Tu-San-Qi (Gynura japonica): the culprit behind pyrrolizidine alkaloid-induced liver injury in China

Herbs and dietary supplement-induced liver injury (HILI) is the leading cause of drug-induced liver injury in China. Among different hepatotoxic herbs, the pyrrolizidine alkaloid (PA)-producing herb Gynura japonica contributes significantly to HILI by inducing hepatic sinusoidal obstruction syndrome (HSOS), a liver disorder characterized by hepatomegaly, hyperbilirubinemia, and ascites. In China, G. japonica has been used as one of the plant species for Tu-San-Qi and is often misused with non-PA-producing Tu-San-Qi (Sedum aizoon) or even San-Qi (Panax notoginseng) for self-medication. It has been reported that over 50% of HSOS cases are caused by the intake of PA-producing G. japonica. In this review, we provide comprehensive information to distinguish these Tu-San-Qi-related herbal plant species in terms of plant/medicinal part morphologies, medicinal indications, and chemical profiles. Approximately 2156 Tu-San-Qi-associated HSOS cases reported in China from 1980 to 2019 are systematically reviewed in terms of their clinical manifestation, diagnostic workups, therapeutic interventions, and outcomes. In addition, based on the application of our developed mechanism-based biomarker of PA exposure, our clinical findings on the definitive diagnosis of 58 PA-producing Tu-San-Qi-induced HSOS patients are also elaborated. Therefore, this review article provides the first comprehensive report on 2214 PA-producing Tu-San-Qi (G. japonica)-induced HSOS cases in China, and the information presented will improve public awareness of the significant incidence of PA-producing Tu-San-Qi (G. japonica)-induced HSOS and facilitate future prevention and better clinical management of this severe HILI.     

Myricetin Selectively Induces Apoptosis on Cancerous Hepatocytes by Directly Targeting Their Mitochondria

Hepatocellular carcinoma (HCC) is the third most common cause of cancer‐related death. In patients for whom HCC could not be detected early, current treatments show poor tolerance and low efficacy. So, alternative therapies with good efficacy are urgently needed. The aim of this research was to evaluate the selective apoptotic effects of myricetin (MYR), a flavonoid compound, on hepatocytes and mitochondria obtained from the liver of HCC rats. In this study, HCC induced by diethylnitrosamine (DEN), as an initiator, and 2‐acetylaminofluorene (2‐AAF), as a promoter. To confirm the HCC induction, serum levels of alpha‐fetoprotein (AFP), AST, AST and ALP and histopathological changes in the liver tissue were evaluated. Rat liver hepatocytes and mitochondria for evaluation of the selective cytotoxic effects of MYR were isolated, and mitochondrial and cellular parameters related to apoptosis signalling were then determined. Our results showed that MYR was able to induce cytotoxicity only in hepatocytes from the HCC but not from the untreated control group. Besides, MYR (12.5, 25 and 50 μM) induced a considerable increase in reactive oxygen species (ROS) level, mitochondrial swelling, mitochondrial membrane permeabilization (MMP) and cytochrome c release only in cancerous but not in untreated normal hepatocyte mitochondria. MYR selectively increased caspase‐3 activation and apoptotic phenotypes in HCC, but not untreated normal hepatocytes. Finally, our finding underlines MYR as a promising therapeutic candidate against HCC and recommends the compound for further studies.     

