Casticin impairs cell growth and induces cell apoptosis via cell cycle arrest in human oral cancer SCC‐4 cells

Casticin, a polymethoxyflavone, present in natural plants, has been shown to have biological activities including anti‐cancer activities. Herein, we investigated the anti‐oral cancer activity of casticin on SCC‐4 cells in vitro. Viable cells, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS) production, and Ca²⁺ production, levels of ΔΨ_m and caspase activity were measured by flow cytometric assay. Cell apoptosis associated protein expressions were examined by Western blotting and confocal laser microscopy. Results indicated that casticin induced cell morphological changes, DNA condensation and damage, decreased the total viable cells, induced G₂/M phase arrest in SCC‐4 cells. Casticin promoted ROS and Ca²⁺ productions, decreases the levels of ΔΨ_m, promoted caspase‐3, ‐8, and ‐9 activities in SCC‐4 cells. Western blotting assay demonstrated that casticin affect protein level associated with G2/M phase arrest and apoptosis. Confocal laser microscopy also confirmed that casticin increased the translocation of AIF and cytochrome c in SCC‐4 cells. In conclusion, casticin decreased cell number through G₂/M phase arrest and the induction of cell apoptosis through caspase‐ and mitochondria‐dependent pathways in SCC‐4 cells.     

Bisdemethoxycurcumin‐induced S phase arrest through the inhibition of cyclin A and E and induction of apoptosis via endoplasmic reticulum stress and mitochondria‐dependent pathways in human lung cancer NCI H460 cells

Curcuminoids are the major natural phenolic compounds found in the rhizome of many Curcuma species. Curcuminoids consist of a mixture of curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). Although numerous studies have shown that curcumin induced cell apoptosis in many human cancer cells, however, mechanisms of BDMC‐inhibited cell growth and ‐induced apoptosis in human lung cancer cells still remain unclear. Herein, we investigated the effect of BDMC on the cell death via the cell cycle arrest and induction of apoptosis in NCI H460 human lung cancer cells. Flow cytometry assay was used to measure viable cells, cell cycle distribution, the productions of reactive oxygen species (ROS) and Ca²⁺, mitochondrial membrane potential (ΔΨ_m) and caspase‐3, ‐8 and ‐9 activity. DNA damage and condension were assayed by Comet assay and DAPI staining, respectively. Western blotting was used to measure the changes of cell cycle and apoptosis associated protein expressions. Results indicated that BDMC significantly induced cell death through induced S phase arrest and induced apoptosis. Moreover, DMC induced DNA damage and condension, increased ROS and Ca²⁺ productions and decreased the levels of ΔΨ_m and promoted activities caspase‐3, ‐8, and ‐9. Western blotting results showed that BDMC inhibited Cdc25A, cyclin A and E for causing S phase arrest, furthermore, promoted the expression of AIF, Endo G and PARP and the levels of Fas ligand (Fas L) and Fas were also up‐regulated. Results also indicated that BDMC increased ER stress associated protein expression such as GRP78, GADD153, IRE1α, IRE1β, ATF‐6α, ATF‐6β, and caspase‐4. Taken together, we suggest that BDMC induced cell apoptosis through multiple signal pathways such as extrinsic, intrinsic and ES tress pathway. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1899–1908, 2016.     

Benzyl isothiocyanate (BITC) induces apoptosis of GBM 8401 human brain glioblastoma multiforms cells via activation of caspase‐8/Bid and the reactive oxygen species‐dependent mitochondrial pathway

