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ABSTRACT: Capsaicin treatment was previously reported to reduce the sensitivity of breast cancer cells, but not normal MCF10A cells, to apoptosis. The present study shows that autophagy is involved in cellular resistance to genotoxic stress, through DNA repair. Capsaicin treatment of MCF-7 cells induced S-phase arrest and autophagy through the AMPKα-mTOR signaling pathway and the accumulation of p53 in the nucleus and cytosol, including a change in mitochondrial membrane potential. Capsaicin treatment also activated δ-H2AX, ataxia telangiectasia mutated (ATM), DNA-dependent protein kinase catalytic subunit (DNA-PKcs), and poly(ADP-ribose) polymerase (PARP)-1. Genetic or pharmacological disruption of autophagy attenuated capsaicin-induced phospho-ATM and phospho-DNA-PKcs and enhanced apoptotic cell death. ATM inhibitors, including Ku55933 and caffeine, and the genetic or pharmacological inhibition of p53 prevented capsaicin-induced DNA-PKcs phosphorylation and stimulated PARP-1 cleavage, but had no effect on microtubule-associated protein light chain 3 (LC3)-II levels. Ly294002, a DNA-PKcs inhibitor, boosted the capsaicin-induced cleavage of PARP-1. In M059K cells, but not M059J cells, capsaicin induced ATM and DNA-PKcs phosphorylation, p53 accumulation, and the stimulation of LC3II production, all of which were attenuated by knockdown of the autophagy-related gene atg5. Ku55933 attenuated capsaicin-induced phospho-DNA-PKcs, but not LC3II, in M059K cells. In human breast tumors, but not in normal tissues, AMPKα, ATM, DNA-PKcs, and PARP-1 were activated and LC3II was induced. The induction of autophagy by genotoxic stress likely contributes to the sustained survival of breast cancer cells through DNA repair regulated by ATM-mediated activation of DNA-PKcs and PARP-1.
Biochemical pharmacology 03/2012; 83(6):747-57. · 4.25 Impact Factor
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ABSTRACT: Proteasome inhibition is a promising approach for cancer treatment; however, the underlying mechanisms involved have not been fully elucidated. Here, we show that proteasome inhibition-induced p38 mitogen-activated protein kinase regulates autophagy and apoptosis by modulating the phosphorylation status of glycogen synthase kinase 3β (GSK3β) and 70kDa ribosomal S6 kinase (p70S6K). The treatment of MDA-MB-231 cells with MG132 induced endoplasmic reticulum stress through the induction of ATF6a, PERK phosphorylation, and CHOP, and apoptosis through the cleavage of Bax and procaspase-3. MG132 caused the phosphorylation of GSK3β at Ser(9) and, to a lesser extent, Thr(390), the dephosphorylation of p70S6K at Thr(389), and the phosphorylation of p70S6K at Thr(421) and Ser(424). The specific p38 inhibitor SB203080 reduced the p-GSK3β(Ser9) and autophagy through the phosphorylation of p70S6K(Thr389); however, it augmented the levels of p-ERK, p-GSK3β(Thr390), and p-70S6K(Thr421/Ser424) induced by MG132, and increased apoptotic cell death. The GSK inhibitor SB216763, but not lithium, inhibited the MG132-induced phosphorylation of p38, and the downstream signaling pathway was consistent with that in SB203580-treated cells. Taken together, our data show that proteasome inhibition regulates p38/GSK(Ser9)/p70S6K(Thr380) and ERK/GSK3β(Thr390)/p70S6K(Thr421/Ser424) kinase signaling, which is involved in cell survival and cell death.
