IL-6/STAT3报告基因系统的构建及抑制IL-6/STAT3信号通路的中药单体的筛选及验证

寻找可抑制IL-6/STAT3信号通路的活化从而抑制肿瘤的生长和恶化的中药单体化合物具有重要意义及发展前景。文中通过基因重组技术构建出一种含有STAT3增强子序列和NanoLuc(NLuc)报告基因序列的新表达载体,并进一步建立受STAT3调控并稳定表达NLuc荧光素酶的细胞系,利用该细胞系定量检测多种中药单体化合物对IL-6/STAT3信号通路的调控作用,并对抑制IL-6/STAT3信号通路的中药单体的效果进行验证。酶切鉴定及测序结果表明报告基因表达载体pQCXIP-STAT3-NLuc构建成功。STAT3转录因子的刺激物白细胞介素-6(IL-6)作用于所构建的稳定表达NLuc的细胞系后出现特异性荧光素酶反应,且作用效果呈良好的剂量依赖性,表明受STAT3调控稳定表达NLuc荧光素酶的细胞系构建成功。Western blotting及Real-time PCR实验结果表明所筛选的中药单体化合物石斛碱及粉防己碱可抑制IL-6/STAT3信号通路并显著下调其下游基因Bcl-2及Bcl-x的表达,且作用呈剂量依赖性。综上所述,文中构建了可高效检测STAT3转录活性的报告基因系统,并利用该系统成功地筛选出可抑制IL-6/STAT3信号通路的中药单体化合物,具有一定的理论和应用价值。     

A trisubstituted pyrazole derivative reduces DMBA-induced mammary tumor growth in rats by inhibiting estrogen receptor-α expression

P16 is the product of cyclin-dependent kinase 2 (CDKN2A) gene and plays multi-pronged roles in the cancer progression. Breast cancer (BC) is the most commonly diagnosed cancer type among females. In the current study, the potential function of P16 in the growth and metastasis of BC was investigated. Firstly, the expression statuses of P16 in different cancer types were investigated using Oncomine database and validated with corresponding cancer cell lines. Afterwards, the expression of P16 was knocked down in BC cell line BT-549 and the effect on the cell proliferation, sensitivity to paclitaxel (TAX), apoptosis, migration, and invasion abilities was assessed using CCK-8, Edu, flow cytometry, scratch, and transwell assays, respectively. The influence of P16 inhibition and P16 overexpression on the activity of IL-6/JAK/STAT3 signaling was explored. Additionally, the effect of P16 inhibition on the tumor growth was verified with a BC xenograft mice model. The abnormal expression of P16 was detected in BC cell line BT-549 as well as colorectal cancer and osteosarcoma cell lines. The inhibition of P16 suppressed the cell proliferation, invasion, and migration abilities while induced the apoptosis and sensitivity to TAX in BT-549 cells. At molecular level, P16 knockdown inhibited the expression of IL6ST and Survivin, and the phosphorylation of JAK2 and STAT3. However, the induced expression of P16 in P16-knockdown BT-549 cells restored the activity of IL-6/JAK2/STAT3 pathway. The results of in vitro assays were confirmed with BC xenograft models: the inhibition of P16 decreased the tumor growth rate. Findings outlined in the current study demonstrated that the inhibition of P16 decreased the growth and metastasis potential of BC cells by inhibiting IL-6/JAK2/STAT3 signaling.     

Induction of tumor promotor-inducible genes in murine 3T3 cell lines and tetradecanoyl phorbol acetate-nonproliferative 3T3 variants can occur through protein kinase C-dependent and -independent pathways.

We isolated a group of genes that are rapidly and transiently induced in 3T3 cells by tetradecanoyl phorbol acetate (TPA). These genes are called TIS genes (for TPA-inducible sequences). Epidermal growth factor (EGF), fibroblast growth factor (FGF), and TPA activated TIS gene expression with similar induction kinetics. TPA pretreatment to deplete protein kinase C activity did not abolish the subsequent induction of TIS gene expression by epidermal growth factor or fibroblast growth factor; both peptide mitogens can activate TIS genes through a protein kinase C-independent pathway(s). We also analyzed TIS gene expression in three TPA-nonproliferative variants (3T3-TNR2, 3T3-TNR9, and A31T6E12A). The results indicate that (i) modulation of a TPA-responsive sodium-potassium-chloride transport system is not necessary for TIS gene induction either by TPA or by other mitogens and (ii) TIS gene induction is not sufficient to guarantee a proliferative response to mitogenic stimulation.     

TIS21/BTG2/PC3 is expressed through PKC-delta pathway and inhibits binding of cyclin B1-Cdc2 and its activity, independent of p53 expression

Signal transduction pathway and a new function of TIS21/BTG2/PC3 were investigated in p53 null U937 cells; Expression of TIS21 by 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulation was mediated by PKC-delta activation, however, was strongly inhibited by cPKC isozymes. When U937 cells were treated with TPA+Go6976, but not TPA+Go6850, the level of TIS21 mRNA was maintained over that of TPA alone. When analyzed by FACS, TPA-induced G2/M arrest was significantly inhibited by Go6850, but not by Go6976, suggesting the involvement of TIS21 and nPKC isozymes. Indeed, PKC-delta was found to be a regulator of the G2/M arrest and TIS21 expression, confirmed by employing rottlerin and dnPKC-delta experiments. In vivo accumulation of TIS21 protein significantly induced cell death through caspase 3 activation, which was supported further by degradations of procaspase 3, full-length PKC-delta, pRB, and p21(WAF1) in TIS21DeltaC expresser. When the cells were synchronized by nocodazole, TIS21 overexpressers inhibited degradations of cyclin A and cyclin B1 in 3 h after release from the synchronization. Furthermore, TIS21 inhibited cyclin B1-Cdc2 binding and its kinase activity in vivo. In summary, TPA-induced TIS21 mRNA expression is mediated by PKC-delta, and TIS21 induces G2/M arrest and cell death by inhibiting cyclin B1-Cdc2 binding and the kinase activity through its binding to Cdc2.     

