Mitochondrial carnitine palmitoyltransferase-II dysfunction: A possible novel mechanism for nonalcoholic fatty liver disease in hepatocarcinogenesis.

Nonalcoholic fatty liver disease (NAFLD) or metabolic-associated fatty liver disease has been characterized by the lipid accumulation with injury of hepatocytes and has become one of the most common chronic liver diseases in the world. The complex mechanisms of NAFLD formation are still under identification. Carnitine palmitoyltransferase-II (CPT-II) on inner mitochondrial membrane (IMM) regulates long chain fatty acid ß-oxidation, and its abnormality has had more and more attention paid to it by basic and clinical research in NAFLD. The sequences of its peptide chain and DNA nucleotides have been identified, and the catalytic activity of CPT-II is affected on its gene mutations, deficiency, enzymatic thermal instability, circulating carnitine level and so on. Recently, the CPT-II dysfunction has been discovered in models of liver lipid accumulation. Meanwhile, the malignant transformation of hepatocyte-related CD44+ stem T cell activation, high levels of tumor-related biomarkers (AFP, GPC3) and abnormal activation of Wnt3a expression as a key signal molecule of the Wnt/ß-catenin pathway run parallel to the alterations of hepatocyte pathology. This review focuses on some of the progress of CPT-II inactivity on IMM with liver fatty accumulation as a possible novel pathogenesis for NAFLD in hepatocarcinogenesis.

Oncogenic Wnt3a is a promising sensitive biomarker for monitoring hepatocarcinogenesis.

BACKGROUND: The effective treatment for hepatocellular carcinoma (HCC) depends on early diagnosis. Previously, the abnormal expression of Wnt3a as the key signaling molecule in the Wnt/ß-catenin pathway was found in HCC cells and could be released into the circulation. In this study, we used rat model of hepatocarcinogenesis to dynamically investigate the alteration of oncogenic Wnt3a and to explore its early monitor value for HCC. METHODS: Sprague-Dawley rats (SD) were fed with diet 2-fluorenylacetamide (2-FAA, 0.05%) for inducing hepatocarcinogenesis, and grouped based on liver morphological alteration by Hematoxylin & Eosin (H&E) staining; rats fed with normal chow were used as normal control (NC). Total RNA and protein were purified from rat livers. Differently expressed genes (DEGs) or Wnt3a mRNA, cellular distribution, and Wnt3a protein levels were analyzed by whole genome microarray with signal logarithm ratio (SLR log2cy5/cy3), immunohistochemistry, and enzyme-linked immunosorbent assay, respectively. RESULTS: Models of rat hepatocarcinogenesis were successfully established based on liver histopathological H&E staining. Rats were divided into the cell degeneration (rDeg), precancerosis (rPre-C) and HCC (rHCC) groups. Total numbers of the up- and down-regulated DEGs with SLR ≥ 8 were 55 and 48 in the rDeg group, 268 and 57 in the rPre-C group, and 312 and 201 in the rHCC group, respectively. Significantly altered genes were involved in cell proliferation, signal transduction, tumor metastasis, and apoptosis. Compared with the NC group, Wnt3a mRNA was increased by 4.6 folds (P < 0.001) in the rDeg group, 7.4 folds (P < 0.001) in the rPre-C group, and 10.4 folds (P < 0.001) in the rHCC group; the positive rates of liver Wnt3a were 66.7% (P = 0.001) in the rDeg group, 100% (P < 0.001) in the rPre-C group, and 100% (P < 0.001) in the rHCC group, respectively. Also, there were significant differences of liver Wnt3a (P < 0.001) or serum Wnt3a (P < 0.001) among different groups. CONCLUSIONS: Overexpression of Wnt3a was associated with rat hepatocarcinogenesis and it should be expected to be a promising monitoring biomarker for HCC occurrence at early stage.

Effects of targeted-edited oncogenic insulin-like growth factor-1 receptor with specific-sgRNA on biological behaviors of HepG2 cells.

