Inhibition of SALL4 suppresses carcinogenesis of colorectal cancer via regulating Gli1 expression

Background: SALL4 is a novel oncogene mediating tumorigenesis in multiple carcinomas. However, its actual role and mechanisms participating in the development of colorectal cancer remains unclear. Methods: Immunohistochemical staining and Western blot were conducted to detect the expression of SALL4 and other molecules. siRNA of SALL4 was transfected to silence SALL4 expression in Caco-2 cell line. Flow cytometry was used for cell cycle and apoptosis analysis. Wound healing and transwell assay were used for invasion test. CCK-8 test was employed for cell proliferation and drug sensitivity assessment. Results: By inhibition of SALL4 expression, the proliferation, invasiveness and drug resistance were dramatically reduced while apoptosis rate was up-regulated. Gli1 was found to decrease its expression in SALL4 silencing cells. Moreover, the inhibition on tumorigenesis of Caco-2 by SALL4 silencing was antagonized by Gli1 up-regulation, suggesting Gli1 as a downstream target of SALL4 in cancer development. Conclusion: SALL4 inhibition limited oncogenesis on colorectal cancer by reducing Gli1 expression.     

干扰HDAC1通过调控STAT3信号通路影响皮肤鳞癌细胞凋亡

目的:探讨干扰组蛋白去乙酰化酶1(HDAC1)对皮肤鳞癌细胞凋亡的影响。方法:皮肤鳞癌细胞A431分别转染HDAC1小干扰RNA(HDAC1 si RNA)和小干扰RNA阴性对照(si RNA NC),RT-PCR和Western blot检测转染后细胞中HDAC1的表达水平,MTT检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测信号转导及转录激活因子3(STAT3)、磷酸化STAT3(p-STAT3)和cleaved caspase-3的蛋白水平。同时用STAT3信号通路抑制剂作用于转染HDAC1 si RNA的A431细胞,MTT法检测细胞活力,流式细胞术检测细胞凋亡,Western blot检测STAT3、p-STAT3和cleaved caspase-3的蛋白水平。结果:HDAC1 si RNA能够抑制A431细胞中HDAC1的m RNA和蛋白表达。干扰HDAC1表达后细胞活力和细胞中p-STAT3水平下降,而细胞凋亡率和细胞中cleaved caspase-3水平升高。STAT3信号通路抑制剂作用后,转染HDAC1 si RNA的A431细胞活力及p-STAT3水平下降,细胞凋亡率及细胞中cleaved caspase-3水平升高。结论:干扰HDAC1表达可能通过调控STAT3信号通路抑制皮肤鳞癌细胞活力,促进皮肤鳞癌细胞凋亡。     

Tubeimoside-1 (TBMS1) inhibits lung cancer cell growth and induces cells apoptosis through activation of MAPK-JNK pathway

Tubeimoside-1 (TBMS1) is a natural compound isolated from tubeimoside, which has anti-tumor properties in some cancer cells, but its mechanisms are unclear. In the present study, we determined if TBMS1 would inhibit cell growth of human lung cancer cell lines. We found that TBMS1 inhibited growth in A549 and PC9 human lung cancer cell. Flow cytometry revealed TBMS1 arrested the cells in the G2/M phase and induced cell apoptosis. Furthermore, results from Western blotting and real-time PCR indicated that decreased the cell proliferation and cell growth-associated protein levels, such as p21, p15 and cyclin B1, TBMS1 up-regulated proapoptotic bax and cleavage of procaspase-3, down-regulated antiapoptotic Mcl-1 and cIAP-1, but did not change expression of PARP and procaspase-8. And TBMS1 also found to increase the phosphorylation, of JNK and p38, suggesting TBMS1 could activate the MAPK-JNK signaling pathway. Luciferase reporter assay revealed that TBMS1 reduced the promoter activity of AP-1, NF-κB and TNFα. In addition, TBMS1 caused the production of reactive oxygen species (ROS). These results provide evidence that TBMS1 may have potential as a novel anti-cancer agent for the treatment of lung cancer. TBMS1 inhibited cell proliferation may through MAPK-JNK signaling pathway.     

