P53 In HepG2 Cells Research Articles

Hepatitis B Virus X Protein Induces Reactive Oxygen Species Generation via Activation of p53 in Human Hepatoma Cells.

Hepatitis B virus (HBV), particularly through the HBx protein, induces oxidative stress during liver infections. This study reveals that HBx increases reactive oxygen species (ROS) via two distinct mechanisms. The first mechanism is p53-independent, likely involving mitochondrial dysfunction, as demonstrated by elevated ROS levels in p53-deficient Hep3B cells and p53-knocked-down HepG2 cells after HBx expression or HBV infection. The increase in ROS persisted even when p53 transcriptional activity was inhibited by pifithrin-α (PFT-α), a p53 inhibitor. The second mechanism is p53-dependent, wherein HBx activates p53, which then amplifies ROS production through a feedback loop involving ROS and p53. The ability of HBx to elevate ROS levels was higher in HepG2 than in Hep3B cells. Knocking down p53 in HepG2 cells lowered ROS levels, while ectopic p53 expression in Hep3B cells raised ROS. HBx-activated p53 downregulated catalase and upregulated manganese-dependent superoxide dismutase, contributing to ROS amplification. The transcriptional activity of p53 was crucial for these effects, as cells with a p53 R175H mutation or those treated with PFT-α generated less ROS. Additionally, HBx variants with Ser-101 increased p53 and ROS levels, whereas variants with Pro-101 did not. These dual mechanisms of HBx-induced ROS generation are likely significant in the pathogenesis of HBV and may contribute to liver diseases, including hepatocellular carcinoma.

Hepatoprotective potential of lamivudine through activation of gamma-glutamyltransferase and down-regulation of p53 in HepG2 cells

The liver is a crucial metabolic organ that aids in detoxification, produces energy, synthesizes essential proteins, and is essential for maintaining good health. Since hepatocytes are the main type of cell found in liver tissue, maintaining their safety is essential for the general health of livers. The hepato-protective qualities of lamivudine shield the liver from stressful situations. The current study aimed to assess lamivudine's protective effects on HepG2 cells subjected to ethanol damage. Lamivudine was given to HepG2 cells after they were cultivated and subjected to ethanol-induced damage. Cell viability was assessed using the MTT, Crystal Violet, and Trypan Blue tests. The results of the MTT experiment demonstrated that lamivudine administration improved the rate of cell viability in a dose-dependent manner at 25, 50, and 100 μg/mL (0.570±0.0205; 0.73 ±0.02; 0.897±0.0206, respectively). According to the Crystal Violet test, lamivμdine-treated cells had higher cell viability than untreated cells; the rates were around 0.38 ± 0.01, 0.516 ± 0.0305, and 0.656 ± 0.041 in a dose dependant manner. The Trypan Blue assay results also confirmed that treatment with lamivudine at 25 μg/mL, 50 μg/mL, and 100 μg/mL led to a increase in the viability of cells compared to the injury group (183.66±5.50; 151±3.61; 115±5, respectively). Pretreatment of HepG2 cells with lamivudine during 100 µg/mL concentration exposure resulted in near-normalization of p53 expression levels (24.73±0.60), GSH levels (28.83±1.16), and in hepatic activity levels of GGT (21.23±1.12) compared to control. In immunofluorescence microscopy, the 100 µg/mL lamivudine group showed the most significant reduction in fluorescence intensity. Lamivudine provided convenient features in protecting HepG2 cells against ethanol-triggered liver damage by enhancing cell survival and antioxidant enzyme activities.

