Phytochemical-induced reactive oxygen species and endoplasmic reticulum stress-mediated apoptosis and differentiation in malignant melanoma cells.

BACKGROUND: Phytochemicals are derived from plants, vegetables and daily products and exert chemopreventive effects. Malignant melanoma is highly metastatic, and melanoma patients can develop chemotherapeutic resistance against conventional melanoma therapies. METHODS: In the present study, we investigated the anti-cancer effect of the phytochemicals kaempferol (Kaem), genistein (Gen), and 3'3-diindolylmethane (DIM) on melanoma cell viability. We also evaluated the altered expression of cell cycle-related genes. We verified the production of intracellular reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress at both the protein and cellular level using a western blot, TUNEL assay, and Dihydrodichlorofluorescein diacetate (DCF-DA) assay. RESULTS: Treatment of A375SM melanoma cells with phytochemicals resulted in inhibition of cell growth. Treatment with phytochemicals increased the gene expression of p21 and decreased the gene expression of cyclin E and/or cyclin B. The three phytochemicals activated the ROS-p38-p53 apoptotic pathway by increasing the level of phosphorylated p38 MAPK and p53, and they activated the ER stress-mediated apoptotic pathway by increasing the level of phosphorylated eIF2α and C/EBP homologous protein (CHOP). Both the ROS-p38-p53 and ER stress-mediated pathway induced the mitochondrial apoptotic pathway by attenuating Bcl-2 expression and upregulating BAX. Detection of morphological changes demonstrated that Kaem and Gen can induce differentiation in A375SM cell line. CONCLUSION: These results indicate that phytochemicals are potentially useful in treatments for melanoma due to their ability to inhibit melanoma cell growth and division via the ROS and ER stress pathway.

Treatment with Phytoestrogens Reversed Triclosan and Bisphenol A-Induced Anti-Apoptosis in Breast Cancer Cells.

Triclosan (TCS) and bisphenol A (BPA) are endocrine-disrupting chemicals that interfere with the hormone or endocrine system and may cause cancer. Kaempferol (Kaem) and 3,3'-diindolylmethane (DIM) are phytoestrogens that play chemopreventive roles in the inhibition of carcinogenesis and cancer progression. In this study, the influence of TCS, BPA, Kaem, and DIM on proliferation and apoptotic abilities of VM7Luc4E2 breast cancer cells were examined. MTT assay revealed that TCS (0.1-10 µM), BPA (0.1-10 µM) and E2 (0.01-0.0001 µM) induced significant cell proliferation of VM7Luc4E2 cells, which was restored to the control (0.1% DMSO) by co-treatment with Kaem (30 µM) or DIM (15 µM). Reactive oxygen species (ROS) production assays showed that TCS and BPA inhibited ROS production of VM7Luc4E2 cells similar to E2, but that co-treatment with Kaem or DIM on VM7Luc4E2 cells induced increased ROS production. Based on these results, the effects of TCS, BPA, Kaem, and DIM on protein expression of apoptosis and ROS production-related markers such as Bax and Bcl-xl, as well as endoplasmic reticulum (ER) stress-related markers such as eIF2α and CHOP were investigated by Western blot assay. The results revealed that TCS, and BPA induced anti-apoptosis by reducing ROS production and ER stress. However, Kaem and DIM effectively inhibited TCS and BPA-induced anti-apoptotic processes in VM7Luc4E2 cells. Overall, TCS and BPA were revealed to be distinct xenoestrogens that enhanced proliferation and anti-apoptosis, while Kaem and DIM were identified as natural chemopreventive compounds that effectively inhibited breast cancer cell proliferation and increased anti-apoptosis induced by TCS and BPA.

Inhibitory effects of 3,3'-diindolylmethane on epithelial-mesenchymal transition induced by endocrine disrupting chemicals in cellular and xenograft mouse models of breast cancer.