Biochemical and Biophysical Research Communications 02/2012; 418(4):759-64. · 2.48 Impact Factor
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ABSTRACT: Orotic acid (OA), an intermediate in pyrimidine metabolism, has been used for a variety of purposes, such as dietary supplements. Although it is well documented that OA induces fatty liver in a species-specific manner, the precise molecular mechanisms remain unclear. The present study investigated the role of the adenosine monophosphate-activated protein kinase (AMPK)-sterol regulatory element-binding protein-1 (SREBP-1) pathway in the OA-induced fatty liver. Treatment with OA suppressed the phosphorylation of AMPK via proteasomal degradation of upstream kinase LKB1 and induced activation of SREBP-1 in both human hepatoma cell lines and primary rat hepatocytes. OA-induced SREBP-1 transcriptional activity was suppressed by cotreatment with aminoimidazole carboxamide ribonucleotide (AICAR) or metformin, or by overexpression of constitutively active AMPK (CA-AMPK) in the human hepatoma cell line. Importantly, in vivo data corroborated these results. Feeding 1% OA with diet decreased the phosphorylation of AMPK and increased the maturation of SREBP-1 and the expression of SREBP-responsive genes in the rat liver. OA-induced lipid accumulation was also completely inhibited by rapamycin. Mouse hepatocytes and mice were resistant to OA-induced lipogenesis because of little if any response in AMPK and downstream effectors. In conclusion, OA induces hepatic lipogenesis, mediated predominantly by the AMPK/SREBP-1 pathway in rat hepatocytes and human hepatoma cell lines.
The Journal of Lipid Research 09/2011; 52(9):1617-25. · 5.56 Impact Factor
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ABSTRACT: Human multidrug-resistance associated protein (MRP1) is known as a cellular efflux pump of heavy metals and anticancer drugs. In our previous study, MRP was found to have involvement in cell protection against cadmium (Cd) toxicity through apoptosis interruption. The purpose of the present study was to investigate the molecular mechanism of MRP1 in Cd resistance. For this purpose, we developed Cd-resistant cells (RWI38) from WI38 human lung epithelial fibroblast cells, which showed a 4-fold resistance to Cd when compared to WI38 cells. WI38 cells elicited endoplasmic reticulum (ER) stress through RNA-dependent protein kinase-like ER kinase (PERK) and the eukaryotic translation initiation factor 2 alpha (eIF2alpha), Chop, and glucose-regulated protein (Grp78). RWI38 cells responding to Cd did not elicit ER stress or mitochondrial apoptosis, but induced autophagy, as demonstrated by Atg5 induction, LC3 conversion, and formation of GFP-LC3 dots. A pharmacological inhibitor of p38 downregulated Cd-induced Atg5 and LC3II. A pharmacological inhibitor of autophagy or silencing of atg5 dephosphorylated p38 and Akt, and downregulated MRP1 and procaspase-3. However, pharmacological inhibition or silencing of mrp-1 had no affect on Cd-induced phosphorylated p38 and LC3II. These data indicate that Cd induces autophagy in RWI38 cells through a mechanism that involves p38 activation, which is involved in cell protection through counterbalance of ER stress and MRP1 induction.
Toxicology 09/2010; 276(1):18-26. · 3.68 Impact Factor
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ABSTRACT: We reported that dihydrocapsaicin (DHC) induces autophagy in a catalase-regulated manner. In this study, we further examined the role of DHC-induced autophagy in lung cell lines. DHC-induced cytotoxicity was higher in WI38 and H1299 cells than in H460 and A549 cells, and was related to the loss of cell membrane integrity. However, apoptotic cells markedly increased in H460 and A549 cells. In WI38 and H1299 cells, DHC-induced catalase was correlated with a decrease of intracellular reactive oxygen species (ROS) and an increase in the level of LC3II, an autophagy marker, and LC3 conversion was attenuated by the catalase inhibitor 3-amino-1,2,4-triazole (3AT) or by knockdown of the catalase gene. In A549 cells, DHC downregulated catalase, led to ROS accumulation, and blocked LC3 conversion. In H460 cells expressing limited amount of catalase, DHC caused ROS accumulation and blocked LC3 conversion. However, H460 cells overexpressing catalase were able to induce autophagy. In contrast to Earle's balanced salt solution and rotenone, H(2)O(2) treatment caused ROS accumulation and did not promote upregulation of catalase and LC3II in lung cell lines. Cytoplasmic vacuolization in WI38 and H1299 cells was blocked by treatment of 3AT and which enhanced caspase-3 activity and LDH release. Suppression of autophagy by 3-methyladenine also enhanced DHC-induced cell death through apoptotic and necrotic cell death. In A549 and H460 cells, treatment of rapamycin attenuated DHC-induced cell death. Collectively, these results suggest that catalase regulates autophagy, which helps protect cells against apoptotic and necrotic cell death.
