败酱草单萜环烯醚酯类对HepG2、MCF7细胞增殖及凋亡的影响

目的 探讨败酱草单萜环烯醚酯类（patrinia monoterpene iridoid ether esters,PMIEE）对HepG2和MCF7细胞增殖抑制和凋亡的影响。方法 HepG2和MCF7细胞经PMIEE作用后,采用CCK8法检测细胞增殖抑制情况;Annexin V-FITC/PI双标记流式细胞术检测细胞凋亡及周期情况;细胞划痕实验检测细胞迁移状况;Western blot法检测Bcl-2、Bax、caspase3、cdc2和CyclinB1的表达情况。结果 CCK8、划痕实验和Annexin V-FITC/PI流式细胞术检测显示,PMIEE对HepG2和MCF7细胞均有显著的增殖抑制、促凋亡率和降低迁移率作用（P<0.05）,呈一定量效关系,且PMIEE对HepG2细胞的周期阻滞以G2/M期为主,MCF7细胞以G0/G1期为主;Western blot结果显示,PMIEE可显著下调2种细胞Bcl-2、cdc2、CyclinB1的表达,上调Bax和caspase3的表达水平。结论 PMIEE可诱导HepG2和MCF7细胞增殖抑制和凋亡,下调Bcl-2、cdc2和CyclinB1表达及上调Bax和caspase3表达,其抗癌的潜在机制可能与此有关。     

Microcystin (-LR) induced testicular cell apoptosis via up-regulating apoptosis-related genes in vivo

Microcystin-LR (MC-LR) can induce apoptosis of a wide range of tissue cells including testicular cells. The purpose of the study was to find out whether the expression and phosphorylation of p53, Bcl-2 protein family proteins, Cyt c, and caspases were involved in the induction of testicular cell apoptosis by MC-LR in mice. Results showed that following exposure to MC-LR, expression of Bax, caspase 3 and caspase 8 was up-regulated. Significant increases in the phosphorylation of both p53 and Bcl-2 were identified after the administration of MC-LR. The administration of MC-LR also resulted in significant increases of c-myc, c-jun, and c-fos. In conclusion, p53, Bcl-2, Bax, Caspase 3 and Caspase 8 are involved in the regulation of MC-LR-induced apoptosis of testicular cells. The overexpression of c-myc, c-jun and c-fos suggests that MC-LR may have carcinogenic potential for testes.     

表没食子儿茶素-3-没食子酸酯通过ERK1/2通路对肝癌HepG2细胞凋亡、增殖、迁移和侵袭的影响

目的 探讨表没食子儿茶素-3-没食子酸酯（EGCG）通过ERK1/2通路对肝癌HepG2细胞凋亡、增殖、迁移和侵袭的影响。方法 将体外培养的肝癌HepG2细胞分为空白组、EGCG组、EGCG+ERK抑制剂组。收集细胞裂解液,CCK-8检测细胞增殖,相差显微镜和Hoechst 33258染色观察细胞形态变化,流式细胞术检测细胞凋亡,RT-qPCR检测ERK mRNA水平,Western blotting分析ERK、磷酸化ERK、Bax和Bcl-2表达水平,并计算Bax/Bcl-2比值。结果 EGCG可以显著抑制肝癌HepG2细胞的增殖、迁移和侵袭（P<0.01）,并促进HepG2细胞的凋亡（P<0.01）;加入ERK通路抑制剂可显著逆转EGCG对HepG2的作用。结论 EGCG可通过ERK1/2信号通路发挥对肝癌HepG2细胞的抑制作用。     

Cytotoxicity,genotoxicity, oxidative stress,and apoptosis in HepG2 cells induced by the imidazole ionic liquid 1‐dodecyl‐3‐methylimidazolium chloride

