Caveolin-1 Regulates Endothelial Adhesion of Lung Cancer Cells via Reactive Oxygen Species-Dependent Mechanism

The knowledge regarding the role of caveolin-1 (Cav-1) protein on endothelium adhesion of cancer cells is unclear. The present study revealed that Cav-1 plays a negative regulatory role on cancer-endothelium interaction. Endogenous Cav-1 was shown to down-regulate during cell detachment and the level of such a protein was conversely associated with tumor-endothelial adhesion. Furthermore, the ectopic overexpression of Cav-1 attenuated the ability of the cancer cells to adhere to endothelium while shRNA-mediated Cav-1 knock-down exhibited the opposite effect. We found that cell detachment increased cellular hydrogen peroxide and hydroxyl radical generation and such reactive oxygen species (ROS) were responsible for the increasing interaction between cancer cells and endothelial cells through vascular endothelial cell adhesion molecule-1 (VCAM-1). Importantly, Cav-1 was shown to suppress hydrogen peroxide and hydroxyl radical formation by sustaining the level of activated Akt which was critical for the role of Cav-1 in attenuating the cell adhesion. Together, the present study revealed the novel role of Cav-1 and underlying mechanism on tumor adhesion which explain and highlight an important role of Cav-1 on lung cancer cell metastasis.     

Hydrogen peroxide inhibits non-small cell lung cancer cell anoikis through the inhibition of caveolin-1 degradation

Anoikis or detachment-induced apoptosis plays an essential role in the regulation of cancer cell metastasis. Caveolin-1 (Cav-1) is a key protein involved in tumor metastasis, but its role in anoikis and its regulation during cell detachment are unclear. We report here that Cav-1 plays a key role as a negative regulator of anoikis through a reactive oxygen species (ROS)-dependent mechanism in human lung carcinoma H460 cells. During cell detachment, Cav-1 is downregulated, whereas ROS generation is upregulated. Hydrogen peroxide and hydroxyl radical are two key ROS produced by cells during detachment. Treatment of the cells with hydrogen peroxide scavengers, catalase and N-acetylcysteine, promoted Cav-1 downregulation and anoikis during cell detachment, indicating that produced hydrogen peroxide plays a primary role in preventing anoikis by stabilizing Cav-1 protein. Catalase and N-acetylcysteine promoted ubiquitination and proteasomal degradation of Cav-1, which is a major pathway of its downregulation during cell anoikis. Furthermore, addition of hydrogen peroxide exogenously to the cells inhibited Cav-1 downregulation by preventing the formation of Cav-1-ubiquitin complex, supporting the inhibitory role of endogenous hydrogen peroxide in Cav-1 degradation during cell detachment. Together, these results indicate a novel role of hydrogen peroxide as an endogenous suppressor of cell anoikis through its stabilizing effect on Cav-1.     

ROS up-regulation mediates Ras-induced changes of cell morphology and motility

Expression of activated Ras causes an increase in intracellular content of reactive oxygen species (ROS). To determine the role of ROS up-regulation in mediation of Ras-induced morphological transformation and increased cell motility, we studied the effects of hydrogen peroxide and antioxidant NAC on morphology of REF52 rat fibroblasts and their ability to migrate into the wound in vitro. Treatment with low dosages of hydrogen peroxide leading to 1.5- to 2-fold increase in intracellular ROS levels induced changes of cell shape, actin cytoskeleton organization, cell adhesions and migration resembling those in Ras-transformed cells. On the other hand, treatment with NAC attenuating ROS up-regulation in cells with conditional or constitutive expression of activated Ras led to partial reversion of morphological transformation and decreased cell motility. The effect of ROS on cell morphology and motility probably results from modulation of activity of Rac1, Rho, and cofilin proteins playing a key role in regulation of actin dynamics. The obtained data are consistent with the idea that ROS up-regulation mediates two key events in Ras-induced morphological transformation and cell motility: it is responsible for Rac1 activation and is necessary (though insufficient) for realization of Ras-induced cofilin dephosphorylation.     