İsatinin HepG2 ve AML12 hücre hatları üzerindeki moleküler etkilerinin araştırılması

Isatin is an indole-derived organic compound. It is a natural component of the Couroupita guianensis plant. It is also the metabolic derivative of the human body hormone adrenaline. Studies have shown the anti-tumoral effects of isatin derivatives. In this study, the cytotoxic effects of isatin on HepG2, a hepatocellular cancer cell line was investigated. Additionally, its cytotoxic and protective-proliferative effects on AML12, a healthy liver cell line was investigated. This evaluation was conducted using MTT, fluorescent staining, wound healing, and real-time polymerase chain reaction analyses. The IC50 values for 48 hours of isatin application were calculated as 186.23 µM for HepG2 and 7.05 mM for AML12. The analysis of wound healing and fluorescent staining at varying doses of HepG2 application revealed suppression of proliferation and triggered apoptosis in HepG2 cells. In contrast, AML12 cells exhibited promoted proliferation under similar conditions. Moreover, the observed upregulation of oxidative stress genes CuZn/Mn-SOD and mitochondrial apoptotic pathway genes Bax, cleaveled-Cas3, APAF1, and p53 in HepG2 cells contrasted with their decreased expression in AML12 cell lines. These results suggest the potential of natural isatin as a promising anti-cancer agent for liver cancer cell lines and as a protective supplement for healthy liver cells.

Doxorubicin Attenuates Free Fatty Acid-Induced Lipid Accumulation via Stimulation of p53 in HepG2 Cells.

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive accumulation of fat in the liver, and there is a global increase in its incidence owing to changes in lifestyle and diet. Recent findings suggest that p53 is involved in the development of non-alcoholic fatty liver disease; however, the association between p53 expression and the disease remains unclear. Doxorubicin, an anticancer agent, increases the expression of p53. Therefore, this study aimed to investigate the role of doxorubicin-induced p53 upregulation in free fatty acid (FFA)-induced intracellular lipid accumulation. HepG2 cells were pretreated with 0.5 μg/mL of doxorubicin for 12 h, followed by treatment with FFA (0.5 mM) for 24 h to induce steatosis. Doxorubicin pretreatment upregulated p53 expression and downregulated the expression of endoplasmic reticulum stress- and lipid synthesis-associated genes in the FFA -treated HepG2 cells. Additionally, doxorubicin treatment upregulated the expression of AMP-activated protein kinase, a key modulator of lipid metabolism. Notably, siRNA-targeted p53 knockdown reversed the effects of doxorubicin in HepG2 cells. Moreover, doxorubicin treatment suppressed FFA -induced lipid accumulation in HepG2 spheroids. Conclusively, these results suggest that doxorubicin possesses potential application for the regulation of lipid metabolism by enhance the expression of p53 an in vitro NAFLD model.

Apoptotic potential of geranyl acetate in HepG2 liver cancer cells

Natural compounds having apoptotic effects are a new potential source for anti-cancer drugs. Numerous plant phytochemicals have proven anti-cancer properties by inducing programmed cell death (apoptosis). The disease known as cancer is characterized by the uncontrollable growth of some body cells and their spread to other body regions. Worldwide, cancer is a major cause of death. The objective of the current study was to assess apoptotic potential of geranyl acetate on HepG2 cancer cell line. Cell death and cell viability in groups were assessed using the MTT, trypan blue and crystal violet to assess anti-proliferative effect. P53 ELISA was carried out to assess apoptosis. When HepG2 cells were exposed to geranyl acetate they exhibited increased cytotoxicity, decreased viability, proliferation, and increased apoptosis. It is concluded that geranyl acetate has the potential to cause apoptosis via P53 in HepG2 cells. It can also prevent cancer cell growth and proliferation.

Context dependent role of p53 during the interaction of hepatocellular carcinoma and endothelial cells.

During the progression of hepatocellular carcinoma (HCC), several angiogenic factors are overexpressed in the hepatic microenvironment, which play a critical role in governing the phenotype of the endothelial cells. Mutation in the p53 gene (TP53) is a common event in HCC that may dysregulate the angiogenic signals. However, their functional messages remain largely unexplored at the onset of metastasis. Role of p53 was studied by siRNA mediated silencing of p53 in HepG2 cells (WTp53), collecting and analyzing their conditioned medium, followed by indirect co-culture with endothelial cells (HUVECs). Gene and protein expression in HCC cells and endothelial cells was studied by RT-qPCR and western blotting respectively. β-catenin protein expression and localization were analyzed by immunocytochemistry. We have studied a cell-to-cell interaction model to investigate the crosstalk of endothelial and hepatoma cells by either knocking down p53 or by using p53 null low metastatic HCC cell line. In the absence of p53, the HCC cells influence the migration and vascular network formation of endothelial cells through paracrine signaling of VEGF. Secretory VEGF activated the VEGF receptor-2 along with the survival signaling in endothelial cells. However, the β-catenin signal is upregulated in endothelial cells only during interaction with metastatic set up irrespective of absence and presence of p53, indicating context-dependent participation of p53 during communication between hepatoma cells and endothelial cells. This study highlights that the role of p53 on cellular responses during interaction of hepatocellular carcinoma and endothelial cells is distinct to cell types and context.