This study purposes to assess the cytotoxicity of 1‐dodecyl‐3‐methylimidazolium chloride ([C₁₂min]Cl) in human hepatocellular carcinoma (HepG2) cells. To this end, HepG2 cells were exposed to a range concentration of [C₁₂min]Cl and evaluated cell viability, genotoxicity, oxidative stress, apoptosis, cell cycle, and apoptosis‐related gene expression to determine cytotoxicity. The outcomes showed that [C₁₂min]Cl curbed HepG2 cell growth and reduced cell viability in a concentration‐ and time‐dependent manner. Moreover, our assay results also revealed that exposure to [C₁₂min]Cl prompted DNA damage and apoptosis, reduced SOD and GSH content, enhanced MDA level, and changed the cell cycle of HepG2 cells. In addition, [C₁₂min] Cl caused alters in the expression levels of p53, Bax, and Bcl‐2, indicating that p53 and Bcl‐2 family may be involved in the cytotoxicity and apoptosis of HepG2 cells induced by [C₁₂min]C1. In summary, these results indicate that [C₁₂min]Cl exerts genotoxicity, physiological toxicity and prompts apoptosis in HepG2 cells, and is not an alleged green solvent.     

3‐Hydroxyflavone enhances the toxicity of ZnO nanoparticles in vitro

It is recently shown that flavonoids might reduce the toxicity of nanoparticles (NPs) due to their antioxidative properties. In this study, the influence of 3‐hydroxyflavone (H3) on the toxicity of ZnO NPs was investigated. H3 increased hydrodynamic size, polydispersity index and absolute value of the zeta potential of ZnO NPs, which indicated that H3 could influence the colloidal aspects of NPs. Surprisingly, H3 markedly decreased the initial concentration of ZnO NPs required to induce cytotoxicity to Caco‐2, HepG2, THP‐1 and human umbilical vein endothelial cells, which suggested that H3 could promote the toxicity of ZnO NPs to both cancerous and normal cells. For comparison, 6‐hydroxyflavone did not show this effect. H3 remarkably increased cellular Zn elements and intracellular Zn ions in HepG2 cells following ZnO NP exposure, and co‐exposure to H3 and NPs induced a relatively higher intracellular reactive oxygen species. Exposure to ZnO NPs at 3 hours induced the expression of endoplasmic reticulum stress markers DDIT3 and XBP‐1 s, which was suppressed by H3. The expression of apoptotic genes BAX and CASP3 was significantly induced by ZnO NP exposure after 3 and 5 hours, respectively, and H3 further significantly promoted CASP3 expression at 5 hours. In combination, the results from this study suggested that H3 affected colloidal stability of ZnO NPs, promoted the interactions between NPs and cells, and altered the NP‐induced endoplasmic reticulum stress–apoptosis signaling pathway, which finally enhanced the cytotoxicity of ZnO NPs.     

复方木鸡颗粒联合顺铂对人肝癌HepG2细胞增殖及凋亡的机制研究

目的探讨复方木鸡颗粒联合顺铂对人肝癌Hep G2细胞增殖、凋亡的影响及其潜在的作用机制。方法体外培养人肝癌Hep G2细胞,取对数生长期细胞,将其消化后随机分为对照组,复方木鸡颗粒高、低(10、5mg/m L)剂量组,顺铂组(1mg/m L)和复方木鸡颗粒(5mg/m L)联合顺铂(1mg/m L)组,采用CCK-8法检测细胞增殖情况,采用Hoehcst染色法检测细胞凋亡情况,采用免疫荧光联合激光共聚焦分别检测各组细胞中凋亡相关蛋白B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、蛋白天冬氨酸蛋白水解酶-9(Caspase-9)、细胞色素C氧化酶(Cyt-C)表达水平。结果与对照组比较,复方木鸡颗粒10、5 mg/mL组及顺铂组、联合组的细胞活力显著降低(P0.01),细胞凋亡显著增加,细胞内Bcl-2蛋白表达显著下调(P0.05、0.01),而Bax、Caspase-9、Cyt-C蛋白显著上调(P0.05、0.01),且联合组在抑制人肝癌Hep G2细胞增殖、促进其凋亡、调控凋亡相关蛋白表达的作用效果最佳。结论复方木鸡颗粒可显著抑制Hep G2细胞增殖并促进其凋亡,其与顺铂联合起到协同增效的效果,其作用机制可能与下调人肝癌Hep G2细胞内抑凋亡蛋白Bcl-2,上调促凋亡蛋白Bax、Caspase-9、Cyt-C表达水平有关。     