Cyanidin-3-O-β-glucoside protects primary mouse hepatocytes against high glucose-induced apoptosis by modulating mitochondrial dysfunction and the PI3K/Akt pathway

Apoptosis is an early event of steatohepatitis in non-alcoholic fatty liver disease (NAFLD), and an increase in oxidative stress induced by hyperglycemia has been linked to an acceleration of apoptosis in hepatocytes. Cyanidin-3-O-β-glucoside (C3G), a classic anthocyanin, has been reported to reduce oxidative stress and attenuate non-alcoholic steatohepatitis in mice. In this study, we evaluated the toxicity of high glucose in primary hepatocytes of mice fed with a high fat diet and amelioration of this toxicity by C3G. Incubation of hepatocytes with 35 mM glucose for 12 h resulted in a significant decrease in cell viability and increase in apoptotic cell death. Furthermore, hyperglycemia-induced mitochondrial depolarization was accompanied by the release of cytochrome c and altered expression of Bax and Bcl-2, suggesting a mitochondria-mediated apoptotic mode of cell death. Pre-incubation with 50 μM C3G induced changes associated with better cell survival and function, including a reduction in reactive species generation, improvement of mitochondrial membrane potential, inactivation of caspase-3 and -9, and down-regulation of the pro-apoptotic Bax protein. We further investigated the role of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways with respect to the anti-apoptotic action of C3G, and our results showed that C3G could activate Akt. Additionally, C3G inactivated c-Jun N-terminal protein kinase (JNK), but not extracellular signal-regulated kinase or p38 MAPK, in glucose-stressed cells. Interestingly, JNK inhibitor enhanced the protective effect of C3G on cell survival. Our results suggest that anthocyanin C3G may exhibit hepatoprotective potential against NAFLD by promoting functional integrity and survival of hepatocytes.     

特异性p38 MAPK抑制剂SB203580对乳鼠小脑颗粒神经元的保护作用

目的　研究p38丝裂原激活蛋白激酶（MAPK）选择性抑制剂SB203580对乳鼠小脑颗粒神经元凋亡的保护作用。方法　SD乳鼠小脑颗粒神经元培养,琼脂糖凝胶电泳,SAPK/JNK分析试剂盒作激酶分析。结果　PI-3-K的特异性抑制剂LY294002诱导小脑颗粒神经元凋亡,但SB203580通过抑制细胞凋亡而促进小脑颗粒神经元的存活,且有浓度依赖性。LY294002诱导凋亡的颗粒神经元中c-Jun的表达量和磷酸化水平均升高,JNK被激活。但是,当小脑颗粒神经元生长在含SB203580的高钾培养基中,c-Jun的表达量、磷酸化水平和JNK的活性都明显的降低。结论　SB203580通过抑制JNK的活性,降低c-Jun的表达和磷酸化水平,对小脑颗粒神经元产生保护作用。     

Molecular mechanisms involved in the adaptive response to cadmium-induced apoptosis in human myelomonocytic lymphoma U937 cells

We examined the molecular mechanisms involved in the adaptive response to cadmium (Cd)-induced apoptosis in human myelomonocytic lymphoma U937 cells. When U937 cells were treated with 50 μM cadmium chloride (CdCl₂) for 12 h, significant apoptosis occurred. This was associated with an increase in intracellular reactive oxygen species (ROS), sustained phosphorylation of JNK, activation of caspase-3, a decrease in Mcl-1 (anti-apoptotic Bcl-2 proteins), and increases in Bim, Noxa and tBid (a pro-apoptotic protein under the Bcl-2 family). No apoptosis occurred when the cells were treated with 1 μM CdCl₂ for 72 h. However, pretreatment with low-dose CdCl₂ dramatically altered the sensitivity of the cells to 50 μM CdCl₂ with inhibition of apoptosis. Concomitantly, there were significant decreases in the generation of intracellular ROS and the activation of JNK. Pretreatment with 1 μM CdCl₂ also attenuated the decrease in Mcl-1 and the increases in Bim, Noxa and tBid induced by 50 μM CdCl₂. In conclusion, pretreatment with low-dose Cd inhibited apoptosis induced by high-dose Cd. The mechanism involves inhibition of intracellular ROS generation and JNK activation, and modulating the balance between the expression of Mcl-1 and its binding partners, Bim, Noxa and tBid.     