UBE2M Drives Hepatocellular Cancer Progression as a p53 Negative Regulator by Binding to MDM2 and Ribosomal Protein L11.

Simple SummaryHerein, the oncogenic role of UBE2M as an E2 NEDD8-conjugating enzyme was explored in hepatocellular carcinoma (HCC) cells, since neddylation plays a critical role in tumorigenesis. To address this issue, human tissue array and TCGA analysis were conducted in HCCs to find overexpression of UBE2M in HCCs. In addition, a differential profile was confirmed in UBE2M-depleted HepG2 cells. Furthermore, UBE2M depletion activated p53 expression and stability, while the ectopic expression of UBE2M disturbed p53 activation and enhanced degradation of exogenous p53 mediated by MDM2 in HepG2 cells via binding to MDM2 and ribosomal protein L11 by immunoprecipitation and immunofluorescence. These findings provide evidence that UBE2M is critically involved in liver cancer progression as a p53 negative regulator by binding to MDM2 and ribosomal protein L11.Though UBE2M, an E2 NEDD8-conjugating enzyme, is overexpressed in HepG2, Hep3B, Huh7 and PLC/PRF5 HCCs with poor prognosis by human tissue array and TCGA analysis, its underlying oncogenic mechanism remains unclear. Herein, UBE2M depletion suppressed viability and proliferation and induced cell cycle arrest and apoptosis via cleavages of PARP and caspase 3 and upregulation of p53, Bax and PUMA in HepG2, Huh7 and Hep3B cells. Furthermore, UBE2M depletion activated p53 expression and stability, while the ectopic expression of UBE2M disturbed p53 activation and enhanced degradation of exogenous p53 mediated by MDM2 in HepG2 cells. Interestingly, UBE2M binds to MDM2 or ribosomal protein L11, but not p53 in HepG2 cells, despite crosstalk between p53 and UBE2M. Consistently, the colocalization between UBE2M and MDM2 was observed by immunofluorescence. Notably, L11 was required in p53 activation by UBE2M depletion. Furthermore, UBE2M depletion retarded the growth of HepG2 cells in athymic nude mice along with elevated p53. Overall, these findings suggest that UBE2M promotes cancer progression as a p53 negative regulator by binding to MDM2 and ribosomal protein L11 in HCCs.

Polyphenols from Broussonetia papyrifera Induce Apoptosis of HepG2 Cells via Inactivation of ERK and AKT Signaling Pathways.

The extract of Broussonetia papyrifera has been proved to have antitumor activity. However, the underlying mechanism remains unclear. This study aimed to elucidate the mechanism of apoptosis of HepG2 cells induced by polyphenols from Broussonetia papyrifera (PBPs). The results revealed that PBPs inhibited the proliferation of HepG2 cells in a dose-dependent and time-dependent manner. Flow cytometry analysis showed that PBPs increased the apoptosis ratio of HepG2 cells significantly. PBPs increased intracellular reactive oxygen species (ROS) production and decreased intracellular superoxide dismutase (SOD) level of HepG2 cells. PBPs induced cell cycle arrest at G1 phase. Western blotting showed that PBPs upregulated the ratio of Bax/Bcl-2 and the expression level of Caspase-3, and activated p53 in HepG2 cells. The inhibition of proliferative relative signals (protein kinase B, PKB/AKT) and survival relative signals (extracellular signal-regulated kinase, ERK) were also observed in PBP-treated HepG2 cells. Our findings suggest that apoptosis of HepG2 cells induced by PBPs is mitochondria-mediated via inactivation of ERK and AKT signaling pathways.

