Ursolic acid induces apoptosis and autophagy in oral cancer cells

Oral squamous cell carcinoma (OSCC) is the fifth common cause of cancer mortality in Taiwan with high incidence and recurrence and needs new therapeutic strategies. In this study, ursolic acid (UA), a triterpenoid, was examined the antitumor potency in OSCC cells. Our results showed that UA inhibited the proliferation of OSCC cells in a dose‐ and time‐dependent manner in both Ca922 and SCC2095 oral cancer cells. UA induced caspase‐dependent apoptosis accompanied with the modulation of various biological biomarkers including downregulating Akt/mTOR/NF‐κB signaling, ERK, and p38. In addition, UA inhibited angiogenesis as evidenced by abrogation of migration/invasion and blocking MMP‐2 secretion in Ca922 cells. Interestingly, UA induced autophagy in OSCC cells, as manifested by LC3B‐II conversion and increased p62 expression and accumulation of autophagosomes. Inhibition by autophagy inhibitor enhanced UA‐mediated apoptosis in Ca922 cells. The experiment provides a rationale for using triterpenoid in the treatment of OSCC.     

Cantharidic acid induces apoptosis through the p38 MAPK signaling pathway in human hepatocellular carcinoma

Cantharidin analogs exhibit anticancer activities, including apoptosis. However, the molecular mechanisms underlying the effects of cantharidic acid (CA), a cantharidin analog, on apoptosis in hepatocellular carcinoma (HCC) cells are unclear. Thus, in this study, we evaluated the anticancer activities of CA by investigating its ability to trigger apoptosis in SK‐Hep‐1 cells. Our data demonstrated that CA effectively inhibited the proliferation of SK‐Hep‐1 cells in a dose‐dependent manner. Furthermore, CA effectively triggered cell cycle arrest and induced apoptosis, as determined by flow cytometric analysis. Western blotting revealed that CA significantly activated proapoptotic signaling including caspase‐3, −8, and −9 in SK‐Hep‐1 cells. Moreover, treatment of SK‐Hep‐1 cells with CA induced the activation of ERK, p38, and c‐Jun N‐terminal kinase. Moreover, the inhibition of p38 by specific inhibitors abolished CA‐induced cell apoptosis. In conclusion, our results indicated that CA induces apoptosis in SK‐Hep‐1 cells through a p38‐mediated apoptotic pathway and could be a new HCC therapeutic agent.     

Fisetin induces apoptosis through mitochondrial apoptosis pathway in human uveal melanoma cells

Fisetin, a diatery flavonoid, been reported that possess anticancer effects in various cancers. The purpose of the study was to investigate the antitumor effects of fisetin in cultured uveal melanoma cell lines and compared with normal retinal pigment epithelial (RPE) cells. MTT assay was used for evaluating cytotoxic effects of fisetin. Flow cytometry study was used for the determination of apoptosis. JC‐1 fluorescent reader was used to determine mitochondrial transmembrane potential changes. The results shown that fisetin dose‐dependently decreased the cell viability of uveal melanoma cells but not influenced the cell viability of RPE cells. Apoptosis of uveal melanoma cells was induced by fisetin efficiently. Fisetin inhibited antiapoptotic Bcl‐2 family proteins and damaged the mitochondrial transmembrane potential. The levels of proapoptotic Bcl‐2 proteins, cytochrome c, and various caspase activities were increased by fisetin. In conclusion, fisetin induces apoptosis of uveal melanoma cells selectively and may be a promising agent to be explored for the treatment of uveal melanoma.     

力达霉素诱导人胃癌BGC823细胞凋亡和抑制裸鼠移植瘤生长

观察力达霉素(LDM)对人胃癌BGC823细胞的诱导凋亡作用及体内抗肿瘤活性。采用MTT法观察LDM对人胃癌BGC823细胞增殖的抑制作用。利用Annexin V-FITC/PI双染结合流式细胞仪和脱氧核糖核酸末端转移酶介导的缺口末端标记技术检测细胞凋亡的改变。采用Western blotting法检测细胞中VEGF蛋白的表达情况。建立裸鼠胃癌皮下移植瘤模型，观察LDM的体内抗肿瘤活性。LDM能够明显抑制BGC823细胞增殖，诱导细胞凋亡，降低细胞VEGF蛋白的表达，抑制胃癌裸鼠移植瘤的生长。LDM剂量0.02和0.04 mg·kg^-1的抑瘤率分别为57%和72%(P<0.01)。LDM可诱导胃癌细胞凋亡并抑制裸鼠移植肿瘤的生长。     

