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…     

Isoliquiritigenin treatment induces apoptosis by increasing intracellular ROS levels in HeLa cells

This study focuses on the relationship between the apoptosis induced by isoliquiritigenin (ISL) and the production of reactive oxygen species (ROS). Cell viability was evaluated using sulforhodamine B assay. The apoptotic rate was determined via flow cytometry. Intracellular ROS level was assessed using the 2,7-dichlorofluorescein probe assay. Poly-ADP-ribose polymerase (PARP) protein expression was examined using Western blot analysis. The results showed that ISL treatment inhibited cell proliferation by inducing apoptosis. The increased apoptotic rate and ROS production induced by ISL were inhibited by the co-treatment of ISL and free radical scavenger N-acetyl-cysteine (NAC), catalase (CAT), and 4,5-dihydroxyl-1,3-benzededisulfonic acid (Tiron). On the contrary, the increased apoptotic rate and the ROS production were compensated by the co-treatment of ISL and l-buthionine-(S,R)-sulfoximine (BSO). ISL treatment increased the degradation of PARP, which was counteracted by antioxidants (NAC or CAT), whereas the combination treatment of ISL and pro-oxidant (BSO) enhanced the PARP degradation induced by ISL. Our findings suggested that ISL treatment induced apoptosis by increasing intracellular ROS levels in HeLa cells.     

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.     

Cardiotoxin III induces apoptosis in T24 cells via reactive oxygen species‐independent mitochondrial death pathway

Cardiotoxin III (CTX III), a basic polypeptide with 60 amino acid residues isolated from Naja naja atra venom, has been reported to have anticancer activity. CTX III inhibited the growth of T24 cells in a time‐ and dose‐dependent manner with an IC₅₀value of 1.7 µg/mL and displayed several features of apoptosis including apoptotic body formation, increase of sub G₁ population, DNA fragmentation, and poly (ADP‐ribose) polymerase (PARP) cleavage. Using apoptosis analysis, measurement of reactive oxygen species (ROS) and assessment of mitochondrial membrane potentials (ΔΨm), CTX III was found to be a potent inducer of apoptosis, transducing apoptotic signals via a decrease in mitochondrial membrane potential (ΔΨm) and release of cytochrome c from mitochondria into cytosol. However, CTX III did not generate reactive oxygen species (ROS). Taken together, the present data indicate that CTX III induces apoptosis in T24 cells through an ROS‐independent mitochondrial dysfunction pathway and resultant cytochrome c release. This is the first report on the mechanism of the anticancer effect of CTX III on T24 cells. Drug Dev. Res. 63:219–224, 2004. © 2004 Wiley‐Liss, Inc.     

Brevifoliol ester induces apoptosis in prostate cancer cells by activation of caspase pathway

Prostate cancer is fourth most abundant cancer type around the globe. Brevifoliol, a rearranged taxoid from Taxus walllichiana needles has been derivatized as C5 esters using Steglich esterification reaction. Seventeen diverse analogues were evaluated against a panel of human cancer cell lines by MTT assay. Among these, two of the semi‐synthetic analogues, that is, 13 and 16 exhibited potent cytotoxicity, selectively against PC‐3, prostate cancer cell lines. In cell cycle analysis, analogue 13 induced S and G2/M phase arrest and induced apoptosis by activating caspase‐3. Compound 13 showed moderate efficacy in in‐vivo Ehrlich ascites carcinoma in Swiss albino mice. Further, compound 13 was found to be safe in Swiss albino mice up to 1,000 mg/kg dose in acute oral toxicity. Brevifoliol ester 13 may further be optimized for better efficacy.     

杨梅素诱导人宫颈癌HeLa细胞凋亡及凋亡通路的研究

目的研究杨梅素体外对人宫颈癌HeLa细胞增殖、凋亡及凋亡通路的影响。方法体外培养人宫颈癌HeLa细胞,MTT和SRB法检测细胞增殖抑制率;流式细胞仪检测细胞凋亡,计算机分析细胞周期;Western Blot法检测caspase-3、caspase-9和survivin含量。结果杨梅素对HeLa细胞的体外增殖具有抑制作用,并具有浓度和时间依耐性;杨梅素体外诱导HeLa细胞凋亡,并将细胞周期阻滞在S期;杨梅素促进caspase-3和caspase-9蛋白表达,抑制survivin蛋白表达。结论杨梅素可抑制HeLa细胞的增殖,诱导其凋亡,其凋亡途径同时涉及细胞内途径和细胞外途径。     