青天葵甲醇提取物通过ERK通路抑制人肝癌细胞增殖并诱导其凋亡

目的 研究青天葵甲醇提取物（Nervilia fordii methanol extracts，NFME）体外对人肝癌SMMC7721和HepG2细胞的凋亡作用及其作用机制。方法 紫外-可见分光光度法测定青天葵中总黄酮和总多酚的含量；MTT法检测不同浓度（0，0.25，0.5，1，1.5，2 mg·mL^-1）NFME处理24 h对SMMC7721、HepG2和LO2细胞生长抑制率的影响；克隆形成试验观察NFME对SMMC7721和HepG2细胞克隆形成率的影响；Hoechst凋亡染色观察NFME对SMMC7721细胞凋亡的影响；流式细胞术检测NFME对SMMC7721细胞凋亡和细胞周期的影响；Western blotting测试NFME作用下caspase3、PARP、ERK1/2和c-Raf蛋白磷酸化水平的变化。结果 NFME中总多酚含量为（4.25±0.46）mg·g^-1，总黄酮含量为（4.72±0.13）mg·g^-1；MTT试验结果显示，与对照组相比，在给药24 h时0.5 mg·mL^-1的NFME就能抑制SMMC7721和HepG2细胞的增殖（P<0.001）；克隆形成试验表明，0.125 mg·mL^-1的NFME能够抑制SMMC7721和HepG2细胞集落的形成（P<0.001）；Hoechst凋亡染色观察到0.25 mg·mL^-1的NFME作用24 h能够观察到SMMC7721细胞发生凋亡（P<0.05）；流式细胞试验结果证实0.5 mg·mL^-1的NFME能够诱导SMMC7721细胞的凋亡（P<0.01），凋亡率14.43%，阻滞细胞周期于S期；Western blotting结果表明NFME能使SMMC7721细胞中caspase3发生剪切，随着给药浓度增加其剪切作用越明显（P<0.05），活化的caspase3剪切下游PARP的表达；同时NFME能够降低SMMC7721细胞中ERK1/2和c-Raf蛋白的磷酸化水平（P<0.05）。结论 NFME能抑制人肝癌SMMC7721和HepG2细胞的活性并诱导其发生凋亡，其作用机制可能与抑制ERK信号通路从而诱导细胞凋亡有关。     

Saikosaponin A,an active glycoside from Radix bupleuri,reverses P-glycoprotein-mediated multidrug resistance in MCF-7/ADR cells and HepG2/ADM cells

1.?The expression and function of P-glycoprotein (P-gp) is associated with the phenotype of multidrug resistance (MDR). Saikosaponin A (SSA) is a triterpenoid saponin isolated from Radix Bupleuri. This study was mainly designed to understand effects of SSA on MDR in MCF-7/ADR and HepG2/ADM cells.

2.?MDR reversal was examined as the alteration of cytotoxic drugs IC50 in resistant cells in the presence of SSA by MTT assay, and was compared with the non-resistant cells. Apoptosis and uptake of P-gp substrates in the tumor cells were detected by flow cytometry. Western blot was performed to assay the expression of P-gp.

3.?Our results demonstrate SSA could increase the chemosensitivity of P-gp overexpressing HepG2/ADM and MCF-7/ADR cells to doxorubicin (DOX), vincristine (VCR) and paclitaxel. SSA promoted apoptosis of MCF-7/ADR cells in the presence of DOX. Moreover, it could also increase the retention of P-gp substrates DOX and rhodamine 123 in MCF-7/ADR cells, and decrease digoxin efflux ratio in Caco-2 cell monolayer. Finally, a mechanistic study showed that SSA reduced P-gp expression without affecting hydrolytic activity of P-gp.

4.?In conclusion, our findings suggest that SSA could be further developed for sensitizing resistant cancer cells and used as an adjuvant therapy together with anticancer drugs to improve their therapeutic efficacies.     