Collective dynamics of cancer cells confined in a confluent monolayer of normal cells

Tumorigenesis often involves specific changes in cell motility and intercellular adhesion. Understanding the collective cancer cell behavior associated with these specific changes could facilitate the detection of malignant characteristics during tumor growth and invasion. In this study, a cellular vertex model is developed to investigate the collective dynamics of a disk-like aggregate of cancer cells confined in a confluent monolayer of normal cells. The effects of intercellular adhesion and cell motility on tumor progression are examined. It is found that the stresses in both the cancer cells and the normal cells increase with tumor growth, resulting in a crowded environment and enhanced cell apoptosis. The intercellular adhesion between cancer cells and normal cells is revealed to promote tumor growth and invasion. The tumor invasion dynamics hinges on the motility of cancer cells. The cancer cells could orchestrate into different collective migration modes, e.g., directional migration and rotational oscillations, dictated by the competition between cell persistence and local coordination. Phase diagrams are established to reveal the competitive mechanisms. This work highlights the role of mechanics in regulating tumor growth and invasion.     

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 promotes pancreatic cancer cell differentiation and restores membranous E-cadherin via suppression of the epithelial-mesenchymal transition

Pancreatic cancer is one of the deadliest cancers due to early rapid metastasis and chemoresistance. Recently, epithelial to mesenchymal transition (EMT) was shown to play a key role in the pathogenesis of pancreatic cancer. To understand the role of caveolin-1 (Cav-1) in EMT, we over-expressed Cav-1 in a pancreatic cancer cell line, Panc 10.05, that does not normally express Cav-1. Here, we show that Cav-1 expression in pancreatic cancer cells induces an epithelial phenotype and promotes cell-cell contact, with increased expression of plasma membrane bound E-cadherin and beta-catenin. Mechanistically, Cav-1 induces Snail downregulation and decreased activation of AKT, MAPK and TGF-beta-Smad signaling pathways. In vitro, Cav-1 expression reduces cell migration and invasion, and attenuates doxorubicin-chemoresistance of pancreatic cancer cells. Importantly, in vivo studies revealed that Cav-1 expression greatly suppresses tumor formation in a xenograft model. Most interestingly, Panc/Cav-1 tumors displayed organized nests of differentiated cells that were totally absent in control tumors. Confirming our in vitro results, these nests of differentiated cells showed reexpression of E-cadherin and beta-catenin at the cell membrane. Thus, we provide evidence that Cav-1 functions as a crucial modulator of EMT and cell differentiation in pancreatic cancer.     

Curcumin sensitizes non-small cell lung cancer cell anoikis through reactive oxygen species-mediated Bcl-2 downregulation

Anoikis, an apoptosis triggered by loss of cell anchorage, has been shown to be a principal mechanism of inhibition of tumor metastasis. Recently, anti-apoptotic Bcl-2 and Cav-1 proteins have been demonstrated to be highly associated with tumor metastasis and apoptosis resistance. Curcumin, a major active component of turmeric, Curcuma longa, has been shown to inhibit neoplastic evolution and tumor progression; however, the underlying mechanisms are unclear. In this study, we investigated the effect of curcumin on cell anoikis as a possible mechanism of anti-tumorigenic action of curcumin, and evaluated the potential role of Bcl-2 and Cav-1 in this process. Our results showed that ectopic expression of either Bcl-2 or Cav-1 induced anoikis resistance of lung carcinoma H460 cells. Curcumin downregulated Bcl-2 protein during anoikis and sensitized the cells to detachment-induced apoptosis, whereas it had no significant effect on Cav-1 protein expression. Bcl-2 down-regulation as well as anoikis enhancement by curcumin were inhibited by superoxide anion scavenger, Mn(III)tetrakis(4-benzoic acid) porphyrin chloride, but were unaffected by other ROS scavengers including catalase and deferoxamine, suggesting that superoxide anion is a key player in the downregulation of Bcl-2 by curcumin. Furthermore, we provided evidence that curcumin decreased Bcl-2 level through ubiquitin-proteasomal degradation which sensitized cells to detachment-induced apoptosis. These findings indicate a novel pathway for curcumin regulation of Bcl-2 and provide a key mechanism of anoikis regulation that may be exploited for metastatic cancer treatment.     

