Caveolin-1 sensitizes cisplatin-induced lung cancer cell apoptosis via superoxide anion-dependent mechanism

Caveolin-1 (Cav-1) expression frequently found in lung cancer was linked with disease prognosis and progression. This study reveals for the first time that Cav-1 sensitizes cisplatin-induced lung carcinoma cell death by the mechanism involving oxidative stress modulation. We established stable Cav-1 overexpressed (H460/Cav-1) cells and investigated their cisplatin susceptibility in comparison with control-transfected cells and found that Cav-1 expression significantly enhanced cisplatin-mediated cell death. Results indicated that the different response to cisplatin between these cells was resulted from different level of superoxide anion induced by cisplatin. Inhibitory study revealed that superoxide anion inhibitor MnTBAP could inhibit cisplatin-mediated toxicity only in H460/Cav-1 cells while had no effect on H460 cells. Further, superoxide anion detected by DHE probe indicated that H460/Cav-1 cells generated significantly higher superoxide anion level in response to cisplatin than that of control cells. The role of Cav-1 in regulating cisplatin sensitivity was confirmed in shRNA-mediated Cav-1 down-regulated (H460/shCav-1) cells and the cells exhibited decreased cisplatin susceptibility and superoxide generation. In summary, these findings reveal novel aspects regarding role of Cav-1 in modulating oxidative stress induced by cisplatin, possibly providing new insights for cancer biology and cisplatin-based chemotherapy.     

Caveolin-1: a critical regulator of lung injury

Caveolin-1 (cav-1), a 22-kDa transmembrane scaffolding protein, is the principal structural component of caveolae. Cav-1 regulates critical cell functions including proliferation, apoptosis, cell differentiation, and transcytosis via diverse signaling pathways. Abundant in almost every cell type in the lung, including type I epithelial cells, endothelial cells, smooth muscle cells, fibroblasts, macrophages, and neutrophils, cav-1 plays a crucial role in the pathogenesis of acute lung injury (ALI). ALI and its severe form, acute respiratory distress syndrome (ARDS), are responsible for significant morbidity and mortality in intensive care units, despite improvement in ventilation strategies. The pathogenesis of ARDS is still poorly understood, and therapeutic options remain limited. In this article, we summarize recent data regarding the regulation and function of cav-1 in lung biology and pathology, in particular as it relates to ALI. We further discuss the potential molecular and cellular mechanisms by which cav-1 expression contributes to ALI. Investigating the cellular functions of cav-1 may provide new insights for understanding the pathogenesis of ALI and provide novel targets for therapeutic interventions in the future.     

Defects in Mitochondrial Fission Protein Dynamin-Related Protein 1 Are Linked to Apoptotic Resistance and Autophagy in a Lung Cancer Model

Evasion of apoptosis is implicated in almost all aspects of cancer progression, as well as treatment resistance. In this study, resistance to apoptosis was identified in tumorigenic lung epithelial (A549) cells as a consequence of defects in mitochondrial and autophagic function. Mitochondrial function is determined in part by mitochondrial morphology, a process regulated by mitochondrial dynamics whereby the joining of two mitochondria, fusion, inhibits apoptosis while fission, the division of a mitochondrion, initiates apoptosis. Mitochondrial morphology of A549 cells displayed an elongated phenotype–mimicking cells deficient in mitochondrial fission protein, Dynamin-related protein 1 (Drp1). A549 cells had impaired Drp1 mitochondrial recruitment and decreased Drp1-dependent fission. Cytochrome c release and caspase-3 and PARP cleavage were impaired both basally and with apoptotic stimuli in A549 cells. Increased mitochondrial mass was observed in A549 cells, suggesting defects in mitophagy (mitochondrial selective autophagy). A549 cells had decreased LC3-II lipidation and lysosomal inhibition suggesting defects in autophagy occur upstream of lysosomal degradation. Immunostaining indicated mitochondrial localized LC3 punctae in A549 cells increased after mitochondrial uncoupling or with a combination of mitochondrial depolarization and ectopic Drp1 expression. Increased inhibition of apoptosis in A549 cells is correlated with impeded mitochondrial fission and mitophagy. We suggest mitochondrial fission defects contribute to apoptotic resistance in A549 cells.     