Benzyl isothiocyanate (BITC) is one of member of the isothiocyanate family which has been shown to induce cancer cell apoptosis in many human cancer cells. In the present study, we investigated the effects of BITC on the growth of GBM 8401 human brain glioblastoma multiforms cells. Results indicated that BITC‐induced cell morphological changes decreased in the percentage of viable GBM8401 cells and these effects are dose‐dependent manners. Results from flow cytometric assay indicated that BITC induced sub‐G1 phase and induction of apoptosis of GBM 8401 cells. Furthermore, results also showed that BITC promoted the production of reactive oxygen species (ROS) and Ca²⁺ release, but decreased the mitochondrial membrane potential (ΔΨ_m) and promoted caspase‐8, ‐9, and ‐3 activates. After cells were pretreated with Z‐IETD‐FMK, Z‐LEHD‐FMK, and Z‐DEVD‐FMK (caspase‐8, ‐9, and ‐3 inhibitors, respectively) led to decrease in the activities of caspase‐8, ‐9, and ‐3 and increased the percentage of viable GBM 8401 cells that indicated which BITC induced cell apoptosis through caspase‐dependent pathways. Western blotting indicated that BITC induced Fas, Fas‐L, FADD, caspase‐8, caspase ‐3, and pro‐apoptotic protein (Bax, Bid, and Bak), but inhibited the ant‐apoptotic proteins (Bcl‐2 and Bcl‐x) in GBM 8401 cells. Furthermore, BITC increased the release of cytochrome c, AIF, and Endo G from mitochondria that led to cell apoptosis. Results also showed that BITC increased GADD153, GRP 78, XBP‐1, and ATF‐6β, IRE‐1α, IRE‐1β, Calpain 1 and 2 in GBM 8401 cells, which is associated with ER stress. Based on these observations, we may suggest that BITC‐induced apoptosis might be through Fas receptor, ROS induced ER stress, caspase‐3, and mitochondrial signaling pathways. Taken together, these molecular alterations and signaling pathways offer an insight into BITC‐caused growth inhibition and induced apoptotic cell death of GBM 8401 cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1751–1760, 2016.     

Capsaicin induces apoptosis in SCC-4 human tongue cancer cells through mitochondria-dependent and -independent pathways

Although there have been advances in the fields of surgery, radiotherapy, and chemotherapy of tongue cancer, the cure rates are still not substantially satisfactory. Capsaicin (trans‐8‐methyl‐N‐vanillyl‐6‐nonenamide) is the major pungent ingredient of hot chili pepper and has been reported to have an antitumor effect on many human cancer cell types. The molecular mechanisms of the antitumor effect of capsaicin are not yet completely understood. Herein, we investigated whether capsaicin induces apoptosis in human tongue cancer cells. Capsaicin decreased the percentage of viable cells in a dose‐dependent manner in human tongue cancer SCC‐4 cells. In addition, capsaicin produced DNA fragmentation, decreased the DNA contents (sub‐G1 phase), and induced G0/G1 phase arrest in SCC‐4 cells. We demonstrated that capsaicin‐induced apoptosis is associated with an increase in reactive oxygen species and Ca²⁺ generations and a disruption of the mitochondrial transmenbrane potential (ΔΨ_m). Treatment with capsaicin induced a dramatic increase in caspase‐3 and ‐9 activities, as assessed by flow cytometric methods. A possible mechanism of capsaicin‐induced apoptosis is involved in the activation of caspase‐3 (one of the apoptosis‐executing enzyme). Confocal laser microscope examination also showed that capsaicin induced the releases of AIF, ATF‐4, and GADD153 from mitochondria of SCC‐4 cells. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

Ouabain induces apoptotic cell death in human prostate DU 145 cancer cells through DNA damage and TRAIL pathways