冬凌草甲素通过诱导人宫颈癌HeLa细胞自噬下调凋亡的机制

研究冬凌草甲素通过诱导人宫颈癌HeLa细胞自噬拮抗凋亡的机制。MTT法测定冬凌草甲素对HeLa细胞的细胞毒作用。通过相差显微镜观察细胞形态学变化，用琼脂糖凝胶电泳检测DNA片段化，用流式细胞仪检测细胞自噬和凋亡水平，用Western blotting检测分析药物对蛋白质表达的影响。冬凌草甲素明显抑制HeLa细胞的增殖，诱导HeLa细胞凋亡，同时诱导HeLa细胞发生自噬。Western blotting检测结果表明，冬凌草甲素作用24 h后，促凋亡蛋白Bax、细胞色素c和控制Bax活力的去乙酰化酶SIRT-1的表达明显改变。冬凌草甲素(64 μmol·L^-1)诱导的自噬通过影响SIRT-1和线粒体途径蛋白的表达下调凋亡。     

Induction of G2/M arrest and apoptosis through mitochondria pathway by a dimer sesquiterpene lactone from Smallanthus sonchifolius in HeLa cells

Dimer sesquiterpene lactones (SLs), uvedafolin and enhydrofolin, against four monomer SLs isolated from yacon, Smallanthus sonchifolius, leaf were the most cytotoxic substances on HeLa cells (IC₅₀ values 2.96–3.17 μM at 24 hours). However, the cytotoxic mechanism of dimer SL has not been elucidated yet. Therefore, in this study, we clarified the in vitro cytotoxic mechanism of uvedafolin on the HeLa cells, and evaluated the cytotoxicity against NIH/3T3 cells which were used as normal cells. In consequence, the dimer SLs had low toxicity for the NIH/3T3 cells (IC₅₀ 4.81–4.98 μM at 24 hours) and then the uvedafolin mediated cell cycle arrest at the G₂/M phase and induced apoptosis on the HeLa cells evidenced by appearance of a subG1 peak. Uvedafolin induced apoptosis was attributed to caspase-9 and caspase-3/7 activities. An effectively induced apoptosis pathway was demonstrated from mitochondria membrane potential change and cytochrome c release to cytosol. These results reveal that uvedafolin induced apoptosis via the mitochondria pathway. The present results indicate the potential of uvedafolin as a leading compound of new anticancer agents.     

厚朴酚通过改变Bax/Bcl-X_L表达和激活caspase诱导人宫颈癌HeLa细胞凋亡

目的研究厚朴酚诱导HeLa细胞凋亡机制。方法MTT法测定厚朴酚对HeLa和人正常胚肺HEL299细胞的细胞毒作用。通过显微镜,荧光染色观察细胞形态学变化,用琼脂糖凝胶电泳检测DNA片断化。用Western印迹法分析药物对蛋白质表达的影响。结果厚朴酚明显抑制HeLa细胞的增殖,诱导HeLa细胞调亡。对HEL299细胞的细胞毒作用弱于HeLa细胞。Caspase3,8,9,10及家族抑制剂可明显抑制厚朴酚诱导的凋亡。激动型Fas抗体CH11对厚朴酚诱导的HeLa细胞死亡有协同作用。Western印迹结果显示厚朴酚作用12h后Bax/BclXL表达比率升高,caspase3前体及其底物ICAD和PARP发生降解,磷酸化p53和p53蛋白表达增加。结论厚朴酚(80μmol·L-1)通过改变Bax/BclXL的表达激活caspase途径诱导HeLa细胞凋亡。     

