Comparison of efficacy of Salmonella typhimurium A1-R and chemotherapy on stem-like and non-stem human pancreatic cancer cells

The XPA1 human pancreatic cancer cell line is dimorphic, with spindle stem-like cells and round non-stem cells. We report here the in vitro IC₅₀ values of stem-like and non-stem XPA1 human pancreatic cells cells for: (1) 5-fluorouracil (5-FU), (2) cisplatinum (CDDP), (3) gemcitabine (GEM), and (4) tumor-targeting Salmonella typhimurium A1-R (A1-R). IC₅₀ values of stem-like XPA1 cells were significantly higher than those of non-stem XPA1 cells for 5-FU (P = 0.007) and CDDP (P = 0.012). In contrast, there was no difference between the efficacy of A1-R on stem-like and non-stem XPA1 cells. In vivo, 5-FU and A1-R significantly reduced the tumor weight of non-stem XPA1 cells (5-FU; P = 0.028; A1-R; P = 0.011). In contrast, only A1-R significantly reduced tumor weight of stem-like XPA1 cells (P = 0.012). The combination A1-R with 5-FU improved the antitumor efficacy compared with 5-FU monotherapy on the stem-like cells (P = 0.004). The results of the present report indicate A1-R is a promising therapy for chemo-resistant pancreatic cancer stem-like cells.     

LncRNA MIR31HG通过诱导细胞周期阻滞抑制食管鳞癌细胞增殖活性

本研究探讨lnc RNA MIR31HG对食管鳞癌细胞增殖活性的影响.利用定量PCR检测MIR31HG在食管鳞癌标本及其癌旁组织、人食管上皮细胞系Het-1A和食管鳞癌细胞系Eca-109、EC-1、KYSE30中的表达;采用过表达质粒pc DNA3.1-MIR31HG在食管鳞癌细胞系中过表达MIR31HG;MTT法和SRB法检测细胞增殖率;细胞周期分析试剂盒检测细胞周期进程;Caspase3活性检测试剂盒分析Caspase3活性;PCR和Western blot法检测p53、Caspase3及Bcl-2的m RNA和蛋白质表达水平.结果显示,食管癌组织中MIR31HG表达水平显著低于癌旁组织(P0.05);与Het-1A细胞相比,Eca-109、EC-1、KYSE30细胞中MIR31HG的表达均显著下调(P0.05),提示MIR31HG可能介导食管癌的发生发展.转染pc DNA3.1-MIR31HG可显著上调食管癌细胞中MIR31HG的m RNA表达(P0.01),且MIR31HG过表达可显著抑制食管癌细胞增殖活性(P0.05),减少S期细胞数(P0.05),增加G1期细胞数(P0.05),提示MIR31HG可能通过阻碍细胞周期G1期~S期进程抑制食管癌细胞增殖活性.此外,MIR31HG过表达显著增加Caspase3活性,增加Caspase3和p53的m RNA和蛋白质表达水平,同时抑制Bcl-2 m RNA和蛋白质表达水平.这表明,MIR31HG可通过抑制食管癌细胞的增殖活性阻碍食管癌的发生发展,这可能为食管癌的诊断和治疗提供新策略.     

TUG1 confers cisplatin resistance in esophageal squamous cell carcinoma by epigenetically suppressing PDCD4 expression via EZH2

Background

Increasing evidence has suggested the involvement of long non-coding RNA taurine upregulated gene 1 (TUG1) in chemoresistance of cancer treatment. However, its function and molecular mechanisms in esophageal squamous cell carcinoma (ESCC) chemoresistance are still not well elucidated. In the present study, we investigate the functional role of TUG1 in cisplatin (DDP) resistance of ESCC and discover the underlying molecular mechanism.

Results

Our study revealed that TUG1 was up-regulated in DDP-resistant ESCC tissues and cells. High TUG1 expression was correlated with poor prognosis of ESCC patients. TUG1 knockdown improved the sensitivity of ECA109/DDP and EC9706/DDP cells to DDP. Moreover, TUG1 could epigenetically suppress PDCD4 expression via recruiting enhancer of zeste homolog 2. PDCD4 overexpression could mimic the functional role of down-regulated TUG1 in DDP resistance. PDCD4 knockdown counteracted the inductive effect of TUG1 inhibition on DDP sensitivity of ECA109/DDP and EC9706/DDP cells. Furthermore, TUG1 knockdown facilitated DDP sensitivity of DDP-resistant ESCC cells in vivo.

