Tetrandrine induces programmed cell death in human oral cancer CAL 27 cells through the reactive oxygen species production and caspase‐dependent pathways and associated with beclin‐1‐induced cell autophagy

Tetrandrine, a bisbenzylisoquinoline alkaloid, is extracted from the root of the Chinese herb Radix Stephania tetrandra S Moore. This compound has antitumor activity in different cancer cell types. In this study, the effects of tetrandrine on human oral cancer CAL 27 cells were examined. Results indicated that tetrandrine induced cytotoxic activity in CAL 27 cells. Effects were due to cell death by the induction of apoptosis and accompany with autophagy and these effects were concentration‐ and time‐dependent manners. Tetrandrine induced apoptosis was accompanied by alterations in cell morphology, chromatin fragmentation, and caspase activation in CAL 27 cells. Tetrandrine treatment also induced intracellular accumulation of reactive oxygen species (ROS). The generation of ROS may play an important role in tetrandrine‐induced apoptosis. Tetrandrine triggered LC3B expression and induced autophagy in CAL 27 cells. Tetrandrine induced apoptosis and autophagy were significantly attenuated by N‐acetylcysteine pretreatment that supports the involvement of ROS production. Tetrandrine induced cell death may act through caspase‐dependent apoptosis with Beclin‐1‐induced autophagy in human oral cancer cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 329–343, 2017.     

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.     

Autophagy potentially protects against 2,3,7,8‐tetrachlorodibenzo‐p‐Dioxin induced apoptosis in SH‐SY5Y cells

The environmental toxicant TCDD may elicit cytotoxic effects by inducing reactive oxygen species (ROS) generation. Autophagy is one of the first lines of defense against oxidative stress damage. Herein, we investigated whether autophagy played a regulatory role in TCDD‐induced neurotoxicity. Here, we showed that TCDD exposure caused marked autophagy in SH‐SY5Y cells, whose dose range was close to that inducing apoptosis. Electron microscopic and Western blot analyses revealed that TCDD induced autophagy at a starting dose of approximate 100 nM. Interestingly, 100–200 nM TCDD exposure resulted in obviously decreased cell viability and evident apoptotic phenotype. Furthermore, the levels of pro‐apoptotic molecules, Bax and cleaved‐PARP, increased significantly, whereas Bcl2 declined after exposed to 100 nM TCDD. In addition, the apoptosis was verified using flow cytometrical analysis. These data strongly suggested that TCDD induced both autophagy and apoptosis at a similar dose range in SH‐SY5Y cells. Interestingly, pretreatment with ROS scavenger, N‐acetyl‐cysteine (NAC), could effectively block both TCDD‐induced apoptosis and autophagy. More surprisingly, inhibition of autophagy with 3‐methyladenine (3MA), remarkably augmented TCDD‐induced apoptosis. The findings implicated that the onset of autophagy might serve as a protective mechanism to ameliorate ROS‐triggered cytotoxic effects in human SH‐SY5Y neuronal cells under TCDD exposure. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1068–1079, 2016.     

Di‐2‐ethylhexyl phthalate (DEHP) induces apoptosis and autophagy of mouse GC‐1 spg cells

As a widely used plasticizer in industry, di‐2‐ethylhexylphthalate (DEHP) can cause testicular toxicity, yet little is known about the potential mechanism. In this study, DEHP exposure dramatically inhibited cellviability and induced apoptosis of mouse GC‐1 spg cells. Furthermore, DEHP significantly increased the levels of autophagy proteins LC3‐II, Beclin1 and Atg5, as well as the ratio ofLC3‐II/LC3‐I. Transmission electron microscopy (TEM) further confirmed that DEHP induced autophagy of mouse GC‐1 spg cells. DEHP was also shown to induceoxidative stress; while inhibition of oxidative stress with NAC could increase cell viability and inhibit DEHP‐induced apoptosis and autophagy. These results suggested that DEHP induced apoptosis and autophagy of mouse GC‐1 spg cells via oxidative stress. 3‐MA, an inhibitor of autophagy, could rescue DEHP‐induced apoptosis. In summary, DEHP induced apoptosis and autophagy of mouse GC‐1 spg cells via oxidative stress, and autophagy might exert a cytotoxic effect on DEHP‐induced apoptosis.     

