Apoptotic Rate Of HepG2 Cells Research Articles

Synergistic Effect of Doxorubicin and Blue Light Irradiation on the Antitumor Treatment of HepG2 Cells in Liver Cancer.

Doxorubicin (DOX) has been an effective antitumor agent for human liver cancer cells; however, an overdose might lead to major side effects appearing in clinical applications. In this work, we present a strategy of combining DOX and blue light (BL) irradiation for the antitumor treatment of HepG2 cells (one typical human liver cancer cell line). It is demonstrated that synergetic DOX and BL can significantly reduce cell proliferation and increase the apoptotic rate of HepG2 cells in comparison to individual DOX treatment. The additional BL irradiation is further helpful for enhancing the inhibition of cell migration and invasion. Analyses of reactive oxygen species (ROS) level and Western blotting reveal that the strategy results in more ROS accumulation, mitochondrial damage, and the upregulation of proapoptotic protein (Bcl-2) and downregulation of antiapoptotic protein (Bax). In addition to the improved therapeutic effect, the non-contact BL irradiation is greatly helpful for reducing the dosage of DOX, and subsequently reduces the side effects caused by the DOX drug. These findings offer a novel perspective for the therapeutic approach toward liver cancer with high efficiency and reduced side effects.

Retraction Note: Matrine promotes liver cancer cell apoptosis by inhibiting mitophagy and PINK1/Parkin pathways.

Matrine is a natural alkaloid isolated from the root and stem of the legume plant Sophora. Its anti-proliferative and pro-apoptotic effects on several types of cancer have been well-documented. However, the role of matrine in regulating mitochondrial homeostasis, particularly mitophagy in liver cancer apoptosis, remains uncertain. The aim of our study was to explore whether matrine promotes liver cancer cell apoptosis by modifying mitophagy. HepG2 cells were used in the study and treated with different doses of matrine. Cell viability and apoptosis were determined by MTT assay, TUNEL staining, western blotting, and LDH release assay. Mitophagy was monitored by immunofluorescence assay and western blotting. Mitochondrial function was assessed by immunofluorescence assay, ELISA, and western blotting. The results of our study indicated that matrine treatment dose-dependently reduced cell viability and increased the apoptotic rate of HepG2 cells. Functional studies demonstrated that matrine treatment induced mitochondrial dysfunction and activated mitochondrial apoptosis by inhibiting protective mitophagy. Re-activation of mitophagy abolished the pro-apoptotic effects of matrine on HepG2 cells. Molecular investigations further confirmed that matrine regulated mitophagy via the PINK1/Parkin pathways. Matrine blocked the PINK1/Parkin pathways and repressed mitophagy, whereas activation of the PINK1/Parkin pathways increased mitophagy activity and promoted HepG2 cell survival in the presence of matrine. Together, our data indicated that matrine promoted HepG2 cell apoptosis through a novel mechanism that acted via inhibiting mitophagy and the PINK1/Parkin pathways. This finding provides new insight into the molecular mechanism of matrine for treating liver cancer and offers a potential target to repress liver cancer progression by modulating mitophagy and the PINK1/Parkin pathways.

Role of the Death Receptor and Endoplasmic Reticulum Stress Signaling Pathways in Polyphyllin I-Regulated Apoptosis of Human Hepatocellular Carcinoma HepG2 Cells.

Polyphyllin has been reported to exhibit anticancer effects against various types of cancer via the proapoptotic signaling pathway. The aim of the present study was to investigate the role of the endoplasmic reticulum stress and death receptor signaling pathways in PPI-induced apoptosis of human hepatocellular carcinoma HepG2 cells. Analysis demonstrated that PPI could significantly inhibit the proliferation and induce apoptosis of HepG2 cells in a dose- and time-dependent manner. Investigation into the molecular mechanism of PPI indicated that PPI notably mediated ER stress activation via IRE-1 overexpression and activation of the caspase-12 to protect HepG2 cells against apoptosis. In addition, PPI markedly induced the expression of death receptors signaling pathways-associated factors, including tumor necrosis factor (TNF) receptor 1/TNF-α and FAS/FASL. Additionally, suppression of the death receptor signaling pathways with a caspase-8 inhibitor, Z-IETD-FMK, revealed an increase in the death rate and apoptotic rate of HepG2 cells. Collectively, the findings of the present study suggested that the ER stress and death receptor signaling pathways were associated with PPI-induced HepG2 cell apoptosis; however, endoplasmic reticulum stress may serve a protective role in this process. The combination of PPI and Z-IETD-FMK may activate necroptosis, which enhances apoptosis. Therefore, the results of the present study may improve understanding regarding the roles of signaling pathways in PPI regulated apoptosis and contribute to the development of novel therapies for the treatment of HCC.