Je-chun-jun induced apoptosis of human cervical carcinoma HeLa cells

AIM: To study the mechanism of Je-Chun-Jun (JCJ)-inducing the apoptosis of the human cervical carcinoma, HeLa cells. METHODS: The cell viability was assessed using MTT assay. The optical density was measured at 570 nm. The caspase activity was measured using 50 mmol/L of fluorogenic substrate, AC-DEVD-AMC (caspase-3), AC-VEID-AMC (caspase-8) or AC-LEHD-AFC (caspase-9). To confirm the expression of proteins, Western blotting was performed. To detect the characteristic of apoptosis chromatin condensation, HeLa cells were stained with Hoechst 33258 in the presence of JCJ. For the cell cycle analysis, HeLa cells were incubated with Propidium iodide (PI) solution. Fluorescence intensity of cell cycle was measured using flow cytometry system. RESULTS: The loss of viability occurred following the exposure of 10 g/L JCJ. Cells treated with 10 g/L JCJ for 3 d exhibited the apoptotic morphology (brightly blue-fluorescent condensed nuclei by Hoechst 33258-staining) and the reduction of cell volume. Cells incubated with JCJ for 48 h were arrested at the G1 phase of cell cycle and their G1 checkpoint related gene products such as cyclin D1 were transiently decreased. We showed that JCJ induced the p38 MAPK activation in HeLa cells. The p38 MAPK inhibitor, SB203580 protected Hela cells from the JCJ-induced death as well as intervened the JCJ-induced accumulation of cells at the G1 phase. In contrast, MEK1 (-ERK upstream) inhibitor, PD98059 had no effect on HeLa cells. CONCLUSION: JCJ induced cell cycle arrest and apoptosis of HeLa cells through p38 MAPK pathway.     

Julibroside J8-induced HeLa cell apoptosis through caspase pathway

The julibroside J₈ was isolated from the Albizia julibrissin and evaluated for antiproliferatived on six cancer cell lines (BGC-823, Bel-7402, HeLa, PC-3MIE8, MDA-MB-435 and LH-60) in vitro. Julibroside J₈ at 100 μg mL^- 1 (46.08 μmol·L^- 1) significantly inhibited growth in the first three cell lines. In addition, in HeLa cells typical apoptotic changes in morphology were observed, and further, nuclear damage was observed by Giemsa staining and DNA fragmentation was exhibited. Effects of julibroside J₈ on induction of DNA fragmentation, caspase-3 activation and downregulation of ICAD expression were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk. In addition, apoptosis induced with julibroside J₈ was associated with an increase in expression of the apoptosis inducer Bax, and a significant reduction in expression of the apoptosis suppressor Bcl-2 in mitochondria. These results suggest that julibroside J₈ induces HeLa death through caspase pathway.     

Capsaicin induces apoptosis in SCC-4 human tongue cancer cells through mitochondria-dependent and -independent pathways

Although there have been advances in the fields of surgery, radiotherapy, and chemotherapy of tongue cancer, the cure rates are still not substantially satisfactory. Capsaicin (trans‐8‐methyl‐N‐vanillyl‐6‐nonenamide) is the major pungent ingredient of hot chili pepper and has been reported to have an antitumor effect on many human cancer cell types. The molecular mechanisms of the antitumor effect of capsaicin are not yet completely understood. Herein, we investigated whether capsaicin induces apoptosis in human tongue cancer cells. Capsaicin decreased the percentage of viable cells in a dose‐dependent manner in human tongue cancer SCC‐4 cells. In addition, capsaicin produced DNA fragmentation, decreased the DNA contents (sub‐G1 phase), and induced G0/G1 phase arrest in SCC‐4 cells. We demonstrated that capsaicin‐induced apoptosis is associated with an increase in reactive oxygen species and Ca²⁺ generations and a disruption of the mitochondrial transmenbrane potential (ΔΨ_m). Treatment with capsaicin induced a dramatic increase in caspase‐3 and ‐9 activities, as assessed by flow cytometric methods. A possible mechanism of capsaicin‐induced apoptosis is involved in the activation of caspase‐3 (one of the apoptosis‐executing enzyme). Confocal laser microscope examination also showed that capsaicin induced the releases of AIF, ATF‐4, and GADD153 from mitochondria of SCC‐4 cells. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2012.     

