TPX2 Is a Prognostic Marker and Contributes to Growth and Metastasis of Human Hepatocellular Carcinoma

Targeting protein for Xenopus kinesin-like protein 2 (TPX2), a microtubule-associated protein, impacts spindle assembly in human cells. Several studies have demonstrated that TPX2 is overexpressed in different types of human cancers and promotes tumor growth and metastasis. In this study, we found that the expression level of TPX2 was obviously higher in hepatocellular carcinoma (HCC) tissues than in matched nontumor tissues. Elevated expressions of TPX2 mRNA were observed in all HCC cell lines (HepG2, Hep3B, SMMC-7721, Bel-7402 and Huh7) as compared with that in a non-transformed hepatic cell line (LO2). Clinical analysis indicated that the positive expression of TPX2 was significantly correlated with venous infiltration, high Edmondson-Steiner grading and advanced TNM tumor stage in HCC. Furthermore, TPX2 was a novel prognostic marker for predicting 5-year overall survival (OS) and disease-free survival (DFS) of HCC patients. In vitro studies found that TPX2 knockdown significantly inhibited cell proliferation and viability in both Hep3B and HepG2 cells. Moreover, TPX2 knockdown obviously slowed down tumor growth in a nude mouse xenograft model. Otherwise, TPX2 knockdown prominently suppressed HCC cell invasion and migration. In conclusion, these results indicate that TPX2 may serve as a prognostic marker and promotes tumorigenesis and metastasis of HCC.     

CXCL10 Decreases GP73 Expression in Hepatoma Cells at the Early Stage of Hepatitis C Virus (HCV) Infection

Golgi protein 73 (GP73), which is up-regulated in hepatocellular carcinoma (HCC), has recently been identified as a novel serum marker for HCC diagnosis. Several reports also noted the increased levels of GP73 expression in chronic liver disease in patients with acute hepatitis of various etiologies, chronic Hepatitis C virus (HCV) infection and alcoholic liver disease. The molecular mechanisms of GP73 expression in HCV related liver disease still need to be determined. In this study, we aimed to evaluate the effect of HCV infection on GP73 expression. GP73 was highly expressed in Huh7, Hep3B, 293T and HUVEC cells, and was low-expressed in HepG2 cells. HCV infection led to down-regulation of GP73 in Huh7 and HepG2/CD81 cells at the early stage of infection. CXCL10 decreased GP73 expression in Huh7 and HepG2 cells. Up-regulation of GP73 was noted in hepatocytes with cytopathic effect at advanced stage of HCV infection, and further research is needed to determine the unknown factors affecting GP73 expression. In conclusion, our study provided additional evidence for the roles of GP73 in liver disease.     

MicroRNA-130b Promotes Cell Aggressiveness by Inhibiting Peroxisome Proliferator-Activated Receptor Gamma in Human Hepatocellular Carcinoma

MircroRNA-130b (miR-130b) is proposed as a novel tumor-related miRNA and has been found to be significantly dysregulated in tumors. In this study, the expression level of miR-130b was found to be obviously higher in hepatocellular carcinoma (HCC) tissues than that in nontumor tissues. Further, miR-130b was expressed at significantly higher levels in aggressive and recurrent tumor tissues. Clinical analysis indicated that high-expression of miR-130b was prominently correlated with venous infiltration, high Edmondson-Steiner grading and advanced tumor-node-metastasis (TNM) tumor stage in HCC. Elevated miR-130b expression was observed in all HCC cell lines (HepG2, SMMC-7721, Huh7, Hep3B and MHCC97H) as compared with that in a nontransformed hepatic cell line (LO2). Furthermore, an inverse correlation between miR-130b and E-cadherin and a positive correlation between miR-130b and Vimentin were observed in HCC tissues. Down-regulation of miR-130b expression reduced invasion and migration in both Hep3B and MHCC97H cells. Peroxisome proliferator-activated receptor gamma (PPAR-γ) was inversely correlated with miR-130b expression in HCC tissues. In addition, down-regulation of miR-130b restored PPAR-γ expression and subsequently suppressed epithelial-mesenchymal transition (EMT) in HCC cells. We identified PPARγ as a direct target of miR-130b in HCC in vitro. Notably, PPAR-γ knockdown abolished down-regulation of miR-130b-inhibited EMT in MHCC97H cells. In conclusion, miR-130b may promote HCC cell migration and invasion by inhibiting PPAR-γ and subsequently inducing EMT.     

