Oridonin induces apoptosis of HeLa cells via altering expression of Bcl-2 ／ Bax and activating caspase-3 ／ ICAD pathway

INTRODUCTIONHerbal medicine, Donglingcao (rabdosiarubescens), has been traditionally used in China for thetreatment of various diseases such as leukemia.Oridonin (Fig 1) is a diterpenoid compound isolated fromRabdosia rubescens (hemsl). It has various pharmaco-logical and physiological effects such as anti-inflammation, anti-bacteria, anti-tumor[1-3] and has beenused for the treatment of human cancers, especiallyFig 1. Chemical structure of oridonin.esophageal carcinoma[4]. This comp…     

Involvement of mitochondria and caspase pathways in N-demethyl-clarithromycin-induced apoptosis in human cervical cancer HeLa cell

AIM: To study the mechanisms by which N-demethyl-clarithromycin (NDC) induces human cervical cancer HeLa cell apoptosis in vitro. METHODS: The viability of N-demethyl-clarithromycin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Measurement of mitochondrial transmembrane potential was analyzed by a FACScan flowcytometer. Caspase-3, poly-(ADP-ribose) polymerase (PARP), caspase-activated DNase (ICAD), Bcl-2, Bax, p53, and SIRT1 protein expression and the release of cytochrome c were detected by Western blot analysis. RESULTS: N-demethyl-clarithromycin, an anti-inflammatory substance, inhibited HeLa cell growth in a dose- and time-dependent manner. N-demethyl-clarithro-mycin induced HeLa cell death through the apoptotic pathways. The pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk) and the caspase-9 inhibitor (z-LEHD-fmk) partially enhanced cell viability induced by N-demethyl-clarithromycin, but the caspase-8 inhibitor (z-IETD-fmk) had almost no effect. Caspase-3 was activated then followed by the degradation of caspase-3 substrates, the inhibitor of ICAD and PARP. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c in the cytosol was increased. N-demethyl-clarithromycin upregulated the expression ratio of mitochondrial Bax/Bcl-2, and significantly increased the expression of the p53 protein. It also downregulated anti-apoptotic protein SIRT1 expression. CONCLUSION: N-demethyl-clarithromycin induced apoptosis in HeLa cells via the mitochondrial pathway.     

Oridonin induces a caspase-independent but mitochondria- and MAPK-dependent cell death in the murine fibrosarcoma cell line L929

Oridonin, an active component isolated from Rabdosia rubescences, has been reported to exhibit antitumor effects, but little is known about its molecular mechanisms of action. In this study, the growth-inhibitory activity of oridonin for L929 cells is in time- and dose-dependent manner. After treatment with various concentrations of oridonin for 12 h, the majority of L929 cells underwent apoptosis as measured by an LDH activity-based assay. Although apoptotic bodies were observed in oridonin-treated L929 cells, DNA fragmentation as a hallmark of apoptosis was not found. The pan-caspase inhibitor, z-VAD, and caspase-3 inhibitor, z-DEVD, sensitized L929 cells to oridonin, however, a PARP inhibitor (DPQ) effectively blocked oridonin-induced cell death. After 12 h treatment, PARP proenzyme was significantly cleaved. This result indicated that oridonin-induced L929 cell death required PARP degradation in a caspase-independent manner. In addition, an MEK/ERK inhibitor (PD98059) markedly blocked oridonin-induced cell death, whereas a p38 inhibitor (SB203580) and JNK inhibitor (SP600125) weakly protected the cells against death. Treatment with 41.2 microM oridonin for 12 h induced significant and persistent ERK activation and p38 inactivation in L929 cells without evident changes in the protein levels. The responsiveness of ERK and p38 to oridonin suggests the involvement of these kinases in this apoptotic process. Moreover, oridonin increased the ratio of Bax/Bcl-2 protein expression, whereas it had no effect on the expression of Bcl-xL. These results indicate that regulation of the Bcl-2 and MAPK families maybe the effector mechanisms of oridonin-induced L929 cell death, independent of the caspase pathway.     

