Silica-induced apoptosis in mouse alveolar macrophages is initiated by lysosomal enzyme activity.

Past studies in our laboratory have shown that silica (-quartz) particle exposure of a mouse alveolar macrophage cell line (MH-S) elicits mitochondrial depolarization and caspase 3 and 9 activation, contributing to apoptosis. However, cellular pathways leading to these outcomes have not been extensively investigated. Initial studies revealed that silica exposure elicits lysosomal permeability after 1 h, as evidenced by leakage of FITC-conjugated dextran and acridine orange. We next evaluated a role for the lysosomal acidic compartment in apoptosis. Cells pretreated with the lysosomotropic weak base ammonium chloride, to increase lysosomal pH, showed decreased caspase activation and apoptotic DNA fragmentation. MH-S cells pretreated with pepstatin A, an inhibitor of lysosomal cathepsin D, showed decreased caspase 9 and 3 activation as well as a decreased percentage of cells that became apoptotic. DNA fragmentation and caspase 9 and 3 activation were also decreased in cells pretreated with despiramine, an inhibitor of lysosomal acidic sphingomyelinase. Silica pretreated with aluminum lactate (to blunt surface active sites) reduced caspase activation and apoptosis. Although aluminum lactate-treated silica still induced lysosomal permeability (by FITC-dextran leakage), one measure of lysosome integrity and function suggested a reduction in the extent and/or nature of lysosomal injury (by acridine orange retention). A role for reactive oxygen species (ROS) was investigated to explore another pathway for silica-induced apoptosis in addition to lysosomal enzymes; however, no role for ROS was apparent. Thus, following silica exposure, lysosomal injury precedes apoptosis, and the apoptotic signaling pathway includes cathepsin D and acidic sphingomyelinase.     

Silica-induced apoptosis in alveolar macrophages: evidence of in vivo thiol depletion and the activation of mitochondrial pathway

Studies have shown that silica induces apoptosis through mechanisms that also regulate the inflammatory responses of lung cells to silica exposure. Although implicated in cell culture studies, the major in vivo pathway through which silica induces apoptosis has not been characterized. The present study is to study the role of mitochondria in silica-induced oxidative stress and apoptosis in vivo. Rats were intratracheally instilled with saline or silica (20 mg/kg) and sacrificed at 3 days post-exposure unless otherwise specified. Alveolar macrophages (AM) were harvested by bronchoalveolar lavage and measured for apoptosis and secretion of inflammatory mediators in the presence or absence of appropriate inhibitors. Concurrent studies were carried out to determine the presence of intracellular reactive oxygen species (ROS) via confocal microscopy, mitochondrial trans-membrane potential by flow cytometry, mitochondrial release of cytochrome c, and the activation of caspase activities in AM by Western blot analysis. Silica was shown to induce elevated levels of intracellular ROS, resulting in a marked decrease in intracellular glutathione (GSH) and cysteine and a sustained presence of apoptotic AM in silica-exposed rats up to two weeks post-exposure. The apoptotic AM were characterized by decreased mitochondrial trans-membrane potential, increased mitochondrial release of cytochrome c, activated caspase 9 (but not caspase 8) and caspase 3 activities, and PARP degradation, comparing to cells from the saline control. Silica induced AM production of IL-1 and TNF-alpha, which may be inhibited by ex vivo treatment of cells with N-acetylcysteine (NAC) or microtubule modifiers such as tetrandrine and taxol. NAC was shown to prevent intracellular GSH depletion and silica-induced production of IL-1beta and TNF-alpha but not apoptosis in AM from silica-exposed rats. These results show that silica-induced apoptosis is mediated through the mitochondrial pathway but not through cellular production of inflammatory cytokines, ROS generation, however, induces both apoptosis and cellular secretion of inflammatory mediators.     