Selective Anti-Cancer Effects of Plasma-Activated Medium and Its High Efficacy with Cisplatin on Hepatocellular Carcinoma with Cancer Stem Cell Characteristics

Hepatocellular carcinoma (HCC) is a major histological subtype of primary liver cancer. Ample evidence suggests that the pathological properties of HCC originate from hepatic cancer stem cells (CSCs), which are responsible for carcinogenesis, recurrence, and drug resistance. Cold atmospheric-pressure plasma (CAP) and plasma-activated medium (PAM) induce apoptosis in cancer cells and represent novel and powerful anti-cancer agents. This study aimed to determine the anti-cancer effect of CAP and PAM in HCC cell lines with CSC characteristics. We showed that the air-based CAP and PAM selectively induced cell death in Hep3B and Huh7 cells with CSC characteristics, but not in the normal liver cell line, MIHA. We observed both caspase-dependent and -independent cell death in the PAM-treated HCC cell lines. Moreover, we determined whether combinatorial PAM therapy with various anti-cancer agents have an additive effect on cell death in Huh7. We found that PAM highly increased the efficacy of the chemotherapeutic agent, cisplatin, while enhanced the anti-cancer effect of doxorubicin and the targeted-therapy drugs, trametinib and sorafenib to a lesser extent. These findings support the application of CAP and PAM as anti-cancer agents to induce selective cell death in cancers containing CSCs, suggesting that the combinatorial use of PAM and some specific anti-cancer agents is complemented mechanistically.     

Evaluating the Effect of Lenvatinib on Sorafenib-Resistant Hepatocellular Carcinoma Cells

Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related deaths worldwide. Sorafenib has been used as a first-line systemic treatment for over a decade. However, resistance to sorafenib limits patient response and presents a major hurdle during HCC treatment. Lenvatinib has been approved as a first-line systemic treatment for advanced HCC and is the first agent to achieve non-inferiority against sorafenib. Therefore, in the present study, we evaluated the inhibition efficacy of lenvatinib in sorafenib-resistant HCC cells. Only a few studies have been conducted on this topic. Two human HCC cell lines, Huh-7 and Hep-3B, were used to establish sorafenib resistance, and in vitro and in vivo studies were employed. Lenvatinib suppressed sorafenib-resistant HCC cell proliferation mainly by inducing G1 cell cycle arrest through ERK signaling. Hep-3B sorafenib-resistant cells showed partial cross-resistance to lenvatinib, possibly due to the contribution of poor autophagic responsiveness. Overall, the findings suggest that the underlying mechanism of lenvatinib in overcoming sorafenib resistance in HCC involves FGFR4-ERK signaling. Lenvatinib may be a suitable second-line therapy for unresectable HCC patients who have developed sorafenib resistance and express FGFR4.     

Identification of 1,2,3,4,6-Penta-O-galloyl-β-d-glucopyranoside as a Glycine N-Methyltransferase Enhancer by High-Throughput Screening of Natural Products Inhibits Hepatocellular Carcinoma

Glycine N-methyltransferase (GNMT) expression is vastly downregulated in hepatocellular carcinomas (HCC). High rates of GNMT knockout mice developed HCC, while overexpression of GNMT prevented aflatoxin-induced carcinogenicity and inhibited liver cancer cell proliferation. Therefore, in this study, we aimed for the identification of a GNMT inducer for HCC therapy. We established a GNMT promoter-driven luciferase reporter assay as a drug screening platform. Screening of 324 pure compounds and 480 crude extracts from Chinese medicinal herbs resulted in the identification of Paeonia lactiflora Pall (PL) extract and the active component 1,2,3,4,6-penta-O-galloyl-β-d-glucopyranoside (PGG) as a GNMT inducer. Purified PL extract and PGG induced GNMT mRNA and protein expression in Huh7 human hepatoma cells and in xenograft tumors. PGG and PL extract had potent anti-HCC effects both in vitro and in vivo. Furthermore, PGG treatment induced apoptosis in Huh7 cells. Moreover, PGG treatment sensitized Huh7 cells to sorafenib treatment. Therefore, these results indicated that identifying a GNMT enhancer using the GNMT promoter-based assay might be a useful approach to find drugs for HCC. These data also suggested that PGG has therapeutic potential for the treatment of HCC.     