Conclusion

TUG1 knockdown overcame DDP resistance of ESCC by epigenetically silencing PDCD4, providing a novel therapeutic target for ESCC.

     

HLA-DRB1 and HLA-DQB1 methylation changes promote the occurrence and progression of Kazakh ESCC

Human leukocyte antigen II (HLA-II) plays an important role in host immune responses to cancer cells. Changes in gene methylation may result in aberrant expression of HLA-II, serving a key role in the pathogenesis of Kazakh esophageal squamous cell carcinoma (ESCC). We analyzed the expression level of HLA-II (HLA-DP, -DQ, and -DR) by immunohistochemistry, as well as the methylation status of HLA-DRB1 and HLA-DQB1 by MassARRAY spectrometry in Xinjiang Kazakh ESCC. Expression of HLA-II in ESCC was significantly higher than that in cancer adjacent normal (ACN) samples (P < 0.05). Decreased HLA-II expression was closely associated with later clinical stages of ESCC (P < 0.05). Hypomethylation of HLA-DRB1 and hypermethylation of HLA-DQB1 was significantly correlated with occurrence of Kazakh ESCC (P < 0.01), and mainly manifested as hypomethylation of CpG9, CpG10-11, and CpG16 in HLA-DRB1 and hypermethylation of CpG6-7 and CpG16-17 in HLA-DQB1 (P < 0.01). Moreover, hypomethylation of HLA-DQB1 CpG6-7 correlated with poor differentiation in ESCCs, whereas hypermethylation of HLA-DRB1 CpG16 and hypomethylation of HLA-DQB1 CpG16-17 were significantly associated with later stages of ESCC (P < 0.05). A significant inverse association between HLA-DRB1 CpG9 methylation and HLA-II expression was found in ESCC (P < 0.05). These findings suggest aberrant HLA-DRB1 and HLA-DQB1 methylation contributes to the aberrant expression of HLA-II. These molecular changes may influence the immune response to specific tumor epitopes, promoting the occurrence and progression of Kazakh ESCC.     

The status of phosphorylated p38 in esophageal squamous cell carcinoma

The p38 mitogen-activated protein kinase (MAPK) is a member of the MAPK family, which is initially found to be activated by stress stimuli, proinflammatory cytokines, and growth factors. However, its role in the pathogenesis of esophageal squamous cell carcinoma (ESCC) is largely unkown, so we investigate the role of phosphorylated p38 (p-p38) MAPK in ESCC. First of all, in vitro cell line ECa109, SB203580 as selective inhibitor of p38, can suppress the growth of esophageal cancer cell in a dose- and time-dependent way, suggesting that ECa109 cell growth and proliferation was closely associated with p-p38. Using western-blot analysis of fresh 16 paired surgically resected ESCC and matched non-tumor adjacent tissues (NAT), we showed that p-p38 was significantly expressed higher in NAT compared to ESCC. Moreover, expressions of p-p38 were further confirmed by 162 paired of formalin-fixed paraffin-embedded (FFPE) ESCC and NAT by immunohistochemistry, the same trend result was obtained through statistical analysis that there was increased expression of p-p38 in NAT as compared with ESCC (P < 0.01), and expression of p-p38 was not significantly associated with lymph nodes metastasis (P > 0.05) and ESCC differentiation degree (P > 0.05). Taken together, all the results we obtained demonstrated that p-p38 plays a key role in the malignant transformation of ESCC.     

