Yingjian Liang

Hypoxia-mediated sorafenib resistance can be overcome by EF24 through Von Hippel-Lindau tumor suppressor-dependent HIF-1α inhibition in hepatocellular carcinoma†‡§

The increasing incidence of hepatocellular carcinoma (HCC) is of great concern not only in the United States but throughout the world. Although sorafenib, a multikinase inhibitor with antiangiogenic and antiproliferative effects, currently sets the new standard for advanced HCC, tumor response rates are usually quite low. An understanding of the underlying mechanisms for sorafenib resistance is critical if outcomes are to be improved. In this study we tested the hypothesis that hypoxia caused by the antiangiogenic effects of sustained sorafenib therapy could induce sorafenib resistance as a cytoprotective adaptive response, thereby limiting sorafenib efficiency. We found that HCCs, clinically resistant to sorafenib, exhibit increased intratumor hypoxia compared with HCCs before treatment or HCCs sensitive to sorafenib. Hypoxia protected HCC cells against sorafenib and hypoxia‐inducible factor 1 (HIF‐1α) was required for the process. HCC cells acquired increased P‐gp expression, enhanced glycolytic metabolism, and increased nuclear factor kappa B (NF‐κB) activity under hypoxia. EF24, a molecule having structural similarity to curcumin, could synergistically enhance the antitumor effects of sorafenib and overcome sorafenib resistance through inhibiting HIF‐1α by sequestering it in cytoplasm and promoting degradation by way of up‐regulating Von Hippel‐Lindau tumor suppressor (VHL). Furthermore, we found that sustained sorafenib therapy led to increased intratumor hypoxia, which was associated with sorafenib sensitivity in HCC subcutaneous mice tumor models. The combination of EF24 and sorafenib showed synergistically effects against metastasis both in vivo and in vitro. Synergistic tumor growth inhibition effects were also observed in subcutaneous and orthotopic hepatic tumors. Conclusion: Hypoxia induced by sustained sorafenib treatment confers sorafenib resistance to HCC through HIF‐1α and NF‐κB activation. EF24 overcomes sorafenib resistance through VHL‐dependent HIF‐1α degradation and NF‐κB inactivation. EF24 in combination with sorafenib represents a promising strategy for HCC. (HEPATOLOGY 2013)

PTEN- and p53-Mediated Apoptosis and Cell Cycle Arrest by FTY720 in Gastric Cancer Cells and Nude Mice

FTY720, a new immunosuppressant, derived from ISP‐1, has been studied for its putative anti‐cancer properties in the recent years. In this study, we have reported that FTY720 greatly inhibited gastric cancer cell proliferation for the first time, and found this effect was associated with G1 phase cell cycle arrest and apoptosis. Results from our Western blotting and Real‐time PCR showed that FTY720 induced obvious PTEN expression in a p53‐independent way, consistent with a substantial decrease in p‐Akt and MDM2. FTY720 dramatically increased the expression of Cip1/p21, p27, and BH3‐only proteins through the accumulation of p53 by PTEN‐mediated inhibition of the PI3K/Akt/MDM2 signaling. Suppression of PTEN expression with siRNA significantly reduced the p53 and p21 levels and activated Akt, resulting in decreased apoptosis and increased cell survival. Furthermore, we have observed an additive effect of FTY720 in killing gastric cancer cells when in combination with Cisplatin, partly through PTEN‐mediated Akt/MDM2 inhibition. In vivo study has also shown that tumor growth was significantly suppressed after FTY720 treatment. In conclusion, our results suggest that FTY720 induces a significant increase of PTEN, which inhibits p‐Akt and MDM2, and then increases the level of p53, thereby inducing G1 phase arrest and apoptosis. We have characterized a novel immunosuppressant, for the first time, which shows potential anti‐tumor effects on gastric cancer by PTEN activation through p53‐independent mechanism, especially in combination with Cisplatin. This PTEN target‐based therapy is worth further investigation and warrants clinical evaluation. J. Cell. Biochem. 111: 218–228, 2010.