Cold Atmospheric Pressure Plasma-Activated Medium Induces Selective Cell Death in Human Hepatocellular Carcinoma Cells Independently of Singlet Oxygen,Hydrogen Peroxide,Nitric Oxide and Nitrite/Nitrate

Cold atmospheric pressure plasma (CAP) and plasma-activated medium (PAM) induce cell death in diverse cancer cells and may function as powerful anti-cancer agents. The main components responsible for the selective anti-cancer effects of CAP and PAM remain elusive. CAP or PAM induces selective cell death in hepatocellular carcinoma cell lines Hep3B and Huh7 containing populations with cancer stem cell markers. Here, we investigated the major component(s) of CAP and PAM for mediating the selective anti-proliferative effect on Hep3B and Huh7 cells. The anti-proliferative effect of CAP was mediated through the medium; however, the reactive oxygen species scavenger N-acetyl cysteine did not suppress PAM-induced cell death. Neither high concentrations of nitrite or nitrite/nitrate nor a low concentration of H₂O₂ present in the PAM containing sodium pyruvate affected the viability of Hep3B and Huh7 cells. Inhibitors of singlet oxygen, superoxide anions, and nitric oxide retained the capacity of PAM to induce anti-cancer effects. The anti-cancer effect was largely blocked in the PAM prepared by placing an aluminum metal mesh, but not a dielectric PVC mesh, between the plasma source and the medium. Hence, singlet oxygen, hydrogen peroxide, nitric oxide, and nitrite/nitrate are not the main factors responsible for PAM-mediated selective death in Hep3B and Huh7 cells. Other factors, such as charged particles including various ions in CAP and PAM, may induce selective anti-cancer effects in certain cancer cells.     

ROS-Mediated Anti-Tumor Effect of Coptidis Rhizoma against Human Hepatocellular Carcinoma Hep3B Cells and Xenografts

Coptidis Rhizoma is the dried rhizome from the Coptis chinensis Franch. that has been shown to have a number of beneficial pharmacological properties including antioxidant, anti-inflammatory, and anti-cancer effects. However, the anti-cancer effects of Coptidis Rhizoma on hepatocellular carcinoma (HCC) remain unclear. In this study, we investigated the anti-cancer properties of Coptidis Rhizoma ethanol extract (CR) in HCC Hep3B cells and in a xenograft mouse model. Our results showed that the CR significantly inhibited cell growth and induced apoptosis in Hep3B cells through increased expression of Bcl-2 associated x-protein (Bax) and cleavage of poly-ADP ribose polymerase (PARP), reduced expression of Bcl-2, and activated caspases. CR also increased the generation of intracellular reactive oxygen species (ROS), which caused a loss of mitochondrial membrane potential (MMP, ΔΨm) and activation of the mitochondria-mediated intrinsic apoptosis pathway. Moreover, N-acetylcysteine (NAC), a ROS inhibitor, markedly blocked the effects of CR on apoptotic pathways. CR also induced the expression of light chain 3 (LC3)-I/II, a key autophagy regulator, whereas CR-mediated autophagy was significantly suppressed by NAC. In addition, pre-treatment with NAC perfectly attenuated the inhibition of cell invasion and migration of CR-stimulated Hep3B cells. Furthermore, oral administration of CR suppressed Hep3B tumor growth in xenograft mice without toxicity, alterations to body weight, or changes in hematological and biochemical profiles. Taken together, our findings suggest that CR has anti-tumor effects that result from ROS generation, and may be a potential pharmacological intervention for HCC.     

