Search

The growing knowledge of ferroptosis has suggested the role and therapeutic potential of ferroptosis in cancer, but has not been translated into effective therapy. Liver cancer, primarily hepatocellular carcinoma (HCC), is highly lethal with limited treatment options. LIFR is frequently downregulated in HCC. Here, by studying hepatocyte-specific and inducible Lifr-knockout mice, we show that loss of Lifr promotes liver tumorigenesis and confers resistance to drug-induced ferroptosis. Mechanistically, loss of LIFR activates NF-κB signaling through SHP1, leading to upregulation of the iron-sequestering cytokine LCN2, which depletes iron and renders insensitivity to ferroptosis inducers. Notably, an LCN2-neutralizing antibody enhances the ferroptosis-inducing and anticancer effects of sorafenib on HCC patient-derived xenograft tumors with low LIFR expression and high LCN2 expression. Thus, anti-LCN2 therapy is a promising way to improve liver cancer treatment by targeting ferroptosis., (© 2021. The Author(s).)

Objective: In the rat, the pancreatic islet transplantation model is an established method to induce hepatocellular carcinomas (HCC), due to insulin-mediated metabolic and molecular alterations like increased glycolysis and de novo lipogenesis and the oncogenic AKT/mTOR pathway including upregulation of the transcription factor Carbohydrate-response element-binding protein (ChREBP). ChREBP could therefore represent an essential oncogenic co-factor during hormonally induced hepatocarcinogenesis., Methods: Pancreatic islet transplantation was implemented in diabetic C57Bl/6J (wild type, WT) and ChREBP-knockout (KO) mice for 6 and 12 months. Liver tissue was examined using histology, immunohistochemistry, electron microscopy and Western blot analysis. Finally, we performed NGS-based transcriptome analysis between WT and KO liver tumor tissues., Results: Three hepatocellular carcinomas were detectable after 6 and 12 months in diabetic transplanted WT mice, but only one in a KO mouse after 12 months. Pre-neoplastic clear cell foci (CCF) were also present in liver acini downstream of the islets in WT and KO mice. In KO tumors, glycolysis, de novo lipogenesis and AKT/mTOR signalling were strongly downregulated compared to WT lesions. Extrafocal liver tissue of diabetic, transplanted KO mice revealed less glycogen storage and proliferative activity than WT mice. From transcriptome analysis, we identified a set of transcripts pertaining to metabolic, oncogenic and immunogenic pathways that are differentially expressed between tumors of WT and KO mice. Of 315 metabolism-associated genes, we observed 199 genes that displayed upregulation in the tumor of WT mice, whereas 116 transcripts showed their downregulated expression in KO mice tumor., Conclusions: The pancreatic islet transplantation model is a suitable method to study hormonally induced hepatocarcinogenesis also in mice, allowing combination with gene knockout models. Our data indicate that deletion of ChREBP delays insulin-induced hepatocarcinogenesis, suggesting a combined oncogenic and lipogenic function of ChREBP along AKT/mTOR-mediated proliferation of hepatocytes and induction of hepatocellular carcinoma.

Autophagy is an important biological process in normal cells. However, how it affects tumor progression still remains poorly understood. Herein, we demonstrated that the oncogenic protein Chromodomain-helicase-DNA-binding-protein 1-like gene (CHD1L) might promote HCC cells migration and metastasis through autophagy. CHD1L could bind to the promotor region of Zinc finger with KRAB and SCAN domain 3 (ZKSCAN3), a pivotal autophagy suppressor, and inhibit its transcription. We established inducible CHD1L conditional knockout cell line (CHD1L-iKO cell) and found that the deletion of CHD1L significantly increased ZKSCAN3 expression both at mRNA and protein level. Deletion of CHD1L impaired the autophagic flux and migration of HCC cells, while specifically inhibiting ZKSCAN3 blocked these effects. Further exploration demonstrated that the enhanced tumor cell migration and metastasis induced by CHD1L was mediated through ZKSCAN3-induced autophagic degradation of Paxillin. In summary, we have characterized a previously unknown function of CHD1L in regulating tumor migration via ZKSCAN3-mediated autophagy in HCC. Further inhibition of CHD1L and its downstream autophagy signaling might shed new light on cancer therapeutics., (© 2021. The Author(s).)

Clinical applications of high-intensity pulsed electric fields have proven useful in ablating solid tumors. However, novel ideas for the development of an effective tumor ablation device are urgently needed. Here, we studied cellular effects of the nanosecond exponential pulse, which is generated by a capacitor-discharging circuit and delivered via a transmission line. Pulses of peak voltage boosted by transmission line oscillation possess high capability to induce swelling and to cause loss of viability in cells. The appropriate parameter of the pulse was selected to investigate the ultrastructural changes in swollen cells, which present smoothened plasma membrane, loss of microvilli, and lowered cytoplasm electron density. We propose the equivalent force field hypothesis to understand the mechanism underlying cell swelling induced by pulsing. Wrinkles on the plasma membrane might indicate recovery from cell swelling, and this was verified by co-culture of pulsed PKH26-Cells with sham-treated PKH67-Cells. We concluded that the ultrastructural changes, such as irregular pores formed on the plasma membrane, were mainly induced by the effect of electric pulse applied on the charged molecules in the membrane., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The landscape of cellular plasticity and sources with relevant niche signals in hepatocellular carcinoma is still obscure. Transient receptor potential vanilloid 1 (TRPV1), a non-selective cation channel, is involved in a variety of malignancies and overexpressed in hepatocellular carcinoma (HCC). We have investigated the role of TRPV1 in HCC from different angles by various experimental techniques, such as in vivo and in vitro experiments, and by bioinformatics analysis of data from genetic models induced by diethylnitrosamine (DEN), mice samples and human HCC samples. We find that TRPV1 knockout promotes to hepatocarcinogenesis and deconstructs the portal triad adjacent to tumor border that is contributed by originations of tumor initiating cells and biliary cells. Epithelial to mesenchymal transition (EMT) is involved and transcription factors Ovol2 and Zeb1 coordinated with Sox 10 drive gene expression in the event which is also confirmed by the expression of these proteins in human HCC samples. Treatment with TRPV1 agonist Capsaicin inhibits the growth of HCC cells in xenograft models. Our findings demonstrate that TRPV1 is a potential therapeutic target in human HCC and exerts effects on cellular plasticity with modulation of Ovol2, Zeb1 and Sox10., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Five cytological types of hepatocellular carcinoma-29 (G-29) grown in the muscle tissue of the thigh of experimental animals were identified by transmission electron microscopy; 89% of these were poorly differentiated type I-III cells. Lithium in a concentration of 20 mM produced a damaging effect on poorly differentiated G-29 cells: the number of cells with zones of intracellular component destruction and volume density of these zones increased, while volume density of cisterns of endoplasmic reticulum decreased. These results suggest that lithium carbonate can cause destructive changes in the heterogeneous population of G-29 cells during in vivo tumor development.

Background: Hepatocellular carcinoma (HCC) is a significant clinical challenge, and the mechanisms underlying HCC pathogenesis remain incompletely understood. Colon cancer associated transcript 1 (CCAT1), is one novel long noncoding RNA (lncRNA) which is upregulated in HCC. Autophagy is a vital process in HCC progression, and it is unknown whether CCAT1 regulates autophagy in HCC., Materials and Methods: Immunofluorescence staining and transmission electron microscopy were used to analyze autophagy activity. Luciferase assay was performed to confirm miRNA-181a-5p (miR-181a-5p) bind CCAT1 and ATG7., Results: CCAT1 levels were higher in tissue and cell lines of HCC. In function research, we found that CCAT1 facilitates HCC cell autophagy and cell proliferation. Our results show that, mechanistically, CCAT1 promotes autophagy through functioning as a sponge for miR-181a-5p, and then regulating ATG7 expression., Conclusion: Our findings indicate CCAT1 may play a role in regulating autophagy by sponging miR-181a-5p in HCC., (© 2019 Wiley Periodicals, Inc.)