Two purified proteins from royal jelly with in vitro dual anti-hepatic damage potency: Major royal jelly protein 2 and its novel isoform X1.

Liver diseases are serious life-threating conditions that should be controlled. Here, we identify a protein fraction from royal-jelly (RJ) that represents the most effective composite against CCl4-induced hepatotoxicity and HepG2 cell growth. Two closely related proteins were purified from this fraction by a new simple method and identified by MALDI-TOF MS as major RJ protein 2 (MRJP2) and its predicted isoform X1. The in silico assessment (3D structures and functions) of these proteins were performed using Iterative Threading ASSEmbly Refinement (I-TASSER) analysis and RAMPAGE program. These two purified proteins were able to relieve the necrotic hepatocytes (by 60.4%) via reducing tumor necrosis factor (TNF)-α, mixed lineage kinase domain-like protein (MLKL) and intracellular reactive species. The latter effects associated with improving hepatocyte functions. Furthermore, they revealed the potent anticancer effect via induction of caspase-dependent apoptosis and controlling the expression of both Bcl-2 and p53 in HepG2 cells. Thus, MRJP2 and its isoform X1 can be a promising dual strategy for fighting hepatic injury and cancer in future animal and human studies.

Hepatitis B virus X protein activates E3 ubiquitin ligase Siah-1 to control virus propagation via a negative feedback loop.

The seven in absentia homologue 1 (Siah-1) protein is an E3 ubiquitin ligase that induces ubiquitin-dependent proteasomal degradation of HBx, the principal regulatory protein of hepatitis B virus (HBV); however, its role in HBV propagation remains unknown. Here, we found that HBx upregulates Siah-1 levels in HepG2 but not in Hep3B cells, in which p53 is absent. For this effect, HBx sequentially activated ataxia telangiectasia mutated kinase and checkpoint kinase 2 via phosphorylation at the Ser-1981 and Thr-68 residues, respectively, which led to the activation of p53 via phosphorylation at the Ser-15 and Ser-20 residues. As a result, HBx was heavily ubiquitinated by Siah-1 and degraded by the ubiquitin-proteasome system in HepG2 cells, whereas this effect was marginal or undetectable in Hep3B cells. Knock-down of p53 in HepG2 cells downregulated Siah-1 levels and subsequently upregulated HBx levels, whereas ectopic p53 expression in Hep3B cells upregulated Siah-1 levels and subsequently downregulated HBx levels. In addition, Siah-1 knock-down impaired the ubiquitination and proteasomal degradation of HBx in HepG2 cells, whereas ectopic Siah-1 expression induced ubiquitin-dependent proteasomal degradation of HBx in Hep3B cells. The effects of HBx on p53 and Siah-1 were exactly reproduced in a 1.2-mer HBV replicon system, mimicking the natural course of HBV infection. In particular, Siah-1 knock-down upregulated the levels of HBx derived from the HBV replicon, resulting in an increase in HBV production. In conclusion, HBx modulates its own protein level via a negative feedback loop involving p53 and Siah-1 to control HBV propagation.

Basal and copper-induced expression of metallothionein isoform 1,2 and 3 genes in epithelial cancer cells: The role of tumor suppressor p53

