Yu Ping Hsiao

Benzyl isothiocyanate (BITC) induces G 2/M phase arrest and apoptosis in human melanoma A375.S2 cells through reactive oxygen species (ROS) and both mitochondria-dependent and death receptor-mediated multiple signaling pathways

Benzyl isothiocyanates (BITC), a member of the isothiocyanate (ITC) family, inhibits cell growth and induces apoptosis in many types of human cancer cell lines. The present study investigated mechanisms underlying BITC-induced apoptosis in A375.S2 human melanoma cancer cells. To observe cell morphological changes and viability, flow cytometric assays, cell counting, and a contrast-phase microscopic examination were carried out in A375.S2 cells after BITC treatment. Cell cycle distribution and apoptosis were assessed with the analysis of cell cycle by flow cytometric assays, DAPI staining, propidium iodide (PI), and annexin V staining. Apoptosis-associated factors such as reactive oxygen species (ROS) formation, loss of mitochondrial membrane potential (ΔΨ(m)), intracellular Ca(2+) release, and caspase-3 activity were evaluated by flow cytometric assays. Abundance of cell cycle and apoptosis associated proteins was determined by Western blotting. AIF and Endo G expression was examined by confocal laser microscope. Results indicated that (1) BITC significantly reduced cell number and induced cell morphological changes in a dose-dependent manner in A375.S2 cells; (2) BITC induced arrest in cell cycle progression at G(2)/M phase through cyclin A, CDK1, CDC25C/Wee1-mediated pathways; (3) BITC induced apoptosis and increased sub-G(1) population; and (4) BITC promoted the production of ROS and Ca(2+) and loss of ΔΨ(m) and caspase-3 activity. Furthermore, BITC induced the down-regulation of Bcl-2 expression and induced up-regulation of Bax in A375.S2 cells. Moreover, BITC-induced cell death was decreased after pretreatment with N-acetyl-l-cysteine (NAC, a ROS scavenger) in A375.S2 cells. In conclusion, the results showed that BITC promoted the induction of G(2)/M phase arrest and apoptosis in A375.S2 human melanoma cells through ER stress- and mitochondria-dependent and death receptor-mediated multiple signaling pathways. These data suggest that BITC has potential as an agent for the treatment of melanoma.

Antiproliferative effects of lactic acid via the induction of apoptosis and cell cycle arrest in a human keratinocyte cell line (HaCaT)

BACKGROUND Alpha-hydroxy acids (AHAs) have been widely used in cosmetic industry. However, knowledge on cytotoxicity of AHAs in human keratinocytes is limited. OBJECTIVE Lactic acid (LA) is one of the most commonly used AHAs in skin care and peeling formulations. We investigated the antiproliferative effects of LA in a human keratinocyte cell line (HaCaT). METHODS HaCaT cells were treated with LA at 7.5 approximately 17.5mM for various time periods. The molecular mechanisms of anti-proliferation through cell cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) stain, flow cytometry, Western blot and confocal microscopy. RESULTS Viability of HaCaT cells decreased on exposure to LA. Flow cytometry showed apoptosis was closely related to the increase of reactive oxygen species (ROS) and calcium release, and to the decline of mitochondrial membrane potential (MMP). Western blotting showed an increase in the levels of P21, P27 and a decrease in the levels of Cyclin E, Cyclin A, and CDK 2, indicating cell cycle arrest at G1/S. The occurrence of apoptosis was proved by the increased expressions of Fas, Bax, caspase-3, -8, and -9, apoptosis-inducing factor (AIF), and endonuclease G (EndoG), and the declined expressions of Bcl-2 and Bcl-xL. In addition, the intracytosolic release of AIF, EndoG, and cytochrome c contributing to the occurrence of apoptosis was demonstrated by confocal microscopy. CONCLUSION We demonstrated that LA had antiproliferative effect in HaCaT cell through the inhibition of cell cycle progression at G1/S, and the induction of programmed cell death through caspase-dependent and caspase-independent pathways.

Triggering Apoptotic Death of Human Malignant Melanoma A375.S2 Cells by Bufalin: Involvement of Caspase Cascade-Dependent and Independent Mitochondrial Signaling Pathways

Bufalin was obtained from the skin and parotid venom glands of toad and has been shown to induce cytotoxic effects in various types of cancer cell lines, but there is no report to show that whether bufalin affects human skin cancer cells. The aim of this investigation was to study the effects of bufalin on human malignant melanoma A375.S2 cells and to elucidate possible mechanisms involved in induction of apoptosis. A375.S2 cells were treated with different concentrations of bufalin for a specific time period and investigated for effects on apoptotic analyses. Our results indicated that cells after exposure to bufalin significantly decreased cell viability, and induced cell morphological changes and chromatin condensation in a concentration-dependent manner. Flow cytometric assays indicated that bufalin promoted ROS productions, loss of mitochondrial membrane potential (ΔΨm), intracellular Ca2+ release, and nitric oxide (NO) formations in A375.S2 cells. Additionally, the apoptotic induction of bufalin on A375.S2 cells resulted from mitochondrial dysfunction-related responses (disruption of the ΔΨm and releases of cytochrome c, AIF, and Endo G), and activations of caspase-3, caspase-8 and caspase-9 expressions. Based on those observations, we suggest that bufalin-triggered apoptosis in A375.S2 cells is correlated with extrinsic- and mitochondria-mediated multiple signal pathways.