Bcl-2 up-regulation and P-p53 down-regulation account for the low sensitivity of murine L929 fibrosarcoma cells to oridonin-induced apoptosis

Drug resistance has been a major limitation to chemotherapy. There are many mechanisms that contribute to such resistance. In our study, we subcloned oridonin-sensitive and low sensitive L929 cells and both types of cells grew at almost the same growth rate. The acquired low sensitivity to oridonin-induced apoptosis was associated with Bcl-2 up-regulation and down-regulation of p53 phosphorylation. The p38 inhibitor SB203580 decreased Bcl-2 expression in the low sensitive L929 cells and made the cells more sensitive to oridonin. Moreover, a higher dose of oridonin promoted p53 phosphorylation, increased Bax expression and subsequently induced death of low sensitive L929 cells, however, it had no effect on Bcl-2 expression. The increased Bcl-2/Bax ratio in oridonin low sensitive L929 cells did not inhibit caspase-9 or -3 activation, but suppressed the cleavage of poly (ADP-ribose) polymerase (PARP), indicating the existence of caspase-9 or -3 independent PARP activation. These results indicated that in L929 cells, there was a relationship among the low sensitivity to oridonin, down-regulation of p53 phosphorylation and Bcl-2 up-regulation.     

冬凌草甲素通过改变Bax/Bcl-xL表达激活caspase-3诱导A375-S2细胞凋亡

目的　研究冬凌草甲素诱导人黑色素瘤A375 S2细胞凋亡的作用原理。方法　形态学观察 ,DNA凝胶电泳法及WesternBlot法。结果　冬凌草甲素能明显诱导A375 S2细胞发生凋亡 ,其作用呈明显的量效关系和时间依赖性。形态学观察可见凋亡小体的形成 ,琼脂糖凝胶电泳可见凋亡典型的DNA梯带 ;caspase 3的抑制剂能阻止caspase 3的活力升高 ;免疫印迹结果显示冬凌草甲素作用A375 S2细胞 12h改变Bax与Bcl xL蛋白的表达 ;且发现caspase 3的底物PARP蛋白在 12h时被降解。结论　冬凌草甲素 (34 3μmol·L-1)诱导A375 S2细胞凋亡 ,这种作用是通过改变了Bax/Bcl xL的表达比率 ,激活caspase 3而实现的。     

4-hydroxynonenal induces apoptosis via caspase-3 activation and cytochrome c release

We investigated the mechanism by which 4-hydroxynonenal (HNE), a major aldehydic product of lipid peroxidation, induces apoptosis in tumor cells. Treatment of human colorectal carcinoma (RKO) cells with HNE-induced poly-ADP-ribose-polymerase (PARP) cleavage and DNA fragmentation in a dose- and time-dependent manner. The induction of PARP cleavage and DNA fragmentation paralleled caspase-2, -3, -8, and -9 activation. Pretreatment of cells with an inhibitor of caspase-3, z-DEVD-fmk, or a broad spectrum caspase inhibitor, z-VAD-fmk, abolished caspase activation and subsequent PARP cleavage. Constitutive expression of high levels of Bcl-2 protected cells from HNE-mediated apoptosis. In addition, Bcl-2 overexpression inhibited cytochrome c release from mitochondria and subsequent caspase-2, -3, and -9 activation. These findings demonstrate that HNE triggers apoptotic cell death through a mitochondrion-dependent pathway involving cytochrome c release and caspase activation. Bcl-2 overexpression protected cells from HNE-induced apoptosis through inhibition of cytochrome c release.     

Melatonin Induces Apoptotic Cell Death via p53 in LNCaP Cells

In this study, we examined whether melatonin promotes apoptotic cell death via p53 in prostate LNCaP cells. Melatonin treatment significantly curtailed the growth of LNCaP cells in a dose- and time-dependent manner. Melatonin treatment (0 to 3 mM) induced the fragmentation of poly(ADP-ribose) polymerase (PARP) and activation of caspase-3, caspase-8, and caspase-9. Moreover, melatonin markedly activated Bax expression and decreased Bcl-2 expression in dose increments. To investigate p53 and p21 expression, LNCaP cells were treated with 0 to 3 mM melatonin. Melatonin increased the expressions of p53, p21, and p27. Treatment with mitogen-activated protein kinase (MAPK) inhibitors, PD98059 (ERK inhibitor), SP600125 (JNK inhibitor) and SB202190 (p38 inhibitor), confirmed that the melatonin-induced apoptosis was p21-dependent, but ERK-independent. With the co-treatment of PD98059 and melatonin, the expression of p-p53, p21, and MDM2 did not decrease. These effects were opposite to the expression of p-p53, p21, and MDM2 observed with SP600125 and SB202190 treatments. Together, these results suggest that p53-dependent induction of JNK/p38 MAPK directly participates in apoptosis induced by melatonin.     