Cytokine induction of tumor necrosis factor receptor 2 is mediated by STAT3 in colon cancer cells

The IL-6/STAT3 and TNFα/NFκB pathways are emerging as critical mediators of inflammation-associated colon cancer. TNF receptor (TNFR) 2 expression is increased in inflammatory bowel diseases, the azoxymethane/dextran sodium sulfate (AOM/DSS) model of colitis-associated cancer, and by combined interleukin (IL) 6 and TNFα. The molecular mechanisms that regulate TNFR2 remain undefined. This study used colon cancer cell lines to test the hypothesis that IL-6 and TNFα induce TNFR2 via STAT3 and/or NFκB. Basal and IL-6 + TNFα-induced TNFR2 were decreased by pharmacologic STAT3 inhibition. NFκB inhibition had little effect on IL-6 + TNFα-induced TNFR2, but did inhibit induction of endogenous IL-6 and TNFR2 in cells treated with TNFα alone. Chromatin immunoprecipitation (ChIP) revealed cooperative effects of IL-6 + TNFα to induce STAT3 binding to a -1,578 STAT response element in the TNFR2 promoter but no effect on NFκB binding to consensus sites. Constitutively active STAT3 was sufficient to induce TNFR2 expression. Overexpression of SOCS3, a cytokine-inducible STAT3 inhibitor, which reduces tumorigenesis in preclinical models of colitis-associated cancer, decreased cytokine-induced TNFR2 expression and STAT3 binding to the -1,578 STAT response element. SOCS3 overexpression also decreased proliferation of colon cancer cells and dramatically decreased anchorage-independent growth of colon cancer cells, even cells overexpressing TNFR2. Collectively, these studies show that IL-6- and TNFα-induced TNFR2 expression in colon cancer cells is mediated primarily by STAT3 and provide evidence that TNFR2 may contribute to the tumor-promoting roles of STAT3.     

Ovarian carcinoma cells inhibit T cell proliferation: suppression of IL-2 receptor beta and gamma expression and their JAK-STAT signaling pathway

Deficient T cell immune function and intracellular signaling in cancer patients may result from effects of tumors or their products on lymphocytes. Recently, it was demonstrated that several ovarian carcinoma cell lines could produce soluble factors that inhibited T cell proliferation. The aim of this study is to assess the effect of supernatants from 3 ovarian carcinoma cell lines (OVCAR3, CAOV3, SKOV3) on signal transduction elements that are linked to the IL-2R and its JAK-STAT pathway. A profound inhibition of proliferation, lower level of IFN-gamma and higher level of IL-10 gene expression were observed when CD8+ T cells were co-cultured with supernatants from 3 ovarian carcinoma cell lines. Cell cycle studies on inhibited CD8+ T cells showed most of them were growth arrested in G0/G1 phase. Western blot analysis showed that tumor supernatants suppressed expression of JAK3 and tyrosine phosphorylation of STAT5. JAK1 was not altered and the inhibition of STAT3 only appeared in OVCAR3 cells. Tumor supernatants also partially blocked induction of IL-2R beta and gamma chains expression. These findings suggest that ovarian carcinoma cells may suppress T cell proliferation through inhibition IL-2 dependent signaling pathways, which may be a mechanism of ovarian carcinoma induced immunosuppression.     

Interleukin-6 Mediates Epithelial–Stromal Interactions and Promotes Gastric Tumorigenesis

Interleukin-6 (IL-6) is a pleiotropic cytokine that affects various functions, including tumor development. Although the importance of IL-6 in gastric cancer has been documented in experimental and clinical studies, the mechanism by which IL-6 promotes gastric cancer remains unclear. In this study, we investigated the role of IL-6 in the epithelial–stromal interaction in gastric tumorigenesis. Immunohistochemical analysis of human gastritis, gastric adenoma, and gastric cancer tissues revealed that IL-6 was frequently detected in the stroma. IL-6–positive cells in the stroma showed positive staining for the fibroblast marker α-smooth muscle actin, suggesting that stromal fibroblasts produce IL-6. We compared IL-6 knockout (IL-6^−/−) mice with wild-type (WT) mice in a model of gastric tumorigenesis induced by the chemical carcinogen N-methyl-N-nitrosourea. The stromal fibroblasts expressed IL-6 in tumors from WT mice. Gastric tumorigenesis was attenuated in IL-6^−/− mice, compared with WT mice. Impaired tumor development in IL-6^−/− mice was correlated with the decreased activation of STAT3, a factor associated with gastric cancer cell proliferation. In vitro, when gastric cancer cell line was co-cultured with primary human gastric fibroblast, STAT3–related genes including COX-2 and iNOS were induced in gastric cancer cells and this response was attenuated with neutralizing anti-IL-6 receptor antibody. IL-6 production from fibroblasts was increased when fibroblasts were cultured in the presence of gastric cancer cell–conditioned media. IL-6 production from fibroblasts was suppressed by an interleukin-1 (IL-1) receptor antagonist and siRNA inhibition of IL-1α in the fibroblasts. IL-1α mRNA and protein were increased in fibroblast lysate, suggesting that cell-associated IL-1α in fibroblasts may be involved. Our results suggest the importance of IL-6 mediated stromal-epithelial cell interaction in gastric tumorigenesis.