Ouabain, a cardiotonic steroid and specific Na⁺/K⁺‐ATPase inhibitor, has a potential to induce cancer cell apoptosis but the mechanisms of apoptosis induced by ouabain are not fully understand. The aim of this study was to investigate the cytotoxic effects of ouabain on human prostate cancer DU 145 cells in vitro. Cell morphological changes were examined by phase contrast microscopy. Cell viability, cell cycle distribution, cell apoptosis, DNA damage, the production of ROS and Ca²⁺, and mitochondrial membrane potential (ΔΨ_m) were measured by flow cytometry assay. Results indicated that ouabain induced cell morphological changes, decreased total cell viability, induced G0/G1 phase arrest, DNA damage, and cell apoptosis, increased ROS and Ca²⁺ production, but decreased the levels of ΔΨ_m in DU 145 cells. Ouabain also increased the activities of caspase‐3, ‐8, and ‐9. Western blotting was used for measuring the alterations of apoptosis‐associated protein expressions in DU 145 cells and results indicated that ouabain increased the expression of DNA damage associated proteins (pATM^Ser1981, p‐H2A.X^Ser139, and p‐p53^Ser15) and ER‐stress‐associated proteins (Grp78, ATF6β, p‐PERK^Thr981, PERK, eIF2A, GADD153, CaMKIIβ, and caspase‐4) in time‐dependently. Furthermore, ouabain increased apoptosis‐associated proteins (DR4, DR5, Fas, Fas Ligand, and FADD), TRAIL pathway, which related to extrinsic pathway, promoted the pro‐apoptotic protein Bax, increased apoptotic‐associated proteins, such as cytochrome c, AIF, Endo G, caspase‐3, ‐8, and ‐9, but reduced anti‐apoptotic protein Bcl‐2 and Bcl‐x in DU 145 cells. In conclusion, we may suggest that ouabain decreased cell viability and induced apoptotic cell death may via caspase‐dependent and mitochondria‐dependent pathways in human prostate cancer DU 145 cells.     

Crude extract of Euphorbia formosana induces apoptosis of DU145 human prostate cancer cells acts through the caspase‐dependent and independent signaling pathway

Prostate cancer is the most frequently diagnosed malignancy in men and the second highest contributor of male cancer mortality. The crude extract of Euphorbia formosana (CEEF) has been used for treatment of different diseases but the cytotoxic effects of CEEF on human cancer cells have not been reported. The purpose of the present experiments was to determine effects of CEEF on cell cycle distribution and induction of apoptosis in DU145 human prostate cancer cells in vitro. Contrast‐phase microscope was used for examining cell morphological changes. Flow cytometric assays were used for cell viability, cell cycle, apoptosis, reactive oxygen species, and Ca²⁺ production and mitochondria membrane potential (ΔΨ_m). Western blotting was used for examining protein expression of cell cycle and apoptosis associated proteins. Real‐time PCR was used for examining mRNA levels of caspase‐3, ‐8, and ‐9, AIF, and Endo G. Confocal laser microscope was used to examine the translocation of AIF, Endo G, and cytochrome in DU145 cells after CEEF exposure. CEEF‐induced cell morphological changes, decreased the percentage of viable cells, and induced S phase arrest and apoptosis in DU145 cells. Furthermore, CEEF promoted RAS and Ca²⁺ production and reduced ΔΨ_m levels. Real‐time QPCR confirmed that CEEF promoted the mRNA expression of caspase‐3 and ‐9, AIF and Endo G and we found that AIF and Endo G and cytochrome c were released from mitochondria. Taken together, CEEF‐induced cytotoxic effects via ROS production, induced S phase arrest and induction of apoptosis through caspase‐dependent and independent and mitochondria‐dependent pathways in DU245 cancer cells. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1600–1611, 2016.     

A novel 7‐azaisoindigo derivative‐induced cancer cell apoptosis and mitochondrial dysfunction mediated by oxidative stress

This research focused on a novel 7‐azaisoindigo derivative [namely N¹‐(n‐butyl)‐7‐azaisoindigo, 7‐AI‐b], and investigated its molecular antitumor mechanism by exploring the means of cell death and the effects on mitochondrial function. 7‐AI‐b inhibited cancer cell proliferation in a dose‐ and time‐dependent way. The morphological and nuclei changes in H₂B‐GFP‐labeled HeLa cells were observed using a live cell system. The results suggested that cell death induced by 7‐AI‐b is closely related to apoptosis. 7‐AI‐b induced release of cytochrome C from mitochondria to cytosol and activation of caspase‐3, showing that the apoptosis is mediated by the mitochondrial pathway. Furthermore, our data indicated that 7‐AI‐b triggers apoptosis through reactive oxygen species (ROS): cellular ROS levels were increased after 3 h exposure of 7‐AI‐b, which was reversed by the ROS scavenger N‐acetyl‐l ‐cysteine. As a consequence, 7‐AI‐b‐mediated cell death, mitochondrial transmembrane potential collapse and ATP level were partly blocked by N‐acetyl‐l ‐cysteine. Further study showed that 7‐AI‐b could induce mitochondrial dysfunction: collapse of the mitochondrial transmembrane potential and reduction of intracellular ATP level. In summary, the novel synthesized 7‐AI‐b was demonstrated to be effective in killing cancer cells via an ROS‐promoted and mitochondria‐ and caspase‐dependent apoptotic pathway. Copyright © 2010 John Wiley & Sons, Ltd.     

