Dalong Yin

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)

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.

EF24 inhibits tumor growth and metastasis via suppressing NF-kappaB dependent pathways in human cholangiocarcinoma

A synthetic monoketone analog of curcumin, termed 3, 5-bis (2-flurobenzylidene) piperidin-4-one (EF24), has been reported to inhibit the growth of a variety of cancer cells both in vitro and in vivo. However, whether EF24 has anticancer effects on cholangiocarcinoma (CCA) cells and the mechanisms remain to be investigated. The aim of our study was to evaluate the molecular mechanisms underlying the anticancer effects of EF24 on CCA tumor growth and metastasis. Cell proliferation, apoptosis, migration, invasion, tumorigenesis and metastasis were examined. EF24 exhibited time- and dose-dependent inhibitory effects on HuCCT-1, TFK-1 and HuH28 human CCA cell lines. EF24 inhibited CCA cell proliferation, migration, and induced G2/M phase arrest. EF24 induced cell apoptosis along with negative regulation of NF-κB- X-linked inhibitor of apoptosis protein (XIAP) signaling pathway. XIAP inhibition by lentivirus mediated RNA interference enhanced EF24-induced apoptosis, while XIAP overexpression reduced it in CCA cells. In vivo, EF24 significantly suppressed the growth of CCA tumor xenografts and tumor metastasis while displaying low toxicity levels. Our findings indicate that EF24 is a potent antitumor agent that inhibits tumor growth and metastasis by inhibiting NF-κB dependent signaling pathways. EF24 may represent a novel approach for CCA treatment.

A preliminary study of ALPPS procedure in a rat model

Associating Liver Partition and Portal vein ligation for Staged hepatectomy (ALPPS) has been reported to be a novel surgical technique that provides fast and effective growth of liver remnant. Despite occasional reports on animal studies, the mechanisms of rapid liver regeneration in ALPPS remains unclear. In the present study, we intend to develop a reproducible rat model to mimick ALPPS and to explore the underlying mechanisms. Rats assigned to the portal vein ligation (PVL), left lateral lobe (LLL) resection, transection and sham groups served as controls. Results indicated that the regeneration rate in the remnant liver after ALPPS was two times relative to PVL, whereas rats with transection alone showed minimal volume increase. The expression levels of Ki-67 and PCNA were about ten-fold higher after ALPPS compared with the transection and LLL resection groups, and four-fold higher compared with the PVL group. The levels of TNF-α, IL-6 and HGF in the regenerating liver remnant were about three-fold higher after ALPPS than the controls. There was a more significant activation of NF-κB p65, STAT3 and Yap after ALPPS, suggesting synergistic activation of the pathways by PVL and transection, which might play an important role in liver regeneration after ALPPS.

Expression and prognostic value of ING3 in human primary hepatocellular carcinoma.

The tumor-suppressor ING3 has been shown to be involved in tumor transcriptional regulation, apoptosis and the cell cycle. Some studies have demonstrated that ING3 is dysregulated in several types of cancers. However, the expression and function of ING3 in human hepatocellular carcinoma (HCC) remains unclear. The aim of this study is to investigate ING3 expression in hepatic tumors and its clinical relevance in hepatic cancer. The expression of ING3 protein was examined in 120 dissected HCC tissues and 47 liver tissues adjacent to the tumor by immunohistochemical assays and confirmed by Western blot analysis in 20 paired frozen tumor and non-tumor liver tissues. The relationship between ING3 staining and clinico-pathological characteristics of HCC was further analyzed. The mRNA expression of ING3 in the dissected tissues was also analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and realtime PCR. Both mRNA and protein concentrations of ING3 were found to be downregulated in the majority of HCC tumors in comparison with matched non-tumor hepatic tissues. Analysis of the relationship between ING3 staining and clinico-pathological characteristics of HCC showed that the low expression of ING3 protein is correlated with more aggressive behavior of the tumor. Kaplan–Meier curves demonstrated that patients with a low expression of ING3 have a significantly increased risk of shortened survival time. In addition, multivariate analysis suggested that the level of ING3 expression may be an independent prognostic factor. Our findings indicate that ING3 may be an important marker for human hepatocellular carcinoma progression and prognosis, as well as a potential therapeutic target.