Induced Liver Cancer Research Articles

Ratlarda Dietilnitrozamin Uygulanarak Oluşturulan Oksidatif ve Nitrozatif Strese Karşı Silimarin’in Karaciğer Koruyucu Etkisi

Liver cancer is the third most common cancer-related death in the World. Hepatocellular carcinoma (HCC) constitutes %70-85 of liver malignant primary tumors. In this study; we aimed to investigate the role of diethylnitrosamine (DEN) in the experimental liver cancer model and to examine the hepatoprotective effect of silymarin in detail using oxidative-nitrosative stress markers with immunohistochemical methods. For this purpose, 50 Wistar Albino male rats were divided into 5 groups with 10 rats. Animals in DEN group received DEN at a dose of 50 mg/kg once a week for 20 weeks. The rats in the DEN +Silymarin group were Silymarin intraperitoneally for 21 weeks 3 times a week at a dose of 100 mg/kg, starting 1 week prior to the administration of DEN. Silymarin was administered to rats in the group of Silymarin 3 times a week intraperitoneally for 20 weeks at a dose of 100 mg/kg. The rats in the control group were fed pellet feed and drinking water for 20 weeks. Animals in Sham group were treated with propylene glycol dissolved in 0,9 % NaCl intraperitoneally 3 times a week for 20 weeks. At the end of 21 weeks, macroscopically different size and number of nodular structures were detected in the DEN and DEN+Silymarin group. The microscopic examination revealed that the nodules were HCC. As a result, DEN induced liver cancer; however, silymarin did not show a statistically significant protective effect in terms of iNOS and Nitrotyrosine expressions.

USP10 Promotes Proliferation of Hepatocellular Carcinoma by Deubiquitinating and Stabilizing YAP/TAZ.

Yes-associated protein (YAP) and its paralog, transcriptional coactivator with PDZ-binding motif (TAZ), play pivotal roles in promoting the progression of hepatocellular carcinoma. However, the regulatory mechanism underpinning aberrant activation of YAP/TAZ in hepatocellular carcinoma remains unclear. In this study, we globally profiled the contribution of deubiquitinating enzymes (DUB) to both transcriptional activity and protein abundance of YAP/TAZ in hepatocellular carcinoma models and identified ubiquitin-specific peptidase 10 (USP10) as a potent YAP/TAZ-activating DUB. Mechanistically, USP10 directly interacted with and stabilized YAP/TAZ by reverting their proteolytic ubiquitination. Depletion of USP10 enhanced polyubiquitination of YAP/TAZ, promoted their proteasomal degradation, and ultimately arrested the proliferation of hepatocellular carcinoma in vitro and in vivo. Expression levels of USP10 positively correlated with the abundance of YAP/TAZ in hepatocellular carcinoma patient samples as well as in N-nitrosodiethylamine (DEN)-induced liver cancer mice models. Collectively, this study establishes the causal link between USP10 and hyperactivated YAP/TAZ in hepatocellular carcinoma cells and provides a rationale for potential therapeutic interventions in the treatment of patients with hepatocellular carcinoma harboring a high level of YAP/TAZ. SIGNIFICANCE: These findings identify USP10 as a DUB of YAP/TAZ and its role in hepatocellular carcinoma progression, which may serve as a potential therapeutic target for hepatocellular carcinoma treatment.

Magnetically responsive peptide coacervates for dual hyperthermia and chemotherapy treatments of liver cancer