辛伐他汀通过线粒体途径诱导K562细胞凋亡

目的观察辛伐他汀诱导K562细胞凋亡不同时间的膜电位(Δψm),caspase-3、9和细胞色素C的改变,以推测其凋亡通路。方法采用浓度为20μmol.L-1的辛伐他汀处理K562细胞24、48、72 h,采用流式细胞技术检测细胞凋亡率和线粒体膜电位,分光光度法检测caspase-3、9蛋白活性,免疫组织化学法检测细胞色素C蛋白。结果浓度为20μmol.L-1辛伐他汀作用K562细胞24、48、72 h后,凋亡率分别为(6.1±0.35)%、(14.15±0.42)%(、30.70±0.65)%,随着凋亡率增加线粒体膜电位降低分别为(39.6±4.80)%,(24.4±2.45)%,(6.0±1.62)%;caspase-3、9蛋白活性与对照组相比上调,细胞浆内细胞色素C升高。结论辛伐他汀诱导K562细胞凋亡时线粒体膜电位下降,caspase-3、9活性增高和细胞色素C释放,推测辛伐他汀诱导K562细胞的凋亡可能经过线粒体凋亡途径。     

Tetracaine induces apoptosis through a mitochondrion-dependent pathway in human corneal stromal cells in vitro

Purpose: Tetracaine is a local anesthetic widely used in ocular diagnosis and ophthalmic surgery and may lead to some adverse effects and complications at a clinical dose. To assess the cytotoxicity and molecular toxicity mechanisms of tetracaine, we used human corneal stromal (HCS) cells as an in vitro model to study the effects of tetracaine on HCS cells.

Materials and methods: The cytotoxicity of tetracaine on HCS cells was investigated by examining the changes of cell growth, morphology, viability and cell cycle progressing when HCS cells were treated with tetracaine at concentrations from 10?g/L to 0.078125?g/L. To prove the hypothesis that the cytotoxicity of tetracaine on HCS cells was related with apoptosis induction, we further detected multiple changes in HCS cells, including plasma membrane (PM) permeability, phosphatidylserine (PS) orientation, genomic DNA integrality, and cell ultrastrcuture after treated with tetracaine. Furthermore, the pro-apoptotic signalling pathway induced by tetracaine was explored through detecting the activation of various caspases, the changes of mitochondrial transmembrane potential (MTP), the expression level of Bcl-2 family proteins and the amount of mitochondria-released apoptosis regulating proteins in cytoplasm.

Results: Tetracaine at concentrations above 0.15625?g/L had a dose- and time-dependent cytotoxicity to HCS cells, which resulted cell growth inhibition, proliferation retardation, morphological abnormalities and decreased viability. Meanwhile, we found that the HCS cells treated with tetracaine had typical features associated with apoptosis, including an increase in PM permeability, PS externalization, DNA fragmentation and apoptotic body formation. Tetracaine not only resulted in caspase-3, caspase-8 and caspase-9 activation and disruption of MTP but also downregulated Bcl-2 and Bcl-xL and upregulated Bad and Bax, along with the upregulation of cytoplasmic cytochrome c (Cyt. c) and apoptosis-inducing factor (AIF).

Conclusions: These results suggested that tetracaine-induced apoptosis might be triggered through Fas death receptors and mediated by Bcl-2 family proteins in the mitochondria-dependent pathway. Our findings identified the cytotoxicity and molecular mechanisms of tetracaine, which could provide a reference value for the safety of this medication and prospective therapeutic interventions in eye clinic.     

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.     