Caspase cascade proceeds rapidly after cytochrome c release from mitochondria in tumor necrosis factor-alpha-induced cell death

The caspase activation cascade and mitochondrial changes are major biochemical reactions in the apoptotic cell death machinery. We attempted to clarify the temporal relationship between caspase activation, cytochrome c release, mitochondrial depolarization, and morphological changes that take place during tumor necrosis factor (TNF)-alpha-induced cell death in HeLa cells. These reactions were analyzed at the single-cell level with 0.5 - 1 min resolution by using green fluorescent protein (GFP)-variant-derived probes and chemical probes. Cytochrome c release, caspase activation, and cellular shrinkage were always observed in this order within 10 min in all dying cells. This sequence of events was thus considered a critical pathway of cell death. Mitochondrial depolarization was also observed in all dying cells observed, but frequently occurred after caspase activation and cellular shrinkage. Mitochondrial depolarization is therefore likely to be a reaction that does not induce caspase activation and subsequent cellular shrinkage. Mitochondrial changes are important for apoptotic cell death; moreover, cytochrome c release, and not depolarization, is a key reaction related to cell death. In addition, we also found that the apoptotic pathway proceeds only when cells are exposed to TNF-alpha. These findings suggest that the entire cell death process proceeds rapidly during TNF-alpha exposure.     

Farnesyltransferase inhibitor BMS-214662 induces apoptosis in myeloma cells through PUMA up-regulation, Bax and Bak activation, and Mcl-1 elimination

We have studied the mechanism of apoptosis elicited by the farnesyltransferase inhibitor (R)-7-cyano-2,3,4,5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulfonyl)-1H-1,4-benzodiazepine (BMS-214662) in human myeloma cell lines. Low concentrations of BMS-214662 efficiently inhibited protein farnesylation but did not affect the activation of Akt. BMS-214662 treatment increased levels of the BH3-only protein PUMA; induced proapoptotic conformational changes of Bax and Bak; reduced Mcl-1 levels; caused mitochondrial transmembrane potential loss; induced cytochrome c release, caspase activation, apoptosis-inducing factor (AIF) nuclear translocation, and phosphatidylserine exposure; and allowed the development of apoptotic morphology. Western blot analysis of cell extracts revealed the activation of caspases 2, 3, 8, and 9 upon treatment with BMS-214662. The general caspase inhibitor Z-VAD-fmk significantly prevented BMS-214662-induced death in U266 and RPMI 8226 cells but not in NCI-H929 cells. A mixture of selective caspase inhibitors for caspases 9 [N-benzyloxycarbonyl-Leu-Glu-His-Asp-fluoromethyl ketone (Z-LEHD-fmk)], 3 (Z-DEVD-fmk), and 6 (Z-VEID-fmk) approached the protective effect of Z-VAD upon cell death. However, Z-VAD-fmk did not prevent BMS-214662-induced Bax and Bak activation and decrease of Mcl-1 levels. According to its effect on cell death, Z-VAD-fmk inhibited nuclear translocation of AIF in RPMI 8226 and U266 but not in NCI-H929 cells. These results suggest that apoptosis triggered by BMS-214662 is initiated by a PUMA/Bax/Bak/Mcl-1-dependent mechanism. In some cell lines, Bax/Bak activation is not sufficient per se to induce mitochondrial failure and release of apoptogenic proteins, and so caspases need to be activated to facilitate apoptosis. After DeltaPsi(m) loss, execution of apoptosis was performed in all cases by a cytochrome c-enabled, caspase-9-triggered, caspase cascade and the nuclear action of AIF.     