Antineoplastic effect of β‐elemene on prostate cancer cells and other types of solid tumour cells

Objectives β‐Elemene, a natural compound extracted from over 50 different Chinese medicinal herbs and plants, has been effective in the treatment of hyperplastic and proliferative disorders such as prostatic hypertrophy, hysteromyoma and neoplasms. Our previous studies have demonstrated that β‐elemene exhibits strong inhibitory activity in ovarian cancer cells. The aim of the present study was to assess the effect of β‐elemene on prostate cancer cells as well as other types of tumour cells and to determine whether the effect of β‐elemene on prostate cancer cell death was mediated through the induction of apoptosis. Methods The MTT assay was used to evaluate the ability of β‐elemene to inhibit cellular proliferation in cancer cells. Cellular apoptosis was assessed by annexin V binding, TUNEL and ELISA‐based assays. Caspase activity was measured using a caspases assay kit. The protein levels of Bcl‐2, caspases, cytochrome c and poly(ADP‐ribose) polymerase (PARP) were analysed by Western blotting. Key findings Here, we showed that β‐elemene had an antiproliferative effect on androgen‐insensitive prostate carcinoma DU145 and PC‐3 cells. Treatment with β‐elemene also inhibited the growth of brain, breast, cervical, colon and lung carcinoma cells. The effect of β‐elemene on cancer cells was dose dependent, with IC50 values ranging from 47 to 95 µg/ml (230–465 µm ). TUNEL assay and flow cytometric analysis using annxin V/propidium iodide staining revealed that the percentage of apoptotic prostate cancer cells was increased by β‐elemene in a dose‐ and time‐dependent manner. Moreover, β‐elemene exposure resulted in a decreased Bcl‐2 protein level, increased cytochrome c release, and activated PARP and caspase‐3, ‐7, ‐9, and ‐10 in prostate cancer cells. Conclusions Overall, these findings suggest that β‐elemene exerts broad‐spectrum antitumour activity against many types of solid carcinoma and supports a proposal of β‐elemene as a new potentially therapeutic drug for castration‐resistant prostate cancer and other solid tumours.     

Synergistic anti-tumour effects of tetrandrine and chloroquine combination therapy in human cancer: a potential antagonistic role for p21

Background and Purpose

Tetrandrine, a bisbenzylisoquinoline alkaloid isolated from the Chinese medicinal herb Stephaniae tetrandrae, has a long history in Chinese clinical applications to treat diverse diseases. Tetrandrine induced apoptosis or, at low concentrations, autophagy of human hepatocellular carcinoma cells. Here we have tested the effects of inhibitors of autophagy such as chloroquine, on the response to low concentrations of tetrandrine in cancer cells.

Experimental Approach

Cultures of several cancer cell lines, including Huh7, U251, HCT116 and A549 cells, were exposed to tetrandrine, chloroquine or a combination of these compounds. Cell viability and content of reactive oxygen species (ROS) were measured and synergy assessed by calculation of the combination index. Western blot and RT-PCR assays were also used along with fluorescence microscopy and histochemical techniques.

Key Results

Combinations of tetrandrine and chloroquine were more cytotoxic than the same concentrations used separately and these effects showed synergy. Such effects involved increased ROS generation and were dependent on caspase-3 but independent of Akt activity. Blockade of tetrandrine-induced autophagy with 3-methyladenine or bafilomycin-A1 induced apoptosis in cancer cells. Lack of p21 protein (p21^−/− HCT116 cells) increased sensitivity to the apoptotic effects of the combination of tetrandrine and chloroquine. In a tumour xenograft model in mice, combined treatment with tetrandrine and chloroquine induced ROS accumulation and cell apoptosis, and decreased tumour growth.

Conclusions and Implications

The combinations of tetrandrine and chloroquine exhibited synergistic anti-tumour activity, in vitro and in vivo. Our results suggest a novel therapeutic strategy for tumour treatment.     

