萝卜硫素对小鼠乳腺癌4T1细胞上皮-间质转化、增殖和迁移的影响研究

背景与目的:组蛋白去乙酰化酶5(histone deacetylase 5,HDAC5)在乳腺癌组织中异常高表达,其与赖氨酸特异性去甲基化酶1(lysine specific demethylase,LSD1)协同促进乳腺癌细胞增殖和迁移.通过萝卜硫素(sulforaphane,SFN)下调HDAC5表达,观察其对小鼠...     

Combination of PTEN and γ-Ionizing Radiation Enhances Cell Death and G2/M Arrest Through Regulation of AKT Activity and p21 Induction in Non–Small-Cell Lung Cancer Cells

PURPOSE: To identify the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) during gamma-ionizing radiation (gamma-IR) treatment for non-small-cell lung cancer cells. METHODS AND MATERIALS: Wild-type PTEN or mutant forms of PTEN plasmids were transfected to construct stable transfectants of the NCI-H1299 non-small-cell lung cancer cell line. Combined effects of PTEN expression and IR treatment were tested using immunoblot, clonogenic, and cell-counting assays. Related signaling pathways were studied with immunoblot and kinase assays. RESULTS: At steady state, stable transfectants showed almost the same proliferation rate but had different AKT phosphorylation patterns. When treated with gamma-IR, wild-type PTEN transfectants showed higher levels of cell death compared with mock vector or mutant transfectants, and showed increased G(2)/M cell-cycle arrest accompanied by p21 induction and CDK1 inactivation. NCI-H1299 cells were treated with phosphosinositide-3 kinase (PI3K)/AKT pathway inhibitor (LY29002), resulting in reduced AKT phosphorylation levels. Treatment of NCI-H1299 cells with LY29002 and gamma-IR resulted in increased cell-cycle arrest and p21 induction. Endogenous wild-type PTEN-containing NCI-H460 cells were treated with PTEN-specific siRNA and then irradiated with gamma-IR: however reduced PTEN levels did not induce cell-cycle arrest or p21 expression. CONCLUSIONS: Taken together, these findings indicate that PTEN may modulate cell death or the cell cycle via AKT inactivation by PTEN and gamma-IR treatment. We also propose that a PTEN-PI3K/AKT-p21-CDK1 pathway could regulate cell death and the cell cycle by gamma-IR treatment.     

人COX-2基因反义真核表达载体对胃癌转移的影响

目的：在成功构建人COX-2基因反义真核表达载体的基础上,研究COX-2基因对人胃癌细胞株SGC-7901转移能力的影响。方法：实验分为COX-2基因反义真核表达载体转染的人胃癌细胞株SGC-7901转染组、COX-2抑制剂SC236处理组（SGC-7901细胞培养基内含100μmol/L SC236）和对照组（SGC-7901细胞）,并分别采用细胞体外侵袭实验,内皮细胞体外迁移实验,观察各组细胞体外转移的情况。采用裸鼠皮下移植瘤抑制实验,用转染和未转染的SGC-7901细胞按每毫升5×10~7个的细胞悬液分别注射于BALB/C-nu/nu无胸腺裸鼠背部皮下,SC236处理组按3mg/（kg·d）剂量于接种SCC-7901细胞10min后,由尾静脉注射,隔日1次。各组6周后处死动物,观察皮下移植瘤的生长情况。并用Western blot法检测各组细胞VEGF及MMP-2蛋白的表达。结果：体外侵袭实验、促内皮细胞体外迁移实验结果显示,与对照组相比,转染组和SC236处理组迁移的肿瘤细胞和内皮细胞均减少（P〈0.01）;裸鼠皮下移植抑制实验结果显示,转染组和SC236处理组荷瘤鼠移植瘤的生长受到抑制;Western blot结果显示,转染组和SC236处理组细胞VEGF及MMP-2蛋白的表达下降。结论：反义COX-2基因能抑制SCG-7901细胞的转移能力,其机制可能与其抑制肿瘤的血管生成、减少MMP-2的表达有关。     

ATP6L promotes metastasis of colorectal cancer by inducing epithelial‐mesenchymal transition

