Secretome of senescent hepatoma cells modulate immune cell fate by macrophage polarization and neutrophil extracellular traps formation.

Presence of dysfunctional senescent hepatocytes is a hallmark feature of liver cirrhosis which finally culminates in liver cancer. We now report the presence of senescent hepatocytes (p21 and p53 positive) in the vicinity of infiltrated immune cells in hepatocellular carcinoma tissue specimens by immunohistochemistry. Hence, we evaluated in vitro, the relevance of senescent hepatoma cells in altering the fate of monocytes and neutrophils by assaying for macrophage polarization and extracellular trap (NETs) formation, respectively. Premature senescence was induced in hepatoma cells (HepG2 and Huh7 cells) by treating cells with doxorubicin. Senescent hepatoma cells showed strong inflammatory phenotype with induced expression of cytokines (IL1ß, IL6, IL8 and IL13) as evaluated by flow cytometry. The senescent secretome from hepatoma cells when incubated with healthy monocytes caused it to differentiate predominantly towards M2 fate (CD80low CD86low CD163high CD206high) when analysed by flow cytometry. This was corroborated by the finding in clinical samples where human hepatocellular carcinoma harbouring senescent hepatocytes showed presence of M2 macrophages, while M1 macrophages were predominant in non-tumorous region. Additionally, the senescent secretome from Huh7 cells enhanced the NETs formation, while HepG2 secretome had an inhibitory effect. In conclusion, the "pro-inflammatory" senescent secretome drives non-inflammatory type M2 macrophage polarization and modulated neutrophil traps which in turn can influence the tumor microenvironment.

Senescent Hepatocytes in Decompensated Liver Show Reduced UPRMT and Its Key Player, CLPP, Attenuates Senescence In Vitro.

BACKGROUND AND AIMS: Non-dividing hepatocytes in end-stage liver disease indicates permanent growth arrest similar to senescence. Identifying senescence in vivo is often challenging and mechanisms inhibiting senescence are poorly understood. In lower organisms mitochondrial unfolded protein response (UPRMT) helps in increasing longevity; however, its role in senescence and liver disease is poorly understood. Aim of this study was to identify hepatocyte senescence and the role of UPRMT in cryptogenic cirrhosis. METHODS: Doxorubicin was used to induce senescence in non-neoplastic hepatocytes (PH5CH8) and hepatoma cells (HepG2 and Huh7). Senescence-associated markers and unfolded protein response was evaluated by fluorescence microscopy, immunoblotting and gene expression. Explants/biopsies from normal, fibrosis, compensated and decompensated cirrhosis without any known etiology were examined for presence of senescence and UPRMT by immunohistochemistry and gene expression. RESULTS: Accumulation of senescent hepatocytes in cryptogenic cirrhosis was associated with reduced proliferation, increased expression of γH2AX and p21, together with loss of LaminB1. Dysfunctional mitochondria and compromised UPRMT were key features of senescent hepatocytes both in vitro and also in decompensated cirrhosis. Intriguingly, compensated cirrhotic liver mounted strong UPRMT, with high levels of mitochondrial protease, CLPP. Overexpression of CLPP inhibited senescence in vitro, by reducing mitochondrial ROS and altering oxygen consumption. CONCLUSIONS: Our results implicate a role of hepatocyte senescence in cryptogenic cirrhosis together with a crucial role of UPRMT in preventing hepatocyte senescence. A compromised UPRMT may shift the fate of cirrhotic liver toward decompensation by exaggerating hepatocyte senescence. Restoring CLPP levels at least in cell culture appears as a promising strategy in mitohormesis, thereby, preventing senescence and possibly improving hepatocyte function.

Placental expression of asialoglycoprotein receptor associated with Hepatitis B virus transmission from mother to child.