BACKGROUND: Insulin-like growth factor-1 receptor (IGF-1R) is over-expressed in hepatocellular carcinoma (HCC). However, the relationship between IGF-1R activation and HCC progression remains unidentified. AIM: To investigate the effects of editing IGF-1R on the biological features of HCC cells. METHODS: Immunohistochemistry analyzed the expressions of IGF-1R and P-glyco protein (P-gp) in HCC tissues and their distal non-cancerous tissues (non-Ca). IGF-1R was edited with Crispr/Cas9 system, screened specific sgRNAs, and then transfected into HepG2 cells. CCK-8, scratch wound test detected cell proliferation, migration, invasion and transwell assays, respectively. Alterations of IGF-1R and P-gp were confirmed by Western blotting. Alterations of anti-cancer drug IC50 values were analyzed at the cell level. RESULTS: The positive rates of IGF-1R (93.6%, χ 2 = 63.947) or P-gp (88.2%, χ2 = 58.448) were significantly higher (P < 0.001) in the HCC group than those (36.6% in IGF-1R or 26.9% in P-gp) in the non-Ca group. They were positively correlated between high IGF-1R and P-gp expression, and they were associated with hepatitis B virus infection and vascular invasion of HCC. Abnormal expressions of circulating IGF-1R and P-gp were confirmed and associated with HCC progression. Biological feature alterations of HCC cells transfected with specific sgRNA showed IGF-1R expression down-regulation, cell proliferation inhibition, cell invasion or migration potential decreasing, and enhancing susceptibility of HepG2 cells to anti-cancer drugs. CONCLUSION: Edited oncogenic IGF-1R was useful to inhibit biological behaviors of HepG2 cells.

Encouraging specific biomarkers-based therapeutic strategies for hepatocellular carcinoma.

The prevention, early discovery and effective treatment of patients with hepatocellular carcinoma (HCC) remain a global medical challenge. At present, HCC is still mainly treated by surgery, supplemented by vascular embolization, radio frequency, radiotherapy, chemotherapy and biotherapy. The application of multikinase inhibitor sorafenib, chimeric antigen receptor T cells, or PD-1/PD-L1 inhibitors can prolong the median survival of HCC patients. However, the treatment efficacy is still unsatisfactory due to HCC metastasis and postoperative recurrence. During the process of hepatocyte malignant transformation, HCC tissues can express and secrete many types of specific biomarkers, or oncogenic antigen molecules into blood, for example, alpha-fetoprotein, glypican-3, Wnt3a (one of the key signaling molecules in the Wnt/ß-catenin pathway), insulin-like growth factor (IGF)-II or IGF-I receptor, vascular endothelial growth factor, secretory clusterin and so on. In addition, combining immunotherapy with non-coding RNAs might improve anti-cancer efficacy. These biomarkers not only contribute to HCC diagnosis or prognosis, but may also become molecular targets for HCC therapy under developing or clinical trials. This article reviews the progress in emerging biomarkers in basic research or clinical trials for HCC immunotherapy.

Oncogenic tuftelin 1 as a potential molecular-targeted for inhibiting hepatocellular carcinoma growth.

