Parental HepG2 Cells Research Articles

HSPB1 is an intracellular antiviral factor against hepatitis B virus

Hepatitis B virus (HBV) is the most common of the hepatitis viruses that cause chronic liver infections in humans and it is considered a major global health problem. However, the mechanisms of HBV replication are complex and not yet fully understood. In this study, the HBV DNA-transfected HepG2.2.15 cell line and its parental HepG2 cell line were analyzed by isobaric tags for relative and absolute quantitation (iTRAQ)-coupled two-dimensional liquid chromatography tandem mass-spectrophotometry (2D LC-MS/MS), a successfully exploited high-throughput proteomic technology. In total, 2,028 unique proteins were identified and 170 proteins were differentially expressed in HepG2.2.15 cells as compared with that in HepG2. Several differentially expressed proteins were further validated by Western blot and real-time quantitative reverse transcription-PCR. Furthermore, the association of HBV replication with heat shock protein B1, one of the highly expressed proteins in HepG2.2.15 cells, was verified. HSPB1 functions as a anti-viral protein during HBV infection by specifically inducing type interferon and some downstream antiviral effectors. This study is the first to report the application of iTRAQ technology to analyze the underlying mechanisms of HBV replication. Many of the differentially expressed proteins identified have not been linked to HBV replication before, and may provide valuable novel insights into HBV replication.

High ADAM8 Expression is Associated with Poor Prognosis in Patients with Hepatocellular Carcinoma

In this study,we investigated the ADAM8 expression in hepatocellular carcinoma (HCC) and its correlation with clinicopathologic features,including the survival of patients with HCC. Furthermore,we examined the biological processes regulated by ADAM8 during the development of using HepG2 cell line as a model system. We used immunohistochemistry to compare ADAM8 protein expression in HCC and normal liver tissues and further analyze the ADAM8 protein expression in clinicopathologically characterized 105 HCC cases.We stably knocked down the endogenous expression level of ADAM8 in HepG2 cells with specific shRNA-expressing lentiviral vector. Following the successful establishment of stable cells,we examined in vitro cell growth by MTT assay,anchorage-independent growth by soft-agar colony formation assay and cell migration/invasion by transwell and boyden chamber assay. And in addition,we also investigated the in vivo tumor growth by xenograft transplantation of HepG2 cells into nude mice. Protein expression level of ADAM8 was markedly higher in HCC tissues than that in the normal liver tissues (P = 0.0058).In addition,high expression of ADAM8 protein was positively correlated with serum AFP elevation,tumor size,histological differentiation,tumor recurrence,tumor metastasis,and tumor stage. Patients with higher ADAM8 expression showed a significantly shorter overall survival time than patients with low ADAM8 expression. Multivariate analysis suggested that ADAM8 expression might be an independent prognostic indicator (p = 0.016) for the survival of patients with HCC. ADAM8-specific shRNA (shADAM8) successfully knocked down its endogenous expression in HepG2 cells. Compared to the parental and control shRNA-transfected (shCtrl) HepG2 cells,the shADAM8 cells exhibited significantly reduced in vitro cell growth,anchorage-independent growth,cell migration and invasion (p < 0.05).In vivo,the xenograft transplants from shADAM8 cells gave rise to much smaller tumors as compared to those from shCtrl cells. High ADAM8 expression is associated with poor overall survival in patients with HCC. Down-regulation of ADAM8 inhibits the growth,anchorage-independent growth,migration and invasion of HepG2 cells. ADAM8 may be a potential target of antiangiogenic therapy for HCC.

Construction and characterization of peroxisome proliferator-activated receptor-gamma co-activator 1 alpha (PGC-1α over-expressing cell line derived from human hepatocyte carcinoma HepG2 cells)