Synergistic phototoxic effects of glycolic acid in a human keratinocyte cell line (HaCaT).

BACKGROUND Glycolic acid (GA) has been widely used in cosmetic agents and superficial chemical peeling in recent years. It has long been concerned that UV irradiation would enhance the photosensitivity of GA on human skin. Therefore, it is mandatory to explore the biologic effects of concomitant exposure of GA and UV irradiation in human keratinocytes. OBJECTIVE The aim of the study is to explore the effects of concomitant exposure of GA and UVB in a human keratinocyte cell line (HaCaT). METHODS We used HaCaT to investigate the effects of GA (5mM), UVB (50mJ/cm(2)), and co-treatment with GA and UVB (GA+UVB) in human keratinocytes. We used a phase contrast microscope to observe morphological changes of the cells, and employed flow cytometry to detect cell viability, cell cycle, and mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) levels. Cell damage was detected by DAPI stain, and Western blot was used to detect the activities of apoptosis- related and cycle checkpoint-related proteins such as Bax, Bcl-2, caspases-3, -4, -9, Endo G, AIF, and p21, p27, p53, cdk2, cyclin E, cyclin A. RESULTS We found that either GA or UVB alone had inhibitory effect on cell proliferation, and co-treatment with GA and UVB had synergistic anti-proliferative effect. GA alone did not affect the cell cycle, and UVB induced HaCaT cells accumulated at S phase, and co-treatment with GA and UVB arrested cells at S phase more prominently. Moreover, all the treatment with GA, UVB, and GA+UVB in HaCaT cells induced apoptosis. We further demonstrated that GA had synergistic apoptotic effect in human keratinocytes. GA and UVB both had effects on the decline of MMP and increase of ROS release, and GA had synergistic increase in the level of ROS in UVB-treated HaCaT cells. Besides, co-treatment with GA and UVB had synergistic effect on apoptosis through the over-expressions of Bax, p21, p53, caspases-3, -4, -9, Endo G and AIF, and confocal microscopy disclosed translocation of AIF and Endo G from cytoplasm to the nucleus. Therefore, apoptosis induced by co-treatment by GA+UVB was initiated and executed by multiple pathways including mitochondria- and ER-dependent, and caspase-dependent and caspase-independent pathways. CONCLUSION We demonstrated that GA, UVB, GA+UVB inhibited proliferation and induced apoptosis in HaCaT cells. The mechanisms of apoptosis induced by co-treatment of GA and UVB involve multiple pathways. The synergistic photo-toxicity may be related to cell cycle arrest and apoptosis in UVB-treated HaCaT cells. These results highlight the potential adverse effects of GA-containing cosmetic agents on human skin.

Crude extract of Rheum palmatum L inhibits migration and invasion of LS1034 human colon cancer cells acts through the inhibition of matrix metalloproteinase‐2/‐9 by MAPK signaling

Crude extract of Rheum palmatum L. (CERP) has been used to treat different diseases in the Chinese population for decades. In this study, we investigated the anti‐metastasis effects of CERP on LS1034 human colorectal cancer cells in vitro and examined potential mechanisms of its effects. CERP significantly inhibited cell migration and invasion of LS1034 cells. We also found that CERP inhibited protein levels of matrix metalloproteinases‐2 (MMP‐2) and matrix metalloproteinases‐9 (MMP‐9), and cytosolic NF‐kB p65, RHO A, ROCK 1. Furthermore, we found CERP inhibited protein levels of GRB2, SOS1, MKK7, FAK, Rho A, ROCK 1, VEGF, PKC, AKT, phosphor‐AKT (Thr308), Cyclin D, iNOS, COX2, NF‐kB p65, p‐ERK1/2, p‐JNK1/2, p‐p38, p‐c‐jun, MMP‐2, MMP‐9, MMP‐1, MMP‐7, MMP‐10, UPA and increased the protein level of Ras in LS1034 cells. In conclusion, our results suggest that CERP may be used as a novel anti‐metastasis agent for the treatment of human colon cancer cells.

Cantharidin induces G2/M phase arrest by inhibition of Cdc25c and cyclin A and triggers apoptosis through reactive oxygen species and the mitochondria-dependent pathways of A375-S2 human melanoma cells

Cantharidin (CTD), a component of natural mylabris (Mylabris phalerata Pallas) was reported to have high cytotoxicity in many human cancer cell lines. However, it was not reported to affect human melanoma A375.S2 cells. In the present study, we found that CTD induced cell morphological changes and decreased the percentage of viable cells and induced G2/M phase arrest and induction of apoptosis in A375.S2 cells. Results also showed that CTD induced the generation of reactive oxygen species (ROS) and Ca2+ and decreased mitochondria membrane potential and lead to the release of cytochrome c, AIF and Endo G. Further investigation revealed that CTD induced A375.S2 cells with an increase of caspase activation and caspase-dependent apoptotic proteins to trigger correlated pathway mechanisms according to western blotting results. Western blotting was used for examining the changes of G2/M phase arrest and apoptosis-associated protein expression and confocal laser microscopy was used to examine the translocation apoptosis-associated protein. Results showed that CTD increased the protein expression of caspase-3, -8 and -9, cytochrome c, Bax, Bid, Endo G and AIF but inhibited the levels of Bcl-2 and Bcl-x. CTD induced ER stress-associated protein expression such as GRP78, IRE1β, ATF6α and caspase-12. Based on those observations, we suggest that CTD may have potential as a novel anti-cancer agent for the treatment of skin cancer.

Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

Malic acid (MA) has been commonly used in cosmetic products, but the safety reports in skin are sparse. To investigate the biological effects of MA in human skin keratinocytes, we investigated the potential cytotoxicity and apoptotic effects of MA in human keratinocyte cell lines (HaCaT). The data showed that MA induced apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells and normal human epidermal keratinocytes (NHEKs). Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX) but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR) was significantly decreased whereas extracellular acidification rate (ECAR) was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways.

Casticin Induced Apoptosis in A375.S2 Human Melanoma Cells through the Inhibition of NF-κ B and Mitochondria-Dependent Pathways in Vitro and Inhibited Human Melanoma Xenografts in a Mouse Model in Vivo

Casticin, a polymethoxyflavone occurring in natural plants, has been shown to have anticancer activities. In the present study, we aims to investigate the anti-skin cancer activity of casticin on melanoma cells in vitro and the antitumor effect of casticin on human melanoma xenografts in nu/nu mice in vivo. A flow cytometric assay was performed to detect expression of viable cells, cell cycles, reactive oxygen species production, levels of [Formula: see text] and caspase activity. A Western blotting assay and confocal laser microscope examination were performed to detect expression of protein levels. In the in vitro studies, we found that casticin induced morphological cell changes and DNA condensation and damage, decreased the total viable cells, and induced G2/M phase arrest. Casticin promoted reactive oxygen species (ROS) production, decreased the level of [Formula: see text], and promoted caspase-3 activities in A375.S2 cells. The induced G2/M phase arrest indicated by the Western blotting assay showed that casticin promoted the expression of p53, p21 and CHK-1 proteins and inhibited the protein levels of Cdc25c, CDK-1, Cyclin A and B. The casticin-induced apoptosis indicated that casticin promoted pro-apoptotic proteins but inhibited anti-apoptotic proteins. These findings also were confirmed by the fact that casticin promoted the release of AIF and Endo G from mitochondria to cytosol. An electrophoretic mobility shift assay (EMSA) assay showed that casticin inhibited the NF-[Formula: see text]B binding DNA and that these effects were time-dependent. In the in vivo studies, results from immuno-deficient nu/nu mice bearing the A375.S2 tumor xenograft indicated that casticin significantly suppressed tumor growth based on tumor size and weight decreases. Early G2/M arrest and mitochondria-dependent signaling contributed to the apoptotic A375.S2 cell demise induced by casticin. In in vivo experiments, A375.S2 also efficaciously suppressed tumor volume in a xenotransplantation model. Therefore, casticin might be a potential therapeutic agent for the treatment of skin cancer in the future.

Casticin Inhibits A375.S2 Human Melanoma Cell Migration/Invasion through Downregulating NF-κB and Matrix Metalloproteinase-2 and -1

Casticin is one of the main components from Fructus Viticis, which is widely used as an anti-inflammatory agent. The mechanism of how casticin affects melanoma cell migration and invasion is still not well known. Here we studied the anti-metastasis effects of casticin on A375.S2 melanoma cells by using a non-lethal concentration. First; we used an adhesion assay to test the A375.S2 cells’ adhesion ability after treatment with casticin. We next investigated the cell migration ability after casticin treatment by using a wound healing assay to prove that the migration of A375.S2 cells can be inhibited by casticin and double checked the results using the transwell-migration assay. The suppressive effects on matrix metalloproteinase-2; and -9 (MMP-2; and -9) activities were examined by gelatin zymography. Furthermore, western blotting was used to investigate the protein level changes in A375.S2 cells. We found that p-EGFR; Ras and p-ERK1/2 are decreased by casticin, indicating that casticin can down-regulate the migration and invasion ability of A375.S2 cells via the p-EGFR/Ras/p-ERK pathway. The NF-κB p65 and p-ERK levels in nuclear proteins are also decreased by treatment with casticin. An EMSA assay also discovered that the NF-κB p65 and DNA interaction is decreased. NF-κB p65 protein level was examined by immunofluorescence staining and also decreased. Our findings suggest that casticin has anti-metastatic potential by decreasing the invasiveness of A375.S2 cells. We also found that casticin suppressed A375.S2 cell proliferation and cell adhesion ability, but did not affect cell death, as examined using cytometry and a collagen adhesion assay. Based on these observations, casticin could be used as an inhibitor of migration and invasion of human melanoma cells in the future.