Liver cancer is an aggressive malignancy associated with high levels of mortality and morbidity. Doxorubicin (Dox) is often used to slow down liver cancer progression; however its efficacy is limited, and its severe side effects prevent its routine use at therapeutic concentrations. We present a biomimetic peptide that coacervates into micro-droplets, within which both Dox and magnetic nanoparticles (MNPs) can be sequestered. These Dox-loaded Magnetic Coacervates (DMCs) can be used for thermo-chemotherapy, with the controlled release of Dox triggered by an external Alternating Magnetic Field (AMF). The DMCs are internalized by the cells via an energy-independent mechanism which is not based on endocytosis. Application of AMF generates a temperature of 45 °C within the DMCs, triggering their disassembly and the simultaneous release of Dox, thereby resulting in dual hyperthermia and chemotherapy for more efficient cancer therapy. In vitro studies conducted under AMF reveal that DMCs are cytocompatible and effective in inducing HepG2 liver cancer cell death. Thermo-chemotherapy treatment against HepG2 cells is also shown to be more effective compared to either hyperthermia or chemotherapy treatments alone. Thus, our novel peptide DMCs can open avenues in theranostic strategies against liver cancer through programmable, wireless, and remote control of Dox release. Statement of SignificanceSimultaneous administration of chemical and thermal therapy (thermo-chemotherapy) is more effective in inducing liver cancer cell death and improving survival rate. Thus, there is a keen interest in developing suitable carriers for thermo-chemotherapy. Coacervate micro-droplets display significant advantages, including high loading capacity, fast self-assembly in aqueous environments, and liquid-like behavior. However, they have not yet been explored as carriers for thermo-chemotherapy. Here, we demonstrate that peptide coacervate micro-droplets can co-encapsulate Dox and magnetic nanoparticles and cross the cell membrane. Applying an alternating magnetic field to cells containing drug-loaded coacervates triggers the release of Dox as well as the localized heating by magnetic hyperthermia, resulting in efficient liver cancer cell death by dual thermo-chemotherapy.

WWC1 and NF2 Prevent the Development of Intrahepatic Cholangiocarcinoma by Regulating YAP/TAZ Activity through LATS in Mice.

Hippo signaling acts as a tumor suppressor pathway by inhibiting the proliferation of adult stem cells and progenitor cells in various organs. Liver-specific deletion of Hippo pathway components in mice induces liver cancer development through activation of the transcriptional coactivators, YAP and TAZ, which exhibit nuclear enrichment and are activated in numerous types of cancer. The upstream-most regulators of Warts, the Drosophila ortholog of mammalian LATS1/2, are Kibra, Expanded, and Merlin. However, the roles of the corresponding mammalian orthologs, WWC1, FRMD6 and NF2, in the regulation of LATS1/2 activity and liver tumorigenesis in vivo are not fully understood. Here, we show that deletion of both Wwc1 and Nf2 in the liver accelerates intrahepatic cholangiocarcinoma (iCCA) development through activation of YAP/TAZ. Additionally, biliary epithelial cell-specific deletion of both Lats1 and Lats2 using a Sox9-CreERT2 system resulted in iCCA development through hyperactivation of YAP/TAZ. These findings suggest that WWC1 and NF2 cooperate to promote suppression of cholangiocarcinoma development by inhibiting the oncogenic activity of YAP/TAZ via LATS1/2.

Spliced X-box binding protein 1 induces liver cancer cell death via activating the Mst1-JNK-mROS signalling pathway.

Previous studies have found that the primary pathogenesis of liver cancer progression is linked to excessive cancer cell proliferation and rapid metastasis. Although therapeutic advances have been made for the treatment of liver cancer, the mechanism underlying the liver cancer progression has not been fully addressed. In the present study, we explored the role of spliced X-box binding protein 1 (XBP1) in regulating the viability and death of liver cancer cells in vitro. Our study demonstrated that XBP1 was upregulated in liver cancer cells when compared to the primary hepatocytes. Interestingly, the deletion of XBP1 could reduce the viability of liver cancer cells in vitro via inducing apoptotic response. Further, we found that XBP1 downregulation was also linked to proliferation arrest and migration inhibition. At the molecular levels, XBP1 inhibition is followed by activation of the Mst1 pathway which promoted the phosphorylation of c-Jun N-terminal kinase (JNK). Then, the active Mst1-JNK pathway mediated mitochondrial reactive oxygen species (mROS) overproduction and then excessive ROS induced cancer cell death. Therefore, our study demonstrated a novel role played by XBP1 in modulating the viability of liver cancer cells via the Mst1-JNK-mROS pathways.