Genistein induces apoptosis in vitro and has antitumor activity against human leukemia HL‐60 cancer cell xenograft growth in vivo

Genistein, a major isoflavone compound in soybeans, has been shown to have biological activities including anti‐cancer activates. In the present, we investigated the anti‐leukemia activity of genistein on HL‐60 cells in vitro. The percentage of viable cell, cell cycle distribution, apoptotic cell death, reactive oxygen species (ROS), and Ca²⁺ production and the level of ΔΨ_m were measured by flow cytometric assay. Cell apoptosis and endoplasmic reticulum (ER) stress associated protein expressions were examined by Western blotting assay. Calpain 1, GRP78, and GADD153 expression were measured by confocal laser microscopy. Results indicated that genistein‐induced cell morphological changes, decreased the total viable cells, induced G₂/M phase arrest and DNA damage and fragmentation (cell apoptosis) in HL‐60 cells. Genistein promoted ROS and Ca²⁺ productions and decreased the level of ΔΨ_m in HL‐60 cells. Western blotting assay demonstrated that genistein increased ER stress‐associated protein expression such as IRE‐1α, Calpain 1, GRP78, GADD153, caspase‐7, caspase‐4, and ATF‐6α at 20‐50 μM treatment and increased apoptosis associated protein expression such as pro‐apoptotic protein Bax, PARP‐cleavage, caspase‐9, and ‐3, but decreased anti‐apoptotic protein such as Bcl‐2 and Bid in HL‐60 cells. Calpain 1, GRP78, and GADD153 were increased in HL‐60 cells after exposure to 40 μM of genistein. In animal xenografted model, mice were intraperitoneally injected with genistein (0, 0.2, and 0.4 mg/kg) for 28 days and the body weight and tumor volume were recorded. Results showed that genistein did not affect the body weights but significantly reduced the tumor weight in 0.4 mg/kg genistein‐treated group. Genistein also increased the expressions of ATF‐6α, GRP78, Bax, Bad, and Bak in tumor. In conclusion, genistein decreased cell number through G₂/M phase arrest and the induction of cell apoptosis through ER stress‐ and mitochondria‐dependent pathways in HL‐60 cells and suppressed tumor properties in vivo.     

Crude extract of Rheum palmatum L induced cell death in LS1034 human colon cancer cells acts through the caspase‐dependent and ‐independent pathways

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 effects of CERP on LS1034 human colorectal cancer cells in vitro and also examined possible mechanisms of cell death. Flow cytometric assays were used to measure the percentage of viable cells, cell cycle distribution including the sub‐G1 phase (apoptosis), the activities of caspase‐8, ‐9, and ‐3, reactive oxygen species (ROS) and Ca²⁺ levels, and mitochondrial membrane potential (ΔΨ_m). DNA damage, nuclei condensation, protein expression, and translocation were examined by Comet assay, 4′‐6‐diamidino‐2‐phenylindole (DAPI) staining, Western blotting, and confocal laser system microscope, respectively. CERP induced apoptosis as seen by DNA fragmentation and DAPI staining in a concentration‐ and time‐dependent manner in cancer cells. CERP was associated with an increase in the Bax/Bcl‐2 protein ratio and CERP promoted the activities of caspase‐8, ‐9, and ‐3. Both ROS and Ca²⁺ levels were increased by CERP but the compound decreased levels of ΔΨ_m in LS1034 cells. Laser confocal microscope also confirmed that CERP promoted the expressions of AIF, Endo G, cytochrome c, and GADD153 to induce apoptosis through mitochondrial‐dependent pathway. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 969–980, 2014.     

