A network pharmacology approach to investigate the anticancer mechanism of cinobufagin against hepatocellular carcinoma via downregulation of EGFR-CDK2 signaling.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers with high mortality and poor prognosis, and the investigation on new approaches and effective drugs for HCC therapy is of great significance. In our study, we demonstrate that treatment with cinobufagin, a natural compound isolated from traditional chinese medicine Chansu, reduces proliferation and the colony formation capacity of the human hepatoma cells in vitro, in addition, cinobufagin induces mitotic arrest in human hepatoma cells. The results of a network pharmacology-based analysis show that EGFR, MAPK1, PTK2, CDK2, MAPK3, ESR1, CDK1, PRKCA, AR, and CSNK2A1 are the key targets involved in the anti-tumor activities of cinobufagin, additionally, several signaling pathways such as proteoglycans in cancer, pathways in cancer, HIF-1 signaling pathway, VEGF signaling pathway, ErbB signaling pathway, and PI3K-AKT signaling pathway are identified as the potential pathways involved in the inhibitory effects of cinobufagin against HCC. Furthermore, at the molecular level, we find that cinobufagin decreases EGFR expression and CDK2 activity in human hepatoma cells. Inhibition of EGFR or CDK2 expression could not only suppress the growth of tumor cells but also enhance the inhibitory effects of cinobufagin on the proliferative potential of human hepatoma cells. We also demonstrate that EGFR positively regulates CDK2 expression. Furthermore, EGFR inhibitor gefitinib or CDK2 inhibitor CVT-313 synergistically enhances anticancer effects of cinobufagin in human hepatoma cells. Taken together, these findings indicate that cinobufagin may exert antitumor effects by suppressing EGFR-CDK2 signaling, and our study suggests that cinobufagin may be a novel, promising anticancer agent for the treatment of HCC.

Discovery of a Carbamoyl Phosphate Synthetase 1-Deficient HCC Subtype With Therapeutic Potential Through Integrative Genomic and Experimental Analysis.

BACKGROUND AND AIMS: Metabolic reprogramming plays an important role in tumorigenesis. However, the metabolic types of different tumors are diverse and lack in-depth study. Here, through analysis of big databases and clinical samples, we identified a carbamoyl phosphate synthetase 1 (CPS1)-deficient hepatocellular carcinoma (HCC) subtype, explored tumorigenesis mechanism of this HCC subtype, and aimed to investigate metabolic reprogramming as a target for HCC prevention. APPROACH AND RESULTS: A pan-cancer study involving differentially expressed metabolic genes of 7,764 tumor samples in 16 cancer types provided by The Cancer Genome Atlas (TCGA) demonstrated that urea cycle (UC) was liver-specific and was down-regulated in HCC. A large-scale gene expression data analysis including 2,596 HCC cases in 7 HCC cohorts from Database of HCC Expression Atlas and 17,444 HCC cases from in-house hepatectomy cohort identified a specific CPS1-deficent HCC subtype with poor clinical prognosis. In vitro and in vivo validation confirmed the crucial role of CPS1 in HCC. Liquid chromatography-mass spectrometry assay and Seahorse analysis revealed that UC disorder (UCD) led to the deceleration of the tricarboxylic acid cycle, whereas excess ammonia caused by CPS1 deficiency activated fatty acid oxidation (FAO) through phosphorylated adenosine monophosphate-activated protein kinase. Mechanistically, FAO provided sufficient ATP for cell proliferation and enhanced chemoresistance of HCC cells by activating forkhead box protein M1. Subcutaneous xenograft tumor models and patient-derived organoids were employed to identify that blocking FAO by etomoxir may provide therapeutic benefit to HCC patients with CPS1 deficiency. CONCLUSIONS: In conclusion, our results prove a direct link between UCD and cancer stemness in HCC, define a CPS1-deficient HCC subtype through big-data mining, and provide insights for therapeutics for this type of HCC through targeting FAO.