For decades, intranuclear inclusions in many normal and neoplastic cells have been considered to be mere invaginations of cytoplasm into the nucleus without any notable function or influence on disease. We investigated such inclusions in 75 specimens of hepatocellular carcinoma (HCC). In this context we demonstrate that these inclusions are true inclusions, completely closed and delimited by the nuclear membrane, containing degenerate cell organelles and lysosomal proteins. Moreover, their occurrence was positively associated with patient survival but not with tumour grade or stage. In a standardised area a mean of 124 inclusions per specimen was present in the tumorous liver tissue in contrast to 5 inclusions in the non-tumorous adjacent section and 89% of all scrutinised HCC showed at least one membrane-bound nuclear inclusion. Ultrastructural characterisation by transmission electron microscopy revealed degenerative materials such as residues of lysosomes, endoplasmic reticulum and Golgi apparatus within the inclusions. Due to the fact that the content of the inclusions appears to be more condensed than cytoplasm and contains fewer intact cell organelles, we assume that they are not mere invaginations of cytoplasm. Three dimensional (3D) reconstruction of isolated and immunofluorescence stained nuclei showed that the inclusions are completely located within the nucleus without any connection to the cytoplasm. The limiting membrane of the inclusions contained lamin B suggesting nuclear membrane origin. The content of the inclusions stained for the autophagy-associated proteins p62, ubiquitin, LC3B, cathepsin B and cathepsin D. Triple immunofluorescence staining followed by 3D reconstruction revealed co-localisation of p62, ubiquitin and LC3B in the same inclusion. Our observations uncover that these inclusions are real inclusions completely surrounded by the nucleus. We propose that the presence of autophagy-associated proteins and proteases within the inclusions contribute to beneficial survival., (© 2019 The Authors. The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

We studied the effect of lithium carbonate on hepatocellular carcinoma-29 cells in CBA male mice after injection in a dose of 20 mM along the tumor periphery. Transmission electron microscopy revealed a decrease in the volume density of the granular endoplasmic reticulum in the cell cytoplasm and an increase in the total numerical and volume density of autophagosomes and autolysosomes and zones of destruction of intracellular organelles. The ability of lithium carbonate to activate intracellular degradation processes in tumor cells and to stimulate cell death can be used to develop new combined strategies in the chemotherapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC), a common liver malignancy worldwide, has high morbidity and mortality. β-Thujaplicin, a tropolone derivative, has been used in some health-care products and clinical adjuvant drugs, but its use for HCC is unknown. In this study, we found that β-Thujaplicin inhibits the growth of HCC cells, but not normal liver cells, with nanomolar potency. Mechanistically, we found that β-Thujaplicin could induce autophagy, as judged by western blot, confocal microscopy, and transmission electron microscopy. Further using β-Thujaplicin combined with an autophagy blocker or agonist treatment HepG2 cells, we found that β-Thujaplicin induced autophagic cell death (ACD) mediated by ROS caused inhibition of the Akt-mTOR signaling pathway. Moreover, β-Thujaplicin triggered HepG2 apoptosis and increased cleaved PARP1, cleaved caspase-3, and Bax/Bcl-2 ratio, which indicated that β-Thujaplicin induced apoptosis mediated by the mitochondrial-dependent pathway. We also found that increased expression of p21 and decreased expression of CDK7, Cyclin D1, and Cyclin A2 participating in β-Thujaplicin caused the S-phase arrest. It seems that β-Thujaplicin exerts these functions by ROS-mediated p38/ERK MAPK but not by JNK signaling pathway activation. Consistent with in vitro findings, our in vivo study verified that β-Thujaplicin treatment significantly reduced HepG2 tumor xenograft growth. Taken together these findings suggest that β-Thujaplicin have an ability of anti-HCC cells and may conducively promote the development of novel anti-cancer agents.

Recent research has tried to use exosomal RNAs (coding and noncoding) as potential diagnostic markers for hepatocellular carcinoma (HCC). Initially, by using bioinformatics, we selected an HCC-exosomal RNA-based biomarker panel. The choice of this panel depends on the integration of Ras-related in brain (RAB11A) gene expression and its competing endogenous network. This network includes long noncoding RNA RP11-513I15.6 (lncRNA-RP11-513I15.6) and microRNA-1262 (miR-1262). Secondly, we tried to validate the expression of this network in the sera of 60 patients with HCC in comparison with 42 chronic hepatitis C virus-infected patients and 18 healthy controls. Then we assessed the diagnostic efficiency of this panel using a receiver operating characteristic curve analysis. The panel of 3 exosomal RNA-based biomarkers (lncRNA-RP11-513I15.6, miR-1262, and RAB11A) showed excellent sensitivity and specificity in discriminating patients with HCC from patients with chronic hepatitis C virus and healthy controls. Among these 3 RNAs, serum RAB11A mRNA was the most independent prognostic factor. The selected circulatory exosomal RNA-based biomarker panel showed its ability to be used as a diagnostic and prognostic biomarker tool for HCC. Moreover, these biomarkers could be therapeutic targets., (© 2018 Wiley Periodicals, Inc.)

Tissue culture medium routinely contains fetal bovine serum (FBS). Here we show that culturing human hepatoma cells in their native, adult serum (human serum, HS) results in the restoration of key morphological and metabolic features of normal liver cells. When moved to HS, these cells show differential transcription of 22-32% of the genes, stop proliferating, and assume a hepatocyte-like morphology. Metabolic analysis shows that the Warburg-like metabolic profile, typical for FBS-cultured cells, is replaced by a diverse metabolic profile consistent with in vivo hepatocytes, including the formation of large lipid and glycogen stores, increased glycogenesis, increased beta-oxidation and ketogenesis, and decreased glycolysis. Finally, organ-specific functions are restored, including xenobiotics degradation and secretion of bile, VLDL and albumin. Thus, organ-specific functions are not necessarily lost in cell cultures, but might be merely suppressed in FBS. The effect of serum is often overseen in cell culture and we provide a detailed study in the changes that occur and provide insight in some of the serum components that may play a role in the establishment of the differentiated phenotype.

Structural changes in the liver of CBA mice were studied during the development of experimental hepatocarcinoma-29 inoculated into the hip. A decrease in the volume density of hepatocyte cytoplasm, mitochondria, endoplasmic reticulum, and lipid inclusions and an increase in the volume density of lysosomal structures during tumor growth were observed. All stages of intracellular autophagy were recorded by the method of electron microscopy. These stages included the appearance of autophagosomes, autophagolysosomes, and secondary lysosomes in the hepatocyte cytoplasm. Fragments of cytoplasm, glycogen rosettes, mitochondria, and fragments of endoplasmic reticulum with ribosomes were found in autophagosomes. The obtained data indicate the development of non-selective autophagy in the liver during distant tumor growth in aimed at the maintenance of intracellular homeostasis in hepatocytes and energy and trophic homeostasis of organism.

Our previous findings showed a good therapeutic effect of the combination of suicide gene HSV-TK, nuclide 131I, and magnetic fluid hyperthermia (MFH) on hepatoma by using magnetic nanoparticles as linkers, far better than any monotherapy involved, with no adverse effects. This combination therapy might be an eligible strategy to treat hepatic cancer. However, it is not clear how the combination regimen took the therapeutic effects. In the current study, to explore the possible mechanisms of radionuclide-gene therapy combined with MFH to treat hepatoma at tissue, cellular, and molecular levels and to provide theoretical and experimental data for its clinical application, we examined the apoptosis induction of the combination therapy and investigated the expression of the proteins related to apoptosis such as survivin, livin, bcl-2, p53, and nucleus protein Ki67 involved in cell proliferation, detected VEGF, and MVD involved in angiogenesis of tumor tissues and analyzed the pathologic changes after treatment. The results showed that the combination therapy significantly induced the hepatoma cell apoptosis. The expression of survivin, VEGF, bcl-2, p53, livin, Ki67, and VEGF proteins and microvascular density (MVD) were all decreased after treatment. The therapeutic mechanisms may be involved in the downregulation of Ki67 expression leading to tumor cell proliferation repression and inhibition of survivin, bcl-2, p53, and livin protein expression inducing tumor cell apoptosis, negatively regulating VEGF protein expression, and reducing vascular endothelial cells, which results in tumor angiogenesis inhibition and microvascular density decrease and tumor cell necrosis. These findings offer another basic data support and theoretical foundation for the clinical application of the combination therapy.