MicroRNA-218 Inhibits Cell Cycle Progression and Promotes Apoptosis in Colon Cancer by Downregulating BMI1 Polycomb Ring Finger Oncogene

Deregulated miRNAs participate in colorectal carcinogenesis. In this study, miR-218 was found to be downregulated in human colorectal cancer (CRC) by miRNA profile assay. miR-218 was silenced or downregulated in all five colon cancer cells (Caco2, HT29, SW620, HCT116 and LoVo) relative to normal colon tissues. miR-218 expression was significantly lower in 46 CRC tumor tissues compared with their adjacent normal tissues (P < 0.001). Potential target genes of miR-218 were predicted and BMI1 polycomb ring finger oncogene (BMI-1), a polycomb ring finger oncogene, was identified as one of the potential targets. Upregulation of BMI-1 was detected in CRC tumors compared with adjacent normal tissues (P < 0.001) and in all five colon cancer cell lines. Transfection of miR-218 in colon cancer cell lines (HCT116, HT29) significantly reduced luciferase activity of the wild-type construct of BMI-1 3′ untranslated region (3′UTR) (P < 0.001), whereas this effect was not seen in the construct with mutant BMI-1 3′UTR, indicating a direct and specific interaction of miR-218 with BMI-1. Ectopic expression of miR-218 in HCT116 and HT29 cells suppressed BMI-1 mRNA and protein expression. In addition, miR-218 suppressed protein expression of BMI-1 downstream targets of cyclin-dependent kinase 4, a cell cycle regulator, while upregulating protein expression of p53. We further revealed that miR-218 induced apoptosis (P < 0.01), inhibited cell proliferation (P < 0.05) and promoted cell cycle arrest in the G2 phase (P < 0.01). In conclusion, miR-218 plays a pivotal role in CRC development through inhibiting cell proliferation and cycle progression and promoting apoptosis by downregulating BMI-1.     

Effect of valproic acid on miRNAs affecting histone deacetylase in a model of anaplastic thyroid cancer

Background

Thyroid cancer is the most common malignant tumor of the endocrine system seen in the thyroid gland. More than 90% of thyroid cancers comprise papillary thyroid cancer (PTC) and follicular thyroid cancer (FTC). Although anaplastic thyroid carcinoma (ATC) accounts for less than 2% of thyroid cancer. But patients’ lifespan after diagnosis is about 6 months. Surgical interventions, radioactive iodine use, and chemotherapy are not sufficient in the treatment of ATC, so alternative therapies are needed.

Methods and results

The WST-1 assay test was performed to evaluate the anti-proliferative effects of Valproic acid (VPA). Also, the effect of VPA on miRNAs affecting histone deacetylase was determined by Quantitative RT-PCR. In the SW1736 cell line, IC50 dose for VPA was found 1.6 mg/ml. In our study, the level of oncogenic genes expression in cells treated with VPA, including miR-184, miR-222-5p, miR-124-3p, and miR-328-3p, decreased. Also, the expression of tumor inhibitory genes including miR-323-5p, miR-182-5p, miR-138-5p, miR-217, miR-15a-5p, miR-29b-3p, miR-324-5p and miR-101-5p increased significantly.

Conclusions

VPA can ad-just countless gene expression patterns, including microRNAs (miRNAs), by targeting histone deacetylase (HDAC). However, further studies are required for more accurate results.

     

CD4 + CD25<Superscript>high</Superscript> Regulatory T Cell Numbers and FOXP3 mRNA Expression in Patients with Advanced Esophageal Cancer Before and After Chemotherapy

We evaluated the changes in CD4 + CD25^high regulatory T (Treg) cells and FOXP3 mRNA expression in patients with advanced esophageal cancer as well as its clinical significance. For this purpose, the frequencies of peripheral blood Treg cells in 68 patients with advanced esophageal cancer and 40 healthy controls were determined by flow cytometry, and FOXP3 mRNA expression in Treg cells of 40 patients was determined by RT–PCR. The data show that Treg cell numbers were significantly higher (P < 0.01) in esophageal cancer patients (1.82 ± 0.54% of CD4 + T cells) as compared with healthy controls (1.52 ± 0.70% of CD4⁺ T cells). Treg cell numbers in the patients were significantly higher (P < 0.05) before chemotherapy (1.82 ± 0.54% of CD4 + T cells) than after chemotherapy (1.66 ± 0.58% of CD4 + T cells). Expression of the FOXP3 mRNA in the patients was significantly lower (P < 0.05) after chemotherapy (0.266 ± 0.028% of CD4 + T cells) than before chemotherapy (0.318 ± 0.027% of CD4 + T cells). It was, therefore, concluded that Treg cell numbers as well as FOXP3 mRNA expression in advanced esophageal cancer patients were significantly decreased after chemotherapy. Notably, FOXP3 gene may thus be involved in regulating the numbers and function of Treg cells in advanced esophageal cancer patients receiving chemotherapy.     