Cisplatin-induced downregulation of SOX1 increases drug resistance by activating autophagy in non-small cell lung cancer cell

SOX1 was aberrant methylated in hepatocellular cancer and non-small cell lung cancer (NSCLC). Long-term cisplatin exposure promotes methylation of SOX1 in ovarian cancer cell, suggesting that SOX1 may be involved in cisplatin resistance. Our aim was to test the hypothesis that cisplatin resistance is associated with alteration of SOX1 expression in NSCLC. Expression of levels of SOX1 was examined using RT-PCR in cisplatin resistance cells and parental cells. The level of SOX1 mRNA in cisplatin resistance cells was markedly reduced when compared to parental cells. Promoter methylation of SOX1 was induced in cisplatin resistance cells. We also found that SOX1 silencing enhanced the cisplatin-mediated autophagy in NSCLC. This study shows that inactivation of SOX1 by promoter hypermethylation, at least in part, is responsible for cisplatin resistance in human NSCLC.     

Wnt1-inducible signaling protein 1 regulates laryngeal squamous cell carcinoma glycolysis and chemoresistance via the YAP1/TEAD1/GLUT1 pathway

Wnt1-inducible signaling protein 1 (WISP1) is a matricellular protein and downstream target of Wnt/β-catenin signaling. This study sought to determine the role of WISP1 in glucose metabolism and chemoresistance in laryngeal squamous cell carcinoma. WISP1 expression was silenced or upregulated in Hep-2 cells by the transfection of WISP1 siRNA or AdWISP1 vector. Ectopic WISP1 expression regulated glucose uptake and lactate production in Hep-2 cells. Subsequently, the expression of glucose transporter 1 (GLUT1) was significantly modulated by WISP1. Furthermore, WISP1 increased cell survival rates, diminished cell death rates, and suppressed ataxia-telangiectasia-mutated (ATM)-mediated DNA damage response pathway in cancer cells treated with cisplatin through GLUT1. WISP1 also promoted cancer cell tumorigenicity and growth in mice implanted with Hep-2 cells. Additionally, WISP1 activated the YAP1/TEAD1 pathway that consequently contributed to the regulation of GLUT1 expression. In summary, WISP1 regulated glucose metabolism and cisplatin resistance in laryngeal cancer by regulating GLUT1 expression. WISP1 may be used as a potential therapeutic target for laryngeal cancer.     

RNA interferences targeting the Fanconi anemia/BRCA pathway upstream genes reverse cisplatin resistance in drug-resistant lung cancer cells

Background

Cisplatin is one of the most commonly used chemotherapy agent for lung cancer. The therapeutic efficacy of cisplatin is limited by the development of resistance.In this study, we test the effect of RNA interference (RNAi) targeting Fanconi anemia (FA)/BRCA pathway upstream genes on the sensitivity of cisplatin-sensitive (A549 and SK-MES-1) and -resistant (A549/DDP) lung cancer cells to cisplatin.

Result

Using small interfering RNA (siRNA), knockdown of FANCF, FANCL, or FANCD2 inhibited function of the FA/BRCA pathway in A549, A549/DDP and SK-MES-1 cells, and potentiated sensitivity of the three cells to cisplatin. The extent of proliferation inhibition induced by cisplatin after knockdown of FANCF and/or FANCL in A549/DDP cells was significantly greater than in A549 and SK-MES-1 cells, suggesting that depletion of FANCF and/or FANCL can reverse resistance of cisplatin-resistant lung cancer cells to cisplatin. Furthermore, knockdown of FANCL resulted in higher cisplatin sensitivity and dramatically elevated apoptosis rates compared with knockdown of FANCF in A549/DDP cells, indicating that FANCL play an important role in the repair of cisplatin-induced DNA damage.

Conclusion

Knockdown of FANCF, FANCL, or FANCD2 by RNAi could synergize the effect of cisplatin on suppressing cell proliferation in cisplatin-resistant lung cancer cells through inhibition of FA/BRCA pathway.     