Objective To explore the role and mechanism of β1 integrin in the regulation of multicellular drug resistance in hepatocellular carcinoma (HCC). Methods This in vitro study used a liquid overlay technique to obtain multicellular spheroids of two human HCC cell lines, HepG2 and Bel-7402. The morphology of the spheroids was observed by optical and electron microscopy. The effects of exposure to 5-fluorouracil (5-FU) and cisplatin (CDDP) on cell proliferation and the induction of apoptosis were assessed in monolayer cells and multicellular spheroids. The levels of β1 integrin and the effects on the focal adhesion kinase (FAK)/protein kinase B (Akt) pathway were evaluated using Western blot analysis, immunofluorescence and flow cytometry. The role of β1 integrin was confirmed by using an inhibitory antibody. Results Cell proliferation inhibition and cell apoptosis induced by 5-FUl and CDDP were abrogated in multicellular spheroids compared with monolayer cells. There were high levels of β1 integrin in multicellular spheroids. β1 integrin inhibitory antibody prevented the formation of multicellular spheroids, coupled with a significant increase in proliferation inhibition and apoptosis induction. β1 integrin inhibitory antibody effectively suppressed activation of both FAK and Akt in multicellular spheroids. Conclusions β1 integrin mediated multicellular drug resistance through the FAK/Akt pathway in HCC spheroids.

Early detection of hepatocellular carcinoma (HCC) is crucial for successful therapy. The present work examined the value of ultrastructural morphometric image analysis of hepatocyte nuclei in patients with chronic hepatitis C virus (HCV) versus HCC cases with chronic HCV and the corresponding surgical tumor-free safe margins (TFMs), to highlight any early predictive signs of neoplastic cellular transformation. This work also performed an immunohistochemical assessment of cytokeratin 19 (CK19) and Ki-67-positive cells to visualize any associated proliferative activity in the examined groups. The results showed significant decrease in the hepatocyte nuclear surface areas in the HCC and TFMs versus those in the HCV cases. The hepatocyte nucleolar surface area was significantly increased in the HCC cases versus that in the HCV cases. This increase was associated with a significant increase in Ki-67-positive cells in the HCC cases compared to those in the other groups. Conversely, the mean number of CK 19-positive cells was significantly reduced in the HCC cases compared to the cell numbers in TFMs and HCV cases with severe hepatic fibrosis. Liver progenitor cells (LPCs) were discerned in the reactive ductules and canaliculo-ductular junctions that characterized TFMs. LPCs were sporadically distributed in the liver lobules and reactive bile ductules in the HCC samples. In conclusion, CK 19 represents an important marker for distinguishing between dysplastic and malignant liver nodules. Electron microscopic morphometric image analysis may be considered as adjunct factor for assessing hepatocyte malignant transformation. Wider scale studies are needed to authenticate these results.

Macroautophagy/autophagy is an important catabolic process mediating cellular homeostasis and plays critical roles in cancer development. Whereas autophagy has been widely studied in various pathological models, little is known about the distribution, clinical significance and regulatory mechanism of this process in human hepatocellular carcinoma (HCC). In the present study, we found that tumor tissues exhibited significantly increased levels of autophagy compared with non-tumor tissues, and cancer cells with higher levels of autophagy were predominantly enriched in the invading edge regions of human HCC. Increased MAP1LC3B/LC3B expression in the invading edge regions was significantly correlated with a higher density of closely located monocytes, and TNF and IL1B derived from tumor-activated monocytes synergistically induced cancer cell autophagy in the invading edge regions of HCC. Monocyte-elicited autophagy induced the epithelial-mesenchymal transition (EMT) of cancer cells and promoted tumor metastasis by activating the NFKB-SNAI1 signaling pathway. Moreover, the increase of LC3B + cancer cells in the invading edge areas was associated with high mortality and reduced survival of patients with HCC. These findings indicated that cancer cell autophagy is regulated by a collaborative interaction between tumor and immune cell components in distinct HCC microenvironments, thus allowing the inflammatory monocytes to be rerouted in a tumor-promoting direction.

Impaired mitochondrial oxidative phosphorylation (OXPHOS) capacity, accompanied by enhanced glycolysis, is a key metabolic feature of cancer cells, but its underlying mechanism remains unclear. Previously, we reported that human hepatoma cells that harbor OXPHOS defects exhibit high tumor cell invasiveness via elevated claudin-1 (CLN1). In the present study, we show that OXPHOS-defective hepatoma cells (SNU354 and SNU423 cell lines) exhibit reduced expression of mitochondrial ribosomal protein L13 (MRPL13), a mitochondrial ribosome (mitoribosome) subunit, suggesting a ribosomal defect. Specific inhibition of mitoribosomal translation by doxycycline, chloramphenicol, or siRNA-mediated MRPL13 knockdown decreased mitochondrial protein expression, reduced oxygen consumption rate, and increased CLN1-mediated tumor cell invasiveness in SNU387 cells, which have active mitochondria. Interestingly, we also found that exogenous lactate treatment suppressed MRPL13 expression and oxygen consumption rate and induced CLN1 expression. A bioinformatic analysis of the open RNA-Seq database from The Cancer Genome Atlas (TCGA) liver hepatocellular carcinoma (LIHC) cohort revealed a significant negative correlation between MRPL13 and CLN1 expression. Moreover, in patients with low MRPL13 expression, two oxidative metabolic indicators, pyruvate dehydrogenase B expression and the ratio of lactate dehydrogenase type B to type A, significantly and negatively correlated with CLN1 expression, indicating that the combination of elevated glycolysis and deficient MRPL13 activity was closely linked to CLN1-mediated tumor activity in LIHC. These results suggest that OXPHOS defects may be initiated and propagated by lactate-mediated mitoribosomal deficiencies and that these deficiencies are critically involved in LIHC development., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

Fibrolamellar carcinoma was first described in 1956. Subsequent large studies failed to identify cases before 1939 (the start of the World War II). This finding, combined with the presence of aryl hydrocarbon receptors on the tumor cells, have suggested that fibrolamellar carcinomas may be caused by environmental exposures that are new since World War II. To investigate this possibility, the surgical pathology files before 1939 were reviewed for hepatocellular carcinomas resected in young individuals. Two cases of fibrolamellar carcinoma were identified, from 1915 to 1924. The diagnosis of fibrolamellar carcinoma was confirmed at the histologic, ultrastructural and proteomic levels. These two fibrolamellar carcinoma cases clarify a key aspect of fibrolamellar carcinoma biology, reducing the likelihood that these tumors result exclusively from post World War II environmental exposures.

Hepatitis C virus represents one of the rising causes of hepatocellular carcinoma (HCC). Although the early diagnosis of HCC is vital for successful curative treatment, the majority of lesions are diagnosed in an irredeemable phase. This work deals with a comparative ultrastructural study of experimentally gradually induced HCC, surgically resected HCC, and potential premalignant lesions from HCV-infected patients, with the prospect to detect cellular criteria denoting premalignant transformation. Among the main detected pathological changes which are postulated to precede frank HCC: failure of normal hepatocyte regeneration with star shape clonal fragmentation, frequent elucidation of hepatic progenitor cells and Hering canals, hepatocytes of different electron density loaded with small sized rounded monotonous mitochondria, increase junctional complexes bordering bile canaliculi and in between hepatocyte membranes, abundant cellular proteinaceous material with hypertrophied or vesiculated rough endoplasmic reticulum (RER), sequestrated nucleus with proteinaceous granular material or hypertrophied RER, formation of lipolysosomes, large autophagosomes, and micro-vesicular fat deposition. In conclusion, the present work has visualized new hepatocytic division or regenerative process that mimic splitting or clonal fragmentation that occurs in primitive creature. Also, new observations that may be of value or assist in predicting HCC and identifying the appropriate patient for surveillance have been reported. Moreover, it has pointed to the possible malignant potentiality of liver stem/progenitor cells. For reliability, the results can be subjected to cohort longitudinal study.