BACKGROUND: Abnormal tuftelin 1 (TUFT1) has been reported in multiple cancers and exhibits oncogenic roles in tumor progression. However, limited data are available on the relationship between TUFT1 and hepatocellular carcinoma (HCC), and the exact biological mechanism of TUFT1 is still poorly understood in HCC. AIM: To investigate TUFT1 expression in HCC and how interfering TUFT1 transcription affects HCC growth. METHODS: TUFT1 in HCC and non-HCC tissues based on databases of the Cancer Genome Atlas and Oncomine were analyzed, and TUFT1 in human HCC tissues on microarray were detected by immunohistochemistry for clinicopathological features, overall survival, and disease-free survival. HCC cells were transfected with constructed vectors of TUFT1 that interfere or over-express TUFT1 for analyzing the biological behaviors of HCC cells. Proliferation, invasion, migration, and apoptosis of cells were detected by cell counting kit-8, scratch assay, transwell tests, and flow cytometry and confirmed by Western blotting, respectively. RESULTS: Abnormal TUFT1 levels in databases expressed in HCC at messenger RNA (mRNA) level and HCC tissues were mainly located in cytoplasm and membrane. The level of TUFT1 expression in the HCC group was significantly higher (χ 2 = 18.563, P < 0.001) than that in the non-cancerous group, closely related to clinical staging, size, vascular invasion of tumor, hepatitis B e-antigen positive, and ascites (P < 0.01) of HCC patients, and negatively to HCC patients' overall survival and disease-free survival (P < 0.001). After interfering with TUFT1 transcription at mRNA level in the MHCC-97H cells by the specific TUFT1-short hairpin RNA, cell proliferation, invasion, and metastasis were significantly inhibited with increasing apoptosis rate. In contrast, proliferation, invasion, and migration were significantly enhanced after over-expression of TUFT1 mRNA in Hep3B cells in vitro. CONCLUSION: Oncogenic TUFT1 was associated with the progression of HCC and could be a potential molecular-target for inhibiting HCC growth.

Dynamic expression of hepatic GP73 mRNA and protein and circulating GP73 during hepatocytes malignant transformation.

BACKGROUND: Hepatic Golgi protein-73 (GP73) expression is related to hepatocellular carcinoma (HCC) progression. The aim of this study was to investigate the dynamic expression of GP73 mRNA and protein during hepatocytes malignant transformation. METHODS: Human GP73 expressions in 88 HCC tissues and their self-control surrounding tissues were examined by immunohistochemistry, and survival time of HCC patients was evaluated by the Kaplan-Meier method. HCC model of Sprague-Dawley rats was made by diet containing 2-fluorenylacetamide. The rats were divided into the control, hepatocyte degeneration, precanceration, and HCC groups to observe GP73 protein and mRNA alterations during hepatocytes malignant transformation. RESULTS: The GP73 expression was significantly higher in the cancerous tissues than that in the surrounding tissues, with shorter survival time, and the positive rates of GP73 protein in human HCC tissues were 53.3% at stage I, 84.0% at stage II, 84.6% at stage III, and 60.0% at stage IV, respectively. The positive rates of hepatic GP73 protein and mRNA in the rat models were none in the control group, 66.7% and 44.4% in the hepatocytes degeneration group, 88.9% and 77.8% in the hepatocytes precanceration group, and 100% in the HCC group, respectively. There was a positive correlation (r = 0.91, P<0.01) between hepatic GP73 and serum GP73 during rat hepatocytes malignant transformation. CONCLUSIONS: Abnormal GP73 expression may be a sensitive and valuable biomarker in hepatocarcinogensis.

Abnormal CD44 activation of hepatocytes with nonalcoholic fatty accumulation in rat hepatocarcinogenesis.

