Effects In Hepatocellular Carcinoma Research Articles

Activated Hepatic Stellate Cells (HSCs) Exert Immunosuppressive Effects in Hepatocellular Carcinoma by Producing Complement C3.

ObjectiveHepatic stellate cells (HSCs) are the important players in liver cirrhosis and liver cancer. They also act as critical mediators of immunosuppression in hepatocellular carcinoma (HCC). In this study, we hypothesized that HSCs promote HCC progression via C3.MethodsC3 in HSCs was knocked down using a shRNA retroviral plasmid. The conditioned medium from HSCs or shC3 HSCs (knockdown of C3 by shRNA in HSCs) was collected to detect their effects on bone marrow (BM) and T cells (including expansion and apoptosis) in vitro, and in an HCC in situ model in mice.ResultsWe found that HSCs promoted T-cell apoptosis and decreased their proliferation, inhibited dendritic cell (DC) maturation, and induced myeloid-derived suppressor cell (MDSC) expansion through the C3 pathway in vitro. In addition, the knockdown of C3 suppressed HSC-promoted HCC development in the orthotopic transplantation tumor model of HCC in mice.ConclusionThese findings provide more insights into the immunomodulatory roles of HSCs in HCC progression and indicate that modulation of the C3 pathway might be a novel therapeutic approach for liver cancer.

TGF-β induced chemoresistance in liver cancer is modulated by xenobiotic nuclear receptor PXR

ABSTRACT Hepatocellular carcinoma appears as an extremely angiogenic solid tumor marked by apoptosis evasion, dysregulated cell cycle and low sensitivity to chemotherapy. TGF-β, a multifunctional cytokine, plays a pleiotropic role in the tumor microenvironment and has implications in cancer drug resistance. The current study provides novel evidence that TGF-β signaling contributes to drug resistance in liver cancer cells by inducing the expression of xenobiotic nuclear receptor PXR. We observed that PXR increases the expression of drug efflux transporters; therefore, accounting for exacerbated drug resistance. Additionally, anti-apoptotic nature of PXR contributes to TGF-β mediated chemoresistance as seen by procaspase-3 and Mcl-1 cellular levels. TGF-β binding to the TGF-β receptor triggers a complex downstream signaling cascade through a non-canonical SMAD-independent ERK pathway that leads to increased PXR expression. Activated ERK activates ETS1 transcription factor which is a critical regulator of endogenous PXR expression in hepatic cells. Loss of function of ETS1 abrogates the TGF-β induced PXR expression. Together these findings indicate that PXR modulates TGF-β induced resistance to chemotherapy in liver cancer cells. This underscores the need for combinatorial approaches with focus on PXR antagonism to improve drug effectiveness in hepatocellular carcinoma. Abbreviations HCC: Hepatocellular Carcinoma; FDA: Food and Drug Administration; TGF-β: Transforming growth factor-β; PXR: Pregnane X receptor; CAR: Constitutive androstane receptor; P-gp/ABCB1: P-glycoproteins/ATP-binding cassette transporter subfamily B member 1; MRP1/ABCC1 and MRP2/ABCC2: Multidrug-resistance associated proteins; BCRP/ABCG2: Breast cancer resistant protein; DMEs: Drug-metabolizing enzymes; CFDA: 5,6-carboxyfluorescein diacetate; ETS1: Transcription factor E26 transformation specific sequence 1.

MiR-383 inhibits cell growth and promotes cell apoptosis in hepatocellular carcinoma by targeting IL-17 via STAT3 signaling pathway

