Activity In Hepatocellular Carcinoma Cells Research Articles

Induction of ROS and DNA damage-dependent senescence by icaritin contributes to its antitumor activity in hepatocellular carcinoma cells

Context: Icaritin (ICT), a prenylflavonoid derivative extracted from the Epimedium (Berberidaceae) genus, has been identified to exhibit antitumor effect in hepatocellular carcinoma (HCC) cells by inducing apoptosis. However, its effect on cellular senescence has not been elucidated.Objective: To investigate the mechanism for low concentrations of ICT exerting antitumor activity through induction of cellular senescence.Materials and methods: Human HepG2 and Huh7 cells were treated with low concentrations of ICT (1 and 2 μM) once per day for a week. Cellular senescence was evaluated through cell viability and senescence-associated-β-galactosidase activity. Cell cycle distribution and ROS levels were measured with flow cytometry. Gene expression was detected using qRT-PCR and western blotting. Fluorescent punctuates formation of γH2AX was analyzed by immunofluorescence.Results: ICT (1 and 2 μM) promoted cellular senescence in HepG2 and Huh7 cells, as observed by enlarged and flattened morphology and increased senescence-associated-β-galactosidase activity (∼7-8-fold and ∼11-12-fold of vehicle controls, respectively), accompanied by significant cell cycle arrest and decrease in DNA synthesis. Mechanistically, ICT-induced senescence occurred through accumulation of ROS (∼1.3-fold and ∼1.8-fold of vehicle controls in response to 1 and 2 μM ICT, respectively), which further resulted in DNA damage response, as evidenced by strong induction of γH2AX through immunofluorescence and western blotting assays. Pharmacological inhibition of ROS production with N-acetylcysteine attenuated ICT-induced γH2AX and senescence-associated-β-galactosidase activity (∼0.28-0.30-fold decrease, p < 0.05).Discussion and conclusions: Induction of cellular senescence by ICT defines a novel anticancer mechanism of ICT and provides a rationale for generalizing the study design to a broader study population to further developing ICT as a novel therapeutic agent for treatment of HCC.

Pseudolaric acid B exhibits anti-cancer activity on human hepatocellular carcinoma through inhibition of multiple carcinogenic signaling pathways

BackgroundPseudolaric acid B (PAB), a diterpene acid isolated from the root bark of Pseudolarix kaempferi, exhibits a potent anti-cancer activity in a variety of tumor cells. PurposeThe present study was designed to evaluate the anti-cancer effects of PAB on hepatocellular carcinoma (HCC) cell lines in vitro, and to explore the underlying mechanism. MethodsThe anti-proliferative activity of PAB on HCC cells were assessed via sulforhodamine B staining, colony formation, cell cycle analysis, respectively. Apoptosis was detected using Annexin V/propidium iodide double staining and diamidino-phenyl-indole staining, respectively. Protein expression regulated by PAB treatment was tested by western blotting. ResultsThe present results showed that PAB significantly inhibited the proliferation of HepG2, SK-Hep-1, and Huh-7 HCC cell lines in vitro with IC50 values of 1.58, 1.90, and 2.06 μM, respectively. Furthermore, PAB treatment repressed the colony formation in HepG2, SK-Hep-1, and Huh-7 HCC cell lines. Flow cytometry analysis revealed that PAB caused an obvious cell cycle arrest in G2/M phase and induced apoptosis with the induction of p21, Bax, cleaved-caspase-3, and cleaved-PARP in human HepG2 and SK-Hep-1 cells. Mechanistically, PAB treatment down-regulated the phosphorylation of STAT3, ERK1/2, and Akt. Moreover, abnormal GSK-3β/β-catenin signaling in HepG2 cells was remarkably suppressed by PAB treatment. Finally, proliferation markers including cyclin D1 and c-Myc, and anti-apoptosis proteins such as Bcl-2 and survivin were also down-regulated by PAB treatment in HepG2 cells. ConclusionTaken together, our results suggest that PAB exerts anti-cancer activity in HCC cells through inhibition of STAT3, ERK1/2, Akt, and GSK-3β/β-catenin carcinogenic signaling pathways, and may be used as a phytomedicine in the treatment of HCC.

