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Background & aims: Only limited therapeutic options are currently available for hepatocellular carcinoma (HCC), making the development of effective alternatives essential. Based on the recent finding that systemic or local hypothyroidism is associated with HCC development in humans and rodents, we investigated whether the thyroid hormone triiodothyronine (T3) could inhibit the progression of HCCs. Methods: Different rat and mouse models of hepatocarcinogenesis were investigated. The effect of T3 on tumorigenesis and metabolism/differentiation was evaluated by transcriptomic analysis, quantitative reverse transcription PCR, immunohistochemistry, and enzymatic assay. Results: A short treatment with T3 caused a shift in the global expression profile of the most aggressive preneoplastic nodules towards that of normal liver. This genomic reprogramming preceded the disappearance of nodules and involved reprogramming of metabolic genes, as well as pro-differentiating transcription factors, including Kruppel-like factor 9, a target of the thyroid hormone receptor β (TRβ). Treatment of HCC-bearing rats with T3 strongly reduced the number and burden of HCCs. Reactivation of a local T3/TRβ axis, a switch from Warburg to oxidative metabolism and loss of markers of poorly differentiated hepatocytes accompanied the reduced burden of HCC. This effect persisted 1 month after T3 withdrawal, suggesting a long-lasting effect of the hormone. The antitumorigenic effect of T3 was further supported by its inhibitory activity on cell growth and the tumorigenic ability of human HCC cell lines. Conclusions: Collectively, these findings suggest that reactivation of the T3/TRβ axis induces differentiation of neoplastic cells towards a more benign phenotype and that T3 or its analogs, particularly agonists of TRβ, could be useful tools in HCC therapy. Lay summary: Hepatocellular carcinoma (HCC) represents an important challenge for global health. Recent findings showed that systemic or local hypothyroidism is associated with HCC development. In rat models, we showed that administration of the thyroid hormone T3 impaired HCC progression, even when given at late stages. This is relevant from a translational point of view as HCC is often diagnosed at an advanced stage when it is no longer amenable to curative treatments. Thyroid hormones and/or thyromimetics could be useful for the treatment of patients with HCC.

Background : Proliferating cell nuclear antigen (PCNA) is a nuclear protein involved in DNA-synthesis and repair. During DNA-synthesis and repair the only active PCNA fraction is tightly bound to DNA. Similarly, during DNA-repair, a fraction of p21 colocalizes with PCNA in a detergent-insoluble form. Aim : The aim of the study was to analyze to what extent the presence of DNA-bound PCNA and p21 correlates with cell proliferation and DNA-repair in hepatocellular carcinoma (HCC). Methods : Twenty-six HCCs and surrounding cirrhosis were studied. The DNA-bound and detergent-soluble fractions of PCNA and p21 were analyzed by immunoblotting. P53 and Ki67-Labeling Index (Ki67-LI) were evaluated by immunocytochemistry. Results : Soluble fractions of PCNA and p21 were found in all samples. One out of 26 cirrhotic samples displayed a DNA-bound fraction of PCNA while no case expressed DNA-bound p21. Fourteen HCCs showed a DNA-bound PCNA fraction. A highly significant correlation was found between Ki67-LI and DNA-bound PCNA but not with detergent-soluble PCNA. DNA-bound p21 and PCNA, indicating ongoing DNA repair activity, were present in 6 of these 14 HCCs and correlated with a high histological grade and high Ki67-LI. Conclusions : Our results suggest that in HCC PCNA participates both in DNA synthesis and repair and that highly proliferating HCCs may display a sustained DNA-repair.

Hepatocellular carcinoma usually arises in cirrhotic livers as a complication of chronic liver disease, and may show a variable trend towards increasing ploidy. The aim of this study was to investigate possible associations between different etiological factors, particularly hepatitis B virus and hepatitis C virus infection, and alteration of DNA-ploidy and nuclearity of neoplastic hepatocytes.DNA-ploidy, the percentage of binucleated cells in the total cell population and the fraction of mononucleated hepatocytes in the polyploid compartment were assessed by image cytometry on cellular suspensions obtained by fine-needle biopsy from 60 hepatocellular carcinomas in patients whose viral status had previously been assessed.Significantly higher DNA-ploidy values (p = 0.005), with a reduction in the percentage of binucleated hepatocytes (p = 0.003) and an increase in the fraction of mononucleated hepatocytes in the polyploid compartment (p0.0001), were found in hepatocellular carcinoma with actual or previous hepatitis B virus infection (including also HCV+ve patients) in comparison to those not associated with hepatitis B virus infection, but not when HCV+ve hepatocellular carcinomas were compared to HCV-ve ones. Statistically significant differences for ploidy values (p0.05), percentage of binucleated hepatocytes (p0.05) and fraction of mononucleated hepatocytes in the polyploid compartment (p = 0.003) were also found between hepatocellular carcinoma associated only to hepatitis B virus infection ("pure" hepatitis B virus cases) and those associated only to hepatitis C virus infection ("pure" hepatitis C virus cases).Hepatocellular carcinoma associated with a previous or actual hepatitis B virus infection shows a peculiar phenotypical appearance, characterized by a trend towards increasing ploidy and reduction of binuclearity.