S-腺苷甲硫氨酸抑制大肠癌细胞生长的实验研究

目的探讨S-腺苷甲硫氨酸(SAM)对人大肠癌细胞生长的抑制作用及抑癌机制。方法用SAM对大肠癌细胞系HT-29进行处理,使其癌基因c-myc和H-ras启动子区域甲基化。用噻唑蓝(MTT)法检测肿瘤细胞生长状态;甲基化特异性聚合酶链反应(PCR)(MSP)法检测c-myc和H-ras启动子区域甲基化状态;细胞免疫荧光染色检测C-MYC和H-RAS蛋白的表达情况。结果大肠癌细胞系HT-29经SAM处理后,癌基因c-myc和H-ras启动子区域重新出现甲基化。经SAM处理的大肠癌细胞组与对照组相比,细胞生长受到明显抑制,差异有统计学意义(P<0.05);C-MYC和H-RAS蛋白表达明显降低,差异有统计学意义(P<0.05)。结论 SAM能使HT-29中癌基因c-myc和H-ras启动子区域重新甲基化,降低C-MYC和H-RAS蛋白的表达水平,且有效抑制了肿瘤细胞的生长,从而为肿瘤治疗提供了新的靶点。     

Prognostic significance of Aurora-A expression in human bladder cancer

Aurora-A is an oncogenic serine/threonine kinase, which plays important roles in tumorigenesis, development and chemoresistance of human cancers. The aim of the study was to detect the expression of Aurora-A gene in bladder cancer tissues and analyze its association with prognosis of bladder cancer patients. RT-PCR was performed to detect the expression of Aurora-A mRNA in 20 cases of bladder cancer and corresponding non-tumor tissue samples. Immunohistochemistry was performed to detect the localization of Aurora-A protein in 96 cases of bladder cancer tissue samples. Associations between Aurora-A protein expression and clinico-pathological factors or survival of bladder cancer patients were statistically analyzed. It was found that the expression levels of Aurora-A mRNA in bladder cancer tissues (1.08 ± 0.24) were significantly higher than those in corresponding non-tumor tissues (0.22 ± 0.07; P < 0.01). Moreover, immunohistochemical staining results showed the localization of Aurora-A protein to be mainly located in the cytoplasm of bladder cancer cells. High levels of Aurora-A protein expression were correlated with pathological stage (P = 0.007), lymph node metastasis (P = 0.014) and venous invasion (P = 0.008), but not with other factors including age, gender, tumor grade and recurrence of superficial cancer. Patients with high expression levels of Aurora-A protein showed lower disease-free and overall survival rates than those with low expression levels (P = 0.0072 and 0.0009, respectively). Univariate and multivariate analysis of prognostic factors in bladder cancer patients indicated that Aurora-A expression was an independent unfavorable prognostic factor (hazard ratio: 0.673; 95% confidence interval: 0.388-0.912; P < 0.001). Our study suggests that overexpression of Aurora-A gene may play an important role in the progression of bladder cancer and that Aurora-A expression is an independent factor for predicting the prognosis of bladder cancer in patients.     

KISS1 expression in colorectal cancer

Kisspeptins, the products of the KISS1 gene, are involved in cancer invasion, migration, metastasis and angiogenesis, while they induce apoptosis in various cancers. Herein, we studied KISS1 expression in colorectal cancer. We analyzed KISS1 expression using immunohistochemistry and image analysis in normal and malignant tissue samples from 60 patients with colorectal adenocarcinoma. The results correlated with various clinicopathological parameters. The expression of KISS1 was much higher in normal than in malignant colonic mucosa. However, among malignant tissues, KISS1 expression was higher in larger tumors (>4 cm) than in smaller ones (≤4 cm) and in stages III and IV than in stages I and II. In addition, it was higher in patients with lymph node metastases. Moreover, KISS1 levels in the normal mucosa and their difference from those in the malignant mucosa were higher in the right part of the large intestine than in the left one. KISS1 expression is reduced during the malignant transformation of the colonic mucosa and there is a difference in the expression pattern between the right and the left part of the large intestine. However, larger and advanced colorectal tumors express higher KISS1 levels than smaller and localized ones.     