Increased ARPP-19 Expression Is Associated with Hepatocellular Carcinoma

The cAMP-regulated phosphoprotein 19 (ARPP-19) plays a key role in cell mitotic G2/M transition. Expression of ARPP-19 was increased in human hepatocellular carcinoma (HCC) compared to adjacent non-tumorous liver tissues in 36 paired liver samples, and the level of ARPP-19 in HCC tissues was positively correlated with the tumor size. To determine the interrelationship between ARPP-19 expression and HCC, we silenced ARPP-19 expression in the human hepatocarcinoma HepG2 and SMMC-7721 cells using lentivirus encoding ARPP-19 siRNA. HepG2 and SMMC-7721 cells with ARPP-19 knockdown displayed lowered cell growth rate, retarded colony formation and increased arrest at the G2/M phase transition. Silencing ARPP-19 in HCC cells resulted in decreased protein levels of phospho-(Ser) CDKs substrates and increased levels of inactivated cyclin division cycle 2 (Cdc2). Therefore, ARPP-19 may play a role in HCC pathogenesis through regulating cell proliferation.     

Albumin Suppresses Human Hepatocellular Carcinoma Proliferation and the Cell Cycle

Many investigations have revealed that a low recurrence rate of hepatocellular carcinoma (HCC) is associated with high serum albumin levels in patients; therefore, high levels of serum albumin are a major indicator of a favorable prognosis. However, the mechanism inhibiting the proliferation of HCC has not yet been elucidated, so we investigated the effect of serum albumin on HCC cell proliferation. Hep3B was cultured in MEM with no serum or containing 5 g/dL human albumin. As control samples, Prionex was added to generate the same osmotic pressure as albumin. After 24-h incubation, the expressions of α-fetoprotein (AFP), p53, p21, and p57 were evaluated with real-time PCR using total RNA extracted from the liver. Protein expressions and the phosphorylation of Rb (retinoblastoma) were determined by Western blot analysis using total protein extracted from the liver. For flow cytometric analysis of the cell cycle, FACS analysis was performed. The percentages of cell cycle distribution were evaluated by PI staining, and all samples were analyzed employing FACScalibur (BD) with appropriate software (ModFit LT; BD). The cell proliferation assay was performed by counting cells with using a Scepter handy automated cell counter (Millipore). The mRNA levels of AFP relative to Alb(−): Alb(−), Alb(+), and Prionex, were 1, 0.7 ± 0.2 (p < 0.001 for Alb(−)), and 1 ± 0.3, respectively. The mRNA levels of p21 were 1, 1.58 ± 0.4 (p = 0.007 for Alb(−) and p = 0.004 for Prionex), and 0.8 ± 0.2, respectively. The mRNA levels of p57 were 1, 4.4 ± 1.4 (p = 0.002 for Alb(−) and Prionex), and 1.0 ± 0.1, respectively. The protein expression levels of Rb were similar in all culture media. The phosphorylation of P807/811 and P780 of Rb protein was reduced in Alb(+). More cells in the G0/G1 phase and fewer cells in S and G2/M phases were obtained in Alb(+) than in Alb(−) (G0/G1: 60.9%, 67.7%, 61.5%; G2/M: 16.5%, 13.1%, 15.6%; S: 22.6%, 19.2%, 23.0%, Alb(−), Alb(+), Prionex, respectively). The same results were obtained in HepG2. Cell proliferation was inhibited in 5 g/dL albumin medium in both HepG2 cells and Hep3B cells in 24 h culture by counting cell numbers. The presence of albumin in serum reduces the phosphorylation of Rb proteins and enhances the expression of p21 and p57, following an increase in the G0/G1 cell population, and suppresses cell proliferation. These results suggest that albumin itself suppresses the proliferation of hepatocellular carcinoma.     

Skeletal Muscle Depletion Predicts the Prognosis of Patients with Hepatocellular Carcinoma Treated with Sorafenib