Free radical biology & medicine 07/2010; 49(2):245-57. · 5.42 Impact Factor
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ABSTRACT: Capsaicin, a pungent compound found in hot chili peppers, induces apoptotic cell death in various cell lines, however, the precise apoptosis signaling pathway is unknown. Here, we investigated capsaicin-induced apoptotic signaling in the human breast cell line MCF10A and found that it involves both endoplasmic reticulum (ER) stress and calpain activation. Capsaicin inhibited growth in a dose-dependent manner and induced apoptotic nuclear changes in MCF10A cells. Capsaicin also induced degradation of tumor suppressor p53; this effect was enhanced by the ER stressor tunicamycin. The proteasome inhibitor MG132 completely blocked capsaicin-induced p53 degradation and enhanced apoptotic cell death. Capsaicin treatment triggered ER stress by increasing levels of IRE1, GADD153/Chop, GRP78/Bip, and activated caspase-4. It led to an increase in cytosolic Ca(2+), calpain activation, loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and caspase-9 and -7 activation. Furthermore, capsaicin-induced the mitochondrial apoptotic pathway through calpain-mediated Bid translocation to the mitochondria and nuclear translocation of apoptosis-inducing factor (AIF). Capsaicin-induced caspase-9, Bid cleavage, and AIF translocation were blocked by calpeptin, and BAPTA and calpeptin attenuated calpain activation and Bid cleavage. Thus, both ER stress- and mitochondria-mediated death pathways are involved in capsaicin-induced apoptosis.
Toxicology 09/2009; 264(3):205-14. · 3.68 Impact Factor
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ABSTRACT: To elucidate the mechanisms involved in adaptation of lung epithelial cells to cadmium (Cd), we established a cell line that exhibits Cd-resistance (RWI38). RWI38 showed approximately 5-fold greater Cd-resistance (MTT assays) than WI38 cells, and cross-resistance to Zn and cisplatin. RWI38 cells also demonstrated an upregulated level of multidrug resistance-associated protein (MRP) and metallothionein (MT) (as shown by Western blot analysis and RT-PCR studies). The protein level of MRP decreased after Cd exposure in WI38 cells, but was sustained at high levels in RWI38 cells, leading led to enhanced calcein efflux. Cd induced Akt phosphorylation in RWI38 but not WI38 cells; this was prevented by probenecid or siRNA for MRP, both of which led to enhanced cell death, as demonstrated by capsase-3 activation and decreased cell viability. These results suggest a functional role for MRP in the regulation of the Akt pathway as well in the efflux pumping of drugs, thereby contributing toward the adaptation of cells to Cd toxicity. The findings of this study could be potentially beneficial in the design of therapeutic targets for Cd-induced tumor progression.
Archives of Pharmacal Research 07/2009; 32(6):883-91. · 1.59 Impact Factor
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ABSTRACT: Although capsaicin, a pungent component of red pepper, is known to induce apoptosis in several types of cancer cells, the mechanisms underlying capsaicin-induced cytotoxicity are unclear. Here, we showed that dihydrocapsaicin (DHC), an analog of capsaicin, is a potential inducer of autophagy. DHC was more cytotoxic than capsaicin in HCT116, MCF-7 and WI38 cell lines. Capsaicin and DHC did not affect the sub-G(1) apoptotic peak, but induced G(0)/G(1) arrest in HCT116 and MCF-7 cells. DHC caused the artificial autophagosome marker GFP-LC3 to redistribute and upregulated expression of autophagy-related proteins. Blocking of autophagy by 3-methyladenine (3MA) as well as siRNA Atg5 induced a high level of caspase-3 activation. Although pretreatment with zVAD completely inhibited caspase-3 activation by 3MA, it did not prevent cell death. DHC-induced autophagy was enhanced by zVAD pretreatment, as shown by increased accumulation of LC3-II protein. DHC attenuated basal ROS levels through catalase induction; this effect was enhanced by antioxidants, which increased both LC3-II expression and caspase-3 activation. The catalase inhibitor 3-amino-1,2,4-triazole (3AT) abrogated DHC-induced expression of LC3-II, overexpression of the catalase gene increased expression of LC3-II protein, and knockdown decreased it. Additionally, DHC-induced autophagy was independent of p53 status. Collectively, DHC activates autophagy in a p53-independent manner and that may contribute to cytotoxicity of DHC.