Microcystin-LR-induced autophagy regulates oxidative stress,inflammation, and apoptosis in grass carp ovary cells in vitro

MC-LR is one of the cyanotoxins produced by fresh water cyanobacteria. Previous studies showed that autophagy played an important role in MC-LR-induced reproduction toxicity. However, information on the toxicological mechanism is limited. In this study, MC-LR could induce autophagy and apoptosis in GCO cells in vitro. In GCO cells that had been exposed to MC-LR, the inhibitor of 3-MA effectively decreased cell viability and damaged cell ultrastructure. Oxidative stress was significantly increased in the 3-MA + MC-LR group, accompanied by significantly increased MDA content and decreased CAT activity and GST, SOD1, GPx, and GR expression levels (P < 0.05). Inflammation was more serious in the 3-MA + MC-LR group than that of MC-LR group, which was evidenced by increasing expression levels of TNFα, IL11, MyD88, TNFR1, TRAF2, JNK, CCL4, and CCL20 (P < 0.05). Interestingly, the significant decrease of Caspase-9, Caspase-7, and Bax expression and significant increase of Bcl-2 and Bcl-2/Bax ratio in 3-MA + MC-LR group compared to MC-LR group, suggesting that extent of apoptosis were reduced. Taken together, these results indicated that MC-LR induced autophagy and apoptosis in GCO cells, however, the inhibition of autophagy decreased the extent of apoptosis, induced more serious oxidative stress and inflammation, which eventually induced cell death. Our findings provided some information for exploring the toxicity of MC-LR, however, the role of autophagy require further study in vivo.     

Nimesulide inhibits the proliferation of HepG2 by up-regulation of Smad4

Objectives:

Hepatocellular carcinoma (HCC) is receiving increased attention. This study was designed to investigate the effect of selective Cyclooxygenase-2 (COX-2) inhibitor, nimesulide, on the expression of Smad4 in human hepatocellular carcinoma HepG2.

Materials and Methods:

HepG2 cells were incubated in various concentrations of nimesulide (25, 50, 100, 200, 400 μmol/L) to detect the effect of proliferation by MTS. The apoptosis of HepG2 was determined by TUNEL; fluorescence microscope was used to observe the expression of Smad4.

Results:

The result showed that nimesulide inhibited the proliferation of HepG2 cell in a concentrations-dependent manner, and promoted the karyopyknosis and fragmentation of HepG2 cell nucleus, induced its apoptosis, the number of fluorescence labeling of Smad4 in Nimesulide group was higher than control group (P<0.05).

Conclusions:

Nimesulide inhibits the proliferation and promotes apoptosis of HepG2 by up-regulation of Smad4 in HepG2.KEY WORDS: Hepatic carcinoma cell, Nimesulide, Smad4     

An ecofriendly synthesized gold nanoparticles induces cytotoxicity via apoptosis in HepG2 cells

Microbes have long been used for the synthesis of a variety of nanoparticles. Hepatocellular carcinoma (HCC) is the primary liver cancer and it is the second leading cause of cancer‐related mortality worldwide. In this study, we have synthesized Enterococcus mediated gold nanoparticles (AuNPs) and investigated their cytotoxic potential against human hepatocellular cancer cell line (HepG2). AuNPs were synthesized using Enterococcus sp. RMAA. HepG2 cells were treated with different concentrations of AuNPs for 24 hours and cytotoxicity was analyzed by MTT ((4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) assay. AuNPs induced reactive oxygen species expression was analyzed by 2′,7′‐dichlorodihydrofluorescein diacetate staining. Morphological changes related to apoptosis was analyzed by annexin V/propidium iodide staining. Protein expression of proliferating cell nuclear antigen (PCNA) was done by western blotting analysis. Bacterial‐mediated AuNPs caused significant cytotoxicity in HepG2 cells. AuNPs treatment also caused the significant expression of ROS and morphological damage related to apoptosis. AuNPs treatments were responsible for the dislocation of cytochrome c from mitochondria to cytosol. The protein expression of PCNA was significantly decreased upon AuNPs treatment. These findings suggest that Enterococcus‐mediated AuNPs can inhibit the proliferation of HepG2 cells via intracellular ROS mediated apoptosis, decreased PCNA expressions, and it may have the potential to treat HCC.     