Quercetin simultaneously induces G0/G1‐phase arrest and caspase‐mediated crosstalk between apoptosis and autophagy in human leukemia HL‐60 cells

Quercetin is a plant‐derived bioflavonoid with high anticancer activity in various tumors. Herein, the molecular mechanisms by which quercetin exerts its anticancer effects against HL‐60 acute myeloid leukemia (AML) cells were investigated. Results showed that quercetin suppressed cell proliferation in the HL‐60 cell line in vitro and in vivo. Quercetin‐induced G ₀/G₁‐phase arrest occurred when expressions of cyclin‐dependent kinase (CDK)2/4 were inhibited and the CDK inhibitors, p16 and p21, were induced. Moreover, quercetin treatment not only activated proapoptotic signaling like poly (ADP ribose) polymerase (PARP)?1 cleavage and caspase activation but also triggered autophagy events as shown by the increased expression of light chain 3 (LC3)‐II, decreased expression of p62, and formation of acidic vesicular organelles. Interestingly, it was found that use of the autophagy inhibitor, 3‐methyladenine, significantly enhanced quercetin‐mediated apoptotic cell death as analyzed by MTS and DNA fragmentation assays. Moreover, pretreatment of HL‐60 cells with the pan‐caspase inhibitor, Z‐VAD‐fmk, dramatically reversed quercetin‐mediated apoptotic and autophagic cell death. Although apoptosis and autophagy are two independent cell death pathways, our findings indicated that quercetin can activate caspases to trigger these two pathways, and both pathways played contrary roles in quercetin‐mediated HL‐60 cell death. In conclusion, besides promoting apoptosis, quercetin also induced cytoprotective autophagy in HL‐60 cells, and inhibition of autophagy may be a novel strategy to enhance the anticancer activity of quercetin in AML.     

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.     

Fisetin‐induced apoptosis of human oral cancer SCC‐4 cells through reactive oxygen species production,endoplasmic reticulum stress,caspase‐, and mitochondria‐dependent signaling pathways

Oral cancer is one of the cancer‐related diseases in human populations and its incidence rates are rising worldwide. Fisetin, a flavonoid from natural products, has been shown to exhibit anticancer activities in many human cancer cell lines but the molecular mechanism of fisetin‐induced apoptosis in human oral cancer cells is still unclear; thus, in this study, we investigated fisetin‐induced cell death and associated signal pathways on human oral cancer SCC‐4 cells in vitro. We examined cell morphological changes, total viable cells, and cell cycle distribution by phase contrast microscopy and flow cytometry assays. Reactive oxygen species (ROS), Ca²⁺, mitochondria membrane potential (ΔΨ_m), and caspase‐8, ‐9, and ‐3 activities were also measured by flow cytometer. Results indicate that fisetin induced cell death through the cell morphological changes, caused G2/M phase arrest, induction of apoptosis, promoted ROS and Ca²⁺ production, and decreased the level of ΔΨ_m and increased caspase‐3, ‐8, and ‐9 activities in SCC‐4 cells. DAPI staining and DNA gel electrophoresis were also used to confirm fisetin‐induced cell apoptosis in SCC‐4 cells. Western blotting also found out that Fisetin increased the proapoptotic proteins such as Bax and Bid and decreased the antiapoptotic proteins such as Bcl‐2. Furthermore, results also showed that Fisetin increased the cytochrome c, AIF, and Endo G release from mitochondria in SCC‐4 cells. We also used ATF‐6α, ATF‐6β, GADD153, and GRP78 which indicated that fisetin induced cell death through ER stress. Based on those observations, we suggest that fisetin induced cell apoptosis through ER stress, mitochondria‐, and caspase‐dependent pathways.     

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.     

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.     

Apoptotic effects of rhein through the mitochondrial pathways,two death receptor pathways,and reducing autophagy in human liver L02 cells

