Haiyong Wang

Bufalin suppresses hepatocellular carcinoma invasion and metastasis by targeting HIF-1α via the PI3K/AKT/mTOR pathway.

It has been reported that there are multiple mechanisms by which bufalin could exert its antimetastatic effect. HIF-1α has been reported to be involved in tumor migration and invasion by regulating EMT. However, it is not known whether bufalin could exert the antimetastatic effect by modulating HIF-1α expression in hepatocellular carcinoma. In the present study, we aimed to evaluate the antimetastatic potential of bufalin in vivo and in vitro. Our results demonstrated that the liver/lung metastases were significantly reduced in bufalin-treated mice, as tested in the orthotopic transplanted and tail vein injection tumor models. Furthermore, the epithelial-to-mesenchymal transition (EMT) was inhibited in bufalin-treated tumors, as reflected the upregulation of E-cadherin, and downregulation of N-cadherin, vimentin, Snail. Similar results were observed in SMMC7721 cells treated with bufalin. Moreover, the transforming growth factor-β1 (TGF-β1)-induced EMT was also abrogated by bufalin. Mechanistically, our study demonstrated that hypoxia-inducible factor-1α (HIF-1α) played an important role in the antimetastatic effect of bufalin in hepatocellular carcinoma. Importantly, HIF-1α expression may be regulated through the inhibition of the PI3K/AKT/mTOR pathway. Taken together, our results suggest that bufalin suppresses hepatic tumor invasion and metastasis and that this process may be related to the PI3K/AKT/mTOR/ HIF-1α axis.

Prognostic nutritional index serves as a predictive marker of survival and associates with systemic inflammatory response in metastatic intrahepatic cholangiocarcinoma

Objective The significance of the prognostic nutritional index (PNI) has been widely reported and confirmed in many types of cancers. However, few studies are available indicating its prognostic power in patients with intrahepatic cholangiocarcinoma (ICC). Thus, we investigated its relationship with overall survival (OS) to evaluate its role in predicting survival in patients with ICC. Patients and methods Between October 2011 and October 2015, 173 consecutive patients with pathologically confirmed locally advanced or metastatic ICC were enrolled. First, the correlations between PNI and clinical factors were analyzed among these patients. Next, univariate and multivariate analyses were conducted to evaluate the association between PNI and OS among these patients with ICC. In addition, the relationships between PNI and three typical systemic inflammatory response (SIR) markers – the neutrophil/lymphocyte ratio (NLR), the platelet/lymphocyte ratio (PLR), and the lymphocyte/monocyte ratio (LMR) – were also assessed. Results A lower PNI was linked with a shorter OS in patients with ICC, as reflected obviously in the Kaplan–Meier analyses. The patients with ICC were divided into the locally advanced group and the metastatic group. Further analyses revealed that PNI is not associated with OS in the locally advanced group. However, in the subgroup of patients with metastatic ICC, a lower PNI significantly correlated with a worsened OS. The OS for patients with a low PNI is 5 months, whereas the OS is 10.17 months for patients with a high PNI. Multivariate analyses revealed that PNI is independently correlated with OS. We finally proved that PNI is negatively proportional to NLR and PLR and positively proportional to LMR. Conclusion Our results demonstrate that decreased PNI signifies a poor OS and is associated with SIR in patients with metastatic ICC. Therefore, it may serve as a valuable predictive marker in patients with metastatic ICC.

Serum liver enzymes serve as prognostic factors in patients with intrahepatic cholangiocarcinoma

Objective Liver functions, reflective of the overall status of the host, have been reported to be important factors affecting the prognosis in many types of cancers. In this study, we explored the influences of liver enzymes albumin (ALB), globulin (GELO), total protein (TP), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), direct bilirubin (DBIL), gamma glutamyltranspeptidase (GGT), and lactate dehydrogenase (LDH) on the overall survival (OS) in a number of 173 patients with intrahepatic cholangiocarcinoma (ICC). Patients and methods Between 2011 and 2015, we enrolled patients with pathologically proven locally advanced or metastatic ICC. The impact of ALB, GELO, TP, ALP, ALT, AST, TBIL, DBIL, GGT, and LDH on OS were analyzed using Kaplan–Meier analysis. Next, the associations between these liver enzymes and OS were evaluated by univariate and multivariate analyses. Finally, the role of these enzymes in OS was evaluated in the subgroups. Results Elevated liver enzymes were linked with OS. We revealed that independent prognostic factors of poor outcome were ALP, TBIL, DBIL, and GGT, whereas ALB is a protective factor in ICC patients. Conclusion Our results demonstrate that these liver enzymes may serve as valuable predictive markers in ICC patients.