The aim was develop stable human cell line stable over-expressing transcription co-activator peroxisome proliferator-activated receptor gamma co-activator 1α (PGC-1α) with restored hepatospecific functions and increased expression of major xenobiotic metabolizing enzymes. Six clones of HepG2-PGC-1α and one control clone HepG2-pcDNA3 were isolated and analyzed for secretion of hepatospecific markers, fibrinogen, albumin and alpha1-antitrypsin. Expression levels of protein and mRNA of hepatocyte nuclear factor (HNF4α), pregnane X receptor (PXR) and aryl hydrocarbon receptor (AhR) were determined. We measured basal and ligand inducible expression of CYP1A1 and CYP3A4. Stably transfected cell line HepG2-PGC-1α derived from HepG2 cells over-expressing PGC-1α displayed increased secretion of fibrinogen, but not albumin or alpha1-antitrypsin compared to parent HepG2 cells. We found increased levels of HNF4α, PXR and AhR proteins but not their mRNAs in HepG2-PGC1 cells. Basal expression of CYP3A4 protein in HepG2-PGC-1α cells was increased but rifampicin-inducible expression of CYP3A4 protein was lowered in comparison with parent HepG2 cells. Induction of CYP3A4 mRNA varied between 1.3 - 1.9 fold in individual clones. Expression of TCDD-inducible CYP1A1 protein was lower in HepG2-PGC-1α cells than in parent HepG2 cells. Induction of CYP1A1 mRNA by TCDD in HepG2-PGC-1α cells was comparable with that in parent HepG2 cells and ranged between 103 - 198 fold. Stable expression of PGC-1α in HepG2 cells restores several hepatospecific functions, such as secretion of fibrinogen, expression of HNF4α1 and xenoreceptors PXR and AhR. However, the expression and induction of key drug-metabolizing enzymes (CYP1A1 and CYP3A4) were not improved.

Overexpression of HOXA1 correlates with poor prognosis in patients with hepatocellular carcinoma

HOXA1 overexpression is sufficient for malignant transformation of nontumorigenic epithelial cells. It is known that HOXA1, which was upregulated in squamous cell carcinomas, affects both cell growth and death. The forced expression of HOXA1 in human breast cancer cells results in increased cell growth activity. However, it has not been reported in hepatocellular carcinoma (HCC). In this study, we used immunohistochemistry to compare HOXA1 protein expression in HCC and normal liver tissues and further analyzed HOXA1 protein expression in 156 clinicopathologically characterized HCC cases. We stably knocked down the endogenous expression level of HOXA1 in HepG2 cells with specific shRNA-expressing lentiviral vector. Following the successful establishment of stable cells, we examined in vitro cell growth by the MTT assay, anchorage-independent growth through a soft agar colony formation assay and cell migration/invasion by transwell and Boyden chamber assay. In addition, we also investigated in vivo tumor growth by xenograft transplantation of HepG2 cells into nude mice. Our results showed that the protein expression level of HOXA1 was markedly higher in HCC tissues than that in normal liver tissue (P = 0.019). In addition, a high expression level of HOXA1 protein was positively correlated with the T classification (P < 0.001), the N classification (P < 0.001), distant metastasis (P = 0.004), and the clinical stage (P < 0.001) of HCC patients. Patients with higher HOXA1 expression showed a significantly shorter overall survival time compared with patients with low HOXA1 expression. Multivariate analysis suggested that HOXA1 expression might be an independent prognostic indicator (P < 0.001) for the survival of patients with HCC. HOXA1-specific shRNA (shHOXA1) successfully knocked down HOXA1 endogenous expression in HepG2 cells. Compared to the parental and control shRNA-transfected (shCtrl) HepG2 cells, the shHOXA1 cells exhibited significantly reduced in vitro cell growth, anchorage-independent growth, and cell migration and invasion (P < 0.05). In vivo, the xenograft transplants from shHOXA1 cells gave rise to much smaller tumors compared with those from shCtrl cells. Collectively, high HOXA1 expression is associated with poor overall survival in patients with HCC. The downregulation of HOXA1 inhibits growth, anchorage-independent growth, and migration and invasion of HepG2 cells.

Insufficient Radiofrequency Ablation Promotes Angiogenesis of Residual Hepatocellular Carcinoma via HIF-1α/VEGFA