Mono(2‐ethylhexyl) phthalate induces apoptosis in p53‐silenced L02 cells via activation of both mitochondrial and death receptor pathways

Mono(2‐ethylhexyl) phthalate (MEHP) is one of the main metabolites of di(2‐ethylhexyl) phthalate. The evidence shows that DEHP may exert its toxic effects primarily via MEHP, which is 10‐fold more potent than its parent compound in toxicity in vitro. MEHP‐induced apoptosis is mediated by either p53‐dependent or ‐independent pathway. However, the detailed mechanism of its toxicity remains unclear. In this study, immortalized normal human liver cell line L02 was chosen, as an in vitro model of nonmalignant liver, to elucidate the role of p53 in MEHP‐induced apoptosis. The cells were treated with MEHP (6.25, 12.50, 25.00, 50.00, and 100.00 μM) for 24 and 36 h, then small interfering RNA (siRNA) was used to specifically silence p53 gene of L02 cells. The results indicated that MEHP caused oxidative DNA damage and apoptosis in L02 cells were associated with the p53 signaling pathway. Further study found that MEHP (50.00 and 100.00 μM) induced apoptosis in p53‐silenced L02 cells, along with the up‐regulations of Fas and FasL proteins as well as increased the Bax/Bcl‐2 ratio and Caspase 3, 8, and 9 activities. Additionally, both FasL inhibitor (AF‐016) and Caspase inhibitor N‐benzyloxycarbonyl‐Val‐Ala‐Asp‐ fluoromethylketone (Z‐VAD‐FMK) could prevent the cell apoptosis induced by MEHP. The findings suggested that MEHP‐induced apoptosis in L02 cells involving a Caspases‐mediated mitochondrial signaling pathway and/or death receptor pathway. p53 was not absolutely necessary for MEHP‐induced L02 cell apoptosis. © 2014 Wiley Periodicals, Inc. Environ Toxicol 30: 1178–1191, 2015.     

ICAM-1 and AMPK regulate cell detachment and apoptosis by N-methyl-N'-nitro-N-nitrosoguanidine, a widely spread environmental chemical, in human hormone-refractory prostate cancers

Poly(ADP-ribose) polymerase-1 (PARP-1), a sensor of DNA damage, plays a crucial role in the regulation of DNA repair. PARP-1 hyperactivation causes DNA damage and cell death. The underlying mechanism is complicated and is through diverse pathways. The understanding of responsible signaling pathways may offer implications for effective therapies. After concentration-response determination of N-Methyl-N′-Nitro-N-Nitrosoguanidine (MNNG, a PARP-1 activating agent and an environmental mutagen) in human hormone-refractory prostate cancers, the data showed that concentrations below 5 μM did not change cell survival but cause a time-dependent up-regulation of intracellular adhesion molecule-1 (ICAM-1) in mRNA, total protein and cell surface levels. Detection of phosphorylation and degradation of IκB-α and nuclear translocation of NF-κB showed that MNNG induced the activation of NF-κB that was responsible for the ICAM-1 up-regulation since PDTC (a NF-κB inhibitor) significantly abolished this effect. However, higher concentrations (e.g., 10 μM) of MNNG induced a 61% detachment of the cells which were apoptosis associated with the activation of AMP-activated protein kinase (AMPK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK). Further identification showed that both AMPK and JNK other than p38 MAPK functionally contributed to cell death. The remaining 39% attached cells were survival associated with high ICAM-1 expression. In conclusion, the data suggest that NF-κB-dependent up-regulation of ICAM-1 plays a key role on cell attachment and survival; whereas, activation of AMPK and JNK participates in cytotoxic signaling pathways in detached cells caused by PARP-1 activation.     