A new meroterpenoid functions as an anti-tumor agent in hepatoma cells by downregulating mTOR activation and inhibiting EMT

Liver cancer, also known as primary liver cancer, is cancer that starts in the liver. JNU-144, a new meroterpenoid purified from Lithospermum erythrorhizon, has exhibited promising anticancer activity; however, the molecular mechanisms of action of JNU-144 on malignant cells remain unclear. Our studies revealed that JNU-144 suppressed cell viability and proliferation in hepatoma cells by downregulating mTOR activation. Meanwhile, JNU-144 activated the intrinsic apoptosis pathway and subsequently triggered apoptotic cell death in SMMC-7721 cells. We also found that JNU-144 inhibited the epithelial–mesenchymal transition in both SMMC-7721 and HepG2 cells through reprogramming of epithelial–mesenchymal transition (EMT)-related gene expression or regulating protein instability. These findings indicate that JNU-144 exerts potent anticancer activity in hepatoma cells and may be developed as a potential therapeutic drug.

Magnolol exerts anticancer activity in hepatocellular carcinoma cells through regulating endoplasmic reticulum stress-mediated apoptotic signaling.

IntroductionMagnolol (Mag), a biologically active compound isolated from the root and stem bark of Magnolia officinalis, has been reported to induce apoptosis in several cancer cell lines in vitro. In the present study, we aimed to determine the anticancer effects of Mag on hepatocellular carcinoma (HCC) cells.Materials and methodsThe HepG2 cells were treated with varying concentrations of Mag (10, 20, and 30 μM) for 48 hours. The effects of Mag on the proliferation, migration, invasion, apoptosis and cell cycle progression of HepG2 cells were respectively detected by MTT assay, transwell assays, and flow cytometric analysis. A HepG2 cell-based tumor-bearing model was established to evaluate the effect of Mag on HCC tumor growth in vivo. The protein expression levels were determined by Western blot analysis.ResultsOur results showed that Mag inhibited the proliferation, migration, and invasion of HepG2 cells in vitro in a dose-dependent manner. In addition, Mag reduced the HCC tumor volume and weight in the mouse xenograft model. Subsequent studies showed that Mag induced apoptosis in HepG2 cells, accompanied by a loss in mitochondrial membrane potential, cytochrome c release, and induction of endoplasmic reticulum stress. Furthermore, inhibition of the endoplasmic reticulum stress by CHOP knockdown restored the effects of Mag in HepG2 cells.ConclusionThe present study highlighted the possibility of using Mag as a novel therapeutic drug for HCC treatment.

HCV-induced EGFR-ERK signaling promotes a pro-inflammatory and pro-angiogenic signature contributing to liver cancer pathogenesis

HCV is a major risk factor for hepatocellular carcinoma (HCC). HCC development in chronically infected HCV patients has until now been attributed to persistent inflammation and interference of viral proteins with host cell signaling. Since activation of the epidermal growth factor receptor (EGFR) presents a crucial step in HCV entry, we aimed at investigating whether EGFR signaling may contribute to the pathogenesis of HCV-related HCC.By applying microarray analysis, we generated a gene expression signature for secreted proteins in HCV-infected hepatoma cells. This gene signature was enriched for inflammatory and angiogenic processes; both crucially involved in HCC development. RT-qPCR analysis, conducted on the entire list of upregulated genes, confirmed induction of 11 genes (AREG, IL8, CCL20, CSF1, GDF15, IGFBP1, VNN3, THBS1 and PAI-1) in a virus titer- and replication-dependent manner. EGFR activation in hepatoma cells largely mimicked the gene signature seen in the infectious HCV model. Further, the EGFR-ERK pathway, but not Akt signaling, was responsible for this gene expression profile. Finally, microarray analysis conducted on clinical data from the GEO database, revealed that our validated gene expression profile is significantly represented in livers of patients with HCV-related liver pathogenesis (cirrhosis and HCC) compared to healthy livers.Taken together, our data indicate that persistent activation of EGFR-ERK signaling in chronically infected HCV patients may induce a specific pro-inflammatory and pro-angiogenic signature that presents a new mechanism by which HCV can promote liver cancer pathogenesis. A better understanding of the key factors in HCV-related oncogenesis, may efficiently direct HCC drug development.