BackgroundThe mechanism of rapid growth of the residual tumor after radiofrequency (RF) ablation is poorly understood. In this study, we investigated the effect of hyperthermia on HepG2 cells and generated a subline with enhanced viability and dys-regulated angiogenesis in vivo, which was used as a model to further determine the molecular mechanism of the rapid growth of residual HCC after RF ablation.Methodology/Principal FindingsHeat treatment was used to establish sublines of HepG2 cells. A subline (HepG2 k) with a relatively higher viability and significant heat tolerance was selected. The cellular protein levels of VEGFA, HIF-1α and p-Akt, VEGFA mRNA and secreted VEGFA were measured, and all of these were up-regulated in this subline compared to parental HepG2 cells. HIF-1α inhibitor YC-1 and VEGFA siRNA inhibited the high viability of the subline. The conditioned media from the subline exerted stronger pro-angiogenic effects. Bevacizumab, VEGFA siRNA and YC-1 inhibited proangiogenic effects of the conditioned media of HepG2 k cells and abolished the difference between parental HepG2 cells and HepG2 k cells. For in vivo studies, a nude mouse model was used, and the efficacy of bavacizumab was determined. HepG2 k tumor had stronger pro-angiogenic effects than parental HepG2 tumor. Bevacizumab could inhibit the tumor growth and angiogenesis, and also eliminate the difference in tumor growth and angiogenesis between parental HepG2 tumor and HepG2 k tumor in vivo.Conclusions/SignificanceThe angiogenesis induced by HIF1α/VEGFA produced by altered cells after hyperthermia treatment may play an important role in the rapid growth of residual HCC after RF ablation. Bevacizumab may be a good candidate drug for preventing and treating the process.

Pharmacological Isolation of Experimental Models of Drug-resistant Hepatocellular Carcinoma Cell Line

Drug resistance is one of the major challenges facing the success of chemotherapy against human hepatocarcinoma (HCC) as well as other types of cancer. Studies with cell lines can serve as initial screening for agents that could modulate drug resistance. Development of a good experimental model of drug-resistant cells is a prerequisite for the success of such cellular studies; but could be laborious and generally time-consuming. Additionally, the high mortality rate associated with advanced HCC calls for a probe into the mechanism of resistance by developing experimental model that mimics clinical method of its treatment. Consequently, we have reported a simplified method of selection of drug-resistant hepatocarcinoma cells from human hepatocellular carcinoma (HEPG2) cell line using pharmacologic agents, cisplatin (CDDP) and 5-fluorouracil (5-FU). HEPG2 cell line was incubated for 24 hours with different concentrations of CDDP (0 - 20 μM) or 5-FU (0 - 100 μM). Cell viability was assayed by CCK-8 (Cell Counting Kit) analysis, and the inhibitory concentrations (IC50) for CDDP and 5-FU were established by dose-dependent cytotoxicity curves. The IC50(s) were confirmed by flow cytometric analysis of cell death due to CDDP or 5-FU. Clinical method of treatment was imitated by treating the parental HEPG2 cell line in pulse, at the optimal concentration of either CDDP or 5-FU for 4 to 6 hours. Induction was repeated 6 times, whilst allowing the cells to attain at least 70% confluence between intervals of induction. The resultant drug-resistant sublines, (HEPG2CR) and (HEPG2FR) were found to be stable after over 3 months of drug withdrawal and maintenance in drug-free medium. This was done with the views of establishing a simple, efficient and direct protocol for the development of good cellular models for the study of drug resistance in liver cancer, with possible application in other cancer types.

In�vivo treatment of HCV core-positive HepG2 cells with the transfer of recombinant caspase-3 using a 2'-5' OAS promoter

Hepatitis C virus (HCV) is one of the most common pathogens causing liver-related morbidity and mortality, which affect 170 million individuals worldwide. There is no vaccine available, and current therapy is only partially effective. In a previous study, we constructed a recombinant caspase-3 expression vector under the 2'-5'-oligoadenylate synthetase gene (OAS) promoter (pGL3-OAS-re-caspase-3) and demonstrated that it is an effective gene therapy for HCV core-positive liver cells in vitro. In the present study, the human hepatoma cell line HepG2 was transfected with the pcDNA3.1-HCV-core-EGFP plasmid and selected by G418. Expression of HCV core protein was confirmed by RT-PCR and immunocytochemistry. Both HepG2-expressing HCV core protein and parental HepG2 cells were inoculated subcutaneously into BALB/c mice, respectively. Tumor-bearing mice were treated with an intratumoral injection of pGL3-OAS-re-caspase-3. The mice were sacrificed after 48 h. The correlation between HCV core and caspase-3 expression in tumor tissues was analyzed by immunohistochemical staining and double-label immunofluorescence staining. The subcutaneous hepatoma in vivo mouse models stably expressing HCV core protein and co-expressing HCV core protein and pGL3-OAS-re-caspase-3 were established. Double-label immunofluorescence staining showed that the percentage of co-expression of both HCV core and caspase-3 was 76 ± 6% in the group treated with pGL3-OAS-re-caspase-3. There was a significant increase in the number of apoptotic cells in the group treated with the pGL3-OAS-re-caspase-3 system by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling and transmission electron microscopy. The results suggest that the pGL3-OAS-re-caspase-3 construct can effectively induce apoptosis in HCV core-positive hepatocytes in vivo. The results presented strongly suggest that the transfer of pGL3-OAS-re-caspase-3 is an effective and promising gene therapy strategy for HCV infection.