Autophagy 12/2008; 4(8):1009-19. · 7.45 Impact Factor
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ABSTRACT: Although reactive oxygen species (ROS) have been implicated in cadmium (Cd)-induced hepatotoxicity, the role of ROS in this pathway remains unclear. Therefore, we attempted to determine the molecular mechanisms relevant to Cd-induced cell death in HepG2 cells. Cd was found to induce apoptosis in the HepG2 cells in a time- and dose-dependent fashion, as confirmed by DNA fragmentation analysis and TUNEL staining. In the early stages, both rapid and transient ROS generation triggered apoptosis via Fas activation and subsequent caspase-8-dependent Bid cleavage, as well as by calpain-mediated mitochondrial Bax cleavage. The timing of Bid activation was coincided with the timing at which the mitochondrial transmembrane potential (MMP) collapsed as well as the cytochrome c (Cyt c) released into the cytosol. Furthermore, mitochondrial permeability transition (MPT) pore inhibitors, such as cyclosporin A (CsA) and bongkrekic acid (BA), did not block Cd-induced ROS generation, MMP collapse and Cyt c release. N-acetylcysteine (NAC) pretreatment resulted in the complete inhibition of the Cd-induced apoptosis via catalase upregulation and subsequent Fas downregulation. NAC treatment also completely blocked the Cd-induced intracellular ROS generation, MMP collapse and Cyt c release, indicating that Cd-induced mitochondrial dysfunction may be regulated indirectly by ROS-mediated signaling pathway. Taken together, a rapid and transient ROS generation by Cd triggers apoptosis via caspase-dependent pathway and subsequent mitochondrial pathway. NAC inhibits Cd-induced apoptosis through the blocking of ROS generation as well as the catalase upregulation.
Toxicology and Applied Pharmacology 06/2006; 212(3):212-23. · 4.45 Impact Factor
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ABSTRACT: The protective effects of betulin (BT) against cadmium (Cd)-induced cytotoxicity have been previously reported. However, the mechanisms responsible for these protective effects are unclear. Therefore, this study investigated the mechanisms responsible for the protection of BT against Cd-induced cytotoxicity in human hepatoma cell lines. The protection of BT against Cd cytotoxicity was more effective in the HepG2 than in the Hep3B cells. The protection of BT on Cd-induced cytotoxicity in the HepG2 cells appeared to be related to the inhibition of apoptosis, as determined by PI staining and DNA fragmentation analysis. The anti-apoptosis exerted by BT involved the blocking of Cd-induced reactive oxygen species (ROS) generation, the abrogation of the Cd-induced Fas upregulation, the blocking of caspase-8-dependent Bid activation, and subsequent inhibition of mitochondrial pathway. The BT pretreatment did not affect the p21 and p53 expression levels, when compared with those of the treated cells with Cd alone. BT induced the transient S phase arrest at an early stage and the G0/G1 arrest at a relatively late stage, but it did not observe the sub-G1 apoptotic peak. In the Hep3B cells, Cd did not induce ROS generation. The BT pretreatment partially inhibited the Cd-induced apoptosis, which was related with the incomplete blockage in caspase-9 or -3 activation, as well as in Bax activation. Taken together, it was found that Cd can induce apoptosis via the Fas-dependent and -independent apoptosis pathways. However, the observed protective effects of BT were clearly more sensitive to Fas-expressing HepG2 cells than to Fas-deficient Hep3B cells.