ObjectivesEmerging microRNAs (miRNAs) are validated to take part in pathological processes, including numerous carcinomas. Currently, we focused on the functional role of miR-383 and interleukin-17 (IL-17) in hepatocellular carcinoma (HCC), and the underlying molecular mechanisms were also the emphases in our research. MethodsWe used reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to measure the expression levels of miR-383 in 45 paired tumor tissues and adjacent non-tumor tissues extracted from patients with hepatocellular carcinoma. These tissues were also stained for IL-17 using immunohistochemical staining. Western blot was performed to detect the protein expressions of following protein-coding genes, including p-Stat3, Stat3 and GAPDH. A dual-luciferase activity was carried out to determine whether IL-17 was the downstream gene of miR-383 in hepatocellular carcinoma development. The colony assay, CCK8 assay, and apoptosis assay were used to explore the detailed regulatory effects of miR-383/IL-17 axis in the cellular processes of hepatocellular carcinoma separately. ResultsmiR-383 was down-regulated significantly in tumor tissues, while IL-17 was up-regulated. IL-17 was certificated to act as the downstream gene of miR-383. Furthermore, overexpression of miR-383 suppressed cell proliferation and promoted apoptosis in hepatocellular carcinoma. However, the raised IL-17 attenuated the inhibition effect of miR-383 in hepatocellular carcinoma. In addition, we found that p-Stat3 was repressed by miR-383, and the up-regulation of IL-17 reversed the suppression effect in hepatocellular carcinoma. ConclusionsmiR-383 may play a anti-tumor role in the pathogenesis of hepatocellular carcinoma by targeting IL-17 through STAT3 signaling pathway. miR-383/IL-17 axis maybe a potent target for the clinical diagnosis and treatment of hepatocellular carcinoma.

Dual Programmed Death Receptor-1 and Vascular Endothelial Growth Factor Receptor-2 Blockade Promotes Vascular Normalization and Enhances Antitumor Immune Responses in Hepatocellular Carcinoma.

Activation of the antitumor immune response using programmed death receptor-1 (PD-1) blockade showed benefit only in a fraction of patients with hepatocellular carcinoma (HCC). Combining PD-1 blockade with antiangiogenesis has shown promise in substantially increasing the fraction of patients with HCC who respond to treatment, but the mechanism of this interaction is unknown. We recapitulated these clinical outcomes using orthotopic-grafted or induced-murine models of HCC. Specific blockade of vascular endothelial receptor 2 (VEGFR-2) using a murine antibody significantly delayed primary tumor growth but failed to prolong survival, while anti-PD-1 antibody treatment alone conferred a minor survival advantage in one model. However, dual anti-PD-1/VEGFR-2 therapy significantly inhibited primary tumor growth and doubled survival in both models. Combination therapy reprogrammed the immune microenvironment by increasing cluster of differentiation 8-positive (CD8+ ) cytotoxic T cell infiltration and activation, shifting the M1/M2 ratio of tumor-associated macrophages and reducing T regulatory cell (Treg) and chemokine (C-C motif) receptor 2-positive monocyte infiltration in HCC tissue. In these models, VEGFR-2 was selectively expressed in tumor endothelial cells. Using spheroid cultures of HCC tissue, we found that PD-ligand 1 expression in HCC cells was induced in a paracrine manner upon anti-VEGFR-2 blockade in endothelial cells in part through interferon-gamma expression. Moreover, we found that VEGFR-2 blockade increased PD-1 expression in tumor-infiltrating CD4+ cells. We also found that under anti-PD-1 therapy, CD4+ cells promote normalized vessel formation in the face of antiangiogenic therapy with anti-VEGFR-2 antibody. We show that dual anti-PD-1/VEGFR-2 therapy has a durable vessel fortification effect in HCC and can overcome treatment resistance to either treatment alone and increase overall survival in both anti-PD-1 therapy-resistant and anti-PD-1 therapy-responsive HCC models.

High throughput circRNA sequencing analysis reveals novel insights into the mechanism of nitidine chloride against hepatocellular carcinoma

Nitidine chloride (NC) has been demonstrated to have an anticancer effect in hepatocellular carcinoma (HCC). However, the mechanism of action of NC against HCC remains largely unclear. In this study, three pairs of NC-treated and NC-untreated HCC xenograft tumour tissues were collected for circRNA sequencing analysis. In total, 297 circRNAs were differently expressed between the two groups, with 188 upregulated and 109 downregulated, among which hsa_circ_0088364 and hsa_circ_0090049 were validated by real-time quantitative polymerase chain reaction. The in vitro experiments showed that the two circRNAs inhibited the malignant biological behaviour of HCC, suggesting that they may play important roles in the development of HCC. To elucidate whether the two circRNAs function as “miRNA sponges” in HCC, we identified circRNA-miRNA and miRNA-mRNA interactions by using the CircInteractome and miRwalk, respectively. Subsequently, 857 miRNA-associated differently expressed genes in HCC were selected for weighted gene co-expression network analysis. Module Eigengene turquoise with 423 genes was found to be significantly related to the survival time, pathology grade and TNM stage of HCC patients. Gene functional enrichment analysis showed that the 423 genes mainly functioned in DNA replication- and cell cycle-related biological processes and signalling cascades. Eighteen hubgenes (SMARCD1, CBX1, HCFC1, RBM12B, RCC2, NUP205, ECT2, PRIM2, RBM28, COPS7B, PRRC2A, GPR107, ANKRD52, TUBA1B, ATXN7L3, FUS, MCM8 and RACGAP1) associated with clinical outcomes of HCC patients were then identified. These findings showed that the crosstalk between hsa_circ_0088364 and hsa_circ_0090049 and their competing mRNAs may play important roles in HCC, providing interesting clues into the potential of circRNAs as therapeutic targets of NC in HCC.