Matrine promotes liver cancer cell apoptosis by inhibiting mitophagy and PINK1/Parkin pathways.

Matrine is a natural alkaloid isolated from the root and stem of the legume plant Sophora. Its anti-proliferative and pro-apoptotic effects on several types of cancer have been well-documented. However, the role of matrine in regulating mitochondrial homeostasis, particularly mitophagy in liver cancer apoptosis, remains uncertain. The aim of our study was to explore whether matrine promotes liver cancer cell apoptosis by modifying mitophagy. HepG2 cells were used in the study and treated with different doses of matrine. Cell viability and apoptosis were determined by MTT assay, TUNEL staining, western blotting, and LDH release assay. Mitophagy was monitored by immunofluorescence assay and western blotting. Mitochondrial function was assessed by immunofluorescence assay, ELISA, and western blotting. The results of our study indicated that matrine treatment dose-dependently reduced cell viability and increased the apoptotic rate of HepG2 cells. Functional studies demonstrated that matrine treatment induced mitochondrial dysfunction and activated mitochondrial apoptosis by inhibiting protective mitophagy. Re-activation of mitophagy abolished the pro-apoptotic effects of matrine on HepG2 cells. Molecular investigations further confirmed that matrine regulated mitophagy via the PINK1/Parkin pathways. Matrine blocked the PINK1/Parkin pathways and repressed mitophagy, whereas activation of the PINK1/Parkin pathways increased mitophagy activity and promoted HepG2 cell survival in the presence of matrine. Together, our data indicated that matrine promoted HepG2 cell apoptosis through a novel mechanism that acted via inhibiting mitophagy and the PINK1/Parkin pathways. This finding provides new insight into the molecular mechanism of matrine for treating liver cancer and offers a potential target to repress liver cancer progression by modulating mitophagy and the PINK1/Parkin pathways.

FPOA induces the apoptosis of HepG2 cells.

The triterpenoid 3-acetoxylanosta-8,24-dien-21-oic acid (FPOA) is isolated from the fruiting body of Fomitopsis pinicola. The present study reports that FPOA exerts cytotoxic activity and describes the molecular mechanism of FPOA-induced apoptosis on human HepG2 hepatoma cells. FPOA exhibited significant cytotoxic effects against HepG2, MCF-7 and HeLa cells. However, FPOA was particularly cytotoxic towards HepG2 cells, with a half maximal inhibitory concentration value of 42.10 µM, thus these cells were taken forward for further analysis. Flow cytometry results demonstrated that FPOA significantly increased the apoptotic rate of HepG2 cells in a dose-dependent manner, explaining its potent cytotoxicity. In addition, western blot analysis revealed that FPOA significantly increased the B-cell lymphoma 2 (Bcl-2)-associated X/Bcl-2 ratio, and cytochrome c, caspase-9 and caspase-3 release, in addition to significantly decreasing poly(ADP) ribose polymerase levels. These observations indicate that FPOA induces the apoptosis of HepG2 cells by activating members of the caspase protein family and triggering the mitochondrial apoptosis signaling pathway. Based on these results, FPOA is a potential agent for the treatment of cancer.

Endoplasmic reticulum stress mediates sulforaphane-induced apoptosis of HepG2 human hepatocellular carcinoma cells.

