Expression of T-STAR gene is associated with regulation of telomerase activity in human colon cancer cell line HCT-116

AIM： To investigate the effects on telomerase activity of transfection of human T-STAR gene full-length sense cDNA or partial antisense cDNA into human colon cancer cell line HCT-116.METHODS： mRNA and protein expression levels of T-STAR gene were determined by RT-PCR and western blot, and telomerase activity was measured by PCR- ELISA, after transfection of T-STAR sense or antisense gene into HCT-116 cells with lipofectamine. RESULTS： T-STAR gene expression was enhanced or knocked down both at mRNA and protein levels, and telomerase activity was significantly increased or decreased. CONCLUSION： The T-STAR gene may participate in regulation of telomerase activity in human colon cancer HCT-116 cells in a parallel fashion.

Pro-apoptotic Effects of OSNQ on Human Colon Cancer SW480 Cells

[Objectives] The aim was to elucidate the pro-apoptosis mechanism of naphthoquinone derivative 2-octyl sulfoxide-1,4-naphthoquinone(OSNQ) on human colon cancer SW480 cells.[Methods]The cytotoxic effect of OSNQ on colon cancer SW480 cells was detected by MTT colorimetry.The pro-apoptotic effect of OSNQ on human colon cancer SW480 cells was detected by Annexin V-FITC/PI double staining.The changes in expression of apoptosis-related proteins were detected by Western blot.[Results]The results of MTT assay showed that OSNQ had a significant cytotoxic effect on colon cancer SW480 cells.The results of Western blot showed that OSNQ induced the apoptosis in colon cancer SW480 cells through promoting the expression of pro-apoptotic caspase-3 and inhibiting the expression of apoptosis-inhibiting protein Bcl-2.[Conclusions] OSNQ has a significant cytotoxic effect on colon cancer SW480 cells,and it induces the apoptosis of colon cancer SW480 cells by AKT signaling pathway.

Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116

The aim of this study is to assess the effects of DNA methylation and historic acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methyla-tion-specific PCR, and the acetylated status of the histones associated with the p21WAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry. We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73, c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

Radiosensitivity of human colon cancer cell enhanced by immunoliposomal docetaxel

AIM:To enhance the radiosensitivity of human colon cancer cells by docetaxel. METHODS: Immunoliposomal docetaxel was prepared by coupling monoclonal antibody against carcinoembryonic antigen to cyanuric chloride at the PEG terminus of liposome. LoVo adenocarcinoma cell line was treated with immunoliposomal docetaxel or/and irradiation. MTT colorimetric assay was used to estimate cytotoxicity of immunoliposomal docetaxel and radiotoxicity. Cell cycle redistribution and apoptosis were determined with flow cytometry. Survivin expression in LoVo cells was verified by immunohistochemistry. D801 morphologic analysis system was used to semi-quantify immunohistochemical staining of survivin. RESULTS: Cytotoxicity was induced by immunoliposomal docetaxel alone in a dose-dependent manner. Immunoliposomal docetaxel yielded a cytotoxicity effect at a low dose of 2 nmol/L. With a single dose irradiation, the relative surviving fraction of LoVo cells showed a dose-dependent response, but there were no significant changes as radiation delivered from 4 to 8 Gy. Compared with liposomal docetaxel or single dose irradiation, strongly radiopotentiating effects of immunoliposomal docetaxel on LoVo cells were observed. A low dose of immunoliposomal docetaxel could yield sufficient radiosensitivity. Immunoliposomal docetaxel were achieved both specificity of the conjugated antibody and drug radiosensitization. Combined with radiation, immunoliposomal docetaxel significantly increased the percentage of G2/M cells and induced apoptosis, but significantly decreased the percentage of cells in G2/G1 and S phase by comparison with liposomal docetaxel. Immunohistochemical analysis showed that the brown stained survivin was mainly in cytoplasm of LoVo cells. Semi-quantitative analysis of the survivin immunostaining showed that the expression of survivin in LoVo cells under irradiation with immunoliposomal docetaxel was significantly decreased. CONCLUSION: Immunoliposomal docetaxel is strongly effective for target radiosensitation in LoVo colon carcinoma cells, and may offer the potential to improve local radiotherapy.