Pixel-level aflatoxin detecting in maize based on feature selection and hyperspectral imaging.

Aflatoxin is highly toxic and is easily found in maize, a little aflatoxin can induce liver cancer. In this paper, we used hyperspectral data in the pixel-level to build the aflatoxin classifying model, each of the pixel have 600 hyperspectral bands and labeled 'clean' or 'contaminated'. We use 3 method to extracted feature bands, one method is to select 4 hyperspectral bands from other articles: 390nm, 440nm, 540nm and 710nm, another method is to use feature extraction PCA to obtain first 5 pcs to shrink the hyperspectral volume, the third method is to use Fscnca, Fscmrmr, Relieff and Fishier algorithm to select top 10 feature bands. After feature band selection or extraction, we put the feature bands into Random Forest (RF) and K-nearest neighbor (KNN) to classify whether a pixel is polluted by aflatoxin. The highest accurate for feature selection is Relieff, it reached the accuracy of 99.38% with RF classifier and 98.77% in KNN classifier. PCA feature extraction with RF classifier also reached a high accuracy 93.83%. And the 600 bands without feature extraction reached the accuracy of 100%. Feature bands selected from other papers could reach an accuracy of 89.51%. The result shows that the feature extraction performs well on its own data set. And if the computing time is not taken into account, we could use full band to classify the aflatoxin due to its high accuracy.

Involvement of hepatic lipid droplets and their associated proteins in the detoxification of aflatoxin B1 in aflatoxin-resistance BALB/C mouse

The highly potent carcinogen, Aflatoxin B1, induces liver cancer in many animals including humans but some mice strains are highly resistant. This murine resistance is due to a rapid detoxification of AFB1. Hepatic lipid droplets (LDs) ultimately impact the liver functions but their potential role in AFB1 detoxification has not been addressed. This study describes the structural and functional impacts on hepatic LDs in BALB/C mice after exposure to 44 (low dose) or 663 (high dose) μg AFB1/kg of body weight. After 7 days, the liver of AFB1-dosed mice did not accumulate any detectable AFB1 or its metabolites and this was associated with a net increase in gene transcripts of the AhR-mediating pathway. Of particular interest, the livers of high-dose mice accumulated many more LDs than those of low-dose mice. This was accompanied with a net increase in transcript levels of LD-associated protein-encoding genes including Plin2, Plin3 and Cideb and an alteration in the LDs lipid profiles that could be likely due to the induction of lipoxygenase and cyclooxygenase genes. Interestingly, our data suggest that hepatic LDs catalyze the in vitro activation of AFB1 into AFB1-exo-8,9-epoxide and subsequent hydrolysis of this epoxide into its corresponding dihydrodiol. Finally, transcript levels of CYP1A2, CYP1B1, GSTA3 and EH1 genes were elevated in livers of high-dose mice. These data suggest new roles for hepatic LDs in the trapping and detoxifying of aflatoxins.

Vicenin-2 Treatment Attenuated the Diethylnitrosamine-Induced Liver Carcinoma and Oxidative Stress through Increased Apoptotic Protein Expression in Experimental Rats.

Liver cancer or hepatocellular carcinoma is considered to be the third leading cause of death among all other cancers. The rate of liver cancer occurrence is high, and the rate of recovery is low. In this study, we investigated the therapeutic efficacy of vicenin-2 against the diethylnitrosamine-induced liver carcinoma in experimental rats. Diethylnitrosamine was widely employed as a carcinogenic agent to stimulate the cancer in animal models. Our results indicated that vicenin-2 administration effectively attenuates the diethylnitrosamine-induced physiological and pharmacological alterations in the experimental rats. Vicenin-2 treatment significantly enhanced the pathological lesions and decreased the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and α-fetoprotein (AFP) in serum. We also observed that vicenin-2 reduced the production of reactive oxygen species, decreased the liver weight, upregulated expression of apoptotic proteins, and decreased the histological changes in the liver, which are induced by the diethylnitrosamine in rats. Moreover, vicenin-2 downregulates antiapoptotic Bcl-2 and Bcl-xL, and upregulates the proapoptotic Bax and caspase. Hence, our results suggested that vicenin-2 had a highly therapeutic effect in reversing diethylnitrosamine-induced liver carcinoma in rats, which might be related to the apoptosis induced by vicenin-2. Therefore vicenin-2 could be a good candidate for future therapeutic use to inhibit chemically induced liver cancer.