Atorvastatin induces mitochondrial dysfunction and cell apoptosis in HepG2 cells via inhibition of the Nrf2 pathway

Atorvastatin (ATO) is a 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitor widely used to treat hypercholesterolemia. However, clinical application is limited by potential hepatotoxicity. Nuclear factor‐erythroid 2‐related factor 2 (Nrf2) is a master regulator of cellular antioxidants, and oxidative stress is implicated in statin‐induced liver injury. This study investigated mechanisms of ATO‐induced hepatotoxicity and potential mitigation by Nrf2 signaling. ATO reduced Nrf2 and antioxidant enzyme superoxide dismutase‐2 (SOD2) expression in human hepatocarcinoma HepG2 cells. ATO also induced concentration‐dependent HepG2 cell toxicity, reactive oxygen species (ROS) accumulation, and mitochondrial dysfunction as evidenced by decreased mitochondrial membrane potential (MMP) and cellular adenosine triphosphate (ATP). Further, ATO induced mitochondria‐dependent apoptosis as indicated by increased Bax/Bcl‐2 ratio, cleaved caspase‐3, mitochondrial cytochrome c release and Annexin V‐fluorescein isothiocyanate/propidium iodide staining. Tert‐butylhydroquinone enhanced Nrf2 and SOD2 expression, and partially reversed ATO‐induced cytotoxicity, ROS accumulation, MMP reduction, ATP depletion and mitochondria‐dependent apoptosis. In conclusion, the present study demonstrates that ATO induces mitochondrial dysfunction and cell apoptosis in HepG2 cells, at least in part, via inhibition of the Nrf2 pathway. Nrf2 pathway activation is a potential prevention for ATO‐induced liver injury.     

p53‐Dependent pathway and the opening of mPTP mediate the apoptosis of co‐cultured Sertoli‐germ cells induced by microcystin‐LR

Microcystin‐LR (MC‐LR), a potent endotoxin, can induce reproductive toxicity. In order to investigate the role and mechanisms of apoptosis (p53‐dependent and mitochondrial pathways) of germ cells induced by MC‐LR, the co‐cultured primary Sertoli‐germ cells from Sprague‐Dawley rats were used for the experiments. Expression levels of proteins, genes, and mitochondrial membrane potential (MMP) were obtained after exposing co‐cultured Sertoli‐germ cells to MC‐LR with or without the addition of the p53 inhibitor, pifithrin‐α (PFT‐α), and MMP inhibitor, cyclosporin A (CsA). Results indicated that MC‐LR could activate p53‐dependent pathway‐associated proteins in Sertoli‐germ cells, leading to a decrease in MMP (indicating the opening of mitochondrial permeability transition pore [mPTP] and the release of Cytochrome‐c [Cyt‐c]) from the mitochondria into the cytoplasm and eventually the induction of apoptosis. PFT‐α inhibited the expression ofp53, ameliorated the MMP of the co‐cultured Sertoli‐germ cells, and prevented the release of Cyt‐c from the mitochondria into the cytoplasm, which reduces the occurrence of apoptosis. Similarly, the decreased release of Cyt‐c from the mitochondria into the cytoplasm and the declined level of apoptosis in Sertoli‐germ cells induced by MC‐LR were observed after the addition of CsA. These results indicated that the apoptosis of the co‐cultured Sertoli‐germ cells induced by MC‐LR was mediated by the p53‐dependent pathway, with the involvement of the opening of mPTP.     