Anti-cancer effect of ethylacetate fraction from Orostachys japonicus on HT-29 human colon cancer cells by induction of apoptosis through caspase-dependent signaling pathway

Objective: To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus(0. japonicus) on HT-29 cancer cells. Methods: The viability of HT-29 cells was assayed by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2 H-tetrazolium(MTS) method. Apoptosis induction and cell cycle inhibition were confirmed by fluorescein isothiocyanate and propidium iodide staining using flow cytometry.Morphological changes in the nucleus were observed, using a fluorescence microscope with4',6-diamidino-2-phenylindole(DAPI) nuclear staining. The expression levels of the upstream and downstream proteins involved in the anti-cancer mechanism were confirmed by Western blotting. Results: After treating HT-29 cells with different concentrations of ethylacetate fraction from O. japonicus, the viability of cells decreased in a concentration-dependent manner,while apoptosis induction and apoptotic body formation increased. Cell cycle analysis showed that the arrest occurred at the sub-G_1 and S phase. Among the upstream and downstream proteins involved in anti-cancer activity, the level of B cell lymphoma-2 decreased, and the bcl-2-associated x protein increased. The level of pro-caspase-3, pro-caspase-8, and pro-caspase-9 decreased, while the level of cleaved-caspase-3, cleaved-caspase-8, and cleaved-caspase-9 increased. Moreover, the phosphorylation, that is, activation of extracellular signal regulated kinase 1/2, Jun-N-terminal kinase, and p38 increased. Conclusions: Combining the above results, it is thought that the survival of HT-29 cells is suppressed by ethylacetate fraction from0. japonicus through mitochondrial regulation-induced caspase cascade activation, induction of apoptosis and cell cycle arrest.

Interaction between Colon Cancer Cells and Human Liver Sinusoidal Endothelial Cells Promotes Liver Metastasis of Tumor Cells

OBJECTIVE To investigate the effect of co-culture between colon cancer cells （SW1116） and human liver sinusoidal endothelial cells （HLSECs） on cancer cell metastasis, and to provide a novel model for studying the mechanism of colon cancer liver metastasis. METHODS HLSECs and SW1116 were co-cultured for 21 rounds in vitro. Transwell migration, gelatin-zymography, CCK-8 proliferation and colony formation assays were used to examine the invasion, proliferation, and colony forming ability of cancer cells. Assays were carried out to examine tumor growth ability and liver metastasis. The associated molecular change was examined by western blotting. RESULTS After 21 selection rounds, colon cancer cells SWl 1161）21 displayed a clear boundary. Compared with the 5W1116 cells, SW1116P21 cells had a greater invasive ability, cell proliferation and colony formation in soft agar. A gelatin-zymography assay showed that the ability of SW1116P21 cells to secrete matrix metalloproteinase-2/9 was significantly greater than that of SWl116 cells. Additionally, the capacity for subcutaneous tumor formation of SW1116P21 was significantly increased. It was found that mice injected with SW1116P21 cells developed significantly more visually observable liver nodules than mice injected with SW1116 cells. Western blotting showed increased vimentin expression and decreased E-cadherin expression in the SW1116P21 cells, compared with the SWl 116 cells. CONCLUSION The interaction between SW1116 and HLSECs may promote tumor cell invasion, proliferation and colony formation in vitro, and tumor formation and liver metastasis in vivo. An epithelial-mesenchymal transition occurs in SWl 116P21 cells, which contributes to the change in the characteristics of tumor cells.

Estradiol agonists inhibit human Lo Vo colorectal-cancer cell proliferation and migration through p53

AIM: To investigate the effects of 17β-estradiol via estrogen receptors (ER) or direct administration of ER agonists on human colorectal cancer.