UHRF1 regulation of the Keap1–Nrf2 pathway in pancreatic cancer contributes to oncogenesis

The cellular defence protein Nrf2 is a mediator of oncogenesis in pancreatic ductal adenocarcinoma (PDAC) and other cancers. However, the control of Nrf2 expression and activity in cancer is not fully understood. We previously reported the absence of Keap1, a pivotal regulator of Nrf2, in ～70% of PDAC cases. Here we describe a novel mechanism whereby the epigenetic regulator UHRF1 suppresses Keap1 protein levels. UHRF1 expression was observed in 20% (5 of 25) of benign pancreatic ducts compared to 86% (114 of 132) of pancreatic tumours, and an inverse relationship between UHRF1 and Keap1 levels in PDAC tumours (n = 124) was apparent (p = 0.002). We also provide evidence that UHRF1‐mediated regulation of the Nrf2 pathway contributes to the aggressive behaviour of PDAC. Depletion of UHRF1 from PDAC cells decreased growth and enhanced apoptosis and cell cycle arrest. UHRF1 depletion also led to reduced levels of Nrf2‐regulated downstream proteins and was accompanied by heightened oxidative stress, in the form of lower glutathione levels and increased reactive oxygen species. Concomitant depletion of Keap1 and UHRF1 restored Nrf2 levels and reversed cell cycle arrest and the increase in reactive oxygen species. Mechanistically, depletion of UHRF1 reduced global and tumour suppressor promoter methylation in pancreatic cancer cell lines, and KEAP1 gene promoter methylation was reduced in one of three cell lines examined. Thus, methylation of the KEAP1 gene promoter may contribute to the suppression of Keap1 protein levels by UHRF1, although our data suggest that additional mechanisms need to be explored. Finally, we demonstrate that K‐Ras drives UHRF1 expression, establishing a novel link between this oncogene and Nrf2‐mediated cellular protection. Since UHRF1 over‐expression occurs in other cancers, its ability to regulate the Keap1–Nrf2 pathway may be critically important to the malignant behaviour of these cancers. © 2015 The Authors. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.     

EGCG inhibits the growth and tumorigenicity of nasopharyngeal tumor-initiating cells through attenuation of STAT3 activation

A subset of cancer cells, termed cancer stem cells (CSCs) or tumor-initiating cells (TICs) could initiate tumors and are responsible for tumor recurrence and chemotherapeutic resistance. In this study, we enriched TICs in nasopharyngeal carcinoma (NPC) by the spheres formation and characterized the stem-like signatures such as self-renewal, proliferation, chemoresistance and tumorigenicity. By this method, we investigated that epigallocathechin gallate (EGCG), the major polyphenol in green tea could target TICs and potently inhibit sphere formation, eliminate the stem-like properties and enhance chemosensitivity in NPC through attenuation of STAT3 activation, which could be important in regulating the stemness expression in NPC. Our results demonstrated that STAT3 pathway plays an important role in mediating tumor-initiating capacities in NPC and suggest that inactivation of STAT3 with EGCG may represent a potential preventive and therapeutic approach for NPC.     

HLJ1高表达对结直肠癌细胞周期和侵袭能力的影响

目的构建并鉴定p CDNA3.0-HLJ1真核表达质粒,检测HLJ1基因表达上调对结直肠癌细胞周期和侵袭能力的影响。方法利用反转录PCR法钓取HLJ1基因片段并与p CDNA3.0载体连接,酶切及测序鉴定p CDNA3.0-HLJ1重组质粒。在结直肠癌细胞SW480和HT29中分别转染p CDNA3.0和p CDNA3.0-HLJ1,应用荧光定量PCR和Western blot法检测HLJ1的表达,分别应用流式细胞仪和细胞侵袭实验检测其对细胞周期和侵袭能力的影响。结果 p CDNA3.0-HLJ1重组质粒构建成功,并在结直肠癌细胞中成功表达。转染p CDNA3.0-HLJ1质粒入结直肠癌细胞后,肿瘤细胞G_1/G_0期比例明显增加(P0.05),侵袭能力明显减弱(P0.05)。结论 p CDNA3.0-HLJ1质粒为进一步研究HLJ1在结直肠癌中的作用提供实验基础。HLJ1将结直肠癌细胞阻滞在G_1期并抑制其侵袭能力。     