Overexpression of Hepatocyte Chemerin-156 Lowers Tumor Burden in a Murine Model of Diethylnitrosamine-Induced Hepatocellular Carcinoma.

The tumor inhibitory potential of the highly active chemerin-156 isoform was described in orthotopic models of hepatocellular carcinoma (HCC). The majority of HCC arises in the fibrotic liver, which was not reproduced in these studies. Here, a potential therapeutic activity of chemerin-156 was evaluated in diethylnitrosamine (DEN)-induced liver cancer, which mimics fibrosis-associated HCC. Mice were infected with adeno-associated virus (AAV) six months after DEN injection to overexpress chemerin-156 in the liver, and animals injected with non-recombinant-AAV served as controls. Three months later, the animals were killed. Both groups were comparable with regard to liver steatosis and fibrosis. Of note, the number of very small tumors was reduced by chemerin-156. Anyhow, the expression of inflammatory and profibrotic genes was similar in larger tumors of control and chemerin-156-AAV-infected animals. Although genes with a role in lipid metabolism, like 3-hydroxy-3-methylglutaryl-coenzym-A--reductase, were overexpressed in tumors of animals with high chemerin-156, total hepatic cholesterol, diacylglycerol and triglyceride levels, and distribution of individual lipid species were normal. Chemerin-156-AAV-infected mice had elevated hepatic and systemic chemerin. Ex vivo activation of the chemerin receptor chemokine-like receptor 1 increased in parallel with serum chemerin, illustrating the biological activity of the recombinant protein. In the tumors, chemerin-155 was the most abundant variant. Chemerin-156 was not detected in tumors of the controls and was hardly found in chemerin-156-AAV infected animals. In conclusion, the present study showed that chemerin-156 overexpression caused a decline in the number of small lesions but did not prevent the growth of pre-existing neoplasms.

Tanshinone IIA mediates SMAD7-YAP interaction to inhibit liver cancer growth by inactivating the transforming growth factor beta signaling pathway.

Tanshinone IIA (TanIIA)-an active constituent of Salvia miltiorrhiza, a traditional Chinese medicinal plant-is known to have blood circulation promotion and anti-tumor properties. Tan IIA can induce tumor cell death and inhibit tumor growth. However, the functions and underling molecular mechanisms of Tan IIA action on human liver cancer cells remain poorly understand. In this study, we found that Tanshinone IIA mediates SMAD7-YAP interaction to induce liver cancer cell apoptosis and inhibit cell growth and migration by inactivating the transforming growth factor beta (TGF-β) signaling pathway. Our findings showed that the Tan IIA-SMAD7-YAP regulatory network might be an effective strategy for liver cancer treatment.

173P - Cancer stem-like phenotypes including immune surveillance and its responsible genes in induced liver cancer stem-like cells