Overexpression of CSN6 promotes the epithelial-mesenchymal transition and predicts poor prognosis in hepatocellular carcinoma

CSN6, as a critical subunit of the constitutive photomorphogenesis 9 (COP9) signalosome (CSN), has been previously reported to be increased in various cancers; however, its effect in hepatocellular carcinoma (HCC) remains unknown, which is the aim of present study, in terms of its explore the expression and role of CSN6 in HCC. QRT-PCR, Western blot and immunohistochemistry (IHC) were used to examine the expression of CSN6. Kaplan-Meier survival analysis and univariate and multivariate Cox analyses were used to investigate the clinical and prognostic significance of CSN6 expression in HCC patients. Furthermore, the biological function of CSN6 on HCC cell proliferation and migration was investigated through CCK-8, transwell migration and invasion assays. Besides, the associations between CSN6 and epithelial-mesenchymal transition (EMT) were determined. CSN6 was increased in HCC tissues, and its overexpression was found to be associated with a poor prognoses for HCC patients. Overexpression of CSN6 promoted processes of HCC cell proliferation, migration, and invasion, while these processes were inhibited when CSN6 was silenced. Additionally, CSN6 was found to promote EMT by inhibiting E-cadherin, which were significantly mitigated via upregulation of Snail as a result of MEK/ERK pathway activation. CSN6 up-regulation may play a contributory role in HCC metastasis and poor prognosis via activation of EMT, and may serve as an independent predictor for HCC prognosis.

Circ-IGF1R has pro-proliferative and anti-apoptotic effects in HCC by activating the PI3K/AKT pathway

Circular RNAs (circRNAs), a novel class of widespread and diverse endogenous RNAs, have been identified as critical regulators of various cancers, including hepatocellular carcinoma (HCC). However, the specific roles of circRNAs in HCC are largely unknown. In this study, we identified a novel circRNA, circ-IGF1R, in HCC tumour tissues and cell lines. Circ-IGF1R levels were found to be significantly upregulated in HCC tissues compared with levels in paired peritumoural tissues. The high expression levels of circ-IGF1R in HCC were associated with tumour size. Moreover, knocking down circ-IGF1R with siRNA significantly attenuated cell proliferation and induced cell apoptosis and cell cycle arrest in vitro. Further investigation revealed that PI3K/AKT signalling pathway activation was involved in the oncogenic functions of circ-IGF1R in HCC. Our study suggests that circ-IGF1R may be a potential target for the prevention and treatment of HCC.

Detection of Total Aflatoxins in Groundnut and Soybean Samples in Yemen Using Enzyme-Linked Immunosorbent Assay

Aflatoxins are fungal toxins that have mutagenic and carcinogenic effects, especially hepatocellular carcinoma effect. This work aimed to investigate the presence of aflatoxins in groundnuts and soybeans that are consumed in Yemen. The samples were collected from three different regions in Yemen (Sana’a, al-Hodeida, and Aden), and they were divided into two groups. The concentration of total aflatoxins was analyzed by enzyme-linked immunosorbent assay (ELISA). Aflatoxins were determined in 89 groundnut and 65 soybean samples. The results showed that 85.39% (76/89) of groundnut and 72.3% (45/65) of soybean samples were contaminated with aflatoxins. In addition, in 49.44% and 27.6% of the groundnut and soybean samples, total aflatoxins exceed the acceptable level of European Commission (4 μg/kg), while in only 6.2% of soybean samples and 22.47% of groundnut samples, total aflatoxins were beyond the maximum limit of FDA/Yemen standards (20 μg/kg). The results showed that the aflatoxin contamination in the groundnut and soybean samples may be considered a significant risk for public health. The present study is the first to report the data on the presence of aflatoxins in groundnut and soybean samples in Yemen.