Saikosaponins and the deglycosylated metabolites exert liver meridian guiding effect through PXR/CYP3A4 inhibition.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Bupleuri (RB), traditionally used to treat inflammatory disorders and infectious diseases, represents one of the most successful and widely used herbal drugs in Asia over the past 2000 years. Being realized the role in regulating metabolism and controlling Yin/Yang, RB is not only chosen specifically for treating liver meridian and the corresponding organs, but also believed to have liver meridian guiding property and help potentiate the therapeutic effects of liver. However, the ingredients in RB with liver meridian guiding property and the underly mechanism have not been comprehensively investigated. AIM OF STUDY: Considering the important role of CYP3A4 in first-pass metabolism and the liver exposure of drugs, the present study aimed to determine whether saikosaponins (SSs) and the corresponding saikogenins (SGs) have a role in inhibiting the catalytic activity of CYP3A4 in human liver microsomes and HepG2 hepatoma cells and whether they could suppress CYP3A4 expression by PXR-mediated pathways in HepG2 hepatoma cells. MATERIALS AND METHODS: The effect of SSs and SGs on CYP3A4-mediated midazolam1'-hydroxylation activities in pooled human liver microsomes (HLMs) was first studied. Dose-dependent experiments were performed to obtain the half inhibit concentration (IC50) values. HepG2 cells were used to assay catalytic activity of CYP3A4, reporter function, mRNA levels, and protein expression. The inhibitory effects of SSa and SSd on CYP3A4 activity are negligible, while the corresponding SGs (SGF and SGG) have obvious inhibitory effects on CYP3A4 activity, with IC50 values of 0.45 and 1.30 µM. The similar results were obtained from testing CYP3A4 catalytic activity in HepG2 cells, which correlated well with the suppression of the mRNA and protein levels of CYP3A4. Time-dependent testing of CYP3A4 mRNA and protein levels, as well as co-transfection experiments using the CYP3A4 promoter luciferase plasmid, further confirmed that SSs and SGs could inhibit the expression of CYP3A4 at the transcription level. Furthermore, PXR protein expression decreased in a concentration- and time-dependent manner after cells were exposed to SSs and SGs. PXR overexpression and RNA interference experiments further showed that SSs and SGs down-regulate the catalytic activity and expression of CYP3A4 in HepG2 may be mainly through PXR-dependent manner. CONCLUSION: SSs and SGs inhibit the catalytic activity and expression of CYP3A4 in a PXR-dependent manner, which may be highly related to the liver meridian guiding property of RB.

Urokinase-type Plasminogen Activator Is a Therapeutic Target for Overcoming Sorafenib Resistance in Hepatoma Cells.

BACKGROUND/AIM: Sorafenib is a multikinase inhibitor approved as a first-line therapy for hepatocellular carcinoma. This study examined the sorafenib resistance mechanism. MATERIALS AND METHODS: Hepatoma HepG2 cells were exposed to sorafenib, and the biological activity of the conditioned media was analyzed using cell proliferation/apoptosis assays, multiplex immunoassays, ELISA, and western blot analyses. The effect of urokinase-type plasminogen activator (uPA) inhibitors or siRNA-mediated gene silencing was examined in culture experiments and a mouse xenograft tumor model. RESULTS: Sorafenib increased uPA secretion, which was abrogated by an Akt inhibitor. The growth-inhibitory effect of sorafenib was significantly enhanced by the uPA inhibitors UK122 and amiloride. Sorafenib-induced apoptosis was increased 2.4-fold in uPA siRNA-transduced cells (p<0.05). Combined therapy with sorafenib and amiloride significantly decreased tumor volumes [mean volume: 759 mm3 (sorafenib) vs. 283 mm3 (sorafenib plus amiloride), p<0.05]. CONCLUSION: uPA may play a critical role in sorafenib resistance.

The clinical value and potential molecular mechanism of the downregulation of MAOA in hepatocellular carcinoma tissues.

BACKGROUND: Hepatocellular carcinoma (HCC) remains one of the most common cancers worldwide and tends to be detected at an advanced stage. More effective biomarkers for HCC screening and prognosis assessment are needed and the mechanisms of HCC require further exploration. The role of MAOA in HCC has not been intensively investigated. METHODS: In-house tissue microarrays, genechips, and RNAsequencing datasets were integrated to explore the expression status and the clinical value of MAOA in HCC. Immunohistochemical staining was utilized to determine MAOA protein expression. Intersection genes of MAOA related co-expressed genes and differentially expressed genes were obtained to perform functional enrichment analyses. In vivo experiment was conducted to study the impact of traditional Chinese medicine nitidine chloride (NC) on MAOA in HCC. RESULTS: MAOA was downregulated and possessed an excellent discriminatory capability in HCC patients. Decreased MAOA correlated with poor prognosis in HCC patients. Downregulated MAOA protein was relevant to an advanced TNM stage in HCC patients. Co-expressed genes that positively related to MAOA were clustered in chemical carcinogenesis, where CYP2E1 was identified as the hub gene. In vivo experiment showed that nitidine chloride significantly upregulated MAOA in a nude mouse HCC model. CONCLUSIONS: A decreased MAOA level is not only correlated with aggressive behaviors in males but also serves as a promising biomarker for the diagnosis and prognosis of HCC patients. Moreover, MAOA may play a role in AFB1 toxic transformation through its synergistic action with co-expressed genes, especially CYP3A4. MAOA also serves as a potential therapy target of NC in HCC patients.