Sdc1 negatively modulates carcinoma cell motility and invasion

During cancer progression, tumor cells eventually invade the surrounding collagen-rich extracellular matrix. Here we show that squamous cell carcinoma cells strongly adhere to Type I collagen substrates but display limited motility and invasion on collagen barriers. Further analysis revealed that in addition to the α2β1 integrin, a second collagen receptor was identified as Syndecan-1 (Sdc1), a cell surface heparan sulfate proteoglycan. We demonstrate that siRNA-mediated depletion of Sdc1 reduced adhesion efficiency to collagen I, whereas knockdown of Sdc4 was without effect. Importantly, silencing Sdc1 expression caused reduced focal adhesion plaque formation and enhanced cell spreading and motility on collagen I substrates, but did not alter cell motility on other ECM substrates. Sdc1 depletion ablated adhesion-induced RhoA activation. In contrast, Rac1 was strongly activated following Sdc1 knockdown, suggesting that Sdc1 may mediate the link between integrin-induced actin remodeling and motility. Taken together, these data substantiate the existence of a co-adhesion receptor system in tumor cells, whereby Sdc1 functions as a key regulator of cell motility and cell invasion by modulating RhoA and Rac activity. Downregulation of Sdc1 expression during carcinoma progression may represent a mechanism by which tumor cells become more invasive and metastatic.     

Hydroxyl radical mediates cisplatin-induced apoptosis in human hair follicle dermal papilla cells and keratinocytes through Bcl-2-dependent mechanism

Induction of massive apoptosis of hair follicle cells by chemotherapy has been implicated in the pathogenesis of chemotherapy-induced alopecia (CIA), but the underlying mechanisms of regulation are not well understood. The present study investigated the apoptotic effect of cisplatin in human hair follicle dermal papilla cells and HaCaT keratinocytes, and determined the identity and role of specific reactive oxygen species (ROS) involved in the process. Treatment of the cells with cisplatin induced ROS generation and a parallel increase in caspase activation and apoptotic cell death. Inhibition of ROS generation by antioxidants inhibited the apoptotic effect of cisplatin, indicating the role of ROS in the process. Studies using specific ROS scavengers further showed that hydroxyl radical, but not hydrogen peroxide or superoxide anion, is the primary oxidative species responsible for the apoptotic effect of cisplatin. Electron spin resonance studies confirmed the formation of hydroxyl radicals induced by cisplatin. The mechanism by which hydroxyl radical mediates the apoptotic effect of cisplatin was shown to involve down-regulation of the anti-apoptotic protein Bcl-2 through ubiquitin-proteasomal degradation. Bcl-2 was also shown to have a negative regulatory role on hydroxyl radical. Together, our results indicate an essential role of hydroxyl radical in cisplatin-induced cell death of hair follicle cells through Bcl-2 regulation. Since CIA is a major side effect of cisplatin and many other chemotherapeutic agents with no known effective treatments, the knowledge gained from this study could be useful in the design of preventive treatment strategies for CIA through localized therapy without compromising the chemotherapy efficacy.     

Hypoxia/reoxygenation: a dynamic regulator of lysyl oxidase-facilitated breast cancer migration

Fluctuating oxygen levels characterize the microenvironment of many cancers and tumor hypoxia is associated with increased invasion and metastatic potential concomitant with a poor prognosis. Similarly, the expression of lysyl oxidase (LOX) in breast cancer facilitates tumor cell migration and is associated with estrogen receptor negative status and reduced patient survival. Here we demonstrate that hypoxia/reoxygenation drives poorly invasive breast cancer cells toward a more aggressive phenotype by up-regulating LOX expression and catalytic activity. Specifically, hypoxia markedly increased LOX protein expression; however, catalytic activity (beta-aminopropionitrile inhibitable hydrogen peroxide production) was significantly reduced under hypoxic conditions. Moreover, poorly invasive breast cancer cells displayed a marked increase in LOX-dependent FAK/Src activation and cell migration following hypoxia/reoxygenation, but not in response to hypoxia alone. Furthermore, LOX expression is only partially dependent on hypoxia inducible factor-1 (HIF-1alpha) in poorly invasive breast cancer cells, as hypoxia mimetics and overexpression of HIF-1alpha could not up-regulate LOX expression to the levels observed under hypoxia. Clinically, LOX expression positively correlates with tumor progression and co-localization with hypoxic regions (defined by HIF-1alpha expression) in ductal carcinoma in situ and invasive ductal carcinoma primary tumors. However, positive correlation is lost in metastatic tumors, suggesting that LOX expression is independent of a hypoxic environment at later stages of tumor progression. This work demonstrates that both hypoxia and reoxygenation are necessary for LOX catalytic activity which facilitates breast cancer cell migration through a hydrogen peroxide-mediated mechanism; thereby illuminating a potentially novel mechanism by which poorly invasive cancer cells can obtain metastatic competency.     