Cantharidic acid induces apoptosis in human nasopharyngeal carcinoma cells through p38‐mediated upregulation of caspase activation

Cantharidic acid (CA) is the hydrolysis product of the acid anhydride cantharidin, which is a natural toxin secreted by several species of blister beetles. Several studies have indicated that as an inhibitor of protein phosphatase 2 (PP2A), CA induces apoptosis in various human cancer cells. However, the effect of CA on human nasopharyngeal carcinoma (NPC) cells and the underlying pathways have not been addressed. In our current study, we tested the hypothesis that CA treatment reduces the viability of human NPC cells (HONE‐1, NPC‐39, and NPC‐BM) by inducing apoptosis. Results indicated that CA markedly reduced cell viability, which was revealed by the upregulation of caspase activation in extrinsic and intrinsic apoptosis pathways as well as the upregulation of extracellular‐signal‐regulated kinase 1/2 (ERK1/2), p38, and c‐Jun N‐terminal kinase 1/2 (JNK1/2) pathways. Coadministration of a p38 inhibitor (SB203580) with CA abolished the activation of caspase proteins. These findings indicated that CA treatment leads to apoptosis in human NPC cells through the upregulation of caspase activation, mediated particularly by the p38 pathway. Hence, CA is a promising therapeutic agent for human NPC.     

Perfluorooctanoic acid induces apoptosis through the p53-dependent mitochondrial pathway in human hepatic cells: A proteomic study

Perfluorooctanoic acid (PFOA) is one of the most commonly used perfluorinated compounds, and exposure to it has been associated with a number of adverse health effects. However, the molecular mechanisms involved in PFOA toxicity are still not well characterized. In the present study, flow cytometry analysis revealed that PFOA induced oxidative stress, cell cycle arrest and apoptosis in human non-tumor hepatic cells (L-02). Furthermore, we investigated the alterations in protein profile within L-02 cells exposed to PFOA, aiming to explore the mechanisms underlying PFOA hepatotoxicity on the proteome level. Of the 28 proteins showing significant differential expression in response to PFOA, 24 were down-regulated and 4 were up-regulated. This proteomic study proposed that the inhibition of some proteins, including GRP78, HSP27, CTSD and hnRNPC may be involved in the activation of p53, which consequently triggered the apoptotic process in L-02 cells. Induction of apoptosis via the p53-dependent mitochondrial pathway is further suggested as one of the key toxicological events occurring in L-02 cells under PFOA stress. We hope these data will shed new light on the molecular mechanisms responsible for PFOA-mediated toxicity in human liver cells, and from such studies useful biomarkers indicative of PFOA exposure could be developed.     

Osthole induces human nasopharyngeal cancer cells apoptosis through Fas–Fas ligand and mitochondrial pathway

Nasopharyngeal carcinoma (NPC) is endemic in Southern China and Southeast Asia. The present study investigated the activity of osthole in suppressing NPC along with the underlying mechanism. Cell growth inhibition was measured using the MTT assay. Apoptosis was detected through 4ʹ,6‐diamidino‐2‐phenylindole staining and flow cytometry. Western blotting was used to identify the signaling pathway. Osthole markedly inhibited cell proliferation and induced apoptosis in the NPC cell line. Western blotting results revealed the increased activation of caspases 3, 8, and 9 and poly (ADP‐ribose) polymerase. Osthole treatment significantly reduced the expression of the antiapoptotic protein Bcl‐2 and increased the expression of the proapoptotic proteins Bax, Bak, BimL, BimS, and t‐Bid. Osthole treatment also increased the expression of Fas, FADD, TNF‐R1, TNF‐R2, DcR2, RIP, and DR5. In addition, osthole treatment significantly increased the expression levels of phosphorylated ERK1/2 and JNK1/2. These results suggested that osthole exerts cytotoxic effects on NPC cell lines mainly through apoptosis mediated by the Fas–Fas ligand and mitochondrial pathway. Osthole could be a potential anticancer agent for NPC.     

科罗索酸上调Bax表达诱导宫颈癌细胞凋亡

目的探讨科罗索酸对宫颈癌细胞Hela的凋亡诱导作用。方法不同浓度(10～50μmol·L~(-1))科罗索酸处理宫颈癌Hela细胞,MTT法检测细胞活性,SPSS12.0统计软件包计算半数抑制浓度(IC_(50)),流式细胞术检测细胞凋亡,荧光检测法检测凋亡酶caspase-3活性,western blot检测细胞蛋白Bax、Bcl-2表达。结果科罗索酸抑制宫颈癌Hela细胞活性随药物浓度及时间的增加而增高,24、48及72 h的IC_(50)分别为45、34、28μmol·L~(-1)。科罗索酸诱导Hela细胞凋亡率明显高于对照组(P<0.01),凋亡酶活性也明显高于对照组(P<0.01),科罗索酸可上调Bax而不影响Bcl-2的表达。结论科罗索酸可抑制宫颈癌Hela细胞活性,诱导细胞凋亡,其机制可能与上调Bax蛋白表达有关。     