BackgroundCancer stem cells are thought to play important roles in carcinogenesis, recurrence, metastasis, and therapy-resistance. We have successfully induced cancer stem-like sphere cells (CSLCs) from hepatoma cell lines using a unique medium. MethodsThe human hepatoma cell line SK-HEP-1 was used as parental cells for CSLC induction. To assess the chemoresistance and liver metastasis, MTS assay and splenic injection were performed, respectively. RNA-seq analysis followed by gene set enrichment analysis (GSEA) was performed with cell line derivatives and clinical specimens of hepatoma. Protein expressions were conducted by western blotting, flow-cytometry, or ELISA. Knock-out experiments was accomplished by CRISPR-Cas9 genome-editing. Following rescue experiments were performed with an expression vector. ResultsThe obtained CSLCs showed increased metastatic potentials and resistance to anti-cancer drugs including molecularly targeted drugs for liver cancer. Regarding immune resistance, CSLCs showed increased proportion of positive cells for PD-L1 and PD-L2. In contrast to decreased membrane-bound MICA/MICB, increased soluble MICA in the medium were observed in CSLCs compared to those from the parental cells. As a result of integrated analysis with RNA-seq, a RAB3B was identified as an up-regulated gene in both CSLCs and poor prognostic liver cancers. Mono-allelic RAB3B knock-out cells showed altered sphere formation, significantly lower chemoresistance, and metastatic potential than that of parental cells, and those were rescued by the complementation of RAB3B. The knock-out cells also showed decreased PD-L1 and PD-L2 expressions in sphere induction medium than those in cells cultured with normal medium. GSEA following RNA-seq with the derivative cells revealed that negative enrichment of G2M checkpoint and positive enrichment of interferon response in CSLCs. ConclusionsOur induced CSLCs showed characteristic phenotypes such as metastatic ability, chemo- and immune-resistances. The up-regulation of RAB3B may plays important roles in CSLCs. Legal entity responsible for the studyRyouichi TSUNEDOMI. FundingJapan Society for the Promotion of Science. DisclosureAll authors have declared no conflicts of interest.

Expression of Clusterin suppresses Cr(VI)-induced premature senescence through activation of PI3K/AKT pathway

Our group found that long-term low-dose exposure to hexavalent chromium [Cr(VI)] in L-02 hepatocytes resulted in premature senescence, which accompanied by the increased expression of Clusterin (CLU), but the functional role of CLU in premature senescence has never been explored. In the present study, the CLU overexpressed or silenced L-02 hepatocytes were established by lentiviral vector transfection. Cell viability assay, cell cycle analysis, western blotting, plate clone formation assay, and confocal microcopy were performed. The results indicated that Cr(VI)-induced premature senescence was associated with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway inhibition, and high expression of CLU in the senescent cells exerted its functional role of promoting cell proliferation. CLU could complex with eukaryotic translation initiation factor 3 subunit I (EIF3I) and prevent its degradation, leading to the increase of AKT activity in Cr(VI)-exposed senescent hepatocytes. Blockage of the PI3K/AKT pathway with its inhibitor LY294002 eliminated the inhibitory effect of CLU on Cr(VI)-induced premature senescence. We concluded that high expression of CLU suppressed Cr(VI)-induced premature senescence through activation of PI3K/AKT pathway, which will provide the experimental basis for the study of Cr(VI)-induced liver cancer, especially for the elucidation of the mechanism of liver cancer cells escaping from senescence.

Chemoprevention of elite tea variety CFT-1 rich in EGCG against chemically induced liver cancer in rats.

Camellia sinensis (L.) O. Kuntze cv. CFT‐1 is an elite tea variety bred by sexual hybridization with a high content of epigallocatechin‐3‐gallate (EGCG) as 134.2 mg/g (which is 2.54‐fold that of the common variety). This study was to evaluate the chemopreventive effects of CFT‐1 green tea infusion (CFT‐1) against N‐nitrosodiethylamine (NDEA)‐induced hepatocarcinogenesis in rats and its mechanisms. The results showed that CFT‐1 had a superior inhibitory effect in NDEA‐initiated hepatocarcinogenesis compared to that of common tea. CFT‐1 significantly reduced the hepatic nodules incidence, size, and number and prevented the hepatic adenoma or hepatocellular carcinoma (HCC) formation. In particular, CFT‐1‐treated animals had the least incidence of HCC (8.33%) followed by common tea treatment (40.00%) and model control rats (87.50%). CFT‐1 treatment significantly ameliorated abnormal liver function enzymes, reduced serum AFP, CEA, TSGF, and TNF‐α levels, inhibited the dickkopf‐related protein‐1 expression, enhanced antioxidant capacity, suppressed the production of livers 8‐hydroxy‐2′‐deoxyguanosine, and regulated hepatic phase I and II xenobiotic‐metabolizing enzymes. Transcriptomic analysis of liver tissue suggested that compared to common tea, administration of CFT‐1 regulated larger gene sets, which were located in several important pathways of antioxidants, inflammatory network, xenobiotic‐metabolizing enzymes, apoptosis, cell proliferation, and metabolism associated with liver tumorigenesis. We identified some genes as potential molecular targets involved in the prevention of CFT‐1 and found that CFT‐1 inhibited inflammation response, proliferation, and accelerated apoptosis by inhibiting NF‐κB and PI3K/Akt pathway. Thus, EGCG‐rich CFT‐1 green tea might be a potential choice for liver cancer prevention/treatment in the future.