BACKGROUND: Prevalence of nonalcoholic fatty liver disease (NAFLD) is rapidly increasing, and NAFLD has become one of the most common chronic liver diseases worldwide. With abnormal CD44 activation, the severe form of NAFLD can progress to liver cirrhosis and hepatocellular carcinoma (HCC). Thus, the molecular mechanism of CD44 in NAFLD needs to be identified. AIM: To investigate the relationship between CD44 activation and malignant transformation of rat hepatocytes under nonalcoholic lipid accumulation. METHODS: Sprague-Dawley rats were fed a high-fat (HF) for 12 wk to entice NAFLD and then with HF plus 2-fluorenylacetamide (0.05%) to induce HCC. Rats were sacrificed every 2 wk, and subsequently divided into the groups based on liver pathological examination (hematoxylin and eosin staining): NAFLD, denaturation, precancerosis, HCC, and control. Liver CD44 mRNA was detected by OneArray. Liver fat as assessed by Oil red O staining or CD44 by immunohistochemical assay was compared with their integral optic density. Serum CD44, alanine aminotransferase, aspartate aminotransferase, triglyceride, total cholesterol, and AFP levels were quantitatively tested. RESULTS: Elevated CD44 was first reported in hepatocarcinogenesis, with increasing expression from NAFLD to HCC at the protein or mRNA level. The CD44 integral optic density values were significantly different between the control group and the NAFLD (t = 25.433, P < 0.001), denaturation (t = 48.822, P < 0.001), precancerosis (t = 27.751, P < 0.001), and HCC (t = 16.239, P < 0.001) groups, respectively. Hepatic CD44 can be secreted into the blood, and serum CD44 levels in HCC or precancerous rats were significantly higher (P < 0.001) than those in any of the other rats. Positive correlations were found between liver CD44 and CD44 mRNA (rs = 0.373, P = 0.043) and serum CD44 (rs = 0.541, P = 0.002) and between liver CD44 mRNA and serum CD44 (rs = 0.507, P = 0.004). Moreover, significant correlations were found between liver CD44 and liver AFP (rs = 0.572, P = 0.001), between serum CD44 and serum AFP (rs = 0.608, P < 0.001), and between CD44 mRNA and AFP mRNA (rs = 0.370, P = 0.044). CONCLUSION: The data suggested that increasing CD44 expression is associated with the malignant transformation of hepatocytes in NAFLD.

Alteration of oncogenic IGF-II gene methylation status associates with hepatocyte malignant transformation.

BACKGROUND: Oncogenic insulin-like growth factor-II (IGF-II) is overexpressed in hepatocellular carcinoma (HCC). The present study aimed to analyze the dynamic alteration of IGF-II CpG site methylation status and its molecular mechanism in HCC progression. METHODS: IGF-II alterations were observed in rat hepatocarcinogenesis models induced by 2-acetylaminofluorene. Liver IGF-II expression was compared by immunohistochemistry or tissue IGF-II specific concentration (nmol/mg protein). Status of human IGF-II promoter 3 (P3) or rat IGF-II P2 CpG site methylation was amplified by methylation-specific polymerase chain reaction (MSP). Serum IGF-II levels were quantitatively detected by an enzyme-linked immunosorbent assay. RESULTS: The levels of hepatic IGF-II expression were significantly elevated in the HCC group (P < 0.001). The unmethylation rate of IGF-II P3 CpG sites was 100% in the HCC-, 52.5% in the paracancerous-, and none (0%) in the distal noncancerous-tissues. Abnormal IGF-II expression was related to differentiation degree, tumor invasion, and positive HBV-DNA (all P < 0.001), with a negative correlation between P3 methylation degree and IGF-II expression. There was a positive correlation between liver IGF-II specific concentration and circulating IGF-II level (r = 0.97, P < 0.001). Significantly negative correlation was found between IGF-II P2 CpG site methylation and circulating IGF-II (rs = -0.89, P < 0.001) or liver IGF-II level (rs = -0.84, P < 0.001). CONCLUSIONS: The increase of serum IGF-II and the alteration of oncogenic gene IGF-II methylation may be biomarkers for HCC diagnosis and DNA methylation may be the therapeutic target of HCC.

Abnormal expression of HMGB-3 is significantly associated with malignant transformation of hepatocytes.

