Zhiguo Rao

M2-Polarized tumor-associated macrophages are associated with poor prognoses resulting from accelerated lymphangiogenesis in lung adenocarcinoma

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.

Alternatively activated RAW264.7 macrophages enhance tumor lymphangiogenesis in mouse lung adenocarcinoma

Tumor‐associated macrophages (TAMs) have been implicated in promoting tumor progression and invasion. The onset and maintenance of tumor angiogenesis and lymphangiogenesis also seem to be partly driven by a group of polarized alternatively activated macrophages (aaMphi) in lung adenocarcinoma. Here, the aaMphi and classically activated macrophages (caMphi) were obtained using RAW264.7 cells via IL‐4 and IFN‐γ + LPS treatment, respectively. Co‐inoculation of aaMphi with Lewis lung carcinoma (LLC) cells promoted tumor growth, increased lymph node metastasis, and reduced the survival in C57BL/6 mice bearing LLC. Furthermore, the effects of the activated macrophages on the lymphangiogenesis‐related properties of lymphatic endothelial cells (LECs) were investigated in vitro. When LECs were cultured in macrophages conditioned medium or in a co‐culture system of macrophages and LECs, aaMphi significantly promoted proliferation, migration, and tube‐like formation of LECs. We identified high VEGF‐C expression in aaMphi and low expression in caMphi as well as unactivated macrophages by ELISA and Western blotting. In LECs, co‐culture with aaMphi resulted in a significant increase of mRNA levels of specific lymphatic marker VEGF receptor‐3 and the homeobox gene Prox‐1, as well as lymphangiogenic factor VEGF‐C rather than VEGF‐D by quantitative RT‐PCR. Furthermore, enhanced LECs migration and capillary formation by co‐culture with aaMphi were significantly inhibited by rVEGF receptor‐3/Fc chimera. In conclusion, these data show that aaMphi play a critical role in tumor‐induced lymphangiogenesis through up‐regulating VEGF‐C and increasing lymphangiogenesis‐related behavior of LECs, which may contribute to lymphatic invasion in lung adenocarcinoma. J. Cell. Biochem. 107: 134–143, 2009.

Berberine Ameliorates Collagen-Induced Arthritis in Rats Associated with Anti-inflammatory and Anti-angiogenic Effects

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammation and joint destruction. In this study, we explored the effect of berberine on rats with bovine type II collagen-induced arthritis (CIA), an animal model for RA. Following treatment, berberine attenuates arthritic scores and suppresses collagen–specific immune responses in CIA rats. Compared with the un-treated CIA group, berberine reversed pathological changes, which showed a significant improvement in synovial hyperplasia and inflammatory infiltration. The expression levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, IL-17 and vascular endothelial growth factor (VEGF) were obviously reduced in the sera of berberine-treated rats (all P < 0.05). Moreover, berberine showed marked inhibition of the expression of VEGF and CD34 (all P < 0.05). Interestingly, berberine significantly suppresses p-ERK, p-p38 and p-JNK activation (all P < 0.05), which may partially explain the anti-RA activity of berberine. These results suggest that berberine ameliorates CIA in rats associated with anti-inflammatory and anti-angiogenic effects, which might be of great therapeutic value for RA.

Matrix metalloproteinase-9 overexpression is closely related to poor prognosis in patients with colon cancer

BackgroundMatrix metalloproteinase-9 (MMP-9) is an important member of the matrix metalloproteinase family and is considered to be involved in the invasion and metastasis of cancer cells. This study analyzed the expression of MMP-9 in colon cancer patients and the relationship between this expression and clinicopathological features and survival.MethodsWe immunohistochemically investigated 68 specimens of colon cancer tissues and corresponding distal normal mucosa tissues using MMP-9 antibody. Then, the correlation between MMP-9 expression and clinicopathological features and its prognostic relevance were determined.ResultsThe expression rate of MMP-9 in colon cancer tissues was significantly higher than that in distal normal mucosa (69.1% versus 2.9%, P < 0.001). Significant correlations were only found between high levels of MMP-9 expression and metastasis of lymph nodes and Dukes’ stage. Overexpression of MMP-9 was associated with shorter survival times in univariate analysis. Multivariate analysis confirmed that MMP-9 expression was an independent prognostic factor.ConclusionsMMP-9 is correlated with the metastasis of lymph nodes, and its elevated expression may be an adverse prognostic indicator for the patients of colon cancer.