Metallothioneins (MTs) are a ubiquitous low-molecular weight, cysteine rich proteins with a high affinity for metal ions. The expression and induction of MTs have been associated with protection against DNA damage, oxidative stress, and apoptosis. Our past research had shown that p53 is an important factor in metal regulation of MTs. The present study was undertaken to explore further the interrelationship between p53 and MTs. We investigated whether silencing of p53 could affect expression pattern of basal and copper induced metallothioneins. The silencing of wild-type p53 (wt-p53) in epithelial breast cancer MCF7 cells affected the basal level of MT-2A RNA, whereas the levels of MT-1A and MT-1X RNA remained largely unchanged. The expression of MT-3 was undetectable in MCF7 with either functional or silenced p53. MCF7 cells with silenced wt-p53 failed to upregulate MT-2A in response to copper and showed a reduced sensitivity toward copper induced cell apoptotic death. Similarly in MCF7-E6 and MDA-MB-231 cells, the presence of inactive/mutated p53 halted MT-1A and MT-2A gene expression in response to copper. Constitutive expression of MT-3 RNA was detectable in the presence of mutated p53 (mtp53). Transient transfection of MDA-MB-231 cells with wt-p53 enabled copper induced upregulation of both MT-1A and MT-2A but not basal level of MT-2A, MT-1E, MT-1X and MT-3. Inactivation of p53 in HepG2 cells amplified the basal expression of studied MT isoforms, including MT-3, as well as copper-induced mRNA expression of MTs except MT-1H and MT-3. Presented data demonstrate a direct relation between p53 and MT-1A and MT-2A and they also indicate that wt-p53 might be a negative regulator of MT-3 in epithelial cancer cells.

Activation of tumor suppressor p53 gene expression by magnetic thymine-imprinted chitosan nanoparticles

Chitosan is a natural biodegradable polysaccharide that has been used to enhance gene delivery, owing to the ease with which chitosan nanoparticles enter the nucleus of cells. To study the effects of nuclear delivery of telomeric gene sequences, which contain thymine, we formed magnetic thymine-imprinted chitosan nanoparticles (TIPs) by the precipitation of chitosan, mixed with thymine and magnetic nanoparticles (to aid in separations). The mean size of the TIPS was 116 ± 18 nm; the dissociation constant for thymine was 21.8 mg mL(-1). We then treated human hepatocellular carcinoma (HepG2) with TIPs nanoparticles bearing bound thymine or a bound telomeric DNA sequence. The expression of the tumor suppressor p53 gene increased when TIPs were applied and decreased when telomere-bound TIPs were applied.

Abstract 5266: RYBP expression predicts survival of patients with hepatocellular carcinoma, and regulates response to chemotherapy

Abstract RYBP (RING 1 and YY1-binding protein) is a newly identified member of the Polycomb group (PcG) proteins. PcG proteins are transcriptional repressors that epigenetically modify chromatin and participate in the establishment and maintenance of cell fates. These proteins are crucial for many biological processes, including self-renewal and differentiation, and cancer. Studies from our group and others suggest that RYBP has non-PcG functions and is downregulated in human cancers, including hepatocellular carcinoma (HCC). However, the potential role of RYBP in HCC is largely unknown. In the present study, for the first time, we systemically investigated the levels of RYBP expression and the linkage between RYBP deregulation and survivals of patients with HCC in 400 pairs of human HCC tissues and matched noncancerous samples. Using in vitro and in vivo HCC models, we determined the effort of RYBP on the survival of several HCC cell lines, including HepG2, Huh7 and Hep3B cells. We first verified that RYBP was downregulated in human HCC tumor specimens and cell lines, and that a low level of RYBP predicted a poor prognosis in HCC patients. We further demonstrated that enforced RYBP expression exerted potent effects against the various cell lines, leading to a significant decrease in cell viability, and inhibited cell colony formation and invasion, and induced apoptosis. We also found that the expression of numerous apoptosis- and metastasis-associated proteins was affected by RYBP. It reduced the expression of p53 in HepG2 cells, and increased Bax, cleaved-PARP and E-cadherin and, conversely, decreased Vimentin expression in all three HCC cells. Moreover, apoptosis was the main mechanism by which the RYBP exerted its cytostatic effects in HCC cells, while knockdown of RYBP showed opposite effects. In addition, we showed that AdRYBP infection inhibited the growth of tumors and induced RYBP expression in HepG2 and Huh7 xenograft models. There were no significant host toxicity and no gross organ abnormalities upon necropsy. We also determined that chemotherapeutic agents induced RYBP protein expression, and that RYBP sensitized HCC tumors to conventional chemotherapy through the induction of apoptosis both in vitro and in vivo. The results of these studies provide initial evidences supporting that the restoration of RYBP expression may be a new approach to targeted therapy for HCC and that RYBP may be a useful biomarker for predicting the prognosis of patients with HCC. (This work was supported by NIH/NCI grants R01 CA112029, R01 CA121211, and R01 CA186662 (to R.Z.); and NSFC 81272725 (to J.F.), NSFC 81372648 (to Q.G.) and Shanghai “Promising Youth Medical Worker” Project 13Y055 (to Q.G.) Citation Format: Wei Wang, Jianwen Cheng, Jiang-Jiang Qin, Sukesh Voruganti, Subhasree Nag, Jia Fan, Qiang Gao, Ruiwen Zhang. RYBP expression predicts survival of patients with hepatocellular carcinoma, and regulates response to chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5266. doi:10.1158/1538-7445.AM2015-5266