In vitro cytotoxicity of cyclodextrin-bonded birch bark extract

Triterpenoids from birch bark, like betulin, seem to have an anticancer potential which needs to be further investigated. Aim of this study was first to explore whether a cyclodextrin-solubilised triterpenoid extract (STE) from birch bark induces selective cytotoxic effects in primary liver cancer cells compared to healthy human hepatocytes. Second, selective cytotoxicity against several tumour cell lines should be analysed. For this purpose, human liver cancer cells derived from mouse xenografts (LIXF 575), healthy human hepatocytes, and 42 different human tumour cell lines were incubated with different concentrations of STE corresponding to 4.3 μM - 137.5 μM betulin (BE). Cytotoxicity was tested with the WST-1 cell proliferation assay, apoptosis with caspase 3/7-activity, and necrosis was determined by the propidiumiodid uptake assay. The pathway of cytotoxic effects was further investigated by immunoblotting of apoptosis inducing factor (AIF) and p53. The monolayer assay was used to analyse selectivity of STE towards different tumour cell lines. STE significantly (p < 0.001) reduced viability and induced apoptosis of LIXF cells in low concentrations corresponding to 8.6 μM BE, while human hepatocytes were affected only in concentrations ≥ 68.8 μM. Cell death occurred in a p53 independent manner, and AIF was not involved. The mean IC50 in the 42 tumour cell lines corresponded to 4.3 μM BE and ranged from 2.05 μM to 8.95 μM BE content. Selectivity was, therefore, rather low. In conclusion, STE exhibits in low concentrations cytotoxicity in a broad spectrum of primary cancer cells and cancer cell lines, which is, at least in LIXF cells, induced by caspase 3/7 mediated apoptosis. STE is far less toxic in hepatocytes. The anticancer potential of STE should be further characterised and also investigated in animal models.     

Butein suppresses bile acid-induced hepatocyte apoptosis through a JNK-dependent but ERK-independent pathway

We investigated the protective effect of butein on glycochenodeoxycholic acid (GCDC)-induced apoptosis in primary cultured rat hepatocytes. Treatment with GCDC at a concentration of 100 microM for 4 h induced apoptosis, and treatment with butein at concentrations of 30 microM inhibited the GCDC-induced apoptosis as shown by the reduced cleavage of poly(ADP-ribose) polymerase, DNA fragmentation, and activation of caspases-3, -8, and -9. c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) play fundamental roles in cell survival, proliferation, and apoptosis. GCDC alone induced ERK and JNK phosphorylation. Butein alone induced ERK activation, and ERK activation was greater in hepatocytes treated with butein and GCDC than in hepatocytes exposed to GCDC alone. Butein treatment reduced JNK activation induced by GCDC. Addition of U0126, an inhibitor of ERK, did not alter the proapoptotic effect of GCDC or the antiapoptotic effect of butein. Addition of SP600125, a specific JNK inhibitor, protected hepatocytes against GCDC-induced apoptosis. These data suggest that butein has a protective effect against GCDC-induced hepatocyte apoptosis and that the protective effect of butein is JNK dependent but ERK independent.     

Silymarin augments human cervical cancer HeLa cell apoptosis via P38/JNK MAPK pathways in serum-free medium

Silymarin was proved to have a protective effect of UV-induced A375-S2 cell apoptosis in our previous research. In this study, its pro-apoptotic and anti-apoptotic activities on human cervical cancer (HeLa) cells in vitro were investigated. Silymarin induced HeLa cell death through both apoptotic and necrotic pathways. At low doses (below 80 μmol l^? 1), it induced cell apoptosis, but caused necrosis at high dose (160 μmol l^? 1). Silymarin induced typical chromatin condensation and nuclear fragmentation as a hallmark of apoptosis. In this case, mitochondrial Bcl-2 family, Bcl-2 and Bax, were not involved in apoptotic effects; however, silymarin-induced cell death was regulated by the activation of p38 and JNK MAPKs. We also found that pan-caspase inhibitor and caspase-3 inhibitor could not antagonise silymarin-induced apoptosis. Therefore, silymarin induced and augmented HeLa cell apoptosis through p38/JNK MAPKs in the serum-free medium.     