Bufalin enhances anti-angiogenic effect of sorafenib via AKT/VEGF signaling.

Sorafenib mainly exerts its anti-hepatoma effect by inhibiting tumor angiogenesis. However, its curative effect is limited. Thus, application of drugs which could augment its anti-angiogenic effect is necessary. Bufalin has been reported to possess anticancer properties. In the present study, we investigated the synergistic anti-angiogenic effect of sorafenib combined with bufalin. The enhanced anti-angiogenic effect of the combination treatment was firstly assessed in nude mice bearing human HCC intradermal tumors. In addition, we found that proliferation was significantly inhibited and the morphology was obviously changed in the combination-treated human umbilical vein endothelial cells (HUVEC) at 48 h of treatment. In addition, the combination treatment was found to suppress vessel formation potently as proved in the tube formation, chick chorioallantoic membrane and rat aortic rings. Mechanistically, HUVEC incubated with the combination treatment showed increased apoptosis, decreased migration, which might account for its capacity against angiogenesis. Vascular endothelial cells have been reported to secrete cytokines to affect angiogenesis. Therefore, suspensions from HUVECs with different treatments were collected as conditioned medium (CM). The combination-treated CM significantly inhibited the migration of HUVEC and blood vessel formation in vitro. Importantly, multiple cytokines associated with angiogenesis were downregulated in the combination-treated CM. Furthermore, we verified that the secretion of VEGF was downregulated and revealed that the reduction might be regulated through the inhibition of the PI3K/AKT pathway. Taken together, our findings demonstrated for the first time that bufalin can enhance anti-angiogenic effect of sorafenib via modulating the AKT/VEGF signaling pathway.

Carbonic anhydrase 2 inhibits epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma

Carbonic anhydrase 2 (CA2) plays vital role in the regulation of ion transport and pH balance and is involved in many biological processes; however, its role in cancer remains obscure. In this study, we identified a novel function of CA2 in facilitating hepatocellular carcinoma (HCC) metastasis. CA2 expression was elevated in Na+-K+-ATPase α1 (ATP1A1)-downregulated HCC cells and was inversely correlated with that of ATP1A1 in HCC. ATP1A1 acted as an oncoprotein whereas CA2 overexpression inhibited cell migration and invasion by reversing epithelial-mesenchymal transition (EMT) in HCC. CA2 downregulation promoted HCC metastasis and invasion whereas ATP1A1 downregulation inhibited HCC metastasis. Because of the opposing effects of CA2 and ATP1A1 in HCC, we examined the role of their correlation in HCC metastasis. CA2 attenuated ATP1A1-triggered tumor growth in vivo and ATP1A1-induced metastasis in vitro. Taken together, the present results suggest that CA2 serves as a suppressor of HCC metastasis and EMT and is correlated with favorable overall survival (OS) in HCC patients.

The appropriate number of ELNs for lymph node negative breast cancer patients underwent MRM: a population-based study