We studied Golgi apparatus disorganizations and reorganizations in human HepG2 hepatoblastoma cells by using the nonmetabolizable glucose analogue 2-deoxy-D-glucose (2DG) and analyzing the changes in Golgi stack architectures by 3D-electron tomography. Golgi stacks remodel in response to 2DG-treatment and are replaced by tubulo-glomerular Golgi bodies, from which mini-Golgi stacks emerge again after removal of 2DG. The Golgi stack changes correlate with the measured ATP-values. Our findings indicate that the classic Golgi stack architecture is impeded, while cells are under the influence of 2DG at constantly low ATP-levels, but the Golgi apparatus is maintained in forms of the Golgi bodies and Golgi stacks can be rebuilt as soon as 2DG is removed. The 3D-electron microscopic results highlight connecting regions that interlink membrane compartments in all phases of Golgi stack reorganizations and show that the compact Golgi bodies mainly consist of continuous intertwined tubules. Connections and continuities point to possible new transport pathways that could substitute for other modes of traffic. The changing architectures visualized in this work reflect Golgi stack dynamics that may be essential for basic cell physiologic and pathologic processes and help to learn, how cells respond to conditions of stress.

Hepatocellular carcinoma (HCC) is one of the aggressive and resistant to drug therapy cancer. It is believed that the development of HCC is correlated with dysregulation of programmed cell death. So, the search for effective inducers of HCC cell death is very important. The aim of the work was to identify structural changes leading to HCC cell death when exposed to nanoscale and original forms of lithium salts. Using light and electron microscopy and flow cytometry, structural features of autophagy and apoptosis in HCC cells after their incubation with various forms of lithium salts has been revealed. It is shown that a more pronounced effect on HCC-29 cells have nanoscale forms of lithium carbonate and lithium citrate. At the same time, nanoscale lithium citrate mainly induced apoptosis, and nanosized form of lithium carbonate, along with apoptosis, induced autophagic death of HCC-29cells.

A direct acting anti-Ebola agent is needed. VP40, a conserved protein across Ebolavirus (EBOV) species has several pivotal roles in the virus life cycle. Inhibition of VP40 functions would lessen the virion integrity and interfere with the viral assembly, budding, and spread. In this study, cell penetrable human scFvs (HuscFvs) that bound to EBOV VP40 were produced by phage display technology. Gene sequences coding for VP40-bound-HuscFvs were subcloned from phagemids into protein expression plasmids downstream to a gene of cell penetrating peptide, i.e., nonaarginine (R9). By electron microscopy, transbodies from three clones effectively inhibited egress of the Ebola virus-like particles from human hepatic cells transduced with pseudo-typed-Lentivirus particles carrying EBOV VP40 and GP genes. Computerized simulation indicated that the effective HuscFvs bound to multiple basic residues in the cationic patch of VP40 C-terminal domain which are important in membrane-binding for viral matrix assembly and virus budding. The transbodies bound also to VP40 N-terminal domain and L domain peptide encompassed the PTAPPEY (WW binding) motif, suggesting that they might confer VP40 function inhibition through additional mechanism(s). The generated transbodies are worthwhile tested with authentic EBOV before developing to direct acting anti-Ebola agent for preclinical and clinical trials., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Aberrant changes in histone post-translational modifications are encountered frequently in diseases like cancer. Although histone H3 post-translational modifications have been extensively studied in context of diseases, the functionally important histone H2A PTM H2A119ub (H2Aub) has not gained much attention. In this study, we report that H2Aub markedly decreases in hepatocellular carcinoma. Usp21, a H2A deubiquitinase, is probably responsible for decrease in H2Aub. In addition, the H2Aub levels showed an inverse correlation with H3S10 phosphorylation (H3S10p) and the proliferative state of the cells. Downregulation of H2Aub is also associated with increased expression of growth factor gene lipocalin 2. Interestingly, we show that treatment of cells with histone deacetylase inhibitor trichostatin A results in increase of H2Aub and decrease in H3S10p. Our work for the first time suggests the in vivo association of H3S10p, H4ac, and H2A119ub with cellular transformation., (© 2016 by the Society for Experimental Biology and Medicine.)

Rationale: Hepatoid adenocarcinoma (HAC) is a rare malignant lung tumor that histologically resembles typical hepatocellular carcinoma (HCC) when it is metastatic to the lung. To date, this clinical entity has not been highlighted in the pulmonary literature., Objective: We present a review of all known cases of HAC, including the relevant clinical and histopathological features important for pulmonologists., Measurements and Main Results: The purpose of this report is to present a new case of HAC, with typical clinical and histologic features of this malignancy, and to summarize findings of previously reported cases. A systematic literature search of the electronic database PUBMED was conducted to identify all cases of hepatoid adenocarcinoma reported in the English literature, between January 1980 and June 2015. HAC and HCC can be distinguished by immunohistochemical staining. HAC usually presents as a large bulky solitary mass in the upper lobe; there is an exceedingly high prevalence in males and most patients with this tumor are smokers. Serum alpha-fetoprotein (AFP) in very high levels has been a distinguishing feature of this tumor. Nodal and distant metastases are common at initial presentation and, as a result, the prognosis is very poor. Resection and long-term survival, however, have been reported., Conclusion: Hepatoid adenocarcinoma, first described as a gastric tumor, has also been described in the lung. It morphologically resembles and must be distinguished from metastatic HCC of the lung. While most tumors produce AFP, the case we present demonstrates that this should not be a criterion for diagnosis., (Copyright © 2016. Published by Elsevier Ltd.)

Combination targeted therapy is a promising cancer therapeutic strategy. Here, using PEI-Mn0.5Zn0.5Fe2O4 nanoparticles (PEI-MZF-NPs) as magnetic media for MFH (magnetic fluid hyperthermia) and gene transfer vector for gene-therapy, a combined therapy, pHRE-Egr1-HSV-TK/(131)I-antiAFPMcAb-GCV/MFH, for hepatoma is developed. AntiAFPMcAb (Monoclonal antibody AFP) is exploited for targeting. The plasmids pHRE-Egr1-HSV-TK are achieved by incorporation of pEgr1-HSV-TK and pHRE-Egr1-EGFP. Restriction enzyme digestion and PCR confirm the recombinant plasmids pHRE-Egr1-HSV-TK are successfully constructed. After exposure to the magnetic field, PEI-MZF-NPs/pHRE-Egr1-EGFP fluid is warmed rapidly and then the temperature is maintained at 43 °C or so, which is quite appropriate for cancer treatment. The gene expression reaches the peak when treated with 200 μCi (131)I for 24 hours, indicating that the dose of 200 μCi might be the optimal dose for irradiation and 24 h irradiation later is the best time to initiate MFH. The in vitro and in vivo experiments demonstrate that pHRE-Egr1-HSV-TK/(131)I-antiAFPMcAb-GCV/MFH can greatly suppress hepatic tumor cell proliferation and induce cell apoptosis and necrosis and effectively inhibit the tumor growth, much better than any monotherapy does alone. Furthermore, the combination therapy has few or no adverse effects. It might be applicable as a strategy to treat hepatic cancer.

