Caspase-3 In HepG2 Cells Research Articles

Study of the antineoplastic effect of modulating apoptosis-related noncoding RNA in human hepatocellular carcinoma cell line (HepG2)

Abstract MiR-421 is considered an important molecule that can prevent tumor growth. Bioinformatics analysis indicated that mRNA caspase-3 gene is a target gene of miR-421. The current study aimed to explore the functional role of miR-421 in hepatocellular carcinoma (HCC) and explore the interaction between miR-421 and caspase-3. To validate bioinformatics data, RT-qPCR was used to detect the expression of miR-421 and caspase-3 in 10 HCC tissues. The results showed miR-421 expression was significantly higher in HCC than non HCC liver tissues (P<0.01), nevertheless caspase-3 gene expression was markedly lower in HCC than non HCC liver tissues (P<0.01). Besides, miR-421 expression was negatively associated with caspase-3 expression. MiR-421 mimic and inhibitor was transfected into HCC cell lines (HepG2). Proliferation assay, showed that low-expression of miR-421 inhibited the proliferation of HCC cells. RT-qPCR was worked for detection the expression levels of miR-421 and caspase-3 in HepG2 cells before and after transfection. The results showed that miR-421 expression in HepG2 cells was significantly lower in miR-421 inhibitor transfected group than in mimic- transfected and control groups (Mock) (P≤ 0.05), and caspase-3 gene expression in HCC tissues was markedly higher in inhibitor transfected group than those transfected by mimic and control group (Mock) (P≤0.05). Thus, miR-421 inhibitor may inhibit the proliferation of HCC cells via over- expression of caspase-3.

Anticancer Activity of Platinum (II) Complex with 2-Benzoylpyridine by Induction of DNA Damage, S-Phase Arrest, and Apoptosis.

To overcome the disadvantages of cisplatin, numerous platinum (Pt) complexes have been prepared. However, the anticancer activity and mechanism of Pt(II) complexed with 2-benzoylpyridine [Pt(II)- Bpy]: [PtCl2(DMSO)L] (DMSO = dimethyl sulfoxide, L = 2-benzoylpyridine) in cancer cells remain unknown. Pt(II)-Bpy was synthesized and characterized by spectrum analysis. Its anticancer activity and underlying mechanisms were demonstrated at the cellular, molecular, and in vivo levels. Pt(II)-Bpy inhibited tumor cell growth, especially HepG2 human liver cancer cells, with a halfmaximal inhibitory concentration of 9.8±0.5μM, but with low toxicity in HL-7702 normal liver cells. Pt(II)- Bpy induced DNA damage, which was demonstrated through a marked increase in the expression of cleavedpoly (ADP ribose) polymerase (PARP) and gamma-H2A histone family member X and a decrease in PARP expression. The interaction of Pt(II)-Bpy with DNA at the molecular level was most likely through an intercalation mechanism, which might be evidence of DNA damage. Pt(II)-Bpy initiated cell cycle arrest at the S phase in HepG2 cells. It also caused severe loss of the mitochondrial membrane potential; a decrease in the expression of caspase-9 and caspase-3; an increase in reactive oxygen species levels; the release of cytochrome c and apoptotic protease activation factor; and the activation of caspase-9 and caspase-3 in HepG2 cells, which in turn resulted in apoptosis. Meanwhile, changes in p53 and related proteins were observed including the upregulation of p53, the phosphorylation of p53, p21, B-cell lymphoma-2-associated X protein, and NOXA; and the downregulation of B-cell lymphoma 2. Moreover, Pt(II)-Bpy displayed marked inhibitory effects on tumor growth in the HepG2 nude mouse model. Pt(II)-Bpy is a potential candidate for cancer chemotherapy.