Niclosamide, an oral antihelmintic drug, exhibits antimetastatic activity in hepatocellular carcinoma cells through downregulating twist-mediated CD10 expression.

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world, especially, in eastern Asia, and its prognosis is poor once metastasis occurs. Niclosamide, a US Food and Drug Administration-approved antihelmintic drug, was shown to inhibit the growth of various cancers including HCC, but the effect of niclosamide on cell motility and the underlying mechanism have not yet been completely defined. The present study demonstrated that niclosamide, at 0-40 nM, concentration-dependently inhibited wound closure and the migratory/invasive capacities of human Huh7 and SK-Hep-1 HCC cells without exhibiting cytotoxicity. A protease array analysis showed that CD10 was dramatically downregulated in Huh7 cells after niclosamide treatment. Western blot and flow cytometric assays further demonstrated that CD10 expression was concentration-dependently downregulated in Huh7 and SK-Hep-1 cells after niclosamide treatment. Mechanistic investigations found that niclosamide suppressed Twist-mediated CD10 transactivation. Moreover, knockdown of CD10 expression by CD10 small interfering RNA in HCC cells suppressed cell migratory/invasive abilities and overexpression of CD10 relieved the migration inhibition induced by niclosamide. Taken together, our results indicated that niclosamide could be a potential agent for inhibiting metastasis of HCC, and CD10 is an important target of niclosamide for suppressing the motility of HCC cells.

Polydatin inhibits cell proliferation, invasion and migration, and induces cell apoptosis in hepatocellular carcinoma.

Polydatin, a small molecule from Polygonum cuspidatum, has many biological functions, particularly anti-cancer effects. However, the anti-cancer effects of polydatin in hepatocellular carcinoma (HCC) have not been examined yet. In the present study, MTT assay, BrdU assay, transwell invasion assay, and wound healing assay were performed to determine cell proliferation, invasion and migration. Flow cytometry and TUNEL assay were used to measure cell apoptosis. Quantitative real-time PCR and western blotting assays were used to determine mRNA and protein expression levels. Xenograft experiment was performed to determine the in vivo anti-tumor effect of polydatin. Immunostaining was performed to analyze the expression of caspase-3 and Ki-67. Our results showed that polydatin inhibited cell proliferation in a concentration-dependent and time-dependent manner in the HCC cell lines. Polydatin also induced cell apoptosis in a concentration-dependent manner possibly via increasing the caspase-3 activity, and up-regulating the protein expression of caspase-3, caspase-9, Bax, and down-regulating the protein expression of Bcl-2. In addition, polydatin treatment had an inhibitory effect on cell proliferation, invasion and migration in HCC cell lines. Polydatin treatment also suppressed the Wnt/beta-catenin signaling activities in HCC cells. Polydatin treatment significantly reduced tumor growth in nude mice inoculated with HepG2 cells, suppressed the expression of Ki-67, and increased caspase-3 expression and TUNEL activity. Our data indicated the important role of polydatin for the suppression of HCC progression.

Upregulation of Mcl-1 inhibits JQ1-triggered anticancer activity in hepatocellular carcinoma cells

Bromodomains and extra-terminal (BET) proteins inhibitors are promising cancer therapeutic agents. However, tumor cells often develop resistance to BET inhibitors, greatly limiting their therapeutic potential. To study the mechanism underlying the resistance of BET inhibitors in hepatocellular carcinoma (HCC) cells, we herein investigated the impact of BET inhibitor JQ1 on the gene expression of Bcl-2 family members by RNA sequencing analysis, and found that acute treatment with JQ1 triggered upregulation of Mcl-1 in HCCLM3 and BEL7402 cell lines. This JQ1-triggered Mcl-1 upregulation was further confirmed by quantitative reverse transcription polymerase chain reaction and western blotting analysis, both at mRNA and protein levels. Inhibition of Mcl-1 by RNA interference dramatically enhanced JQ1-triggered caspase-3 activation, cleavage of poly (ADP-ribose) polymerase and apoptotic cell death induction in multiple HCC cell lines. Moreover, JQ1 in combination with cyclin-dependent kinase inhibitor flavopiridol at a subtoxic concentration that reduced expression of Mcl-1, triggered massive apoptotic cell death in HCCLM3 and BEL7402 cell lines. Together, these data suggest that Mcl-1 is a major contributor to BET inhibitor-resistance in HCC cells, and that combining drugs capable of down-regulating Mcl-1 may promote therapeutic potential in human HCC.