AIM: To explore the relationship between dynamic expression of high mobility group box-3 (HMGB3) and malignant transformation of hepatocytes. METHODS: Expression of HMGB family proteins were observed in rat hepatocarcinogenesis models induced with 2-acetylaminofluorene. Alterations of HMGB3 were analyzed at the mRNA level by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and at the protein level by immunohistochemistry or Western blotting. HMGB3 in human liver cancer tissues were evaluated using bioinformatics databases from GEO, TCGA, and Oncomine. A specific HMGB3-shRNA was used to knock down HMGB3 expression in order to investigate its effects on proliferation and cell cycle in vitro and in vivo. RESULTS: Elevated HMGB3 levels were first reported in hepatocarcinogenesis, with increasing expression from normal liver to cancer. Bioinformatic databases showed that HMGB3 expression in hepatocellular carcinoma tissues was significantly higher than that in normal liver tissues. Higher HMGB3 expression was discovered in liver cancer cells compared with LO2 cells in vitro. According to gene set enrichment analysis, HMGB3 mRNA levels were correlated with cell cycle and DNA replication pathways. Knocking down HMGB3 by specific shRNA significantly inhibited proliferation of HepG2 cells by cell cycle arrest and downregulating DNA replication related genes (cyclin B1, FEN1, and PCNA) at the mRNA and protein level. Furthermore, silencing HMGB3 significantly inhibited xenograft tumor growth (measured by Ki67) in vivo. CONCLUSION: HMGB3 is involved in malignant transformation of hepatocytes and could be a useful biomarker for diagnosis and a potential target for therapy of liver cancer.

Expression of hepatic Wnt5a and its clinicopathological features in patients with hepatocellular carcinoma.

BACKGROUD: Wingless-type MMTV integration site family member 5a (Wnt5a) is involved in carcinogenesis. However, little data are available in Wnt5a signaling with hepatocellular carcinoma (HCC). In the present study, we investigated the expression of hepatic Wnt5a in HCC and the role of Wnt5a in HCC progression and outcome. METHODS: Wnt5a expression and cellular distribution in HCCs and their matched paracancerous tissues from 87 patients were analyzed with tissue microarray and immunohistochemistry and compared with hepatic Wnt3a signaling. Wnt5a expression was categorized into low or high based on immunohistochemistry. Overall survival rate of HCC patients was estimated in correlation with the hepatic Wnt5a level using Kaplan-Meier method; the survival difference between patients with low and those with high Wnt5a was compared with log-rank test; and prognostic analysis was carried out with Cox regression. RESULTS: Total incidence of Wnt5a expression in the HCC tissues was 70.1%, which was significantly lower (χ2 = 13.585, P < 0.001) than that in their paracancerous tissues (88.5%). Significant difference of Wnt5a intensity was found between HCC and their paracancerous tissues (Z = 8.463, P < 0.001). Wnt5a intensity was inversely correlated with Wnt3a signaling (r = -0.402, P < 0.001) in HCC tissues. A decrease of Wnt5a expression in relation to the clinical staging from stage I to IV and low or no staining at advanced HCC were observed. Wnt5a level was related to periportal embolus (χ2 = 11.069, P < 0.001), TNM staging (χ2 = 8.852, P < 0.05), 5-year survival (χ2 = 4.961, P < 0.05), and confirmed as an independent prognosis factor of HCC patients (hazard ratio: 1.957; 95% confidence interval: 1.109-3.456; P < 0.05). CONCLUSIONS: The decrease of hepatic Wnt5a signaling is associated with HCC progression and poor prognosis.

Cancer survival in Qidong between 1972 and 2011: A population-based analysis.

Population-based cancer survival is an improved index for evaluating the overall efficiency of cancer health services in a given region. The current study analysed the observed survival and relative survival of leading cancer sites from a population-based cancer registry between 1972 and 2011 in Qidong, China. A total of 92,780 incident cases with cancer were registered and followed-up for survival status. The main sites of the cancer types, based on the rank order of incidence, were the liver, stomach, lung, colon and rectum, oesophagus, breast, pancreas, leukaemia, brain and central nervous system (B and CNS), bladder, blood [non-Hodgkin's lymphoma (NHL)] and cervix. For all malignancies combined, the 5-year observed survival was 13.18% and the relative survival was 15.80%. Females had higher observed survival and relative survival (19.32 and 22.71%, respectively) compared with males (9.63 and 11.68%, respectively). The cancer sites with the highest five-year relative survival rates were the female breast, bladder, cervix and colon and rectum; followed by NHL, stomach, B and CNS cancer and leukaemia. The poorest survival rates were cancers of oesophagus, lung, pancreas and liver. Higher survival rates were observed in younger patients compared with older patients. Cancers of the oesophagus, female breast and bladder were associated with higher survival in middle-aged groups. Improved survival rates in the most recent two 5-year calendar periods were identified for stomach, lung, colon and rectum, oesophagus, female breast and bladder cancer, as well as leukaemia and NHL. The observations of the current study provide the opportunity for evaluation of the survival outcomes of frequent cancer sites that reflects the changes and improvement in a rural area in China.