Study on the effect and mechanism of hepatitis B virus X protein transactivates gene 4 in HepG2 cell apoptosis

Objective: To investigate the effect and mechanism of XTP4 gene in apoptotic hepatoblastoma HepG2 cell line. Methods: HepG2 cells were transiently transfected with small interfering RNA of XTP4 genes, plasmid pcDNA3.1/myc-His(-) A-XTP4, and hepatitis B virus X protein transactivated x gene 4 (HBX protein trans-activate gene4, XTP4) and their respective negative controls. After 48h, the overexpression and interference expression condition of XTP4 in HepG2 cells were detected by Western blot. HepG2 cells apoptosis was detected by flow cytometry. The expression levels of apoptosis-related proteins P53, Bcl-2, Bax and Caspase-3 in HepG2 cells were detected by Western blot, and Bcl-2/Bax ratio was calculated. The chemiluminescence assay was used to detect activity of caspase-3 in HepG2 cells. The measured data were presented as (x ± s), and independent sample t-test was used for comparison between the two groups. Results: HepG2 cells had successfully achieved the overexpression and interference expression of XTP4 protein. Compared with the control group, the overexpression of XTP4 in HepG2 cells had significantly decreased the number of apoptotic cells (P < 0.05), and increased Bcl-2/Bax (P < 0.05) ratio, but decreased the expression of P53 protein (P < 0.05). The protein expression of Caspase-3 and activity of caspase-3 was decreased (P < 0.05). However, interference with XTP4 expression in HepG2 cells had significantly increased the number of apoptotic cells (P < 0.05) and decreased Bcl-2/Bax (P < 0.05) ratio, but increased the expression of P53 protein (P < 0.05). The protein expression of Caspase-3 and activity of caspase-3 was increased (P<0.05). Conclusion: In HepG2 apoptosis XTP4 has inhibitory effect, and its effect on inhibiting HepG2 apoptosis may be achieved by regulating the Bcl-2/Bax ratio, and the P53 protein may be involved.

Ethylacetate extract from Tetrastigma hemsleyanum inhibits proliferation and induces apoptosis in HepG2 and SMMC-7721 cells.

PurposeThis study aimed to investigate the effect of ethylacetate extract from Tetrastigma hemsleyanum (EET) on the proliferation and apoptosis of HepG2 and SMMC-7721 cells and determine the underlying mechanisms.Materials and methodsHepG2 and SMMC-7721 cells were cultured in vitro until the exponential growth phase and then treated with different concentrations of EET for 24 h. We performed a colony forming assay to determine colony forming ability, CCK8 assay to detect cell proliferation, Annexin V–FITC/PI double staining to analyze cell apoptosis, and Western blot to measure the protein expression of Caspase-3, Bcl-2, and Bax.ResultsEET significantly inhibited the proliferation of HepG2 and SMMC-7721 cells in a concentration- and time-dependent manner (P<0.05). After treatment with 0, 50, 100, 150, 200, and 250 μg/mL EET for 24 h, HepG2 the proliferation rates were 100.00%±0.00%, 90.33%±1.76%, 67.67%±0.88%, 47.33%±0.88%, 37.00%±0.00%, and 30.33%±0.67%, respectively, and 100.00%±0.00%, 18.25%±1.05%, 19.99%±0.59%, 23.42%±0.46%, 29.70%±0.79%, and 29.8%±0.41% for SMMC-7721 cells, respectively. After treatment with 0, 50, 100, 150, 200, and 250 μg/mL EET for 24 h, the apoptotic rates were 11.08%±0.72%, 27.44%±0.51%, 32.92%±0.41%, 26.20%±0.47%, 22.92%±0.24%, and 55.60%±0.08%, for HepG2 cells, respectively, and 59.18%±0.17%, 41.24%±2.05%, 52.54%±0.39%, 50.54%±1.08%, and 57.44%±1.93% for SMMC-7721 cells, respectively. Compared with the treatment groups, the control group showed a significantly lower apoptotic rate (47.91%±1.09%, P<0.05). EET at the different concentrations downregulated the protein expression of Caspase-3 in HepG2 cells and upregulated it in SMMC-7721 cells; it also downregulated the protein expression of Bcl-2 in HepG2 and SMMC-7721 cells and upregulated the protein expression of Bax in HepG2 and SMMC-7721 cells.ConclusionThese findings suggest that EET exerts antiproliferative and proapoptotic effects against HepG2 and SMMC-7721 cells mediated by downregulation or upregulation of Caspase-3 expression. Our study may help to develop EET for the pharmacological treatment of hepatoblastoma or hepatocellular carcinoma.