Overexpression of a disintegrin and metalloproteinase 21 is associated with motility, metastasis, and poor prognosis in hepatocellular carcinoma

Cell motility plays an important role in intrahepatic metastasis of hepatocellular carcinoma (HCC), and predicts poor prognosis in patients. The present study investigated the role of a disintegrin and metalloproteinases (ADAMs) in HCC, since these proteins are known to be associated with cell motility. We confirmed the expression of 12 ADAMs with putative metalloproteinase activity in HCC cells, and established a KYN-2 HCC cell line stably expressing short interfering RNA against ADAM21 to investigate the effect of ADAM21 deficiency on HCC cell motility and metastasis in vitro and in vivo. We also examined ADAM21 expression in a cohort of 119 HCC patients by immunohistochemistry. ADAM21 was overexpressed in KYN-2 cells, and its knockdown reduced invasion, migration, proliferation, and metastasis relative to controls. In clinical specimens, ADAM21 positivity was associated with vascular invasion, large tumor size, high histological grade, and lower overall and recurrence-free survival as compared to cases that were negative for ADAM21 expression. A multivariate analysis revealed that ADAM21 positivity was an independent risk factor for overall (P = 0.003) and recurrence-free (P = 0.001) survival. These results suggest that ADAM21 plays a role in HCC metastasis and can serve as a prognostic marker for disease progression.

Hepatitis C virus core protein potentiates proangiogenic activity of hepatocellular carcinoma cells.

Increased angiogenic activity has been demonstrated in hepatitis C virus (HCV)-related hepatocellular carcinoma (HCC), but the mechanism was unclear. To study the role of HCV core protein, we used tube formation and Matrigel plug assays to assess the proangiogenic activity of an HCC cell line, HuH7, and 2 of its stable clones—HuH7-core-high and HuH7-core-low, with high and low HCV core protein expression, respectively. In both assays, HuH7-core-high and HuH7-core-low cells dose-dependently induced stronger angiogenesis than control cells. HuH7 cells with HCV core protein expression showed increased mRNA and protein expression of vascular endothelial growth factor (VEGF). VEGF inhibition by bevacizumab reduced the proangiogenic activity of HuH7-core-high cells. The promotor region of VEGF contains the binding site of activator protein-1 (AP-1). Compared with controls, HuH7-core-high cells had an increased AP-1 activity and nuclear localization of phospho-c-jun. AP-1 inhibition using either RNA knockdown or AP-1 inhibitors reduced the VEGF mRNA expression and the proangiogenic activity of HuH7-core-high cells. Among 131 tissue samples from HCC patients, HCV-related HCC revealed stronger VEGF expression than did hepatitis B virus-related HCC. In conclusion, increased VEGF expression through AP-1 activation is a crucial mechanism underlying the proangiogenic activity of the HCV core protein in HCC cells.

Molecular mechanisms governing microRNA-125a expression in human hepatocellular carcinoma cells

MicroRNA-125a-5p (miR-125a) is a vertebrate homolog of lin-4, the first discovered microRNA, and plays a fundamental role in embryo development by downregulating Lin-28 protein. MiR-125a is also expressed in differentiated cells where it generally acts as an antiproliferative factor by targeting membrane receptors or intracellular transductors of mitogenic signals. MiR-125a expression is downregulated in several tumors, including hepatocellular carcinoma (HCC) where it targets sirtuin-7, matrix metalloproteinase-11, VEGF-A, Zbtb7a, and c-Raf. In this study, we have isolated the transcription promoter of human miR-125a and characterized its activity in HCC cells. It is a TATA-less Pol II promoter provided with an initiator element and a downstream promoter element, located 3939 bp upstream the genomic sequence of the miRNA. The activity of the promoter is increased by the transcription factor NF-kB, a master regulator of inflammatory response, and miR-125a itself was found to strengthen this activation through inhibition of TNFAIP3, a negative regulator of NF-kB. This finding contributes to explain the increased levels of miR-125a observed in the liver of patients with chronic hepatitis B.