Fermented Herbal Decoction Selectively Targeting Human Cancer Cell Line and Human Pathogenic Microorganism

Introduction: Prolonged immuno-suppressed status promised to induce internal growth of malignant cell and infectious agent, yet, only a small part of affected individuals seek medical attention or berried by commercially over-flowed fake information. Several studies have described complementary and alternative medicine as effective strategies for improving anti-infectious agent including malignant cell. The purpose of this study was to investigate the effect of a fermented herbal decoction (FHD) both in vitro and in vivo to malignant cells and microorganism by regulating leukocyte subset proportioning FHD as dietary material. Methods: In this approach of alternative study, selective anti-cancer effect by fermented decoction was tried to show first in vitro system both, cancer cell and virus strain. The fermented herbal decoction consisting of 80 sorts of herbs and fruits. The selective toxicity was set up and then for immunological factors in animal and human. The most important factor is to reduce side effect for a normal cell. Results: First, FHD was proved as safe by animal test. FHD regulated also the proportion of granulocyte and lymphocyte ratio both animal and human. In vitro culture showed selective toxicity by FHD against human melanoma and leukemia cell line but reduced toxicity was showed by normal cell line. As for the anti-virus activity, anti-virus effect was tested on the feeder layer of human fibroblast cell, after 9 days of culture. Second, FHD inhibits colon cancer growth in 3-methylholanthrene induced cancer in rat. Conclusion: The present results suggest that our fermented herbal decoction showed selective anti-cancer activities and anti-virus activities, together with the regulative effect on the immune system.

抗肿瘤抗生素C1027对人结肠癌HCT-8细胞凋亡相关基因表达的影响

目的：研究抗肿瘤抗生素Ｃ１０２７ 对人结肠癌ＨＣＴ８ 细胞凋亡相关基因表达的影响。方法：利用ｃＤＮＡ 排列（ｃＤＮＡａｒｒａｙｓ）、Ｎｏｒｔｈｅｒｎ 杂交、ＲＴＰＣＲ 检测技术。结果：用１０ ｎｍｏｌ·Ｌ－ １ Ｃ１０２７ 处理ＨＣＴ８ 细胞８ ｈ 后，利用ｃＤＮＡ 排列检测，发现Ｃ１０２７ 可以促进ＴＲＩＰ，ＴＲＡＦ３ ，ＤＲ４ ，ＭＣＨ６ ，ＭＣＨ４，Ａｐｏ３ ，ＤＲ５，ＡＢＬＬ，ＳＴＡＴ１ 等基因的表达，抑制ｒｈｏＣ的表达。用Ｎｏｒｔｈｅｒｎ 杂交及ＲＴＰＣＲ 也得到了同样结果。结论：Ｃ１０２７ 可能通过调节ＴＮＦ 受体家族有关的凋亡信号传导通路，诱导细胞凋亡。

RNA干扰PTEN基因对HCT-8细胞增殖能力的影响

目的:观察小干扰RNA(siRNA)对结肠癌相关基因PTEN表达的抑制作用及其对结肠癌细胞增殖能力的影响。方法:构建携带有针对PTEN基因序列的siRNA真核绿色荧光表达载体重组质粒pGPU6/GFP/Neo-PTEN-1、pGPU6/GFP/Neo-PTEN-2、pGPU6/GFP/Neo-PTEN-3和pGPU6/GFP/Neo-PTEN-4,脂质体法转染入结肠癌HCT-8细胞(分别为p1、p2、p3和p4组),同时设转染阴性对照质粒组(p5组)和亲本细胞组(p6组),实时PCR方法检测PTEN mRNA表达,MTT法分析细胞增殖活性变化。结果:通过荧光显微镜观察计数,细胞转染率为58%。实时PCR方法检测结果显示,siRNA重组质粒pGPU6/GFP/Neo-PTEN-1、pGPU6/GFP/Neo-PTEN-2、pGPU6/GFP/Neo-PTEN-3和pGPU6/GFP/Neo-PTEN-4的抑制率分别为48.3%、76.5%、29.4%和61.2%,与p6组比较差异均有统计学意义(P<0.05)。MTT结果表明,细胞增殖抑制能力明显增强。结论:PTEN基因在肿瘤的发生发展中可能起到一定的作用。