MiR-26a regulates cell cycle and anoikis of human esophageal adenocarcinoma cells through Rb1-E2F1 signaling pathway

Resistance to anoikis, the subtype of apoptosis induced by lack of matrix adhesion, contributes to malignant transformation and development of metastasis. MicroRNAs play key regulatory roles in tumorigenesis and metastasis. In this study, we described that miR-26a, which is usually downregulated in tumor cells, is involved in the acquisition of anoikis-resistance of human esophageal adenocarcinoma (EA) cells. Results of qRT-PCR in clinical samples showed that downregulated miR-26a expression is related to tumorigenesis and metastasis of EA. In vitro experiments determined that miR-26a directly participates in the regulation of cell cycle and anoikis of human EA OE33 cells. Further, we identified that Rb1 is the direct functional target of miR-26a, and revealed that the reduction of miR-26a expression leads to increased Rb1 protein level and thus inhibits the function of E2F1, by which it influences the phenotypes of cell cycle and anoikis. The findings we reported here presented the evidence that miR-26a may be involved in regulation of anoikis-resistance of EA cells. Targeting miR-26a may provide a novel strategy to inhibit metastasis.

M2-polarized macrophages promote metastatic behavior of Lewis lung carcinoma cells by inducing vascular endothelial growth factor-C expression

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 two-dimensional 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.

Clinicopathological significance and prognostic value of MMP-13 expression in colorectal cancer

Abstract The purpose of this study was to evaluate the association of matrix metalloproteinase-13 (MMP-13) expression with clinicopathological features and prognosis in colorectal cancer (CRC) patients. CRC tissues and distal normal mucosa tissues of 158 CRC patients were detected by immunohistochemistry. The correlations between MMP-13 expression, the patients’ clinicopathological features, and overall survival rate were analyzed. It was found that positive expression rate of MMP-13 in distal normal mucosa tissues was significantly lower than that in CRC tissues (36.7% vs 60.8%, p < 0.001). Poor histological differentiation, advanced clinical stage and lymph node metastasis were significantly correlated with the MMP-13 expression in CRC. The overall survival rate of the MMP-13-negative group was significantly higher than the positive group (Log-rank test = 12.452, p < 0.001). Collectively, we found that MMP-13 was correlated with progression and metastasis of CRC and could be used as a prognostic marker in CRC.

IL-6 upregulation contributes to the reduction of miR-26a expression in hepatocellular carcinoma cells

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.

Antisense Oligonucleotide Targeting Matrix Metalloproteinase-7 (MMP-7) Changes the Ultrastructure of Human A549 Lung Adenocarcinoma Cells

Antisense oligonucleotide (ASODN) targeting specific gene can be capable of potently downregulating proliferation and invasion in human cancer cells. However, the underlying mechanisms are less well defined. Here the authors show that matrix metalloproteinase-7 (MMP-7) ASODN changes the ultrastructure of human A549 lung adenocarcinoma cells. Transfection of MMP-7 ASODN significantly lowered the expression of MMP-7 protein in A549 cells. Decreased microvilli, endoplasmic reticulum dilation, swelling of mitochondria, and formation of apoptotic bodies were observed by transmission electron microscope. Collectively, the findings identified the morphological mechanism that MMP-7 ASODN inhibited proliferation and invasion in A549 cells.

Co-transduction of ribosomal protein L23 enhances the therapeutic efficacy of adenoviral-mediated p53 gene transfer in human gastric cancer

Induction of murine double minute 2 (MDM2) expression is thought to be a determinant of resistance to p53 gene therapy for cancer. Previous studies have revealed that ribosomal protein L23 (RPL23) inhibits MDM2-mediated p53 degradation through direct binding to MDM2. In addition, ectopically expressed RPL23 was reported to interact with MDM2 in both the nucleus and cytoplasm, by which RPL23 indirectly inhibited MDM2-p53 binding. Based on the known molecular properties of the RPL23 protein, it was speculated that co-transduction of RPL23 may protect wild‑type p53 protein from MDM2-mediated inactivation and, thus, improve the effect of delivering therapeutic exogenous p53. To test this hypothesis, we constructed a bicistronic adenoviral vector expressing both the RPL23 and p53 genes (Ad-RPL23/p53) and compared its tumor-suppressor activity in human gastric cancer with that of a single gene vector for p53 (Ad-p53). In the in vivo and in vitro experiments, we observed that treatment with Ad-RPL23/p53 resulted in a stronger antitumor response compared to that obtained using Ad-p53. Moreover, the antitumor response of the bicistronic adenovirus was obtained not only in MGC803 cells (endogenous mutant p53) but also in MKN45 cells (endogenous wild‑type p53) which were initially resistant to p53 gene transfer, indicating that co-transduction of RPL23 also expanded the utility of p53 gene therapy. Furthermore, in an orthotopic nude mouse model of human gastric cancer, we found that the survival benefit was greater after Ad-RPL23/p53 treatment than after Ad-p53. Taken together, the data presented here demonstrate that co-transduction of RPL23 enhances the therapeutic efficacy of adenoviral-mediated p53 gene transfer in models of human gastric cancer and support the use of this strategy for cancer treatment.