Structural basis for alpha fetoprotein‐mediated inhibition of caspase‐3 activity in hepatocellular carcinoma cells

Alpha-fetoprotein (AFP) is an early serum growth factor in the foetal liver development and hepatic carcinogenesis; However, the precise biological role of cytoplasmic AFP remains elusive. Although we recently demonstrated that cytoplasmic AFP might interact with caspase-3 and inhibit the signal transduction of apoptosis in human hepatocellular carcinoma (HCC) cells, the details of this interaction are not clear. To reveal the molecular relationship between AFP and caspase-3, we performed molecular docking, co-immunoprecipitation (Co-IP), laser confocal microscopy, site-directed mutagenesis and functional experiments to analyse the key amino acid residues in the binding site of caspase-3. The results of Co-IP, laser confocal microscopy and functional analyses were consistent with the computational model. We also used the model to explain why AFP cannot bind to caspase-8. These results provide the molecular basis for the AFP-mediated inhibition of caspase-3 activity in HCC cells. Altogether, we found that AFP interacts with caspase-3 through precise amino acids, namely loop-4 residues Glu-248, Asp-253 and His-257. The results further demonstrated that AFP plays a critical role in the inhibition of the apoptotic signal transduction that mediated by caspase-3. Thus, AFP might represent a novel biotarget for the therapy of HCC patients.

Regulation of IRE1α by the small molecule inhibitor 4μ8c in hepatoma cells

The unfolded protein response (UPR) is activated in response to impairments of the folding environment in the endoplasmic reticulum (ER). The most conserved arm of the UPR, inositol-requiring ER-to-nucleus signaling protein (IRE1α), has been linked to the regulation of a diverse array of cellular processes including ER-associated degradation, inflammatory signaling, cell proliferation and membrane biogenesis. Recent studies have utilized the selective, small molecule inhibitor, 4μ8c, to examine the role of IRE1α endoribonuclease (RNase) activity in various cell types including multiple myeloma, mouse embryonic fibroblasts and pancreatic beta cells [1-5]. In the present study we utilized this inhibitor to examine the role of IRE1α RNase activity in hepatoma cells (H4IIE), specifically concentrating on cell proliferation and the identification of potential off target effects under both unstressed and stressed conditions. Experiments were performed in H4IIE hepatoma cells in the absence (control conditions (LG)) or presence (LG + Thapsigargin (Thap)) of ER stress. The presence of 4μ8c decreased IRE1α RNase activity, based on reduced splicing of X-box binding protein-1 (XBP1s) and regulated IRE1α-dependent decay of mRNA in both treatments and at concentrations ranging from 10-90 μM. Cell proliferation was significantly reduced at higher concentrations (> 60 μM 4μ8c) in unstressed cells and displayed a dose-response relationship with 4μ8c in both treatments. The presence of 4μ8c did not promote cytoxicity in either of the treatment conditions but higher concentrations of the inhibitor (60 μM) were associated with apparent off-target or compensatory responses that were not observed at 10 μM. Overall, the small-molecule inhibitor, 4μ8c is an effective inhibitor of IRE1α RNase activity in H4IIE cells. Potential off-target effects associated with this inhibitor require the use of multiple inhibitor concentrations in all experiments.

Identification of sphingosine kinase 1 (SphK1) as a primary target of icaritin in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is a highly aggressive neoplasm. We aim to explore the anti-HCC activity by a natural prenylflavonoid icaritin. Icaritin was cytotoxic and pro-apoptotic when added to established (HepG2, KYN-2 and Huh-7 lines) and primary human HCC cells. At the signaling level, icaritin inhibited sphingosine kinase 1 (SphK1) activity in HCC cells, which led to pro-apoptotic ceramide production and JNK1 activation. SphK1 inhibition or silence (by shRNA/microRNA) mimicked icaritin-mediated cytotoxicity, and almost nullified icaritin's activity in HepG2 cells. Reversely, exogenous over-expression of SphK1 sensitized icaritin-induced HepG2 cell apoptosis. In vivo, oral administration of icaritin dramatically inhibited HepG2 xenograft growth in SCID mice. Further, SphK1 activity in icaritin-treated tumors was largely inhibited. In summary, icaritin exerts potent anti-HCC activity in vitro and in vivo. SphK1 inhibition could be the primary mechanism of its actions in HCC cells.