BACKGROUND: Asialoglycoprotein receptor expression on hepatocytes has been associated with endocytosis, binding and uptake of hepatitis B virus. The role of asialoglycoprotein receptor in hepatitis B virus vertical transmission and its expression on placenta has not yet been studied. PATIENTS AND METHODS: Thirty-four HBsAg+ve and 13 healthy pregnant mothers along with their newborns were enrolled. The former were categorized into transmitting and non-transmitting mothers based on their newborns being hepatitis B surface antigen and hepatitis B virus DNA positive. Expression of asialoglycoprotein receptor and hepatitis B surface antigen in placenta and isoform of asialoglycoprotein receptor on dendritic cell in peripheral and cord blood dendritic cells were analysed using flowcytometry, immune histochemistry, immune florescence and qRT-PCR. RESULTS: Twelve HBsAg+ve mothers transmitted hepatitis B virus to their newborns whereas the rest (n = 22) did not. Hepatitis B virus-transmitting mothers showed increased expression of asialoglycoprotein receptor in trophoblasts of placenta. Immunofluorescence microscopy revealed colocalization of hepatitis B surface antigen and asialoglycoprotein receptor in placenta as well as in DCs of transmitting mothers. There was no significant difference in the expression of asialoglycoprotein receptor on peripheral blood mononuclear cells or chord blood mononuclear cells between the 2 groups. However, hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed increased mRNA levels of isoform of asialoglycoprotein receptor on dendritic cell in peripheral blood mononuclear cells. Hepatitis B virus-transmitting mothers and their HBsAg+ve newborns showed an increased expression of isoform of asialoglycoprotein receptor on dendritic cell on circulating dendritic cells compared to hepatitis B virus non-transmitting mothers and their negative newborns. CONCLUSIONS: This study revealed that increased expression of asialoglycoprotein receptor in placenta and colocalization with hepatitis B surface antigen strongly indicates its role in intrauterine transmission of hepatitis B virus. Asialoglycoprotein receptor-blocking strategy can be used for therapeutic intervention of vertical transmission.

Differentially regulated gene expression in quiescence versus senescence and identification of ARID5A as a quiescence associated marker.

In multicellular organisms majority of the cells remain in a non-dividing states of either quiescence (reversible) or senescence (irreversible). In the present study, gene expression signatures unique to quiescence and senescence were identified using microarray in osteosarcoma cell line, U2OS. It was noted that certain genes and pathways like NOD pathway was shared by both the growth arrest conditions. A major highlight of the present study was increased expression of number of chemokines and cytokines in both quiescence and senescence. While senescence-associated secretory phenotype (SASP) is well known, the quiescence-associated secretory phenotype (QASP) is relatively unknown and appeared novel in this study. ARID5A, a subunit of SWI/SNF complex was identified as a quiescence associated gene. The endogenous expression of ARID5A increased during serum starved condition of quiescence. Overexpression of ARID5A resulted in more number of cells in G0/G1 phase of cell cycle. Further ARID5A overexpressing cells when subjected to serum starvation showed a pronounced secretory phenotype. Overall, the present work has identified gene expression signatures which can distinguish quiescence from senescence.

Quantitative fibrosis estimation by image analysis predicts development of decompensation, composite events and defines event-free survival in chronic hepatitis B patients.

The extent of fibrosis is a major determinant of the clinical outcome in patients with chronic liver diseases. We undertook this study to explore the degree of fibrosis in baseline liver biopsies to predict clinical outcomes in chronic hepatitis B (CHB) patients. Fibrosis quantification was done by image analysis on Masson's trichrome-stained sections and correlated with clinical and biochemical parameters, liver stiffness and hepatic vein pressure gradient (n = 96). Follow-up information collected related to clinical outcome. A total of 964 cases was analyzed. Median quantitative fibrosis (QF) was 3.7% (interquartile range, 1.6%-9.7%) with substantial variation in various stages. Median QF was F0, 1% (0.7%-1.65%); F1, 3.03% (2.07%-4.0%); F2, 7.1% (5.6%-8.7%); F3, 12.7% (10.15%-16.7%); F4, 26.9% (20.3%-36.4%). QF positively correlated with METAVIR staging, liver stiffness measurement, and hepatic vein pressure gradient. Eighty-nine cases developed liver-related events: decompensation, hepatocellular carcinoma, liver transplantation and death. Cox regression analysis after adjusting for METAVIR staging-QF, albumin, and AST for composite events; QF and albumin for decompensation; and only QF for hepatocellular carcinoma-were found to be significant predictors of clinical outcomes. QF categorized into five stages: QF1, 0%-5%; QF2, 5.1%-10%; QF3, 10.1%-15%; QF4, 15.1%-20%; QF5, >20.1%. In patients with advanced stages of QF, probability of event-free survival found to be low. Quantitative fibrosis in baseline liver biopsy predicts progression of the disease and disease outcome in CHB patients. QF defines the probability of event-free survival in CHB cases.

Expression/localization patterns of sirtuins (SIRT1, SIRT2, and SIRT7) during progression of cervical cancer and effects of sirtuin inhibitors on growth of cervical cancer cells.