Prognostic Role of Apelin Receptor Expression in Hepatocellular Carcinoma Treated With Curative Surgical Resection.

apelin and apelin receptor (APJR) are involved in the regulation of angiogenesis, and their high expression is related to poor outcomes in several cancer types. Recently, several positive results on APJR antagonists in cancer treatment have been reported at the preclinical level. The aim of this study was to evaluate the prognostic effect of APJR expression on hepatocellular carcinoma (HCC) survival. We evaluated APJR expression in 288 curatively resected HCCs using immunohistochemistry and investigated the correlation with clinicopathological features. High APJR expression was significantly associated with the presence of microvascular invasion (p<0.001), intrahepatic metastasis (p=0.004), and early recurrence (p=0.029). The high-expression group showed shorter recurrence-free survival (p<0.001) and overall survival (p=0.001) than the low-expression group. In multivariate analysis, high APJR expression was an independent predictor of shorter recurrence-free survival (Hazard Ratio 1.49; 95% confidence interval 1.08-2.05, p=0.016). We described-high APJR expression and its prognostic effect in HCC. Emerging target agents may be applicable in patients with HCC and high APJR expression.

The establishment and application of a preoperative predictive nomogram for hepatocellular carcinoma with microvascular invasion

Objective To establish a preoperative nomogram model in predicting microvascular invasion (MVI) and to test its predictive effectiveness in hepatocellular carcinoma (HCC). Methods This retrospective study was conducted on 798 patients with HCC, including 690 males and 108 females, aged (49.8±10.9) years old who underwent curative hepatectomy in the Guangxi Medical University Affiliated Tumor Hospital between January 2014 and December 2017 were retrospectively analyzed. The patients were divided into the model group (n=579) and the validation group (n=219) according to the periods of the operation time. Independent risk factors of MVI were identified by univariate and multivariate logistic regression analysis in the model group, and a nomogram model was established according to the independent risk factors. The accuracy of the nomogram model in predicting MVI was detected in the two groups by the computer consistency coefficient (C-index) and calibration graph method. The predictive value was evaluated by receiver operating characteristic curve. Results Histopathological diagnosis revealed 278 patients with MVI and no MVI in the 301 patients of HCC out of the 579 patients in the model group. In the validation group, there were 119 patients with MVI and 100 patients with no MVI out of the 219 patients. Total bilirubin >15 μmol/L(OR=1.519, 95%CI: 1.041~2.217), alkaline phosphatase >60 U/L(OR=1.681, 95%CI: 1.059~2.670), alpha-fetoprotein >200 ng/L (OR=2.192, 95%CI: 1.531~3.134) and tumor maximum diameter (OR=1.120, 95%CI: 1.057~1.187) were the independent risk factors of MVI on multivariate analysis. After establishment of the nomogram model using the independent risk factors, the C-indexes were 0.680 and 0.773 respectively in the model group and the validation group. In the calibration graph, the standard curve properly fitted with the predicting calibration curve. The predicted value of MVI obtained was in good agreement with the observed value. The ROC curve analysis nomogram model predicted the low performance of MVI. Conclusion The nomogram model in predicting MVI in patients with HCC was successfully established. The model offered certain guiding significance in the clinical treatment of HCC. Key words: Carcinoma, hepatocellular; Risk factors; Microvascular invasion; Nomogram

Sustained virologic response to direct-acting antiviral therapy in patients with chronic hepatitis C and hepatocellular carcinoma: A systematic review and meta-analysis