Bioactivity-guided isolation of β-Carboline alkaloids with potential anti-hepatoma effect from Picrasma quassioides (D. Don) Benn

β-Carboline alkaloids in Picrasma quassioides (D. Don) Benn. have been proven to possess inhibitory activity against various cancer cells. However, their effect on hepatocellular carcinoma and structure-activity relationships (SAR) have not been systematically reported. In this work, bioactivity-directed fractionation of P. quassioides led to the separation of active fraction A2–2. A total of 39 β-carbolines, including 4 new ones (1–4), were obtained from the active fraction. Moreover, all the isolated compounds were identified in the active fraction A2–2 by LC-MS. The cytotoxicity on HepG2 and Hep3B cells of all compounds was screened by MTT assay, and the SAR were established. The SAR were also supported by the apoptosis ratio of HepG2 cells using flow cytometry analysis after treatment with potential compounds 1, 2, 9, 10, 12, 29, 36 and 38. It suggested that these active compounds caused death of hepatoma cells through apoptosis induction. In addition, further study revealed that compounds 12, 29, 36 significantly activated caspase-3 in HepG2 cells.

Gastrodin Protects against Ethanol-Induced Liver Injury and Apoptosis in HepG2 Cells and Animal Models of Alcoholic Liver Disease.

This study aims to investigate the protective effects of gastrodin (GSTD), a natural compound isolated from the root of Gastrodia elata BL., on ethanol-induced liver injury and apoptosis in HepG2 cells and animal models. For in vitro studies, GSTD was used to pre-treat the cells for 4 h followed by 600 mM of ethanol co-administration for 24 h. Alcoholic liver disease (ALD) of Sprague-Dawley (SD) rats was induced by chronic ethanol-feeding plus a single dose (5 g/kg) of acute ethanol administration, GSTD at different doses were co-administered for 8 weeks. For acute liver injury experiment of ICR mice, GSTD (100 mg/kg/d) was pre-treated for 3 d followed by ethanol administration (5 g/kg) for 3 times. The results showed that GSTD protects HepG2 cells from ethanol-induced toxicity, injury, and apoptosis significantly. Co-administered with ethanol, GSTD prevented the loss of mitochondrial membrane potential, reduced the release cytochrome c from mitochondria, and inhibited the activation of caspase-3 in HepG2 cells. In SD rats induced by chronic ethanol-feeding, GSTD significantly restored liver function and ameliorated pathological changes of the liver. In rat liver, GSTD greatly suppressed the activation of caspase-3 and inhibited hepatocellular apoptosis. In ethanol-induced acute liver injury of ICR mice, GSTD reduced liver acetaldehyde and suppressed the up-regulation of alcohol dehydrogenase (ADH) and CYP2E1 significantly. Our results demonstrate that GSTD is efficacious in protecting liver cells from ethanol-induced injury and apoptosis; it may be useful for the development of novel agents for the treatment of ALD in the future.

Toxic effect of nonylphenol on the marine macroalgae Gracilaria lemaneiformis (Gracilariales, Rhodophyta): antioxidant system and antitumor activity.