Abstract 680: Antitumor activity of CDDP and nuclear receptor PXR

Abstract The goal of this study was to evaluate the effect of the possibility of compound that regulate the function of nuclear receptor as agent enhancing the activity of CDDP. Cell lines used in this study were HepG2 and some CDDP-resistant HepG2 cells that were established in our laboratory. These CDDP-resistant HepG2 cells show IC50 values several times higher than that of HegG2 cells. Changes in gene expression were assessed in RT-PCR assays, and the ability of apoptosis induction was assessed as caspase-3 activity. Gene expressions of nuclear receptors in HepG2 cells after exposure to CDDP were examined over time. Interestingly, IC90 dose of CDDP induced significant reduction in PXR mRNA expression with remarkable apoptosis, but IC50 dose of CDDP did no apoptosis and no significant change in PXR mRNA expression. Also, six established CDDP-resistant HepG2 cells show increased PXR mRNA expression relative to parent HepG2 cells. Therfore, PXR agonist and antagonist were used to elucidate if PXR could modulate the sensitivity of cells to CDDP. HepG2 cells exposured to PXR agonist rifampicin for 24 hr showed increased PXR mRNA expression. In HepG2 cells, pre-exposure to rifampicin followed by exposure to CDDP and coexposure to rifampicin and CDDP reduced induction of apoptosis compared to exposure to CDDP alone. Moreover, almost total suppression of apoptosis induced by CDDP was achieved by pre-exposure for 48 hr followed by coexposure to rifampicin for 24 hr. PXR antagonist leflunomide caused a slight enhancement in apoptosis induced by CDDP in HepG2 cells and a significant enhancement in apoptosis induced by CDDP in CDDP-resistant HepG2 cells. We concluded that nuclear receptor PXR is an important factor which regulates the expression of genes that affect on the antitumor activity of CDDP and that PXR antagonist can be a candidate as an agent augmenting the antitumor activity of CDDP. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 680. doi:10.1158/1538-7445.AM2011-680

Hepatoma cell line HepG2.2.15 demonstrates distinct biological features compared with parental HepG2

To investigate the biological features of hepatitis B virus (HBV)-transfected HepG2.2.15 cells. The cell ultrastructure, cell cycle and apoptosis, and the abilities of proliferation and invasion of HBV-transfected HepG2.2.15 and the parent HepG2 cells were examined by electron microscopy, flow cytometry, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and trans-well assay. Oncogenicity of the two cell lines was compared via subcutaneous injection and orthotopic injection or implantation in nude mice, and the pathological analysis of tumor formation was performed. Two cytoskeletal proteins were detected by Western blotting. Compared with HepG2 cells, HepG2.2.15 cells showed organelle degeneration and filopodia disappearance under electron microscope. HepG2.2.15 cells proliferated and migrated slowly in vitro, and hardly formed tumor and lung metastasis in nude mice. Flow cytometry showed that the majority of HepG2.2.15 cells were arrested in G1 phase, and apoptosis was minor in both cell lines. Furthermore, the levels of cytoskeletal proteins F-actin and Ezrin were decreased in HepG2.2.15 cells. HepG2.2.15 cells demonstrated a lower proliferation and invasion ability than the HepG2 cells due to HBV transfection.