Gankyrin promotes tumor growth and metastasis through activation of IL‐6/STAT3 signaling in human cholangiocarcinoma

Although gankyrin is involved in the tumorigenicity and metastasis of some malignancies, the role of gankyrin in cholangiocarcinoma (CCA) is unclear. In this study we investigated the expression of gankyrin in human CCA tissues and cell lines. The effects of gankyrin on CCA tumor growth and metastasis were determined both in vivo and in vitro. The results showed that gankyrin was overexpressed in CCA tissues and cell lines. Gankyrin expression was associated with CCA histological differentiation, TNM stage, and metastasis. The multivariate Cox analysis revealed that gankyrin was an independent prognostic indicator for overall survival. Gankyrin overexpression promoted CCA cell proliferation, migration, and invasion, while gankyrin knockdown inhibited CCA tumor growth, metastasis, and induced Rb‐dependent senescence and G1 phase cell cycle arrest. Gankyrin increased the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and promoted the nuclear translocation of p‐STAT3. Suppression of STAT3 signaling by small interfering RNA (siRNA) or STAT3 inhibitor interfered with gankyrin‐mediated carcinogenesis and metastasis, while interleukin (IL)‐6, a known upstream activator of STAT3, could restore the proliferation and migration of gankyrin‐silenced CCA cells. The IL‐6 level was decreased by gankyrin knockdown, while increased by gankyrin overexpression. Gankyrin regulated IL‐6 expression by way of facilitating the phosphorylation of Rb; meanwhile, rIL‐6 treatment increased the expression of gankyrin, suggesting that IL‐6 was regulated by a positive feedback loop involving gankyrin in CCA. In the xenograft experiments, gankyrin overexpression accelerated tumor formation and increased tumor weight, whereas gankyrin knockdown showed the opposite effects. The in vivo spontaneous metastasis assay revealed that gankyrin promoted CCA metastasis through IL‐6/STAT3 signaling pathway. Conclusion: Gankyrin is crucial for CCA carcinogenesis and metastasis by activating IL‐6/STAT3 signaling pathway through down‐regulating Rb protein. (Hepatology 2014;59:935–946)

Reciprocal activation between ATPase inhibitory factor 1 and NF‐κB drives hepatocellular carcinoma angiogenesis and metastasis

Hepatocellular carcinoma (HCC) is a highly vascularized tumor with frequent extrahepatic metastasis. Active angiogenesis and metastasis are responsible for rapid recurrence and poor survival of HCC. However, the mechanisms that contribute to tumor metastasis remain unclear. Here we evaluate the effects of ATPase inhibitory factor 1 (IF1), an inhibitor of the mitochondrial H(+)‐adenosine triphosphate (ATP) synthase, on HCC angiogenesis and metastasis. We found that increased expression of IF1 in human HCC predicts poor survival and disease recurrence after surgery. Patients with HCC who have large tumors, with vascular invasion and metastasis, expressed high levels of IF1. Invasive tumors overexpressing IF1 were featured by active epithelial‐mesenchymal transition (EMT) and increased angiogenesis, whereas silencing IF1 expression attenuated EMT and invasion of HCC cells. Mechanistically, IF1 promoted Snai1 and vascular endothelial growth factor (VEGF) expression by way of activating nuclear factor kappa B (NF‐κB) signaling, which depended on the binding of tumor necrosis factor (TNF) receptor‐associated factor 1 (TRAF1) to NF‐κB‐inducing kinase (NIK) and the disruption of NIK association with the TRAF2‐cIAP2 complex. Suppression of the NF‐κB pathway interfered with IF1‐mediated EMT and invasion. Chromatin immunoprecipitation assay showed that NF‐κB can bind to the Snai1 promoter and trigger its transcription. IF1 was directly transcribed by NF‐κB, thus forming a positive feedback signaling loop. There was a significant correlation between IF1 expression and pp65 levels in a cohort of HCC biopsies, and the combination of these two parameters was a more powerful predictor of poor prognosis. Conclusion: IF1 promotes HCC angiogenesis and metastasis by up‐regulation of Snai1 and VEGF transcription, thereby providing new insight into HCC progression and IF1 function. (Hepatology 2014;60:1659–1673)