腺病毒介导miR-99a过表达抑制人结肠癌HCT-8细胞的生长抑制及其作用机制

目的探讨miR-99a过表达对人结肠癌HCT-8细胞的生长抑制及其作用机制。方法构建miR-99a的重组腺病毒Ad5-miR-99a,并感染人结肠癌HCT-8细胞。采用甲基化特异性PCR检测miR-99a、抑癌基因SOCS1和P16的甲基化水平;采用实时荧光定量PCR和Western Blot检测miR-99a、DNAC5胞嘧啶甲基转移酶、SOCS1和P16的表达情况;采用MTT法、细胞集落形成和流式细胞术检测HCT-8细胞的体外增殖凋亡情况;采用裸鼠体内荷瘤实验检测HCT-8细胞体内成瘤性。结果成功构建miR-99a腺病毒载体Ad5-miR-99a。Ad5-miR-99a感染HCT-8细胞后,Ad5-miR-99a组细胞中miR-99a表达显著高于空载腺病毒组和空白对照组(P<0.05),而其甲基化水平显著低于空载腺病毒组和空白对照组(P<0.05);Ad5-miR-99a组细胞中DNAC5胞嘧啶甲基转移酶表达显著低于空载腺病毒组和空白对照组(P<0.05);Ad5-miR-99a组细胞中SOCS1和P16表达显著高于空载腺病毒组和空白对照组(P<0.05),而其甲基化水平显著低于空载腺病毒组和空白对照组(P<0.05)。MTT法、细胞集落形成、流式细胞术和裸鼠体内荷瘤实验显示,Ad5-miR-99a组细胞的体外增殖和体内成瘤能力显著低于空载腺病毒组和空白对照组(P<0.05)。结论 miR-99a可显著抑制人结肠癌HCT-8细胞的体外增殖和体内成瘤能力,其机制可能与miR-99a过表达导致DNMT1表达升高,降低抑癌基因SOCS-1和P16甲基化程度,使得SOCS-1和P16高表达,从而发挥抑制肿瘤细胞生长的作用。

人结肠腺癌细胞HCT-8绿色荧光标记肿瘤模型的建立

目的筛选表达绿色荧光蛋白基因的人的单克隆结肠腺癌细胞系,为体内监测肿瘤的早期生长建立一种新的肿瘤动物模型。方法以脂质体2000介导chicken-βactin-GFP-NEO转染人结肠腺癌细胞HCT-8,经梯度浓度G418筛选获得稳定表达绿色荧光蛋白的细胞克隆并扩大培养。BALB/CA-nu裸鼠皮下接种1×106个发光细胞使其成瘤,活体荧光成像系统观察肿瘤的生长情况。结果获得了稳定表达GFP的人结肠腺癌细胞株,将其接种到裸鼠体内可成瘤,利用活体成像系统观察了肿瘤的生长过程,肿瘤的发光随着观察时间的延长而增加。结论绿色荧光蛋白能够在人结肠腺癌细胞HCT-8中长期稳定表达,用绿色荧光蛋白标记的人结肠腺癌细胞HCT-8建立的裸鼠肿瘤模型为进一步研究结肠肿瘤和相应的药物筛选提供了一种简便、可行的新方法。

姜烯酚6衍生物M14通过调控Keap1/Nrf2信号通路诱导人结直肠癌HCT-8细胞凋亡

目的探讨姜烯酚6衍生物M14(S6-M14)诱导人结直肠癌HCT-8细胞凋亡的作用及可能的分子机制。方法MTT法检测不同浓度的姜烯酚6衍生物M14对人结直肠癌HCT-8细胞增殖的影响;ATP显色法检测姜烯酚6衍生物M14促进人结直肠癌HCT-8细胞凋亡的剂量依赖效应;Western blot检测胞质内Keap1和Nrf2蛋白及核内Nrf2蛋白的表达。结果0.25、0.5、1、2、4 mmol·L^(-1)S6-M14均可抑制人结直肠癌HCT-8细胞的增殖(P均<0.001);姜烯酚6衍生物M14剂量依赖性地诱导人结直肠癌HCT-8细胞凋亡;姜烯酚6衍生物M14下调人结直肠癌HCT-8细胞胞质内Keap1和Nrf2蛋白表达,促进Nrf2蛋白的核易位(P均<0.05)。结论姜烯酚6衍生物M14抑制人结直肠癌HCT-8细胞增殖、诱导细胞凋亡,调控Keap1/Nrf2信号通路,有潜在的预防结直肠癌发生的作用。

