Search

Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC-1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose-dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl-2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl-2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma., (© 2018 Wiley Periodicals, Inc.)

Background: Norcantharidin, a modified pure compound from blister beetles, was previously demonstrated to induce apoptosis of cancer cells. This study investigated its anti-cancer activity in prostate cancer cells and the mechanisms involved., Methods: Two human prostate cancer cell lines, 22Rv1 and Du145, were treated with norcantharidin at concentrations ranging from 3 to 30μg/ml. Cytotoxic effect of norcantharidin was determined by use of the 3-(4,5-dimethylthiazol-yl)-diphenyl tetrazoliumbromide (MTT) assay. The effects of apoptosis were evaluated by cell death assay, Caspase-3, -8, -9 activity and cytochrome c release. The apoptotic related protein expressions (Bcl-2 family and inhibitor of apoptosis proteins) were determined using western blotting., Results: An MTT assay revealed that norcantharidin induced cytotoxicity against both prostate cancer cells in dose- and time-dependent manners. Treatment with norcantharidin at 3μg/ml or higher significantly increased oligonucleosomal formation with concomitant appearance of PARP cleavage, implicating the induction of apoptosis. Norcantharidin intrinsically elevated cytosolic cytochrome c levels and activated caspase-3, -8, and -9. Extrinsically, it upregulated the expression of not only the death receptors Fas and DR5 in 22Rv1 cells, but also of RIP and TRADD adaptor proteins in Du145 cells. Mechanistically, norcantharidin increased ratios of pro-/anti-apoptotic proteins and decreased expression of IAP family member proteins, including cIAP1 and survivin, regardless of the distinct status of androgen receptor expression in both cells., Conclusions: Norcantharidin exhibited cytotoxicity against 22Rv1 and Du145 prostate cancer cells by inducing both intrinsic and extrinsic apoptotic pathways and could thus potentially be a remedy for prostate cancer., (Copyright © 2016 Institute of Pharmacology, Polish Academy of Sciences. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.)

Butein is a polyphenol, one of the compounds of chalcones, which are flavonoids that are widely biosynthesized in plants, and exhibits different pharmacological activities. Plants containing butein have been used in Chinese traditional medicine. Recently, it has been reported that butein suppresses proliferation and triggers apoptosis in various human cancer cells in vitro and in vivo. The aim of this study was to investigate its pro-apoptotic effect and mechanisms in two cultured human ovarian cancer cells (ES-2 and TOV-21G). The effects of butein on cell viability were assessed by a MTT assay at 3, 10, 30, and 100 μ/M. The apoptotic pathway related factors, including the mitochondrial transmembrane potential (MTP), cytochrome c, caspase cascade, and Bcl-2 family proteins, were examined. MTT assay revealed that butein was cytotoxic to both ovarian cancer cells in a dose- and time-dependent manner. JC-1 flow cytometry, cytochrome c, and caspase activity assays revealed that butein damaged the MTP, increased the level of cytosol cytochrome c and the activities of caspase-3, -8, and -9 in the two ovarian cancer cells. Western blot analysis revealed that butein down-regulated the anti-apoptotic proteins Bcl-2 and Bcl-xL and increased the pro-apoptotic proteins Bax and Bad. These findings suggest that butein-induced apoptosis in ovarian cancer cells via the activation of both extrinsic and intrinsic pathways. In addition, butein also down-regulated the expressions of the inhibitor of apoptosis (IAP) proteins, XIAP, survivin, CIAP-1, and CIAP-2. This indicates that the inhibition of IAP proteins was also involved in butein-induced apoptosis. The results of our study suggest that butein may be a promising anticancer agent in treating ovarian cancer.