ATP6L, the C subunit of the V‐ATPase V0 domain, is involved in regulating the acidic tumor micro‐environment and may promote tumor progression. However, the expression and functional role of ATP6L in tumors have not yet been well explored. In this study, we found that ATP6L protein overexpression was related to colorectal cancer histological differentiation (P < 0.001), presence of metastasis (P < 0.001) and recurrence (P = 0.02). ATP6L expression in the liver metastatic foci was higher than in the primary foci (P = 0.04). ATP6L expression was notably concomitant with epithelial‐mesenchymal transition (EMT) immunohistochemical features, such as reduced expression of the epithelial marker E‐cadherin (P = 0.021) and increased expression of the mesenchymal marker vimentin (P = 0.004). Results of in vitro and in vivo experiments showed that ATP6L expression could alter cell morphology, regulate EMT‐associated protein expression, and enhance migration and invasion. The effect of ATP6L on metastasis was further demonstrated in a tail vein injection mice model. In addition, the mouse xenograft model showed that ATP6L‐overexpressing HCT116 cells grew into larger tumor masses, showed less necrosis and formed more micro‐vessels than the control cells. Taken together, our results suggest that ATP6L promotes metastasis of colorectal cancer by inducing EMT and angiogenesis, and is a potential target for tumor therapy.     

上皮间质转化与肿瘤干细胞

上皮-间质转化(EMT)是生物胚胎发育的基础过程,与肿瘤细胞的原位侵袭和远处转移也密切相关.EMT与肿瘤干细胞(CSC)关系密切.CSC有较强的自我更新和致瘤能力以及细胞分化潜能.通过对CSC标志物的鉴定,深入研究其耐药性,可为肿瘤治疗提供新径.     

Knockdown of CD44 inhibits the invasion and metastasis of hepatocellular carcinoma both in vitro and in vivo by reversing epithelial-mesenchymal transition

Mounting evidence has shown that induction of epithelial-mesenchymal transition (EMT) contributes to the the expression of CSC (cancer stem cell) markers. However, whether and how CSC markers could be involved in regulating EMT has rarely been reported. CD44, being one of the most commonly used CSC markers in hepatocellular carcinoma (HCC), has been demonstrated to act as a multidomain, transmembrane platform that serves to integrate a wide variety of extracellular signals. Therefore, we determined to seek whether CD44 is necessary for the EMT process in HCC. First, we noticed that CD44 expression was associated with the mesenchymal phenotype in HCC cell lines, and knocking down CD44 with lentivirus-mediated shRNA in HCC cell lines resulted in the mesenchymal-epithelial-transition (MET) and the subsequent impaired migration and invasion in vitro. Moreover, in a metastatic mice model established by tail vein injection of luciferase labelled MHCC97-H cells, we confirmed that CD44 knockdown resulted in the decreased metastasis of HCC cells. Furthermore, we found that the induction of MET by CD44 inhibition might be achieved, at least in part, by repressing the ERK/Snail pathway.     

Harmine suppresses breast cancer cell migration and invasion by regulating TAZ-mediated epithelial-mesenchymal transition

Breast cancer is a highly lethal disease due to cancer metastasis. Harmine (HM), a β-carboline alkaloid, is present in various medicinal plants. Our previous study demonstrated that HM suppresses cell proliferation and migration by regulating TAZ in breast cancer cells and accelerates apoptosis. Epithelial-mesenchymal transition (EMT) plays an important role in the development of breast cancer by inducing the characteristics of cancer stem cells, cancer metastasis and recurrence. Overexpression of TAZ was shown to mediate EMT in breast cancer cells. We aimed to investigate whether HM inhibits EMT and metastasis of breast cancer cells by targeting TAZ. In this study, the cells treated with HM or with downregulated expression of TAZ showed an increase in epithelial markers and decrease in mesenchymal markers in breast cancer cells. Consistently, the breast cancer cells treated with HM or with downregulated expression of TAZ showed suppressed migration and proliferation. Moreover, TAZ overexpression reversed EMT and metastasis induced by HM in breast cancer cells. Thus, HM suppresses EMT and metastasis and invasion by targeting TAZ in breast cancer cells. HM can be used as an anticancer drug for breast cancer treatment and chemoprevention.     