Sulforaphane (SFN) is a naturally occurring chemopreventive agent, which effectively inhibits proliferation of HepG2 human hepatocellular carcinoma cells via mitochondria‑mediated apoptosis. Endoplasmic reticulum stress is considered the most important cause of cell apoptosis; therefore, the present study aimed to determine whether the endoplasmic reticulum pathway was involved in SFN-induced apoptosis of HepG2 cells. An MTT assay was used to detect the inhibitory effects of SFN on HepG2 cells. Fluorescence microscopy was used to observe the morphological changes in apoptotic cells, and western blot analysis was conducted to detect the expression of binding immunoglobulin protein (Bip)/glucose-regulated protein78 (GRP78), X‑box binding protein‑1 (XBP‑1) and BH3 interacting domain death agonist (Bid). Furthermore, flow cytometry was used to determine the apoptotic rate of HepG2 cells, and the protein expression of C/EBP homologous protein (CHOP)/growth arrest‑ and DNA damage‑inducible gene153 (GADD153) and caspase-12 in HepG2 cells. The results indicated that SFN significantly inhibited the proliferation of HepG2 cells; the half maximal inhibitory concentration values were 32.03±0.96, 20.90±1.96 and 13.87±0.44µmol/l, following treatment with SFN for 24, 48and72h, respectively. Following 48h of SFN treatment (10, 20and40µmol/l), the apoptotic rates of HepG2 cells were 31.8, 61.3and77.1%, respectively. Furthermore, after 48h of exposure to SFN, the cells presented typical morphological alterations of apoptosis, as detected under fluorescence microscopy. Treatment with SFN for 48h also significantly upregulated the protein expression levels of Bip/GRP78, XBP‑1, caspase‑12, CHOP/GADD153 and Bid in HepG2 cells. In conclusion, endoplasmic reticulum stress may be considered the most important mechanism underlying SFN-induced apoptosis in HepG2 cells.

On-demand drug release of ICG-liposomal wedelolactone combined photothermal therapy for tumor

In the study, a new photoresponsive nano drug delivery system was developed by encapsulating indocyanine green into liposomes (ICG-liposomal wedelolactone), which could improve the water solubility and bioavailability of wedelolactone. The hyperthermia, produced by ICG under near-infrared (NIR) light irradiation, promoted wedelolactone release rapidly from the carriers. The release amount of ICG-liposomal wedelolactone under NIR irradiation reached up to 96.74% over 8h, achieving the drug of on-demand release. Moreover, the growth of HepG2 cells was obviously inhibited by ICG-liposomal wedelolactone under NIR, and the early apoptotic rate of HepG2 cells was 33.74%. The tumor inhibition rate was 81% in the mice bearing tumor treated with the drug system. The results proved that ICG-liposomal wedelolactone, as a novel drug delivery system to co-delivery chemotherapeutic agents and photothermal agents, achieved synergetic effect of chemotherapy and photothermotherapy, which will have an enormous potential in future cancer therapy.

Anticancer effect of SN-38 combined with sorafenib on hepatocellular carcinoma in vitro and its mechanism

To investigate the anticancer effect and its mechanism of SN-38 combined with sorafenib on hepatocellular cancer cell lines HepG-2 and BEL-7402. SRB colorimetry was employed to measure the viability of HepG-2 and BEL-7402 cells after the treatment of SN-38 with sorafenib. Propidium iodide flow cytometric assay and DAPI staining were used to evaluate the apoptosis of HCC cells. Western blotting was conducted to detect the expression level of apoptosis-related and DNA damage-related proteins. SRB colorimetry showed the synergistic anticancer activities of SN-38 combined with sorafenib, with a combination index of <0.9. The apoptotic rates of HepG-2 cells in control, 60 nmol/L SN-38, 2.5μmol/L sorafenib and combination groups were 4.25%±2.45%, 28.95%±10.75%, 3.49%±2.49% and 53.19%±11.21%, respectively(P<0.05). Western blotting showed that the combination of these two drugs increased the enzymolysis of PARP, Caspase-8 and Caspase-3, and promoted the expression levels of p53, p21 and γ-H2AX significantly. SN-38 and sorafenib have synergistic anticancer activity on hepatocellular carcinoma cells in vitro with the augmentation of apoptosis.

MicroRNA-122 mimic transfection contributes to apoptosis in HepG2 cells.

