Varisa Pongrakhananon

Regulation of lung cancer cell migration and invasion by reactive oxygen species and Caveolin-1

The acquired capability of tumor cells to migrate and invade neighboring tissues is associated with high metastatic potential and advanced stage of cancers. Recently, signaling molecules such as reactive oxygen species (ROS) and caveolin-1 (Cav-1) have been implicated in the aggressive behavior of cancer cells. However, the roles of specific ROS in cancer cell migration and Cav-1 regulation are unclear. We demonstrate here that Cav-1 plays an important role in the migration and invasion of human lung carcinoma H460 cells and that these effects are differentially regulated by cellular ROS. Using various known inhibitors and donors of ROS, we found that different ROS have different effects on Cav-1 expression and cell migration and invasion. Superoxide anion and hydrogen peroxide down-regulated Cav-1 expression and inhibited cell migration and invasion, whereas hydroxyl radical up-regulated the Cav-1 expression and promoted cell migration and invasion. The down-regulating effect of superoxide anion and hydrogen peroxide on Cav-1 is mediated through a transcription-independent mechanism that involves protein degradation via the ubiquitin-proteasome pathway. These results indicate the essential role of different ROS in cancer cell motility and through Cav-1 expression, which may provide a key mechanism controlling tumor progression and metastasis. The up-regulation of Cav-1 and cell motility by hydroxyl free radical suggests an important role of this ROS as a positive regulator of tumor progression.

Carbon nanotubes induce malignant transformation and tumorigenesis of human lung epithelial cells.

Carcinogenicity of carbon nanotubes is a major concern but has not been well addressed due to the lack of experimental models. Here, we show that chronic exposure to single-walled carbon nanotubes causes malignant transformation of human lung epithelial cells. The transformed cells induce tumorigenesis in mice and exhibit an apoptosis resistant phenotype characteristic of cancer cells. This study provides new evidence for carbon nanotube-induced carcinogenesis and indicates the potential role of p53 in the process.

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.

Curcumin sensitizes lung cancer cells to cisplatin-induced apoptosis through superoxide anion-mediated Bcl-2 degradation.

The purpose of this study was to investigate the sensitizing effect of curcumin on cisplatin-induced apoptosis in non-small cell lung cancer (NSCLC) H460 cells. Curcumin was shown to induce superoxide anion generation, down-regulate anti-apoptotic Bcl-2 protein, and subsequently sensitize cells to cisplatin-induced apoptosis. Co-treatment of the cells with curcumin and cisplatin resulted in increased apoptosis and reversal of Bcl-2-mediated cisplatin resistance. The mechanism by which curcumin down-regulates Bcl-2 and sensitizes cells to cisplatin-induced apoptosis involves proteasomal degradation of Bcl-2. These findings indicate a novel pathway for curcumin regulation of Bcl-2, which could benefit the development of a cisplatin sensitizing agent.

Caveolin-1 regulates Mcl-1 stability and anoikis in lung carcinoma cells

Both caveolin-1 (Cav-1) and Mcl-1 have been implicated in the regulation of cancer cell anoikis, but their relationship and underlying mechanisms of regulation are not known. The present study demonstrated for the first time that Cav-1 regulates Mcl-1 through protein-protein interaction and inhibits its downregulation during cell anoikis in human lung cancer cells. Immunoprecipitation and immunocytochemistry studies showed that Cav-1 interacted with Mcl-1 and prevented it from degradation via the ubiquitin-proteasome pathway. Mcl-1 and Mcl-1-Cav-1 complex were highly elevated in Cav-1-overexpressing cells but were greatly reduced in Cav-1 knockdown cells. Consistent with this finding, we found that Mcl-1 ubiquitination was significantly attenuated by Cav-1 overexpression but increased by Cav-1 knockdown. Together, our results indicate a novel role of Cav-1 in anoikis regulation through Mcl-1 interaction and stabilization, which provides a new insight to the pathogenesis of metastatic lung cancer and its potential treatment.

Long-term nitric oxide exposure enhances lung cancer cell migration.

Nitric oxide (NO) found in the vicinity of lung cancer cells may play a role in the regulation of cancer cell behaviors. To explore the possible effects of NO on cell motility, human lung cancer cells were exposed to nontoxic concentrations of NO for 0–14 days, and the migratory characteristics of the cells were determined. The present study found that long-term treatment with NO significantly enhanced cell migration in a dose- and time-dependent manner. Furthermore, we found that the increased migratory action was associated with the increased expression of caveolin-1 (Cav-1), which in turn activated the focal adhesion kinase (FAK) and ATP-dependent tyrosine kinase (Akt) pathways. Notably, the NO-treated cells exhibited an increased number of filopodia per cell, as well as an increase in the levels of cell division cycle 42 (Cdc42) protein. Together, these results indicate that extended NO exposure has a novel effect on cell migration through a Cav-1-dependent mechanism, a finding that strengthens our understanding of cancer biology.

Ouabain downregulates Mcl-1 and sensitizes lung cancer cells to TRAIL-induced apoptosis.