NDRG2 overexpression suppresses hepatoma cells survival during metabolic stress through disturbing the activation of fatty acid oxidation

Because of the high nutrient consumption and inadequate vascularization, solid tumor constantly undergoes metabolic stress during tumor development. Oncogenes and tumor suppressor genes participated in cancer cells' metabolic reprogramming. N-Myc downstream regulated gene 2 (NDRG2) is a recently identified tumor suppressor gene, but its function in cancer metabolism, particularly during metabolic stress, remains unclear. In this study, we found that NDRG2 overexpression significantly reduced hepatoma cell proliferation and enhanced cell apoptosis under glucose limitation. Moreover, NDRG2 overexpression aggravated energy imbalance and oxidative stress by decreasing the intracellular ATP and NADPH generation and increasing ROS levels. Strikingly, NDRG2 inhibited the activation of fatty acid oxidation (FAO), which preserves ATP and NADPH purveyance in the absence of glucose. Finally, mechanistic investigation showed that NDRG2 overexpression suppressed the glucose-deprivation induced AMPK/ACC pathway activation in hepatoma cells, whereas the expression of a constitutively active form of AMPK abrogated glucose-deprivation induced AMPK activation and cell apoptosis. Thus, as a negative regulator of AMPK, NDRG2 disturbs the induction of FAO genes by glucose limitation, leading to dysregulation of ATP and NADPH, and thus reduces the tolerance of hepatoma cells to glucose limitation.

The microRNA-99 family modulates hepatitis B virus replication by promoting IGF-1R/PI3K/Akt/mTOR/ULK1 signaling-induced autophagy.

MicroRNAs are small highly conserved noncoding RNAs that are widely expressed in multicellular organisms and participate in the regulation of various cellular processes including autophagy and viral replication. Evidently, microRNAs are able to modulate host gene expression and thereby inhibit or enhance hepatitis B virus (HBV) replication. The miR-99 family members are highly expressed in the liver. Interestingly, the plasma levels of miR-99 family in the peripheral blood correspond with HBV DNA loads. Thus, we asked whether the miR-99 family regulated HBV replication and analyzed the underlying molecular mechanism. Compared with primary hepatocytes, miR-99 family expression was downregulated in hepatoma cells. Transfection of miR-99a, miR-99b, and miR-100 markedly increased HBV replication, progeny secretion, and antigen expression in hepatoma cells. However, miR-99 family had no effect on HBV transcription and HBV promoter activities, suggesting that they regulate HBV replication at posttranscriptional steps. Consistent with bioinformatic analysis and recent reports, ectopic expression of miR-99 family attenuated IGF-1R/Akt/mTOR pathway signaling and repressed insulin-stimulated activation in hepatoma cells. Moreover, the experimental data demonstrated that the miR-99 family promoted autophagy through mTOR/ULK1 signaling and thereby enhanced HBV replication. In conclusion, the miR-99 family promotes HBV replication posttranscriptionally through IGF-1R/PI3K/Akt/mTOR/ULK1 signaling-induced autophagy.

Substrate stiffness promotes latent TGF-β1 activation in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) was usually coupled with increased stiffness of the extracellular matrix (ECM) and elevated level of transforming growth factor-β1 (TGF-β1). However, the mechanism by which substrate rigidity modulated TGF-β1 signaling transduction remained unknown. This paper investigated the molecular mechanism of how matrix stiffness regulating TGF-β1 signaling in HCC cells. By means of stiffness tunable collagen I-coated polyacrylamide (PA) gels, we found that the expressions of β1 integrin, p-FAK Y397 and p-Smad2 upregulated on stiffer gels as well as the content of TGF-β1 in culture media of HCC cells, which were inhibited by RGD blocking peptides, Y-27632 (ROCK inhibitor) or Blebbistatin (myosin II inhibitor). Cellular traction force was also significantly higher when plated on stiffer substrates but dramatically decreased after treatment with Y-27632 or Blebbistatin. Furthermore, the upregulation of p-Smad2 in the HCC cells on stiffer PA gels induced by exogenetic latent TGF-β1 was downregulated in the presence of RGD peptides. The nuclear translocation of Smad2 induced by latent TGF-β1 was inhibited by Y-27632 or Blebbistatin. Our results suggested that the extracellular matrix stiffness regulated latent TGF-β1 activation by cytoskeletal tension in HCC cells, showing that matrix stiffness was a key regulator involving the TGF-β1 activity in HCC cells. The current study presented a mechanism of how hepatocirrhosis developed into liver cancer.