There is currently a requirement for effective treatment strategies for human hepatocellular carcinoma (HCC), a leading cause of cancer‑associated mortality. MicroRNA-122 (miR-122), a repressor of the endogenous apoptosis regulator Bcl‑w, is frequently downregulated in HCC. Thus, it is hypothesized that the activation of miR‑122 may induce selective hepatocellular apoptosis via caspase activation in a model of HCC. In the present study, an miR‑122 mimic transfection was performed in HepG2 cells, and used to investigate the role and therapeutic potential of miR‑122 in the regulation of HCC‑derived cell lines. The apoptotic rates of HepG2 cells were significantly increased following miR‑122 mimic transfection. Reverse transcription‑polymerase chain reaction analysis revealed that Bcl‑w mRNA was significantly reduced, while the mRNA levels of caspase‑9 and caspase‑3 were markedly increased. The immunocytochemistry results supported the mRNA trends. Collectively, the present results suggest that endogenous miR‑122 contributes to HepG2 apoptosis and that transfection of mimic miR‑122 normalizes apoptotic levels in a model of HCC.

Photothermal treatment of liver cancer with albumin-conjugated gold nanoparticles initiates Golgi Apparatus-ER dysfunction and caspase-3 apoptotic pathway activation by selective targeting of Gp60 receptor.

We present a method of enhanced laser thermal ablation of HepG2 cells based on a simple gold nanoparticle (GNP) carrier system such as serum albumin (Alb), and demonstrate its selective therapeutic efficacy compared with normal hepatocyte cells. HepG2 or hepatocytes were treated with Alb-GNPs at various concentrations and various incubation times, and further irradiated using a 2 W, 808 nm laser. Darkfield microscopy and immunochemical staining was used to demonstrate the selective internalization of Alb-GNPs inside the HepG2 cells via Gp60 receptors targeting. The postirradiation apoptotic rate of HepG2 cells treated with Alb-GNPs ranged from 25.8% (for 5 μg/mL) to 48.2% (for 50 μg/mL) at 60 seconds, while at 30 minutes the necrotic rate increased from 35.7% (5 μg/mL) to 52.3% (50 μg/mL), P-value <0.001. Significantly lower necrotic rates were obtained when human hepatocytes were treated with Alb-GNPs in a similar manner. We also showed by means of immunocytochemistry that photothermal treatment of Alb-conjugated GNPs in liver cancer initiates Golgi apparatus–endoplasmic reticulum dysfunction with consequent caspase-3 apoptotic pathway activation and cellular apoptosis. The presented results may become a new method of treating cancer cells by selective therapeutic vectors using nanolocalized thermal ablation by laser heating.

Cecropin-P17, an analog of Cecropin B, inhibits human hepatocellular carcinoma cell HepG-2 proliferation via regulation of ROS, Caspase, Bax, and Bcl-2.

Cecropin-P17 is a peptide derived from Cecropin B. In this study, we investigated the effects and relative mechanisms of Cecropin-P17 in a human liver cancer cell line (HepG-2) in vitro and in vivo. A cell viability assay, Annexin V/propidium iodide assay, western blot, flow cytometry, quantitative real-time polymerase chain reaction, and a tumor-xenograft model were applied to elucidate the mechanism exerted by Cecropin-P17 on HepG-2 cells. Cecropin-P17 significantly inhibited the proliferation of HepG-2 cells and demonstrated low cytotoxicity to normal liver cells in vitro. The apoptotic rate of HepG-2 cells was increased after Cecropin-P17 treatment together with increased production of reactive oxygen species. Moreover, Cecropin-P17 stimulated caspase-3, caspase-9, and Bax and inhibited Bcl-2 on both the transcriptional and translational levels. Finally, Cecropin-P17 significantly suppressed tumor growth in a HepG-2-bearing nude mouse model. All of these results indicated that Cecropin-P17 could be a potential agent for the treatment of liver cancer.

Notch1 downregulation combined with interleukin-24 inhibits invasion and migration of hepatocellular carcinoma cells.