The roles of endoplasmic reticulum stress and mitochondrial apoptotic signaling pathway in quercetin‐mediated cell death of human prostate cancer PC‐3 cells

Prostate cancer has its highest incidence and is becoming a major concern. Many studies have shown that traditional Chinese medicine exhibited antitumor responses. Quercetin, a natural polyphenolic compound, has been shown to induce apoptosis in many human cancer cell lines. Although numerous evidences show multiple possible signaling pathways of quercetin in apoptosis, there is no report to address the role of endoplasmic reticulum (ER) stress in quercetin‐induced apoptosis in PC‐3 cells. The purpose of this study was to investigate the effects of quercetin on the induction of the apoptotic pathway in human prostate cancer PC‐3 cells. Cells were treated with quercetin for 24 and 48 h and at various doses (50–200 μM), and cell morphology and viability decreased significantly in dose‐dependent manners. Flow cytometric assay indicated that quercetin at 150 μM caused G0/G1 phase arrest (31.4–49.7%) and sub‐G1 phase cells (19.77%) for 36 h treatment and this effect is a time‐dependent manner. Western blotting analysis indicated that quercetin induces the G0/G1 phase arrest via decreasing the levels of CDK2, cyclins E, and D proteins. Quercetin also stimulated the protein expression of ATF, GRP78, and GADD153 which is a hall marker of ER stress. Furthermore, PC‐3 cells after incubation with quercetin for 48 h showed an apoptotic cell death and DNA damage which are confirmed by DAPI and Comet assays, leading to decrease the antiapoptotic Bcl‐2 protein and level of ΔΨ_m, and increase the proapoptotic Bax protein and the activations of caspase‐3, ‐8, and ‐9. Moreover, quercetin promoted the trafficking of AIF protein released from mitochondria to nuclei. These data suggest that quercetin may induce apoptosis by direct activation of caspase cascade through mitochondrial pathway and ER stress in PC‐3 cells. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 428–439, 2014.     

The crude extract of Corni Fructus induces apoptotic cell death through reactive oxygen species‐modulated pathways in U‐2 OS human osteosarcoma cells

Crude extract of Corni Fructus (CECF) has been used in Traditional Chinese medicine for the treatment of different diseases for hundreds of years. The purpose of this study was to investigate the cytotoxic effects of CECF on U‐2 OS human osteosarcoma cells. Flow cytometry was used for measuring the percentage of viable cells, cell‐cycle distribution, apoptotic cells in sub‐G1 phase, reactive oxygen species (ROS), Ca²⁺ levels, and mitochondrial membrane potential (ΔΨ_m). Comet assay and 4′‐6‐diamidino‐2‐phenylindole staining were used for examining DNA damage and condensation. Western blotting was used to examine apoptosis‐associated protein levels in U‐2 OS cells after exposed to CECF. Immunostaining and confocal laser system microscope were used to examine protein translocation after CECF incubation. CECF decreased the percentage of viability, induced DNA damage and DNA condensation, G₀/G₁ arrest, and apoptosis in U‐2 OS cells. CECF‐stimulated activities of caspase‐8, caspase‐9, and caspase‐3, ROS, and Ca²⁺ production, decreased ΔΨ_m levels of in U‐2 OS cells. CECF increased protein levels of caspase‐3, caspase‐9, Bax, cytochrome c, GRP78, AIF, ATF‐6α, Fas, TRAIL, p21, p27, and p16 which were associated with cell‐cycle arrest and apoptosis. These findings suggest that CECF triggers apoptosis in U‐2 OS cells via ROS‐modulated caspase‐dependent and ‐independent pathways. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 1020–1031, 2014.     