Mitochonic acid-5 attenuates TNF-α-mediated neuronal inflammation via activating Parkin-related mitophagy and augmenting the AMPK–Sirt3 pathways

Mitochondrial dysfunction has been found to be associated with neuronal inflammation; however, no effective drug is available to attenuate neuroinflammation via sustaining mitochondrial function. In the current study, experiments were performed to understand the beneficial effects of mitochonic acid 5 (MA-5) on tumor necrosis factor-α (TNF-α)-mediated neuronal injury and mitochondrial damage. Our data illustrated that MA-5 pretreatment reduced inflammation response induced by TNF-α in CATH.a cells. Molecular investigations demonstrated that MA-5 pretreatment repressed oxidative stress, inhibited endoplasmic reticulum stress, sustained cellular energy metabolism, and blocked cell apoptosis induced by TNF-α stress. Further, we found that MA-5 treatment elevated the expression of Sirtuin 3 (Sirt3) and this effect was dependent on the activation of AMP-activated protein kinase (AMPK) pathway. Blockade of AMPK abolished the promotive action of MA-5 on Sirt3 and thus mediated mitochondrial damage and cell death. Besides, we also found that MA-5 treatment augmented Parkin-related mitophagy and increased mitophagy promoted CATH.a cells survival via improving mitochondrial function. Knockdown of Parkin abolished the beneficial action of MA-5 on mitochondrial homeostasis and CATH.a cell survival. Altogether, our results confirm that MA-5 is an effective drug to attenuate neuroinflammation via sustaining mitochondrial damage and promoting CATH.a cell survival. The protective action of MA-5 on neuronal damage is associated with Parkin-related mitophagy and the activation of AMPK–Sirt3 pathways.     

Inhibitory role of TRIP-Br1 oncoprotein in anticancer drug-mediated programmed cell death via mitophagy activation

Chemotherapy has been widely used as a clinical treatment for cancer over the years. However, its effectiveness is limited because of resistance of cancer cells to programmed cell death (PCD) after treatment with anticancer drugs. To elucidate the resistance mechanism, we initially focused on cancer cell-specific mitophagy, an autophagic degradation of damaged mitochondria. This is because mitophagy has been reported to provide cancer cells with high resistance to anticancer drugs. Our data showed that TRIP-Br1 oncoprotein level was greatly increased in the mitochondria of breast cancer cells after treatment with various anticancer drugs including staurosporine (STS), the main focus of this study. STS treatment increased cellular ROS generation in cancer cells, which triggered mitochondrial translocation of TRIP-Br1 from the cytosol via dephosphorylation of TRIP-Br1 by protein phosphatase 2A (PP2A). Up-regulated mitochondrial TRIP-Br1 suppressed cellular ROS levels. In addition, TRIP-Br1 rapidly removed STS-mediated damaged mitochondria by activating mitophagy. It eventually suppressed STS-mediated PCD via degradation of VDACI, TOMM20, and TIMM23 mitochondrial membrane proteins. TRIP-Br1 enhanced mitophagy by increasing expression levels of two crucial lysosomal proteases, cathepsins B and D. In conclusion, TRIP-Br1 can suppress the sensitivity of breast cancer cells to anticancer drugs by activating autophagy/mitophagy, eventually promoting cancer cell survival.     

DNA damage responsive miR-33b-3p promoted lung cancer cells survival and cisplatin resistance by targeting p21WAF1/CIP1

Cisplatin is the most potent and widespread used chemotherapy drug for lung cancer treatment. However, the development of resistance to cisplatin is a major obstacle in clinical therapy. The principal mechanism of cisplatin is the induction of DNA damage, thus the capability of DNA damage response (DDR) is a key factor that influences the cisplatin sensitivity of cancer cells. Recent advances have demonstrated that miRNAs (microRNAs) exerted critical roles in DNA damage response; nonetheless, the association between DNA damage responsive miRNAs and cisplatin resistance and its underlying molecular mechanism still require further investigation. The present study has attempted to identify differentially expressed miRNAs in cisplatin induced DNA damage response in lung cancer cells, and probe into the effects of the misexpressed miRNAs on cisplatin sensitivity. Deep sequencing showed that miR-33b-3p was dramatically down-regulated in cisplatin-induced DNA damage response in A549 cells; and ectopic expression of miR-33b-3p endowed the lung cancer cells with enhanced survival and decreased γH2A.X expression level under cisplatin treatment. Consistently, silencing of miR-33b-3p in the cisplatin-resistant A549/DDP cells evidently sensitized the cells to cisplatin. Furthermore, we identified CDKN1A (p21) as a functional target of miR-33b-3p, a critical regulator of G1/S checkpoint, which potentially mediated the protection effects of miR-33b-3p against cisplatin. In aggregate, our results suggested that miR-33b-3p modulated the cisplatin sensitivity of cancer cells might probably through impairing the DNA damage response. And the knowledge of the drug resistance conferred by miR-33b-3p has great clinical implications for improving the efficacy of chemotherapies for treating lung cancers.     