To confirm the anti-invasion and anti-migration effects of down-regulation of Notch1 combined with interleukin (IL)-24 in hepatocellular carcinoma (HCC) cells. γ-secretase inhibitors (GSIs) were used to down-regulate Notch1. HepG2 and SMMC7721 cells were seeded in 96-well plates and treated with GSI-I or/and IL-24 for 48 h. Cell viability was measured by MTT assay. The cellular and nuclear morphology was observed under a fluorescence microscope. To further verify the apoptotic phenotype, cell cultures were also analyzed by flow cytometry with Annexin V-FITC/propidium iodide staining. The expression of Notch1, SNAIL1, SNAIL2, E-cadherin, IL-24, XIAP and VEGF was detected by Western blot. The invasion and migration capacities of HCC cells were detected by wound healing assays. Notch1 and Snail were down-regulated by RNA interference, and the target proteins were analyzed by Western blot. To investigate the mechanism of apoptosis, we analyzed HepG2 cells treated with siNotch1 or siCON plus IL-24 or not for 48 h by caspase-3/7 activity luminescent assay. GSI-I at a dose of 2.5 μmol/L for 24 h caused a reduction in cell viability of about 38% in HepG2 cells. The addition of 50 ng/mL IL-24 in combination with 1 or 2.5 μmol/L GSI-I reduced cell viability of about 30% and 15%, respectively. Treatment with IL-24 alone did not induce any cytotoxic effect. In SMMC7721 cells with the addition of IL-24 to GSI-I (2.5 μmol/L), the reduction of cell viability was only about 25%. Following GSI-I/IL-24 combined treatment for 6 h, the apoptotic rate of HepG2 cells was 47.2%, while no significant effect was observed in cells treated with the compounds employed separately. Decreased expression of Notch1 and its associated proteins SNAIL1 and SNAIL2 was detected in HepG2 cells. Increased E-cadherin protein expression was noted in the presence of IL-24 and GSI-I. Furthermore, the increased GSI-I and IL-24 in HepG2 cell was associated with downregulation of MMP-2, XIAP and VEGF. In the absence of treatment, HepG2 cells could migrate into the scratched space in 24 h. With IL-24 or GSI-I treatment, the wound was still open after 24 h. And the distance of the wound closure strongly correlated with the concentrations of IL-24 and GSI-I. Treatment of Notch-1 silenced HepG2 cells with 50 ng/mL IL-24 alone for 48 h induced cytotoxic effects very similar to those observed in non-silenced cells treated with GSI-I/IL-24 combination. Caspase-3/7 activity was increased in the presence of siNotch1 plus IL-24 treatment. Down-regulation of Notch1 by GSI-I or siRNA combined with IL-24 can sensitize apoptosis and decrease the invasion and migration capabilities of HepG2 cells.

ASPP2 gene silence promotes hepatocellular carcinoma cell proliferation via regulating autophagy under starvation

Objective To study the effects of apoptosis-stimulating protein of p53-2(ASPP2) gene on hepatocellular carcinoma(HCC) cell proliferation and apoptosis under starvation and the related mechanism.MethodsLentivirus encoding shRNA against ASPP2 was constructed to knockdown ASPP2 expression in hepatoma cell HepG2.Cell proliferation and apoptosis were observed by transmission electron microscopy,MTS analysis,flow cytometry analysis and morphologic changes. The influence of silencing ASPP2 gene on the proliferation and apoptosis under amino acid-starvation and serum-deprivation culture was observed;autophagy inhibitor 3-methyladenine(3-MA) was added in the experiment so as to detect the involvement of autophagy in the changes induced by ASPP2 down-regulation. ResultsTransmission electron microscopy showed cytoplasmic accumulation of autophagosomes when ASPP2 was knocked down under amino acid-starvation and serum-deprivation(P0.05),with increased GFP-LC3 dots(P0.05).MTS analysis showed that silence of ASPP2 gene greatly enhanced the proliferation of HepG2 cells(P0.05),which could be inhibited by addition of 3-MA(P0.05).Microscope observation showed that silence of ASPP2 gene promoted the anti-apoptotic ability of HepG2 cells,which was reversed by treatment with 3-MA.Early sign of apoptosis was observed in shASPP2+3-MA group. Annexin Ⅴ-PI double staining showed that ASPP2 silence decreased the apoptotic rate of HepG2 cells from(38±5)% to(15±4)%(P0.05),and 3-MA treatment increased it to(36±3)%(P0.05).ConclusionDown-regulation of ASPP2 expression may facilitate the survival and proliferation of HCC cells through activating autophagy under starvation.