Alcohol Induces RNA Polymerase III-dependent Transcription through c-Jun by Co-regulating TATA-binding Protein (TBP) and Brf1 Expression

Chronic alcohol consumption is associated with steatohepatitis and cirrhosis, enhancing the risk for hepatocellular carcinoma. RNA polymerase (pol) III transcribes a variety of small, untranslated RNAs, including tRNAs and 5S rRNAs, which determine the biosynthetic capacity of cells. Increased RNA pol III-dependent transcription, observed in transformed cells and human tumors, is required for oncogenic transformation. Given that alcohol consumption increases risk for liver cancer, we examined whether alcohol regulates this class of genes. Ethanol induces RNA pol III-dependent transcription in both HepG2 cells and primary mouse hepatocytes in a manner that requires ethanol metabolism and the activation of JNK1. This regulatory event is mediated, at least in part, through the ability of ethanol to induce expression of the TFIIIB components, Brf1, and the TATA-binding protein (TBP). Induction of TBP, Brf1, and RNA pol III-dependent gene expression is driven by enhanced c-Jun expression. Ethanol promotes a marked increase in the direct recruitment of c-Jun to TBP, Brf1, and tRNA gene promoters. Chronic alcohol administration in mice leads to enhanced expression of TBP, Brf1, tRNA, and 5S rRNA gene transcription in the liver. These alcohol-dependent increases are more pronounced in transgenic animals that express the HCV NS5A protein that display increased incidence of liver tumors. Together, these results identify a new class of genes that are regulated by alcohol through the co-regulation of TFIIIB components and define a central role for c-Jun in this process.

Overexpressed HDGF as an independent prognostic factor is involved in poor prognosis in Chinese patients with liver cancer

BackgroundHepatoma-derived growth factor (HDGF) is involved in the hepatocarcinogenesis. In this study, we investigated the HDGF expression in hepatocellular carcinoma (HCC) and its correlation with clinicopathologic features, including the survival of patients with HCC. Furthermore, we examined the biological processes regulated by HDGF during the development of using HepG2 cell line as a model system.Methodswe used immunohistochemistry to compare HDGF protein expression in HCC and normal liver tissues and further analyze the HDGF protein expression in clinicopathologically characterized 137 HCC cases. We stably knocked down the endogenous expression level of HDGF in HepG2 cells with specific shRNA-expressing lentiviral vector. Following the successful establishment of stable cells, we examined in vitro cell growth by MTT assay, anchorage-independent growth by soft-agar colony formation assay and cell migration/invasion by transwell and boyden chamber assay. And in addition, we also investigated the in vivo tumor growth by xenograft transplantation of HepG2 cells into nude mice.ResultsProtein expression level of HDGF was markedly higher in HCC tissues than that in the normal liver tissues(P = 0.011). In addition, high expression of HDGF protein was positively correlated with T classification(p < 0.001), N classification (p < 0.001), and clinical stage (p < 0.001) of HCC patients. Patients with higher HDGF expression showed a significantly shorter overall survival time than did patients with low HDGF expression. Multivariate analysis suggested that HDGF expression might be an independent prognostic indicator(p < 0.001) for the survival of patients with HCC. HDGF-specific shRNA (shHDGF) successfully knocked down its endogenous expression in HepG2 cells. Compared to the parental and control shRNA-transfected (shCtrl) HepG2 cells, the shHDGF cells exhibited significantly reduced in vitro cell growth, anchorage-independent growth, cell migration and invasion (p < 0.05). In vivo, the xenograft transplants from shHDGF cells gave rise to much smaller tumors as compared to those from shCtrl cells.ConclusionHigh HDGF expression is associated with poor overall survival in patients with HCC. Down-regulation of HDGF inhibits the growth, anchorage-independent growth, migration and invasion of HepG2 cells.

EDEM1 accelerates the trimming of 1,2-linked mannose on the C branch of N-glycans