Mitochondrial carnitine palmitoyl transferase-II inactivity aggravates lipid accumulation in rat hepatocarcinogenesis.

AIM: To investigate the dynamic alteration of mitochondrial carnitine palmitoyl transferase II (CPT-II) expression during malignant transformation of rat hepatocytes. METHODS: Sprague-Dawley male rats were fed with normal, high fat (HF), and HF containing 2-fluorenylacetamide (2-FAA) diet, respectively. According to the Hematoxylin and Eosin staining of livers, rats were divided into control, fatty liver, degeneration, precancerous, and cancerous groups. Liver lipids were dyed with Oil Red O, CPT-II alterations were analyzed by immunohistochemistry, and compared with CPT-II specific concentration (µg/mg protein). Levels of total cholesterol (Tch), triglyceride (TG), and amino-transferases [alanine aminotransferase (ALT), aspartate aminotransferase (AST)] were determined by the routine methods. RESULTS: After intake of HF and/or HF+2-FAA diets, the rat livers showed mass lipid accumulation. The lipid level in the control group was significantly lower than that in other groups. The changes of serum TG and Tch levels were abnormally increasing, 2-3 times more than those in the controls (P < 0.05). During the rat liver morphological changes from normal to cancer development process with hepatocyte injury, serum AST and ALT levels were significantly higher (4-8 times, P < 0.05) than those in the control group. The specific concentration of CPT-II in liver tissues progressively decreased during hepatocyte malignant transformation, with the lowest CPT-II levels in the cancer group than in any of the other groups (P < 0.05). CONCLUSION: Low CPT-II expression might lead to abnormal hepatic lipid accumulation, which should promote the malignant transformation of hepatocytes.

Reversal of multidrug resistance of hepatocellular carcinoma cells by metformin through inhibiting NF-κB gene transcription.

AIM: To interfere with the activation of nuclear factor-κB (NF-κB) with metformin and explore its effect in reversing multidrug resistance (MDR) of hepatocellular carcinoma (HCC) cells. METHODS: Expression of P-glycoprotein (P-gp) and NF-κB in human HepG2 or HepG2/adriamycin (ADM) cells treated with pCMV-NF-κB-small interference RNA (siRNA) with or without metformin, was analyzed by Western blot or fluorescence quantitative PCR. Cell viability was tested by CCK-8 assay. Cell cycle and apoptosis were measured by flow cytometry and Annexin-V-PE/7-AnnexinV apoptosis detection double staining assay, respectively. RESULTS: P-gp overexpression in HepG2 and HepG2/ADM cells was closely related to mdr1 mRNA (3.310 ± 0.154) and NF-κB mRNA (2.580 ± 0.040) expression. NF-κB gene transcription was inhibited by specific siRNA with significant down-regulation of P-gp and enhanced HCC cell chemosensitivity to doxorubicin. After pretreatment with metformin, HepG2/ADM cells were sensitized to doxorubicin and P-gp was decreased through the NF-κB signaling pathway. The synergistic effect of metformin and NF-κB siRNA were found in HepG2/ADM cells with regard to proliferation inhibition, cell cycle arrest and inducing cell apoptosis. CONCLUSION: Metformin via silencing NF-κB signaling could effectively reverse MDR of HCC cells by down-regulating MDR1/P-gp expression.