Toxicology 04/2006; 220(1):1-12. · 3.68 Impact Factor
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ABSTRACT: CD24 is a small, heavily glycosylated glycosylphosphatidylinositol-linked cell surface protein, which is expressed in hematologic malignancies and in a large variety of solid tumors. It appears to function as a ligand of P-selectin, an adhesion molecule present in activated platelets and endothelial cells. The authors aimed at evaluating CD24 protein expression in adenoma and adenocarcinoma of the stomach, colon, gallbladder, ovary, and breast to establish a correlation with clinicopathologic data. Staining was evaluated using four degrees of positivity (negative, weakly, moderately, strongly positive), and the staining patterns (membranous vs. intracytoplasmic) were analyzed for statistical analysis. The present study clearly demonstrates that CD24 is abundantly expressed in adenocarcinoma compared to adenoma of the colon and breast. Moreover, the positivity degree of CD24 expression increases with positive nodal status in advanced gastric carcinoma. Intracytoplasmic CD24 expression was found to be highly associated with adenocarcinoma of the colon, gallbladder, and ovary compared to the adenoma group of those organs, and with the positive nodal status compared to the negative nodal status of the colonic adenocarcinoma. We conclude that the degree of positivity and the staining pattern of CD24 constitute an important molecular marker for various epithelial neoplasms, which could help to define malignant transformation and to predict lymph node metastasis.
Pathology - Research and Practice 02/2005; 201(7):479-86. · 1.21 Impact Factor
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ABSTRACT: Previous reports have demonstrated that cadmium (Cd) may induce cell death via apoptosis, but the mechanism responsible for cellular death is not clear. In this study, we investigated the signaling pathways implicated in Cd-induced apoptosis in lung epithelial fibroblast (WI 38) cells. Apoptotic features were observed using terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, propidium iodide staining and DNA laddering. A treatment of cadmium caused the caspase-8-dependent Bid cleavage, the release of cytochrome c (Cyt c), activation of caspase-9 and -3, and PARP cleavage. A caspase-8 specific inhibitor prevented the Bid cleavage, caspase-3 activation and cell death. Alternatively, we observed that full-length Bax was cleaved into 18-kDa fragment (p18/Bax); this was initiated after 12 h and by 36 h the full-length Bax protein was totally cleaved to the p18/Bax, which caused a drastic release of Cyt c from mitochondria. The p18/Bax was detected exclusively in the mitochondrial fraction, and it originated from mitochondrial full-length Bax, but not from the cytosol full-length Bax. Cd also induced the activation of the mitochondrial 30-kDa small subunit of calpain that was preceded by Bax cleavage. Cd induced the upregulation of Bcl-2 and the degradation of p53 protein. N-acetyl cysteine effectively inhibited the Cd-induced DeltaPsim reduction, indicating ROS acts upstream of mitochondrial membrane depolarization. Taken together, our results suggest that Cd-induced apoptosis was thought to be mediated at least two pathways; caspase-dependent Bid cleavage, and the other is calpain-mediated mitochondrial Bax cleavage. Moreover, we found that the function of Bid and Bax was not dependent of Bcl-2, and that ROS can also contribute in the Cd-induced cell death.
Biochemical Pharmacology 12/2004; 68(9):1845-55. · 4.70 Impact Factor
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ABSTRACT: Carbofuran is an anti-acetylcholinesterase insecticide regarded as a relatively safe chemical based on extensive toxicological data. However, the N-nitroso metabolite of carbofuran has been reported to be genotoxic. We previously observed that N-nitrosocarbofuran (NOCF) induces apoptosis and cell cycle arrest in Chinese hamster lung (CHL) fibroblasts. To extend our initial observations, we investigated the molecular mechanism of NOCF-induced apoptosis. Treatment of cells with NOCF caused dose-dependent upregulation of cytosolic factors, such as Bax and Bid, and release of cytochrome c, which were accompanied by activation of caspase-9. We also observed activation of caspase-8 and caspase-3, and subsequent cleavage of poly(ADP-ribose) polymerase. A broad-spectrum caspase inhibitor and a caspase-8-specific inhibitor completely blocked caspase-3 activation and cell death induced by NOCF. These results suggest that the mitochondrial pathway is primarily involved in the NOCF-induced apoptosis of CHL cells.