As a phytoestrogen, 3,3'-diindolylmethane (DIM) plays a chemopreventive role by inhibiting cancer progression. In this study, we examined the effects of 17ß-estradiol (E2), two endocrine disrupting chemicals (EDCs), triclosan (TCS) and bisphenol A (BPA), and DIM on epithelial-mesenchymal transition (EMT) and metastatic behaviors of estrogen receptor (ER)-positive MCF-7 breast cancer cells. An in vitro assay revealed that E2 (10-9 M), TCS (10-5-10-7 M), and BPA (10-5-10-7 M) induced MCF-7 cell proliferation compared to a control through the ER pathway. In addition, E2, TCS, and BPA changed the cell morphology from the epithelial to the mesenchymal phenotype and increased the migration and invasion capacity of MCF-7 cells via ER; however, co-treatment with DIM (20 µM) effectively suppressed E2, TCS, and BPA-induced cell proliferation, EMT, migration, and invasion of MCF-7 cells. Western blot assay revealed that DIM regulated the protein expression of EMT- and metastasis-related genes toward the inhibition of these processes. Moreover, E2, TCS, and BPA increased the protein expression of CXCR4, which is a receptor of chemokine CXCL12 that is positively involved in breast cancer metastasis via an ER-dependent pathway. Conversely, DIM and a CXCR4 antagonist (AMD3100) decreased CXCR4 protein expression, which led to inhibition of the EMT process, indicating that DIM may suppress E2, TCS or BPA-induced EMT, migration, and invasion of MCF-7 breast cancer cells by suppressing CXCR4 protein expression. These in vitro effects of E2, TCS, BPA, and DIM were also identified in a xenografted mouse model transplanted with MCF-7 breast cancer cells. Taken together, DIM is a potent chemopreventive compound for preventing metastatic behaviors of breast cancer cells induced by EDCs with cancer-related toxicity.

Effects of bisphenol compounds on the growth and epithelial mesenchymal transition of MCF-7 CV human breast cancer cells.

Bisphenol-A (BPA) has been considered as an endocrine disrupting chemical (EDC) because it can exert estrogenic properties. For bisphenol-S (BPS) and bisphenol-F (BPF) that are BPA analogs and substitutes, their risk to estrogen-dependent cancer has been reported rarely compared with the numerous cases of BPA. In this study, we examined whether BPA, BPS, and BPF can lead to the proliferation, migration, and epithelial mesenchymal transition (EMT) of MCF-7 clonal variant (MCF-7 CV) breast cancer cells expressing estrogen receptors (ERs). In a cell viability assay, BPA, BPS, and BPF significantly increased proliferation of MCF-7 CV cells compared to control (DMSO) as did 17ß-estradiol (E2). In Western blotting assay, BPA, BPS, and BPF enhanced the protein expression of cell cycle progression genes such as cyclin D1 and E1. In addition, MCF-7 CV cells lost cell to cell contacts and acquired fibroblast-like morphology by the treatment of BPA, BPS, or BPF for 24 hours. In cell migration assay, BPA, BPS, and BPF accelerated the migration capability of MCF-7 CV cells as did E2. In relation with the EMT process, BPA, BPS, and BPF increased the protein expression ofN-cadherin, while they decreased the protein expression of E-cadherin. When BPA, BPS, and BPF were co-treated with ICI 182,780, an ER antagonist, proliferation effects were reversed, the expression of cyclin D1 and cyclin E1 was downregulated, and the altered cell migration and expression ofN-cadherin and E-cadherin by BPA, BPS, and BPF were restored to the control level. Thus, these results imply that BPS and BPF also have the risk of breast cancer progression as much as BPA in the induction of proliferation and migration of MCF-7 CV cells by regulating the protein expression of cell cycle-related genes and EMT markersvia the ER-dependent pathway.

Potential Anti-proliferative and Immunomodulatory Effects of Marine Microalgal Exopolysaccharide on Various Human Cancer Cells and Lymphocytes In Vitro.