Antrodia camphorata is a Chinese herb indigenous to Taiwan. Previous reports demonstrated that it could induce apoptosis in some cancer cells. The purpose of this study was to investigate the apoptotic effect of the crude extract of A. camphorata in cervical cancer cells. Two human cervical cancer cell lines, HeLa and C-33A, were treated with extract of A. camphorata (10-1000 μg/mL). We found that A. camphorata extract was cytotoxic to both cervical cancer cells in a dose- and time-dependent manner as examined by MTT assay. Treatment with A. camphorata extract at 400 μg/mL induced a 2.3- and 4.4-fold increase in oligonucleosome formation from the cleaved chromosomal DNA in HeLa and C-33A cells, respectively. A. camphorata extract also activated caspase-3, -8, and -9 activities and increased the cytosolic level of cytochrome c in both cell lines as the dosage increased. Furthermore, A. camphorata extract increased expressions of Bak, Bad and Bim, while decreasing expressions of Bcl-2 and Bcl-xL of the Bcl-2 family proteins in HeLa and C-33A cells. The expression of IAP proteins, XIAP and survivin, was also decreased in both cervical cancer cells after treatment with A. camphorata. Our in vitro study suggests that A. camphorata is cytotoxic to cervical cancer cells through both extrinsic and intrinsic apoptotic mechanisms. It could be used as a novel phytotherapeutic agent or auxiliary therapy in the treatment of cervical cancer.

Purpose: To study the cytotoxic effects and related signaling pathways of butein on human uveal melanoma cells in vitro., Materials and Methods: Three human uveal melanoma cell lines (M17, SP6.5, and C918), retinal pigment epithelial (RPE) cells and scleral fibroblasts were treated with butein at different dosages. The effects of butein on cell viability were assessed by using the MTT assay. Cell apoptosis was determined using annexin V-FITC/ethidium homodimer III flow cytometry. Mitochondrial transmembrane potential changes were assessed by using the JC-1 fluorescent reader, cytosol cytochrome c levels, and the activities of caspase-3, -8, and -9 were measured by using an enzyme-linked immunosorbent assay or colorimetric assay., Results: Butein reduced the cell viability of cultured human uveal melanoma cells in a dose-dependent manner (10, 30, and 100 μM), with IC50 at 13.3 μM and 15.8 μM in SP6.5 and M17 cell lines, respectively. Similar effects were also found in a highly aggressive and metastatic C918 cell line (IC50 16.7 μM). Butein at lower concentrations (10-30 μM) selectively reduced the cell viability of uveal melanoma cells, without affecting cell viability of RPE cells and fibroblasts. Butein-induced apoptosis of melanoma cells, increased mitochondrial permeability and the level of cytosol cytochrome c, caspase-9 and -3 activities (but not caspase-8) in a dose-dependent manner., Conclusions: Butein has selectively potent pro-apoptotic effects on cultured human uveal melanoma cells via the intrinsic mitochondrial pathway.

Introduction: Antrodia camphorata is a Chinese herb. Recently, several reports demonstrated that it had growth-inhibiting effects on some cancer cells. In this study, we investigated whether the crude extract of A. camphorata could inhibit the growth of ovarian cancer cells and examined the possible mechanisms involved. We also examined whether the cytotoxic effect of paclitaxel on ovarian cancer cells would be affected by A. camphorata., Materials and Methods: Two human ovarian cancer cell lines, SKOV-3 and TOV-21G, were treated with A. camphorata (3-300 μg/mL). An MTT assay was used to test its cytotoxic effect. The apoptosis-related factors including the activity of caspase-3, -8, and -9 and the cytochrome c level released from mitochondria were analyzed. The expression of Bcl-2 family proteins (Bcl-2, Bcl-xL, Bax, Bim, Bad, and Bak) was examined by Western blot analysis. Cell lines were further treated with paclitaxel or paclitaxel plus A. camphorata to examine the cytotoxic efficiency., Results: The MTT assay revealed that A. camphorata was cytotoxic to both the ovarian cancer cells in a dose- and time-dependent manner. Activities of caspase-3, -8, and -9 and release of mitochondrial cytochrome c increased in both ovarian cancer cell lines with increased dose of A. camphorata. Western blot analysis of Bcl-2 family proteins revealed an increased expression of Bad in SKOV-3 cells, whereas increased expression of Bim and Bak and decreased expression of Bcl-xL were noted in TOV-21G cells. In addition, the cytotoxic effect of paclitaxel on SKOV-3 and TOV-21G cells was increased significantly with the addition of A. camphorata (P < 0.01) by MTT assay., Conclusions: These in vitro results suggest that A. camphorata causes a cytotoxic effect on ovarian cancer cells through the induction of apoptosis. It may also enhance the antitumor effect of paclitaxel. Further studies with the ultimate goal of conducting clinical trials are warranted.