Organic solvent-induced proximal tubular cell apoptosis via caspase-9 activation

Long-term exposure to solvents is associated with apoptosis, which is implicated in the development and progression of tubulo-interstitial fibrosis and chronic renal failure. In our previous study, we demonstrated that toluene and p-xylene as the most commonly used organic solvents induced proximal tubular cells apoptosis. This study was conducted to assess the apoptotic pathway of toluene and p-xylene induced proximal tubular apoptosis. This was assessed by measuring the caspase-9 activity LLC-PK1 cells exposed to both compounds. A model of proximal tubular cell (LLC-PK1) cytotoxicity exposed to 1 mM of either p-xylene or toluene was compared to untreated control for caspase-9 activity and Bax/Bcl-2 protein level. Furthermore, DNA fragmentation in the presence of caspase-9 inhibitor (Z-LEHD-FMK) in a dose-dependent manner was assessed. Both compounds induced caspase-9 activity, which was accompanied by up-regulation of Bax, whereas Bcl-2 level did not change. DNA fragmentation induced by both solvents was inhibited by caspase-9 inhibitor in dose-dependent manner. This data suggest that p-xylene or toluene induces nephrotoxicity via mitochondrial caspase-9 pathway. This mechanism involves up-regulation of the apoptotic protein, Bax.     

Ginsenoside Rh2 induces apoptosisvia activation of caspase-1 and -3 and up-regulation of bax in human neuroblastoma

In human neuroblastoma SK-N-BE(2) cells undergoing apoptotic death induced by ginsenoside Rh2, a dammarane glycoside that was isolated from Panax ginseng C. A. Meyer, caspase-1 and caspase-3 were activated. The expression of Bax was increased in the cells treated with ginsenoside Rh2, whereas Bcl-2 expression was not altered. Treatment with caspase-1 inhibitor, Ac-YVAD-CMK, or caspase-3 inhibitor, Z-DEVD-FMK, partially inhibited ginsenoside Rh2-induced cell death but almost suppressed the cleavage of the 116 kDa PARP into a 85 kDa fragment. When the levels of p53 were examined in this process, p53 accumulated rapidly in the cells treated early with ginsenoside Rh2. These results suggest that activation of caspase-1 and -3 and the up-regulation of Bax are required in order for apoptotic death of SK-N-BE(2) cells to be induced by ginsenoside Rh2, and p53 plays an important role in the pathways to promote apoptosis.     

Interferon-alpha enhances sensitivity of human osteosarcoma U2OS cells to doxorubicin by p53-dependent apoptosis

Aim： To determine whether interferon-or （IFNα） can enhance doxorubicin sensitivity in osteosarcoma cells and its molecular mechanism. Methods： Cell viability was evaluated using 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide assay. Apoptosis was studied using Flow cytometry analysis, Hoechst33258 staining, DNA fragmentation assay, as well as the activation of caspase-3 and poly （ADP-ribose） polymerase. Protein expression was detected by Western blotting. The dependence of p53 was determined using p53-siRNA transfection. Results： IFNα increased doxorubicin-induced cytotoxicity to a much greater degree through apoptosis in human osteosarcoma p53-wild U2OS cells, but not p53-mutant MG63 cells. IFNα markedly upregulated p53, Bax, Mdm2, and p21, downregulated Bcl-2, and activated caspase-3 and PARP cleavage in response to doxorubicin in U2OS cells. Moreover, the siRNA-mediated silencing of p53 significantly reduced the IFNα/doxorubicin combination-induced cytotoxicity and PARP cleavage. Conclusion： IFNα enhances the sensitivity of human osteosarcoma U2OS cells to doxorubicin by p53-dependent apoptosis. The proper combination with IFNα and conventional chemotherapeutic agents may be a rational strategy for improving the treatment of osteosarcoma with functional p53.     

Cadmium induces apoptotic cell death in WI 38 cells via caspase-dependent Bid cleavage and calpain-mediated mitochondrial Bax cleavage by Bcl-2-independent pathway

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.     