The objective of the present work was to evaluate the toxic effect of nonylphenol (NP) on the antioxidant response and antitumor activity of Gracilaria lemaneiformis. An obvious oxidative damage was observed in this study. The thallus exposed to NP showed 1.2-2.0-fold increase in lipid peroxide and displayed a maximum level of 16.58μmolg-1 Fw on 0.6mgL-1 for 15-day exposure. The activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) enhanced significantly by 1.1-3.2-fold and subsequently diminished at the high concentrations and prolonged exposure. The results of DNA damage in comet assay also supported that NP was obviously toxic on G. lemaneiformis with increasing the percentage of tail DNA in a dose-dependent manner. Furthermore, the ethanol extract of G. lemaneiformis (EEGL) did exhibit antitumor potential against HepG-2 cells. While decreased in cell inhibition, ROS generation, apoptosis, and caspase-3 in HepG-2 cells treated with the EEGL were observed when G. lemaneiformis was exposed to NP for 15days, and which were related to exposure concentration of NP. These suggested that NP has strongly toxic effect on the antitumor activity of G. lemaneiformis. The results revealed in this study imply that macroalgae can be useful biomarkers to evaluate marine pollutions.

The Protective Effect of Antioxidant Enriched Fractions from Colored Potatoes Against Hepatotoxic Oxidative Stress in Cultured Hepatocytes and Mice

Herein, we prepared standardized antioxidant-enriched fractions from red (AFR) and purple (AFP) colored potatoes and evaluated their protective effects against hepatotoxicity induced by oxidative stress. The major compound contained in both AFR and AFP was 5-caffeoylquinic acid. Experiments in vitro showed that AFR and AFP protected against HepG2 cell death induced by tert-butyl hydroperoxide (t-BHP) as determined by cell viability assay. AFR and AFP recovered alanine aminotransferase (ALT) and aspartate aminotransferase (AST) enzyme activities, which increased by t-BHP treatment in HepG2 cells. AFR and AFP also inhibited the cleavage of PARP and caspase-3 in HepG2 cells. Moreover, in vivo experiments in mice showed that AFR and AFP protected against t-BHP-induced hepatotoxicity and recovered serum ALT and AST enzyme activities. AFR and AFP also rescued serum antioxidant enzyme activities, catalase and glutathione peroxidase, which were disturbed by t-BHP treatment. Our data clearly demonstrate that AFR and AFP protect against hepatic damage induced by oxidative stress. Practical Applications Colored potatoes contain plentiful amounts of antioxidant compounds that can be developed into dietary supplements for the improvement of human health. We prepared antioxidant-enriched fractions from red and purple colored potatoes (AFR and AFP, respectively) to be used as hepatoprotective agents. Based on chemical profiling analysis, we found that 5-caffeoylquinic acid can serve as a marker for the standardization of AFR and AFP as functional food materials. AFR and AFP strongly protected against oxidative stress and injury both in cultured hepatocytes and mice. AFR and AFP prevented the cell death of hepatocytes, and they also rescued hepatic damage markers ALT and AST both in cell and animal experiments. Generally, AFP showed higher hepatoprotective activity than AFR. Taken together, AFR and AFP may be promising candidates as functional foods for improving human liver function.

The Effect of ADAM8 on the Proliferation and Apoptosis of Hepatocytes and Hepatoma Carcinoma Cells.

This study was undertaken to evaluate the effect of ADAM8 on the proliferation and apoptosis of hepatocytes and hepatoma carcinoma cells during hepatocellular carcinoma (HCC) progression. The expression of ADAM8 was significantly increased with good correlation of PCNA expression increasing and cells apoptosis decreasing during the progression of HCC in the liver of mice. Proliferation experiment in vitro showed that recombinant ADAM8 could induce the expression of PCNA in L02 cells, but not in HepG2 cells. Apoptosis experiment in vitro showed that recombinant ADAM8 did not induce or inhibit the expression of apoptosis-related factors Bcl2, Bax, and Caspase3 in L02 cells, but significantly induced the expression of Bcl2, inhibited the expression of Bax and Caspase3 in HepG2 cells. In conclusion, our study suggested that ADAM8 could promote the proliferation of normal hepatocytes and render hepatoma carcinoma cells more resistant to apoptosis to play important roles during the progression of HCC. ADAM8; Proliferation; Apoptosis.

Sasanquasaponin from Camellia oleifera Abel. induces apoptosis via Bcl-2, Bax and caspase-3 activation in HepG2 cells.