The epigallocatechin gallate derivative Y6 reduces the cardiotoxicity and enhances the efficacy of daunorubicin against human hepatocellular carcinoma by inhibiting carbonyl reductase 1 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Green tea is the most ancient and popular beverage worldwide and its main constituent epigallocatechin-3-gallate (EGCG) has a potential role in the management of cancer through the modulation of cell signaling pathways. However, EGCG is frangible to oxidation and exhibits low lipid solubility and bioavailability, and we synthesized a derivative of EGCG in an attempt to overcome these limitations. AIM OF THE STUDY: The anthracycline antibiotic daunorubicin (DNR) is a potent anticancer agent. However, its severe cardiotoxic limits its clinical efficacy. Human carbonyl reductase 1 (CBR1) is one of the most effective human reductases for producing hydroxyl metabolites and thus may be involved in increasing the cardiotoxicity and decreasing the antineoplastic effect of anthracycline antibiotics. Accordingly, in this study, we investigated the co-therapeutic effect of Y6, a novel and potent adjuvant obtained by optimization of the structure of EGCG. MATERIAL AND METHODS: The cellular concentrations of DNR and its metabolite DNRol were measured by HPLC to determine the effects of EGCG and Y6 on the inhibition of DNRol formation. The cytotoxic effects of EGCG and Y6 were tested by MTT assay in order to identify non-toxic concentrations of them. To understand their antitumor and cardioprotective mechanisms, hypoxia-inducible factor-1α (HIF-1α) and CBR1 protein expression was measured via Western blotting and immunohistochemical staining while gene expression was analyzed using RT-PCR. Moreover, PI3K/AKT and MEK/ERK signaling pathways were analyzed via Western blotting. HepG2 xenograft model was used to detect the effects of EGCG and Y6 on the antitumor activity and cardiotoxicity of DNR in vivo. Finally, to obtain further insight into the interactions of Y6 and EGCG with HIF-1α and CBR1, we performed a molecular modeling. RESULTS: Y6(10 µg/ml or 55 mg/kg) decreased the expression of HIF-1α and CBR1 at both the mRNA and protein levels during combined drug therapy in vitro as well as in vivo, thereby inhibiting formation of the metabolite DNRol from DNR, with the mechanisms being related to PI3K/AKT and MEK/ERK signaling inhibition. In a human carcinoma xenograft model established with subcutaneous HepG2 cells, Y6(55 mg/kg) enhanced the antitumor effect and reduced the cardiotoxicity of DNR more effectively than EGCG(40 mg/kg). CONCLUSIONS: Y6 has the ability to inhibit CBR1 expression through the coordinate inhibition of PI3K/AKT and MEK/ERK signaling, then synergistically enhances the antitumor effect and reduces the cardiotoxicity of DNR.

Anticancer Action of Psilostachyin-A in 5-Fluorouracil-Resistant Human Liver Carcinoma are Mediated Through Autophagy Induction, G2/M Phase Cell Cycle Arrest and Inhibiting Extracellular-Signal-Regulated Kinase/Mitogen Activated Protein Kinase (ERK/MAPK) Signaling Pathway.