Involvement of activities of mitogen-activated protein kinases (MAPKs) and signal transducers and activators of transcription (STATs) remains unsolved in norcantharidin-associated breast cancer cell apoptosis. This study investigated the anti-cancer effect of norcantharidin and its underlying mechanism in two human breast cancer cell lines, estrogen receptor (ER)- HS-578T and ER+ MCF-7 cells. Norcantharidin induced potent cytotoxicity and arrested cell growth through increasing phosphorylation of Chk1, Chk2 and total p21(Waf1/Cip1) and reducing cyclin B and cdc25c expression. It also induced apoptosis through extrinsic death receptor and intrinsic mitochondrial pathways by cytochrome c release, caspase activation, oligonucleosome appearance, PARP cleavage, and aberration of Bcl-2 family protein expression and phosphorylation. Although norcantharidin did not affect STAT1, STAT3, and STAT5 protein expression, it suppressed STAT3 and STAT5 phosphorylation in HS-578T cells, whereas it up-regulated STAT1 phosphorylation and down-regulated STAT5 phosphorylation in MCF-7 cells. Moreover, norcantharidin activated MAPK family member proteins, extracellular signal-regulated kinase (ERK), p38(MAPK) and c-Jun N-terminal kinase (JNK), were all phosphorylated by treatment. Pretreatment with selective kinase inhibitors significantly attenuated the norcantharidin-induced cytotoxicity in breast cancer cells. These findings suggest the potential involvement of MAPK and STAT pathways in norcantharidin-induced apoptogenesis. Norcantharidin may be an effective anti-cancer drug against breast cancer., (Copyright © 2011. Published by Elsevier Ltd.)

Norcantharidin exhibits cytotoxicity in many cancer cell lines, including colorectal cancer (CRC) cells. Its cytotoxic potency on primary CRC cells and other normal constituent cells of the human body remains elusive. This study investigates whether norcantharidin differentially exhibits cytotoxicity on primarily isolated CRC cells and dermal fibroblasts. The in vitro chemosensitivity of norcantharidin was measured using a MTT tetrazolium assay and compared with 73 primary tumor cells from surgically excised colorectal tumors, six human CRC cell lines and dermal fibroblasts. Observations of cytotoxicity to primary tumor cells reveal significant differences among genders and histological types; however, drug-induced chemosensitivity was not correlated with age or clinical stages of CRC patients. Norcantharidin had a similar cytotoxic effect on primary tumor cells and CRC cell lines in a dose-dependent manner. In contrast, normal fibroblasts were more resistant to norcantharidin-induced cytotoxicity than CRC cells. DAPI staining results demonstrated that norcantharidin caused CRC cell apoptosis by nuclear fragmentation and chromatin condensation. The release of cytochrome c and the triggering of caspase-3, -8 and -9 activation mediated apoptotic induction. Conversely, pretreatment with caspases or mitogen-activated protein kinase (MAPK) inhibitors significantly suppressed norcantharidin-induced CRC cytotoxicity. These in vitro results suggest that norcantharidin may be a safe and effective anti-cancer drug for CRC., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Uveal melanoma, the most common primary intraocular malignancy in adults, is highly resistant to most chemotherapeutic drugs. Arsenic trioxide (ATO) is known to inhibit ocular melanoma cell growth. However, the effects of ATO on human uveal melanoma cells are poorly understood. Therefore, this study evaluated the mechanisms of ATO and its inhibiting effects on a human uveal melanoma cell line (SP6.5). An MTT assay indicated that, compared to human fibroblasts, ATO had a stronger inhibiting effect on SP6.5 cell proliferation in a dose- and time-dependent manner. The apoptosis ratio in SP6.5 cells, which was indicated by cell DNA fragmentation, was 4.1- to 7.7-fold higher after ATO-treatment. The ATO treatment substantially increased the activities of caspase-3 and caspase-9, but not of caspase-8. These findings were consistent with the protein expression observed by Western blots. ATO also significantly enhanced expression of Bax and cytochrome c proteins but suppressed those of Bcl-2. Therefore, ATO-induced apoptosis in uveal melanoma cells occurs mainly through the mitochondrial pathway rather than through the death receptor pathway. This report is the first to evaluate the complete mitochondria-dependent apoptotic pathway of ATO in uveal melanoma cells. These results can be used to improve the clinical effectiveness of ATO treatment for uveal melanoma.