Involvement of both endoplasmic reticulum- and mitochondria-dependent pathways in cardiotoxin III-induced apoptosis in HL-60 cells

Cardiotoxin (CTX) III, a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. In the present study, we investigated the mechanisms underlying the anticancer activity of CTX III in human leukaemia (HL-60 cells). Cardiotoxin III activated the endoplasmic reticulum (ER) pathway of apoptosis in HL-60 cells, as indicated by increased levels of calcium and glucose-related protein 78 (Grp78), and triggered the subsequent activation of micro-calpain and caspase 12. In addition, CTX III initiated the mitochondrial apoptotic pathway in HL-60 cells, as evidenced by an increased Bax/Bcl-2 ratio, the release of cytochrome c and activation of caspase 9. In the presence of 50 micromol/L Z-ATAD-FMK (a caspase 12 inhibitor) and 100 micromol/L Z-LEHD-FMK (a caspase 9 inhibitor), the CTX III-mediated activation of caspase 9 and caspase 3 was significantly reduced. There was no significant effect of the caspase 12 inhibitor Z-ATAD-FMK on mitochondrial cytochrome c release. Cardiotoxin III-mediated activation of caspase 12 was not abrogated in the presence of the caspase 9 inhibitor Z-LEHD-FMK, indicating that caspase 12 activation was not downstream of caspase 9. These results indicate that CTX III induces cell apoptosis via both ER stress and a mitochondrial death pathway.     

Reactive oxygen species-independent rapid initiation of mitochondrial apoptotic pathway by chelerythrine

Chelerythrine, formerly identified as a protein kinase C inhibitor, has also been shown to inhibit the anti-apoptotic Bcl-2 family proteins. However, recent studies have now demonstrated that chelerythrine can induce the loss of mitochondrial membrane potential (ΔΨ_m), a membrane permeability transition (MPT), and the subsequent activation of the mitochondrial apoptotic pathway, even in the cells deficient in Bax and Bak. This suggests the existence of an alternative Bax/Bak-independent pathway for apoptosis. The generation of reactive oxygen species (ROS) from the mitochondrial electron transport chain (ETC) is also implicated in the cytotoxity elicited by chelerythrine. In our current study, we show that chelerythrine induces the rapid apoptotic death of H9c2 cardiomyocyte-derived cells within 8 min of treatment. The proteolytic activation of caspase9 and caspase3, crucial mediators of the mitochondrial apoptotic pathway, are also observed within 6 min of exposure to this drug. The generation of ROS is detected but at only marginal levels in the treated cells. The inhibition of the mitochondrial ETC by rotenone and malonate had almost no effects on ROS generation but in both cases effectively inhibited both cell death and the caspase activation induced by chelerythrine. Hence, chelerythrine initiates the rapid mitochondrial apoptotic death of H9c2 cardiomyoblastoma cells in a manner that is likely independent of the generation of ROS from mitochondria.     

TRAIL的诱导肿瘤细胞凋亡与临床

TRAIL是最近发现的属肿瘤坏死因子家族新成员 ,它能选择性地杀伤多种肿瘤细胞而对正常细胞没有细胞毒性。它的作用机制主要通过细胞膜的表面死亡受体DR4和DR5介导细胞的凋亡信号 ,激活细胞内的多种诱导细胞凋亡蛋白的表达。主要的途径是通过TRAIL与死亡受体结合后促进DISC的形成和caspase8的激活 ,然后启动非依赖线粒体的凋亡途径和线粒体的凋亡途径。非线粒体凋亡途径通过激活的csapase8直接激活caspase3,从而启动细胞的凋亡。线粒体依赖途径通过激活的tBID导致细胞色素C的释放 ,与Apaf1和caspase9形成凋亡小体导致细胞的凋亡。通过以往的研究表明 ,非融合表达的TRAIL能明显诱导多种肿瘤细胞的凋亡 ,对正常人和猴的肝细胞没有细胞毒性 ,提示非融合表达的人TRAIL蛋白可能是未来最有发展前途的抗肿瘤药物     

Mechanism of dibucaine-induced apoptosis in promyelocytic leukemia cells (HL-60)