BACKGROUND Liver cancer is one of the most common malignancies around the world and one of the major causes of cancer related mortality. The objective of this study was to evaluate the anticancer effect of the natural compound psilostachyin-A on 5-fluorouracil-resistant human liver carcinoma cells and its effects on autophagy, cell cycle, caspase activation, and the ERK/MAPK signaling pathway. MATERIAL AND METHODS Cell Counting Kit 8 (CCK-8) assay was used to evaluate the effects on HepG2 cell viability at different doses of psilostachyin-A. Cell cycle analysis was performed using flow cytometry, and Transwell assay was used to check effects on cell invasion. Transmission electron microscopic studies were done to evaluate autophagy induced by psilostachyin-A, and the western blot method was carried out to evaluate the effects on autophagy and the ERK/MAPK signaling pathway. RESULTS CCK-8 assay revealed that the psilostachyin-A reduced the cell viability of HepG2 cancer cells in a dose dependent manner. Psilostachyin-A also reduced the colony forming potential of HepG2 cells, concentration dependently. The IC50 of psilostachyin was found to be 25 µM. The anticancer effects of psilostachyin-A were due to the induction of autophagy which was accompanied by enhancement of LC3B II expression. Psilostachyin also caused cell cycle arrest by enhancing the accumulation of HepG2 cells in the G2/M phase. Transwell assay showed that psilostachyin-A suppressed the invasion of HepG2 cells. The results also showed that psilostachyin-A could block the ERK/MAPK pathway, indicative of the cytotoxic effects of psilostachyin-A on liver cancer. CONCLUSIONS These preliminary observations suggested that psilostachyin-A might prove beneficial in the treatment of liver cancer.

Targeting AMP-activated kinase impacts hepatocellular cancer stem cells induced by long-term treatment with sorafenib.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer death worldwide. HCC treatment is hindered by the frequent emergence of chemoresistance to the multikinase inhibitor sorafenib, which has been related to the presence of cancer stem cells (CSCs) that self-renew and often escape therapy. The key metabolic sensor AMP-activated kinase (AMPK) has recently been recognized as a tumour growth regulator. In this study, we aimed to elucidate the role of AMPK in the development of a stem cell phenotype in HCC cells. To this end, we enriched the CSC population in HCC cell lines that showed increased expression of drug resistance (ALDH1A1, ABCB1A) and stem cell (CD133, Nanog, Oct4, alpha fetoprotein) markers and demonstrated their stemness phenotype. These cells were refractory to sorafenib-induced cell death. We report that sorafenib-resistant cells had lower levels of total and phosphorylated AMPK as well as its downstream substrate, ACC, compared with the parental cells. Interestingly, AMPK knockdown with siRNA or inhibition with dorsomorphin increased the expression of stem cell markers in parental cells and blocked sorafenib-induced cell death. Conversely, the upregulation of AMPK, either by transfection or by pharmacological activation with A-769662, decreased the expression of ALDH1A1, ABCB1A, CD133, Nanog, Oct4, and alpha fetoprotein, and restored sensitivity to sorafenib. Analysis of the underlying mechanism points to hypoxia-inducible factor HIF-1α as a regulator of stemness. In vivo studies in a xenograft mouse model demonstrated that stem-like cells have greater tumourigenic capacity. AMPK activation reduced xenograft tumour growth and decreased the expression of stem cell markers. Taken together, these results indicate that AMPK may serve as a novel target to overcome chemoresistance in HCC.

A phase 1b study of transforming growth factor-beta receptor I inhibitor galunisertib in combination with sorafenib in Japanese patients with unresectable hepatocellular carcinoma.

Background Galunisertib inhibits type I transforming growth factor-beta receptor serine/threonine kinase. The primary objective of this study was to evaluate the safety and tolerability of galunisertib in combination with sorafenib in Japanese patients with unresectable hepatocellular carcinoma. Patients and methods This open-label, dose-escalation, multicenter, nonrandomized phase 1b study consisted of two dose levels of galunisertib, 160 or 300 mg/day, in combination with sorafenib 800 mg/day. Galunisertib 80 mg or 150 mg was administered orally twice daily for 14 days followed by 14 days of rest plus sorafenib 400 mg administered orally twice daily for 28 days. The dose-limiting toxicity evaluation was 28 days after the first dose. Safety measures, pharmacokinetics, and antitumor activity were assessed. Results Fourteen patients, 7 at each galunisertib dose, were enrolled and treated. Three dose-limiting toxicities were reported for 2 patients. The most common treatment-emergent adverse events (TEAEs) were hypophosphatemia (14 patients [100%]), palmar-plantar erythrodysesthesia syndrome (12 patients [85.7%]), and decreased platelet count (10 patients [71.4%]). The most common grade ≥ 3 TEAEs were hypophosphatemia (10 patients [71.4%]) and palmar-plantar erythrodysesthesia syndrome (7 patients [50.0%]). No grade 5 TEAEs were reported. The pharmacokinetic profile of galunisertib in combination with sorafenib was similar to that previously reported for galunisertib. Eleven patients had a best overall response of stable disease, and 1 patient achieved a partial response by hepatocellular carcinoma-specific modified RECIST. Conclusions These data are consistent with the known safety profile for galunisertib and sorafenib and confirm tolerability of the recommended dose of galunisertib (150 mg twice daily for 14 days) in combination with sorafenib in Japanese patients with unresectable hepatocellular carcinoma.