Novel 2,5-disubstituted-1,3,4-oxadiazole derivatives induce apoptosis in HepG2 cells through p53 mediated intrinsic pathway

A series of novel 1,3,4-oxadiazole derivatives (OSD, OCOD, ONOD, OPD, COD, PMOD, and PCOD) were synthesized and characterized. Their structures were confirmed on the basis of IR, NMR and mass spectroscopy and molecular weights were found in the range 300–325g/mol. Cancerous cell lines (MCF-7, HepG2) and non-cancerous cell lines (Chang liver cells) were treated with these compounds for 48h, which caused dose dependent decrease in the cell viability. From the seven derivatives, OSD was found to be most potent with IC50 value close to 50μM on all tested cell lines. Hence, this compound was selected for mechanistic study on HepG2 cell lines. Fluorescent cell staining and DNA fragmentation study of 50μM OSD on HepG2 cells, showed events marked by apoptosis such as nuclear fragmentation, cytoplasm shrinkage and DNA damage. Further, the cells with same treatment were quantified for apoptosis using annexin V-PI flow cytometric technique. The percentage of apoptotic cells was significantly higher (p<0.05) after OSD treatment compared to control cells. OSD induced a significant increase (p<0.05) in the expression of the tumor suppressor p53 in HepG2 cells. The constitutive expression of anti-apoptotic protein Bcl-2 significantly decreased (p<0.05) after treatment, while the expression of proapoptotic protein Bax significantly increased (p<0.05). The change in Bax to Bcl-2 ratio suggested involvement of Bcl-2 family in induction of apoptosis. Furthermore, the levels of caspase-9 and caspase-3 were significantly (p<0.05) up regulated in HepG2 cells after OSD treatment. The data suggest that 1,3,4-oxadiazole derivatives induce apoptosis mediated by intrinsic pathway of apoptosis. The findings strengthen the potential of the 1,3,4-oxadiazole scaffold OSD, as an agent with chemotherapeutic and cytostatic activity in human hepatocellular carcinoma in vitro.

Transcriptional regulation of the tumor suppressor FHL2 by p53 in human kidney and liver cells.

Four and a Half LIM protein 2 (FHL2) is a LIM domain only protein that is able to form various protein complexes and regulate gene transcription. Recent findings showed that FHL2 is a potential tumor suppressor gene that was down-regulated in hepatocellular carcinoma (HCC). Moreover, FHL2 can bind to and activate the TP53 promoter in hepatic cells. In this study, the activity of the two promoters of FHL2, 1a and 1b, were determined in the human embryonic kidney cell line HEK293 and the activation of these two promoters by p53 was investigated. Our results showed that the 1b promoter has a higher activity than the 1a promoter in HEK 293 cells but the 1a promoter is more responsive to the activation by p53 when compared with the 1b promoter. The regulation of FHL2 by p53 was further confirmed in liver cells by the overexpression of p53 in Hep3B cells and the knockdown of p53 in HepG2 cells. Combining promoter activity results of truncated mutants and predictions by bioinformatics tools, a putative p53 binding site was found in the exon 1a of FHL2 from +213 to +232. The binding between the p53 protein and the putative p53 binding site was then validated by the ChIP assay. Furthermore, the expression of FHL2 and TP53 were down-regulated in majority of HCC tumour samples (n = 41) and significantly correlated (P = 0.026). Finally, we found that the somatic mutation 747 (G→T), a hot spot mutation of the TP53 gene, is potentially associated with a higher expression of FHL2 in HCC tumour samples. Taken together, this is the first in-depth study about the transcriptional regulation of FHL2 by p53.