Engagement of I-branching {beta}-1, 6-N-acetylglucosaminyltransferase 2 in breast cancer metastasis and TGF-{beta} signaling

In this study, we have showed that GCNT2, a gene-encoding glucosaminyl (N-acetyl) transferase 2, I-branching enzyme, is overexpressed in highly metastatic breast cancer cell lines of human and mouse origin and basal-like breast tumor samples. GCNT2 expression is also significantly correlated to the metastatic phenotype in breast tumor samples. Functional studies showed that ectopic expression of GCNT2 enhances cell detachment, adhesion to endothelial cells, cell migration and invasion in vitro, and lung metastasis of breast cancer cells in vivo. Knockdown of GCNT2 expression decreases cell migration and invasion in vitro and lung metastasis in vivo. We have further shown the involvement of GCNT2 in the epithelial-to-mesenchymal transition (EMT). Specifically, the expression of E-cadherin is significantly changed upon GCNT2 expression at the protein level but not at the RNA level. Moreover, we have shown that GCNT2 is a direct target of the TGF-β-smad pathway and that change in GCNT2 expression modulates EMT induced by TGF-β1 treatment. Finally, we have shown that diminution of the glycosyltransferase activity of I-branching β-1, 6-N-acetylglucosaminyl transferase 2 (GCNT2) abrogates its cell migration and invasion-promoting function and synergistic effect with TGF-β to induce EMT. Our study for the first time showed that GCNT2 is a novel gene contributing to breast cancer metastasis with preferential expression in basal-like breast cancer. Moreover, we discovered that involvement of GCNT2 in EMT and TGF-β signaling, and further glycosylation modification of E-cadherin by GCNT2, are the underlying integrative mechanisms for breast cancer metastasis, implying that blocking TGF-β/GCNT2 signaling is a promising approach for targeting metastatic breast cancer.     

上皮间质转化与肿瘤干细胞的关系

目前越来越多的研究显示上皮间质转化(EMT)参与了肿瘤干细胞的形成以及肿瘤细胞的浸润、迁移和转移,不仅增强了癌细胞的侵袭和转移能力,还可使细胞获得自我更新等干细胞特性,促进肿瘤干细胞的产生.因此,研究EMT与常见干细胞之间的关系对于寻找控制肿瘤侵袭和转移的有效途径有非常重要的意义,亦可为肿瘤的治疗提供新的靶点.     

Excessive activation of IL-33/ST2 in cancer-associated fibroblasts promotes invasion and metastasis in ovarian cancer

Ovarian cancer is highly prevalent and has high mortality rates due to metastasis and relapse. The cross communication between cancer-associated fibroblasts (CAFs) and cancer-associated macrophages (CAMs) in the ovarian tumor microenvironment leads to cancer cell invasion and metastasis. However, the role of overproduction of IL-33/ST2 in the CAFs of ovarian cancer is still unclear. The expression of IL-33, ST2, apoptosis-related proteins and epithelial-mesenchymal transition (EMT) markers was measured by western blotting. Primary normal fibroblasts and CAFs from ovarian cancerous tissue were isolated and cultured in vitro, and the medium was used to stimulate blood-derived monocytes. Flow cytometry analysis was used to detect the frequency of M2-like macrophages in blood-derived monocytes from patients with ovarian cancer. Cell invasion were evaluated using Transwell assays. A xenograft model was used to study tumor growth in ST2-knockout and wild-type NOD-SCID mice. The results demonstrated higher expression of IL-33 and ST2 in carcinoma tissues compared with in para-carcinoma tissues, and there was a survival improvement associated with elevated IL-33. IL-33 and culture supernatants from CAFs, rather than normal ovarian fibroblasts, led to a higher expression of M2 macrophage marker genes in human blood-derived monocytes. Invasion and migration were aggravated in COC1 cells co-cultured with CAF-induced CAMs, and the EMT marker genes were upregulated. It was reported that EMT marker genes were downregulated and tumor volumes were significantly reduced in ST2-deficient mice. Overall, the IL-33/ST2 axis in ovarian cancer might integrate IL-33-expressing CAFs with M2 type-like CAMs, which aggravated invasion and metastasis by promoting EMT.     