Oncogenic Wnt3a expression as an estimable prognostic marker for hepatocellular carcinoma.

AIM: To investigate member 3a of Wingless-type MMTV integration site family (Wnt3a) expression in cancerous and surrounding tissues and the relationship between clinicopathologic features of hepatocellular carcinoma (HCC) and Wnt3a expression. METHODS: Wnt3a expression and cellular distribution and clinicopathologic characteristics in cancerous tissue and matched surrounding tissues were analyzed in 80 HCC patients from January 2006 to August 2008 by tissue microarrays and immunohistochemistry. The overall and disease-free survival rates were estimated using the Kaplan-Meier method and compared with the log-rank test. The prognostic analysis was carried out with univariate and multivariate Cox regressions models. RESULTS: The incidence of oncogenic Wnt3a expression in the cancerous group was up to 96.25% (77 of 80), which was significantly higher (χ(2) = 48.818, P < 0.001) than that in the surrounding group (46.25%, 37 of 80). Brown Wnt3a staining gradually increased with clinical staging that showed very strong staining in advanced HCC. The clinicopathologic features of high Wnt3a expression in HCC were related to poorly-differentiated grade (χ(2) = 20.211, P < 0.001), liver cirrhosis (χ(2) = 8.467, P < 0.004), hepatitis B virus (HBV) infection (χ(2) = 12.957, P < 0.001), higher tumor-node-metastasis stage (χ(2) = 22.960, P < 0.001), and 5-year survival rate (χ(2) = 15.469, P < 0.001). CONCLUSION: Oncogenic Wnt3a expression associated with HBV infection and cirrhotic liver might be an independent prognostic factor for HCC.

Effective targeting of colorectal cancer cells using TORC1/2 kinase inhibitors in vitro and in vivo.

AIM: We investigated the effects of TORC1/2 kinase inhibitors on colorectal cancer (CRC) cell lines. MATERIALS & METHODS: Using selective TORC1/2 inhibitors, rapamycin and PP242, we assessed their effect on the growth of CRC cells in vitro and tumor growth in vivo. RESULTS: Rapamycin and PP242 inhibit proliferation and induce apoptosis of CRC cells. They also enhance proapoptotic effect of conventional chemo drug doxorubicin in CRC cells in vitro. When combined with doxorubicin, rapamycin and PP242 almost completely inhibit tumor growth in vivo. Rapamycin and PP242 inhibit phosphorylation of Akt, ribosomal S6 kinase, 4EBP1 and mTOR. CONCLUSION: Our study suggests rapamycin and PP242 may be a useful therapeutic agent and inhibiting mTOR signaling pathway represents a new targeted therapy for CRC.

Chemosensitization of HepG2 cells by suppression of NF-κB/p65 gene transcription with specific-siRNA.

AIM: To investigate small interfering RNA (siRNA)-mediated inhibition of nuclear factor-kappa B (NF-κB) activation and multidrug-resistant (MDR) phenotype formation in human HepG2 cells. METHODS: Total RNA was extracted from human HepG2 or LO2 cells. NF-κB/p65 mRNA was amplified by nested reverse transcription polymerase chain reaction and confirmed by sequencing. NF-κB/p65 was analyzed by immunohistochemistry. Specific-siRNA was transfected to HepG2 cells to knock down NF-κB/p65 expression. The effects on cell proliferation, survival, and apoptosis were assessed, and the level of NF-κB/p65 or P-glycoprotein (P-gp) was quantitatively analyzed by enzyme-linked immunosorbent assay. RESULTS: HepG2 cells express NF-κB/p65 and express relatively less phosphorylated p65 (P-p65) and little P-gp. After treatment of HepG2 cells with different doses of doxorubicin, the expression of NF-κB/p65, P-p65, and especially P-gp were dose-dependently upregulated. After HepG2 cells were transfected with NF-κB/p65 siRNA (100 nmol/L), the expression of NF-κB/p65, P-p65, and P-gp were downregulated significantly and dose-dependently. The viability of HepG2 cells was decreased to 23% in the combination NF-κB/p65 siRNA (100 nmol/L) and doxorubicin (0.5 µmol/L) group and 47% in the doxorubicin (0.5 µmol/L) group (t = 7.043, P < 0.001). CONCLUSION: Knockdown of NF-κB/p65 with siRNA is an effective strategy for inhibiting HepG2 cell growth by downregulating P-gp expression associated chemosensitization and apoptosis induction.