Hypocrellin B-mediated sonodynamic action induces apoptosis of hepatocellular carcinoma cells

ObjectiveThe present study aims to investigate apoptosis of hepatocellular carcinoma cells induced by hypocrellin B-mediated sonodynamic action. MethodsThe hypocrellin B concentration was kept constant at 2.5μM and cells from the hepatocellular carcinoma HepG2 cell line were exposed to ultrasound with an intensity of 0.46W/cm2 for 8s. Cell cytotoxicity was quantified using an MTT assay 24h after sonodynamic therapy (SDT) of hypocrellin B. Apoptosis was investigated using a flow cytometry with Annexin V-FITC and propidium iodine staining. Intracellular reactive oxygen species (ROS) levels were detected using a flow cytometry with 2,7-dichlorodihydrofluorecein diacetate (DCFH-DA) staining. ResultsThe cytotoxicity of hypocrellin B-mediated sonodynamic action on HepG2 cells was significantly higher than those of other treatments including ultrasound alone, hypocrellin B alone and sham treatment. Flow cytometry showed that hypocrellin B-induced sonodynamic action markedly enhanced the apoptotic rate of HepG2 cells. Increased ROS was observed in HepG2 cells after being treated with hypocrellin B-mediated sonodynamic action. ConclusionsOur data demonstrated that hypocrellin B-mediated sonodynamic action remarkably induced apoptosis of HepG2 cells, suggesting that apoptosis is an important mechanism of cell death induced by hypocrellin B-mediated SDT.

Enhanced laser thermal ablation for the in vitro treatment of liver cancer by specific delivery of multiwalled carbon nanotubes functionalized with human serum albumin.

The main goal of this investigation was to develop and test a new method of treatment for human hepatocellular carcinoma (HCC). We present a method of carbon nanotube-enhanced laser thermal ablation of HepG2 cells (human hepatocellular liver carcinoma cell line) based on a simple multiwalled carbon nanotube (MWCNT) carrier system, such as human serum albumin (HSA), and demonstrate its selective therapeutic efficacy compared with normal hepatocyte cells. Both HepG2 cells and hepatocytes were treated with HSA–MWCNTs at various concentrations and at various incubation times and further irradiated using a 2 W, 808 nm laser beam. Transmission electron, phase contrast, and confocal microscopy combined with immunochemical staining were used to demonstrate the selective internalization of HSA–MWCNTs via Gp60 receptors and the caveolin-mediated endocytosis inside HepG2 cells. The postirradiation apoptotic rate of HepG2 cells treated with HSA–MWCNTs ranged from 88.24% (for 50 mg/L) at 60 sec to 92.34% (for 50 mg/L) at 30 min. Significantly lower necrotic rates were obtained when human hepatocytes were treated with HSA–MWCNTs in a similar manner. Our results clearly show that HSA–MWCNTs selectively attach on the albondin (aka Gp60) receptor located on the HepG2 membrane, followed by an uptake through a caveolin-dependent endocytosis process. These unique results may represent a major step in liver cancer treatment using nanolocalized thermal ablation by laser heating.

The research of tumor necrosis factor-related apoptosis-inducing ligand in combination with sodium butyrate on hepatoma cell HepG2

Objective To investigate the inhibitory effects of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) combined with sodium butyrate (NaB) on hepatic carcinoma cell line HepG-2.Methods different concentrations of NaB( 1.0,2.5,5.0 mmol/L)and TRAIL ( 10,100,500,1000ng/ml) solely or combined with both added to the culture plates including HepG-2 suspension,inhibitory action of NaB and TRAIL on HepG-2 were Measured by MTT assays; The apoptotic rate of HepG-2 cells and the mRNA expressions of DR4 ,DR5 in HepG-2 cells were respectively detected by flow cytometry analysis and Real-time RT-PCR.Results By using different concentrations of TRAIL,the inhibitory rate of HepG-2 growth and HepG-2 apoptotic rate were ( 4.5 ± 1.3 ) % and 3.2 ± 0.7 ) % respectively,compared with control group,the differences were not significant statistically ( P ＞ 0.05 ).There was a dose-dependent inhibition of cell growth of HepG-2 by NaB ( P ＜ 0.05 ).The combination of the NaB and TRAIL can significantly increase the inhibitory rate of HepG-2 growth and HepG-2 apoptotic rate ( P ＜ 0.05 ).Expression level of DR5 mRNA of HepG-2 deal with NaB were higher than control group.Conclusion HepG-2 cells were resistant to apoptosis induced by TRAIL,NaB could enhance the sensitivity of Hep-2 cells to TRAIL by up-regulating the mRNA expressions of DR5 in HepG-2 cells. Key words: Apoptosis; TRAIL; Hepatoma; Sodium butyrate