Ibuprofen enhances the anticancer activity of cisplatin in lung cancer cells by inhibiting the heat shock protein 70

Hsp70 is often overexpressed in cancer cells, and the selective cellular survival advantage that it confers may contribute to the process of tumour formation. Thus, the pharmacological manipulation of Hsp70 levels in cancer cells may be an effective means of preventing the progression of tumours. We found that the downregulation of Hsp70 by ibuprofen in vitro enhances the antitumoural activity of cisplatin in lung cancer. Ibuprofen prominently suppressed the expression of Hsp70 in A549 cells derived from lung adenocarcinoma and sensitized them to cisplatin in association with an increase in the mitochondrial apoptotic cascade, whereas ibuprofen alone did not induce cell death. The cisplatin-dependent events occurring up- and downstream of mitochondrial disruption were accelerated by treatment with ibuprofen. The increase in cisplatin-induced apoptosis caused by the depletion of Hsp70 by RNA interference is evidence that the increased apoptosis by ibuprofen is mediated by its effect on Hsp70. Our observations indicate that the suppression of Hsp70 by ibuprofen mediates the sensitivity to cisplatin by enhancing apoptosis at several stages of the mitochondrial cascade. Ibuprofen, therefore, is a potential therapeutic agent that might allow lowering the doses of cisplatin and limiting the many challenge associated with its toxicity and development of drug resistance.     

Metabolic reprogramming of cancer-associated fibroblasts by TGF-β drives tumor growth: Connecting TGF-β signaling with “Warburg-like” cancer metabolism and L-lactate production

We have previously shown that a loss of stromal Cav-1 is a biomarker of poor prognosis in breast cancers. Mechanistically, a loss of Cav-1 induces the metabolic reprogramming of stromal cells, with increased autophagy/mitophagy, mitochondrial dysfunction and aerobic glycolysis. As a consequence, Cav-1-low CAFs generate nutrients (such as L-lactate) and chemical building blocks that fuel mitochondrial metabolism and the anabolic growth of adjacent breast cancer cells. It is also known that a loss of Cav-1 is associated with hyperactive TGF-β signaling. However, it remains unknown whether hyperactivation of the TGF-β signaling pathway contributes to the metabolic reprogramming of Cav-1-low CAFs. To address these issues, we overexpressed TGF-β ligands and the TGF-β receptor I (TGFβ-RI) in stromal fibroblasts and breast cancer cells. Here, we show that the role of TGF-β in tumorigenesis is compartment-specific, and that TGF-β promotes tumorigenesis by shifting cancer-associated fibroblasts toward catabolic metabolism. Importantly, the tumor-promoting effects of TGF-β are independent of the cell type generating TGF-β. Thus, stromal-derived TGF-β activates signaling in stromal cells in an autocrine fashion, leading to fibroblast activation, as judged by increased expression of myofibroblast markers, and metabolic reprogramming, with a shift toward catabolic metabolism and oxidative stress. We also show that TGF-β-activated fibroblasts promote the mitochondrial activity of adjacent cancer cells, and in a xenograft model, enhancing the growth of breast cancer cells, independently of angiogenesis. Conversely, activation of the TGF-β pathway in cancer cells does not influence tumor growth, but cancer cell-derived-TGF-β ligands affect stromal cells in a paracrine fashion, leading to fibroblast activation and enhanced tumor growth. In conclusion, ligand-dependent or cell-autonomous activation of the TGF-β pathway in stromal cells induces their metabolic reprogramming, with increased oxidative stress, autophagy/mitophagy and glycolysis, and downregulation of Cav-1. These metabolic alterations can spread among neighboring fibroblasts and greatly sustain the growth of breast cancer cells. Our data provide novel insights into the role of the TGF-β pathway in breast tumorigenesis, and establish a clear causative link between the tumor-promoting effects of TGF-β signaling and the metabolic reprogramming of the tumor microenvironment.     