Glycoprotein folding and degradation in the endoplasmic reticulum (ER) is mediated by the ER quality control system. Mannose trimming plays an important role by forming specific N-glycans that permit the recognition and sorting of terminally misfolded conformers for ERAD (ER-associated degradation). The EDEM (ER degradation enhancing alpha-mannosidase-like protein) subgroup of proteins belonging to the Class I alpha1,2-mannosidase family (glycosylhydrolase family 47) has been shown to enhance ERAD. We recently reported that overexpression of EDEM3 enhances glycoprotein ERAD with a concomitant increase in mannose-trimming activity in vivo. Herein, we report that overexpression of EDEM1 produces Glc(1)Man(8)GlcNAc(2) isomer C on terminally misfolded null Hong Kong alpha1-antitrypsin (NHK) in vivo. Levels of this isomer increased throughout the chase period and comprised approximately 10% of the [(3)H]mannose-labeled N-glycans on NHK after a 3-h chase. Furthermore, overexpression of EDEM1 E220Q containing a mutation in a conserved catalytic residue essential for alpha1,2-mannosidase activity did not yield detectable levels of Glc(1)Man(8)GlcNAc(2) isomer C. Yet, the same extent of NHK ERAD-enhancement was observed in both EDEM1 and EDEM1 E220Q overexpressing cells. This can be attributed to both wild-type and mutant EDEM1 inhibiting aberrant NHK dimer formation. We further analyzed the N-glycan profile of total cellular glycoproteins from HepG2 cells stably overexpressing EDEM1 and found that the relative amount of Man(7)GlcNAc(2) isomer A, which lacks the terminal B and C branch mannoses, was increased compared to parental HepG2 cells. Based on this observation, we conclude that EDEM1 activity trims mannose from the C branch of N-glycans in vivo.

Transcriptional analysis of doxorubicin‐induced cytotoxicity and resistance in human hepatocellular carcinoma cell lines

Hepatoma is either intrinsically resistant to chemotherapy or response to it but later develop resistance. The aim of this study was to clarify the relationship of treatment with doxorubicin (Dox) in hepatoma HepG2 cells and drug resistance developed by Dox. We have analysed the bioactivities and gene expression profiles of multidrug resistant (MDR) HepG2/DR cell line and its parental HepG2 cell, which were exposure to Dox. We confirmed that Dox-induced apoptosis of HepG2 cells in a time-dependent manner; cDNA microarray and hierarchical cluster analysis demonstrate that the features of the transcriptional programme of the later response to Dox in HepG2 cells and MDR HepG2/DR cells have a common character, which is upregulation of stress response, cytoskeleton, ubiquitin-proteasome pathway and repressed G-protein signal transduction system; differentially expressed genes in MDR HepG2/DR such as drug transporters and tumour-associated antigens were verified at the levels of mRNA by semiquantitative reverse transcriptase-polymerase chain reaction. These results reveal novel co-ordinated changes that occurred in resistant HepG2 cells to survive from cell apoptosis elicited by Dox treatment.

Overexpression of Akt/PKB modulates N‐myristoyltransferase activity in cancer cells

N-myristoyltransferase (NMT) catalyses the myristoylation reaction. Since NMT activity is elevated in various cancers and activated Akt/PKB leads to cell survival, we were interested in studying if activation of Akt/PKB has any effect on NMT. Overexpression of constitutively active Akt/PKB in HepG2 cells (HepG2-CA-Akt/PKB) led to an approximately 50% reduction of NMT compared with parental HepG2 cells. Reduced NMT activity in HepG2-CA-Akt/PKB was found to be due to the NMT1 phosphorylation. We determined NMT activity in various human breast cancer cell lines with differing metastatic potentials and pseudo-normal breast cells (HBL-100). Tumourigenic or metastatic breast cancer cell lines such as MDA-MB-231, MDA-MB-435, and Hs 578T displayed reduced NMT activity. Western blot analysis revealed that NMT1 is phosphorylated in these breast cancer cells. Furthermore, patients' breast cancer tissue array revealed strong positivity and high intensity for NMT in malignant breast tissues compared with normal breast cells. A gradation in the NMT staining was observed for grade I, II, and III infiltrating ductal carcinoma breast tissues. These studies demonstrate that overexpression of Akt/PKB results in NMT1 phosphorylation and that NMT1 is phosphorylated in breast cancer cells. Immunohistochemical analysis suggests that NMT may prove to be an added diagnostic biomarker for breast cancer.