PTEN antagonises Tcl1/hnRNPK-mediated G6PD pre-mRNA splicing which contributes to hepatocarcinogenesis

Background Mounting epidemiological evidence supports a role for phosphatase and tensin homologue (PTEN)-T cell leukaemia 1 (Tcl1) signalling deregulation in hepatocarcinogenesis. Objective To determine the molecular and biochemical mechanisms by which the PTEN/Tcl1 axis regulates the pentose phosphate pathway (PPP) in hepatocellular carcinoma (HCC). Methods We compared levels of PTEN and glucose-6-phosphate dehydrogenase (G6PD) mRNA in human HCC and healthy liver tissue. We measured PPP flux, glucose consumption, lactate production, nicotinamide adenine dinucleotide phosphate (NADPH) levels and lipid accumulation. We investigated the PTEN/Tcl1 axis using molecular biology, biochemistry and mass spectrometry analysis. We assessed proliferation, apoptosis and senescence in cultured cells, and tumour formation in mice. Results We showed that PTEN inhibited the PPP pathway in human liver tumours. Through the PPP, PTEN suppressed glucose consumption and biosynthesis. Mechanistically, the PTEN protein bound to G6PD, the first and rate-limiting enzyme of the PPP and prevented the formation of the active G6PD dimer. Tcl1, a coactivator for Akt, reversed the effects of PTEN on biosynthesis. Tcl1 promoted G6PD activity and also increased G6PD pre-mRNA splicing and protein expression in a heterogeneous nuclear ribonucleoprotein (hnRNPK)-dependent manner. PTEN also formed a complex with hnRNPK, which inhibited G6PD pre-mRNA splicing. Moreover, PTEN inactivated Tcl1 via glycogen synthase kinase-3β (GSK3β)-mediated phosphorylation. Importantly, Tcl1 knockdown enhanced the sensitivity of HCC to sorafenib, whereas G6PD knockdown inhibited hepatocarcinogenesis. Conclusions These results establish the counteraction between PTEN and Tcl1 as a key mechanism that regulates the PPP and suggest that targeting the PTEN/Tcl1/hnRNPK/G6PD axis could open up possibilities for therapeutic intervention and improve the prognosis of patients with HCC.

FTY720 inhibits proliferation and epithelial-mesenchymal transition in cholangiocarcinoma by inactivating STAT3 signaling

BackgroundInterleukin 6 (IL-6)-mediated signal transducers and activators of transcription 3 (STAT-3) phosphorylation (activation) is aberrantly sustained in cholangiocarcinoma cells resulting in enhanced myeloid cell leukemia 1 (Mcl-1) expression and resistance to apoptosis. FTY720, a new immunosuppressant, derived from ISP-1, has been studied for its putative anti-cancer properties. This study aimed to elucidate the mechanism by which FTY720 mediates antitumor effects in cholangiocarcinoma (CC) cells.MethodsThree CC cell lines were examined, QBC939, TFK-1, and HuCCT1. The therapeutic effects of FTY720 were evaluated in vitro and in vivo. Cell proliferation, apoptosis, cell cycle, invasive potential, and epithelial- mesenchy-mal transition (EMT) were examined.ResultsFTY720 greatly inhibited CC cells proliferation and EMT in vitro and in vivo, and this effect was associated with dephosphorylation of STAT3tyr705. FTY720 induced apoptosis and G1 phase arrest in CC cells, and inhibited invasion of CC cells. Western blot analysis showed that FTY720 induced cleavage of caspases 3, 8 and 9, and of PARP, in a dose-dependent manner, consistent with a substantial decrease in p-STAT3, Bcl-xL, Bcl-2, survivin, cyclin D1, cyclin E, N-cadherin, vimentin, VEGF and TWIST1. In vivo studies showed that tumor growth and metastasis were significantly suppressed after FTY720 treatment.ConclusionsThese results suggest that FTY720 induces a significant decrease in p-STAT3, which inhibits proliferation and EMT of CC cells, and then induces G1 phase arrest and apoptosis. We have characterized a novel immunosuppressant, which shows potential anti-tumor effects on CC via p-STAT3 inhibition. FTY720 merits further investigation and warrants clinical evaluation.