Honokiol induces paraptosis and apoptosis and exhibits schedule-dependent synergy in combination with imatinib in human leukemia cells

Honokiol, an active component isolated and purified from Chinese traditional herb magnolia, has been shown to inhibit growth and induce apoptosis in different cancer cell lines. This study shows that honokiol can induce a cell death distinct from apoptosis at lower concentrations. The death was characterized by cytoplasmic vacuolization with the endoplasmic reticulum swelling and accompanied by apoptosis at higher concentrations in NB4 and K562 cells. The two death processes may be in sequence at lower concentrations and in parallel with the increase of honokiol concentration. Membrane-associated cytotoxicity was involved in honokiol-induced paraptosis and apoptosis. Furthermore, honokiol inhibited concentration-dependent cell adhesion to extracellular matrix for NB4 cells. In addition, the cytotoxicity of honokiol combined treatment with imatinib was schedule- and concentration-dependent and the sequential administration of honokiol before imatinib appeared to be more beneficial in K562 cells. Taken together, the data suggest that honokiol induced a novel cell death pathway and there was cross-talk between apoptotic and non-apoptotic programmed cell death caused by honokiol in leukemia cells. Moreover, honikiol exhibited schedule-dependent synergy in combination with imatinib and sequential administration of imatinib followed by honokiol could be the optimal sequence to combine these two drugs in K562 cells.     

Antitumor activity of RT2 peptide derived from crocodile leukocyte peptide on human colon cancer xenografts in nude mice

RT2, derived from the leukocyte peptide of Crocodylus siamensis, can kill human cervical cancer cells via apoptosis induction, but no evidence has shown in vivo. In this study, we investigated the antitumor effect of RT2 on human colon cancer xenografts in nude mice. Twenty‐four mice were injected subcutaneously with human colon cancer HCT 116 cells. Eleven days after cancer cell implantation, the mice were treated with intratumoral injections of phosphate buffered saline (PBS) or RT2 (0.01, 0.1, and 1 mg/mouse) once every 2 days for a total of 5 times. The effect of a 10‐day intratumoral injection of RT2 on body weight, biochemical, and hematological parameters in BALB/c mice showed no significant difference between the groups. Tumor volume showed a significant decrease only in the treatment group with RT2 (1 mg/mouse) at day 6 (P < .05), day 8 (P < .01), and day 10 (P < .01) after the first treatment. The protein expression levels of cleaved poly (ADP‐ribose) polymerase (PARP), apoptosis‐inducing factor (AIF), and the p53 tumor suppressor protein (p53) in xenograft tumors increased after treatment with RT2 (1 mg/mouse) compared to those in the PBS‐injected group. Moreover, RT2 increased the expression of Endo G and Bcl‐2 family proteins. Therefore, the peptide RT2 can inhibit tumor growth via the induction of apoptosis in an in vivo xenograft model.     

YC-1 induces apoptosis of human renal carcinoma A498 cells in vitro and in vivo through activation of the JNK pathway

Background and purpose:

The aim of this study was to elucidate the mechanism of YC-1{3-(5′-hydroxy methyl-2′-furyl)-1-benzylindazole}-induced human renal carcinoma cells apoptosis and to evaluate the potency of YC-1 in models of tumour growth in mice.

Experimental approach:

YC-1-mediated apoptosis was assessed by analysis of MTT, SRB, DAPI staining and flow cytometry analysis. Knockdown of JNK protein was achieved by transient transfection using siRNA. The mechanisms of action of YC-1 on different signalling pathways involved were studied using western blot. Fas clustering was analysed by confocal microscopy and in vivo efficacy was examined in a A498 xenograft model.