Marine microalgal exopolysaccharides (EPSs) have drawn great attention due to their biotechnological potentials such as anti-viral, anti-oxidant, anti-lipidemic, anti-proliferative, and immunomodulatory activities, etc. In the present study, the EPS derived from microalgae Thraustochytriidae sp.-derived mutant GA was investigated for its anti-proliferation and immunomodulation. Anti-cancer efficacy of the microalgal EPS was examined for the alterations in cell proliferation and cell cycle-related gene expression that occur in three types of human cancer cell lines, BG-1 ovarian, MCF-7 breast, and SW-620 colon cancer cell lines, by its treatment. Alterations in immunoreactivity by the microalgal EPS were examined by measuring its influence on the growth of T and B lymphocytes and cytokine production of T cells. In cell viability assay, the microalgal EPS inhibited cancer cell growth at the lowest concentration of 10-11 dilution and in a dose-responsive manner within the range of dilution of 10-11~10-3. In addition, the protein expression of cell cycle progression genes such as cyclin D1 and E in these cancer cell lines was significantly reduced by the microalgal EPS in a dose- and a time-dependant manner. In cell proliferation assay using T and B cells, the microalgal EPS induced B cell proliferation even at the lowest dilution of 10-11, but not T cells. In cytokine assay, the microalgal EPS decreased the formation of IL-6 and INF-γ at 10-3 dilution compared to the control and had no significant effects on TNF-α. Collectively, these findings suggest that the EPS derived from microalgae Thraustochytriidae sp. GA has an anti-proliferative activity against cancer cells and an immunomodulatory effect by having an influence on B cell proliferation and cytokine secretion of T cells.

Kaempferol, a phytoestrogen, suppressed triclosan-induced epithelial-mesenchymal transition and metastatic-related behaviors of MCF-7 breast cancer cells.

As a phytoestrogen, kaempferol is known to play a chemopreventive role inhibiting carcinogenesis and cancer progression. In this study, the influences of triclosan, an anti-bacterial agent recently known for an endocrine disrupting chemical (EDC), and kaempferol on breast cancer progression were examined by measuring their effects on epithelial-mesenchymal transition (EMT) and metastatic-related behaviors of MCF-7 breast cancer cells. Morphological changes of MCF-7 cells were observed, and a wound-healing assay was performed after the treatment of triclosan and kaempferol. The effects of triclosan and kaempferol on protein expression of EMT-related markers such as E-cadherin, N-cadherin, Snail, and Slug and metastasis-related markers such as cathepsin B, D, MMP-2 and -9 were investigated by Western blot assay. In microscopic observations, triclosan (10-6M) or E2 (10-9M) induced transition to mesenchymal phenotype of MCF-7 cells compared with the control. Co-treatment of ICI 182,780 (10-8M), an ER antagonist, or kaempferol (25µM) with E2 or triclosan restored the cellular morphology to an epithelial phenotype. In a wound-healing scratch and a transwell migration assay, triclosan enhanced migration and invasion of MCF-7 cells, but co-treatment of kaempferol or ICI 182,780 reduced the migration and invasion ability of MCF-7 cells to the control level. In addition, kaempferol effectively suppressed E2 or triclosan-induced protein expressions of EMT and metastasis promoting markers. Taken together, triclosan may be a distinct xenoestrogenic EDC to promote EMT, migration, and invasion of MCF-7 breast cancer cells through ER. On the other hand, kaempferol can be an alternative chemopreventive agent to effectively suppress the metastatic behavior of breast cancer induced by an endogenous estrogen as well as exogenous xenoestrogenic compounds including triclosan.

Effects of microalgal polyunsaturated fatty acid oil on body weight and lipid accumulation in the liver of C57BL/6 mice fed a high fat diet.

Dietary polyunsaturated fatty acids (PUFAs), which are abundant in marine fish oils, have recently received global attention for their prominent anti-obesogenic effects. Among PUFAs, eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), which are n-3 long-chain PUFAs widely referred to as omega-3 oils, were reported to prevent the development of obesity in rodents and humans. In the present study, we evaluated the anti-obesity effects of microalgal oil on high-fat induced obese C57BL/6 mice, compared with commercial omega-3 fish oil and vegetable corn oil. Microalgal oil is an inherent mixture of several PUFAs, including EPA, DHA and other fatty acids produced from a marine microalgal strain of Thraustochytriidae sp. derived mutant. It was found to contain more PUFAs (>80%) and more omega-3 oils than commercial omega-3 fish oil (PUFAs >31%) and corn oil (PUFAs 59%). All three types of oils induced weight loss in high-fat-induced obese mice, with the loss induced by microalgal oil being most significant at 9 weeks (10% reduction). However, the oils tested did not improve blood lipid levels, although microalgal oil showed an apparent inhibitory effect on lipid accumulation in the liver. These findings may be attributed to the higher PUFA content, including omega-3 oils of microalgal oil than other oils. Collectively, these findings suggest that microalgal oil, derived from Thraustochytriidae sp. derived mutant, is a prominent candidate for replacement of omega-3 fish oils based on its apparent anti-obesity effect in vivo.