Differential responses of human hepatocytes to the non-nucleoside HIV-1 reverse transcriptase inhibitor nevirapine

Nevirapine is a non-nucleoside reverse transcriptase (RT) inhibitor used for the treatment of AIDS and the prevention of mother-to-child transmission of HIV-1. Despite its therapeutic benefits, treatment with nevirapine has been associated with significant incidences of liver and dermal toxicity. The present study examined the effects of nevirapine on cell growth and death in human hepatocyte HepG2 cells and THLE2 cells and the possible pathways involved in these effects. The concentrations of nevirapine inhibiting 50% cell growth were similar for both cell lines. Nevirapine (0-250 µM) treatment caused a slight increase in the amount of lactate dehydrogenase released into the medium. Apoptotic cell death did not contribute to the decrease in viable cells. Exposing of HepG2 cells to nevirapine caused G2/M phase arrest, and the activity of senescence-associated β-galactosidase was not altered. In THLE2 cells, the percentage of cells in G1/G0 phase was increased and cellular senescence was induced in a concentration-dependent manner. Endogenous non-telomeric RT activity was not detected in either cell line. Western blot analysis indicated lower levels of p53 and phospho-p53 (ser15) in HepG2 cells as compared to THLE2 cells; no significant changes in p53 or phospho-p53 (ser15) were noted with nevirapine treatment. These data demonstrate that nevirapine inhibits cell growth, induces cell cycle arrest at different phases, and has different effects on cellular senescence in HepG2 cells and THLE2 cells. The differential responses appear to be related to differences in the basal levels of p53 in the HepG2 cells and THLE2 cells.

Novel molecular, cytotoxical, and immunological study on promising and selective anticancer activity of Mung bean sprouts

BackgroundThe anticancer and immunomodulatory activity of mung bean sprouts (MBS) and the underlying mechanisms against human cervical and hepatocarcinoma cancer cells were explored.MethodsMBS cytotoxicity and MBS-induced anticancer cytokines, TNF-α and IFN-β from cancer cells, and immunological cytokines, IL-4, IFN-γ, and IL-10 from peripheral mononuclear cells (PMNC) were assessed by MTS and ELISA assays. Apoptotic cells were investigated by flow cytometry. The expression level of apoptotic genes (Bax, BCL-2, Capsases 7–9) and cell cycle regulatory genes (cyclin D, E, and A) and tumor suppressor proteins (p27, p21, and p53) was assessed by real-time qPCR in the cancer cells treated with extract IC50.ResultsThe cytotoxicity on normal human cells was significantly different from HeLa and HepG2 cells, 163.97 ± 5.73, 13.3 ± 0.89, and 14.04 ± 1.5 mg/ml, respectively. The selectivity index (SI) was 12.44 ± 0.83 for HeLa and 11.94 ± 1.2 for HepG2 cells. Increased levels of TNF-α and IFN-β were observed in the treated HeLa and HepG2 culture supernatants when compared with untreated cells. MBS extract was shown to be an immunopolarizing agent by inducing IFNγ and inhibiting IL-4 production by PBMC; this leads to triggering of CMI and cellular cytotoxicity. The extract induced apoptosis, in a dose and time dependent manner, in treated HeLa and HepG2, but not in untreated, cells (P < 0.05). The treatment significantly induced cell cycle arrest in G0/G1 in HeLa cells. The percentage of cells in G0/G1 phase of the treated HeLa cells increased from 62.87 ± 2.1%, in untreated cells, to 80.48 ± 2.97%. Interestingly, MBS IC50 induced the expression of apoptosis and tumor suppressor related genes in both HeLa and HepG2 cells. MBS extract succeeded in inducing cdk-inhibitors, p21, p53, and p27 in HeLa cells while it induced only p53 in HepG2 cells (P < 0.05). This is a clue for the cell type- specific interaction of the studied extract. These proteins inhibit the cyclin-cdk complexes apart from the presence of some other components that might stimulate some cyclins such as cyclin E, A, and D.ConclusionMBS extract was shown to be a potent anticancer agent granting new prospects of anticancer therapy using natural products.