The effect of hepatocellular carcinoma (HCC) on the response to interferon-free direct-acting antiviral (DAA) therapy in patients with chronic hepatitis C (CHC) infection remains unclear. Using a systematic review and meta-analysis approach, we aimed to investigate the effect of DAA therapy on sustained virologic response (SVR) among patients with CHC and either active, inactive or no HCC. PubMed, Embase, Web of Science, and the Cochrane Central Register of Controlled Trials were searched from 1/1/2013 to 9/24/2018. The pooled SVR rates were computed using DerSimonian-Laird random-effects models. We included 49 studies from 15 countries, comprised of 3,341 patients with HCC and 35,701 without HCC. Overall, the pooled SVR was lower in patients with HCC than in those without HCC (89.6%, 95% CI 86.8-92.1%, I2 = 79.1% vs. 93.3%, 95% CI 91.9-94.7%, I2 = 95.0%, p = 0.0012), translating to a 4.8% (95% CI 0.2-7.4%) SVR reduction by meta-regression analysis. The largest SVR reduction (18.8%) occurred in patients with active/residual HCC vs. inactive/ablated HCC (SVR 73.1% vs. 92.6%, p = 0.002). Meanwhile, patients with HCC who received a prior liver transplant had higher SVR rates than those who did not (p <0.001). Regarding specific DAA regimens, patients with HCC treated with ledipasvir/sofosbuvir had lower SVR rates than patients without HCC (92.6%, n = 884 vs. 97.8%, n = 13,141, p = 0.026), but heterogeneity was high (I2 = 84.7%, p <0.001). The SVR rate was similar in patients with/without HCC who were treated with ombitasvir/paritaprevir/ritonavir ± dasabuvir (n = 101) (97.2% vs. 94.8%, p = 0.79), or daclatasvir/asunaprevir (91.7% vs. 89.8%, p = 0.66). Overall, SVR rates were lower in patients with HCC, especially with active HCC, compared to those without HCC, though heterogeneity was high. Continued efforts are needed to aggressively screen, diagnose, and treat HCC to ensure higher CHC cure rates. There are now medications (direct-acting antivirals or "DAAs") that can "cure" hepatitis C virus, but patients with hepatitis C and liver cancer may be less likely to achieve cure than those without liver cancer. However, patients with liver cancer are also more likely to have advanced liver disease and risk factors that can decrease cure rates, so better controlled studies are needed to confirm these findings.

Signal transducer and activator of transcription 3 promotes the Warburg effect possibly by inducing pyruvate kinase M2 phosphorylation in liver precancerous lesions.

BACKGROUNDStudy shows that signal transducer and activator of transcription 3 (STAT3) can increase the Warburg effect by stimulating hexokinase 2 in breast cancer and upregulate lactate dehydrogenase A and pyruvate dehydrogenase kinase 1 in myeloma. STAT3 and pyruvate kinase M2 (PKM2) can also be activated and enhance the Warburg effect in hepatocellular carcinoma. Precancerous lesions are critical to human and rodent hepatocarcinogenesis. However, the underlying molecular mechanism for the development of liver precancerous lesions remains unknown. We hypothesized that STAT3 promotes the Warburg effect possibly by upregulating p-PKM2 in liver precancerous lesions in rats.AIMTo investigate the mechanism of the Warburg effect in liver precancerous lesions in rats.METHODSA model of liver precancerous lesions was established by a modified Solt-Farber method. The liver pathological changes were observed by HE staining and immunohistochemistry. The transformation of WB-F344 cells induced with N-methyl-N’-nitro-N-nitrosoguanidine and hydrogen peroxide was evaluated by the soft agar assay and aneuploidy. The levels of glucose and lactate in the tissue and culture medium were detected with a spectrophotometer. The protein levels of glutathione S-transferase-π, proliferating cell nuclear antigen (PCNA), STAT3, and PKM2 were examined by Western blot and immunofluorescence.RESULTSWe found that the Warburg effect was increased in liver precancerous lesions in rats. PKM2 and p-STAT3 were upregulated in activated oval cells in liver precancerous lesions in rats. The Warburg effect, p-PKM2, and p-STAT3 expression were also increased in transformed WB-F344 cells. STAT3 activation promoted the clonal formation rate, aneuploidy, alpha-fetoprotein expression, PCNA expression, G1/S phase transition, the Warburg effect, PKM2 phosphorylation, and nuclear translocation in transformed WB-F344 cells. Moreover, the Warburg effect was inhibited by stattic, a specific inhibitor of STAT3, and further reduced in transformed WB-F344 cells after the intervention for PKM2.CONCLUSIONThe Warburg effect is initiated in liver precancerous lesions in rats. STAT3 activation promotes the Warburg effect by enhancing the phosphorylation of PKM2 in transformed WB-F344 cells.