Roles of Dietary Phytoestrogens on the Regulation of Epithelial-Mesenchymal Transition in Diverse Cancer Metastasis.

Epithelial-mesenchymal transition (EMT) plays a key role in tumor progression. The cells undergoing EMT upregulate the expression of cell motility-related proteins and show enhanced migration and invasion. The hallmarks of EMT in cancer cells include changed cell morphology and increased metastatic capabilities in cell migration and invasion. Therefore, prevention of EMT is an important tool for the inhibition of tumor metastasis. A novel preventive therapy is needed, such as treatment of natural dietary substances that are nontoxic to normal human cells, but effective in inhibiting cancer cells. Phytoestrogens, such as genistein, resveratrol, kaempferol and 3,3'-diindolylmethane (DIM), can be raised as possible candidates. They are plant-derived dietary estrogens, which are found in tea, vegetables and fruits, and are known to have various biological efficacies, including chemopreventive activity against cancers. Specifically, these phytoestrogens may induce not only anti-proliferation, apoptosis and cell cycle arrest, but also anti-metastasis by inhibiting the EMT process in various cancer cells. There have been several signaling pathways found to be associated with the induction of the EMT process in cancer cells. Phytoestrogens were demonstrated to have chemopreventive effects on cancer metastasis by inhibiting EMT-associated pathways, such as Notch-1 and TGF-beta signaling. As a result, phytoestrogens can inhibit or reverse the EMT process by upregulating the expression of epithelial phenotypes, including E-cadherin, and downregulating the expression of mesenchymal phenotypes, including N-cadherin, Snail, Slug, and vimentin. In this review, we focused on the important roles of phytoestrogens in inhibiting EMT in many types of cancer and suggested phytoestrogens as prominent alternative compounds to chemotherapy.

Immortalization of human corneal epithelial cells using simian virus 40 large T antigen and cell characterization.

INTRODUCTION: Primary cultures of human corneal epithelial (HCE) cells usually cease to grow after four or five passages. This result in a small cell yield for experiments such as the eye irritancy test represents a serious problem for human and animal corneal epithelial research. In the present study, we established an HCE cell line immortalized by simian virus 40 (SV40), a polyomavirus, and characterized the inherent morphologic and cytologic cell properties. METHODS: Primary cultured HCE cells were infected with a SV40 large T antigen (SV40 T)-expressing retrovirus, and were selected using G418 solution, an aminoglycoside antibiotic. To ensure that the immortalized cell lines express SV40 T and cytokeratin-3, a corneal epithelial-specific marker, we conducted reverse-transcription (RT)-PCR and Western blot analysis. RESULTS: These cell lines continued to grow for more than 50 generations, exhibiting a cobble stone-like appearance similar to normal HCE cells and an increased proliferation rate compared to primary cultured HCE cells. RT-PCR results showed that the immortalized cell lines expressed SV40 T while the primary cultured cells did not. In the Western blot assay, protein levels of phosphorylated (Ser15) p53 protein were significantly decreased in the immortalized cell lines while the expression of total p53 protein was constant. In addition, expression of p21(cip1), a cell cycle protein, was down-regulated in the immortalized cells. Moreover, a cornea epithelium-specific marker, cytokeratin-3 (CK-3), was expressed at equal levels in the immortalized cells and primary HCE cells. DISCUSSION: Taken together, these results indicate that immortalized HCE cell lines were successfully established using the SV40-retroviral vector. These cells may be an excellent model for detecting the adverse effects of standard toxic materials and could replace the traditional eye irritation test as an animal-free alternative method.