Ethnopharmacological Relevance: Shen-Ling-Bai-Zhu Powder (SLBZP) is a classic traditional Chinese medical formula that has been used for several decades in the treatment of patients with gastrointestinal malignancies. Whether SLBZP is best employed as single agent or adjunctive therapy has yet to be determined as does the mechanism whereby SLBZP exerts its anti-tumor effects., Aim of the Study: To investigate the effects of SLBZP alone and in combination with Cytoxan (CTX) on tumor growth, malignant cell apoptosis and Akt/Nuclear Factor kappa B (NF-КB) signaling in a murine model of hepatocellular carcinoma (HCC) receiving chemotherapy., Materials and Methods: Sixty-four adult mice developed HCC following subcutaneous inoculation with H22 hepatocellular carcinoma cells. Seven days later, all received chemotherapy with CTX (200mg/kg) once. Mice were then randomized into eight study groups (N=8/group). Three groups were treated with different concentrations of SLBZP alone (6.00, 3.00, 1.5g/kg), three with SLBZP (6.00, 3.00, 1.5g/kg) plus CTX (20mg/kg), one with CTX (20mg/kg) alone (positive control), and one with physiologic saline (untreated, negative control). All groups were treated for 14 days. Tumor size, histology and serum or tissue levels and/or mRNA expression of PDGF-BB, VEGF, Ang-1, Ang-2, NF-КB, B-cell lymphoma-2 (Bcl-2); B-cell lymphoma-extra large (Bcl-xL); X-linked inhibitor of apoptosis (XIAP), Survivin, Caspase-3, Caspase-9, Caspase-7, Akt and phosphorylated Akt expression were documented at the end of treatment., Results: Compared to untreated negative controls, tumor sizes were decreased in the CTX alone, SLBZP (M)+CTX and SLBZP (H)+CTX groups (-52%,-53% and -58% respectively). Tumor cell density was decreased in all treated groups but most apparent in the SLBZP (H)+CTX group. Electron microscopic evidence of apoptosis was also most apparent in this group. Serum and/or tissue levels and expression of PDGF-BB, VEGF, Ang-1, Ang-2, their downstream signaling proteins and anti-apoptotic markers were lowest and pro-apoptotic markers highest in SLBZP (H)+CTX treated mice., Conclusions: In this chemotherapy-induced animal model of HCC, SLBZP was most efficacious as adjunctive therapy and appears to act by inhibiting tumor growth promoters and anti-apoptotic proteins while enhancing pro-apoptotic proteins., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Context 2a,-3a,-24-Trihydroxyurs-12-en-28-oic acid (TEO, a corosolic acid analogue) is a triterpenoid saponin isolated from Actinidia valvata Dunn (Actinidiaceae), a well-known traditional Chinese medicine. Objective This study investigated the anti-proliferation and inducing apoptosis effects of TEO in three human hepatocellular carcinoma (HCC) cell lines. Materials and methods Cytotoxic activity of TEO was determined by the MTT assay at various concentrations from 2.5 to 40 μg/mL in BEL-7402, BEL-7404 and SMMC-7721 cell lines. Cell morphology was assessed by acridine orange/ethidium bromide and 4'-6-diamidino-2-phenylindole dihydrochloride staining and fluorescence microscopy. Cell-cycle distribution and DNA damage were determined by flow cytometry and comet assay. Mitochondrial dysfunction was assessed by JC-1 staining and transmission electron microscopy. Apoptosis changes were explored by Western blot, TNF-α and caspase-3, -8, -9 assays. Results TEO exhibited inhibition effects on BEL-7402, BEL-7404 and SMMC-7721 cells treated for 24 h, the IC50 values were 34.6, 30.8 and 30.5 μg/mL, respectively. TEO (40 μg/mL)-treated three cell lines increased by more than 21% in the G1 phase and presented the morphological change and DNA damage. TEO also declined the mitochondrial membrane potential and altered mitochondrial ultra-structure. Furthermore, caspase-3, caspase-8, caspase-9 and TNF-α were also activated. Mechanism investigation showed that TEO could decrease anti-apoptotic Bcl-2 protein expression, increase proapoptotic Bax and Bid proteins expressions and increase Bax/Bcl-2 ratio. Conclusion Our results demonstrate for the first time that TEO inhibited growth of HCC cell lines and induced G1 phase arrest. Moreover, proapoptotic effects of TEO were mediated through the activation of TNF-α, caspases and mitochondrial pathway.

Galangin suppresses proliferation and induces apoptosis and autophagy in hepatocellular carcinoma (HCC) cells, but the precise mechanism is not clear. In this study, we demonstrated that galangin induced autophagy, enhanced the binding of SIRT1-LC3 and reduced the acetylation of endogenous LC3 in HepG2 cells. But this autophagy was inhibited by inactivation of SIRT1 meanwhile, galangin failed to reduce the acetylation of endogenous LC3 after SIRT1 was knocked-down. Collectively, these findings demonstrate a new mechanism by which galangin induces autophagy via the deacetylation of endogenous LC3 by SIRT1.

Objective: To investigate the anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc and its possible molecular mechanisms in vitro and in vivo., Methods: Transonic alcohol-chloroform extraction method was used to extract toosendanin from the bark of Melia toosendan Sieb. et Zucc, and the content of toosendanin in the crude extract was measured by high performance liquid chromatography (HPLC). Anti-cancer effects of crude extract from Melia toosendan Sieb. et Zucc were investigated in in vivo and in vitro studies. In the in vitro experiment, human hepatocellular carcinoma cell lines SMMC-7721 and Hep3B were co-incubated with toosendanin crude extract of different concentrations, respectively. In the in vivo experiment, BALB/c mice were subcutaneously inoculated with mouse hepatocellular carcinoma H22 cells and treated with crude extract., Results: HPLC revealed the content of toosendanin was about 15%. Crude extract from Melia toosendan Sieb. et Zucc inhibited cancer cells growth in a dose- and time-dependent manner. The 50% inhibitory concentration (IC50, 72 h) was 0.6 mg/L for SMMC-7721 cells and 0.8 mg/L for Hep3B cells. Both high-dose [0.69 mg/(kg d)] and low-dose [0.138 mg/(kg d)] crude extract could markedly suppress cancer growth, and the inhibition rate was greater than 50%. Hematoxylin and eosin staining showed necrotic area in cancers and transmission electron microscopy displayed necrotic and apoptotic cancer cells with apoptotic bodies. Immunohistochemistry showed that the expression of Bax and Fas increased and the expression of Bcl-2 reduced., Conclusions: Toosendanin extract has potent anti-cancer effects via suppressing proliferation and inducing apoptosis of cancer cells in vivo and in vitro. The mechanism of apoptosis involves in mitochondrial pathway and death receptor pathway.

The multifactorial and multistage pathogenesis of hepatocellular carcinoma (HCC) has fascinated a wide spectrum of scientists for decades. While a number of major risk factors have been identified, their mechanistic roles in hepatocarcinogenesis still need to be elucidated. Many tumor suppressor genes (TSGs) have been identified as being involved in HCC. These TSGs can be classified into two groups depending on the situation with respect to allelic mutation/loss in the tumors: the recessive TSGs with two required mutated alleles and the haploinsufficient TSGs with one required mutated allele. Hepatitis B virus (HBV) is one of the most important risk factors associated with HCC. Although mice cannot be infected with HBV due to the narrow host range of HBV and the lack of a proper receptor, one advantage of mouse models for HBV/HCC research is the numerous and powerful genetic tools that help investigate the phenotypic effects of viral proteins and allow the dissection of the dose-dependent action of TSGs. Here, we mainly focus on the application of mouse models in relation to HBV-associated HCC and on TSGs that act either in a recessive or in a haploinsufficient manner. Discoveries obtained using mouse models will have a great impact on HCC translational medicine.

Gelsolin (GSN), which is a Ca(2+)-dependent actin filament severing and capping protein, plays a critical role in the cancer progress and has the potential for providing a novel thread for cancer therapy. In current study, we demonstrate the roles of GSN on anti-apoptosis of hepatocarcinoma cells by transcriptome RNA-seq method. Then flow cytometry (FCM), in-cell immunoblotting and transmission electron microscopy (TEM) were used to examine the GSN regulatory cell apoptosis. The results revealed GSN significantly suppresses apoptosis-associated functional categories through down-regulating apoptosis-associated genes in 5 apoptosis terms and 6 relevant KEGG pathways. FCM showed a significant lower apoptotic rate in GSN-SMMC7721 (P<0.05). In-cell immunoblotting detected discrepant expression of the apoptosis factors among GSN expressed/shRNA transfectants (P<0.05). TEM observed the discernible apoptosis morphology. Above results suggest a negative relationship between GSN expression and hepatocarcinoma cell apoptosis. GSN overexpression suppresses apoptosis while down-regulated GSN promotes apoptosis. The possible mechanism could be associated with the regulation of GSN on the apoptosis-associated pathways and the apoptosis factors caspase 3 and bcl-2.