Nutlin-3 overcomes arsenic trioxide resistance and tumor metastasis mediated by mutant p53 in Hepatocellular Carcinoma

BackgroundArsenic trioxide has been demonstrated as an effective anti-cancer drug against leukemia and solid tumors both in vitro and in vivo. However, recent phase II trials demonstrated that single agent arsenic trioxide was poorly effective against hepatocellular carcinoma (HCC), which might be due to drug resistance.MethodsMutation detection of p53 gene in arsenic trioxide resistant HCC cell lines was performed. The therapeutic effects of arsenic trioxide and Nutlin-3 on HCC were evaluated both in vitro and in vivo. A series of experiments including MTT, apoptosis assays, co-Immunoprecipitation, siRNA transfection, lentiviral infection, cell migration, invasion, and epithelial-mesenchy-mal transition (EMT) assays were performed to investigate the underlying mechanisms.ResultsThe acquisition of p53 mutation contributed to arsenic trioxide resistance and enhanced metastatic potential of HCC cells. Mutant p53 (Mutp53) silence could re-sensitize HCC resistant cells to arsenic trioxide and inhibit the metastatic activities, while mutp53 overexpression showed the opposite effects. Neither arsenic trioxide nor Nutlin-3 could exhibit obvious effects against arsenic trioxide resistant HCC cells, while combination of them showed significant effects. Nutlin-3 can not only increase the intracellular arsenicals through inhibition of p-gp but also promote the p73 activation and mutp53 degradation mediated by arsenic trioxide. In vivo experiments indicated that Nutlin-3 can potentiate the antitumor activities of arsenic trioxide in an orthotopic hepatic tumor model and inhibit the metastasis to lung.ConclusionsAcquisitions of p53 mutations contributed to the resistance of HCC to arsenic trioxide. Nutlin-3 could overcome arsenic trioxide resistance and inhibit tumor metastasis through p73 activation and promoting mutant p53 degradation mediated by arsenic trioxide.

Liver abscesses in adult patients with and without diabetes mellitus: an analysis of the clinical characteristics, features of the causative pathogens, outcomes and predictors of fatality: a report based on a large population, retrospective study in China

In China, there are four types of liver abscesses (LAs) that meet the clinical criteria. Pyogenic liver abscesses (PLAs) and amoebic liver abscesses (ALAs) are two of the most common types of abscesses, followed by fungal liver abscesses (FLAs) and hydatid secondary liver abscesses (HsLAs). Diabetes mellitus (DM) is associated with the development of PLAs. However, there is a lack of population-based studies that have evaluated the underlying relationship between LAs (mainly PLAs and FLAs) and DM. We conducted a retrospective study based on a large population to identify the potential differences and factors that affect the mortality of PLA patients in DM and non-DM groups. Our results revealed that the prevalence of DM is 44.3% (158/357) in PLA patients and 35.3% (18/51) in FLA patients. Compared with the non-DM patients, statistically significant differences were found in DM patients according to symptomatology, clinical manifestations, laboratory findings, microbiological characteristics, antimicrobial resistance, clinical treatments and outcomes in relation to mortality. In addition, the status of antibiotic resistance to E. coli and K. pneumoniae, which were isolated from the patient samples, is severe in the area in which the study was conducted. Regarding the treatment of PLAs, our study indicated that broad-spectrum antimicrobial therapy and drug combinations should be recommended and initiated before the pathogens are cultured and identified. In the clinic, therapies that combine percutaneous drainage with antibiotics and surgery with antibiotics are the two most useful strategies for treating an LA. These two combined treatments resulted in satisfactory cure rates. In the DM and non-DM groups, the cure rates for percutaneous drainage with antibiotics were 90.3% and 92.0%, respectively, and the cure rates for surgery with antibiotics were 93.9% and 95.2%, respectively.