LncRNA CADM1-AS1 inhibits cell-cycle progression and invasion via PTEN/AKT/GSK-3β axis in hepatocellular carcinoma.

Purpose: CADM1-AS1 (cell adhesion molecule 1 antisense RNA 1, long non-coding RNA), was firstly characterized in renal clear cell carcinoma, and exhibits a tumor suppressor role. However, its clinical relevance and exact effects in hepatocellular carcinoma (HCC) remain unknown. Therefore, in this study, we aimed to assess the clinical significance and function of CADM1-AS1 in HCC.Methods: We detected CADM1-AS1 expression in liver cancer tissue samples and cell lines, and analyzed the association between CADM1-AS1 expression and clinical parameters in 90 liver cancer patients. Moreover, we conducted gain-of-function and loss-of-function studies in liver cancer cell to explore the biological function and molecular mechanism of CADM1-AS1.Results: CADM1-AS1 expression was reduced in HCC. Clinical data showed that this downregulation was associated with advanced tumor stage, high TNM stage and reduced survival in HCC patients. CADM1-AS1 overexpression inhibited HCC cells proliferation, migration and invasion, while inducing G0/G1 phase arrest. Meanwhile, we revealed that CADM1-AS1 inhibited the phosphorylation of AKT and GSK-3β. Furthermore, our study showed that CADM1-AS1 decreased the cell cycle associated proteins expression of cyclinD, cyclinE, CDK2 CDK4, CDK6, and enhanced the levels of p15, p21 and p27. More importantly, SC79, a specific activator for AKT;, apparently attenuated the effects of CADM1-AS1 on above cell-cycle associated proteins, confirming that CADM1-AS1 inhibited cell cycles through the AKT signaling pathway. And we also found the CADM1-AS1 has antitumor effect in vivo by a xenograft HCC mouse model. In conclusion, the present findings show that the CADM1-AS1 inhibits proliferation of HCC by inhibiting AKT/GSK-3β signaling pathway, then upregulate p15, p21, p27 expression and downregulate cyclin, CDK expression to inhibit the G0/G1 to S phase transition both in vitro and in vivo.Conclusion: CADM1-AS1 functions as a tumor-suppressive lncRNA. This study reveals a molecular pathway involving PTEN/AKT/GSK-3β which regulates HCC cell-cycle progression.

High Throughput circRNA Sequencing Analysis Reveals Novel Insights into the Mechanism of Nitidine Chloride Against Hepatocellular Carcinoma

Background: Nitidine chloride (NC) has been demonstrated to have an anticancer effect in hepatocellular carcinoma (HCC). However, the mechanism of action of NC against HCC remains largely unclear. Methods: Three pairs of NC-treated and NC-untreated HCC xenograft tumour tissues were collected for circRNA sequencing. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to corroborate the expression of differently expressed circRNAs. Weighted gene co-expression network analysis (WGCNA) was used to identify the weighted gene co-expression network. Findings: A total of 298 circRNAs were differently expressed between NC-treated and NC-untreated HCC xenograft tissues, with 188 upregulated and 109 downregulated, among which hsa_circ_0088364, hsa_circ_0090049 and hsa_circ_0102434 were validated by RT-qPCR. To elucidate the mechanisms of action of the three circRNAs in HCC, we predicted the circRNA-miRNA and miRNA-mRNA interactions by using the CircInteractome and miRwalk, respectively. In total, 2519 miRNA-correlated differently expressed genes in HCC were selected for WGCNA analysis. Module Eigengene blue with 234 genes were found to be significantly related to the survival time, pathology grade and TNM stage of patients with HCC. Gene functional enrichment analyses showed that these genes mainly functioned in cell adhesion and apoptosis. Eight hubgenes (AGAP1, FBXL19, IGSF3, ITPR3, KIAA1522, NCDN, SLC38A1 and TCTN2) of the 234 genes were identified. All of these genes were linked to the survival outcome in patients with HCC. Interpretation: These findings showed that the crosstalk between hsa_circ_0088364, hsa_circ_0090049 and hsa_circ_0102434 and their competing mRNAs may play important roles in HCC, providing interesting clues into the potential of circRNAs as therapeutic targets of NC in HCC. Funding Statement: This study was supported by the National Natural Science Foundation of China (NSFC81860717, NSFC81560489, NSFC81860717 and NSFC81560489), the Natural Science Foundation of Guangxi, China (2017GXNSFAA198017 and 2018GXNSFAA294025), the Guangxi Medical University Training Program for Distinguished Young Scholars, Medical Excellence Award Funded by the Creative Research Development Grant from the First Affiliated Hospital of Guangxi Medical University (Gang Chen and Yi-wu Dang), the Guangxi Degree and Postgraduate Education Reform and Development Research Projects, China, and the Guangxi Zhuang Autonomous Region Health and Family Planning Commission Self-financed Scientific Research Project (Z20180979) Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals (the Shanghai SLAC Laboratory Animal of China, 2015) and were approved by the Ethics Committee of the First Affiliated Hospital of Guangxi Medical University (Nanning, China).