Toxicity of thallium on isolated rat liver mitochondria: The role of oxidative stress and MPT pore opening

Thallium(I) is a highly toxic heavy metal; however, up to now, its mechanisms are poorly understood. The authors' previous studies showed that this compound could induce reactive oxygen species (ROS) formation, reduced glutathione (GSH) oxidation, membrane lipid peroxidation, and mitochondrial membrane potential (MMP) collapse in isolated rat hepatocyte. Because the liver is the storage site of thallium, it seems that the liver mitochondria are one of the important targets for hepatotoxicity. In this investigation, the effects of thallium on mitochondria were studied to investigate its mechanisms of toxicity. Mitochondria were isolated from rat liver and incubated with different concentrations of thallium (25–200 µM). Thallium(I)‐treated mitochondria showed a marked elevation in oxidative stress parameters accompanied by MMP collapse when compared with the control group. These results showed that different concentrations of thallium (25–200 µM) induced a significant (P < 0.05) increase in mitochondrial ROS formation, ATP depletion, GSH oxidation, mitochondrial outer membrane rupture, mitochondrial swelling, MMP collapse, and cytochrome c release. In general, these data strongly supported that the thallium(I)‐induced liver toxicity is a result of the disruptive effect of this metal on the mitochondrial respiratory complexes (I, II, and IV), which are the obvious causes of metal‐induced ROS formation and ATP depletion. The latter two events, in turn, trigger cell death signaling via opening of mitochondrial permeability transition pore and cytochrome c expulsion. © 2013 Wiley Periodicals, Inc. Environ Toxicol 30: 232–241, 2015.     

Apoptosis induced by para‐phenylenediamine involves formation of ROS and activation of p38 and JNK in chang liver cells

para‐phenylenediamine (p‐PD) is a suspected carcinogen, but it has been widely used as a component in permanent hair dyes. In this study, the mechanism of p‐PD‐induced cell death in normal Chang liver cells was investigated. The results demonstrated that p‐PD decreased cell viability in a dose‐dependent manner. Cell death via apoptosis was confirmed by enhanced DNA damage and increased cell number in the sub‐G1 phase of the cell cycle, using Hoechst 33258 dye staining and flow cytometry analysis. Apoptosis via reactive oxygen species generation was detected by the dichlorofluorescin diacetate staining method. Mitogen‐activated protein kinase (MAPK) activation was assessed by western blot analysis and revealed that p‐PD activated not only stress‐activated protein kinase (SAPK)/c‐Jun N‐terminal kinases (JNK) and p38 MAPK but also extracellular signal‐regulated kinase (ERK). Cytotoxicity and apoptosis induced by p‐PD were markedly enhanced by ERK activation and selectively inhibited by ERK inhibitor PD98059, thus indicating a negative role of ERK. In contrast, inhibition of p38 MAPK activity with the p38‐specific inhibitor SB203580 moderately inhibited cytotoxicity and apoptosis induction by p‐PD. Similarly, SP600125, an inhibitor of SAPK/JNK, moderately inhibited cytotoxicity and apoptosis induced by p‐PD, thus implying that p38 MAPK and SAPK/JNK had a partial role in p‐PD‐induced apoptosis. Western blot analysis revealed that p‐PD significantly increased phosphorylation of p38 and SAPK/JNK and decreased phosphorylation of ERK. In conclusion, the results demonstrated that SAPK/JNK and p38 cooperatively participate in apoptosis induced by p‐PD and that a decreased ERK signal contributes to growth inhibition or apoptosis. © 2012 Wiley Periodicals, Inc. Environ Toxicol 29: 981–990, 2014.     

Ketoconazole-induced JNK phosphorylation and subsequent cell death via apoptosis in human osteosarcoma cells

This study examined the effect of ketoconazole on viability, apoptosis, mitogen-activated protein kinases (MAPKs) and Ca²⁺ levels in MG63 osteosarcoma cells. Ketoconazole at 20–200 μM decreased cell viability via apoptosis as demonstrated by propidium iodide staining and activation of caspase-3. Immunoblotting suggested that ketoconazole induced phosphorylation of ERK and JNK, but not p38, MAPKs. Ketoconazole-induced cell death and apoptosis were partially reversed by the selective JNK inhibitor SP600125, but not by the selective ERK inhibitor PD98059, suggesting that ketoconazole’s cytotoxic action was via JNK, but not via ERK and p38 MAPKs. Ketoconazole at a concentration of 100 μM induced [Ca²⁺]_i increases. Chelation of intracellular Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) totally inhibited ketoconazole-induced [Ca²⁺]_i increases without reversing ketoconazole-induced cell death. Collectively, in MG63 cells, ketoconazole induced cell death and apoptosis via evoking JNK phosphorylation in a Ca²⁺-independent manner.     