Endoplasmic reticulum stress in murine liver and kidney exposed to microcystin-LR

To investigate the effect of microcystin-LR (MC-LR) on apoptosis based on the endoplasmic reticulum stress (ERS) pathway in mouse liver and kidney, male ICR mice were intraperitoneally injected with 20 μg kg⁻¹ body weight MC-LR for 21 days, and mRNA and protein levels of ERS special molecules in liver and kidney were analyzed using quantitative real-time PCR and western blotting. MC-LR significantly improved mRNA and protein expression of C/EBP homologous protein (CHOP) and cleaved caspase-12 in liver, whereas it inhibited expression of CHOP and caspase-12 in kidney. MC-LR also induced significant down-regulation of B-cell lymphoma/leukemia-2 (Bcl-2) mRNA expression in liver and weak up-regulation in kidney. These results indicated the involvement of the ERS pathway in MC-LR-induced apoptosis of hepatic cells but not in renal cells of mice. The weight changes and histological damage of liver and kidney were in accordance with the appearance of ERS. Our results indicate that ERS plays an important role in hepatic cell apoptosis induced by MC-LR, and is considered as a new pathway of liver toxicity. Its relative special genes might be considered as potentially new biomarkers used for risk assessment of MC-LR in the environment.     

MC-LR Exposure Leads to Subfertility of Female Mice and Induces Oxidative Stress in Granulosa Cells

Health risk of human exposure to microcystin-leucine arginine (MC-LR) has aroused more and more attention over the past few decades. In the present study, MC-LR was orally administered to female mice at 0, 1, 10 and 40 μg/L for three and six months. We found that chronic exposure to MC-LR at environmental levels could stimulate follicle atresia and lead to decreased developmental follicles, accompanied by a reduction of gonadosomatic index (GSI). In line with the irregular gonadal hormone level and estrus cycles, subfertility of female mice was also confirmed by analyzing numbers of litters and pups. The in vitro study suggested that granulosa cells could uptake MC-LR and should be the target of the toxicant. Oxidative stress in granulose cells induced by MC-LR promoted follicle atresia and eventually leads to female subfertility.     

Ellagic acid induces apoptosis in TSGH8301 human bladder cancer cells through the endoplasmic reticulum stress‐ and mitochondria‐dependent signaling pathways

To investigate the effects of ellagic acid on the growth inhibition of TSGH8301 human bladder cancer cells in vitro, cells were incubated with various doses of ellagic acid for different time periods. The phase‐contrast microscope was used for examining and photographing the morphological changes in TSGH8301 cells. Flow cytometric assay was used to measure the percentage of viable cells, cell cycle distribution, apoptotic cells, ROS, mitochondrial membrane potential (ΔΨm), Ca²⁺, caspase‐9 and ‐3 activities in TSGH8301 cells after exposure to ellagic acid. Western blotting was used to examine the changes of cell cycle and apoptosis associated proteins levels. Results indicated that ellagic acid induced morphological changes, decreased the percentage of viable cells through the induction of G0/G1 phase arrest and apoptosis, and also showed that ellagic acid promoted ROS and Ca²⁺ productions and decreased the level of ΔΨm and promoted activities of caspase‐9 and ‐3. The induction of apoptosis also confirmed by annexin V staining, comet assay, DAPI staining and DNA gel electrophoresis showed that ellagic acid induced apoptosis and DNA damage in TSGH8301 cells. Western blotting assay showed that ellagic acid promoted p21, p53 and decreased CDC2 and WEE1 for leading to G0/G1 phase arrest and promoting BAD expression, AIF and Endo G, cytochrome c, caspase‐9 and ‐3 for leading to apoptosis in TSGH8301 cells. On the basis of these observations, we suggest that ellagic acid induced cytotoxic effects for causing a decrease in the percentage of viable cells via G0/G1 phase arrest and induction of apoptosis in TSGH8301 cells. © 2013 Wiley Periodicals, Inc. Environ Toxicol 29: 1262–1274, 2014.     