Glutathione depletion is necessary for apoptosis in lymphoid cells independent of reactive oxygen species formation

Changes in the intracellular redox environment of cells have been reported to be critical for the activation of apoptotic enzymes and the progression of programmed cell death. Glutathione (GSH) depletion is an early hallmark observed in apoptosis, and we have demonstrated that GSH efflux during death receptor-mediated apoptosis occurs via a GSH transporter. We now evaluate the relationship between GSH depletion, the generation of reactive oxygen species (ROS), and the progression of apoptosis. Simultaneous single cell analysis of changes in GSH content and ROS formation by multiparametric FACS revealed that loss of intracellular GSH was paralleled by the generation of different ROS including hydrogen peroxide, superoxide anion, hydroxyl radical, and lipid peroxides. However, inhibition of ROS formation by a variety of antioxidants showed that GSH loss was independent from the generation of ROS. Furthermore, GSH depletion was observed to be necessary for ROS generation. Interestingly, high extracellular thiol concentration (GSH and N-acetyl-cysteine) inhibited apoptosis, whereas, inhibition of ROS generation by other non-thiol antioxidants was ineffective in preventing cell death. Finally, GSH depletion was shown to be a necessary for the progression of apoptosis activated by both extrinsic and intrinsic signaling pathways. These results document a necessary and critical role for GSH loss in apoptosis and clearly uncouple for the first time GSH depletion from ROS formation.     

Caveolin-1 Is Up-Regulated by GLI1 and Contributes to GLI1-Driven EMT in Hepatocellular Carcinoma

Caveolin-1 (Cav-1) has been recently identified to be over-expressed in hepatocellular carcinoma (HCC) and promote HCC cell motility and invasion ability via inducing epithelial-mesenchymal transition (EMT). However, the mechanism of aberrant overexpression of Cav-1 remains vague. Here, we observed that Cav-1 expression was positively associated with GLI1 expression in HCC tissues. Forced expression of GLI1 up-regulated Cav-1 in Huh7 cells, while knockdown of GLI1 decreased expression of Cav-1 in SNU449 cells. Additionally, silencing Cav-1 abolished GLI1-induced EMT of Huh7 cells. The correlation between GLI1 and Cav-1 was confirmed in tumor specimens from HCC patients and Cav-1 was found to be associated with poor prognosis after hepatic resection. The relationship between protein expression of GLI1 and Cav-1 was also established in HCC xenografts of nude mice. These results suggest that GLI1 may be attributed to Cav-1 up-regulation which plays an important role in GLI1-driven EMT phenotype in HCC.     

Protein kinase Cdelta signaling downstream of the EGF receptor mediates migration and invasiveness of prostate cancer cells

Tumor progression to the invasive phenotype occurs secondary to upregulated signaling from growth factor receptors that drive key cellular responses like proliferation, migration, and invasion. We hypothesized that Protein kinase Cdelta (PKCdelta)-mediated transcellular contractility is required for migration and invasion of prostate tumor cells. Two invasive human prostate cancer cell lines, DU145 cells overexpressing wildtype human EGFR (DU145WT) and PC3 cells, were studied. PKCdelta is overexpressed in these cells relative to normal prostate epithelial cells, and is activated downstream of EGFR leading to cell motility via modulation of myosin light chain activity. Abrogation of PKCdelta using Rottlerin and specific siRNA significantly decreased migration and invasion of both cell lines in vitro. Both PKCdelta and phosphorylated PKCdelta protein levels were higher in human prostate cancer tissue relative to normal donor prostate as assessed by Western blotting and immunohistochemistry. Thus, we conclude that PKCdelta inhibition can limit migration and invasion of prostate cancer cells.     