Dibucaine, a local anesthetic, inhibited the growth of promyelocytic leukemia cells (HL-60) without inducing arrest of the cell cycle and differentiation to granulocytes. Typical DNA fragmentation and DNA ladder formation were induced in a concentration- and time-dependent manner. The half-maximal concentration of dibucaine required to induce apoptosis was 100 microM. These effects were prevented completely by the pan-caspase inhibitor z-Val-Ala-Asp-(OMe)-fluoromethylketone (z-VAD-fmk), thereby implicating the cysteine aspartase (caspase) cascade in the process. Dibucaine activated various caspases, such as caspase-3, -6, -8, and -9 (-like) activities, but not caspase-1 (-like) activity, and induced mitochondrial membrane depolarization and the release of cytochrome c (Cyt.c) from mitochondria into the cytosol. Processing of pro-caspase-3, -8, and -9 by dibucaine was confirmed by western blot analysis. Bid, a death agonist member of the Bcl-2 family, was processed by caspases following exposure of cells to dibucaine. However, 100 microM dibucaine scarcely inhibited oxidative phosphorylation, but it induced membrane permeability transition in isolated rat liver mitochondria. Taken together, these data suggest that dibucaine induced apoptosis of HL-60 cells through activation of the caspase cascade in conjunction with Cyt.c release induced by a processed product of Bid and depolarization of the mitochondrial membrane potential.     

Licochalcone A induces apoptotic cell death via JNK/p38 activation in human nasopharyngeal carcinoma cells

Licochalcone A is widely studied in different fields and possesses antiasthmatic, antibacterial, anti‐inflammatory, antioxidative, and anticancer properties. Its antimalignancy activity on renal, liver, lung, and oral cancer has been explored. However, limited studies have been conducted on the inhibitory effects of licochalcone A in human nasopharyngeal carcinoma cells. We determined cell viability using MTT assay. Cell cycle distribution and apoptotic cell death were measured via flow cytometry. Caspase activation and mitogen‐activated protein kinase‐related proteins in nasopharyngeal cancer cells in response to licochalcone A were identified by Western blot analysis. Results indicated that licochalcone A reduces cell viability and induces apoptosis, as evidenced by the upregulation of caspase‐8 and caspase‐9, caspase‐3 activation, and cleaved‐poly ADP‐ribose polymerase expression. Treatment with licochalcone A significantly increases ERK1/2, p38, and JNK1/2 activation. Co‐administration of a JNK inhibitor (JNK‐IN‐8) or p38 inhibitor (SB203580) abolishes the activation of caspase‐9, caspase‐8, and caspase‐3 protein expression during licochalcone A treatment. These findings indicate that licochalcone A exerts a cytostatic effect through apoptosis by targeting the JNK/p38 pathway in human nasopharyngeal carcinoma cells. Therefore, licochalcone A is a promising therapeutic agent for the treatment of human nasopharyngeal cancer cells.     

The apoptotic effect of brucine from the seed of Strychnos nux-vomica on human hepatoma cells is mediated via Bcl-2 and Ca2+ involved mitochondrial pathway.

In an attempt to dissect the mechanism of Strychnos nux-vomica, a commonly used Chinese folk medicine in the therapy of liver cancer, the cytotoxic effects of four alkaloids in Strychnos nux-vomica, brucine, brucine N-oxide, strychnine, and isostrychnine, on human hepatoma cells (HepG2) were screened by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrasolium bromide (MTT) assay. Brucine, among the four alkaloids, exhibited the strongest toxic effect, the mechanism of which was found to cause HepG2 cell apoptosis, since brucine caused HepG2 cell shrinkage, the formation of apoptotic bodies, DNA fragmentation, cell cycle arrest, as well as phosphatidylserine externalization, all of which are typical characteristics of apoptotic programmed cell death. Brucine-induced HepG2 cell apoptosis was caspase dependent, with caspase-3 activated by caspase-9. Brucine also caused the proteolytic processing of caspase-9. In addition, brucine caused depolarization of the mitochondrial membrane of HepG2 cells, the inhibition of which by cyclosporine A completely abrogated the activation of casapses and release of cytochrome c in brucine-treated HepG2 cells. These findings suggested a pivotal role of mitochondrial membrane depolarization in HepG2 cell apoptosis elicited by brucine. Furthermore, brucine induced a rapid and sustained elevation of intracellular [Ca2+], which compromised the mitochondrial membrane potential and triggered the process of HepG2 cell apoptosis. Finally, Bcl-2 was found to predominately control the whole event of cell apoptosis induced by brucine. The elevation of [Ca2+]i caused by brucine was also suppressed by overexpression of Bcl-2 protein in HepG2 cells. From the facts given above, Ca2+ and Bcl-2 mediated mitochondrial pathway were found to be involved in brucine-induced HepG2 cell apoptosis.     