Pyruvate Dehydrogenase Kinase 4 Deficiency Results in Expedited Cellular Proliferation through E2F1-Mediated Increase of Cyclins

Hepatocellular carcinoma (HCC) is a common form of cancer with prevalence worldwide. There are many factors that lead to the development and progression of HCC. This study aimed to identify potential new tumor suppressors, examine their function as cell cycle modulators, and investigate their impact on the cyclin family of proteins and cyclin-dependent kinases (CDKs). In this study, the pyruvate dehydrogenase kinase (PDK)4 gene was shown to have potential tumor suppressor characteristics. PDK4 expression was significantly downregulated in human HCC. Pdk4-/- mouse liver exhibited a consistent increase in cell cycle regulator proteins, including cyclin D1, cyclin E1, cyclin A2, some associated CDKs, and transcription factor E2F1. PDK4-knockdown HCC cells also progressed faster through the cell cycle, which concurrently expressed high levels of cyclins and E2F1 as seen in the Pdk4-/- mice. Interestingly, the induced cyclin E1 and cyclin A2 caused by Pdk4 deficiency was repressed by arsenic treatment in mouse liver and in HCC cells. E2f1 deficiency in E2f1-/- mouse liver or knockdown E2F1 using small hairpin RNAs in HCC cells significantly decreased cyclin E1, cyclin A2, and E2F1 proteins. In contrast, inhibition of PDK4 activity in HCC cells increased cyclin E1, cyclin A2, and E2F1 proteins. These findings demonstrate that PDK4 is a critical regulator of hepatocyte proliferation via E2F1-mediated regulation of cyclins.

Indole-3- carbinol enhances sorafenib cytotoxicity in hepatocellular carcinoma cells: A mechanistic study.

Sorafenib is the only chemotherapeutic agent currently approved for unresectable hepatocellular carcinoma (HCC). However, poor response rates have been widely reported. Indole-3-carbinol (I3C) is a potential chemopreventive phytochemical. The present study aimed to explore the potential chemomodulatory effects of I3C on sorafenib in HCC cells as well as the possible underlying mechanisms. I3C exhibited a greater cytotoxicity in HepG2 cells compared to Huh-7 cells (p < 0.0001). Moreover, the co-treatment of HepG2 cells with I3C and sorafenib was more effective (p = 0.002). Accordingly, subsequent mechanistic studies were carried on HepG2 cells. The results show that the ability of I3C to enhance sorafenib cytotoxicity in HCC cells could be partially attributed to increasing the apoptotic activity and decreasing the angiogenic potentials. The combination had a negative effect on epithelial-mesenchymal transition (EMT). Increased NOX-1 expression was also observed which may indicate the involvement of NOX-1 in I3C chemomodulatory effects. Additionally, the combination induced cell cycle arrest at the G0/G1 phase. In conclusion, these findings provide evidence that I3C enhances sorafenib anti-cancer activity in HCC cells.

An oncolytic adenovirus that expresses the HAb18 and interleukin 24 genes exhibits enhanced antitumor activity in hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is characterized by alterations in multiple genes. High expression of CD147 on the surface of HCC cells promotes proliferation. The monoclonal antibody HAb18 recognizes CD147. We constructed an oncolytic adenoviral vector to express HAb18 (ZD55-HAb18) in HCC cells. Interleukin 24 (IL24) was co-expressed through the use of an F2A linker. ZD55-HAb18-IL24 decreased HCC cell viability to a greater degree than either ZD55-HAb18 or ZD55-IL24 alone. ZD55-HAb18-IL24 also induced apoptosis and autophagy in PLC/PRF/5 HCC cells. Intratumoral injection of ZD55-HAb18-IL24 repressed tumor growth in a PLC/PRF/5 xenograft model. Our results suggest that antibody-antitumor gene conjugation elicited a stronger antitumor effect than the antibody alone, and that this strategy could broaden the applications of antibody-based therapies in HCC.