表没食子儿茶素没食子酸酯诱导人肝癌细胞HepG2凋亡与TGF-β1-Smad通路激活有关

Objective To investigate the cytotoxic effect of epigallocatechin gallate(EGCG)on human hepatocellular carcinoma cell line HepG2 cells and corresponding changes of TGF-β1-Smad pathway.Methods The cytotoxic effect of EGCG on HepG2 cells was determined by MTT assay.Cell cycle and apoptosis rate were detected by flow cytometry.RT-PCR and luciferase assay were used to verify whether TGF-β1-Smad signaling pathway is intact in HepG2.The mRNA expression of Smad 2,Smad3,Smad4 and Smad7 was detected by real-time PCR.Results EGCG induced apoptosis in the HepG2 ceils in a time-and concentration-dependent manner.The proportion of G1 phase cells was increased gradually as the concentration increased.However,the percentage of cells in S phase was decreased gradually.Annexin V/PI assay demonstrated that early apoptosis increased as the concentration increased,and late apoptosis also increased,when treated with high-concentration EGCG.The intact TGF-β1-Smad pathway was verified by luciferase assay and RT-PCR.There was no significant effect of EGCG on mRNA level of Smad 2,Smad 3,and Smad 4 in HepG2 cells,but downregulated mRNA level of Smad 7.Conclusion EGCG can reduce apoptosis in human hepatocellular carcinoma cell line HepG2 cells.The activation of TGF-β1-Smad signaling pathway may be involved in its cytotoxicity mechanisms.     

在BEP2D细胞恶性转化过程中TGF-β1对Smad7表达的调节

背景与目的:细胞逃避转化生长因子-β(TGF-β)诱导的对细胞生长、增殖的抑制是许多肿瘤发生的一个重要机制.Smad7是TGF-β信号转导通路的抑制型Smads,它可阻断TGF-β信号在胞浆内的传导,其紊乱是TGF-β信号转导通路紊乱的机制之一.本研究旨在分析在细胞恶性转化过程中,Smad7基因表达是否发生紊乱,TGF-β1对Smad7基因的调控功能有无发生变化,以探索细胞发生恶性转化的原因.方法:培养BEP2D细胞及BERP35T-2细胞,于收获前60min和90min加入不同剂量的TGF-β1,提取细胞总RNA,分别以未加TGF-β1的细胞组作为对照,用Northernblot杂交比较两组细胞Smad7mRNA表达的差异以及细胞对TGF-β1细胞因子刺激的反应性.同时提取BEP2D及BERP35T-2细胞蛋白,用Westernblot方法比较两组细胞内源性TGF-β1表达的差异.结果:Smad7mRNA表达水平恶性转化细胞高于永生化细胞;加了TGF-β1细胞因子后,BEP2D细胞Smad7mRNA表达增高,BERP35T-2细胞表达水平改变不明显.而内源性TGF-β1的表达水平,BERP35T-2细胞稍高于BEP2D细胞.结论:Smad7在辐射致肺癌细胞系中的过表达及对TGF-β1应答的降低可能是辐射诱发肺癌发生的机制之一.     

Expression of Preprotachykinin-I （PPT-I）, Neurokinin-1 （NK-1） and Neurokinin-2 （NK-2） in Breast Cancer Cells Improves Tumor Cell Survival in Bone Marrow in the Early Stage of Metastasis

OBJECTIVE To study the potential relationship between the expression of PPT-I, NK-1, NK2 and the development of breast cancer cells in bone marrow stroma and to provide evidence of potential molecular mechanisms of breast cancer patients. of bone metastasis in early stage METHODS The cocultures of breast cancer cell line T-47D and marrow-derived mesenchymal stem cells （MSC） were established with equal numbers. T-47D cells were separated from the coculture system at 48 h and 96 h after coculture by MACS magnetic cell sorting （MicroBeads）. The expression of PPT-I, NK-1, NK-2 in T-47D was then examined before and after coculture by real-time PCR and by Western blot. Alterations in cellular ultrastructure of T-47D cells were detected before and after coculture under electron microscope. Finally, changes in cell cycle distribution were examined by flow cytometry, and growth curves from before and after coculture were drawn and analyzed. RESULTS Following coculture of T-47D and MSC, the expression of PPT-I mRNA and protein was significantly upregulated, while the expression of NK-1 and NK-2 mRNA and protein was greatly downregulated. The analysis of cell cycle distribution by flow cytometry showed that the proportion of T-47D during S phase was increased, and the duration of the G2/M phase was sharply decreased. Under electron microscope, we observed that the synthesis of hereditary material was increased, but the hepatin granules were shown prominent stacking in T-47D cells. These results suggest that although the synthesis of DNA was increased, the proliferation of cells was inhibited after coculture. The cell growth curve confirmed the findings from the observation under the electron microscope and flow cytometry. CONCLUSION Tumor cells could survive through the upregulation in expression of preprotachykinin-I gene during early bone metastasis in breast cancer. The phenomenon of growth suppression in breast cancer cells after coculture in the current study could be induced by downregulation in expression of NK-1 and NK-2.     