LncRNA XIST promotes human lung adenocarcinoma cells to cisplatin resistance via let-7i/BAG-1 axis

Long noncoding RNAs (lncRNAs) have been identified as oncogenes or tumor suppressors that are involved in tumorigenesis and chemoresistance. LncRNA XIST expression is upregulated in several cancers, however, its biologic role in the development of the chemotherapy of human lung adenocarcinoma (LAD) has not been elucidated. This study aimed to observe the expression of LncRNA XIST in LAD and to evaluate its biologic role and clinical significance in the resistance of LAD cells to cisplatin. LncRNA XIST expression was markedly increased in cisplatin-resistant A549/DDP cells compared with parental A549 cells as shown by qRT-PCR. LncRNA XIST overexpression in A549 cells increased their chemosensitivity to cisplatin both in vitro and in vivo by protecting cells from apoptosis and promoting cell proliferation. By contrast, LncRNA XIST knockdown in A549/DDP cells decreased the chemoresistance. We revealed that XIST functioned as competing endogenous RNA to repress let-7i, which controlled its down-stream target BAG-1. We proposed that XIST was responsible for cisplatin resistance of LAD cells and XIST exerted its function through the let-7i/BAG-1 axis. Our findings suggested that lncRNA XIST may be a new marker of poor response to cisplatin and could be a potential therapeutic target for LAD chemotherapy.     

Shear stress promotes anoikis resistance of cancer cells via caveolin-1-dependent extrinsic and intrinsic apoptotic pathways

Circulating tumor cells (CTCs) need to acquire resistance to anoikis to survive after they experience fluid shear stress in the circulatory and lymphatic systems. However, the mechanism by which tumor cells resist anoikis under shear stress conditions remains unknown. Here, we found that the application of low shear stress (LSS; 2 dyn/cm²) to human breast carcinoma cells (MDA-MB-231) resulted in increased anoikis resistance when tumor cells were grown under anchorage-independent conditions. Caveolin-1 (Cav-1), the major component of plasma membrane caveolae, was overexpressed in LSS-treated cells and prevented tumor cells from anoikis, while depletion of Cav-1 restored sensitivity to anoikis. LSS-induced dissociation of Cav-1–Fas inhibited formation of the death-inducing signaling complex, caspase-8 activation, and rendered tumor cells resistant to anoikis. Likewise, LSS blocked the mitochondrial pathway through promotion of integrin β1–focal adhesion kinase-mediated multicellular aggregation and suppression of truncated BID translocation mediated crosstalk between the extrinsic and intrinsic apoptotic pathways. Our findings provide insights into the mechanisms by which LSS induces anoikis resistance in breast carcinoma cells through inhibition of Cav-1-dependent extrinsic and intrinsic apoptotic pathways, and serves as a potential therapeutic target for CTCs and metastatic breast cancer.     

Dynamin 1-like-dependent mitochondrial fission initiates overactive mitophagy in the hepatotoxicity of cadmium

How cadmium (Cd) induces mitochondrial loss in the context of its hepatotoxic effects remains enigmatic. The purpose of the study was to investigate whether mitophagy contributes to mitochondrial loss in cadmium-induced hepatotoxicity and to determine the potential mechanism. In normal human liver L02 cells, we observed that Cd treatment led to a significant increase in LC3-II formation, the number of GFP-LC3 puncta and lysosomal colocalization with mitochondria. These results were associated with mitochondrial loss and bioenergetic deficit. Additionally, the abrogation of excessive mitophagy by ATG5 siRNA treatment efficiently suppressed the mitochondrial loss and cytotoxicity of Cd. Before overactivating mitophagy, Cd induced excessive mitochondrial fragmentation as a result of increasing dynamin 1-like (DNM1L) expression and enhancing the DNM1L mitochondrial translocation. Moreover, reversing the excessive mitochondrial fragmentation via the administration of DNM1L siRNA significantly inhibited the observed overactivation of mitophagy in Cd-induced hepatotoxicity. Notably, the selective DNM1L inhibitor Mdivi-1 blocked abnormal mitophagy and subsequently ameliorated Cd-induced hepatotoxicity in vivo. Together, our data indicated that Cd induces mitochondrial loss via the overactivation of mitophagy following DNM1L-dependent mitochondrial fragmentation. The balanced activity of DNM1L and mitophagy signaling may be a potential therapeutic approach to treat Cd-induced hepatotoxicity.     