Glycycoumarin Sensitizes Liver Cancer Cells to ABT-737 by Targeting De Novo Lipogenesis and TOPK-Survivin Axis.

Glycycoumarin (GCM) is a representative of bioactive coumarin compounds isolated from licorice, an edible and medicinal plant widely used for treating various diseases including liver diseases. The purpose of the present study is to examine the possibility of GCM as a sensitizer to improve the efficacy of BH3 mimetic ABT-737 against liver cancer. Three liver cancer cell lines (HepG2, Huh-7 and SMMC-7721) were used to evaluate the in vitro combinatory effect of ABT-737/GCM. HepG2 xenograft model was employed to assess the in vivo efficacy of ABT-737/GCM combination. Results showed that GCM was able to significantly sensitize liver cancer cells to ABT-737 in both in vitro and in vivo models. The enhanced efficacy by the combination of ABT-737 and GCM was attributed to the inactivation of T-LAK cell-originated protein kinase (TOPK)-survivin axis and inhibition of de novo lipogenesis. Our findings have identified induction of TOPK-survivin axis as a novel mechanism rendering cancer cells resistant to ABT-737. In addition, ABT-737-induced platelet toxicity was attenuated by the combination. The findings of the present study implicate that bioactive coumarin compound GCM holds great potential to be used as a novel chemo-enhancer to improve the efficacy of BH3 mimetic-based therapy.

Au-Se-Bond-Based Nanoprobe for Imaging MMP-2 in Tumor Cells under a High-Thiol Environment.

The gold nanosensors based on the Au-S bond have been widely applied to biochemical detections. However, signal distortion caused by biothiols has been seldom mentioned and urgently needs to be solved. Herein, we designed a novel but easily assembled gold nanoprobe by coupling a selenol-modified peptide with FITC onto the gold nanoparticle's surface via an Au-Se bond for fluorescence imaging of a tumor marker matrix, metalloproteinases 2 (MMP-2). Compared to the Au-S probes, the Au-Se probes display high thermal stability and a very good anti-interference ability toward glutathione under simulated physiological conditions. More importantly, the Au-Se nanoprobe exhibits a high-fidelity fluorescent signal toward MMP-2, effectively avoiding interference caused by high levels of thiol compounds in vivo. In addition, in vivo experiments further proved that no significant signal intensity change for the tumor cells treated by the Au-Se probes was observed before and after eliminating glutathione. Hence, we believe such Au-Se probes with in vivo glutathione interfering resistance offer new routes and perspectives in biology and medicine in the future.

EGFR-PI3K-PDK1 pathway regulates YAP signaling in hepatocellular carcinoma: the mechanism and its implications in targeted therapy.

The epidermal growth factor receptor (EGFR) pathway and Hippo signaling play an important role in the carcinogenesis of hepatocellular carcinoma (HCC). However, the crosstalk between these two pathways and its implications in targeted therapy remains unclear. We found that the activated EGFR signaling could bypass RhoA to promote the expression of YAP(Yes-associated protein), the core effector of the Hippo signaling, and its downstream target Cyr61. Further studies indicated that EGFR signaling mainly acted through the PI3K-PDK1 (Phosphoinositide 3-kinase-Phosphoinositide-dependent kinase-1) pathway to activate YAP, but not the AKT and MAPK pathways. While YAP knockdown hardly affected the EGFR signaling. In addition, EGF could promote the proliferation of HCC cells in a YAP-independent manner. Combined targeting of YAP and EGFR signaling by simvastatin and the EGFR signaling inhibitors, including the EGFR tyrosine kinase inhibitor (TKI) gefitinib, the RAF inhibitor sorafenib and the MEK inhibitor trametinib, presented strong synergistic cytotoxicities in HCC cells. Therefore, the EGFR-PI3K-PDK1 pathway could activate the YAP signaling, and the activated EGFR signaling could promote the HCC cell growth in a YAP-independent manner. Combined use of FDA-approved inhibitors to simultaneously target YAP and EGFR signaling presented several promising therapeutic approaches for HCC treatment.