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a prerequisite for cancer progression, and TRAIL resistance is prevalent in lung cancer. Ouabain, a recently identified human hormone, has shown therapeutic promise by potentiating the apoptotic response of metastatic lung cancer cells to TRAIL. Nontoxic concentrations of ouabain are shown to increase caspase-3 activation, poly(ADP-ribose) polymerase (PARP) cleavage, and apoptosis of H292 cells in response to TRAIL. While ouabain had a minimal effect on c-FLIP, Bcl-2, and Bax levels, we show that it possesses an ability to downregulate the antiapoptotic Mcl-1 protein. The present study also reveals that the sensitizing effect of ouabain is associated with its ability to generate reactive oxygen species (ROS), and hydrogen peroxide is identified as the principle ROS triggering proteasomal Mcl-1 degradation. In summary, our results indicate a novel function for ouabain in TRAIL-mediated cancer cell death through Mcl-1 downregulation, thereby providing new insight into a potential lung cancer treatment as well as a better understanding of the physiological activity of ouabain.

Gigantol, a Bibenzyl from Dendrobium draconis, Inhibits the Migratory Behavior of Non-Small Cell Lung Cancer Cells

Lung cancer is one of the most common causes of cancer death due to its high metastasis potential. The process of cancer migration is an early step that is required for successful metastasis. The discovery and development of natural compounds for cancer therapy have garnered increasing attention in recent years. Gigantol (1) is a bibenzyl compound derived from the Thai orchid, Dendrobium draconis. It exhibits significant cytotoxic activity against several cancer cell lines; however, until recently, the role of 1 on tumor metastasis has not been characterized. This study demonstrates that 1 suppresses the migratory behavior of non-small cell lung cancer H460 cells. Western blot analysis reveals that 1 down-regulates caveolin-1 (Cav-1), activates ATP-dependent tyrosine kinase (phosphorylated Akt at Ser 473), and cell division cycle 42 (Cdc42), thereby suppressing filopodia formation. The inhibitory effect of 1 on cell movement is also exhibited in another lung cancer cell line, H292, but not in normal human keratinocytes (HaCat). The inhibitory activity of 1 on lung cancer migration suggests that this compound may be suitable for further development for the treatment of cancer metastasis.

Ouabain Suppresses the Migratory Behavior of Lung Cancer Cells

The migratory capability of cancer cells is one of the most important hallmarks reflecting metastatic potential. Ouabain, an endogenous cardiac glycoside produced by the adrenal gland, has been previously reported to have anti-tumor activities; however, its role in the regulation of cancer cell migration remains unknown. The present study has revealed that treatment with ouabain at physiological concentrations is able to inhibit the migratory activities of human lung cancer H292 cells. The negative effects of ouabain were found to be mediated through the suppression of migration regulatory proteins, such as focal adhesion kinase (FAK), ATP-dependent tyrosine kinase (Akt), and cell division cycle 42 (Cdc42). We found that the observed actions of ouabain were mediated via a reactive oxygen species (ROS)-dependent mechanism because the addition of ROS scavengers (N-acetylcysteine and glutathione) could reverse the effect of ouabain on cell migration. Furthermore, ouabain was shown to inhibit the spheroidal tumor growth and decrease the cancer cell adhesion to endothelial cells. However, the compound had no significant effect on anoikis of the cells. Together, these findings shed light on the understanding of cancer cell biology by exploring the novel function of this endogenous human substance.

Nitric oxide induces cancer stem cell-like phenotypes in human lung cancer cells

Even though tremendous advances have been made in the treatment of cancers during the past decades, the success rate among patients with cancer is still dismal, largely because of problems associated with chemo/radioresistance and relapse. Emerging evidence has indicated that cancer stem cells (CSCs) are behind the resistance and recurrence problems, but our understanding of their regulation is limited. Rapid reversible changes of CSC-like cells within tumors may result from the effect of biological mediators found in the tumor microenvironment. Here we show how nitric oxide (NO), a key cellular modulator whose level is elevated in many tumors, affects CSC-like phenotypes of human non-small cell lung carcinoma H292 and H460 cells. Exposure of NO gradually altered the cell morphology toward mesenchymal stem-like shape. NO exposure promoted CSC-like phenotype, indicated by increased expression of known CSC markers, CD133 and ALDH1A1, in the exposed cells. These effects of NO on stemness were reversible after cessation of the NO treatment for 7 days. Furthermore, such effect was reproducible using another NO donor, S-nitroso-N-acetylpenicillamine. Importantly, inhibition of NO by the known NO scavenger 2-(4-carboxy-phenyl)-4,4,5,5 tetramethylimidazoline-1-oxy-3-oxide strongly inhibited CSC-like aggressive cellular behavior and marker expression. Last, we unveiled the underlying mechanism of NO action through the activation of caveolin-1 (Cav-1), which is upregulated by NO and is responsible for the aggressive behavior of the cells, including anoikis resistance, anchorage-independent cell growth, and increased cell migration and invasion. These findings indicate a novel role of NO in CSC regulation and its importance in aggressive cancer behaviors through Cav-1 upregulation.