Observational Study of PD-L1, TGF-β, and Immune Cell Infiltrates in Hepatocellular Carcinoma.

Introduction: Hepatocellular carcinoma (HCC) typically develops in cirrhotic livers, with increased programed death ligand 1 (PD-L1) and transforming growth factor beta (TGF-β) activity implicated in immunosuppression.Methods: In an observational study of HCC liver samples, we determined the incidence of PD-L1 and immune cell (IC) infiltrates, and signs of TGF-β activity. HCCs were characterized by the incidence and distribution of PD-L1+ cells, and CD8+, CD68+, and FoxP3+ infiltrating ICs in HCC and surrounding liver. Gene expression signatures (GESs) associated with TGF-β activity and ICs were evaluated by RNAseq.Results: In non-neoplastic cirrhotic and non-cirrhotic liver, PD-L1 occurred on sinusoidal lining cells (mostly Kupffer cells), endothelial cells and ICs. In HCC, PD-L1+ tumor cells were rare. Most PD-L1+ cells were identified as ICs. CD8+, CD68+, and FoxP3+ ICs were associated with HCC, particularly in the invasive margin. CD8+ cell incidence correlated with PD-L1+ cells, consistent with PD-L1 being upregulated in response to pre-existing cytotoxic T-lymphocyte activity. TGFB1 mRNA levels and TGF-β activation GES correlated with the strength of the tumor-associated macrophage GES.Conclusion: Inhibition of PD-L1+ ICs and TGF-β activity and their respective immunomodulatory pathways may contribute to antitumor effects in HCC.

Sinoporphyrin sodium based sonodynamic therapy induces anti-tumor effects in hepatocellular carcinoma and activates p53/caspase 3 axis

Sonodynamic therapy (SDT) is a noninvasive therapeutic method via the activation of certain chemical sensitizers using low intensity ultrasound. In this work, we evaluated the antitumor effect of sinoporphyrin sodium (DVDMS) mediated SDT (DVDMS-SDT) on Hepatocellular carcinoma (HCC) cell lines both in vitro and in vivo. The results indicated that DVDMS-SDT was significantly more efficacious than PpIX-SDT in treating hepatocellular cell line Hep-G2. DVDMS-SDT also increased the ratio of cells in the G2/M phase and decreased the CDK1 and Cyclin B1 protein level. DVDMS-SDT markedly increased intracellular reactive oxygen species (ROS) in vitro. The increased ROS production up-regulated the expression of p53 and Bax, and down-regulated Bcl-2 expression, which led to the activation of caspase-3, ultimately initiated cell apoptosis. These effects could be partially reversed by the ROS scavenger N-acetylcysteine (NAC). In vivo experiments revealed that the DVDMS-SDT resulted in an effective inhibition of tumor growth and prolonged the survival time of tumor-bearing mice. More importantly, no obvious signs of side effects were observed. These results suggested that DVDMS-SDT is very effective in treating Hepatocellular carcinoma without side effects. The primary mechanism of SDT is due to the increased ROS activated the p53/Caspase 3 axis of apoptosis.

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.

Effects of rosmarinic acid on immunoregulatory activity and hepatocellular carcinoma cell apoptosis in H22 tumor-bearing mice.