Cell surface regulation of silica-induced apoptosis by the SR-A scavenger receptor in a murine lung macrophage cell line (MH-S).

Scavenger receptors (SR) are responsible for recognition of ligands as diverse as oxidized LDL (endogenous) to respirable particulates (exogenous). A number of recent studies have suggested that these SR ligands induce apoptosis of macrophages. However, the mechanism by which SR triggers apoptosis is not understood. This study used a murine alveolar macrophage cell line (MH-S) to investigate the role of the SR in caspase activation. The presence of SR on MH-S cells was confirmed by FACS analysis and was similar to the distribution found on murine alveolar macrophages. The activity of caspases 1, 3, and 6 was measured following a 6-h exposure to crystalline silica with and without blockers of the SR. Caspase activities were determined by hydrolysis of specific chromogenic substrates and formation of an active enzymatic form (Western for active caspase 3). Silica stimulated significant caspase activity, apoptosis, and necrosis of MH-S cells, which was attenuated by 2F8 (a blocking antibody) and polyinosinic acid (a nonspecific SR antagonist). The results indicate that the SR are necessary for caspase activation and subsequent apoptosis (as well as necrosis) caused by silica in macrophage cells.     

Antibody Against N‐terminal Domain of Phospholipid Scramblase 1 Induces Apoptosis in Colorectal Cancer Cells Through the Intrinsic Apoptotic Pathway

Phospholipid scramblase 1 involve in biological processes including phospholipid movement, proliferation, and apoptosis. Treatment with an antiphospholipid scramblase 1 antibody (NP1) has been demonstrated to inhibit cell proliferation in colorectal cancer. This study aimed to explore the role of NP1 treatment in the apoptosis of colorectal cancer cells. Results showed that NP1 treatment significantly increases the apoptosis of colorectal cancer cells via the activation of caspase 8, caspase 9, and caspase 3. Moreover, pretreatment with a caspase 8 inhibitor did not fully prevent the apoptotic effects of NP1. Taken together, these data indicate NP1 induces cell apoptosis primary through the intrinsic apoptotic pathway. NP1 may serve as a potential therapeutic agent.     

Titanium dioxide nanoparticles cause apoptosis in BEAS-2B cells through the caspase 8/t-Bid-independent mitochondrial pathway

To understand the underlying mechanism for apoptosis induced by titanium dioxide nanoparticles (TNP), human airway epithelial cell line was cultured to investigate the relevant apoptosis pathways. Our results showed that the levels of reactive oxygen species and morphological apoptosis increased in a dose-dependent manner whereas cell viability decreased in a similar manner in response to TNP exposure in the BEAS-2B cells. The activities of caspase 3 and PARP were also increased in parallel to the morphological apoptosis. Levels of caspase 9 increased significantly whereas there were no detectable changes in caspase 8 and t-Bid in the TNP treated cells. Caspase 9 inhibition blocked the TNP-induced activation of caspase 3 significantly. The levels of bax, cytochrome C, p53 and bcl-2 also changed reflecting the activation of intrinsic apoptosis pathway. Our results provide solid evidence that apoptosis in BEAS-2B cells exposed to TNP occurred via a mitochondrial apoptosis pathway independent of caspase 8/t-Bid pathway.     