The present study aimed to elucidate the molecular mechanisms underlying the induction of cytotoxic effects by sasanquasaponin (SQS) in HepG2 cells. Following SQS treatment, time- and dose-dependent increases in the apoptotic rate were observed. The induction of cell death by SQS mainly occurs via programmed cell death, as indicated by Annexin V-fluorescein isothiocyanate and propidium iodide staining, where up to 30% apoptotic cells were detected following 12 h SQS treatment. Reverse transcription-polymerase chain reaction analysis demonstrated that SQS treatment upregulated B-cell lymphoma-2 (Bcl-2)-associated x protein and caspase-3 mRNA expression and downregulated Bcl-2 mRNA expression. Greater alterations in Bax, Bcl-2 and caspase-3 expression were observed with increasing treatment duration. The decrease in Bcl-2, increase in Bax and, finally, the activation of caspase-3 in HepG2 cells indicated that the apoptotic process induced by SQS was irreversible. The results of the present study therefore suggested that SQS induced HepG2 cell apoptosis via the activation of mitochondrial apoptotic pathways.

Resveratrol Differentially Regulates NAMPT and SIRT1 in Hepatocarcinoma Cells and Primary Human Hepatocytes

Resveratrol is reported to possess chemotherapeutic properties in several cancers. In this study, we wanted to investigate the molecular mechanisms of resveratrol-induced cell cycle arrest and apoptosis as well as the impact of resveratrol on NAMPT and SIRT1 protein function and asked whether there are differences in hepatocarcinoma cells (HepG2, Hep3B cells) and non-cancerous primary human hepatocytes. We found a lower basal NAMPT mRNA and protein expression in hepatocarcinoma cells compared to primary hepatocytes. In contrast, SIRT1 was significantly higher expressed in hepatocarcinoma cells than in primary hepatocytes. Resveratrol induced cell cycle arrest in the S- and G2/M- phase and apoptosis was mediated by activation of p53 and caspase-3 in HepG2 cells. In contrast to primary hepatocytes, resveratrol treated HepG2 cells showed a reduction of NAMPT enzymatic activity and increased p53 acetylation (K382). Resveratrol induced NAMPT release from HepG2 cells which was associated with increased NAMPT mRNA expression. This effect was absent in primary hepatocytes where resveratrol was shown to function as NAMPT and SIRT1 activator. SIRT1 inhibition by EX527 resembled resveratrol effects on HepG2 cells. Furthermore, a SIRT1 overexpression significantly decreased both p53 hyperacetylation and resveratrol-induced NAMPT release as well as S-phase arrest in HepG2 cells. We could show that NAMPT and SIRT1 are differentially regulated by resveratrol in hepatocarcinoma cells and primary hepatocytes and that resveratrol did not act as a SIRT1 activator in hepatocarcinoma cells.

Bear bile powder (熊胆粉) induces apoptosis of human hepatocellular carcinoma cells via mitochondrion-dependent pathway

To evaluate the effect of Bear Bile Powder(, BBP) on the growth and apoptosis of HepG2 human hepatocellular carcinoma cells, and investigate the possible molecular mechanisms mediating its anti-cancer activity. HepG2 cells were treated with 0.4-1.0 mg/mL of BBP for 24, 48 and 72 h. The viability of HePG2 cells was determined by MTT assay. Cellular morphology was observed via phase-contrast microscopy. Fluorescence-activated cell sorting analysis with Annexin-V/propidium idodide and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazol-carbocyanine iodide (JC-1) staining was performed to determine cell apoptosis and the loss of mitochondrial membrane potential, respectively. Activation of caspase-9 and -3 was evaluated by a colorimetric assay. The treatment with 0.4-1 mg/mL of BBP for 24, 48, or 72 h respectively reduced cell viability significantly by 7%-60%, 20%-90% or 25%-98%, compared with the untreated control cells (P<0.01). In addition, BBP treatment induced morphological changes in HepG2 cells. Furthermore, after treated with 0, 0.4, 0.6, 0.8 and 1.0 mg/mL of BBP, apoptosis cells (including early and late apoptotic cells) were 18.0%±1.3%, 34.9%±2.2%, 33.9%±2.8%, 37.4%±2.8% and 46.0%±2.5%, respectively (P<0.05); and the percentage of cells with reduced JC-1 red fluorescence were 6.6%±0.8%, 8.5%±0.8%, 13.5%±1.6%, 17.6%±2.3% and 46.7%±3.6%, respectively (P<0.01). Finally, BBP treatment significantly and dose-dependently induced activation of both caspase-9 and caspase-3 in HepG2 cells (P<0.05). BBP could inhibit the growth of HepG2 hepatocellular cancer cells through mitochondrion-mediated apoptosis, which may, in part, explain its anti-cancer activity. BBP may be a potential novel therapeutic agent for the treatment of hepatocellular carcinoma.