Circulating exosomal miR-92b: Its role for cancer immunoediting and clinical value for prediction of posttransplant hepatocellular carcinoma recurrence.

A recurrence of hepatocellular carcinoma (HCC) after living donor liver transplantation (LDLT) is one of the major concerns reflecting the higher mortality of HCC. This study aimed to explore the impact of circulating exosomes on HCC development and recurrence. One-shot transfusion of hepatoma serum to naïve rats induced liver cancer development with gradual elevation of alpha-fetoprotein (AFP), but exosome-free hepatoma serum failed to induce AFP elevation. The microarray analysis revealed miR-92b as one of the highly expressing microribonucleic acids in hepatoma serum exosomes. Overexpression of miR-92b enhanced the migration ability of liver cancer cell lines with active release of exosomal miR-92b. The hepatoma-derived exosomal miR-92b transferred to natural killer (NK) cells, resulting in the downregulation of CD69 and NK cell-mediated cytotoxicity. Furthermore, higher expression of miR-92b in serum exosomes was confirmed in HCC patients before LDLT, and its value at 1 month after LDLT was maintained at a higher level in the patients with posttransplant HCC recurrence. In summary, we demonstrated the impact of circulating exosomes on liver cancer development, partly through the suppression of CD69 on NK cells by hepatoma-derived exosomal miR-92b. The value of circulating exosomal miR-92b may predict the risk of posttransplant HCC recurrence.

Abstract 3455: TNFAIP8 increases drug resistance and steatosis in liver cancer by inducing autophagy

Abstract Tumor necrosis factor-α-inducible protein 8 (TNFAIP8) is a TNF-α inducible anti-apoptotic protein which facilitates tumor growth and progression in several cancers. The molecular mechanisms how TNFAIP8 modulates the progression of liver cancer remain elusive. The present study investigated the role of TNFAIP8 in the liver cancer cell growth, drug resistance/survival and cell steatosis. Tissue microarray data from patients demonstrated an increased expression of TNFAIP8 protein during development of different stages of liver cancer compared with normal liver tissues. To test the correlation between TNFAIP8 expression and liver cancer progression, we transiently/stably expressed TNFAIP8 protein in liver cancer cell lines. Expression of TNFAIP8 in liver cancer cells HepG2 and SK-Hep1 increased both cell growth/survival and drug resistance against anti-liver cancer drugs sorafenib and regorafenib by inhibition of apoptosis. In addition, TNFAIP8 knockdown sensitized tumor cell lines to sorafenib and regorafenib. Expression of TNFAIP8 modulates AKT/mTOR pathway and induced autophagy and thus increased drug resistance. Moreover, TNFAIP8 induced liver cancer cell steatosis by increasing the expression of L-FABP1, SCD1, ACC, and PPARγ proteins which involved in lipid synthesis. We also demonstrate that chronic EtOH induced steatosis in mice liver was associated with increased TNFAIP8 expression and autophagy suggesting that TNFAIP8 mediate EtOH induced steatosis. In addition, increased expression of TNFAIP8 and hepatic steatosis was observed in patients with a history of alcohol use compared with no history of alcohol use. Taken together our data suggested that TNFAIP8 increases drug resistance/survival, EtOH-induced cell steatosis and early liver cancer development by the induction of autophagy. Citation Format: Suresh Niture, Maxwell A. Gyamfi, Deepak Kumar. TNFAIP8 increases drug resistance and steatosis in liver cancer by inducing autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3455.