Humic acid induces G1 phase arrest and apoptosis in cultured vascular smooth muscle cells

Humic acid (HA) in well water used by the inhabitants for drinking is one of the possible etiological factors for Blackfoot disease (BFD). In this study, the ability of HA to inhibit cell cycle progression and induce apoptosis in cultured smooth muscle cells (SMCs; A7r5) was investigated. Treatment of the SMCs at various HA concentrations (25-200 microg/mL) resulted in sequences of events marked by apoptosis, as shown by loss of cell viability, morphology change, and internucleosomal DNA fragmentation. HA-induced apoptotic cell death that is associated with loss of mitochondrial membrane potential (Delta Psi m), cytochrome c translocation, caspase-3, -8, and -9 activation, poly ADP-ribose polymerase (PARP) degradation, dysregulation of Bcl-2 and Bax, and upregulation of p53 and phospholyrated p53 (p-p53) in SMCs. Flow cytometry analysis demonstrated that HA blocked cell cycle progress in the G1 phase in SMCs. This blockade of cell cycle was associated with reduced amounts of cyclin D1, CDK4, cyclin E, CDK2, and hyperphosphorylated retinoblastoma protein (pRb) in a time-dependent manner. Apparent DNA strand breaks (DNA damage) were also detected in a dose-dependent manner using Single-cell gel electrophoresis assay (comet assay). Furthermore, HA induced dose-dependent elevation of reactive oxygen species (ROS) level in SMCs, and antioxidant vitamin C and Trolox effectively suppressed HA-induced DNA damage and dysregulation of Bcl-2/Bax. Our findings suggest that HA-induced DNA damage, cell cycle arrest, and apoptosis in SMCs may be an underlying mechanisms for the atherosclerosis and thrombosis observed in the BFD endemic region.     

Induction of apoptosis and expression of apoptosis related genes in human epithelial carcinoma cells by Helicobacter pylori VacA toxin.

Virulence factors produced by Helicobacter pylori have been known to be associated with serious gastroduodenal diseases. The aims of this study were to clarify the apoptosis-inducing properties of vacuolating cytotoxin (VacA) and examine the expression of apoptosis related proteins in human epithelial carcinoma cells expressing (AGS) or lacking (Kato III) p53. The midregion VacA homolog from H. pylori strain Q35 (Korean isolate) was cloned, expressed and sequenced. Recombinant VacA (VacA(418-799)) inhibited cell growth and induced apoptosis in gastric epithelial cells. Treatment with VacA(418-799) resulted in morphological changes and DNA fragmentation. Cell cycle analysis revealed subdiploid cells suggesting apoptosis, which was confirmed by the activation of caspase-3 and cleavage of PARP. VacA(418-799) also mediated a prolongation of the cell cycle progression in G1 phase. Furthermore, VacA(418-799) increased the expression of p53, p21(waf1/cip1) and Bax in AGS cells, but not in Kato III cells and did not affect the phosphorylation of Rb in both cell lines. These results indicate that recombinant VacA of H. pylori induces apoptosis in both Kato III and AGS cells, regardless of p53 status and suggest that VacA(418-799) mediate the development of gastric diseases through cell cycle arrest in the G1 phase. VacA(418-799) induction of apoptosis is associated with up-regulation of p53, p21(waf1/cip1), Bax in AGS cells and activation of caspase-3 in both cell lines.     

Olaquindox induces apoptosis through the mitochondrial pathway in HepG2 cells

Olaquindox is used in China as feed additive for growth promotion in pigs. Recently, we have demonstrated that olaquindox induced genome DNA damage and oxidative stress in HepG2 cells. The aim of this study was to explore the molecular mechanism of cell cycle arrest and apoptosis induced by olaquindox in HepG2 cells. In the present study olaquindox induced cell cycle arrest to the S phase and dose-dependent apoptotic cell death in HepG2 cells, indicated by accumulation of sub-G1 cell population, nuclear condenstion, DNA fragmentation, caspases activation and PARP cleavage. Meanwhile, the data showed that olaquindox triggered ROS-mediated apoptosis in HepG2 cells correlated with both the mitochondrial DNA damage and nuclear DNA damage, collapse of Δψ_m, opening of mPTP, down-regulation of Bcl-2 and up-regulation of Bax. Furthermore, we also found that olaquindox increased the expression of p53 protein and induced the release of cytochrome C from mitochondria to cytosol. In conclusion, olaquindox induced apoptosis of HepG2 cells through a caspase-9 and -3 dependent mitochondrial pathway, involving p53, Bcl-2 family protein expression, Δψ_m disruption and mPTP opening.     