RNA干扰IDH2基因对HCT-8细胞增殖能力的影响

目的:研究RNA干扰(RNAi)对结肠癌HCT-8细胞IDH2基因表达的抑制作用及干扰后对HCT-8细胞增殖的影响,为结肠癌的基因靶向治疗提供理论依据。方法:实验分siRNA干扰质粒转染组、阴性质粒对照组和HCT-8细胞空白对照组。构建靶向基因IDH2的siRNA真核绿色荧光载体重组质粒PGPU6/GFP/Neo-IDH2,利用FuGENE HD转染剂转染HCT-8细胞,通过荧光定量PCR检测IDH2mRNA的表达情况及MTT法检测结肠癌HCT-8细胞增殖变化。结果:在荧光显微镜下进行细胞计数,细胞转染率为74%。实时荧光定量PCR检测,siRNA重组质粒PGPU6/GFP/Neo-IDH2对IDH2基因表达的抑制率为85.02%,与空白对照组比较差异有统计学意义(P<0.05)。MTT检测,干扰质粒转染组细胞的增殖能力明显低于空白对照组(P<0.05)。结论:抑制IDH2基因的表达可影响结肠癌细胞的增殖能力,提示IDH2基因在结肠癌的发生发展中起重要作用,抑制IDH2基因的表达可能成为一种治疗结肠癌的方法。

Identification of an anticancer compound against HT-29 cells from Phellinus linteus grown on germinated brown rice

Objective:To isolate and identify the anticancer compound against proliferation of human colon cancer cells from ethyl acetate(EtOAc)extract ol Phellinus linteus grown on germinated brown rice(PB).Methods:EtOAc extract of PB was partitioned with n-hexane,EtOAc,and water-saturated n-butanol.Anticancer compound of n-hexane layer was isolated and identified by HPLC and NMR,respectively.Cytotoxicity against HT-29 cells was tested by SRB assay.Results:The n-hexane layer obtained after solvent fractionation of PB EtOAc extracts showed a potent anticancer activity against the HT-29 cell line.Atractylenolide I,a eudesmane-type sesquiterpene lactone,a major anticancer substance of PB,was isolated from the n-hexane layer by silica gel column chromatography and preparative-HPLC.This structure was elucidated by one-and two-dimensional NMR spectroscopic data.Atractylenolide I has not been reported in mushrooms or rice as of yet.The isolated compound dose-dependently inhibited the growth of HT-29 human colon cancer cells.Conclusions:Atractylenolide I might contribute to the anticancer effect of PB.

Oxidative stress-induced mitochondrial dysfunction in a normal colon epithelial cell line

AIM To determine how a normal human colon cell line reacts to microbial challenge as a way to study oxidative stress-induced responses associated with inflammatory bowel disease.METHODS Normal human colon epithelial cells(ATCC?CRL.1790?)were stimulated with either heat killed E.coli or heat killed murine cecal contents(HKC)and examined for several relevant biomarkers associated with inflammation and oxidative stress including cytokine production,mitochondrial autophagy and oxidant status.TNFα,IL-1βand IL-8 protein concentrations were measured within the supernatants.Fluorescent microscopy was performed to quantify the production of reactive oxygen species(ROS)using an oxidation responsive fluorogenic probe.Mitochondrial morphology and mitochondrial membrane potential was assessed by dual staining using COXIV antibody and a dye concentrating in active mitochondria.Mitochondrial ROS scavenger was used to determine the source of ROS in stimulated cells.Autophagy was detected by staining for the presence of autophagic vesicles.Positive controls for autophagy and ROS/RNS experiments were treated with rapamycin and chloroquine.Mitochondrial morphology,ROS production and autophagy microscopy experiments were analyzed using a custom acquisition and analysis microscopy software(Image J).RESULTS Exposing CRL.1790 cells to microbial challenge stimulated cells to produce several relevant biomarkers associated with inflammation and oxidative stress.Heat killed cecal contents treatment induced a 10-12fold increase in IL-8 production by CRL.1790 cells compared to unstimulated controls at 6 and 12 h(P<0.001).Heat killed E.coli stimulation resulted in a4-5 fold increase in IL-8 compared to the unstimulated control cells at each time point(P<0.001).Both heat killed E.coli and HKC stimulated robust ROS production at 6(P<0.001),and 12 h(P<0.01).Mitochondrial morphologic abnormalities were detected at 6 and12 h based on reduced mitochondrial circularity and decreased mitochondrial membrane potential,P<0.01.Microbial stimulation also induced significant autophagy at 6 and 12 h,P<0.01.Lastly,blocking mitochondrial ROS generation using mitochondrial specific ROS scavenger reversed microbial challenge induced mitochondrial morphologic abnormalities and autophagy.CONCLUSION The findings from this study suggest that CRL.1790cells may be a useful alternative to other colon cancer cell lines in studying the mechanisms of oxidative stress events associated with intestinal inflammatory disorders.