Nab‐paclitaxel interrupts cancer‐stromal interaction through C‐X‐C motif chemokine 10‐mediated interleukin‐6 downregulation in vitro

Cancer‐associated fibroblasts (CAF), derived from stroma of cancer tissues, interact with cancer cells and play an important role in cancer initiation, growth, and metastasis. Nab‐paclitaxel (nab‐PTX) is a 130 nm albumin‐binding paclitaxel and recommended for many types of cancer chemotherapy. The nab‐PTX stromal‐disrupting effect during pancreatic cancer treatment has been reported. The aim of the present study was to determine the role of nab‐PTX in cancer cells and CAF interaction. Cancer cells (MIA PaCa‐2 and Panc‐1) were cocultured with CAF or treated with CAF conditioned medium, after which their migration and invasion ability, epithelial‐mesenchymal transition (EMT)‐related marker expression and C‐X‐C motif chemokine 10 (CXCL10) expression and secretion were detected. Nab‐PTX treatment was carried out during the coculture system or during preparation of CAF conditioned medium. Then cancer cell migration and invasion ability, EMT‐related marker expression, CXCL10 expression and secretion, and interleukin‐6 (IL‐6) expression and secretion by CAF were checked After coculture with CAF, migration and invasion ability of cancer cells increased. CAF also downregulated E‐cadherin and upregulated N‐cadherin and vimentin expression in cancer cells. During coculture or stimulation with cancer cell‐cultured medium, CAF significantly increased IL‐6 expression and secretion. However, nab‐PTX in the coculture system canceled CAF‐induced migration and invasion promotion and EMT‐related gene changes. Moreover, nab‐PTX increased CXCL10 expression of cancer cells which blocked CAF IL‐6 expression and secretion. Nab‐PTX treatment could increase CXCL10 expression of cancer cells which blocks CAF cancer cell migration and invasion‐promoting effect by inhibiting IL‐6 expression.     

Characterization of an in vitro model system to explore control of tumor invasion of EMT6 and 4THM breast tumors by CD200:CD200R interactions

Background and purpose

In BALB/c mice with transplantable breast tumors, we showed that CD200R1KO mice were cured of local and metastatic growth of EMT6 cells following surgical resection of localized tumor and immunization with irradiated cells along with CpG as adjuvant. On the other hand, wild-type (WT) animals treated in the same fashion develop pulmonary and liver metastases within 20 days of surgery. To develop an in vitro system which would mimic the in vivo model and allow exploration of factors controlling tumor invasion as a precursor to in vivo metastasis, we have developed and characterized a two-phase culture system.

Methods

Bone marrow mesenchymal stromal cells (BMMSCs) from WT, CD200KO or CD200R1KO mice were admixed with T lymphocytes from tumor-immunized mice and cultured in collagen gels. Tumor cells were subsequently seeded in fresh medium above this gel 1d later. We then investigated the regulation of tumor invasion from the liquid to the gel layer. Tumor cells were measured in the gel layer following collagenase digestion and cultured at limiting dilution—an aliquot of the digest was also analyzed for cytokine levels in ELISA.

Results

BMMSCs from WT, CD200KO and CD200R1KO mice all augmented seeding/growth of EMT6 and 4THM tumor cells into the collagen matrix. Inclusion of IL-6 and IL-17 in the gel matrix was associated with increased invasion of tumor cells into this layer. Inclusion of DLN cells from EMT6 immune or 4THM immune mice further modified tumor invasion, with increased tumor numbers seen using stromal elements from CD200 and CD200R1KO mice and DLN from 4THM immune, while CD200R1KO-derived DLN of EMT6 immune mice attenuated tumor invasion, despite inclusion of IL-6/IL-17 in the gel layer.

Conclusion

Multiple factors can regulate tumor invasion, including micro-environmental stromal elements, IL-6/IL-17, and signals from tumor-derived DLN cells.