冬凌草甲素通过激活caspase诱导HeLa细胞凋亡

目的　研究冬凌草甲素诱导人子宫颈癌HeLa细胞凋亡的作用机制。方法　采用形态学观察、DNA凝胶电泳及LDH法。结果　冬凌草甲素能显著诱导HeLa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家族抑制剂 ,caspase 1和 3抑制剂能抑制冬凌草甲素诱导HeLa细胞的凋亡 ,6 8 7μmol·L-1冬凌草甲素作用HeLa细胞 12h使caspase 3的活力提高 2倍。 结论　适宜浓度以下 ,冬凌草甲素 (6 8 7μmol·L-1)诱导HeLa细胞凋亡具有浓度与时间依赖性 ,大剂量 (137 4 μmol·L-1)时 ,冬凌草甲素诱导HeLa细胞坏死的程度强于凋亡 ,且通过激活caspase途径诱导HeLa细胞凋亡。     

AlCl3 induces lymphocyte apoptosis in rats through the mitochondria–caspase dependent pathway

To investigate apoptosis mechanisms in lymphocytes induced by aluminum trichloride (AlCl₃) through the mitochondria–caspase dependent pathway, the spleen lymphocytes of rats were cultured with RPMI‐1640 medium and exposed to AlCl₃·6H₂O in the final concentrations of 0 (control group, CG), 0.3 (low‐dose group, LG), 0.6 (mid‐dose group, MG), and 1.2 (high‐dose group, HG) mmol·L^?1 for 24 h, respectively. Mitochondrial transmembrane potential (ΔΨm), cytochrome C (Cyt C) protein expression in cytoplasm, Caspase‐3 and Caspase‐9 activity, Bcl‐2, Bax, Caspase‐3 and Caspase‐9 mRNA expressions, DNA ladder and lymphocytes apoptosis index were detected. The results showed that Cyt C protein expression in cytoplasm, Caspase‐3 and Caspase‐9 activity, Bcl‐2, Bax, Caspase‐3 and Caspase‐9 mRNA expressions, the ratio of Bcl‐2 and Bax mRNA expression, lymphocytes apoptosis index increased, while ΔΨm decreased in the AlCl₃‐treated groups compared with those in CG. The results indicate that AlCl₃ induces lymphocyte apoptosis in rats through the mitochondria–caspase dependent pathway. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 385–394, 2016.     

氧化还原调控在紫檀芪诱导HeLa细胞内质网凋亡途径中的作用

目的探讨细胞氧化还原调控在紫檀芪引起HeLa细胞凋亡过程中对内质网途径的作用。方法 SRB法评价紫檀芪对HeLa细胞的细胞毒活性。采用细胞形态学和生物化学方法检测细胞凋亡。通过荧光法对细胞内活性氧/GSH的变化进行分析。通过氧化还原相关抑制剂分析凋亡过程中的决定因素。通过半定量RT-PCR表达分析来确定内质网胁迫在紫檀芪诱导HeLa细胞凋亡中的作用。结果紫檀芪对HeLa细胞的IC50为80μmol·L-1。凋亡是紫檀芪对He-La细胞毒活性的主要表现。随着紫檀芪浓度由5μmol·L-1增加至160μmol·L-1,HeLa细胞氧化还原平衡发生明显变化。紫檀芪在80μmol·L-1～120μmol·L-1可引起内质网胁迫分子表达。抑制细胞内过氧化氢的产生会减轻紫檀芪引起的内质网胁迫并最终抑制了紫檀芪所诱导的细胞凋亡。结论细胞内氧化还原平衡的变化在紫檀芪引起HeLa细胞凋亡的内质网途径方面起了决定性的作用。     

Cardiotoxin III induces apoptosis in K562 cells through a mitochondrial-mediated pathway

1. Cardiotoxin (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 on human leukaemia K562 cells. 2. Cardiotoxin III was found to inhibit the growth of K562 cells in a time- and dose-dependent manner, with an IC(50) value of 1.7 mug/mL, and displayed several features of apoptosis, including apoptotic body formation, an increase in the sub-G(1) population, DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage. 3. Investigation of the mechanism of CTX III-induced apoptosis revealed that treatment of K562 cells with CTX III resulted in the loss of mitochondrial membrane potential, cytochrome c release from mitochondria into the cytosol and activation of caspase-9 and caspase-3 and the subsequent cleavage of the caspase-3 substrate PARP; however, CTX III did not generate reactive oxygen species (ROS). 4. Taken together, the results indicate that CTX III induces apoptosis in K562 cells through an ROS-independent mitochondrial dysfunction pathway.     