Relationship between Expression of Cell Adhesion Molecules and the Metastatic Mechanism in Invasive Micropapillary Carcinoma of the Breast

OBJECTIVE To explore the expression and the function of cell adhesion molecules in invasive micropapillary carcinoma (IMPC) of the breast, and to investigate the metastatic mechanism of IMPC. METHODS The expression of E-cadherin, α-catenin and β-catenin was detected by imrnunohistochemical staining in 64 cases of IMPC, and compared with that of invasive ductal carcinoma (IDC). RESULTS E-cadherin and β-catenin were mainly expressed on the cell membrane of tumors, and cc-catenin was expressed in the cytoplasm and/or on the cell membrane. The expression of E-cadherin in IMPC was significantly higher than that in IDC. Furthermore, the expression of E-cadherin was mainly on the intercellular contact surface of the tumor cell clusters in IMPC, while that on the outer surface of the tumor cell clusters decreased or could not be detected. The degree of lymph nodes metastases in IMPC was significantly higher than that in IDC. The co-expressions of α-catenin and β-catenin in cases of lymph nodes metastases along with the expression of E-cadherin in IMPC were significantly higher than that in IDC. CONCLUSIONS These findings indicated that the adhesiveness of the intercellular contact surfaces of tumor clusters in IMPC was strong, while that of the outer surface of tumor clusters was decreased or lost. It is suggested that the adhesive characteristic of the cells in IMPC might play an important role in its higher metastatic potential.     

TPX2 knockdown suppressed hepatocellular carcinoma cell invasion via inactivating AKT signaling and inhibiting MMP2 and MMP9 expression

Objective： Targeting protein for Xenopus kinesin-like protein 2 （TPX2） is a nuclear proliferation-related protein that plays a critical role in the formation of mitotic spindle. High expression of TPX2 has been observed in several types of tumors. However, the role of TPX2 in hepatocellular carcinoma （HCC） remains unclear. Our study aimed to investigate the effect of TPX2 on HCC cell invasion. Methods： The immortalized normal human liver cell line L02 and six HCC cell lines including SMMC- 7721, BEL-7402, Huh-7, HepG2, Hep3B and SKHepl were subjected to qRT-PCR and western blot for TPX2 mRNA and protein, respectively. Furthermore, TPX2 small interfering RNA （siRNA） was used to knock down TPX2 expression in SMMC-7721 and HepG2 cells. Cell proliferation and invasion were determined by MTT and transwell assays. Otherwise, expression of p-AKT, MMP2 and MMP9 were evaluated by western blot in SMMC-7721 cells. Results： The expression of TPX2 in HCC cell lines was markedly higher than that in normal human liver cell line. TPX2 knockdown using a specific TPX2-siP, NA reduced the number of invaded cells and inhibited cell proliferation in SMMC-7721 and HepG2 cells. Furthermore, TPX2 knockdown resulted in inactivation of AKT signaling and down-regulation of MMP2 and MMP9 expression in SMMC-7721 cells. Conclusions： Our study identified that TPX2 might contribute to tumor cell invasion through activating AKT signaling and subsequently increasing MMP2 and MMP9 in HCC.     