Clinically relevant radioresistant cells efficiently repair DNA double‐strand breaks induced by X‐rays

Radiotherapy is one of the major therapeutic modalities for eradicating malignant tumors. However, the existence of radioresistant cells remains one of the most critical obstacles in radiotherapy and radiochemotherapy. Standard radiotherapy for tumor treatment consists of approximately 2 Gy once a day, 5 days a week, over a period of 5-8 weeks. To understand the characteristics of radioresistant cells and to develop more effective radiotherapy, we established a novel radioresistant cell line, HepG2-8960-R with clinical relevance from parental HepG2 cells by long-term fractionated exposure to 2 Gy of X-rays. HepG2-8960-R cells continued to proliferate with daily exposure to 2 Gy X-rays for more than 30 days, while all parental HepG2 cells ceased. After exposure to fractionated 2 Gy X-rays, induction frequencies of micronuclei and remaining foci of gamma-H2AX in HepG2-8960-R were less than those in HepG2. Flow cytometric analysis revealed that the proportion of cells in S- and G2/M-phase of the cell cycle was higher in HepG2-8960-R than in HepG2. These suggest that the response of clinically relevant radioresistant (CRR) cells to fractionated radiation is not merely an accumulated response to each fractionated radiation. This is the first report on the establishment of a CRR cell line from an isogenic parental cell line.

Let-7a microRNA suppresses therapeutics-induced cancer cell death by targeting caspase-3

MicroRNAs (miRNA) are endogenously expressed non-coding RNAs that regulate gene expression post-transcriptionally. Let-7a miRNA is a founding member in the let-7 family and its down-regulation in association with over-expression of RAS and HMGA2 oncogenes has previously been reported. In the present study, caspase-3, the executioner caspase, was confirmed to be the target of let-7a as ectopic expression of let-7a decreased the luciferase activity of a reporter construct containing the 3' untranslated region of caspase-3 and at the same time repressed the enzyme expression in human squamous carcinoma A431 cells and hepatocellular carcinoma HepG2 cells. Moreover, let-7a was over-expressed while caspase-3 was down-regulated in A10A cells, a doxorubicin-resistant A431 subline. Enforced let-7a expression increased the resistance in A431 cells and HepG2 cells to apoptosis induced by therapeutic drugs such as interferon-gamma, doxorubicin and paclitaxel. On the other hand, down-regulation of let-7a by the anti-let-7a inhibitor increased the doxorubicin-induced apoptosis in A431 parent cells, A10A cells and HepG2 cells while the increase was suppressed by caspase-3 inhibitor. Both anti-let-7a inhibitor and caspase-3 inhibitor however failed to affect the drug-induced apoptosis in human breast cancer MCF7 cells, the cells that do not express caspase-3. Therefore, let-7a by targeting caspase-3 may play a functional role in modulating drug-induced cell death in human cancer cells.

Pregnane X Receptor Protects HepG2 Cells from BaP-Induced DNA Damage

Pregnane X receptor (PXR) is a nuclear receptor that coordinately regulates transcriptional expression of both phase I and phase II metabolizing enzymes. PXR plays an important role in the pharmacokinetics of a broad spectrum of endogenous and xenobiotic compounds and appears to have evolved in part to protect organisms from toxic xenobiotics. Metabolism of benzo[a]pyrene (BaP), a well-established carcinogen and ubiquitous environmental contaminant, can result in either detoxification or bioactivation to its genotoxic forms. Therefore, PXR could modulate the genotoxicity of BaP by changing the balance of the metabolic pathways in favor of BaP detoxification. To examine the role of PXR in BaP genotoxicity, BaP-DNA adduct formation was measured by 32P-postlabeling in BaP-treated parental HepG2 cells and human PXR-transfected HepG2 cells. The presence of transfected PXR significantly reduced the level of adducts relative to parental cells by 50-65% (p < 0.001), demonstrating that PXR protects liver cells from genotoxicity induced by exposure to BaP. To analyze potential PXR-regulated detoxification pathways in liver cells, a panel of genes involved in phase I and phase II metabolism and excretion was surveyed with real-time quantitative reverse transcription PCR. The messenger RNA levels of CYP1A2, GSTA1, GSTA2, GSTM1, UGT1A6, and BCRP (ABCG2) were significantly higher in cells overexpressing PXR, independent of exposure to BaP. In addition, the total GST enzymatic activity, which favors the metabolic detoxification of BaP, was significantly increased by the presence of PXR (p < 0.001), independent of BaP exposure. Taken together, these results suggest that PXR plays an important role in protection against DNA damage by polycyclic aromatic hydrocarbons (PAHs) such as BaP, and that these protective effects may be through a coordinated regulation of genes involved in xenobiotic metabolism.