Placenta growth factor, PLGF, influences the motility of lung cancer cells, the role of Rho associated kinase, Rock1

Placenta growth factor (PlGF) is a member of the VEGF family and has been implicated in the aggressive capacity of solid tumours, partly via its impact on angiogenesis. The present study determined the direct biological function of endogenous PlGF in lung cancer cells. From the human non-small cell lung cancer cell line A549 which expressed good level of PlGF, we created sublines within which PlGF expression was knockdown by way of anti-PlGF ribozyme transgenes. Remarkable reductions of both PlGF mRNA and protein by the ribozyme transgenes were revealed in A549 transfectants (A549(DeltaPlGF)) using RT-PCR and Western blotting respectively. A549(DeltaPlGF) cells exhibited significantly reduced migration and adhesion compared with the wild-type (A549(WT)) and the empty plasmid control (A549(pEF/His)) cells. Immunocytochemistry and Western blotting further revealed that the expression of ROCK1, Rho associated kinase, was also reduced in A549(DeltaPlGF) cells, in comparison with wild-type and control cells. In addition, A549(DeltaPlGF) cells lost its response to a ROCK inhibitor, which otherwise strongly inhibited the motility of A549(WT) and A549(pEF/His) cells. These data indicate that PlGF directly regulates the motility of human lung cancer cells and that this regulation critically dependent on ROCK-1. The study further indicates that PlGF is a potential therapeutic target in lung cancer.     

斑马鱼窖蛋白-1基因cDNA克隆及功能初步研究

窖蛋白-1(Cav-1)是胞膜窖的主要结构蛋白, 可与多种信号分子相互作用, 调节细胞的增殖、分化和凋亡, 其异常表达与多种人体疾病的发生和发展密切相关, 而在斑马鱼发育中的功能尚不很清楚。研究克隆出斑马鱼窖蛋白-1基因两个亚型的全长cDNA, 与其他物种窖蛋白-1的氨基酸序列进行比较, 发现该蛋白在脊椎动物中非常保守。利用逆转录多聚酶链反应检测发现, 在斑马鱼多个成年组织中窖蛋白-1的两个亚型均有转录表达。利用胚胎整体原位杂交检测组织或器官特异基因的时空表达变化发现, 过表达或利用Morpholino反义寡聚核苷酸(MO)抑制cav-1α的表达可影响脊索和体节的发育, 而过表达或MO抑制cav-1β可导致肝脏发育的异常;此外, 过表达或MO抑制cav-1α或-1β均可影响斑马鱼神经系统的发育。因此, 斑马鱼Cav-1在维持组织器官的生理功能和调控胚胎的正常发育中起着重要作用。       

Wnt/β-catenin signaling regulates cancer stem cells in lung cancer A549 cells

Wnt/β-catenin signaling plays an important role not only in cancer, but also in cancer stem cells. In this study, we found that β-catenin and OCT-4 was highly expressed in cisplatin (DDP) selected A549 cells. Stimulating A549 cells with lithium chloride (LiCl) resulted in accumulation of β-catenin and up-regulation of a typical Wnt target gene cyclin D1. This stimulation also significantly enhanced proliferation, clone formation, migration and drug resistance abilities in A549 cells. Moreover, the up-regulation of OCT-4, a stem cell marker, was observed through real-time PCR and Western blotting. In a reverse approach, we inhibited Wnt signaling by knocking down the expression of β-catenin using RNA interference technology. This inhibition resulted in down-regulation of the Wnt target gene cyclin D1 as well as the proliferation, clone formation, migration and drug resistance abilities. Meanwhile, the expression of OCT-4 was reduced after the inhibition of Wnt/β-catenin signaling. Taken together, our study provides strong evidence that canonical Wnt signaling plays an important role in lung cancer stem cell properties, and it also regulates OCT-4, a lung cancer stem cell marker.