Rhein (4,5‐dihydroxyanthraquinone‐2‐carboxylic acid) is a major component of many medicinal herbs such as Rheum palmatum L. and Polygonum multiflorum. Despite being widely used, intoxication cases associated with rhein‐containing herbs are often reported. Currently, there are no available reports addressing the effects of rhein on apoptosis in human liver L02 cells. Thus, the aim of this study is to determine the cytotoxic effects and the underlying mechanism of rhein on human normal liver L02 cells. In the present study, the methyl thiazolyl tetrazolium assay demonstrated that rhein decreased the viability of L02 cells in dose‐dependent and time‐dependent ways. Rhein was found to trigger apoptosis in L02 cells as shown by Annexin V‐fluoresceine isothiocyanate (FITC) apoptosis detection kit and cell mitochondrial membrane potential (MMP) assay, with nuclear morphological changes demonstrated by Hoechst 33258 staining. Detection of intracellular superoxide dismutase activity, lipid oxidation (malondialdehyde) content, and reactive oxygen species (ROS) levels showed that apoptosis was associated with oxidative stress. Moreover, it was observed that the mechanism implicated in rhein‐induced apoptosis was presumably via the death receptor pathway and the mitochondrial pathway, as illustrated by upregulation of TNF‐α, TNFR1, TRADD, and cleaved caspase‐3, and downregulation of procaspase‐8, and it is suggested that rhein may increase hepatocyte apoptosis by activating the increase of TNF‐α level. Meanwhile, rhein upregulates the expression of Bax and downregulates the expression of procaspase‐9 and ‐3, and it is suggested that the mitochondrial pathway is activated and rhein‐induced apoptosis may be involved. In addition, we also want to explore whether rhein‐induced apoptosis is related to the autophagic changes induced by rhein. The results showed that rhein treatment increased P62 and decreased LC3‐II and beclin‐1, which means that autophagy was weakened. The results of our studies indicated that rhein induced caspase‐dependent apoptosis via both the Fas death pathway and the mitochondrial pathway by generating ROS, and meanwhile the autophagy tended to weaken.     

Sinularin induces oxidative stress‐mediated G2/M arrest and apoptosis in oral cancer cells

Soft corals‐derived natural product, sinularin, was antiproliferative against some cancers but its effect and detailed mechanism on oral cancer cells remain unclear. The subject of this study is to examine the antioral cancer effects and underlying detailed mechanisms in terms of cell viability, oxidative stress, cell cycle analysis, and apoptosis analyses. In MTS assay, sinularin dose‐responsively decreased cell viability of three oral cancer cells (Ca9‐22, HSC‐3, and CAL 27) but only little damage to oral normal cells (HGF‐1). This cell killing effect was rescued by the antioxidant N‐acetylcysteine (NAC) pretreatment. Abnormal cell morphology and induction of reactive oxygen species (ROS) were found in sinularin‐treated oral cancer Ca9‐22 cells, however, NAC pretreatment also recovered these changes. Sinularin arrested the Ca9‐22 cells at G2/M phase and dysregulated the G2/M regulatory proteins such as cdc2 and cyclin B1. Sinularin dose‐responsively induced apoptosis on Ca9‐22 cells in terms of flow cytometry (annexin V and pancaspase analyses) and western blotting (caspases 3, 8, 9) and poly (ADP‐ribose) polymerase (PARP). These apoptotic changes of sinularin‐treated Ca9‐22 cells were rescued by NAC pretreatment. Taken together, sinularin induces oxidative stress‐mediated antiproliferation, G2/M arrest, and apoptosis against oral cancer cells and may be a potential marine drug for antioral cancer therapy.     

mTOR inhibition by rapamycin protects against deltamethrin‐induced apoptosis in PC12 Cells

The autophagy pathway can be induced and upregulated in response to intracellular reactive oxygen species (ROS). In this study, we explored a novel pharmacotherapeutic approach involving the regulation of autophagy to prevent deltamethrin (DLM) neurotoxicity. We found that DLM‐induced apoptosis in PC12 cells, as demonstrated by the activation of caspase‐3 and ‐9 and by nuclear condensation. DLM treatment significantly decreased dopamine (DA) levels in PC12 cells. In addition, we observed that cells treated with DLM underwent autophagic cell death, by monitoring the expression of LC3‐II, p62, and Beclin‐1. Exposure of PC12 cells to DLM led to the production of ROS. Treatment with N‐acetyl cysteine (NAC) effectively blocked both apoptosis and autophagy. In addition, mitogen‐activated protein kinase (MAPK) inhibitors attenuated apoptosis as well as autophagic cell death. We also investigated the modulation of DLM‐induced apoptosis in response to autophagy regulation. Pretreatment with the autophagy inducer, rapamycin, significantly enhanced the viability of DLM‐exposed cells, and this enhancement of cell viability was partially due to alleviation of DLM‐induced apoptosis via a decrease in levels of cleaved caspase‐3. However, pretreatment of cells with the autophagy inhibitor, 3‐methyladenine (3MA), significantly increased DLM toxicity in these cells. Our results suggest that DLM‐induced cytotoxicity is modified by autophagy regulation and that rapamycin protects against DLM‐induced apoptosis by enhancing autophagy. Pharmacologic induction of autophagy by rapamycin may be a useful treatment strategy in neurodegenerative disorders. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 109–121, 2017.     