Aims: Cyclin-dependent kinases (CDKs) play essential roles in the intracellular control of the cell cycle. It has been postulated that roscovitine, a potent CDK2, CDK5, and CDC2 inhibitor, might inhibit cellular proliferation by arresting the cell cycle. This in vitro study investigated the antiproliferative and apoptotic effects of roscovitine in cultured rabbit retinal pigment epithelial (RPE) cells., Methods: Experiments using rabbit RPE from young pigmented rabbits were carried out using roscovitine dissolved in dimethylsulfoxide at concentrations ranging from 1 to 100 micromol. Cell proliferation was measured by an MTT assay. The cell cycle response of RPE cells to roscovitine was analyzed by flow cytometry of propidium iodide-stained nuclei. Proteins related to DNA damage in the RPE cells were then assayed by Western blot., Results: Roscovitine inhibited proliferation of RPE cells in a dose-dependent manner. Cell cycle analysis after treatment demonstrated an accumulation of cells arrested in the S- and G2/M phases. Flow cytometry showed that 40 microM of roscovitine increased the cell population in the sub-G1 peak, which is considered a marker of cell death by apoptosis. Western blot analysis revealed Bcl-2 decreased and Bax increased after treatment of RPE cells with roscovitine., Conclusions: This study of the response of RPE cells to roscovitine demonstrated a bidirectional relationship between cell cycle control and apoptosis.

Butein is a chalcone, a flavonoid that is widely biosynthesized in plants. Butein has been identified to possess varied pharmacological activity and is extractable from traditional Chinese medicinal herbs, therefore applicable for disease treatment. Recently, in vitro and in vivo studies have shown that butein may induce apoptotic cell death in various human cancer cells. In this study we investigated the apoptotic effect of butein and the underlying mechanisms in human cervical cancer cells. Two cell lines, C-33A and SiHa cells, were treated with butein at different dosages for different durations. The effect of butein on cell viability was assessed by MTT assay, which revealed that butein exerted cytotoxicity in both cervical cancer cells in a dose- and time-dependent fashion. Apoptotic pathway-related factors in the butein-treated cervical cancer cells were then examined. JC-1 flow cytometry, cytochrome c assay, and caspase activity assays demonstrated that butein disturbed mitochondrial transmembrane potential, and increased cytosolic cytochrome c levels and caspase activities in both cervical cancer cells. Western blot analysis revealed that butein downregulated anti-apoptotic protein Bcl-xL and led to proteolytic cleavage of poly (ADP-ribose) polymerase. In addition, butein decreased expressions of the inhibitor of apoptosis (IAP) proteins, including X-linked IAP, survivin, and cellular IAP-1. The findings of this study suggest that butein can decrease cervical cancer cell viability via a pro-apoptotic effect, which involves inhibition of the IAP proteins and activation of both extrinsic and intrinsic pro-apoptotic pathways. Therefore, butein may be applicable for cervical cancer treatment. [ABSTRACT FROM AUTHOR]

Abstract: Oxidative damage from reactive oxygen species (ROS) has been implicated in many diseases, including age-related macular degeneration, in which the retinal pigment epithelium (RPE) is considered a primary target. The aim of this study was to determine whether erythropoietin (EPO) protects cultured human RPE cells against oxidative damage and to identify the pathways that may mediate protection. EPO (1 IU/ml) significantly increased the viability of oxidant-treated RPE cells, decreased the release of the inflammatory cytokines tumor necrosis factor-α and interleukin-1β, recovered the RPE cells'' barrier integrity disrupted by oxidative stress, prevented oxidant-induced cell DNA fragmentation and membrane phosphatidylserine exposure, and also reduced the levels of oxidant-induced intracellular ROS and restored cellular antioxidant potential, total antioxidant capacity, glutathione peroxidase, and superoxide dismutase and decreased malondialdehyde, the end product of lipid peroxidation. EPO inhibited caspase-3-like activity. Protection by EPO was partly dependent on the activation of Akt1 and the maintenance of the mitochondrial membrane potential. No enhanced or synergistic protection was observed during application of Z-DEVD-FMK (caspase-3 inhibitor) combined with EPO compared with cultures exposed to EPO and H2O2 alone. Together, these results suggest that EPO could protect against oxidative injury-induced cell death and mitochondrial dysfunction in RPE cells through modulation of Akt1 phosphorylation, mitochondrial membrane potential, and cysteine protease activity. [Copyright &y& Elsevier]