Apoptosis in human hepatoma HepG2 cells induced by corn peptides and its anti-tumor efficacy in H22 tumor bearing mice

This study was aimed at evaluating the anti-tumor mechanism of corn peptides (CPs). In vitro, the results showed that CPs significantly inhibited cell viability in both a dose- and a time-dependent manner. CPs treatment induced S cell-cycle arrest and caused apoptotic death in HepG2 cells. It was observed that CPs caused the increased in Bax/Bcl-2 ratio and triggered the activation of Cleaved-caspase-3, p53 in HepG2 cells. In vivo, the results showed that CPs could not only inhibit the growth of the tumor, but also enhance the spleen index, as well as the level of IL-2, IFN-γ and TNF-α. Moreover, CPs could prolong the survival time in H22-bearing mice. This study demonstrated that CPs was an apoptosis inducer in HepG2 cells, that it could effectively inhibit hepatocellular carcinoma in vivo via enhancement of host immune system function, and that it could be a safe and effective anticancer, bioactive agent or functional food.

Quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapeutic agents

Quinacrine has been widely explored in treatment of malaria, giardiasis, and rheumatic diseases. We find that quinacrine stabilizes p53 and induces p53-dependent and independent cell death. Treatment by quinacrine alone at concentrations of 10–20 mM for 1–2 d cannot kill hepatocellular carcinoma cells, such as HepG2, Hep3B, Huh7, which are also resistant to TRAIL. However, quinacrine renders these cells sensitive to treatment by TRAIL. Co-treatment of these cells with quinacrine and TRAIL induces overwhelming cell death within 3–4 h. Levels of DR5, a pro-apoptotic death receptor of TRAIL, are increased upon treatment with quinacrine, while levels of Mcl-1, an anti-apoptotic member of the Bcl-2 family, are decreased. While the synergistic effect of quinacrine with TRAIL appears to be in part independent of p53, knockdown of p53 in HepG2 cells by siRNA results in more cell death after treatment by quinacrine and TRAIL. The mechanism by which quinacrine sensitizes hepatocellular carcinoma cells to TRAIL and chemotherapies, and the potential for clinical application currently are being further explored. Lastly, quinacrine synergizes with chemotherapeutics, such as adriamycin, 5-FU, etoposide, CPT11, sorafenib, and gemcitabine, in killing hepatocellular carcinoma cells in vitro and the drug enhances the activity of sorafenib to delay tumor growth in vivo.

MDM2 antagonist can inhibit tumor growth in hepatocellular carcinoma with different types of p53 in vitro

Nutlin-3, a selective small-molecule inhibitor of the p53-MDM2 interaction, has been shown to have antitumor activities in various tumors with wild-type p53. However, its effect on hepatocellular carcinoma (HCC) with different types of p53 remains unclear. This study is designed to determine nutlin-3's antitumor efficacy and underlying mechanisms of action in human HCC cells. Cell viability assay, cell cycle analysis, apoptosis assay, western blot, co- immunoprecipitation and siRNA experiments were analyzed in three human HCC cells. Anti-tumoral effects of nutlin-3 targeting the p53 and p73 pathways were evaluated in HCC cell lines. Nutlin-3 exerted the greatest anti-tumoral effect to three human HCC cells with wild-type p53, mutant p53 and p53-null. Nutlin-3 not only upregulated p53 in HepG2 cells, but also p73 in Huh7 and Hep3B cells, and disrupted p53-MDM2 and p73-MDM2 complexes in HCC cells. The compound inhibited cell proliferation, induced G0/G1 phase arrest, decreased the levels of CyclinD1, CyclinE, CDK2, CDK4, PCNA and E2F-1, and increased the levels of p21 and p27. It also induced apoptosis, increased the Bax/Bcl-2 ratio, then activated caspase-9 and caspase-3. Nutlin-3 has significant anticancer effects against human HCC cells, regardless of p53 status, indicating that it is a promising therapy for human hepatocellular carcinoma.