Arsenic induces apoptosis by the lysosomal‐mitochondrial pathway in INS‐1 cells

Recently, long term arsenic exposure was considered to be associated with an increased risk of diabetes mellitus. While a relation of cause‐and‐effect between apoptosis of pancreatic β‐cells and arsenic exposure, the precise mechanisms of these events remains unclear. The aim of this study was to explore arsenic‐induced pancreatic β‐cell apoptosis and the mechanisms of through the possible link between lysosomal and the mitochondrial apoptotic pathway. After exposure to 10 μM of arsenic, the reactive oxygen species (ROS) level was significantly increased at 12 h, while the mitochondrial membrane potential was reduced at 24 h and the lysosomal membrane integrity was disrupted at 48 h. A significant increase in protein expression for cytochrome c was also observed using Western blot analysis after exposure to arsenic for 48 h. To further demonstrate that arsenic reduced the lysosomal membrane integrity, cells pretreated with NH₄Cl and exposed to arsenic harbored a lower fluorescence increase than cells that were only exposed to arsenic. In addition, apoptosis was mesured using Hoechst 33342/PI dual staining by microscopy and annexin V‐FITC/propidium iodide dual staining by flow cytometry. The results show an increased uptake of the arsenic dose and the cells changed from dark blue to light blue, karyopyknosis, nuclear chromatin condensation, side set or fracture, and a correlation was found between the number of apoptotic cells and arsenic dose. The result of present study suggest that arsenic may induce pancreatic β‐cell apoptosis through activation of the lysosome‐mitochondrial pathway. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 133–141, 2016.     

Astragalus saponins induce apoptosis in human gastric adenocarcinoma cells via a caspase 3-dependent pathway

Objective Many Asian countries including China,Japan and Korea have very high incidence of gastric cancer,in which about 42% cases occur in mainland China.The precise targets and underlying mechanisms are not well understood.Our previous study revealed that Astragalus saponins(AST)showed promising effects on the suppression of the growth of HT-29 human colon cancer cells and tumor xenograft by inhibiting cell proliferation and promoting apoptosis.In the present study,we investigated the anti-carcinogenic effects of AST in AGS human gastric adenocarcinoma cells and attempted to elucidate the underlying mechanisms.Methods Growth inhibition of AGS cells was determined by using the MTT viability test.Involvement of different members of the apoptotic cascade and other growth-related factors was explored by assessment of their protein expression using Western blot analysis.Distribution of cells in different phases of the cell cycle was assessed by flow cytometry.Results Our data indicate that AST induced growth-inhibition and apoptosis in AGS cells by activating caspase 3 with subsequent poly(ADP-ribose)polymerase(PARP)cleavage.Cell cycle arrest at the G2/M phase had been observed in AST-treated AGS cells.The anti-proliferative effect of AST was associated with modulation of cyclin B1 and p21.We then demonstrate that AST could downregulate the expression of VEGF,of which interaction with its receptors is important for angiogenesis during tumor formation.Conclusions Our findings suggest that AST is an effective agent in gastric cancer treatment by inducing cell cycle arrest and apoptosis,of which anti-angiogenesis could be an alternative mode of action.     

新的微管抑制剂YB-13诱导HeLa细胞凋亡及其机制

目的研究吲哚3-草酰胺衍生物YB-13在体外试验中诱导宫颈癌细胞株(HeLa细胞)发生凋亡及其作用机制。方法采用MTT法、细胞生长曲线、细胞集落、荧光显微镜、DNAladder和流式细胞仪等方法进行凋亡检测,用RT-PCR方法检测凋亡过程中相关基因表达的变化;间接免疫荧光观察YB-13对细胞骨架的影响,观察YB-13对微管蛋白聚合和解聚。结果吲哚3-草酰胺衍生物YB-13在体外试验中对HeLa细胞的杀伤力强,荧光显微镜观察到了凋亡小体;DNAladder法检测到凋亡时DNA降解形成的梯带,流式细胞仪检测到了细胞凋亡峰,同时观察到细胞周期的变化。在凋亡过程中,凋亡相关基因bcl-2家族中bcl-2表达下调而bax表达上调;实验观察到YB-13能影响HeLa细胞的细胞骨架,并且YB-13可影响微管蛋白聚合和解聚。结论YB-13在体外试验中能诱导HeLa细胞发生凋亡,其作用机制可能与bax基因表达上调、bcl-2基因表达下调以及对微管蛋白的抑制作用有关。