Cytotoxicity of natural products and derivatives toward MCF-7 cell monolayers and cancer stem-like mammospheres

Although cancer stem-like cells (CSCs) are rare, they can enter a non-proliferative or dormant state and resist therapy. Furthermore, quiescent CSCs are responsible for metastases that can appear after curative surgical treatment of a primary tumor. Because of drug resistance of CSCs, the development of novel therapies is urgently required that specifically target CSCs.PurposeThe aim of the present study was to investigate the potential of a panel of natural products and derivatives to inhibit CSC-enriched mammospheres of MCF-7 breast cancer cells.MethodsCD44^high/CD24^low cells were identified by flow cytometry and maintained as mammospheres. As a control, we used two clinically established anticancer drugs (5-fluorouracil and docetaxel). A panel of natural products, shikonin, two cajanin stilbene acid (CSA) derivatives and artesunate were tested by resazurin assay on CSC-enriched mammospheres and MCF-7 monolayer cells. Besides, cellular shikonin uptake experiments were performed by flow cytometry.ResultsWe found two CSA derivatives (Nos. 6 and 19) to be active cancer stem-like MCF-7 mammospheres. Especially, CSA derivative No. 19 clearly showed collateral sensitivity in mammospheres compared to monolayer cells.ConclusionPhytochemicals which provoke collateral sensitivity in cancer-stem like cells are worth more detailed investigations in the future, since there is a great potential for improved chemotherapy to eradicate tumors and prolong cancer patients’ survival times.     

HLA-DRB1 and HLA-DQB1 methylation changes promote the occurrence and progression of Kazakh ESCC

Human leukocyte antigen II (HLA-II) plays an important role in host immune responses to cancer cells. Changes in gene methylation may result in aberrant expression of HLA-II, serving a key role in the pathogenesis of Kazakh esophageal squamous cell carcinoma (ESCC). We analyzed the expression level of HLA-II (HLA-DP, -DQ, and -DR) by immunohistochemistry, as well as the methylation status of HLA-DRB1 and HLA-DQB1 by MassARRAY spectrometry in Xinjiang Kazakh ESCC. Expression of HLA-II in ESCC was significantly higher than that in cancer adjacent normal (ACN) samples (P < 0.05). Decreased HLA-II expression was closely associated with later clinical stages of ESCC (P < 0.05). Hypomethylation of HLA-DRB1 and hypermethylation of HLA-DQB1 was significantly correlated with occurrence of Kazakh ESCC (P < 0.01), and mainly manifested as hypomethylation of CpG9, CpG10-11, and CpG16 in HLA-DRB1 and hypermethylation of CpG6-7 and CpG16-17 in HLA-DQB1 (P < 0.01). Moreover, hypomethylation of HLA-DQB1 CpG6-7 correlated with poor differentiation in ESCCs, whereas hypermethylation of HLA-DRB1 CpG16 and hypomethylation of HLA-DQB1 CpG16-17 were significantly associated with later stages of ESCC (P < 0.05). A significant inverse association between HLA-DRB1 CpG9 methylation and HLA-II expression was found in ESCC (P < 0.05). These findings suggest aberrant HLA-DRB1 and HLA-DQB1 methylation contributes to the aberrant expression of HLA-II. These molecular changes may influence the immune response to specific tumor epitopes, promoting the occurrence and progression of Kazakh ESCC.     

Integrated analysis of DNA methylation and mRNA expression profiling reveals candidate genes associated with cisplatin resistance in non-small cell lung cancer