Effects of the Fas/Fas‐L pathway on fluoride‐induced apoptosis in SH‐SY5Y cells

The mechanisms underlying fluoride‐induced apoptosis in neurons still remain unknown. To investigate apoptosis, caspase‐3 activity, and mRNA expression of Fas, Fas‐L, and caspases (‐3 and ‐8) induced by fluoride, human neuroblastoma (SH‐SY5Y) cells were incubated with 0, 20, 40, and 80 mg/L sodium fluoride (NaF) for 24 h in vitro. The data show that cell viability in the 40 and 80 mg/L fluoride groups were significantly lower than that of the control group. The percentages of apoptosis in the 40 and 80 mg/L fluoride groups were markedly higher than those in the control group, and they increased with the increase in fluoride concentration. The activity of caspase‐3 and mRNA expression levels for Fas, Fas‐L, and caspases (‐3 and ‐8) in the 40 and 80 mg/L fluoride groups were significantly higher than those in the control group. An agonistic anti‐Fas monoclonal antibody (CH‐11) significantly augmented apoptosis induction by fluoride, showing a synergistic effect, while a Fas‐blocking antibody (ZB4) partly inhibited fluoride‐induced apoptosis of SH‐SY5Y cells. The results indicate that fluoride exposure could induce apoptosis in SH‐SY5Y cells, and the Fas/Fas‐L signaling pathway may play an important role in the process. © 2009 Wiley Periodicals, Inc. Environ Toxicol 26: 86–92, 2011.     

Reactive oxygen species‐dependent JNK downregulated olaquindox‐induced autophagy in HepG2 cells

Autophagy plays an important role in response to intracellular and extracellular stress to sustain cell survival. However, dysregulated or excessive autophagy may lead to cell death, known as “type II programmed cell death,” and it is closely associated with apoptosis. In our previous study, we proposed that olaquindox induced apoptosis of HepG2 cells through a caspase‐9 dependent mitochondrial pathway. In this study, we investigated autophagy induced by olaquindox and explored the crosstalk between apoptosis and autophagy in olaquindox‐treated HepG2 cells. Olaquindox‐induced autophagy was demonstrated by the accumulation of monodansylcadervarine, as well as elevated expression of autophagy‐related MAP‐LC3 and Beclin 1 proteins. The autophagy inhibitor 3‐methyladenine significantly increased the apoptotic rate induced by olaquindox, which was correlated with increased ratio of Bax/Bcl‐2. The further studies showed that olaquindox increased the levels of reactive oxygen species (ROS), and antioxidant N‐acetyl‐L ‐cysteine (NAC) effectively blocked the accumulation of ROS but failed to block autophagy. Moreover, olaquindox induced the activation of c‐Jun N‐terminal protein kinase (JNK), and JNK inhibitor SP600125 failed to block autophagy. Instead, olaquindox‐induced autophagy was enhanced by NAC or SP600125. Meanwhile, JNK activation was remarkably blocked by NAC, indicating that ROS may be the upstream signaling molecules of JNK activation and involved in the negative regulation of olaquindox‐induced autophagy. These results suggest that olaquindox induces autophagy in HepG2 cells and that olaquindox‐induced apoptosis can be enhanced by 3‐methyladenine. Olaquindox‐induced autophagy in HepG2 cells is upregulated by Beclin 1 but downregulated by ROS‐dependent JNK. Copyright © 2014 John Wiley & Sons, Ltd.     

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.     