Long-term effects of rapamycin treatment on insulin mediated phosphorylation of Akt/PKB and glycogen synthase activity

Protein kinase B (Akt/PKB) is a Ser/Thr kinase that is involved in the regulation of cell proliferation/survival through mammalian target of rapamycin (mTOR) and the regulation of glycogen metabolism through glycogen synthase kinase 3β (GSK-3β) and glycogen synthase (GS). Rapamycin is an inhibitor of mTOR. The objective of this study was to investigate the effects of rapamycin pretreatment on the insulin mediated phosphorylation of Akt/PKB phosphorylation and GS activity in parental HepG2 and HepG2 cells with overexpression of constitutively active Akt1/PKB-α (HepG2-CA-Akt/PKB). Rapamycin pretreatment resulted in a decrease (20–30%) in the insulin mediated phosphorylation of Akt1 (Ser 473) in parental HepG2 cells but showed an upregulation of phosphorylation in HepG2-CA-Akt/PKB cells. Rictor levels were decreased (20–50%) in parental HepG2 cells but were not significantly altered in the HepG2-CA-Akt/PKB cells. Furthermore, rictor knockdown decreased the phosphorylation of Akt (Ser 473) by 40–60% upon rapamycin pretreatment. GS activity followed similar trends as that of phosphorylated Akt and so with rictor levels in these cells pretreated with rapamycin; parental HepG2 cells showed a decrease in GS activity, whereas as HepG2-CA-Akt/PKB cells showed an increase in GS activity. The changes in the levels of phosphorylated Akt/PKB (Ser 473) correlated with GS and protein phoshatase-1 activity.

Polyphyllin D induces mitochondrial fragmentation and acts directly on the mitochondria to induce apoptosis in drug-resistant HepG2 cells

We previously showed that polyphyllin D (PD) produced a stronger apoptotic effect in R-HepG2 with multi-drug resistance (MDR) than that in its parent HepG2 cells without MDR. In this study, PD was found to elicit mitochondrial fragmentation in live cells by using total internal reflection fluorescence microscopy (TIRFM). When mitochondria were isolated and treated directly with PD, a stronger swelling, deeper transmembrane depolarization, and more apoptosis-inducing factor (AIF) release were observed from the mitochondria of R-HepG2 than that of HepG2. These observations suggest that PD is a potent anti-cancer agent that bypasses MDR and elicits apoptosis via mitochondrial injury.

Microsomal Triglyceride Transfer Protein Activity Is Not Required for the Initiation of Apolipoprotein B-containing Lipoprotein Assembly in McA-RH7777 Cells

We previously demonstrated that the N-terminal 1000 amino acid residues of human apolipoprotein (apo) B (designated apoB:1000) are competent to fold into a three-sided lipovitellin-like lipid binding cavity to form the apoB "lipid pocket" without a structural requirement for microsomal triglyceride transfer protein (MTP). Our results established that this primordial apoB-containing particle is phospholipid-rich (Manchekar, M., Richardson, P. E., Forte, T. M., Datta, G., Segrest, J. P., and Dashti, N. (2004) J. Biol. Chem. 279, 39757-39766). In this study we have investigated the putative functional role of MTP in the initial lipidation of apoB:1000 in stable transformants of McA-RH7777 cells. Inhibition of MTP lipid transfer activity by 0.1 microm BMS-197636 and 5, 10, and 20 microm of BMS-200150 had no detectable effect on the synthesis, lipidation, and secretion of apoB:1000-containing particles. Under identical experimental conditions, the synthesis, lipidation, and secretion of endogenous apoB100-containing particles in HepG2 and parental untransfected McA-RH7777 cells were inhibited by 86-94%. BMS-200150 at 40 microm nearly abolished the secretion of endogenous apoB100-containing particles in HepG2 and parental McA-RH cells but caused only 15-20% inhibition in the secretion of apoB: 1000-containing particles. This modest decrease was attributable to the nonspecific effect of a high concentration of this compound on hepatic protein synthesis, as reflected in a similar (20-25%) reduction in albumin secretion. Suppression of MTP gene expression in stable transformants of McA-RH7777 cells by micro-interfering RNA led to 60-70% decrease in MTP mRNA and protein levels, but it had no detectable effect on the secretion of apoB:1000. Our results provide a compelling argument that the initial addition of phospholipids to apoB:1000 and initiation of apoB-containing lipoprotein assembly occur independently of MTP lipid transfer activity.