisplatin sensitivity in non-small cell lung cancer through Pgp downregulation

Cisplatin resistance is one of the main causes of chemotherapy failure and tumor progression in non-small cell lung cancer (NSCLC). Emodin has been demonstrated to induce NSCLC cell apoptosis and act as a potential cancer therapeutic agent. However, whether emodin could affect NSCLC cell sensitivity toward cisplatin remains unclear. The present study aimed to determine the effect of emodin and cisplatin combination on the chemosensitivity of NSCLC cells. A549 and H460 cells were treated with different concentrations of cisplatin and/or emodin. Cell Counting Kit-8, fluorescence microscopy, immunofluorescence assays and flow cytometry were used to determine cell proliferation, drug efflux, DNA damage level and cell apoptosis, respectively. P-glycoprotein (Pgp) and multidrug resistance-associated protein 1 (MRP1) expression was detected by western blotting. The results demonstrated that emodin and cisplatin inhibited the proliferation of A549 and H460 cells. Furthermore, emodin inhibited the drug efflux in A549 and H460 cells in a dose-dependent manner. In addition, emodin enhanced cisplatin-induced apoptosis and DNA damage in A549 and H460 cells. Emodin also decreased Pgp expression in A549 and H460 cells in a dose-dependent manner; however, it had no effect on MRP1 expression. Taken together, the results from the present study demonstrated that emodin can increase A549 and H460 cell sensitivity to cisplatin by inhibiting Pgp expression. Emodin may therefore be considered as an effective adjuvant for cisplatin treatment.     

Acquisition of an enhanced aggressive phenotype in human lung cancer cells selected by suboptimal doses of cisplatin following cell deattachment and reattachment

Chemotherapy is one major approach for treating non-small cell lung carcinoma (NSCLC). However, the progression-free survival rate depends on whether there is tumor metastasis after drug treatment. The biological behavior for its characteristics remains to be clarified. Here, we treated A549 and H1299 NSCLC cell lines with cisplatin, doxorubicin and gemcitabine at the IC(50) dose. Most attached cells were surviving cells (A549-A and H1299-A), whereas only a small portion of detached cells survived and reattached to tissue culture plates (A549-R and H1299-R) for further growth. Using cisplatin, a series of H1299 sublines (H1299-R2～H1299-R5) were also generated by the same selection procedure. Drug treatment increased the migratory ability of A549-R and H1299-R cells. A serial selection could enhance the invasiveness of cells. Cisplatin treatment inhibited the adhesion ability of H1299-R cells compared with their H1299 and H1299-A counterparts. H1299-R cells exhibited increased drug resistance to cisplatin and increased expression of ABCG2, CD133 and CD44. Compared with mice subcutaneously injected with H1299 cells, mice subcutaneously injected with H1299-R cells showed an increase in the number of metastatic lung nodules. We conclude that H1299-R cells selected by suboptimal doses of cisplatin following detachment from and reattachment to the tissue culture plate acquire an enhanced malignant phenotype. Therefore, they provide a more faithful lung cancer model associated with biological aggressiveness for studying clinically recurrent cancers after chemotherapy.