Paraoxon induces apoptosis in EL4 cells via activation of mitochondrial pathways

The toxicity of organophosphorus compounds, such as paraoxon (POX), is due to their anticholinesterase action. Recently, we have shown that, at noncholinergic doses (1 to 10 nM), POX (the bioactive metabolite of parathion) causes apoptotic cell death in murine EL4 T-lymphocytic leukemia cell line through activation of caspase-3. In this study, by employing caspase-specific inhibitors, we extend our observations to elucidate the sequence of events involved in POX-stimulated apoptosis. Pretreatment of EL4 cells with the caspase-9-specific inhibitor zLEHD-fmk attenuated POX-induced apoptosis in a dose-dependent manner, whereas the caspase-8 inhibitor zIETD-fmk had no effect. Furthermore, the activation of caspase-9, -8, and -3 in response to POX treatment was completely inhibited in the presence of zLEHD-fmk, implicating the involvement of caspase 9-dependent mitochondrial pathways in POX-stimulated apoptosis. Indeed, under both in vitro and in vivo conditions, POX triggered a dose- and time-dependent translocation of cytochrome c from mitochondria into the cytosol, as assessed by Western blot analysis. Investigation of the mechanism of cytochrome c release revealed that POX disrupted mitochondrial transmembrane potential. Neither this effect nor cytchrome c release was dependent on caspase activation, since the general inhibitor of the caspase family zVAD-fmk did not influence both processes. Finally, POX treatment also resulted in a time-dependent up-regulation and translocation of the proapoptotic molecule Bax to mitochondria. Inhibition of this event by zVAD-fmk suggests that the activation and translocation of Bax to mitochondria is subsequent to activation of the caspase cascades. The results indicate that POX induces apoptosis in EL4 cells through a direct effect on mitochondria by disrupting its transmembrane potential, causing the release of cytochrome c into the cytosol and subsequent activation of caspase-9. Inhibition of this specific pathway might provide a useful strategy to minimize organophosphate-induced poisoning.     

Induction of apoptosis in human hepatoblastoma cells by tetrandrine via caspase-dependent Bid cleavage and cytochrome c release

Tetrandrine, a bis-benzylisoquinoline alkaloid from the root of Stephania tetrandra, induces apoptosis in human T-cell lines, lung carcinoma and hepatoblastoma cells. However, the mechanisms by which tetrandrine inhibits tumor cell growth are poorly understood. The purpose of the present study was to investigate the intracellular signaling mechanism of tetrandrine-induced apoptosis in HepG2 cells. The induction of apoptosis was determined by morphological analysis and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay. Treatment of cells with tetrandrine caused the upregulation of p53, downregulation of Bcl-X(L), cleavage of Bid and Bax, and release of cytochrome c, which were accompanied by activation of caspases 9, 3 and 8. The activation of caspases 9 and 3 preceded that of caspase 8. A broad-spectrum caspase inhibitor and a caspase 8-specific inhibitor completely blocked tetrandrine-induced Bid processing, cytochrome c release, activation of caspase 3, and cell death. These findings and data showing the early release of cytochrome c, cleavage of Bid and downregulation of Bcl-X(L) suggest that the mitochondrial pathway is primarily involved in tetrandrine-induced apoptosis. The activation of caspase 8 after early caspases 9 and 3 activation might act as an amplification loop for activation of upstream signals such as Bid cleavage or cytochrome c release. These data suggest that tetrandrine may constitute a plausible therapeutic for hepatocellular carcinoma.     

