A recent study showed that miR-26a is downregulated in hepatocellular carcinoma tissues and that this downregulation is an independent predictor of survival. Interestingly, the same study also reported that miR-26a downregulation causes a concomitant elevation of IL-6 expression. Because miR-26a expression was found to be transcriptionally downregulated by oncogene c-Myc in various cancers, and the expression of c-Myc was increased by IL-6 stimulation, we hypothesized that IL-6 contributes to reduction of miR-26a in hepatocellular carcinoma. Serum IL-6 was measured by ELISA and miR-26a was detected by qRT-PCR. The data of 30 patients with hepatocellular carcinoma who had undergone surgical tumor resection revealed that serum IL-6 could be considered to be a predictor of survival up to 5 years for hepatocellular carcinoma patients (log-rank test, P < 0.05). We observed that the serum IL-6 concentration was inversely correlated with miR-26a expression in cancerous tissues (Pearson correlation test, r = -0.651, P < 0.01). Furthermore, by in vitro experiments with HepG2 cells, we showed that IL-6 stimulation can lead to miR-26a suppression via c-Myc activation, whereas in normal hepatocyte LO2 cells incubation with IL-6 had no significant effect on miR-26a expression. Taken together, these results indicate that miR-26a reduction in hepatocellular carcinoma might be due to IL-6 upregulation.

OBJECTIVE: Sorafenib is an oral multikinase inhibitor that has been proven effective as a single-agent therapy in hepatocellular carcinoma, and there is a strong rationale for investigating its use in combination with other agents. Vitamin K2 is nearly non-toxic to humans and has been shown to inhibit the growth of hepatocellular carcinoma. In this study, we evaluated the effects of a combination of sorafenib and vitamin K2 on the growth of hepatocellular carcinoma cells. METHODS: Flow cytometry, 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide) and nude mouse xenograft assays were used to examine the effects of sorafenib and vitamin K2 on the growth of hepatocellular carcinoma cells. Western blotting was used to elucidate the possible mechanisms underlying these effects. RESULTS: Assays for 3-(4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2H-tetrazolium bromide) revealed a strong synergistic growth-inhibitory effect between sorafenib and vitamin K2. Flow cytometry showed an increase in cell cycle arrest and apoptosis after treatment with a combination of these two drugs at low concentrations. Sorafenib-mediated inhibition of extracellular signal-regulated kinase phosphorylation was promoted by vitamin K2, and downregulation of Mcl-1, which is required for sorafenib-induced apoptosis, was observed after combined treatment. Vitamin K2 also attenuated the downregulation of p21 expression induced by sorafenib, which may represent the mechanism by which vitamin K2 promotes the inhibitory effects of sorafenib on cell proliferation. Moreover, the combination of sorafenib and vitamin K2 significantly inhibited the growth of hepatocellular carcinoma xenografts in nude mice. CONCLUSIONS: Our results determined that combined treatment with sorafenib and vitamin K2 can work synergistically to inhibit the growth of hepatocellular carcinoma cells. This finding raises the possibility that this combined treatment strategy might be promising as a new therapy against hepatocellular carcinoma, especially for patients with poor liver tolerance.

OBJECTIVES: Tumor-associated macrophages that generally exhibit an alternatively activated (M2) phenotype have been linked to tumor progression and metastasis. However, the role of M2-polarized macrophages in the growth and metastasis of lung adenocarcinoma remains enigmatic. The aim of this study was to explore the effect of M2 macrophages on the proliferation and migration of mouse Lewis lung carcinoma cells and tumor-induced lymphangiogenesis. METHODS: Trypan blue staining and the Transwell migration assay were performed to evaluate the effects of activated (M1 or M2) macrophages on the proliferation and migration of Lewis cells. Furthermore, vascular endothelial growth factor-C expression in Lewis cells and nitric oxide secretion from activated macrophages were detected during the co-culture assay. Following treatment with activated macrophages, lymphatic endothelial cells differentiated into capillary-like structures, and the induction of Lewis cell migration was assessed using a twodimensional Matrigel-based assay. RESULTS: In the co-culture Transwell system, the proliferation and migration of Lewis cells were promoted by M2 macrophages. Moreover, the co-culture significantly increased the expression of vascular endothelial growth factor-C by Lewis cells and reduced the secretion of nitric oxide from M2 macrophages, which subsequently led to the capillary morphogenesis of lymphatic endothelial cells. Interestingly, following co-culture with Lewis cells, the function of RAW264.7 cells was polarized toward that of the M2 macrophage phenotype. CONCLUSION: M2-polarized macrophages promoted the metastatic behavior of Lewis cells by inducing vascular endothelial growth factor-C expression. Thus, the interruption of signaling between M2 macrophages and Lewis cells may be considered to be a new therapeutic strategy.

OBJECTIVES: Tumor-associated macrophages have been implicated in promoting tumor growth, progression and metastasis. However, the activated phenotype (M1 or M2) of tumor-associated macrophages remains unknown in solid tumors. Therefore, this study examined the density and prognostic significance of M2-polarized tumor-associated macrophages in lung adenocarcinoma. METHODS: Tumor specimens from 65 lung adenocarcinoma patients were assessed by ELISA for Th1/Th2 cytokine concentrations. The activated phenotype (M1 or M2) of tumor-associated macrophages was determined utilizing immunofluorescence staining. Additionally, to evaluate lymphangiogenesis, peritumoral lymphatic microvessel density was measured using D2-40. The correlation between tumor-associated macrophage subtype and overall patient survival was analyzed using the Kaplan-Meier method and compared using the log-rank test. RESULTS: A shift toward Th2 cytokine expression was detected within lung adenocarcinoma microenvironments. Approximately 79.71±16.27% of tumor-associated macrophages were M2 polarized; the remaining 20.35±5.31% were M1 polarized. The infiltration of M2-polarized macrophages was significantly associated with P-TNM staging and lymph node metastasis. The peritumoral lymphatic microvessel density was significantly higher in the high M2-polarized tumor-associated macrophage group than in the low M2-polarized tumor-associated macrophage group. A significant difference in overall patient survival was detected not only between patients with tumors with high and low macrophage counts but also between patients with tumors with high and low counts of M2-polarized macrophages. CONCLUSION: Tumor-associated macrophages in lung adenocarcinoma have an M2-polarized subtype and are associated with poor prognoses, perhaps resulting from accelerated lymphangiogenesis and lymph node metastasis.