The aim of this study was to determine whether skeletal muscle depletion predicts the prognosis of patients with hepatocellular carcinoma (HCC) that is being treated with sorafenib. We evaluated 40 consecutive HCC patients who received sorafenib treatment. The skeletal muscle cross-sectional area was measured by computed tomography at the third lumbar vertebra (L3), from which the L3 skeletal muscle index (L3 SMI) was obtained. The factors contributing to overall survival, sorafenib dose reduction, and discontinuation of sorafenib were analyzed using the Cox proportional hazards model. L3 SMI (p = 0.020) and log (α-fetoprotein (AFP)) (p = 0.010) were identified as independent prognostic factors in HCC patients treated with sorafenib. The initial dose of sorafenib (p = 0.008) was an independent risk factor for sorafenib dose reduction, and log (AFP) (p = 0.008) was the only significant risk factor for the discontinuation of this drug. L3 SMI was not a risk factor for either dose reduction (p = 0.423) or the discontinuation (p = 0.132) of sorafenib. A multiple linear regression analysis determined the following relationship between skeletal muscle mass (assessed as L3 SMI) and the explanatory factors: L3 SMI = −0.1896 × (Age) − 10.3441 × (Child-Pugh score) − 9.3922 × (log (AFP)) + 1.6139 × (log (AFP)) × (Child-Pugh score) + 112.9166. Skeletal muscle depletion is inversely associated with age, Child-Pugh score, and log (AFP). Moreover, it is an independent prognostic factor for HCC patients treated with sorafenib.     

The Role of Receptor for Advanced Glycation End Products (RAGE) in the Proliferation of Hepatocellular Carcinoma

The receptor for advanced glycation end products (RAGE) is oncogenic and overexpressed in human cancers, but its role in hepatocellular carcinoma remains unclear. Here we demonstrated that RAGE is overexpressed in primary hepatocellular carcinoma (PHC) compared to adjacent para-neoplastic liver samples. Serum endogenous secretory RAGE levels were also increased in PHC patients (p < 0.01). Moreover, we demonstrated that RAGE regulates cellular proliferation in Hepatocellular carcinoma (HCC). Knockdown of RAGE by specific siRNA inhibited cellular growth in the hepatocellular carcinoma cell line, Huh7, whereas the RAGE ligand, high mobility group box 1 protein (HMGB1) increased cellular proliferation. In addition, knockdown of RAGE by siRNA arrested cells in the G1 phase and inhibited DNA synthesis (p < 0.01), while HMGB1 protein decreased the number of cells in the G1 phase and increased the number in the S phase (p < 0.05). Furthermore, quantitative real time RT-PCR (qRT-PCR) and Western Blot results demonstrated that RAGE and HMGB1 positively regulate NF-κB p65 expression in Huh7 cells. These studies suggest that RAGE and RAGE ligands are important targets for therapeutic intervention in hepatocellular carcinoma.     

Secretory NPC2 Protein-Mediated Free Cholesterol Levels Were Correlated with the Sorafenib Response in Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor in the world. Sorafenib is the first-line drug for patients with advanced HCC. However, long-term treatment with sorafenib often results in reduced sensitivity of tumor cells to the drug, leading to acquired resistance. Identifying biomarkers which can predict the response to sorafenib treatment may represent a clinical challenge in the personalized treatment era. Niemann-Pick type C2 (NPC2), a secretory glycoprotein, plays an important role in regulating intracellular free cholesterol homeostasis. In HCC patients, downregulation of hepatic NPC2 is correlated with poor clinical pathological features through regulating mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) activation. This study aimed to investigate the roles of secretory NPC2-mediated free cholesterol levels as biomarkers when undergoing sorafenib treatment and evaluate its impact on acquired sorafenib resistance in HCC cells. Herein, we showed that NPC2 downregulation and free cholesterol accumulation weakened sorafenib’s efficacy through enhancing MAPK/AKT signaling in HCC cells. Meanwhile, NPC2 overexpression slightly enhanced the sorafenib-induced cytotoxic effect. Compared to normal diet feeding, mice fed a high-cholesterol diet had much higher tumor growth rates, whereas treatment with the free cholesterol-lowering agent, hydroxypropyl-β-cyclodextrin, enhanced sorafenib’s tumor-inhibiting ability. In addition, sorafenib treatment induced higher NPC2 secretion, which was mediated by inhibition of the Ras/Raf/MAPK kinase (MEK)/ERK signaling pathway in HCC cells. In both acquired sorafenib-resistant cell and xenograft models, NPC2 and free cholesterol secretion were increased in culture supernatant and serum samples. In conclusion, NPC2-mediated free cholesterol secretion may represent a candidate biomarker for the likelihood of HCC cells developing resistance to sorafenib.