     

Anti-Metastatic Potential of a Novel Xanthone Sourced by Swertia chirata Against In Vivo and In Vitro Breast Adenocarcinoma Frameworks

Background: The Anticancer property of Swertia chirata has been well established. It forms a rich source of compounds to which its anticancer property can be attributed, among the compounds found in S. chirata xanthones form an important group. Among the most abundant xanthones found in S. chirata, 1,5,8-trihydroxy-3-methoxy xanthone (TMX) was found to be most effective. As metastasis is the underlying cause of most cancer-related deaths, in this study, we evaluated the anti-metastatic potential of TMX against adenocarcinoma both in vivo and in vitro. Materials and Methods: In vivo anti-metastatic potential was proved by histological evidence of different organs, giemsa staining of bone marrow, subcutaneous re-injection of the aberrant bone marrow cells into the right flank of the mice to observe the formation of tumors and analyzing the markers related to metastasis by immunohistochemistry (IHC) and western blot. In vitro validation of anti-metastatic potential was carried out against human breast adenocarcinoma cell line MCF-7 by primarily analyzing the migratory property of cells through scratch wound healing assay and the ability of cells to form colonies. The re-validation part was performed by western blot of markers related to metastasis and real-time analysis of EMT related markers. Results: In vivo, TMX treatment restricted metastasis of EAC induced solid tumor to liver, lung, bone marrow, and validation of this finding was achieved by down regulation of metastatic and EMT markers.  In vitro, TMX treatment restricted migratory and colony forming ability of MCF-7 cells by down regulating metastatic and EMT markers. Conclusion: It was proved from our study that TMX treatment successfully reduced the metastatic potential of EAC induced solid tumor, with in vitro validation TMX on the MCF-7 cell line.     

UCH-L1 promotes cancer metastasis in prostate cancer cells through EMT induction

Ubiquitin C-terminal hydrolse-L1 (UCH-L1) is a deubiquitinating enzyme (DUB) that cleaves the ubiquitin (ub) moiety from ub precursors or protein substrates. The correlation between UCH-L1 and cancer has been reported in various tissues, but the role of UCH-L1 in prostate cancer has not been thoroughly researched. Here we found that UCH-L1 is specifically highly expressed in the metastatic DU145 prostate cancer cell line, but not in the benign or weakly metastatic prostate cancer cells. To determine the role of UCH-L1 in prostate cancer metastasis, we constructed UCH-L1-knockdown DU145 and UCH-L1 or the active site mutant form of UCH-L1 (UCH-L1 C90S) expressing RWPE1 stable cells. Notably, the expression of UCH-L1 in RWPE1 cells promotes epithelial-to-mesenchymal transition (EMT), and this is an important process for cancer cell invasion and metastasis. On the contrary, knockdown of UCH-L1 in DU145 cells induces MET, the reverse program of EMT. Furthermore, the change of EMT status caused by altering the UCH-L1 level affects the migration and invasiveness of prostate cancer cells. Our results indicate that UCH-L1 promotes prostate cancer metastasis through EMT induction and UCH-L1 could be a novel diagnostic and therapeutic target for prostate cancer treatment.     

Establishment and characterization of a cell line, EH-GB2, derived from hepatic metastasis of gallbladder cancer

Gallbladder cancer is a fatal neoplasia with an extremely low survival rate. Liver invasion and metastasis are the most common causes of death; however, the metastatic mechanism is still unclear, and no effective treatment methods are available. To provide comprehensive and profound approaches in investigating the metastatic mechanism and treatment methods, new cell lines derived from liver metastasis are urgently needed. A hepatic metastasis lesion was obtained from a 65-year-old patient, and was treated using a primary culture method to establish a novel gallbladder cancer cell line. Different in?vitro/in?vivo methods were used to characterize the phenotypes of this cell line. The gallbladder cancer cell line was named EH-GB2, with a roughly 48-h doubling time. The cell line represents stronger colony formation and migration abilities than the control group. The cells showed complicated chromosomal abnormalities. EH-GB2 cells showed epithelial-to-mesenchymal transition (EMT) and the mRNA expression levels of E-cadherin and integrin were decreased, and those of vimentin, Snail, Twist, matrix metalloproteinase-1 (MMP-1) and MMP-2 were increased in comparison with control cells. The in?vivo study demonstrated that EH-GB2 cells show significant tumorigenicity in nude mice. The EH-GB2 established gallbladder cancer cell line is useful for future studies of gallbladder cancer development, progression, metastasis and therapy.