Aims/Methods: The relationship between AgNOR protein expression and doubling time was evaluated in 20 untreated nodules of hepatocellular carcinoma arising in cirrhotic liver. AgNOR protein quantity within the lesion was defined by image cytometry on histological sections from frozen biopsies obtained under ultrasound-guidance, selectively stained for AgNOR proteins. Tumour doubling time was calculated 6 months after diagnosis by measuring the volume variations of the nodules over a fixed period by "real time" ultrasonography. Results: The doubling time of nodules characterized by high AgNOR protein area values ( > 5.50 μm 2 , corresponding to the median AgNOR protein value) was shorter than that of nodules with low AgNOR protein area values ( 2 ). A highly significant difference in the mean doubling time values between the two groups (6.31±2.68 (E.S.) versus 15.92±3.03 (E.S.) months, respectively; p =0.009) was found. Moreover, when the relationship between AgNOR protein and doubling time values was tested by linear regression analysis, a significant inverse correlation was observed ( r =−0.68; p Conclusions: Our results indicate that AgNOR protein quantity represents a reliable parameter for predicting the tumour growth rate of untreated hepatocellular carcinoma nodules. Among the procedures commonly employed for the assessment of cell proliferation, the evaluation of the AgNOR parameter seems to be particularly suitable for kinetic analysis of ultrasound-guided fine-needle liver biopsies.

Background: MicroRNAs (miRNAs) are aberrantly expressed in cancers and evidences support their potential use in cancer treatment. MiR-519d belongs to the Chromosome 19 MiRNA Cluster (C19MC), which was recognized as overexpressed in placenta and breast cancer. This study aims to assay miR-519d expression pattern in Hepatocellular Carcinoma (HCC), to identify miR-519d targets and to characterize its role in hepatocarcinogenesis and response to anti-cancer treatments. Methods: Real-Time RT-PCR analysis of miR-519d was performed in 56 HCCs and cirrhosis, in normal livers and HCC cells. Transfection experiments with miR-519d or anti-miR-519d were performed in HCC cells and modulation of CDKN1A/p21 expression was evaluated by western blot (WB) and RT-PCR. Luciferase assay was performed to evaluate the interaction between miR-519d and 3’UTR region of CDKN1A/p21. Cell cycle modulation was analysed by FACS in miR-519d overexpressing and CDKN1A/p21 silenced cells. Apoptotic markers were evaluated by WB and FACS analysis in miR-519d and CDKN1A/p21 siRNA transfected cells following Doxorubicin treatment. Results: MiR-519d is overexpressed in 51.7% of HCCs. Upon miR-519d enforced expression, a decrease of CDKN1A/p21 was observed in HepG2 cells. Conversely, miR-519d silencing determined an increase of CDKN1A/p21 in SNU475 cells. Co-transfection of CDKN1A/p21-3’UTR reporter vector with miR-519d determined a decrease of the luciferase activity in HepG2 cells, proving a direct interaction between miR-519d and CDKN1A/p21 3’UTR. Cell cycle analysis showed a decrease of G1 phase in both miR-519d overexpressing and CDKN1A/p21 silenced HepG2 cells. Conversely, a cell cycle arrest in G1 phase was observed following miR-519d silencing in SNU475 cells. Doxorubicin treatment determined an increased apoptotic cell death, assessed by FACS-Annexin V and WB, following miR-519d overexpression and CDKN1A/p21 inhibition in HepG2 cells. Conclusions: Here we report the overexpression of miR-519d in HCC and identify CDKN1A/p21 among its targets. We provide evidences that miR-519d exerts an oncogenic activity by promoting proliferation of HCC cells through CDKN1A/p21 inhibition. We demonstrated that miR-519d overexpression is associated with a stronger response to Doxorubicin, suggesting miR-519d as a promising molecular marker for the optimization of HCC treatment.