Chrysophanol‐induced cell death (necrosis) in human lung cancer A549 cells is mediated through increasing reactive oxygen species and decreasing the level of mitochondrial membrane potential

Chrysophanol (1,8‐dihydroxy‐3‐methylanthraquinone) is one of the anthraquinone compounds, and it has been shown to induce cell death in different types of cancer cells. The effects of chrysophanol on human lung cancer cell death have not been well studied. The purpose of this study is to examine chrysophanol‐induced cytotoxic effects and also to investigate such influences that involved apoptosis or necrosis in A549 human lung cancer cells in vitro. Our results indicated that chrysophanol decreased the viable A549 cells in a dose‐ and time‐dependent manner. Chrysophanol also promoted the release of reactive oxygen species (ROS) and Ca²⁺ and decreased the levels of mitochondria membrane potential (ΔΨ_m) and adenosine triphosphate in A549 cells. Furthermore, chrysophanol triggered DNA damage by using Comet assay and DAPI staining. Importantly, chrysophanol only stimulated the cytocheome c release, but it did not activate other apoptosis‐associated protein levels including caspase‐3, caspase‐8, Apaf‐1, and AIF. In conclusion, human lung cancer A549 cells treated with chrysophanol exhibited a cellular pattern associated with necrotic cell death and not apoptosis in vitro. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 740–749, 2014.     

Casticin induced apoptotic cell death and altered associated gene expression in human colon cancer colo 205 cells

Casticin, a polymethoxyflavone, derived from natural plant Fructus Viticis exhibits biological activities including anti‐cancer characteristics. The anti‐cancer and alter gene expression of casticin on human colon cancer cells and the underlying mechanisms were investigated. Flow cytometric assay was used to measure viable cell, cell cycle and sub‐G1 phase, reactive oxygen species (ROS) and Ca²⁺ productions, level of mitochondria membrane potential (ΔΨ_m) and caspase activity. Western blotting assay was used to detect expression of protein level associated with cell death. Casticin induced cell morphological changes, decreased cell viability and induced G2/M phase arrest in colo 205 cells. Casticin increased ROS production but decreased the levels of ΔΨ_m, and Ca²⁺, increased caspase‐3, ‐8, and ‐9 activities. The cDNA microarray indicated that some of the cell cycle associated genes were down‐regulated such as cyclin‐dependent kinase inhibitor 1A (CDKN1A) (p21, Cip1) and p21 protein (Cdc42/Rac)‐activated kinase 3 (PAK3). TNF receptor‐associated protein 1 (TRAP1), CREB1 (cAMP responsive element binding protein 1) and cyclin‐dependent kinase inhibitor 1B (CDKN1B) (p27, Kip1) genes were increased but matrix metallopeptidase 2 (MMP‐2), toll‐like receptor 4 (TLR4), PRKAR2B (protein kinase, cAMP‐dependent, regulatory, type II, bet), and CaMK4 (calcium/calmodulin‐dependent protein kinase IV) genes were inhibited. Results suggest that casticin induced cell apoptosis via the activation of the caspase‐ and/or mitochondria‐dependent signaling cascade, the accumulation of ROS and altered associated gene expressions in colo 205 human colon cancer cells.     

Sulforaphane‐induced apoptosis in human leukemia HL‐60 cells through extrinsic and intrinsic signal pathways and altering associated genes expression assayed by cDNA microarray

Sulforaphane (SFN), one of the isothiocyanates, is a biologically active compound extracted from cruciferous vegetables, and has been shown to induce cytotoxic effects on many human cancer cells including human leukemia cells. However, the exact molecular mechanism and altered gene expression associated with apoptosis is unclear. In this study, we investigated SFN‐induced cytotoxic effects and whether or not they went through cell‐cycle arrest and induction of apoptosis and further examined molecular mechanism and altered gene expression in human leukemia HL‐60 cells. Cell viability, cell‐cycle distribution, sub‐G1 (apoptosis), reactive oxygen species (ROS) and Ca²⁺ production, levels of mitochondrial membrane potential (ΔΨ_m), and caspase‐3, ?8, and ?9 activities were assayed by flow cytometry. Apoptosis‐associated proteins levels and gene expressions were examined by Western blotting and cDNA microarray assays, respectively. Results indicated that SFN decreased viable cells, induced G2/M phase arrest and apoptosis based on sub‐G1 phase development. Furthermore, SFN increased ROS and Ca²⁺ production and decreased the levels of ΔΨ_m and activated caspase‐3, ?8, and ?9 activities in HL‐60 cells. SFN significantly upregulated the expression of BAX, Bid, Fas, Fas‐L, caspase‐8, Endo G, AIF, and cytochrome c, and inhibited the antiapoptotic proteins such as Bcl‐x and XIAP, that is associated with apoptosis. We also used cDNA microarray to confirm several gene expressions such as caspase ?8, ?3, ?4, ?6, and ?7 that are affected by SFN. Those results indicated that SFN induced apoptosis in HL‐60 cells via Fas‐ and mitochondria‐dependent pathways. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 311–328, 2017.     