二甲双胍与顺铂联合诱导人结肠癌HCT-8细胞凋亡的研究

目的研究二甲双胍联合顺铂对HCT-8人类结肠癌细胞凋亡的影响及其可能机制。方法将HCT-8人类结肠癌细胞分成二甲双胍组(MET)、顺铂组(DDP)、二甲双胍联合顺铂组(MET+DDP)、空白对照组(CK) 4组,MTT实验观察4组细胞于24 h、48 h、72 h时的增殖能力;流式细胞术观察各组细胞48 h的凋亡比例;Western blotting观察各组细胞48 h时凋亡相关蛋白Bcl-2、Bax、caspase-3(激活型)以及AKT/GSK-3通路蛋白的表达水平。结果该实验中MET+DDP组的细胞增殖能力弱于其它三组(P<0. 05);细胞凋亡比例高于其它三组(P<0. 05); Bax、caspase-3(激活型)表达量高于其它组群,Bcl-2、P-AKT、P-GSK-3表达量低于其它组群,AKT、GSK-3在4组中表达量相对恒定(P>0. 05)。结论二甲双胍、顺铂可能通过下调AKT/GSK-3信号通路,改变Bcl-2家族蛋白的表达活性从而促进HCT-8人类结肠癌细胞的凋亡,并促进p-caspase-3剪切转化为caspase-3(激活型);二甲双胍与顺铂可协同发挥促HCT-8人类结肠癌细胞凋亡的作用。

黑水虻抗菌肽HI-3对人结肠癌HCT-8细胞谷氨酰胺和谷氨酸代谢通路的影响

【目的】研究黑水虻Hermetia illucens抗菌肽HI-3对人结肠癌HCT-8细胞氨基酸代谢的影响,以丰富对其抑癌机理的认识。【方法】采用CCK-8法测定不同浓度(80,160和320μg/mL)抗菌肽HI-3对HCT-8细胞的抑制率;利用GC-MS进行HCT-8细胞代谢物测定,通过基于R软件的通路分析找出氨基酸含量差异最显著的氨基酸代谢通路并筛选出该通路靶标酶。320μg/mL HI-3处理HCT-8细胞后,利用酶活性检测试剂盒测定靶标酶谷氨酰胺酶(GLS)活性;利用RT-qPCR和Western blot技术分别对HCT-8细胞的GLS基因进行mRNA及蛋白表达水平的测定;利用生化试剂盒和ELISA试剂盒检测HCT-8细胞内谷氨酰胺和谷氨酸代谢通路涉及的重要代谢物谷氨酰胺(Gln)、谷氨酸(Glu)、谷胱甘肽(GSH)、α-酮戊二酸(α-KG)和ATP含量的变化。【结果】浓度为80,160和320μg/mLHI-3对HCT-8细胞的抑制率分别为33.85%±3.50%,46.26%±0.90%和55.53%±1.70%,且抑制率随HI-3浓度升高而增大。320μg/mL HI-3处理对谷氨酰胺和谷氨酸代谢通路的影响最大,其中氨基酸代谢物含量与阴性对照组(0μg/mL HI-3)相比差异最为显著;这一通路中的靶标酶GLS活性及其GLS的mRNA和蛋白表达水平均极显著低于阴性对照组;另外与此通路相关的重要代谢物Gln,Glu,GSH,α-KG和ATP含量与阴性对照组相比亦显著减少。【结论】浓度为320μg/mL黑水虻抗菌肽HI-3对HCT-8细胞谷氨酰胺和谷氨酸代谢通路影响最为显著,并能通过阻碍该通路来显著抑制HCT-8细胞的增殖。