EFLDO induces apoptosis in hepatic cancer cells by caspase activation in vitro and suppresses tumor growth in vivo.

To study the apoptosis induced by EFLDO (ent-3α-formylabieta-8(14), 13(15)-dien-16,12ß-olide), extracted from the Euphorbia lunulata Bge, in the HepG2 cell line and to study the antitumor activity of this compound in vivo, Cell viability and migration were evaluated with CCK-8 (2-(2-methoxy-4-nitrophenyl)-3- (4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) and wound healing assays, respectively. In addition, the cell cycle was examined using flow cytometry after propidium iodide (PI) staining. Apoptosis was analyzed by using the Annexin V/PI staining assay. Pro-caspase activation and apoptosis protein expression were evaluated by western blotting. A HepG2 xenograft model in nude mice was also established to study the antitumor activity of EFLDO in vivo. Immunohistochemical analysis was used to detect the expression of Ki67 in the tumors in situ. EFLDO could induce dose- and time-dependent apoptosis in HepG2 human hepatic cancer cells. Activation of caspases 3, 8, and 9 played an important role in EFLDO-induced apoptosis in vitro. Decreased levels of Bcl-2 and Survivin and increased level of BAX were also involved in this process. Furthermore, EFLDO could inhibit HepG2 tumor growth in nude mice, and the proliferation characteristics, reflected by the Ki67 index, were suppressed significantly. The results indicated that EFLDO could induce apoptosis in hepatic cancer cells by caspase activation in vitro and suppress tumor growth in vivo.

Outcomes of treatment with sorafenib in Egyptian patients with hepatocellular carcinoma: a retrospective cohort study.

BACKGROUND: Sorafenib is the standard of care, first line treatment for advanced HCC. This study aims to evaluate real-life efficacy and safety of sorafenib in Egyptian patients with Hepatocellular carcinoma (HCC). METHODS: This retrospective cohort study was conducted in the medical oncology department at Maadi Armed Forces Medical Compound. Patients with advanced HCC who received sorafenib between January and December 2015 were included (130 patients). RESULTS: The median overall survival of patients with HCC treated with sorafenib was 5 months (CI: 4.166-5.834), and progression free survival was 4 months (CI: 3.479-4.521). Disease control rate was 45.44% with 2 patients experiencing complete remission (1.2%). The adverse events rate was 76.1% for toxicities of all grades; with hand and foot syndrome being the most common (32.3% of any grade) and liver dysfunction the most common grade III toxicity (13.8%). Treatment was stopped for radiological progression based on modified RECIST criteria in 47 patients (36.3%), 18 patients stopped the treatment for intolerable toxicity. At the end of treatment upon radiological progression, 51 patients (39.2%) were still classified as Child A class of cirrhosis. CONCLUSION: Sorafenib use should be limited to patients with Child A, PS 0-1, and low disease burden.

Metformin and insulin impact on clinical outcome in patients with advanced hepatocellular carcinoma receiving sorafenib: Validation study and biological rationale.

PURPOSE: In 2015, we published a study on a small series of patients with hepatocellular carcinoma (HCC) treated chronically with metformin for type II diabetes mellitus (DM2) who showed a poorer response to sorafenib. The aim of the present study was to validate the prognostic significance of metformin in HCC patients treated with sorafenib, providing a biological rationale for the mechanism of resistance to sorafenib in patients on chronic metformin therapy, and to clarify the role of sirtuin-3 (SIRT-3), a protein involved in metabolic diseases and acknowledged as a tumour suppressor in HCC, in this resistance. PATIENTS AND METHODS: We analysed 279 patients consecutively treated with sorafenib for the clinical analysis. Of the 86 (30%) patients with DM2, 52 (19%) were on chronic treatment with metformin and 34 (12%) with insulin. We included 43 patients with HCC for the biological study: 19 (44.1%) were diabetic and 14 (73.7%) of these received metformin for DM2. SIRT-3 expression was investigated by immunohistochemistry (IHC) in formalin-fixed and paraffin-embedded (FFPE) samples. RESULTS: In HCC patients undergoing chronic treatment with metformin, the use of sorafenib was associated with poor progression-free survival (PFS) and overall survival (OS) (1.9 and 6.6 months, respectively) compared to 3.7 months and 10.8 months, respectively, for patients without DM2 and 8.4 months and 16.6 months, respectively, for patients on insulin (P < .0001). We also observed that SIRT-3 protein expression was significantly higher in patients treated with metformin than in those not taking this medication (65% versus 25%, respectively) (P = .013). CONCLUSIONS: Our findings could be attributed to increased tumour aggressiveness and resistance to sorafenib caused by chronic treatment with metformin.