Reactive oxygen species accumulation contributes to gambogic acid-induced apoptosis in human hepatoma SMMC-7721 cells

It is reported that gambogic acid (GA), the main active compound of gamboge which is a dry resin extracted from Garcinia hanburyi tree, has potent antitumor activity both in vivo and in vitro. Activation of mitochondrial apoptotic pathway in cancer cells is one effective therapy for cancer treatment. In the present study, we focus on the effect of GA on induction of reactive oxygen species (ROS) accumulation and triggering the mitochondrial signaling pathway in human hepatoma SMMC-7721 cells. The results indicated that GA induced ROS accumulation and collapse of mitochondrial membrane potential in SMMC-7721 cells in a concentration-dependent manner and subsequently induced that release of Cytochrome c and apoptosis-inducing factor from mitochondria to cytosol, which inhibited ATP generation and induced apoptosis in the cells. Moreover, GA elevated the phosphorylation of c-Jun-N-terminal protein kinase (JNK) and p38, which was the downstream effect of ROS accumulation. Furthermore, N-acetylcysteine, a ROS production inhibitor, partly reversed the activation of JNK and p38 and the induction of apoptosis in GA-treated cells. Collectively, our study demonstrated that accumulation of ROS played an important role in GA-induced mitochondrial signaling pathway, which provided further theoretical support for the application of GA as a promising anticancer agent.     

c-Jun NH2-terminal kinase-induced proteasomal degradation of c-FLIPL/S and Bcl2 sensitize prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine

Tetrandrine, a constituent of Chinese herb Stephania tetrandra, causes cell death in prostate cancer, but the molecular mechanisms leading to apoptosis is not known. Here we demonstrated that tetrandrine selectively inhibits the growth of prostate cancer PC3 and DU145 cells compared to normal prostate epithelial PWR-1E cells. Tetrandrine-induced cell death in prostate cancer cells is caused by reactive oxygen species (ROS)-mediated activation of c-Jun NH₂-terminal kinase (JNK1/2). JNK1/2-mediated proteasomal degradation of c-FLIP_L/S and Bcl₂ proteins are key events in the sensitization of prostate cancer cells to Fas- and mitochondria-mediated apoptosis by tetrandrine. Tetrandrine-induced JNK1/2 activation caused the translocation of Bax to mitochondria by disrupting its association with Bcl₂ which was accompanied by collapse of mitochondrial membrane potential (MMP), cytosolic release of cytochrome c and Smac, and apoptotic cell death. Additionally, tetrandrine-induced JNK1/2 activation increased the phosphorylation of Bcl₂ at Ser70 and facilitated its degradation via the ubiquitin-mediated proteasomal pathway. In parallel, tetrandrine-mediated ROS generation also caused the induction of ligand-independent Fas-mediated apoptosis by activating procaspase-8 and Bid cleavage. Inhibition of procaspase-8 activation attenuated the cleavage of Bid, loss of MMP and caspase-3 activation suggest that tetrandrine-induced Fas-mediated apoptosis is associated with the mitochondrial pathway. Furthermore, most of the signaling effects of tetrandrine on apoptosis were significantly attenuated in the presence of antioxidant N-acetyl-l-cysteine, thereby confirming the involvement of ROS in these events. In conclusion, the results of the present study indicate that tetrandrine-induced apoptosis in prostate cancer cells is initiated by ROS generation and that both intrinsic and extrinsic pathway contributes to cell death.     