Sirtuins belong to the family of class III histone deacetylases; its role in neoplasia is controversial as both tumor-suppressive and promoting functions have been reported. There are very few reports available, where expressions of sirtuin isoforms are comprehensively analyzed during neoplasia. Therefore, in the present study, the expression of SIRT1, SIRT2, and SIRT7 during different stages of cervical cancer progression was analyzed. The normal cervical epithelium showed feeble expression of sirtuin isoforms, SIRT1, SIRT2, and SIRT7. A significant increase in SIRT1 expression was noted in the cytoplasm as well as in the nucleus of proliferative layers of cervical epithelium in squamous intraepithelial lesions (SIL); however, in the squamous cell carcinomas (SCC), a heterogeneous pattern of SIRT1 expression varying from low to high was noted. A progressive increase in the expression of both SIRT2 and SIRT7 was noted during cancer progression in the following order: normal < preneoplasia < cancer. Cervical cancer cell lines, HeLa and SiHa, showed higher levels of SIRT1 and SIRT2 in comparison to the immortalized cell counterpart, HaCaT. Specific inhibitors of SIRT1 (Ex527) and SIRT2 (AGK2) impaired the growth of the cervical cancer cells, SiHa, but not of the HaCaT cells. SIRT1 inhibition caused cell death, while SIRT2 inhibition resulted in cell cycle arrest. In conclusion, we report the overexpression of SIRT2 and SIRT7 proteins in cervical cancer and suggest probable application of sirtuin inhibitors as therapeutic targets. Further, a specific increase in the levels of SIRT1 in intraepithelial lesion makes it a promising candidate for identification of preneoplastic changes.

Hepatitis B virus induces cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma by targeting programmed cell death protein4 (PDCD4) and phosphatase and tensin homologue (PTEN).

Hepatitis B viral infection-induced hepatocellular carcinoma is one of the major problems in the developing countries. One of the HBV proteins, HBx, modulates the host cell machinery via several mechanisms. In this study we hypothesized that HBV enhances cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma. HBx gene was over-expressed, and miRNA-21 expression and cell proliferation were measured in Huh 7 and Hep G2 cells. miRNA-21 was over-expressed in these cells, cell proliferation and the target proteins were analyzed. To confirm the role of miRNA-21 in HBx-induced proliferation, Hep G 2.2.1.5 cells (a cell line that expresses HBV stably) were used for miRNA-21 inhibition studies. HBx over-expression enhanced proliferation (3.7- and 4.5-fold increase; n = 3; p<0.01) and miRNA-21 expression (24- and 36-fold increase, normalized with 5S rRNA; p<0.001) in Huh 7 and Hep G2 cells respectively. HBx also resulted in the inhibition of miRNA-21 target proteins, PDCD4 and PTEN. miRNA-21 resulted in a significant increase in proliferation (2- and 2.3-fold increase over control cells; p<0.05 in Huh 7 and Hep G2 cells respectively) and decreased target proteins, PDCD4 and PTEN expression. Anti-miR-21 resulted in a significant decrease in proliferation (p<0.05) and increased miRNA-21 target protein expression. We conclude that HBV infection enhances cell proliferation, at least in part, via HBx-induced miRNA-21 expression during hepatocellular carcinoma progression.

From cirrhosis to hepatocellular carcinoma: new molecular insights on inflammation and cellular senescence.

Sequential progression from chronic liver disease to fibrosis and to cirrhosis culminates in neoplasia in hepatocellular carcinoma (HCC). The preneoplastic setting of the cirrhotic background provides a conducive environment for cellular transformation. The role of classical inflammation in cirrhosis is widely known, but the exact mechanism linking inflammation and cancer remains elusive. Recent studies have elucidated roles for NF-κB, STAT3 and JNK as possible missing links. In addition, the "inflammasome" (a multiprotein complex and sensor of cellular damage) is a recently identified player in this field. The hallmarks of cirrhosis include necroinflammation, deposition of extracellular matrix and shortening of telomeres, leading to senescence and regeneration. Additionally, the accumulation of genetic/epigenetic changes propels atypical cells toward a malignant phenotype. This review provides recent information on the classical inflammatory pathway, together with a spotlight on inflammasomes and the immunomodulatory role of cellular senescence during the progression from cirrhosis to HCC. Moreover, lacunae in the current knowledge were identified and key questions raised on whether the observed adaptive responses are beneficial or detrimental to tissue homeostasis in a complex organ like liver.