In this study, we examined the mechanism underlying the effect of Saururus chinensis Baill (saururaceae) on hepatocellular carcinoma HepG2 cells. HepG2 cells and Chang cells were exposed to various concentrations of S. chinensis Baill extract (SC-E) for 24 h. SC-E affected more significantly HepG2 cells than Chang cells in terms of cell viability and ATP production. Therefore, current study examined detailed mechanism how SC-E affected HepG2 cell survival. We found that SC-E (75 and 150 μg/ml) induced apoptosis via oxidative stress. SC-E also caused CCAAT-enhancer-binding protein homologous protein (CHOP) activation by dissociating the binding immunoglobulin protein (BiP) from inositol-requiring 1α (IRE1α) in the endoplasmic reticulum (ER) and induced Bax, cytochrome c release to cytosol, caspase-3 activation, and poly ADP ribose polymerase (PARP) cleavage, resulting in HepG2 cell apoptosis. Furthermore, SC-E caused ER Ca(2+) leakage into the cytosol; ER dilation and mitochondrial membrane damage were observed in transmission electron microscopy (TEM). Taken together, our results demonstrated that SC-E induced cancer cell apoptosis specifically through ER stress., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Purpose: Most transplant centers use chemoembolisation as locoregional bridge therapy for hepatocellular carcinoma (HCC) before liver transplantation (LT). Chemoembolisation using beads loaded with doxorubicin (DEBDOX) is a promising technique that enables delivery of a large quantity of drugs against HCC. We sought to assess the imaging-histologic correlation after DEBDOX chemoembolisation., Materials and Methods: All consecutive patients who had undergone DEBDOX chemoembolisation before receiving liver graft for HCC were included. Tumour response was evaluated according to Response Evaluation Criteria in Solid Tumours (RECIST) and modified RECIST (mRECIST) criteria. The result of final imaging made before LT was correlated with histological data to predict tumour necrosis., Results: Twenty-eight patients underwent 43 DEBDOX procedures for 45 HCC. Therapy had a significant effect as shown by a decrease in the mean size of the largest nodule (p = 0.02) and the sum of viable part of tumour sizes according to mRECIST criteria (p < 0.001). An objective response using mRECIST criteria was significantly correlated with mean tumour necrosis ≥90 % (p = 0.03). A complete response using mRECIST criteria enabled accurate prediction of complete tumour necrosis (p = 0.01). Correlations using RECIST criteria were not significant., Conclusion: Our data confirm the potential benefit of DEBDOX chemoembolisation as bridge therapy before LT, and they provide a rational basis for new studies focusing on recurrence-free survival after LT. Radiologic evaluation according to mRECIST criteria enables accurate prediction of tumour necrosis, whereas RECIST criteria do not.

Aim: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action., Methods: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level., Results: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface., Conclusion: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity.

Introduction: The impact of tumor cells on tumor-infiltrating lymphocytes (TILs) in cancer development is not yet clarified. Our study analyzed the distribution and prognostic value of CD4+ and CD8+ T lymphocytes in hepatocellular carcinoma (HCC) and liver metastases (LM)., Material and Methods: Archival tissue specimens of 35 HCC and 39 LM patients were immunohistochemically processed. The number of intratumoral (IT) and peritumoral (PT) CD4+ and CD8+ T cells was quantitatively analyzed., Results: We noted large variances of T lymphocyte subpopulations. Similar number of CD4+ and CD8+ lym-phocytes was present in HCC, whereas in LM the number of CD8+ cells was approximately two times higher than CD4+ lymphocytes. A significant prevalence of T cells in PT over IT areas was observed. The prognostic value was demonstrated only for PT CD8+ lymphocytes in LM, their reduced number being associated with shorter survival., Conclusions: The differences between proportions of T lymphocytes within tumor and its environment might be explained by proapoptotic effect of cancer cells on TILs.

Purpose: To retrospectively determine the correlation between heptic tumor signal intensity on gadoxetic acid-enhanced and diffusion-weighted MR images and histopathological grading of hepatocellular carcinoma (HCC)., Methods: We retrospectively reviewed the MR images of 79 patients with 141 surgically resected HCCs. The signal intensity and its relationship with histopathological grade were assessed. We measured the apparent diffusion correlation (ADC) values and calculated arterial enhancement ratios, washout ratios, and relative intensity ratios of HCCs relative to the surrounding liver parenchyma in gadoxetic-enhanced MR images in order to determine their relationship to the histological grade., Results: Morphological evaluation showed that larger tumor size and extrahepatic extension were associated with higher histologic grade (p < 0.01). Multivariate logistic regression showed that low ADC value and low relative intensity ratio in the arterial phase (RIRa) predict high histological grade. ADC value (cut-off 1.7 × 10(-3) mm(2)/s, sensitivity 82.4%, specificity 83.2%) was the best predictor of well-differentiated HCC, and RIRa (cut-off 0.93, sensitivity 81.4%, specificity 93.9%) was superior to ADC for predicting poorly differentiated HCC., Conclusion: Relative low arterial enhancement on gadoxetic acid-enhanced MR images and low ADC are predictive of worse histological grades of HCC.

Background: Functional disturbances of the endoplasmic reticulum (ER) lead to activation of the unfolded protein response (UPR), which is involved in the consecutive steps of carcinogenesis. In human hepatocellular carcinoma (HCC), the UPR is shown to be activated; however, little is known about the UPR kinetics and effects of UPR modulation in HCC., Methods: We sequentially monitored the UPR over time in an orthotopic mouse model for HCC and explored the effects of UPR modulation on cell viability and proliferation in vitro and in the mouse model., Results: The expression of ER-resident chaperones peaked during tumor initiation and increased further during tumor progression, predominantly within the nodules. A peak in Ire1 signaling was observed during tumor initiation. The Perk pathway was activated during tumor progression, and the proapoptotic target Chop was upregulated from week 5 and continued to rise, especially in the tumors. The Atf6 pathway was modestly activated only after tumor initiation. Consistent with the UPR activation, electron microscopy demonstrated ER expansion and reorganization in HCC cells in vivo. Strikingly, under ER stress or hypoxia, the Perk inhibitor and not the Ire1 inhibitor reduced cell viability and proliferation via escalating proteotoxic stress in vitro. Notably, the Perk inhibitor significantly decreased tumor burden in the mouse model., Conclusion: We provide the first evaluation of the UPR dynamics in a long-term cancer model and identified a small molecule inhibitor of Perk as a promising strategy for HCC therapy.

To investigate the effect of γ-terpineol on cell proliferation and apoptosis of human hepatoma BEL-7402 cells to elucidate its molecular mechanism. Here, BEL-7402 cells were treated with various concentrations (40, 80, 160, 320 and 640 μg/ml) of γ-terpineol for 48 h, cell proliferation was determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromides (MTT) assay. Cell colony inhibition was determined by soft agar assay. Apoptosis and possible molecular mechanisms were evaluated by morphological observation, flow cytometry analysis, and DNA fragmentation assay. The γ-terpineol significantly suppressed BEL-7402 cell proliferation in a dose-dependent manner. Characteristic morphological and biochemical changes associated with apoptosis such as cells shrinkage, deformation and vacuolization of mitochondria, nuclear chromatin condensation and fragmentation, formation of apoptotic bodies were observed after BEL-7402 cells treated with γ-terpineol for 24 h and 48 h. Cell cycle were displayed by flow cytometry analysis, the γ-terpineol treatment resulted in accumulation of cells at G₁ or S phase and a blockade of cell proliferation compared to control group. Treating BEL-7402 cells with 320 μg/ml of γ-terpineol for 36 h and 48 h, a typical apoptotic "DNA ladder" was observed using DNA fragmentation assay. The present study demonstrated that possible anti-cancer mechanism of γ-terpineol on human hematomas cells is through inducing cell apoptosis to suppress tumor cell growth.