Bufalin inhibits migration and invasion in human hepatocellular carcinoma SK‐Hep1 cells through the inhibitions of NF‐kB and matrix metalloproteinase‐2/‐9‐signaling pathways

Metastasis plays an important role in mortality of cancer patients. Migration and invasion are the major characteristics of tumor metastasis. The induction of matrix metalloproteinases (MMPs) such as MMP‐2 and ‐9 are particularly important for the invasiveness of various cancer cells. Bufalin, a class of toxic steroids, was purified from the skin glands of Bufo gargarizans or Bufo melanostictus; it is known to inhibit proliferation of human cancer cells. In this study, we investigated the antiinvasive mechanisms of bufalin in the human hepatocellular cancer cell line SK‐Hep1. Bufalin significantly reduced serum‐induced cell invasion and migration. Furthermore, bufalin markedly inhibited MMP‐2 and ‐9 activity, mRNA expression and protein levels in SK‐Hep1 cells. Bufalin attenuated phosphoinisitide‐3‐kinase (PI3K) and phosphorylation of AKT which was associated with reduced levels of nuclear factor kappa B (NF‐κB). Bufalin also suppressed protein levels of FAK and Rho A, VEGF, MEKK3, MKK7, and uPA and it diminished NF‐κB translocation. Based on these observations, we propose that bufalin is acts as an antiinvasive agent by inhibiting MMP‐2 and ‐9 and involving PI3K/AKT and NF‐κB pathways. Bufalin is a potential therapeutic agent that may have efficacy in preventing the invasion and metastasis of malignant liver tumors. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 74–82, 2015.     

l‐Carnitine protects against 1,4‐benzoquinone‐induced apoptosis and DNA damage by suppressing oxidative stress and promoting fatty acid oxidation in K562 cells

Widespread occupational and environmental exposure to benzene is unavoidable and poses a public health threat. Studies of potential interventions to prevent or relieve benzene toxicity are, thus, essential. Research has shown l ‐carnitine (LC) has beneficial effects against various pathological processes and diseases. LC possesses antioxidant activities and participates in fatty acid oxidation (FAO). In this study, we investigated whether 1,4‐benzoquinone (1,4‐BQ) affects LC levels and the FAO pathway, as well as analyzed the influence of LC on the cytotoxic effects of 1,4‐BQ. We found that 1,4‐BQ significantly decreased LC levels and downregulated Cpt1a, Cpt2, Crat, Hadha, Acaa2, and Acadvl mRNA expression in K562 cells. Subsequent assays confirmed that 1,4‐BQ decreased cell viability and increased apoptosis and caspase‐3, ‐8, and ‐9 activities. It also induced obvious oxidative stress and DNA damage, including an increase in the levels of reactive oxygen species and malondialdehyde, tail DNA%, and olive tail moment. Additionally, the mitochondrial membrane potential was significantly reduced. Cotreatment with LC (500 μmol/L) relieved these alterations by reducing oxidative stress and increasing the protein expression levels of Cpt1a and Hadha, particularly in the 20 μmol/L 1,4‐BQ group. Thus, our results demonstrate that 1,4‐BQ causes cytotoxicity, reduces LC levels, and downregulates the FAO genes. In contrast, LC exhibits protective effects against 1,4‐BQ‐induced apoptosis and DNA damage by decreasing oxidative stress and promoting the FAO pathway.     

Carnosic acid induces autophagic cell death through inhibition of the Akt/mTOR pathway in human hepatoma cells

The therapeutic goal of cancer treatment is now geared towards triggering tumour‐selective cell death with autophagic cell death being required for the chemotherapy of apoptosis‐resistant cancer. In this study, Carnosic acid (CA), a polyphenolic diterpene isolated from Rosemary (Rosemarinus officinalis), significantly induced autophagic cell death in HepG2 cells. Ca treatment caused the formation of autophagic vacuoles produced an increasing ratio of LC3‐II to LC3‐I in a time‐ and dose‐dependent manner but had no effect on the levels of autophagy‐related protein ATG6 and ATG13 expression. Autophagy inhibitors, 3‐methyladenine (3‐MA), chloroquine and bafilomycin A1, or ATG genes silencing in HepG2 cells significantly inhibited CA‐induced autophagic cell death. The CA treatment decreased the levels of phosphorylated Akt and mTOR without any effects on PI3K or PTEN. Most importantly, overexpression of Akt and knockdown of PTEN attenuated autophagy induction in CA‐treated cells. Taken together, our results indicated that CA induced autophagic cell death through inhibition of the Akt/mTOR pathway in human hepatoma cells. These findings suggest that CA has a great potential for the treatment of hepatoma via autophagic induction. Copyright © 2014 John Wiley & Sons, Ltd.