Polyp pT1 colorectal cancer

With the introduction of NHS Bowel Cancer Screening Programme, coupled with advances made in endoscopic equipment and techniques, there is a higher detection rate for early pT1 polyp colorectal cancers (CRC). The current clinical trend is towards a conservative approach without surgical resection, if felt that there is successful endoscopic excision and without unfavourable histology parameters. Nonetheless, the further management of malignant polyps, observation or resection, remains controversial.     

Elevated expression of UHRF1 predicts unfavorable prognosis for patients with hepatocellular carcinoma

Aims: The present study was designed to evaluate the different expression of ubiquitin-like with PHD and ring finger domains 1 (UHRF1) in hepatocellular carcinoma (HCC) tissues and the adjacent normal tissues, further explore the correlation between UHRF1 expression and the prognosis of HCC patients. Methods: The UHRF1 expression at protein level in HCC tissues and the adjacent normal tissues were measured by high performance liquid chromatography (HPLC). Chi-square test was used to estimate the relationship between UHRF1 expression and clinicopathologic characteristics of HCC patients. The overall survival of HCC patients with diverse expression of UHRF1 was measured by Kaplan-Meier analysis. Cox regression analysis was conducted to judge the prognostic value of UHRF1 in HCC patients. Results: The UHRF1 was over-expressed in HCC tissues compared with the adjacent normal tissues according to the outcome of HPLC (P<0.001). Besides, the UHRF1 expression was tightly related to distant metastasis, cancer area, and HBV (P<0.05), but shared no correlation with gender, cirrhosis, and bilirubin (P>0.05). Patients with high UHRF1 expression had a shorter overall survival time than those with low UHRF1 expression (P<0.001). Cox regression analysis showed that UHRF1 was significantly linked with the prognosis of HCC patients (P=0.002, HR=5.807, 95% CI=1.901-17.742). Conclusion: UHRF1 was over-expressed in HCC tissues compared to the adjacent normal tissues and UHRF1 expression shared significant relevance with distant metastasis, cancer area and HBV. It could be an important and independent prognostic biomarker for HCC patients.     

Relationship between T cell receptor clonotype and PD-1 expression of tumor-infiltrating lymphocytes in colorectal cancer

Adoptive T cell therapy using tumor-specific T cells or TCR-modified T cells is a promising next-generation immunotherapy. The major source of tumor-reactive T cells is PD-1⁺ tumor-infiltrating lymphocytes (TILs). In contrast, PD-1⁻ TILs have received little attention. Here, we analyzed the TCR-β repertoires of PD-1⁻ and PD-1⁺ CD8⁺ TILs derived from colorectal cancer and breast cancer. Approximately 40–60% of the PD-1⁺ population consisted of oligoclonal populations in both colorectal cancer and breast cancer. In contrast, approximately 37% of the PD-1⁻ population consisted of an oligoclonal population in colorectal cancer, whereas 14% of them were oligoclonal in breast cancer. In colorectal cancer, the TCR repertoires of PD-1⁻CD8⁺ TILs and PD-1⁺CD8⁺ TILs hardly overlapped. Interestingly, clonally expanded CD8⁺ TILs in primary tumors and the metastases expressing the same clonotypic TCR showed the same phenotype regarding the PD-1-expression. These results suggest that the intrinsic properties of TCRs determine the fate of TILs in terms of whether they become PD-1⁺ or PD-1⁻ in the tumor microenvironment. Further functional analysis of TCRs in TILs will allow us to better understand the regulatory mechanisms for PD-1 expression on TILs and may contribute to tumor immunotherapy.     