In Vivo and In Vitro Suppression of Hepatocellular Carcinoma by EF24, a Curcumin Analog

The synthetic compound 3,5-bis(2-flurobenzylidene)piperidin-4-one (EF24) is a potent analog of curcumin that exhibits enhanced biological activity and bioavailability without increasing toxicity. EF24 exerts antitumor activity by arresting the cell cycle and inducing apoptosis, suppressing many types of cancer cells in vitro. The antiproliferative and antiangiogenic properties of EF24 provide theoretical support for its development and application to liver cancers. We investigated the in vitro and in vivo activities of EF24 on liver cancer to better understand its therapeutic effects and mechanisms. EF24 induced significant apoptosis and G2/M-phase cell cycle arrest in mouse liver cancer cell lines, Hepa1-6 and H22. The expression levels of G2/M cell cycle regulating factors, cyclin B1 and Cdc2, were significantly decreased, pp53, p53, and p21 were significantly increased in EF24-treated cells. In addition, EF24 treatment significantly reduced Bcl-2 concomitant with an increase in Bax, enhanced the release of cytochrome c from the mitochondria into the cytosol, resulting in an upregulation of cleaved-caspase-3, which promoted poly (ADP-ribose) polymerase cleavage. EF24-treated cells also displayed decreases in phosphorylated Akt, phosphorylated extracellular signal-regulated kinase and vascular endothelial growth factor. Our in vitro protein expression data were confirmed in vivo using a subcutaneous hepatocellular carcinoma (HCC) tumor model. This mouse HCC model confirmed that total body weight was unchanged following EF24 treatment, although tumor weight was significantly decreased. Using an orthotopic HCC model, EF24 significantly reduced the liver/body weight ratio and relative tumor areas compared to the control group. In situ detection of apoptotic cells and quantification of Ki-67, a biomarker of cell proliferation, all indicated significant tumor suppression with EF24 treatment. These results suggest that EF24 exhibits anti-tumor activity on liver cancer cells via mitochondria-dependent apoptosis and inducing cell cycle arrest coupled with antiangiogenesis. The demonstrated activities of EF24 support its further evaluation as a treatment for human liver cancers.

FCN2 inhibits epithelial–mesenchymal transition-induced metastasis of hepatocellular carcinoma via TGF-β/Smad signaling

Hepatocellular carcinoma (HCC) is currently still a major cause of cancer-related deaths. Identifying early metastatic biomarkers and therapeutic targets for HCC is of great importance. Emerging evidence suggest that epithelial-mesenchymal transitions (EMTs) play important roles in tumor metastasis and recurrence. Understanding molecular mechanisms that regulate the EMT process is crucial for improving HCC. In this study, we find Ficolin-2 (FCN2) plays an essential role in metastasis and EMT of HCC. FCN2 expression is downregulated in HCC cells and tissues. Low level of FCN2 in HCCs is correlated with aggressive metastatic features, and would be a prognostic factor for overall disease-free survival of HCC patients. Ectopic expression of FCN2 markedly inhibits HCC cells migration, invasion as well as EMT in vitro and in vivo. Moreover, TGF-β is found contribute to the function of FCN2 in suppressing metastasis and EMT of HCC. Collectively, our data suggest that FCN2 may have prognostic value in HCC metastasis. Additionally, the FCN2/ TGF-β/EMT axis identified in this study provides novel insight into the mechanisms of HCC metastasis, which may facilitate the development of new therapeutics against HCC.