Chrysotile asbestos causes AEC apoptosis via the caspase activation in vitro and in vivo

To determine whether alveolar epithelial cell (AEC) apoptosis via caspase activation is involved in asbestos-induced lung injury, we examined apoptosis, caspase-3 and-9 activation using chrysotile asbestos exposure models in vitro and in vivo. Apoptotic cells were assessed in A549 cells with terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method after 48 hours exposure of chrysotile asbestos (5 to 100 ug/cm2). Asbestos exposure induced a dose-dependent increase of apoptotic cells, and both pretreatment with Z-LEHD-FMK (caspase-3 inhibitor) or Z-DEMK-FMK (caspase-9 inhibitor) significantly suppressed asbestos-induced apoptosis. Expression of cleaved caspase-3 and-9 increased significantly from 18 to at least 48 hours after asbestos exposure. In vivo study, either 1 or 2 mg of chrysotile asbestos were administered into rat lungs intratracheally, and the lungs were obtained 3 days, 1 and 2 weeks, 1, 3 and 6 months after the administration. Asbestos exposure increased the number of apoptotic cells and the activation of cleaved caspase-3 and -9 most at 3 days in a dose dependent manner, and continued to increase them until at least 6 months after asbestos exposure. Apoptotic cells and cleaved caspase-3 and -9 positive cells were mainly observed in AECs. These findings suggest that AEC apoptosis via caspase-3 and -9 activation is involved in asbestos-induced lung injury.     

Mechanisms of cardiotoxin lll-induced apoptosis in human colorectal cancer colo205 cells

Cardiotoxin III (CTX III) is a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom. This is the first report on the mechanism of the anticancer effect of CTX III in human colorectal cancer Colo205 cells. 2. Cardiotoxin III-induced Colo205 cell apoptosis was confirmed by DNA fragmentation (DNA ladder and sub-G1 formation) with an IC(50) of 4 mg/mL at 48 h. 3. Further mechanistic analysis demonstrate that CTX III induced the loss of mitochondrial membrane potential (Dym), cytochrome c release from mitochondria into the cytosol and activation of capase-9, caspase 3, as well as markedly enhancing the expression of Bax, but not Bcl-2, protein in the cells. Moreover, the CTX III-induced apoptosis was significantly blocked by the broad-spectrum caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. 4. However, CTX III did not generate the formation of reactive oxygen species and anti-oxidants, including N-acetylcysteine, and catalase could not block CTX III-induced apoptosis in the Colo205 cells. 5. Taken together, these results suggest that CTX III may induce apoptosis through a mitochondrial- and caspase-dependent mechanism and alteration of Bax/Bcl-2 ratio in human colorectal Colo205 cancer cells.     

Dihydroartemisinin exhibits antitumor activity toward hepatocellular carcinoma in vitro and in vivo

Dihydroartemisinin (DHA), a semi-synthetic derivative of artemisinin isolated from the traditional Chinese herb Artemisia annua L., has been shown to exhibit inhibitory effects on human cancer cells. However, its antitumor ability toward hepatocellular carcinoma (HCC) has not been studied. In this study, we demonstrated that DHA significantly inhibited HCC cell growth in vitro and in vivo via inducing G2/M cell cycle arrest and apoptosis. The induction of p21 and the inhibition of cyclin B and CDC25C contributed to DHA-induced G2/M arrest. DHA-induced apoptosis was associated with mitochondrial membrane depolarization, release of cytochrome c, activation of caspases, and DNA fragmentation. Activation of caspase 9 and caspase 3, but not caspase 8, was detected in DHA-treated cells. Attenuation of apoptosis in cells pretreated with Z-VAD-FMK suggested the involvement of caspase cascade. Furthermore, p53 facilitated apoptosis caused by DHA. Bcl-2 family proteins were also responsible for DHA-induced apoptosis. DHA exposure decreased Mcl-1 expression but increased the levels of Noxa and active Bak. Bak was released from the Mcl-1/Bak complex due to the decline of Mcl-1. Further study revealed that Mcl-1 was rapidly degraded in DHA-treated cells and that DHA-induced apoptosis was largely inhibited by overexpression of Mcl-1 or RNAi-mediated decrease of Bak and Noxa. In a HCC-xenograft mouse model, the intraperitoneal injection of DHA resulted in significant inhibition of HCC xenograft tumors. Taken together, our data, for the first time, demonstrate the potential antitumor activity of DHA in HCC.