miR-197-3p调控DMBT1抑制甲状腺癌细胞系增殖、迁移和侵袭

目的探讨miR-197-3p是否通过靶向调控恶性脑瘤缺失1基因(DMBT1)影响甲状腺癌细胞增殖、迁移和侵袭。方法RT-qPCR检测健康人甲状腺细胞Nthy-ori 3-1和甲状腺癌细胞SW579、CGTHW-1中miR-197-3p表达;MTT法检测SW579细胞增殖;Transwell小室法检测SW579细胞迁移和侵袭;双荧光素酶报告基因实验验证miR-197-3p是否靶向DMBT1;Western blot检测细胞DMBT1、cyclinD1、p21、MMP-2和E-cadherin蛋白表达。结果与Nthy-ori 3-1细胞比较,SW579和CGTHW-1细胞中miR-197-3p相对表达量升高(P<0.05);抑制miR-197-3p表达后,SW579细胞的增殖、迁移和侵袭能力明显受到抑制,细胞中cyclinD1蛋白和MMP-2蛋白表达降低而p21蛋白和E-cadherin蛋白表达升高;SW579细胞中miR-197-3p靶向负调控DMBT1的表达;过表达DMBT1明显抑制SW579细胞增殖、迁移和侵袭,而抑制DMBT1则能逆转miR-197-3p对SW579细胞增殖、迁移和侵袭的影响。结论miR-197-3p通过靶向调控DMBT1的表达,抑制甲状腺癌细胞增殖、迁移和侵袭。     

Zinc finger of the cerebellum 5 promotes colorectal cancer cell proliferation and cell cycle progression through enhanced CDK1/CDC25c signaling

IntroductionColorectal cancer (CRC), mostly caused by external or environmental factors, is the third most common and lethal cancer worldwide. Although a large number of investigations have been carried out to reveal the evolution of CRC, the underlying mechanisms of CRC remain unclear.Material and methodsExpression of zinc finger of the cerebellum 5 (ZIC5) in CRC tissues and cell models was measured by qRT-PCR and IHC. Cell transfection was carried out for ZIC5 overexpression or knockdown. The MTT assay was applied to examine the capacity of glioma cell proliferation. Wound healing assay and tumor invasion assay were used to test the capacity of glioma cell migration and invasion respectively. Cell cycle analysis and western blot were used to verify the apoptosis rates of CRC cells upon ZIC5 overexpression or downregulation. A further tumor Xenograft study was used to examine the effects of ZIC5 on tumor malignancy in vivo.ResultsCell models using HCT116 and SW620 cells were established to study the ZIC5 function upon ZIC5 overexpression of knockdown. Consistently, we discovered that ZIC5 also significantly increased in Chinese CRC patients. In addition, ZIC5 promoted CRC cell proliferation through increasing the proportion of cells maintained in the S phase. ZIC5 overexpression facilitated the capacity of CRC cell migration and invasion. Inhibition of ZIC5 mitigated such malignant effects.ConclusionsCollectively, investigations of the ZIC5 in CRC provided a new insight into CRC diagnosis, treatment, prognosis and next-step translational therapeutic developments from bench to clinic.     

A clinicopathological analysis of KISS1 and KISS1R expression in colorectal cancer

Kisspeptins, the products of the KISS1 gene have tumor suppressing and antimetastatic properties. We aimed to study KISS1 and KISS1R expression in colorectal cancer. We analyzed KISS1 and KISS1R expression using immunohistochemistry and image analysis in normal and malignant tissue samples from 111 patients with colorectal adenocarcinoma. KISS1 expression was much higher in the normal than in the malignant colonic mucosa. Regarding malignant tissues, KISS1 levels were higher in larger tumors, in stage III and IV cancers, in cancers with lymph node metastasis and in tumors located in the distal part of the large intestine. Patients with greater KISS1 levels had worse prognosis. No KISS1R expression was detected in normal or malignant tissues or in liver metastases. KISS1 expression is reduced during the malignant transformation of the colonic mucosa. However, larger and advanced colorectal cancers express more KISS1, without reaching the former normal levels, and increased KISS1 levels are associated with worse prognosis. Finally, neither the normal nor the malignant colonic epithelial cells produce KISS1R.