Characterization of E2F3a function in HepG2 liver cancer cells

E2F3a is a transcription factor that has been shown to be overexpressed in liver cancer tissues. To characterize the function of E2F3a in hepatocellular carcinoma (HCC), effects of ectopic overexpression of E2F3a on cell cycle, apoptosis, and gene expression of HepG2 cells were studied. E2F3a significantly enhances the apoptotic rate of HepG2 cells by 33% but only has minor effects on cell proliferation. By using microarray analyses, we identified 162 target genes (160 upregulated and 2 downregulated) of the E2F3a. Differential expression of 11 genes was further confirmed by real-time PCR. Eight of these 11 genes, including XAF1, CEACAM1, STAT1, ATF3, TNFSF10, KLF6, CLDN1, and TAP1, were confirmed to be upregulated by more than twofold. Functional enrichments of differentially expressed genes retrieved 21 apoptosis-related genes and 32 transcriptional regulation-related genes. These results suggest that E2F3a induces apoptosis in HepG2 cells and plays important roles in regulating transcription. Finally, positive correlation was found between E2F3a and CEACAM1 mRNA levels in clinically well-differentiated human HCC specimens.

The molecular mechanisms of HepG2 apoptosis induced by Hedyotis diffusa

The present study was undertaken to investigate the molecular mechanisms of inhibitory effects on proliferation and apoptosis inducing effect of Hedyotis diffusa (EHD) in HepG2. The viability and proliferation of HepG2 cell co‐culturing with EHD extraction were measured by MTT. Apoptosis frequency of HepG2 cell co‐culturing with EHD extraction was detected by FCM. The expression of bcl‐2 and caspase‐3 were detected by immunohistochemical analysis. Results: EHD inhibited the growth of HepG2 cells in time and dose‐dependent way, obvious morphological and biomolecular changes of apoptosis of HepG2 cells were detected after 48h EHD treatment evidenced by microscope observation and typical DNA ladder, EHD increases the apoptotic rates of HepG2 cells in dose‐dependent way after 48h EHD treatment, immunohistochemical analysis showed that EHD decreased bcl‐2 expression and increased caspase‐3 expression in dose‐dependent way after 48h EHD treatment. EHD inhibits the proliferation of HepG2 cells and induces apoptosis of HepG2 cells which is time‐dependent and dose‐dependent way. The expression of bcl‐2 and caspase‐3 was involved in this mechanism.

Antitumor Activity of the Aqueous Extract from Sedum sarmentosum Bunge In Vitro

Sedum sarmentosum Bunge, a Chinese herb, is mainly used for the treatment of chronic viral hepatitis in China, The aim of this work was to examine the cytotoxic activity of S. sarmentosum (aqueous extract, AE) against a human hepatoma cell line (HepG2) in culture. Cell-proliferation ability was determined by the MTT method. Cell-cycle changes and earlier period apoptotic rate of HepG2 cells were detected by flow cytometry. Apoptosis of cultured HepG2 cells induced by AE were observed with a classic laddering pattern on agarose gel electrophoresis. The mRNA levels of Bcl-2 and vascular endothelial growth factor (VEGF) were determined by reverse-transcriptase polymerase chain reaction. The protein expressions of Bcl-2, VEGF, and p-STAT3 were valued by immunocytochemistry. The application of AE to the HepG2 cell culture caused a significant, dose-dependent inhibition of cancer cell growth. It was found that the AE treatment induced apoptosis of the cancer cells, although no changes were found after AE treatment for 48 hours in the HepG2 cell cycle. The mRNA and protein expressions of Bcl-2 and VEGF and the protein level of p-STAT3 were significantly decreased after the AE treatment for 48 hours. This study suggests that AE of S. sarmentosum has potential in preventing and inhibiting effects on hepatocellular carcinoma, which is associated with apoptosis of the cancer cells.