Rosmarinic acid (RA) is a natural polyphenolic compound that exists in many medicinal species of Boraginaceae and Lamiaceae. The previous studies have revealed that RA had therapeutic effects on hepatocellular carcinoma (HCC) in the H22-xenograft models by inhibiting the inflammatory cytokines and NF-κB p65 pathway in the tumor microenvironment. However, its molecular mechanisms of immunoregulation and pro-apoptotic effect in HCC have not been fully explored. In the present study, RA at 75, 150, and 300 mg/kg was given to H22 tumor-bearing mice via gavage once a day for 10 days. The results showed that RA can effectively inhibit the tumor growth through regulating the ratio of CD4+/CD8+ and the secretion of interleukin (IL)-2 and interferon-γ, inhibiting the expressions of IL-6, IL-10 and signal transducer and activator of transcription 3, thereby up-regulating Bax and Caspase-3 and down-regulating Bcl-2. The underlying mechanisms involved regulation of immune response and induction of HCC cell apoptosis. These results may provide a more comprehensive perspective to clarify the anti-tumor mechanism of RA in HCC.

The long noncoding RNA NORAD enhances the TGF-β pathway to promote hepatocellular carcinoma progression by targeting miR-202-5p.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers with common features of invasion and metastasis. Recent evidence indicate that the long noncoding RNA NORAD is a potential oncogene and is significantly upregulated in several cancers. However, the general biological role and clinical value of NORAD in HCC remains unknown. Here, NORAD expression was measured in 29 paired tumor and paratumor tissues via quantitative real‐time polymerase chain reaction (qPCR). The effects of NORAD on HCC cell malignant potential were investigated via NORAD overexpression and knockdown both in vitro and in vivo. The mechanism of competitive endogenous RNAs (ceRNAs) was acquired and identified by bioinformatics analyses and luciferase assays. Moreover, the impact of NORAD level on the transforming growth factor β (TGF‐β) pathway was further determined by qPCR. We found that HCC tissues had a high level of NORAD compared with the paratumor tissues, and NORAD upregulation was associated with the shorter overall survival of patients with HCC. Furthermore, NORAD overexpression was demonstrated to promote HCC cell migration and invasion. Mechanically, NORAD might function as a ceRNA to regulate miR‐202‐5p, which served as a tumor‐suppressing microRNA via the TGF‐β pathway. We address that NORAD has a tumor‐promoting effect in HCC and describes a novel mechanism whereby NORAD regulates the TGF‐β pathway as a ceRNA of Homo sapiens (hsa)‐miR‐202‐5p.

Overexpressing lncRNA SNHG16 inhibited HCC proliferation and chemoresistance by functionally sponging hsa-miR-93.

BackgroundLong noncoding RNAs (lncRNAs) have been identified as prognostic biomarkers and functional regulators in human cancers. The present study aimed to determine the expressions and functions of an lncRNA, Small Nucleolar RNA Host Gene 16 (SNHG16), in hepatocellular carcinoma (HCC).Patients and methodsSNHG16 expressions were tested by quantitative real-time PCR (qRT-PCR) in HCC cell lines, as well as 43 pairs of HCC tissues and pair-matched healthy hepatic tissues. It was overexpressed in Hep3B and HuH7 cells. The effects of SNHG16 overexpression in HCC in vitro proliferation, 5-fluorouracil (5-FU) chemoresistance, and in vivo tumor growth were tested. A potential microRNA (miRNA) sponge target of SNHG16, hsa-miR-93, was tested by luciferase reporter assay and qRT-PCR. In addition, hsa-miR-93 was upregulated in SNHG16-overexpressed HCC cells to examine its effect on SNHG16-mediated cancer cell functional regulation in HCC.ResultsSNHG16 levels were markedly downregulated in both HCC cell lines and HCC tissues. Lentivirus-mediated SNHG16 overexpression inhibited HCC cell proliferation, 5-FU chemoresistance, and in vivo tumor growth. Hsa-miR-93 was confirmed to be directly sponging on SNHG16. Its upregulation in HCC cells reversed SNHG16 overexpression and induced tumor-suppressing effects in HCC cells.ConclusionOur data demonstrate that SNHG16 plays a critical role in HCC development via functionally sponging hsa-miR-93.