A standardized extract from Paeonia lactiflora and Astragalus membranaceus induces apoptosis and inhibits the proliferation, migration and invasion of human hepatoma cell lines

Paeonia lactiflora and Astragalus membranaceus are two traditional Chinese medicines, which are commonly used in Chinese herb prescription to treat liver diseases. The protective effects of the extract prepared from the roots of Paeonia lactiflora and Astragalus membranaceus (PAE) on liver fibrosis have been demonstrated in previous studies. However, its effect on hepatocellular carcinoma (HCC) has not been investigated to date. In this study, the effects of PAE on the apoptosis, proliferation, migration and invasion of the human hepatoma cell lines HepG2 and SMMC-7721 were investigated. Our data demonstrated that treatment with PAE (50-200 mg/l) caused an inhibitory effect on the proliferation of the hepatoma cell lines HepG2 and SMMC-7721. Furthermore, PAE induced apoptosis of HepG2 cells and SMMC-7721 cells, which was demonstrated by PI staining. In addition, immunocytochemistry and western blotting showed that PAE significantly decreased the expression of Bcl-2, while the expression of Bax and cleaved caspase-3 in HepG2 cells and SMMC-7721 cells was significantly increased after treatment with PAE. These results clearly demonstrated that PAE induced hepatoma cell apoptosis through increasing the Bax-to-Bcl-2 ratio and upregulating the activation of caspase-3. In addition, the results of wound healing assay and Matrigel invasion assay showed that PAE displayed inhibitory activity on the migration and invasion of HCC cells. Taken together, the present data provides evidence that PAE is a potent antineoplastic drug candidate for the treatment of HCC.

Telekin Induces Apoptosis Associated with the Mitochondria-Mediated Pathway in Human Hepatocellular Carcinoma Cells

Telekin, a eudesmane-type sesquiterpene lactone compound isolated from Chinese folk medicine Carpesium divaricatum, has been reported to strongly inhibit the proliferation of cancer cells. In this study, the involvement of a mitochondria-mediated pathway in the pro-apoptotic action of telekin was investigated in human hepatocellular carcinoma cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays showed that telekin exhibited excellent anti-proliferation activity in hepatocellular carcinoma cells and low cytotoxicity to normal hepatocyte cells. Telekin-induced apoptosis was characterized by chromatin condensation, formation of apoptotic bodies, and exposure of phosphatidylserine on the extracellular surface, as revealed by 4,6-diamidino-2-phenylindole (DAPI) nuclear staining and flow cytometry. Flow cytometry analysis showed that telekin induced the loss of mitochondrial membrane potential (MMP), as well as increased the levels of intracellular free calcium and reactive oxygen species (ROS). Additionally, Western blot results demonstrated that telekin induced the decrease in Apaf-1 and Bcl-2 expression, increase in Bax expression, release of cytochrome C, and activation of caspase-9 and caspase-3 in HepG-2 cells. These findings indicate that telekin activates the mitochondria-mediated apoptotic pathway in hepatocellular carcinoma cells and may merit further investigation as a potential therapeutic agent for the treatment of hepatocellular carcinoma.