Tetrandrine induces apoptosis Via caspase‐8, ‐9, and ‐3 and poly (ADP ribose) polymerase dependent pathways and autophagy through beclin‐1/ LC3‐I,II signaling pathways in human oral cancer HSC‐3 cells

Tetrandrine is a bisbenzylisoquinoline alkaloid that was found in the Radix Stephania tetrandra S Moore. It had been reported to induce cytotoxic effects on many human cancer cells. In this study, we investigated the cytotoxic effects of tetrandrine on human oral cancer HSC‐3 cells in vitro. Treatments of HSC‐3 cells with tetrandrine significantly decreased the percentage of viable cells through the induction of autophagy and apoptosis and these effects are in concentration‐dependent manner. To define the mechanism underlying the cytotoxic effects of tetrandrine, we investigated the critical molecular events known to regulate the apoptotic and autophagic machinery. Tetrandrine induced chromatin condensation, internucleosomal DNA fragmentation, activation of caspases‐3, ‐8, and ‐9, and cleavage of poly (ADP ribose) polymerase (PARP) that were associated with apoptosis, and it also enhanced the expression of LC3‐I and ‐II that were associated with the induction of autophagy in human squamous carcinoma cell line (HSC‐3) cells. Tetrandrine induced autophagy in HSC‐3 cells was significantly attenuated by bafilomycin A1 (inhibitor of autophagy) pre‐treatment that confirmed tetrandrine induced cell death may be associated with the autophagy. In conclusion, we suggest that tetrandrine induced cell death may be through the induction of apoptosis as well as autophagy in human oral cancer HSC‐3 cells via PARP, caspases/Becline I/LC3‐I/II signaling pathways. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 395–406, 2016.     

Ellagic acid induces apoptosis in TSGH8301 human bladder cancer cells through the endoplasmic reticulum stress‐ and mitochondria‐dependent signaling pathways

To investigate the effects of ellagic acid on the growth inhibition of TSGH8301 human bladder cancer cells in vitro, cells were incubated with various doses of ellagic acid for different time periods. The phase‐contrast microscope was used for examining and photographing the morphological changes in TSGH8301 cells. Flow cytometric assay was used to measure the percentage of viable cells, cell cycle distribution, apoptotic cells, ROS, mitochondrial membrane potential (ΔΨm), Ca²⁺, caspase‐9 and ‐3 activities in TSGH8301 cells after exposure to ellagic acid. Western blotting was used to examine the changes of cell cycle and apoptosis associated proteins levels. Results indicated that ellagic acid induced morphological changes, decreased the percentage of viable cells through the induction of G0/G1 phase arrest and apoptosis, and also showed that ellagic acid promoted ROS and Ca²⁺ productions and decreased the level of ΔΨm and promoted activities of caspase‐9 and ‐3. The induction of apoptosis also confirmed by annexin V staining, comet assay, DAPI staining and DNA gel electrophoresis showed that ellagic acid induced apoptosis and DNA damage in TSGH8301 cells. Western blotting assay showed that ellagic acid promoted p21, p53 and decreased CDC2 and WEE1 for leading to G0/G1 phase arrest and promoting BAD expression, AIF and Endo G, cytochrome c, caspase‐9 and ‐3 for leading to apoptosis in TSGH8301 cells. On the basis of these observations, we suggest that ellagic acid induced cytotoxic effects for causing a decrease in the percentage of viable cells via G0/G1 phase arrest and induction of apoptosis in TSGH8301 cells. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1262–1274, 2014.     