Zinc Salts Block Hepatitis E Virus Replication by Inhibiting the Activity of Viral RNA-Dependent RNA Polymerase.

Hepatitis E virus (HEV) causes an acute, self-limiting hepatitis in healthy individuals and leads to chronic disease in immunocompromised individuals. HEV infection in pregnant women results in a more severe outcome, with the mortality rate going up to 30%. Though the virus usually causes sporadic infection, epidemics have been reported in developing and resource-starved countries. No specific antiviral exists against HEV. A combination of interferon and ribavirin therapy has been used to control the disease with some success. Zinc is an essential micronutrient that plays crucial roles in multiple cellular processes. Zinc salts are known to be effective in reducing infections caused by few viruses. Here, we investigated the effect of zinc salts on HEV replication. In a human hepatoma cell (Huh7) culture model, zinc salts inhibited the replication of genotype 1 (g-1) and g-3 HEV replicons and g-1 HEV infectious genomic RNA in a dose-dependent manner. Analysis of a replication-defective mutant of g-1 HEV genomic RNA under similar conditions ruled out the possibility of zinc salts acting on replication-independent processes. An ORF4-Huh7 cell line-based infection model of g-1 HEV further confirmed the above observations. Zinc salts did not show any effect on the entry of g-1 HEV into the host cell. Furthermore, our data reveal that zinc salts directly inhibit the activity of viral RNA-dependent RNA polymerase (RdRp), leading to inhibition of viral replication. Taken together, these studies unravel the ability of zinc salts in inhibiting HEV replication, suggesting their possible therapeutic value in controlling HEV infection.IMPORTANCE Hepatitis E virus (HEV) is a public health concern in resource-starved countries due to frequent outbreaks. It is also emerging as a health concern in developed countries owing to its ability to cause acute and chronic infection in organ transplant and immunocompromised individuals. Although antivirals such as ribavirin have been used to treat HEV cases, there are known side effects and limitations of such therapy. Our discovery of the ability of zinc salts to block HEV replication by virtue of their ability to inhibit the activity of viral RdRp is important because these findings pave the way to test the efficacy of zinc supplementation therapy in HEV-infected patients. Since zinc supplementation therapy is known to be safe in healthy individuals and since high-dose zinc is used in the treatment of Wilson's disease, it may be possible to control HEV-associated health problems following a similar treatment regimen.

Astragalin Reduces Hexokinase 2 through Increasing miR-125b to Inhibit the Proliferation of Hepatocellular Carcinoma Cells in Vitro and in Vivo.

Astragalin (ASG) can be found in a variety of food components. ASG exhibits cytotoxic effects on several different types of malignant cells. However, its effects on hepatocellular carcinoma (HCC) cells and the underlying molecular mechanisms have remained to be fully elucidated. Here, we revealed that ASG remarkably suppressed the proliferation of HCC cells. In HCC cells, ASG inhibited glucose glycolysis and promoted oxidative phosphorylation, resulting in a surge of reactive oxygen species (ROS). Mechanistically, ASG suppressed the expression of hexokinase 2 (HK2). This event was indispensible for ASG-mediated metabolic reprogramming, ROS accumulation, and subsequent growth arrest. Our further investigations unveiled that ASG repressed HK2 expression via increasing miR-125b. In vivo experiments showed that gavage of ASG decreased the proliferation of Huh-7 HCC xenografts in nude mice and inhibited the growth of transplanted H22 HCC cells in Kunming mice. Declined HCC tumor growth in vivo was associated with boosted miR-125b and reduced expression of HK2 in tumor tissues. Collectively, our results demonstrated that ASG is able to suppress the proliferation of HCC cells both in vitro and in vivo. Inhibition of HK2 through upregulating miR-125b and subsequent metabolic reprogramming is implicated in the antiproliferative effects of ASG on HCC cells.