Epigallocatechin-3-gallate induces apoptosis in human hepatocellular carcinoma cells

To investigate the effects of epigallocatechin-3-gallate (EGCG) on human hepatocellular carcinoma (HCC) cells and mechanism thereof. Human HCC cells of the lines HepG2 and SMMC-7721 were cultured and treated with of EGCG of the concentrations of 6.25, 12.5, 25, 50, 100, 200, and 400 microg/ml respectively for 24 h and 48 h. The cell viability was assessed by 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Trypan blue staining was used to count the cells. Flow cytometry was conducted to detect the cell apoptosis. The protein levels of Bcl-2, an anti-apoptosis factor, and cyclooxygenase-2 (COX-2), an up-regulator of Bcl-2. The activities of caspase-9 and caspase-3 hat promote the apoptosis of HCC cells, were measured using colorimetric method. RT-PCR was used to detect the mRNA expression of COX-2 and Bcl-2 family. The viabilities of the HepG2 and SMMC-7721 cells treated with EGCG of the concentrations of 50 - 400 microg/ml for 48 h reduced to 93.8% +/- 2.8%, 62.3% +/- 5.4%, 33.9% +/- 2.5%, and 17.6% +/- 3.2% respectively, all significantly lower than that of the control group [(100.0% +/- 2.8%), all P < 0.05]; and the viabilities of the SMMC-772 cells treated with EGCG of the concentrations of 50 - 400 microg/ml for 48 h reduced to 49.6% +/- 3.5%, 30.3% +/- 3.8%, 17.7% +/- 2.2%, and 13.0% +/- 2.5% respectively, all significantly lower than that of the control group [(100.0% +/- 0.8%), all P < 0.05]. After treatment with 100 microg/ml EGCG for 24 h, 48 h, 72 h, and 96 h, the live HepG2 cell numbers were (8.0 +/- 1.5), (22.0 +/- 3.1), (37.0 +/- 5.4), and (61.0 +/- 8.7) 10(4) respectively, all significantly lower than those of the control cells [(15.0 +/- 2.5), (45.0 +/- 5.3), (86.0 +/- 11.0), and (210.0 +/- 23.0) 10(4) respectively, all P < 0.05]; and the live SMMC-7721 cell numbers were (7.0 +/- 2.2), (13.0 +/- 2.5), (20.0 +/- 3.7), and (31.0 +/- 4.0) 10(4) respectively, all significantly lower than those of the control cells [(15.0 +/- 2.5), (45.0 +/- 5.3), (86.0 +/- 11.0), and (210.0 +/- 23.0) 10(4) respectively, all P < 0.05]. The apoptotic rates of HepG2 cells treated with EGCG of the concentrations of 50, 100, and 200 microg/ml for 12 h were 8.7% +/- 0.4%, 18.1% +/- 1.1%, and 22.1% +/- 1.8% respectively, all significantly higher than that of the control group (3.3% +/- 0.3%, P < 0.05); and the apoptotic rates of SMMC-7721 cells were 5.9% +/- 0.3%, 7.8% +/- 0.6%, and 12.2% +/- 0.8% respectively, all significantly higher than that of the control group (3.7% +/- 0.4%, P < 0.05). After treatment with EGCG of the concentrations of 100 and 200 microg/ml for 12 h, the caspase-9 activities of the HepG2 cells increased to (1.8 +/- 0.4) and (2.5 +/- 0.4) respectively, both significantly higher than that of the control group (1.0 +/- 0.1, both P < 0.05); and the caspase-3 activities of the HepG2 cells increased to (2.0 +/- 0.4) and (2.8 +/- 0.5) respectively, both significantly higher than that of the control group (1.0 +/- 0.2, P < 0.05) ; and the caspase-9 activities of the SMMC-7721 cells increased to (1.7 +/- 0.4) and (2.5 +/- 0.4), both significantly higher than that of the control group (1.0 +/- 0.1, both P < 0.05), and the caspase-3 activities of the SMMC-7721 cells increased to (1.9 +/- 0.4) and (2.6 +/- 0.3) respectively, both significantly higher than that of the control group [ (1.0 +/- 0.2), both P < 0.05]. When the concentration of EGCG was over 200microg/ml, it down-regulated the expression of COX-2 and Bcl-2 in both cell lines, however, EGCG resulted in no significant changes of Bcl-xl, Bax, Bad, and Bid. EGCG induces apoptosis in HCC cells through down-regulation of COX-2 and Bcl-2 and consequently activating caspase-9 and caspase-3.