沉默Rock2对肝癌细胞增殖及凋亡作用的研究

目的:探讨沉默Rock2基因对人肝癌细胞Huh-7和HepG2增殖和凋亡作用的影响.方法:实验分为空白对照组、干扰无意义组、转染PBS组及干扰Rock2组.将Rock2干扰质粒shRock2转染到人肝癌细胞Huh-7和HepG2中,通过实时荧光定量PCR检测Rock2 mRNA的表达水平;Western blot检测Rock2蛋白的表达水平;MTT比色法检测沉默Rock2后对Huh-7和HepG2细胞增殖抑制的影响;流式细胞术检测沉默Rock2对Huh-7和HepG2细胞周期及早期凋亡的变化.结果:将shRock2转染肝癌细胞系Huh-7和HepG2后,Rock2 mRNA以及Rock2蛋白的表达水平均明显下降.沉默Rock2表达后,MTT结果显示Huh-7和HepG2细胞较对照组细胞增殖能力明显减弱(P＜0.01);Huh-7和HepG2细胞G0/G1期细胞比例明显升高,而S期和G2/M期细胞比例明显降低,与对照组比较差异有统计学意义(P＜0.01);肝癌细胞的早期凋亡较对照组明显增加(P＜0.01).结论:沉默Rock2能明显抑制肝癌细胞系Huh-7和HepG2的增殖,并诱导其早期凋亡,提示Rock2可作为肝癌基因治疗的一个新的分子靶点.     

Cytotoxic potentials of S. cumini methanolic seed kernel extract in human hepatoma HepG2 cells

Syzygium cumini (Myrtaceae) is commonly called as Jamun or Jambolan. It has antidiabetic, anti‐inflammatory, antipyretic, and antioxidant activities. Hepatocellular carcinoma is the most frequent and deadliest cancers worldwide. We investigated the cytotoxic potentials of S. cumini methanolic seed kernel extract against human hepatoma HepG2 cells. HepG2 cells were treated with 10, 20, and 40 μg/mL of seed kernel extract for 24 hours and cytotoxic analysis was performed by MTT assay. S. cumini induced apoptosis related morphological changes in HepG2 cells were analyzed by annexin V and propidium iodide double staining. Nuclear fragmentation and chromatin condensation were analyzed by Hoechst nuclear staining. Mitochondrial membrane potential (MMP) was investigated by 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolyl‐carbocyanine iodide (JC‐1) staining. Protein expressions of hepatocyte nuclear factor‐1α (HFN‐1α) was performed using western blotting. S. cumini treatments caused a significant and a concentration‐dependent increase in the cytotoxicity of HepG2 cells. S. cumini treatments increased the percentage of cells in an early and late apoptosis stage. This treatment also caused chromatin condensation and nuclear fragmentation. Further, S. cumini treatments decreased MMP and also caused a significant downregulation of HFN‐1α protein expression. The present study demonstrated that S. cumini seed extract induced apoptosis in HepG2 cells through decrease in MMP and downregulation of HFN‐1α.     

MAPK signaling pathways regulate mitochondrial-mediated apoptosis induced by isoorientin in human hepatoblastoma cancer cells

Isoorientin (ISO) (CAS RN: 4261-42-1) is a flavonoid compound that can be extracted from several plant species, such as Phyllostachys pubescens, Patrinia, and Drosophyllum lusitanicum. ISO is able to induce apoptosis through mitochondrial dysfunction and inhibition of PI3K/Akt signaling pathway in HepG2 cells, however, the effects of ISO on MAPK signaling pathways remain unknown. The present study investigated the effects of ISO on this pathway, and the roles of MAPK kinases on mitochondrial-mediated apoptosis in HepG2 cells. The results showed that ISO induced cell death in a dose- and time-dependent manner, and induction apoptosis is main cause for ISO-induced cytotoxicity in HepG2 cells. ISO significantly inhibited the levels of ERK1/2 kinase and increased the expression of JNK and p38 kinases. Furthermore, U0126 (an ERK1/2 inhibitor) significantly enhanced the ISO-induced the Bax/Bcl-2 ratio, the release of cytochrome c to the cytosol fraction, and the levels of cleaved caspase-3. While SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor) markedly prevented the expression of these proteins induced by ISO. Furthermore, the ROS inhibitor (NAC) notably promoted the inhibited effect of ISO on the ERK1/2 kinase. NAC also suppressed the p-JNK and p-p38, but failed to reverse the effects of ISO. These results demonstrated for the first time that ISO induces apoptosis in HepG2 cells through inactivating ERK1/2 kinase and activating JNK and p38 kinases, and ROS stimulated by ISO is able to activate the MAPK singaling pathway as the upstream signaling molecules. Initiating event of the mitochondrial-mediated apoptosis induced by ISO is MAPK signals.     