Magnolol induces apoptosis via activation of both mitochondrial and death receptor pathways in A375-S2 cells

Magnolol inhibited proliferation of human malignant melanoma A375-S2 cells. The drug induced oligonucleosomal fragmentation of DNA in A375-S2 cells and increased caspase-3, 8, 9 activities followed by the degradation of caspase-3 substrates, inhibitor of caspase dependent DNase (ICAD) and poly-(ADP-ribose) polymerase (PARP). Pan-caspase inhibitor (z-VADfmk), caspase-3 inhibitor (z-DEVD-fmk), capase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD-fmk) and caspase-10 inhibitor (z-AEVD-fmk) inhibited magnolol-induced A375-S2 cell apoptosis. The level of anti-apoptotic mitochondrial protein Bcl-2 was up-regulated while the level of pro-apoptotic protein Bax was down-regulated. Taken together, our results indicate that magnolol induces apoptosis by activation of both mitochondrial and death receptor pathways in A375-S2 cells.     

The heterocyclic amine, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole induces apoptosis in cocultures of rat parenchymal and nonparenchymal liver cells.

In this study, we investigated the mechanism of apoptosis by 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole (Trp-P-1) in cocultures of parenchymal and nonparenchymal liver cells, since the liver consists of various cell types and they cooperatively respond to chemicals. It was found that cocultures were more susceptible to cell death by Trp-P-1 than culture of each cell type alone. In cocultures, Trp-P-1 induced DNA fragmentation accompanied by the activation of 18-kDa endonuclease. Trp-P-1 (30 microM) caused a rapid increase in Bid protein level in mitochondria and the leakage of cytochrome c from mitochondria into the cytosol 15 min after treatment. On the other hand, an increase in Bax protein and a decrease in Bcl-2 protein were detected in the mitochondrial fraction 2 h after treatment following the increases in p53 protein level and DNA binding activity of NF-kappa B. Caspase-8 was activated within 30 min followed by the activation of downstream caspases as measured using the corresponding peptide substrates. The activation of caspases was also confirmed by cleavage of caspase-3, poly(ADP-ribose)polymerase, and protein kinase C-delta as analyzed by Western blotting. A peptide inhibitor of caspase-8 diminished DNA ladder formation and the activation of downstream caspases, but a caspase-9 inhibitor and pyrrolidinedithiocarbamate as an inhibitor of NF-kappa B showed only partial inhibition, suggesting that caspase-8 is the apical caspase in the cascade. These results led to the conclusion that Trp-P-1 mainly drives the caspase-8-mediated pathway that involves Bid, accompanied by a delay in the p53/NF-kappa B-mediated side pathway that involves Bax, Bcl-2, and caspase-9.     

p53-mediated cell cycle arrest and apoptosis induced by shikonin via a caspase-9-dependent mechanism in human malignant melanoma A375-S2 cells

Natural products regulate cell growth in response to oncogene activation that induces cell cycle arrest and apoptosis in tumor cell lines. We investigated the mechanisms of caspase activation in human malignant melanoma, A375-S2 cells, by the natural product shikonin, which was isolated from the plant Lithospermum erythrorhizon SIEB. et ZUCC. Shikonin inhibited cell growth in a time- and dose-dependent manner, which might be mediated through up-regulation of p53 and down-regulation of cyclin-dependent protein kinase 4. Caspase activation was detected in shikonin-induced cell apoptosis, which involved in a post-mitochondrial caspase-9-dependent pathway. Decreased Bcl-2 protein levels and increased Bax protein levels were positively correlated with elevated expression of p53 protein. Apoptosis-inducing factor, another apoptotic protein of mitochondria, partially contributed to shikonin-induced release of cytochrome c. Taken together, shikonin-induced DNA damage activates p53 and caspase-9 pathways.     

Lead-induced apoptosis in PC 12 cells: involvement of p53, Bcl-2 family and caspase-3

It has been reported that lead could induce apoptosis in a variety of cell types. Although mitochondrion is regarded as the most pertinent pathway in mediating apoptosis, the exact mechanisms of lead-induced apoptosis are still largely unknown. Furthermore, there is little information about expressions and regulations of Bax, Bcl-2, and p53 in lead-induced apoptosis, which are critical regulators of mitochondrial stability. The present study was undertaken to determine whether lead could induce DNA damage and apoptosis in PC 12 cells, and the involvement of Bax, Bcl-2, p53, and caspase-3 in this process. The results showed that lead could induce DNA damage and apoptosis in PC 12 cells, accompanying with upregulation of Bax and downregulation of Bcl-2. Additionally, the expression of p53 increased, and caspase-3 was activated. Therefore, it suggests that lead can induce activation of p53 by DNA damage, which may lead to imbalance of Bax/Bcl-2 and mitochondrial dysfunction. Subsequently, after activation of caspase-3, lead-induced apoptosis occurres.