Coordination-Driven Self-Assembly of Triazole-Based Apoptosis-Inducible Metallomacrocycles.

Ru(II)-metallomacrocycles containing 4-pyridyl-1,2,3-triazole moiety were realized by coordination-driven self-assembly. All new compounds were characterized by electrospray ionisation mass spectrometry, elemental analysis, and 1H and 13C NMR spectroscopic techniques. The molecular structure of metallomacrocycle 8 was determined by single-crystal X-ray crystallography. The anticancer activities of metallomacrocycles 5–8 were evaluated by cytotoxicity, cell cycle analysis, and related protein expression. Metallomacrocycle 7 showed the highest cytotoxicity in HepG2 human hepatocellular carcinoma cells. In addition, apoptotic HepG2 cells were analyzed when metallomacrocycle 7 was treated. Our results suggest that metallomacrocycle 7 induces liver cancer cell death by increasing apoptosis and cell cycle arrest and that it has potential use as an agent for the treatment of human hepatocellular carcinoma.

Chronic administration of diethylnitrosamine to induce hepatocarcinogenesis and to evaluate its synergistic effect with other hepatotoxins in mice

Hepatocellular carcinoma (HCC) arises after a long period of exposition to etiological factors that might be either independent or collectively contributing. Several rodent models resemble human HCC; however, the major limitation of these models is the lack of chronic injury that reproducibly mimics the molecular alterations as it occurs in humans. Thus, we hypothesized that chronic administration of different DEN treatments identifies the best-fit dose to induce the HCC and/or to determine whether small DEN doses act synergistically with other known hepatotoxins to induce HCC in mice. C57BL/6 J male mice were intraperitoneally injected twice a week for 6 weeks with different DEN doses ranging from 2.5 to 40 mg/kg body weight; then, selected doses (2.5, 5 and 20 mg/kg) for 6, 10, 14, and 18 weeks. We demonstrated that DEN at 20 mg/kg promoted reactive oxygen species and 4-hydroxynonenal production, cell proliferation inflammatory infiltrate, and fibrosis, which in turn induced liver cancer by week 18. These parameters were established by evaluating histopathological changes, HCC markers such as glutathione S-transferase placental-1 (Gstp1), Cytokeratin-19 (Ck19) and prostaglandin reductase-1 (Ptgr1); that of Cyp2e1, a DEN metabolizing enzyme; and the expression of the proliferation marker Ki67. While DEN at 2.5 and 5 mg/kg increased Gstp1 and Ck19, DEN at 20 mg/kg decreased them and Cyp2e1 expression and activity. In summary, our results demonstrate that DEN chronically administrated at 20 mg/kg induces the HCC, while DEN at 2.5 and 5 mg/kg could be useful in elucidating its synergistic effect with other hepatotoxic agents in mice.

Oridonin derivatives as potential anticancer drug candidates triggering apoptosis through mitochondrial pathway in the liver cancer cells

The biological function of the natural ent-kaurene diterpenoid isolated from genus Isodon, oridonin, has been intensively studied. However, its mechanism studies and clinical applications were hampered by its moderate biological activities. In order to enlarge the applied range of oridonin and explore its mechanism of action, a series of derivatives were designed and synthesized based on the structure of oridonin. Some of the derivatives were significantly more potent than oridonin against four cancer cell lines. Especially, the most potent compound 20 markedly inhibited the proliferation of well differentiated HepG2 and poorly differentiated PLC/PRF/5 cells, with IC50 values as low as 1.36 μM and 0.78 μM respectively, while the IC50 values of oridonin are 8.12 μM and 7.41 μM. We found that compound 20 inhibited liver cancer cell proliferation via arresting cell cycle at G1 phase. Moreover, it induced liver cancer cell apoptosis by decreasing the mitochondrial membrane potential, increasing intracellular reactive oxygen species level and inducing the expression of apoptosis-related proteins. Furthermore, compound 20 significantly inhibited growth of PLC/PRF/5 xenograft tumors in nude mice and had no observable toxic effect. Altogether, these results indicated that compound 20 is a promising lead for liver cancer therapeutics.