山茱萸多糖通过上调Klotho表达和抑制PI3K/AKT通路对肝癌HepG2细胞增殖、凋亡的影响

目的探究山茱萸多糖通过上调Klotho表达和抑制PI3K/AKT通路对肝癌HepG2细胞增殖、凋亡的影响。方法分别以6.25、12.5、25 mg/mL山茱萸多糖作用于人肝癌细胞株HepG2,CCK8法检测细胞增殖能力,采用Hoechst 33258荧光染色以及流式AnnexinV-FITC/PI双染法检测细胞凋亡率;通过免疫印迹法检测增殖相关蛋白Ki67、凋亡相关蛋白Bax、Bcl-2、Caspase-3以及PI3K/Akt通路相关蛋白水平,并检测Klotho蛋白表达水平。结果与对照组相比,6.25、12.5、25 mg/mL山茱萸多糖组HepG2细胞克隆形成数显著下降(P0.05),且随山茱萸多糖浓度升高克隆形成数显著下降;与对照组相比,6.25、12.5、25mg/mL山茱萸多糖组HepG2细胞凋亡率显著升高(P0.05),且随山茱萸多糖浓度升高细胞凋亡率逐渐升高(P0.05);与对照组相比,6.25、12.5、25mg/mL山茱萸多糖组HepG2细胞中Bax、cleaved-caspase-3、Klotho蛋白表达升高(P0.05),Ki67、Bcl-2、p-PI3K/PI3K、p-Akt/Akt蛋白表达下降(P0.05),且呈剂量相关性。结论山茱萸多糖可能通过上调Klotho表达抑制PI3K/AKT通路活化,抑制HepG2细胞增殖,促进HepG2细胞凋亡。     

Glycoborinine induces apoptosis through mitochondrial pathway in HepG2 cells

Glycoborinine (GB), a natural carbazole alkaloid isolated from Glycosmis pentaphylla, has been shown to be a potential molecule against cancer cells. In this study, the cell-signaling pathway of its anti-tumor activity was investigated. MTT assay result showed that GB inhibited HepG2 cell proliferation in a dose- and time-dependent manner and 50% inhibiting concentration (IC₅₀) of GB-induced cell death was 39.7 μM for a period of 48 h. GB-induced HepG2 apoptosis was confirmed by Hochest 33258 staining and PI staining. The level of reactive oxygen species (ROS) was measured with H₂DCF-DA staining and the change of mitochondrial membrane potential (△Ψ^m) was analyzed with tetrechloro-tetraethylbenzimidazolcarbocyanine iodide (JC-1) probe. Results showed that GB at 12.5, 25, and 50 μM promoted ROS production. GB induced HepG2 apoptosis through a mitochondrial apoptotic pathway, which was demonstrated by GB-induced increase in the ratio of Bax/Bcl-2, cytochrome C release, the ratio of cleaved caspase-3/procaspase-3, and the ratio of cleaved poly ADP-ribose polymerase (cleaved PARP)/poly ADP-ribose polymerase (PARP). To summarize, this study demonstrated that GB could induce HepG2 apoptosis through the mitochondrial-dependent pathway, which might provide a promising approach to cure liver cancer with GB.     

Isoniazid-induced apoptosis in HepG2 cells: Generation of oxidative stress and Bcl-2 down-regulation

Isoniazid (INH) is a first-line antibiotic used in the treatment of infections caused by Mycobacterium tuberculosis. However it has a serious limitation of being hepatotoxic. Delineating the mechanism underlying INH-induced hepatotoxicity may be beneficial in devising ways to counteract its toxic manifestations. Studies in human hepatoma HepG2 cells have indicated that INH exposure causes induction of apoptosis. This study was aimed at identifying the key components/pathways of the INH-induced apoptotic pathway using HepG2 cells. HepG2 cells were exposed to increasing concentrations of INH (6.5, 13, 26, and 52?mM). Hydrogen peroxide (0.3?mM) served as positive control. After incubating for specific time intervals cells were harvested and evidences of cytotoxicity, oxidative stress, and apoptosis were sought. The findings indicated that INH exposure causes increased ROS generation along with alteration in levels of enzymatic antioxidants such as Superoxide dismutase, Catalase, and Glucose-6-Phosphate dehydrogenase. Altered Bcl-2/Bax content, cytochrome-c translocation, caspase activation, and DNA fragmentation emphasized involvement of apoptosis.