Mono-2-ethylhexyl phthalate induced loss of mitochondrial membrane potential and activation of Caspase3 in HepG2 cells

L02 and HepG2 cells were exposed to mono-(2-ethylhexyl) phthalate (MEHP) at concentrations of 6.25–100μM. After 48h treatment, MEHP decreased HepG2 cell viability in a concentration-dependent manner and L02 cell viability in the 50 and 100μM groups (p<0.01). Furthermore, at 24 and 48h after treatment, MEHP decreased the glutathione levels of HepG2 cells in all treatment groups and in the ΔΨm in L02 and HepG2 cells with MEHP≥25μM (p<0.05 or p<0.01). At 24h after treatment, MEHP induced activation of caspase3 in all treated HepG2 and L02 cells (p<0.05 or p<0.01) except the 100μM MEHP treatment group. The increase in the Bax to Bcl-2 ratio suggests that Bcl-2 family involved in the control of MEHP-induced apoptosis in these two cell types. The data suggest that MEHP could induce apoptosis of HepG2 cells through mitochondria- and caspase3-dependent pathways.

The Growth Suppressing Effects of Girinimbine on Hepg2 Involve Induction of Apoptosis and Cell Cycle Arrest

Murraya koenigii is an edible herb widely used in folk medicine. Here we report that girinimbine, a carbazole alkaloid isolated from this plant, inhibited the growth and induced apoptosis in human hepatocellular carcinoma, HepG2 cells. The MTT and LDH assay results showed that girinimbine decreased cell viability and increased cytotoxicity in a dose-and time-dependent manner selectively. Girinimbine-treated HepG2 cells showed typical morphological features of apoptosis, as observed from normal inverted microscopy and Hoechst 33342 assay. Furthermore, girinimbine treatment resulted in DNA fragmentation and elevated levels of caspase-3 in HepG2 cells. Girinimbine treatment also displayed a time-dependent accumulation of the Sub-G0/G1 peak (hypodiploid) and caused G0/G1-phase arrest. Together, these results demonstrated for the first time that girinimbine could effectively induce programmed cell death in HepG2 cells and suggests the importance of conducting further investigations in preclinical human hepatocellular carcinoma models, especially on in vivo efficacy, to promote girinimbine for use as an anticancer agent against hepatocellular carcinoma.

In vitro and in vivo anticancer activity evaluation of ursolic acid derivatives

Twenty-three ursolic acid (1) derivatives 2– 24 (ten novel compounds 8– 10, 14– 17 and 22– 24) modified at the C-3 and the C-28 positions were synthesized, and their structures were confirmed by IR, 1H NMR, MS, and elemental analysis. The single crystals of compounds 15 and 17 were obtained. The cytotoxic activity of the derivatives was evaluated against HepG2, BGC-823, SH-SY5Y, HeLa and HELF cells by the MTT assay. The induction of apoptosis and affects on the cell cycle distribution with compound 14 were assessed by fluorescence microscopy, flow cytometry and the activity of caspase-3 in HepG2 cells. Compounds 14– 17 had more significant antiproliferative ability against the four cancer cell lines and low cytotoxicity to human embryonic lung fibroblast cells (HELF). Compounds 11, 14– 16, 21 and 23 were particularly active against HepG2 cell growth. Compound 14 was selected to investigate cell apoptosis and cell cycle distribution. Flow cytometric analysis and morphologic changes of the cell exhibited that treatment of HepG2 cells with compound 14 led to cell apoptosis accompanied by cell cycle arrest at the S phase in a dose-dependent manner. Furthermore, the activity of the caspase-3 enzyme was increased in the treated cells. In vivo studies using H22 xenografts in Kunming mice were conducted with compound 14 at doses of 50, 100 and 150 mg/kg body weight. The results revealed that the medium dosage group (100 mg/kg) showed significant anticancer activity (45.6 ± 4.3%) compared to the control group.