Curcumin Upregulates Antioxidant Defense, Lon Protease, and Heat-Shock Protein 70 Under Hyperglycemic Conditions in Human Hepatoma Cells.

Sirtuin 3 (SIRT3) regulates mitochondrial antioxidant (AO) defense and improves mitochondrial disorders. Curcumin protects mitochondria; however, the mechanisms need investigation. We postulated that curcumin increases AO defense under hyperglycemic conditions in HepG2 cells through SIRT3-mediated mechanisms. Cell viability was determined in HepG2 cells cultured with 5 mM glucose, 19.9 mM mannitol, vehicle control, 10 mM glucose, and 30 mM glucose in the absence or presence of curcumin for 24 h. SIRT3, nuclear factor-kappa B (NF-κB), heat-shock protein 70 (Hsp70), and Lon protein expressions were determined using western blot. Transcript levels of SIRT3, peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α), cAMP response element-binding protein (CREB), glutathione peroxidase 1 (GPx1), and superoxide dismutase 2 (SOD2) were measured by quantitative polymerase chain reaction. Cell viability, SIRT3 protein expression, transcript levels of SIRT3, PGC-1α, CREB, GPx1, and SOD2 and protein expressions of NF-κB, Lon, and Hsp70 were significantly increased in the curcumin-treated hyperglycemic groups. Since curcumin and SIRT3 both improve mitochondrial function and AO defense, SIRT3 may be involved in the protective effects of curcumin.

Quantum mechanical model for the anticarcinogenic effect of extremely-low-frequency electromagnetic fields on early chemical hepatocarcinogenesis.

Using the conventional Haberkorn approach, it is evaluated the recombination of the radical pair (RP) singlet spin state to study theoretically the cytoprotective effect of an extremely-low-frequency electromagnetic field (ELF-EMF) on early stages of hepatic cancer chemically induced in rats. The proposal is that ELF-EMF modulates the interconversion rate of singlet and triplet spin states of the RP populations modifying the products from the metabolization of carcinogens. Previously, we found that the daily treatment with ELF-EMF 120 Hz inhibited the number and area of preneoplastic lesions in chemical carcinogenesis. The singlet spin population is evaluated diagonalizing the spin density matrix through the Lanczos method in a radical pair mechanism (RPM). Using four values of the interchange energy, we have studied the variations over the singlet population. The low magnetic field effect as a test of the influence over the enzymatic chemical reaction is evaluated calculating the quantum yield. Through a bootstrap technique the range is found for the singlet decay rate for the process. Applying the quantum measurements concept, we addressed the impact toward hepatic cells. The result contributes to improving our understanding of the chemical carcinogenesis process affected by charged particles that damage the DNA.

The Inhibitory Effect of C-phycocyanin Containing Protein Extract (C-PC Extract) on Human Matrix Metalloproteinases (MMP-2 and MMP-9) in Hepatocellular Cancer Cell Line (HepG2).

Spirulina platensis :have been studied for several biological activities. In the current study C-phycocyanin containing protein extract (C-PC extract) of Spirulina platensis have been studied for its effect on human matrix metalloproteinases (MMP-1, MMP-2 and MMP-9) and tissue inhibitors of MMPs (TIMP-1 and TIMP-2). In the present study, breast cancer cell line (MDA-MB 231) and hepatocellular cancer cell line (HepG2) were examined for inhibition of MMPs at different levels of expression after C-PC extract treatment. Herein, we have demonstrated that C-PC extract significantly reduced activity of MMP-2 by 55.13% and MMP-9 by 57.9% in HepG2 cells at 15 µg concentration. Additionally, the treatment has reduced mRNA expression of MMP-2 and MMP-9 at 20 µg concentration by 1.65-folds and 1.66-folds respectively. The C-PC extract treatment have also downregulated a mRNA expression of TIMP-2 by 1.12 folds at 20 µg concentration in HepG2 cells. Together, these results indicate that C-PC, extract successfully inhibited MMP-2 and -9 at different levels of expression and TIMP-2 at a mRNA expression level; however, extract did not have any effect on MMP-1 expressed in MDA-MB231 and TIMP-1 expressed in HepG2 cells as well as the exact mechanism of inhibition of MMP-2, MMP-9 and TIMP-2 remained unclear.