Shear stress stimulates integrin β1 trafficking and increases directional migration of cancer cells via promoting deacetylation of microtubules

In egress routes of malignancy, cancer cells are constantly subjected to shear stress imposed by blood/lymph flow. Increasing evidence points toward the regulatory roles of shear stress in tumor cell adhesion and motility. Although it is known that integrin endocytic trafficking governs focal adhesion (FA) turnover and cell migration, the effect and biological consequences of low shear stress (LSS) on integrin trafficking remain unclear. Here, we identified the critical role of integrin β1 trafficking and caveolin-1 (Cav-1) mediated endocytosis in LSS-induced cell directional migration. LSS altered the distribution of integrin β1 in MDA-MB-231 cells and significantly promoted its internalization and recycling, which in turn facilitated FA turnover and directional cell migration. Furthermore, LSS induced cytoskeleton remodeling, which was required for internalization of integrin β1. LSS down-regulated the acetylation level of microtubules (MTs) via activating ROCK/HDAC6 pathway, resulting in elevation of MTs dynamics, Cav-1 motility, and Cav-1-dependent integrin β1 recycling. We also showed that high HDAC6 expression was a ROCK-dependent prognostic factor, which was correlated with poor outcomes in breast cancer patients. Taken together, these results defined a novel mechanism by which LSS enhanced integrin β1 trafficking via actin cytoskeleton remodeling and ROCK/HDAC6 mediated deacetylation of MTs, thereby promoting FAs turnover and directional cell migration.     

Fenton反应及其可能的活性产物

活性氧对许多生物分子,如脂质、蛋白质和DNA等均可引起损伤,它与许多疾病过程相联系.由超氧阴离子自由基和过氧化氢所引起的许多损伤被认为与它们转变为反应活性更强的组分有关,这些组分包括羟自由基及可能的高价铁组分.实验材料及理论结果表明,当铁盐与过氧化氢混合时,除羟自由基产生以外,高价铁组分也被认为同时产生.Fenton试剂的活性中间体是一亲核加合物,其反应活性及其产物不同于游离态羟自由基的反应活性及产物.Fenton试剂的产物分布依赖于不同的过渡金属离子、不同的配位体、不同的反应底物以及不同的溶剂基体效应.     

Deletion of caveolin scaffolding domain alters cancer cell migration

Caveolin-1 (Cav-1) is an integral membrane protein that plays an important role in proliferative and terminally differentiated cells. As a structural component of Caveolae, Cav-1 interacts with signaling molecules via a caveolin scaffolding domain (CSD) regulating cell signaling. Recent reports have shown that Cav-1 is a negative regulator in tumor metastasis. Therefore, we hypothesize that Cav-1 inhibits cell migration through its CSD. HeLa cells were engineered to overexpress Cav-1 (Cav-1 OE), Cav-1 without a functional CSD (?CSD), or enhanced green fluorescent protein (EGFP) as a control. HeLa cell migration was suppressed in Cav-1 OE cells while ?CSD showed increased migration, which corresponded to a decrease in the tight junction protein, zonula occludens (ZO-1). The migration phenotype was confirmed in multiple cancer cell lines. Phosphorylated STAT-3 was decreased in Cav-1 OE cells compared to control and ?CSD cells; reducing STAT-3 expression alone decreased cell migration. ?CSD blunted HeLa proliferation by increasing the number of cells in the G2/M phase of the cell cycle. Overexpressing the CSD peptide alone suppressed HeLa cell migration and inhibited pSTAT3. These findings suggest that Cav-1 CSD may be critical in controlling the dynamic phenotype of cancer cells by facilitating the interaction of specific signal transduction pathways, regulating STAT3 and participating in a G2/M checkpoint. Modulating the CSD and targeting specific proteins may offer potential new therapies in the treatment of cancer metastasis.