Aim: To investigate the roles of Golgi protein (GP) 73 in the regulation of cell proliferation and apoptosis., Methods: Stealth RNAi targeting GP73 gene sequence was used to silence its expression in Hep G2 cells and Bel7402 cells. Stealth RNAi effects were assessed by reverse transcriptase polymerase chain reaction and ELISA. Cell proliferation assay and cell cycle analysis were assessed by MTT assay and flow cytometry. Apoptosis was assessed by flow cytometry and transmission electron microscopy. Apoptosis-related proteins were assessed by western immunoblot analysis., Results: Stealth RNAi targeting GP73 gene sequence markedly reduced the expression of GP73 gene. The reduction of GP73 in Hep G2 cells and Bel7402 cells inhibited cell proliferation and induced apoptosis, however, terminal apoptosis occurred in Hep G2 cells, but early apoptosis occurred in Bel7402 cells. Reduced expression of GP73 gene might lead to a reduction in Bcl-2/Bax ratio, an increase in cytochrome c, but a reduction in capase-3., Conclusion: GP73 might play an important role in proliferation and apoptosis in hepatocellular carcinoma cells.

We report a case of typical fibrolamellar hepatocellular carcinoma (FL-HCC) in a 16-year-old Japanese boy. This is very rare malignancy in Japan. The patient was referred for investigation of a large hepatic tumor and the results of tests for hepatitis B virus surface antigen and hepatitis C virus antibody were negative. Liver function test results and serum alpha-fetoprotein (AFP) levels were normal; however, the prothrombin induced by vitamin K absence/antagonist II was elevated. Computed tomography (CT) showed a large lobulated heterogeneously enhanced tumor in the posterior section of the liver. We diagnosed FL-HCC and performed posterior sectionectomy of the liver. The resected specimen contained a light brown and green tumor with a central fibrous scar, 10.0 cm in diameter. Microscopic and electron microscopic examinations revealed the typical features of FL-HCC. Immunohistochemically, the tumor cells were positive for cytokeratin (CK) 7, but negative for CK19 and AFP. The patient was alive without recurrence 48 months after surgery. Following this case report, we summarize the clinical features of the Japanese cases documented in the literature.

The biosynthetic nanoscale peptide Cecropin A is postulated to disrupt microbial phospholipid membranes by forming stable or transient pores. We demonstrated previously that green fluorescent protein (GFP), driven by the survivin promoter, was expressed highly in HepG2 but not in LO2 cells when they were transfected with the recombinant plasmid reporter vector (pSURV-GFP). To investigate the selective killing effect of this survivin promoter-driven peptide toxin gene system on hepatocellular carcinoma (HCC) cells in vitro, the recombinant plasmid pSURV-Cecropin A was constructed. HepG2 and LO2 cells were then transfected with the recombinant plasmid, which was driven by the survivin promoter, and the effects of Cecropin A were evaluated. Forty-eight hours after transfection with pSURV-Cecropin A, the growth of HepG2 cells was inhibited significantly. This finding was confirmed further by immunoblotting, which revealed consistently suppressed expression of proliferating cell nuclear antigen (PCNA) and cysteinyl aspartate specific proteinase-3 (caspase-3). Data demonstrated that the plasmid carrying the gene for the Cecropin A fusion protein was constructed successfully, and that its specific expression in HepG2 cells could provide the basis for targeted gene therapy in HCC. The identification of novel gene therapies for cancer is highly desirable to reduce drug toxicity and improve therapeutic outcomes..

MicroRNAs (miRNAs) are a large family of post‑transcriptional regulators of gene expression that control a number of developmental and cellular processes in eukaryotic organisms and are ~23 nucleotides in length. miRNA‑122 is an abundant liver‑specific miRNA, implicated in fatty acid and cholesterol metabolism, as well as in hepatitis C viral replication and is frequently suppressed in primary hepatocellular carcinomas. In the current study, the Hep3B cell line with stable overexpression of miR‑122 was successfully established through gene transfection methods and drug screening. miR‑122 was observed to alter cell morphology in vitro by stable overexpression in Hep3B cells. This alteration was viewed by light microscopy and transmission electron microscopy. These alterations included increases in the cell volume, the appearance of lipid granules and vacuoles, thickening of nuclear membrane, swelling of the mitochondria, cytoplasm vacuolization and a more prominent nucleolus. Furthermore, the study provided novel evidence that miR‑122 function was dependent upon its expression level. In addition, it was observed to negatively regulate mitochondria.

Compared with single nodular liver cancer, the prominent biological characteristics of multinodular liver cancer include rapid progression and short survival. Here, we developed a multinodular liver cancer model in mice and assessed the biological characteristics of the resulting neoplasms. H22 hepatoma cells at a dose of 2 × 10(5)/mouse, suspended in 1.6 mL, 0.8 mL, or 200 µL saline were injected via the tail vein of BALB/c mice at a velocity of 200 µL per second. The mice were sacrificed at different time points after injection. And at the time of death the liver, lungs, spleen, kidneys and heart were removed for morphological study. The biological characteristics of the tumor nodules were evaluated by immunohistochemistry. In the mice treated with a large volume injection of H22 cells, by day 7, there was a 100% occurrence of multinodular tumors in the livers, determined by histology. At the time of death, there were 100%, 100%, 37.5% and 37.5% occurrences of tumors in the lungs, kidneys, spleen and heart, respectively. The neoplastic cells in the liver nodules showed pleomorphism, and exhibited high expression of proliferating cell nuclear antigen (PCNA), c-myc, vascular endothelial growth factor (VEGF) and matrix metalloproteinase 2 (MMP-2). In mice treated with a small or medium volume injection, no tumor cells were identified in the livers, spleen, kidneys or heart at any of the examined time points. By day 7 and at the time of death, there was a 100% occurrence of tumor in the lungs. A multinodular liver cancer model in mice was achieved using a large volume injection of H22 cells.

Tea polyphenols have strong antioxidant and antitumor activities. However, these health benefits are limited due to their poor in vivo stability and low bioavailability. Chitosan nanoparticles as delivery systems may provide an alternative approach for enhancing bioavailability of poorly absorbed drugs. In this study, tea polyphenol-loaded chitosan nanoparticles have been prepared using two different chitosan biomaterials, and their antitumor effects were evaluated in HepG2 cells, including cell cytotoxicity comparison, cell morphology analysis, cell apoptosis and cell cycle detection. The results indicated that the tea polyphenol-loaded chitosan nanoparticles showed a branch shape and heterogeneous distribution in prepared suspension. MTT assay suggested that tea polyphenol-loaded chitosan nanoparticles could inhibit the proliferation of HepG2 cells, and the cytotoxicity rates were increased gradually and appeared an obvious dose-dependent relationship. Transmission electron microscope images showed that the HepG2 cells treated with tea polyphenol-loaded chitosan nanoparticles exhibited some typical apoptotic features, such as microvilli disappearance, margination of nuclear chromatin, intracytoplasmic vacuoles and the mitochondrial swelling. In addition, the tea polyphenol-loaded chitosan nanoparticles had relatively weak inhibitory effects on HepG2 cancer cells compared with tea polyphenols. Tea polyphenols not only induced cancer cell apoptosis, but also promoted their necrosis. However, tea polyphenol-loaded chitosan nanoparticles exhibited their antitumor effects mainly through inducing cell apoptosis. Our results revealed that the inhibition effects of tea polyphenol-loaded chitosan nanoparticles on tumor cells probably depended on their controlled drug release and effective cell delivery. The chitosan nanoparticles themselves as the delivery carrier showed limited antitumor effects compared with their encapsulated drugs., (Copyright © 2013. Published by Elsevier B.V.)