Functional and binding kinetic studies make a distinction between OX(1) and OX(2) orexin receptor antagonists

Mdivi-1 is an inhibitor of dynamin related protein 1- (drp1)-mediated mitochondrial fission. However, the mechanisms through which this compound interacts directly with ion currents in heart cells remain unknown. In this study, its effects on ion currents and membrane potential in murine HL-1 cardiomyocytes were investigated. In whole-cell recordings, the addition of mdivi-1 decreased the amplitude of tail current (I(tail)) for the rapidly activating delayed-rectifier K? current (I(Kr)) in a concentration-dependent manner with an IC?? value at 11.6 μM, a value that resembles the inhibition requirement for mitochondrial division. It shifted the activation curve of I(tail) to depolarized voltages with no change in the gating charge. However, mdivi-1 did not alter the rate of recovery from current inactivation. In cell-attached configuration, mdivi-1 inside the pipette suppressed the activity of acetylcholine-activated K? channels without modifying the single-channel conductance. Mdivi-1 (30 μM) slightly depressed the peak amplitude of Na? current with no change in the overall current-voltage relationship. Under current-clamp recordings, addition of mdivi-1 resulted in prolongation for the duration of action potentials (APs) and to increase the firing of spontaneous APs in HL-1 cells. Similarly, in pituitary GH? cells, mdivi-1 was effective in directly suppressing the amplitude of ether-à-go-go-related gene-mediated K? current. Therefore, the lengthening of AP duration and increased firing of APs caused by mdivi-1 can be primarily explained by its inhibition of these K? channels enriched in heart cells. The observed effects of mdivi-1 on ion currents were direct and not associated with its inhibition of mitochondrial division.     

Tanshinone IIA protects rat primary hepatocytes against carbon tetrachloride toxicity via inhibiting mitochondria permeability transition

Tanshinone IIA (Tan IIA), one of the key components of Salvia milthorrhiza Bunge (Lamiaceae), is used to treat liver disease. The present study was carried out to investigate the possible mechanisms involved in the hepatoprotective effects of Tan IIA on carbon tetrachloride (CCl₄)-induced hepatocyte toxicity. In cultures treated with 1 or 2 μM CCl₄, Tan IIA (10–75 μM) significantly increased hepatocyte survival rates. However, only at a concentration of 75 μM could Tan IIA partially reverse the CCl₄ (3 μM)-induced decrease of survival rate (34?±?3% vs. 18?±?3%, n?=?8, p?<?0.01). In isolated mitochondria energized with succinate, Tan IIA could inhibit the large swelling effect induced by CCl₄ (1 and 2 μM). Base on these results, Tan IIA could protect rat primary cultured hepatocytes from CCl₄-induced toxicity partially by the inhibitory effect on the opening of mitochondrial permeability transition (MPT).     

MG132, a proteasome inhibitor-induced calf pulmonary arterial endothelial cell growth and death,are changed by MAPK inhibitors

MG132, as a proteasome inhibitor, has been shown to induce apoptotic cell death through the formation of reactive oxygen species (ROS). In this study, we investigated the effects of MAPK inhibitors on MG132-treated calf pulmonary artery endothelial cells (CPAECs) in relation to cell death, ROS, and glutathione (GSH). MG132 inhibited the growth of CPAEC and also induced apoptosis, which was accompanied by the loss of mitochondrial membrane potential (MMP; ΔΨ_m). MG132 increased ROS levels and GSH-depleted cell numbers in CPAEC. Treatment with MAPK (MEK, JNK, and p38) inhibitors showed a slight enhancement of cell-growth inhibition by MG132. All the MAPK inhibitors decreased cell death by MG132. Especially, the JNK inhibitor showed a strong effect. They all did not affect ROS levels and GSH depletion in MG132-treated CPAEC, but increased ROS and GSH levels in MG132-untreated CPAEC. In conclusion, MG132 induced apoptosis in CPAEC, which was accompanied by ROS increase and GSH depletion. The changes of MG132-induced CPAEC growth inhibition and death by MAPK inhibitors were not tightly correlated to ROS and GSH levels.