Toxicology 10/2004; 201(1-3):51-8. · 3.68 Impact Factor
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ABSTRACT: Cadmium is an environmentally widely dispersed and highly toxic heavy metal that has been classified as a human carcinogen. Using the suppression subtractive hybridization technique, we identified previously 29 cadmium-inducible genes, primarily involved in inflammation, cell survival and apoptosis. Among these genes, we are particularly interested in Nor-1, because this gene belongs to the Nur77 family, which plays a key role in the apoptotic processes of a variety of cells and tissues, including the lung. In the present study, we characterized the induction of the Nur77 family genes in the lungs after cadmium exposure. Nur77, Nor-1 and Nurr1 were all induced after cadmium treatment in a dose- and time-dependent manner in WI-38 and A549 lung cell lines. Treatment with inhibitors of signaling pathways, such as PD98059 and H89, almost completely blocked the expression of Nur77, indicating that the extracellular signal-regulated kinase and protein kinase A signaling pathways are important in cadmium-induced Nur77 expression. When a plasmid encoding dominant-negative Nur77 was transfected into A549 cells, cadmium-induced apoptotic changes, such as chromosomal condensation and Bax expression, were significantly reduced, suggesting that the expression of Nur77 plays an important role in cadmium-induced apoptosis. Furthermore, the number of apoptotic cells and the expression of Nur77 was increased in lung tissues collected from cadmium-treated (30 micromol/kg body wt) Wistar rats. Taken together, these results demonstrate that cadmium induces the expression of Nur77 family genes, leading to apoptosis in lung cells, which may cause pulmonary toxicity in response to cadmium exposure.
Carcinogenesis 09/2004; 25(8):1467-75. · 5.70 Impact Factor
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ABSTRACT: This research team found in previous studies, that the ginseng saponin metabolite IH901 induces apoptosis in HepG2 cells via a mitochondrial-mediated pathway, which resulted in the activation of caspase-9 and subsequently of caspase-3 and -8. Based on these results, the involvement of the Fas/Fas ligand (FasL) death-receptor pathway, in IH901-induced apoptosis in HepG2 cells, was investigated. Levels of Fas and the Fas ligand (FasL) mRNA or protein were not increased by IH901, rather they were decreased significantly at 18 h post treatment. Soluble FasL (sFasL) was detectable by immunoprecipitation analysis in the medium of HepG2 cells treated with IH901. Increased levels of sFasL were inversely correlated with the levels of FasL. Preincubation of HepG2 cells with antagonistic anti-Fas antibody showed little protective effect, if any, on IH901-induced cell death. At a 30 microM (24 and 48 h) and 40 microM (24 h) concentration of IH901, the cytotoxic effect of IH901 was less then 50%, anti-Fas antibody prevented IH901-induced cell death. However, at a 60 microM (24 and 48 h) and 40 microM (48 h) concentration of IH901, cell death rates were about 80% or more and most of the chemopreventive and chemotherapeutic effects of IH901 were manifested. Blocking the Fas receptor did not influence IH901-induced cell death. These results indicate that the Fas/FasL system is engaged, but not required for IH901-induced cell death, at pharmacologically significant concentrations.
Archives of Pharmacal Research 05/2004; 27(4):402-6. · 1.59 Impact Factor
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ABSTRACT: 20-O-(beta-D-glucopyranosyl)-20(S)-protopanaxadiol (IH901), an intestinal bacterial metabolite of ginseng saponin formed from ginsenosides Rb1, Rb2, and Rc, is suggested to be a potential chemopreventive agent. Here, we show that IH901 induces apoptosis in human hepatoblastoma HepG2 cells. IH901 led to an early activation of procaspase-3 (12 h posttreatment), and the activation of caspase-8 became evident only later (18 h posttreatment). Caspase activation was a necessary requirement for apoptosis because caspase inhibitors significantly inhibited cell death by IH901. Treatment of HepG2 cells with IH901 also induced the cleavage of cytosolic factors such as Bid and Bax and translocation of truncated Bid (tBid) to mitochondria. A time-dependent release of cytochrome c from mitochondria was observed, which was accompanied by activation of caspase-9. A broad-spectrum caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), and a specific inhibitor for caspase-8, N-benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk), abrogated Bid processing and translocation, and caspase-3 activation. Cytochrome c release was inhibited by zVAD-fmk, however, the inhibition by zIETD-fmk was not complete. The activation of caspase-8 was inhibited not only by zIETD-fmk but also by zVAD-fmk. The results, together with the kinetic change of caspase activation, indicate that activation of caspase-8 occurred downstream of caspase-3 and -9. Our data suggest that the activation of caspase-8 after early caspase-3 activation might act as an amplification loop necessary for successful apoptosis. Primary hepatocytes isolated from normal Sprague-Dawley rats were not affected by IH901 (0-60 microM). The very low toxicity in normal hepatocytes and high activity in hepatoblastoma HepG2 cells suggest that IH901 is a promising experimental cancer chemopreventive agent.