TrxR1, Gsr, and oxidative stress determine hepatocellular carcinoma malignancy

Thioredoxin reductase-1 (TrxR1)-, glutathione reductase (Gsr)-, and Nrf2 transcription factor-driven antioxidant systems form an integrated network that combats potentially carcinogenic oxidative damage yet also protects cancer cells from oxidative death. Here we show that although unchallenged wild-type (WT), TrxR1-null, or Gsr-null mouse livers exhibited similarly low DNA damage indices, these were 100-fold higher in unchallenged TrxR1/Gsr-double-null livers. Notwithstanding, spontaneous cancer rates remained surprisingly low in TrxR1/Gsr-null livers. All genotypes, including TrxR1/Gsr-null, were susceptible to N-diethylnitrosamine (DEN)-induced liver cancer, indicating that loss of these antioxidant systems did not prevent cancer cell survival. Interestingly, however, following DEN treatment, TrxR1-null livers developed threefold fewer tumors compared with WT livers. Disruption of TrxR1 in a marked subset of DEN-initiated cancer cells had no effect on their subsequent contributions to tumors, suggesting that TrxR1-disruption does not affect cancer progression under normal care, but does decrease the frequency of DEN-induced cancer initiation. Consistent with this idea, TrxR1-null livers showed altered basal and DEN-exposed metabolomic profiles compared with WT livers. To examine how oxidative stress influenced cancer progression, we compared DEN-induced cancer malignancy under chronically low oxidative stress (TrxR1-null, standard care) vs. elevated oxidative stress (TrxR1/Gsr-null livers, standard care or phenobarbital-exposed TrxR1-null livers). In both cases, elevated oxidative stress was correlated with significantly increased malignancy. Finally, although TrxR1-null and TrxR1/Gsr-null livers showed strong Nrf2 activity in noncancerous hepatocytes, there was no correlation between malignancy and Nrf2 expression within tumors across genotypes. We conclude that TrxR1, Gsr, Nrf2, and oxidative stress are major determinants of liver cancer but in a complex, context-dependent manner.

Epigenetic silencing of ALX4 regulates microcystin-LR induced hepatocellular carcinoma through the P53 pathway

Recent studies have shown that microcystin-LR (MC-LR) is one of the principal factors that cause liver cancer. Previously we have found that Aristaless-like Homeobox 4 (ALX4) was differentially expressed in MC-LR-induced malignant transformed L02 cells. However, the expression regulation, role and molecular mechanism of ALX4 during the process of liver cancer induced by MC-LR are still unclear. The expression of ALX4 was detected by quantitative reverse-transcription PCR and Western blot in MC-LR induced malignantly transformed cell and rat models. Methylation status of ALX4 promoter region was evaluated by methylation-specific PCR and bisulfite genomic sequencing. The anti-tumor effects of ALX4 on MC-LR induced liver cancer were identified in vitro and in vivo. ALX4 expression was progressively down-regulated in MC-LR-induced malignantly transformed L02 cells and the MC-LR exposed rat models. ALX4 promoter regions were highly methylated in malignantly transformed cells, while treatment with demethylation agent 5-aza-dC significantly increased ALX4 expression. Functional studies showed that overexpression of ALX4 inhibits cell proliferation, migration, invasion and metastasis in vitro and in vivo, blocks the G1/S phase and promotes the apoptosis. Conversely, knockdown of ALX4 promotes cell proliferation, migration and invasion. Mechanism study found that ALX4 exerts its antitumor function through the P53 pathway, C-MYC and MMP9. More importantly, ALX4 expression level showed a negative relation with serum MC-LR levels in patients with hepatocellular carcinoma. Our results suggested that ALX4 was inactivated by DNA methylation and played a tumor suppressor function through the P53 pathway in MC-LR induced liver cancer.