Triptolide induced cell death through apoptosis and autophagy in murine leukemia WEHI‐3 cells in vitro and promoting immune responses in WEHI‐3 generated leukemia mice in vivo

Triptolide, a traditional Chinese medicine, obtained from Tripterygium wilfordii Hook F, has anti‐inflammatory, antiproliferative, and proapoptotic properties. We investigated the potential efficacy of triptolide on murine leukemia by measuring the triptolide‐induced cytotoxicity in murine leukemia WEHI‐3 cells in vitro. Results indicated that triptolide induced cell morphological changes and induced cytotoxic effects through G0/G1 phase arrest, induction of apoptosis. Flow cytometric assays showed that triptolide increased the production of reactive oxygen species, Ca²⁺ release and mitochondrial membrane potential (ΔΨ_m), and activations of caspase‐8, ‐9, and ‐3. Triptolide increased protein levels of Fas, Fas‐L, Bax, cytochrome c, caspase‐9, Endo G, Apaf‐1, PARP, caspase‐3 but reduced levels of AIF, ATF6α, ATF6β, and GRP78 in WEHI‐3 cells. Triptolide stimulated autophagy based on an increase in acidic vacuoles, monodansylcadaverine staining for LC‐3 expression and increased protein levels of ATG 5, ATG 7, and ATG 12. The in vitro data suggest that the cytotoxic effects of triptolide may involve cross‐talk between cross‐interaction of apoptosis and autophagy. Normal BALB/c mice were i.p. injected with WEHI‐3 cells to generate leukemia and were oral treatment with triptolide at 0, 0.02, and 0.2 mg/kg for 3 weeks then animals were weighted and blood, liver, spleen samples were collected. Results indicated that triptolide did not significantly affect the weights of animal body, spleen and liver of leukemia mice, however, triptolide significant increased the cell populations of T cells (CD3), B cells (CD19), monocytes (CD11b), and macrophage (Mac‐3). Furthermore, triptolide increased the phagocytosis of macrophage from peripheral blood mononuclear cells (PBMC) but not effects from peritoneum. Triptolide promoted T and B cell proliferation at 0.02 and 0.2 mg/kg treatment when cells were pretreated with Con A and LPS stimulation, respectively; however, triptolide did not significant affect NK cell activities in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 550–568, 2017.     

Nonylphenol‐induced apoptotic pathways in SCM1 human gastric cancer cells

Environmental chemicals may affect human health by disrupting endocrine function. Many endocrine disrupting chemicals (EDCs) are estrogen‐like molecules that are classified as xenoestrogens (XEs). One XE, nonylphenol, is used as a surfactant or plasticizer and exhibits biotoxicity when accumulated in the body via the food chain. The aim of the present study was to clarify the role of nonylphenol‐induced SCM1 apoptosis by measuring cultured human gastric cancer cell (SCM1) death. Using WST‐1 reduction and propidium iodide‐staining assays, nonylphenol treatment was found to activate caspase‐3 and mitogen‐activated protein kinases (MAPKs), major markers in apoptotic pathways. Nonylphenol also activated the phosphorylation of extracellular signal‐regulated kinase (ERK), c‐Jun NH_2‐terminal kinase (JNK), and p38 mitogen‐activated protein kinase (p38 MAPK). However, only SB203580 (a p38MAPK inhibitor) partially inhibited nonylphenol‐induced apoptosis. Nonylphenol induced a [Ca²⁺]_i rise by causing extracellular Ca²⁺ influx and intracellular Ca²⁺ release from the endoplasmic reticulum, and its effects on SCM1 cell death were prevented by pretreatment with the Ca²⁺ chelator BAPTA/AM. These results suggest that nonylphenol caused Ca²⁺‐dependent apoptosis via the activation of p38 MAPK‐associated caspase‐3 in SCM1 cells. Drug Dev Res 2009. © 2009 Wiley‐Liss, Inc.     

Fisetin induces apoptosis through mitochondrial apoptosis pathway in human uveal melanoma cells

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.