DNA methylation plays a critical role during the development of acquired chemoresistance. The aim of this study was to identify candidate DNA methylation drivers of cisplatin (DDP) resistance in non-small cell lung cancer (NSCLC). The A549/DDP cell line was established by continuous exposure of A549 cells to increasing concentrations of DDP. Gene expression and methylation profiling were determined by high-throughput microarrays. Relationship of methylation status and DDP response was validated in primary tumor cell culture and the Cancer Genome Atlas (TCGA) samples. Cell proliferation, apoptosis, cell cycle, and response to DDP were determined in vitro and in vivo. A total of 372 genes showed hypermethylation and downregulation in A549/DDP cells, and these genes were involved in most fundamental biological processes. Ten candidate genes (S100P, GDA, WISP2, LOXL1, TIMP4, ICAM1, CLMP, HSP8, GAS1, BMP2) were selected, and exhibited varying degrees of association with DDP resistance. Low dose combination of 5-aza-2′-deoxycytidine (5-Aza-dC) and trichostatin A (TSA) reversed drug resistance of A549/DDP cells in vitro and in vivo, along with demethylation and restoration of expression of candidate genes (GAS1, TIMP4, ICAM1 and WISP2). Forced expression of GAS1 in A549/DDP cells by gene transfection contributed to increased sensitivity to DDP, proliferation inhibition, cell cycle arrest, apoptosis enhancement, and in vivo growth retardation. Together, our study demonstrated that a panel of candidate genes downregulated by DNA methylation induced DDP resistance in NSCLC, and showed that epigenetic therapy resensitized cells to DDP.     

Isolation of Stem-Like Cancer Cells in Primary Endometrial Cancer Using Cell Surface Markers CD133 and CXCR4

Endometrial cancer (EC) is the most common familiar gynecologic malignant tumor identified in the female reproductive system and has been increasing yearly. In this study, we will identify the surface markers and stem cell markers related with cancer stem cells (CSCs) of EC. Tissue samples were obtained from endometrial cancer patients during surgical procedures. Single cells were isolated from the tissues for culturing, transfection into nude mice, and histopathology analysis. RT-PCR demonstrated that the cultured cells strongly expressed stemness-related genes, such as c-Myc, Sox-2, Nanog, Oct 4A, ABCG2, BMI-1, CK-18, Nestin and β-actin. The expression of surface markers CD24, CD133, CD47, CD29, CD44, CXCR4, SSEA3 and SSEA4, CD24, and CD133 and chemokine markers such as CXCR4 were measured by flow cytometry. Then the double percentage of CD133+CXCR4+ cells constituted 7.2% and 9.3% in EC cells originated from two different patients, respectively. The CD133+CXCR4+ primary endometrial cancer cells grew faster, exhibited high expression of mRNA of stemness-related genes, produced more spheres, and had higher clonogenic ability than other subpopulations. They are also more resistant to anti-cancer drugs than other subpopulations. These findings indicate that CD133+CXCR4+ cells may possess some characteristics of CSCs in primary endometrial cancer. These cell surface markers may be useful for the development of drugs against CSC molecular targets or as a predictive marker for poor prognosis in primary endometrial cancer.     

大肠癌组织中联合检测USP22和BMI-1表达的临床意义

目的:探讨大肠癌组织中联合检测USP22和BMI-1表达的临床意义。方法:应用定量RT-PCR检测82例大肠癌手术切除标本及相应的癌旁组织中USP22及BMI-1 mRNA的表达水平,并分析其表达的差异,研究其表达水平与大肠癌患者临床病理特征及预后的关系。结果:USP22、BMI-1 mRNA在大肠癌及癌旁组织中均可被检出,大肠癌组织中USP22、BMI-1 mRNA的相对表达水平均显著高于癌旁组织(P0.01),且二者之间呈显著正相关(r=0.708,P0.01)。USP22、BMI-1 mRNA的高表达均与大肠癌的美国癌症分期联合委员会(AJCC)分期密切相关(P0.01)。COX回归分析显示USP22及BMI-1的共表达可作为大肠癌患者预后的独立预测因素(P0.01)。结论:USP22和BMI-1的共同激活可促进大肠癌的进展,并预示预后不良。     

B7-H4 expression associates with cancer progression and predicts patient’s survival in human esophageal squamous cell carcinoma