Key results:

YC-1 displayed cytotoxicity in renal carcinoma cells at 10⁻⁷–10⁻⁸ M. Increased condensation of chromatin was observed and an increase in the cell population in subG1 phase. Moreover, YC-1 triggered mitochondria-mediated and caspase-dependent pathways. YC-1 significantly induced Fas ligand expression, but did not modify either the protein levels of death receptors or ligands. In addition, Fas clustering in cells responsive to YC-1 was observed, suggesting involvement of a Fas-mediated pathway. Furthermore, YC-1 markedly induced phosphorylation of JNK and a JNK inhibitor, SP600125, and siRNA JNK1/2 significantly reversed YC-1-induced cytotoxicity and protein expression. We suggest that YC-1 induced JNK phosphorylation, the upregulation of FasL and Fas receptor clustering to promote the activation of caspases 8 and 3, resulting in apoptosis. Finally, we demonstrated the antitumour effect of YC-1 in vivo.

Conclusions and implications:

These data suggest that YC-1 is a good candidate for development as an anticancer drug.     

冬凌草甲素通过PI3K/Akt通路诱导MDA-MB-231细胞的凋亡

目的：研究冬凌草甲素对人乳腺癌MDA-MB-231细胞增殖产生的影响,初步探讨其作用机理。方法：体外培养人乳腺癌MDA-MB-231细胞,采用6、12、24μmol/L冬凌草甲素对其进行处理,采用倒置显微镜进行细胞形态学观察,MTT比色法检测细胞存活率,流式细胞术检测细胞凋亡率,Western blotting检测凋亡相关蛋白procaspase-3、PARP及Akt、p-Akt、p-GSK3β表达的变化。结果：冬凌草甲素作用MDA-MB-231细胞24h后,可观察到细胞凋亡的形态学改变,以24μmol/L组最为明显。实验组与对照组相比,细胞存活率显著降低、凋亡率显著升高（P〈0.01）,具有时间和剂量依赖性,凋亡相关蛋白procaspase-3下调,caspase-3底物PARP被逐步剪切,并伴随p-Akt、p-GSK3β蛋白水平下调（P〈0.05）。结论：冬凌草甲素可有效抑制人乳腺癌MDA-MB-231细胞的增殖,诱导其凋亡,机制与PI3K/Akt通路的抑制有关。     

Galangin induces apoptosis in hepatocellular carcinoma cells through the caspase 8/t-Bid mitochondrial pathway

This study has investigated whether galangin, a flavonol derived from Alpinia officinarum Hance and used as food additives in southern China, induces apoptosis in hepatocellular carcinoma cells (HCCs) by activation of the caspase-8 and Bid pathway. The apoptosis of HCCs was evaluated by in situ uptake of propidium iodide and Hoechst 33258. Protein expressions were detected by Western blotting. Caspase-8 activity was measured using colorimetric method. To confirm the galangin-induced apoptotic pathway, inhibition of caspase-8 activity by Z-IETD-FMK, knockdown of Bid expression with siRNA, and overexpression of Bcl-2 in cells were carried out, respectively. The results show that galangin has significantly induced apoptosis in HCC lines. The caspase-8 is activated, and the cleavage of Bid results in the increase in tBid. The galangin-induced apoptosis is attenuated by Z-IETD-FMK, Bid siRNA, and Bcl-2 overexpression, respectively. However, Bcl-2 fails to suppress caspase-8 activation and the cleavage of Bid. This study has demonstrated that galangin induces apoptosis in HCCs by activating caspase 8/t-Bid mitochondrial pathway. Although Bcl-2 overexpression attenuates galangin-mediated apoptosis of HCCs, it is not mediated by the inhibition of tBid generation and caspase-8 activation.     

Dichloroacetonitrile induces cytotoxicity through oxidative stress-mediated and p53-dependent apoptosis pathway in LO2 cells

Dichloroacetonitrile (DCAN), one of the disinfection byproducts of water chlorination, induces cell proliferation and apoptosis; however, the detailed mechanism remains unclear. Oxidative stress participates in various biological processes, including DNA damage and cytotoxicity. To explore whether oxidative stress mediated DCAN-induced cell proliferation and apoptosis, we assessed the effect of redox imbalance and apoptosis in LO2 cells. We observed increase of reactive oxygen species and malondialdehyde and increased apoptosis by 13.6% in 500?μM DCAN compared with the control group. We also observed a decrease of antioxidant ability damage including glutathione, superoxide dismutase, and total antioxidant capacity depletion. Furthermore, DCAN might activate oxidative stress-mediated apoptosis pathway via up-regulation of p53 expression and caspase-3 activity. Therefore, we conclude that DCAN may activate apoptotic signals via p53 up-regulation and oxidative stress-mediated apoptosis in LO2 cells.