(-)-Epigallocatechin-3-O-gallate(EGCG), the highest catechins from green tea, has promisingly been found to sensitize the efficacy of several chemotherapy agents like doxorubicin (DOX) in hepatocellular carcinoma (HCC) treatment. However, the detailed mechanisms by which EGCG augments the chemotherapeutic efficacy remain unclear. Herein, this study was designed to determine the synergistic impacts of EGCG and DOX on hepatoma cells and particularly to reveal whether the autophagic flux is involved in this combination strategy for the HCC. Electron microscopy and fluorescent microscopy confirmed that DOX significantly increased autophagic vesicles in hepatoma Hep3B cells. Western blot and trypan blue assay showed that the increasing autophagy flux by DOX impaired about 45% of DOX-induced cell death in these cells. Conversely, both qRT-PCR and western blotting showed that EGCG played dose-dependently inhibitory role in autophagy signaling, and that markedly promoted cellular growth inhibition. Amazingly, the combined treatment caused a synergistic effect with 40 to 60% increment on cell death and about 45% augmentation on apoptosis versus monotherapy pattern. The DOX-induced autophagy was abolished by this combination therapy. Rapamycin, an autophagic agonist, substantially impaired the anticancer effect of either DOX or combination with EGCG treatment. On the other hand, using small interference RNA targeting chloroquine autophagy-related gene Atg5 and beclin1 to inhibit autophagy signal, hepatoma cell death was dramatically enhanced. Furthermore, in the established subcutaneous Hep3B cells xenograft tumor model, about 25% reduction in tumor growth as well as 50% increment of apoptotic cells were found in combination therapy compared with DOX alone. In addition, immunohistochemistry analysis indicated that the suppressed tendency of autophagic hallmark microtubule-associated protein light chain 3 (LC3) expressions was consistent with thus combined usage in vitro. Taken together, the current study suggested that EGCG emerges as a chemotherapeutic augmenter and synergistically enhances DOX anticancer effects involving autophagy inhibition in HCC.

Tumor hypoxia underlies treatment failure and yields more aggressive and metastatic cancer phenotypes. Although therapeutically targeting these hypoxic environments has been proposed for many years, to date no approaches have shown the therapeutic value to gain regulatory approval. Here, we demonstrated that a novel hypoxia-activated prodrug, Q6, exhibits potent antiproliferative efficacy under hypoxic conditions and induces caspase-dependent apoptosis in 2 hepatocellular carcinoma (HCC) cell lines, with no obvious toxicity being detected in 2 normal liver cell lines. Treatment with Q6 markedly downregulated HIF1A [hypoxia inducible factor 1, α subunit (basic helix-loop-helix transcription factor)] expression and transcription of the downstream target gene, VEGFA (vascular endothelial growth factor A). This dual hypoxia-targeted modulation mechanism leads to high potency in suppressing tumor growth and vascularization in 2 in vivo models. Intriguingly, it is the autophagy-dependent degradation pathway that plays a crucial role in Q6-induced attenuation of HIF1A expression, rather than the proteasome-dependent pathway, which is normally regarded as the predominant mechanism underlying posttranslational regulation of HIF1A. Inhibition of autophagy, either by short interfering RNA (siRNA) or by chemical inhibitors, blocked Q6-induced HIF1A degradation. Autophagic degradation of HIF1A was further confirmed by the observation that HIF1A coimmunoprecipitated with the ubiquitin-binding adaptor protein, SQSTM1, which is degraded through autophagy. Additionally, silencing of SQSTM1 inhibited Q6-induced HIF1A degradation. These findings suggest that the novel hypoxia-targeted agent, Q6, has potential clinical value in the therapy of HCC. Furthermore, the identification of autophagy as a crucial regulator of HIF1A provides new insights into hypoxia-related treatments.

Aim: Rosin, the traditional Chinese medicine, is reported to be able to inhibit skin cancer cell lines. In this report, we investigate the inhibitory effect against HCC cells of QC2, the derivative of rosin's main components dehydroabietic acid., Methods: MTT assay was used to determine the cytotoxicity of QC2. Morphological changes were observed by time-lapse microscopy and transmission electron microscopy and the cytoskeleton changes were observed by laser-scanning confocal microscopy. Cytomembrane integrity and organelles damage were confirmed by detection of the reactive oxygen (ROS), lactate dehydrogenase (LDH), and mitochondrial membrane potential (Δψm). The underlying mechanism was manifested by Western blotting. The oncotic cell death was further confirmed by detection of oncosis related protein calpain., Results: Swelling cell type and destroyed cytoskeleton were observed in QC2-treated HCC cells. Organelle damage was visualized by transmission electron microscopy. The detection of ROS accumulation, increased LDH release, and decreased ATP and Δψm confirmed the cell death. The oncotic related protein calpain was found to increase time-dependently in QC2-treated HCC cells, while its inhibitor PD150606 attenuated the cytotoxicity., Conclusions: Dehydroabietic acid derivative QC2 activated oncosis related protein calpain to induce the damage of cytomembrane and organelles which finally lead to oncosis in HCC cells.

Background: Tumor-derived exosomes have been viewed as a source of tumor antigens that can be used to induce anti-tumor immune responses. In the current study, we aim to investigate the regulatory effect of the epigenetic drug MS-275 on hepatoma G2 (HepG2) cell-derived exosomes, especially for their immunostimulatory properties and alteration of some non-specific immune protein expression, such as heat shock protein (HSP) 70, major histocompatibility complex (MHC) class I polypeptide-related sequence A (MICA) and MICB., Methods: MS-275 was used to modulate the secretion of exosomes in human HepG2 cells, and exosomes from untreated HepG2 cells served as negative controls. RT-PCR was used to test the expression of HSP70, MICA and MICB in HepG2 cells. Immunogold labeling of exosomes and western blotting analysis were carried out to compare the expression of HSP70, MICA and MICB proteins in exosomes with or without MS-275 treatment. A natural killer (NK) cell cytotoxicity assay and peripheral blood mononuclear cell (PBMC) proliferation assay were used to evaluate the effect of MS-275 on the immunostimulatory ability of exosomes., Results: Immunogold labeling and western blot analysis showed that modification of MS-275 increased the expression of HSP70 and MICB in exosomes. RT-PCR showed the mRNA levels of HSP70 and MICB were upregulated in HepG2 cells and were consistent with their protein levels in exosomes. The exosomes modified by MS-275 could significantly increase the cytotoxicity of NK cells and proliferation of PBMC (P < 0.05)., Conclusions: The non-specific immune response of exosomes derived from HepG2 cells could be enhanced with treatment by the histone deacetylase inhibitor (HDACi) drug MS-275; this could provide a potential tumor vaccine strategy against liver cancer.

Background: Autophagy is an important adaptive survival mechanism, which has been postulated to be involved in cancer metastasis. The purpose of this study was to investigate autophagy in metastasis of hepatocellular carcinoma (HCC)., Methods: Immunohistochemical analysis of autophagic activity in metastatic and paired primary HCC tissues using LC3 as autophagosome marker was performed in samples from 216 HCC patients diagnosed with metastasis (including 158 intravascular, 42 intrabiliary, 8 lymph node, 4 bone and 4 lung metastases). Then a mouse model of pulmonary metastasis was established using a highly metastatic HCC cell line (HCCLM3). Autophagy in pulmonary metastases and paired primary tumors were analyzed by LC3 immunohistochemistry, transmission electron microscopy (TEM) and western blot analysis. Further, mouse model of pulmonary metastasis and in vitro cell migration, invasion and detachment models were established using a stable GFP-LC3-expressing HCCLM3 cell line (HCCLM3-GFP-LC3). Autophagic alterations during metastatic colonization, migration, invasion and detachment were determined by GFP-LC3 analysis and western blot analysis., Results: LC3 immunohistochemistry of metastases and primary tumors from HCC patients revealed significantly higher LC3 expression in metastases than primary HCC, which suggested a higher level of autophagy in HCC metastases. Further immunohistochemical, TEM, western blot and in vivo GFP-LC3 analyses of lung metastases and primary tumors in mouse model of pulmonary metastasis confirmed that metastatic colonies displayed higher level of autophagy than primary tumors and the early metastatic colonies displayed highest level. The dynamic monitoring of autophagy in cell migration, invasion and detachment showed that autophagy did not significantly alter in those processes., Conclusions: Autophagy is activated in metastatic colonization but not in invasion, migration and detachment of HCC cells. Autophagy may play a role in HCC metastasis via promoting metastatic colonization of HCC cells.