Whether number of examed lymph nodes (ELNs) would bring survival benefit for patients with negative lymph nodes after modified radical mastectomy (MRM) is uncertain. In our study, using the Surveillance Epidemiology and End Results (SEER) database between 2004 and 2009, we screened the appropriate patients with negative lymph nodes underwent MRM. The Cox proportional hazard analysis was used to determine the effect of number of ELNs on cancer specific survival (CSS). The results showed that the number of ELNs was not an independent prognostic factor on CSS (P = 0.940). Then the X-tile mode was used to determine the appropriate threshold for ELNs count. The results showed that 9 was the appropriate cut-off point. Next, the log-rank χ2 test was used to analyze the CSS based on different subgroup variables. The results showed that some subgroup variables including age < 50/ ≥ 50, grade I/III, AJCC T1/T2, ER positive/negative and PR positive/negative ,demonstrated significant CSS benefits among the patients with the number of ELNs ≤ 9 (all, P < 0.05). However, three subgroup variables including grade II, AJCC T3 and AJCC T4, the patients with the number of ELNs ≤ 9 did not bring significant CSS benefits (all, P > 0.1). In conclusion, our study demonstrated that the number of ELNs was not an independent prognostic factor on CSS, and 9 can be selected as the appropriate cut-off point of ELNs for patients with negative lymph nodes who underwent MRM.

Bufalin suppresses cancer stem-like cells in gemcitabine-resistant pancreatic cancer cells via Hedgehog signaling

Cancer stem cells (CSCs) are important in cancer, as these cells possess enhanced tumor-forming capabilities and are resistant to current anticancer therapies. Agents with the ability to suppress CSCs are likely to provide novel opportunities for combating tumor proliferation and metastasis. The present study aimed to evaluate the effects of bufalin on pancreatic CSCs in vivo and in vitro. Using a serum-free suspension culture, tumor spheres were enriched in a gemcitabine-resistant human pancreatic cancer cell line, which had a higher percentage of CSCs, and western blotting, flow cytometry, and colony and tumor formation assays were used to demonstrate that these sphere cells exhibited CSC characteristics. Using these cancer stem-like cells as a model, the present study examined the effect of bufalin on pancreatic CSCs. It was demonstrated that bufalin inhibited the number of tumor spheres, and western blotting and immunohistochemical assays showed that the expression levels of CD24 and epithelial specific antigen (ESA) were downregulated by bufalin. Furthermore, in a subcutaneous xenograft model of implanted gemcitabine-resistant MiaPaCa2 cells, bufalin inhibited tumor growth and prolonged the duration of tumor formation. Additionally, the expression levels of CD24 and ESA were inhibited in the bufalin-treated mice. Notably, in another cancer model injected with tumor cells via the tail vein, fewer metastatic lesions were detected in the group in which tumor cells were pretreated with bufalin in vitro, compared with those without pretreatment. Of note, the Hedgehog (Hh) signaling pathway was found to be inhibited in the bufalin-treated cells. Taken together, these results suggested that bufalin suppressed pancreatic CSCs in gemcitabine-resistant MiaPaCa2 cells, and the Hh signaling pathway may be involved in this process.

Synergistic anticancer effects of bufalin and sorafenib by regulating apoptosis associated proteins

As one of the most recognized and well-known drugs for hepatocellular carcinoma (HCC), the antitumor effect of sorafenib against HCC remains to be improved. Bufalin has displayed an antitumor effect in HCC; however, whether the enhanced antitumor effect may be generated with their combined treatment remains unclear. Therefore, in the present study, their combined effects on HCC proliferation and apoptosis were investigated. It was revealed that either bufalin or sorafenib suppressed PLC/PRF/5 and SMMC-7721 cell proliferation in a concentration-dependent manner following incubation for 24 h, and the inhibitory effect was augmented with their combined treatment. The synergistic effect peaked in HCC cells treated with 20 nM bufalin and 10 µM sorafenib. In addition, cell cycle and terminal deoxynucleotidyl transferase dUTP nick-end labelling assays revealed that bufalin also enhanced sorafenib-induced apoptosis. Colony formation assay demonstrated that combined treatment significantly suppressed HCC proliferation compared with treatment with either of them alone. Furthermore, B-cell lymphoma 2-associated X protein, caspase 7 and poly-(adenosine diphosphate-ribose) polymerase were upregulated in HCC cells with combined treatment. Taken together, the results of the present study revealed that the treatment of sorafenib combined with bufalin synergistically suppressed HCC proliferation and induced apoptosis. Therefore, bufalin combined with sorafenib may be a favorable treatment strategy for patients with HCC.