A retrospective cohort study including 112 patients suffering from esophageal squamous cell carcinoma (ESCC) was performed to investigate the expression of B7-H4 in ESCC and determine its association with patient’s clinicopathological parameters and survival. Expression levels of B7-H4 on tumor cells and densities of tumor infiltrating lymphocytes (TILs) in the surgical specimens of ESCC tissues were characterized using immunohistochemical assays. Uni- and multivariate analyses were performed to evaluate the prognostic value of B7-H4 expression levels and densities of TILs in tumor sections. Positive B7-H4 immunostaining was observed in 107 of 112 (95.5%) of ESCC tissue sections. We further divided all patients into two major subgroups, a lower B7-H4 expression group with 46 patients and a higher B7-H4 expression group with 66 patients. We found that expression levels of B7-H4 on tumor cells were significantly correlated with patient’s gender (P = 0.0288), distant metastasis (P = 0.0500), and TNM stage (P = 0.0258). Moreover, tumor cell B7-H4 expression was inversely correlated with densities of CD3⁺ T cells in tumor nest (P = 0.0424) and CD8⁺ T cells in tumor stroma (P = 0.0229). The overall survival rate of the patients with higher B7-H4 expression was significantly worse than that of the patients with lower B7-H4 expression (P = 0.0105, Hazard Ratio: 1.854, 95%CI:1.152–2.902). Markers of cell-mediated immune responses such as CD3, CD8, and T-bet were associated with better patient survival. The present study demonstrated that B7-H4 expression in human ESCC is associated with cancer progression, reduced tumor immunosurveillance and worse patient outcomes. B7-H4 can serve as a novel prognostic predictor for human ESCC and a potential target for the immune therapy against this malignancy.     

Downregulation of BMI-1 enhances 5-fluorouracil-induced apoptosis in nasopharyngeal carcinoma cells

5-Fluorouracil (5-FU) is an important chemotherapeutic agent for nasopharyngeal carcinoma (NPC). However, drug resistance may occur after several cycles of 5-FU-based chemotherapy. The oncogene B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI-1) has been shown to be involved in the protection of cancer cells from apoptosis. In this study, 5-FU treatment could increase the percentage of apoptotic NPC cells among BMI-1/RNAi-transfected cells than that among cells transfected with the empty vector. The 50% inhibitory concentration (IC₅₀) values of 5-FU were significantly decreased to a greater extent in the cells transfected with BMI-1/RNAi. Most importantly, the expression of phospho-AKT and the anti-apoptotic protein BCL-2 were downregulated in the cells in which BMI-1 expression was inhibited, whereas the apoptosis-inducer BAX was observed to be upregulated. Abrogation of AKT pathway by a PI3K inhibitor could not further increase the sensitivity to 5-FU in the cells with reduced BMI-1 expression. Taken together, BMI-1 depletion enhanced the chemosensitivity of NPC cells by inducing apoptosis; which is associated with inhibition of the PI3K/AKT pathway.     

Curcumin reverses cis-platin resistance and promotes human lung adenocarcinoma A549/DDP cell apoptosis through HIF-1α and caspase-3 mechanisms

Curcumin, a yellow pigment derived from Curcuma longa Linn, has been favored by the Eastern as dietary ingredients for centuries. During the past decade, extensive investigations have revealed curcumin sensitized various chemotherapeutic agents in human breast, colon, pancreas, gastric, liver, brain and hematological malignant disorders in vivo and in vitro. Several pathways and specific targets including NF-κB, STAT3, COX-2, Akt and multidrug resistant protein have been identified to facilitate curcumin as a chemosensitizer. Recent studies suggest HIF-1α participated in the development of drug resistance in cancer cells and targeting HIF-1α either by RNAi or siRNA successfully overcame chemotherapeutic resistance. To investigate the mechanism basis of curcumin as a chemosensitizer in lung cancer, we examined curcumin's effects on HIF-1α in cis-platin (DDP) sensitive A549 and resistant A549/DDP cell lines by RT-PCR and Western blot. HIF-1α in A549/DDP cells was found to be overexpressed at both mRNA and protein levels together with a poor response to DDP. Results from transient transfection and flow cytometry showed the HIF-1α abnormality contributed to DDP resistance in A549/DDP lung cancer cells. Combined curcumin and DDP treatment markedly inhibited A549/DDP cells proliferation, reversed DDP resistance and triggered apoptotic death by promoting HIF-1α degradation and activating caspase-3, respectively. Expression of HIF-1α-dependent P-gp also seemed to decrease as response to curcumin in a dose-dependent manner. Our findings shed light on drug resistant reversing effect of curcumin in lung cancer cells by inhibiting HIF-1α expression and activating caspase-3.