Inhibition of proliferation of human breast cancer MCF-7 cells by small interference RNA against LRP16 gene

Objective: Our previous studies have firstly demonstrated that 17β-E2 up-regulates LRP16 gene expression in human breast cancer MCF-7 cells, and ectopic expression of the LRP16 gene promotes MCF-7 cells proliferation. Here, the effects of the LRP16 gene expression on growth of MCF-7 human breast cancer cells and the mechanism were further studied by establishing two stably LRP16-inhibitory MCR-7 cell lines. Methods: Hairpin small interference RNA (siRNA)strategy, bY which hairpin siRNA was released by U6 promoter and was mediated by pLPC-based retroviralvector, was adopted to knockdown endogenous LRP16 level in MCF-7 cells. And the hairpin siRNA against green fluorescence protein (GFP) was used as the negative control. The suppressant efficiency of the LRP16 gene expression was confirmed by Nothern blot.Cell proliferation assay and soft agar colony formation assay were used to determine the status of the cell sproliferation. Cell cycle checkpoints including cyclin E and cyclin D1 were examined by Western blot. Results:The results from cell proliferation assays suggested that down-regulation of LRP16 gene expression is capable of inhibiting MCF-7 breast cancer cell growth and down-regulation of the LRP16 gene expression is able to inhibit anchorage-independent growth of breast cancer cells in soft agar. We also demonstrated that cyclin E and cyclin D1 proteins were much lower in the LRP16-inhibitory cells than in the control cells.Conclusion: These data suggest that LRP16 gene play an important role in MCF-7 cells proliferation by regulating the pathway of the G1/S transition and may function as an important modulator in regulating the process of tumorigenesis in human breast.     

γ-Secretase Inhibitor, DAPT Inhibits Self-renewal and Stemness Maintenance of Ovarian Cancer Stem-like Cells In Vitro

Objective: The Notch signaling pathway plays an important role in the stem cell signaling network and contributes to tumorigenesis. However, the functions of Notch signaling in ovarian cancer stem cells (OCSCs) are not well understood. We aimed to investigate the effects of Notch blockade on self-renewal and stemness maintenance of OCSCs. Methods: Ovarian cancer stem-like cells were enriched from ovarian cancer cell lines in serum-free medium. A γ-secretase inhibitor, (DAPT), was used to block Notch signaling. MTT assays were performed to assess self-renewal and proliferation inhibition, flow cytometry was performed to analyze cell surface marker and immunofluorescence, Western Blot and Real-time RT-PCR assays were performed to detect Oct4 and Sox2 protein and mRNA expression of the Ovarian cancer stem-like cells treated with DAPT. Results: Notch blockade markedly inhibits self-renewal and proliferation of ovarian cancer stem-like cells, significantly downregulates the expression of OCSCs-specific surface markers, and reduces protein and mRNA expression of Oct4 and Sox2 in OCSC-like cells. Conclusion: Our results suggest that Notch signaling is not only critical for the self-renewal and proliferation of OCSCs, but also for the stemness maintenance of OCSCs. The γ-secretase inhibitor is a promising treatment targeting OCSCs.     

Expression of epidermal growth factor receptor in different salivary adenoid cystic carcinoma cell lines

Objective： To investigate the expression of epidermal growth factor receptor, a receptor tyrosine protein kinase, in the subcellular fractions of human salivary adenoid cystic carcinoma cell lines SACC-83 and SACC-LM. Methods： Low metastatic and high metastatic cells of the adenoid cystic carcinoma, SACC-83 and SACC-LM, were cultured. Their subcellular fractions were extracted. The expression of epidermal growth factor receptor was detected with Western blot method, and the results of protein expression were quantitatively analyzed by FluorChem V2.0 software. Results： The results of Western blot analysis indicated that, EGFR expression on the membrane of SACC-83 cells was significantly higher than that of SACC-LM cells, but its expression in cytoplasm was significantly less in the former than the later （P〈0.01）. In SACC-83 cell line, EGFR was over-expressed in membrane （P〈0.01）, but in SACC-LM cell line, EGFR was over-expressed in cytoplasm （P〈0.01）. Conclusion： The results suggest that the obtaining of metastasis ability is related to the high expression of EGFR protein in cytoplasm, so the molecular targeting therapy to EGFR may be an ideal treatment for the invasion and metastasis of salivary adenoid cystic carcinoma.     