Toxicology and Applied Pharmacology 03/2004; 194(3):221-9. · 4.45 Impact Factor
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ABSTRACT: 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.
Biochemical Pharmacology 10/2003; 66(5):725-31. · 4.70 Impact Factor
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ABSTRACT: The enzyme complex 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4-isomerase (3beta-HSD) is involved in the biosynthesis of all classes of active steroids. The expression of 3b-HSD in human uterine endometrium during the menstrual cycle and decidua was examined in an effort to understand its role during ova implantation. 3beta-HSD was weakly expressed in the glandular epithelium of the proliferative phase and moderately expressed in the glandular epithelium of secretory phase of the endometrium. In the decidua of the ectopic pregnancy, 3beta-HSD was strongly expressed. The human uterine endometrial 3beta-HSD was identified as being the same type as the placental 3beta-HSD by RT-PCR and sequence analysis. In addition to the expression of 3beta-HSD, P450scc was expressed in the decidua of the ectopic pregnancy. These results suggest that pregnenolone might be synthesized from cholesterol by P450scc de novo and then, it is converted to progesterone by 3beta-HSD in the uterine endometrium. The data implies that the endometrial 3beta-HSD can use not only the out-coming pregnenolone from the adrenal gland but also the self- made pregnenolone to produce progesterone. The de novo synthesis of progesterone in the endometrium might be a crucial factor for implantation and maintenance of pregnancy.
Experimental and Molecular Medicine 07/2003; 35(3):160-6. · 2.48 Impact Factor
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ABSTRACT: Cholestatic liver injury results from the accumulation of toxic bile salts within the liver. The aim of the present study was to examine the temporal changes in expression and immunolocalization of protein associated with apoptosis in cholestatic rat liver. Rats were anesthetized and cholestasis was induced by double ligation of the common bile duct and sectioning between the ligatures. The animals were euthanized at day 3 and at weeks 1, 2, 4, and 6 after bile duct ligation (BDL). Apoptotic cell death was increased fivefold after 3 days of BDL, decreased over 2 weeks, and remained constant thereafter as has been demonstrated by TUNEL staining. Western blot analysis for Bax, Bcl-2, cytochrome c, and p53 were performed. Results show that total cellular Bax protein was increased 3 days after BDL and decreased over time thereafter. We observed the translocation of Bax to mitochondria and subsequent release of cytochrome c. According to our immunohistochemical data, nuclear p53 increased 3 days after BDL, but cytoplasmic sequestration of p53 was observed after 1 week. The expression of c-Myc was inhibited by 3 days, but increased at later stages following BDL. Bcl-2 was increased over time in BDL rats. Our data suggest toxic bile salts-induced hepatocellular apoptosis is related to differential expression of Bcl-2 family member protein and release of cytochrome c. Cellular localization of p53 plays an important role in apoptotic death of hepatocytes in BDL rats.
Archive für Toxikologie 03/2003; 77(2):110-5. · 4.67 Impact Factor
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ABSTRACT: Tetrandrine is a bisbenzylisoquinoline alkaloid derived from the root of Stephania tetrandra S. Moore, which has been reported to elicit in vitro cytotoxic effects on HeLa cells, and in vivo suppressive effects on mouse ascites tumors. In the present study, we examined the antiproliferative and apoptosis-inducing activity of tetrandrine in HepG2 cells, a human hepatoma cell line. Tetrandrine showed potent cytotoxic activity in HepG2 cells (IC(50)=9.0+/-1.0 micro M) following incubation for 48 h. Dose-dependent induction of apoptosis was observed by agarose gel electrophoresis and flow cytometric analysis. Treatment of HepG2 cells with tetrandrine resulted in the activation of caspase-3 protease, and subsequent proteolytic cleavage of poly(ADP-ribose) polymerase. These results suggest that tetrandrine is potentially useful as a chemotherapeutic/chemopreventive agent in hepatocellular carcinoma.
Journal of Ethnopharmacology 08/2002; 81(2):225-9. · 3.01 Impact Factor