Glypican-3 is a biomarker and a therapeutic target of hepatocellular carcinoma.

BACKGROUND: The carcinogenesis of hepatocellular carcinoma (HCC) is a multi-factorial, multi-step and complex process. Early diagnosis and effective treatments are of utmost importance. This review summarized the recent studies of oncofetal glypican-3 (GPC-3), a membrane-associated heparan sulfate proteoglycan, in the diagnosis and treatment of HCC. DATA SOURCES: English-language reports published from June 2001 to September 2014 were searched from MEDLINE. The key words searched included: GPC-3, biomarker, target and HCC. The sensitivity, specificity, positive and negative predictive values were extracted, and the effect of GPC-3 targeted therapy on HCC was also evaluated. RESULTS: GPC-3 plays a crucial role in HCC cell proliferation and metastasis. It mediates oncogenesis involving signaling pathways during hepatocyte malignant transformation. GPC-3 expression is increased in atypical hyperplasia and cancerous tissues. GPC-3 levels in HCC patients are related to HBV infection, TNM stage, periportal cancerous embolus, and extrahepatic metastasis. The diagnostic accuracy of the combination of serum GPC-3 and alpha-fetoprotein in HCC is up to 94.3%. Down-regulation of GPC-3 with specific siRNA or anti-GPC-3 antibody alters cell migration, metastasis and invasion behaviors. The nude mice xenograft tumor growth is inhibited by silencing GPC-3 gene transcription. CONCLUSION: Oncofetal GPC-3 is a highly specific biomarker for the diagnosis of HCC and a promising target molecule for HCC gene therapy.

[Down-regulated clusterin expression enhances sensitivity of hepatoma cells to anti-cancer drugs].

OBJECTIVE: To investigate the relationship between and underlying mechanistic pathway of clusterin (CLU) and chemo-resistance ofhepatocellular carcinoma (HCC) cells. METHODS: CLU protein expression in HCC cell lines (Hep3B, SMMC7721, PLC, and HepG2) and HepG2/ADM cells was quantified by western blotting. Four short-hairpin (sh)RNAs designed to block CLU-mRNA were generated, screened by RT-PCR, and transfected into the cells to determine effects of CLU on cell viability and apoptosis. Effects of CLU blockade on drug efflux pump activity were measured by flow cytometry. RESULTS: CLU was found to be over-expressed in HCC cell lines and HepG2/ADM cells. The four shRNAs inhibited CLU-mRNA as follows (vs. levels in untransfected cells): shRNA-1: 73.68% (q =23.011, P < 0.01), shRNA-2: 39.26% (q =11.991, P < 0.01), shRNA-3: 62.36% (q =19.392, P < 0.01), and shRNA-4: 55.35% (q =17.149, P < 0.01). shRNA-mediated depletion of CLU led to increased sensitivity to anti-cancer drugs and increased doxorubicin-induced apoptosis in HepG2/ADM cells, as evidenced by the apoptosis ratio of the shRNA-1 group of 39.28% vs. the apoptosis ratio of the untransfected control group of 4.92%. Silencing of CLU also decreased drug etflux pump activity, and the level of MDR1/P-gp expression was significantly reduced (shRNA-1 group vs.untransfected control group: q =14.604, P < 0.01). CONCLUSION: CLU repression may enhance sensitivity of HCC cells to anti-cancers drugs and represents a potential molecular-target for reversal of multidrug-resistant HCC.