CK2 inhibitor CX4945 reverses cisplatin-resistance of lung cancer A549/DDP cells北大核心CSCD

Objective: To investigate the effect of CK2 (casein kinase 2) inhibitor CX4945 on the cisplatin (DDP)-resistance of lung cancer A549/DDP cells and the underlying molecular mechanism. Methods: The CCK-8 assay was used to detect the half maximal inhibitory concentration (IC50) of DDP in lung cancer A549 and A549/DDP cells, and to compare the DDP-resistance of two cell lines. The effect of CX4945 on DDP-resistance of A549/DDP cells was tested by CCK-8 method. Western blotting was used to detect the expressions of Wnt signaling pathway-related proteins (CK2α, β-catenin and cyclin D1), drug resistance-related proteins [multidrug resistance-associated protein 1 (MRP1) and lung resistance-related protein (LRP)] and apoptosis-related protein [cleaved caspase-3 (c-caspase-3)] in A549 and A549/DDP cells treated with DDP or not. The A549/DDP cells were treated with no drug (as the control group), CX4945, DDP and their combination (as CX4945+DDP group), then the expressions of Wnt signaling pathway-, drug resistance- and apoptosis-related proteins were detected by Western blotting, and the apoptosis of A549/DDP cells was detected by FCM method. Results: The IC50 value of DDP in A549/DDP cells was 4.59 times higher than that in A549 cells, and the DDP-resistance of A549/DDP cells was decreased by CX4945 pretreatment (P < 0.001). The expression levels of CK2α, β-catenin, cyclin D1, MRP1 and LRP proteins were significantly increased in A549/DDP cells as compared with A549 cells (all P < 0.001), and the levels of these proteins in A549/DDP cells were further increased after DDP treatment (all P < 0.001). In A549 cells after treatment with DDP, the expression levels of CK2α, β-catenin and cyclin D1 proteins were reduced (all P < 0.01), but the levels of MRP1 and LRP proteins were not significantly changed (both P > 0.05). Compared with the control group and DDP group, the expression levels of β-catenin, cyclin D1, MRP1 and LRP proteins in A549/DDP cells of CX4945 group and CX4945+DDP group were significantly declined (all P < 0.01). In addition, the apoptosis rate of A549/DDP cells and the expression level of c-caspase-3 in CX4945+DDP group were significantly higher than those in the control group and DDP group (all P < 0.001). Conclusion: CK2 inhibitor CX4945 can reverse the DDP resistance of lung cancer A549/ DDP cells through blocking Wnt signal pathway and decreasing the expressions of drug resistance-relatied proteins. © 2019 by TUMOR.     

Expression of CD44s mRNA in Gastric Carcinoma

Objective： To study the expression of CD44s mRNA in the occurrence, development and invasion of gastric carcinoma （GC）. Methods： The expressions of CD44s mRNA in 66 cases of GC, 25 cases of superficial gastritis and 25 cases of atypical hyperplasia were examined by in situ hybridization （ISH）. Results： There was no expression of CD44s mRNA in the group of superficial gastritis; the positive rate was 20%（5/25） in the group of atypical hyperplasia and 62.12%（41/66） in the group of gastric carcinoma. The positive rate in poor differentiation group was significantly higher than that in well differentiation group （P〈0.05）, and the positive rate of lymph node metastasis group was significantly higher than that in negative lymph node metastasis group（P〈0.05）. Conclusion： The expression of CD44s mRNA was related to cell differentiation degree and lymph node metastasis, the activation of CD44s gene was related to strong invasion of cancer cells and poor prognosis.     

Influence of PPAR -γligand on radiation - induced PAI - 1 and TGF -βmRNA in fibroblast cells