Expression of nucleus accumbens-1 in colon cancer negatively modulates antitumor immunity

BACKGROUND Nucleus accumbens-1(NAC-1)is highly expressed in a variety of tumors,including colon cancer,and is closely associated with tumor recurrence,metastasis,and invasion.AIM To determine whether and how NAC-1 affects antitumor immunity in colon cancer.METHODS NAC-1-siRNA was transfected into RKO colon cancer cells to knock down NAC expression;tumor cells with or without knockdown of NAC-1 were treated with CD8+T cells to test their cytocidal effect.The level of the immune checkpoint programmed death receptor-1 ligand(PD-L1)in colon cancer cells with or without knockdown of NAC-1 was analyzed using Quantitative real-time polymerase chain reaction and Western blotting.A double luciferase reporter assay was used to examine the effects of NAC-1 on the transcription of PD-L1.Mice bearing MC-38-OVA colon cancer cells expressing NAC-shRNA or controlshRNA were treated with OT-I mouse CD8+T cells to determine the tumor response to immunotherapy.Immune cells in the tumor tissues were analyzed using flow cytometry.NAC-1,PD-L1 and CD8+T cells in colon cancer specimens from patients were examined using immunohistochemistry staining.RESULTS Knockdown of NAC-1 expression in colon cancer cells significantly enhanced the cytocidal effect of CD8+T cells in cell culture experiments.The sensitizing effect of NAC-1 knockdown on the antitumor action of cytotoxic CD8+T cells was recapitulated in a colon cancer xenograft animal model.Furthermore,knockdown of NAC-1 in colon cancer cells decreased the expression of PD-L1 at both the mRNA and protein levels,and this effect could be rescued by transfection of an RNAi-resistant NAC-1 expression plasmid.In a reporter gene assay,transient expression of NAC-1 in colon cancer cells increased the promoter activity of PD-L1,indicating that NAC-1 regulates PD-L1 expression at the transcriptional level.In addition,depletion of tumoral NAC-1 increased the number of CD8+T cells but decreased the number of suppressive myeloid-derived suppressor cells and regulatory T cells.CONCLUSION Tumor expression of NAC-1 is a negative determinant of immunotherapy.

PD-L1和CD8在HER2阳性乳腺癌患者中的表达及其与病理参数相关性分析

目的通过评估程序性死亡受体配体1(programmed cell death-ligand 1,PD-L1)和分化群8(cluster of differentiation 8,CD8)在肿瘤微环境中的表达情况及其与患者临床病理特征的相关性,明确PD-L1和CD8在判断人表皮生长因子受体2(human epidermal growth factor receptor 2,HER2)阳性乳腺癌患者预后方面的作用。方法选取2016年10月至2018年10月山东省滕州市中心人民医院收治的HER2阳性乳腺癌患者65例,所有患者均为女性。癌组织切片经免疫组织化学方法标记PD-L1和CD8;收集患者的临床病理资料,包括年龄、组织学分级、P53、KI-67、雌激素受体、孕激索受体、肿瘤淋巴结转移分类(tumor node metastasis classification,TNM)分期在内的7项与预后相关的临床病理参数。分别分析PD-L1和CD8的表达是否和上述临床病理参数相关。结果PD-L1阳性表达率为21.5%,PD-L1阳性表达与组织学分级和TNM分期相关,差异有统计学意义(χ^(2)=6.250,P=0.044;χ^(2)=13.730,P=0.001);CD8阳性表达率为21.5%,CD8表达与组织学分级和TNM分期相关,差异有统计学意义(χ^(2)=8.023,P=0.018;χ^(2)=6.117,P=0.047)。结论PD-L1和CD8表达与乳腺癌组织学分级和TNM分期相关,可能是HER2阳性乳腺癌患者重要的预后标志物。