Objective To observe the influence of peroxisome proliferator activated receptor-γligand (PPAR-γ, pioglatazone) on expression of PAI-1 and TGF-β mRNA and proliferation in fibroblast cells before and after X-ray radiation, and to study the effect of PPAR-γon normal cells during radiation induced fibrosis process. Methods RT-PCR method was used to measure PPAR-γgene expression in L929 cells.After X-ray irradiation of 10 Gy,4 Gy or 2 Gy, the expressions of PAI-1 and TGF-β mRNA in mouse lung fibroblast cells (L929) were measured using RT-PCR. After X-ray irradiation and pioglatazone treatment,the influence of pioglatazone on PAI-1 and TGF-β was measured using RT-PCR method. MTT method was used to test cell proliferation after the treatment of irradiation and pioglatazone. Results PPAR-γ mRNA expression was observed in L929 cells. Expression of PAI-1 and TGF-β mRNA reached the highest level 483.40,P =0. 090) ). At 48 h after the treatment of pioglatazone and 10 Gy radiation, pioglatazone decreased 0. 36, 0. 34 and 0. 32( F = 3.90, P = 0. 040) ). The inhibitory effect was significantly increased when L9292. 50,P =0. 005)). Conclusions X-ray irradiation can increase the expression of PAI-1 and TGF-β in L929 cells. Pioglatazone can decrease the expression of radiation-induced PAI-1 and TGF-β, and restrain the fibroblast proliferation.     

B7-H4 expression is elevated in human U251 glioma stem-like cells and is inducible in monocytes cultured with U251 stem-like cell conditioned medium

Previous studies indicated that B7-H4, the youngest B7 family, negatively regulates T cell-mediated immunity and is significantly overexpressed in many human tumors. Tumor stem cells are purported to play a role in tumor renewal and resistance to radiation and chemotherapy. However, the link between B7-H4 and tumor stem cells is unclear. In this study, we investigated B7-H4 expression in the medium of human glioma U251 cell cultures. Immunofluorescence results showed that U251 cells cultured in serum-free medium （supplemented with 2% B27, 20 ng/mL epidermal growth factor, 20 ng/mL basic fibroblast growth factor） maintained stem-like cell characteristics, including expression of stem cell marker CD133 and the neural progenitor cell markers nestin and SOX2. In contrast, U251 cells cultured in serum-containing medium highly expressed differentiation marker glial fibrillary acidic protein. Flow cytometry analysis showed serum-free medium-cultured U251 cells expressed higher intracellular B7-H4 than serum- containing medium-cultured U251 cells （24%-35% vs. 8%-11%, P 〈 0.001）. Immunofluorescence in purified monocytes from normal human peripheral blood mononuclear cells revealed moderate expression of BT-H4 after stimulation with conditioned medium from U251 cells cultured in serum-containing medium. Moreover, conditioned medium from U251 stem-like cells had a significant stimulation effect on B7-H4 expression compared with serum-containing conditioned medium （P 〈 0.01）. Negative costimulatory molecule B7-H4 was preferentially expressed in U251 stem-like cells, and conditioned medium from these cells more effectively induced monocytes to express BT-H4 than conditioned medium from U251 cells cultured in the presence of serum. Our results show that U251 stem-like cells may play a more crucial role in tumor immunoloregulation with high expression of B7-H4.     

RELATIONSHIP BETWEEN THE EXPRESSIONS OF LMP1 AND E-CADHERIN/β-CATENIN IN NASOPHARYNGEAL CARCINOMA

Objective:To compare the expression of Epstein-Barr virus encoded LMP1 and E-cadherin/β-catenin in primary and metastatic nasopharyngeal carcinoma (NPC) for the purpose of understanding their relationship. Methods: Twenty-two pairs of biopsies taken from the nasopharynx and cervical lymph node(s) of the same patient with nasopharyngeal carcinoma were collected. The expression of LMP1, E-cadherin and β-catenin was observed on immunostained slides using LSAB method. Results:The expression rate of LMP1 in the 22 metastatic tumors (86.36%, 19/22) was significantly higher than that in the 22 primary growths (68.18%, 15/22), P&lt;0.05. The mean expression percentages of E-cadherin and β-catenin in metastatic tumors (50.11%(22.53% and 66.36(21.05%, respectively) were significantly lower than those in primary growths (71.52(24.34 % and 79.40%(15.05%, respectively), P&lt;0.05. There was a positive correlation between the expressions of E-cadherin and β-catenin either in primary growths or metastatic tumors. Conclusion: The LMP1 is more likely to be expressed in metastatic neoplastic cells of NPC than in primary carcinoma cells, and on the contrary the expression of E-cadherin/β-catenin in metastatic cells was decreased. Accordingly, the LMP1 might have the ability to downregulate the expression of E-cadherin/β- catenin, resulting in enhancement of the invasive capacity of metastatic NPC cells.