Alterations in APC, BECN1, and TP53 gene expression levels in colon cancer cells caused by monosodium glutamate / Alterações nos níveis de expressão gênica APC, BECN1 e TP53 em células de câncer de cólon causadas por glutamato monossódico

Colorectal cancer (CRC) is a disease with high incidence worldwide. As of 2018, it is the second leading cause of cancer deaths in the world. In Saudi Arabia, the incidence of this disease has been increasing in the younger population. Both genetic and lifestyle factors may have contributed to its increased incidence and pathogenesis. Monosodium glutamate (MSG) is a food flavor enhancer that can be found in many commercial foods, and it can sometimes be used as a substitute to table salt. MSG has been investigated for its possible genotoxicity, yielding controversial results. In the present study, the effect of MSG on cell viability and its effect on expression of APC, BECN1, and TP53 genes in SW620 and SW480 colon cancer cell lines were studied. TP53 is a tumor suppressor gene that functions in modifying DNA errors and/or inducing apoptosis of damaged cells, and both APC and BECN1 genes are involved in CRC and are of importance in cellular growth and metastasis. Cancer cell viability was analyzed using MTT assay, and the results showed a significant increase in the number of viable cells after 24h of treatment with MSG with different concentrations (0.5, 1.0, 10, 50, and 100mM). Moreover, gene expression results showed a significant increase in the expression levels of APC and BECN1 under specified conditions in both cell lines; conversely, TP53 showed a significant decrease in expression in SW620 cells. Thus, it can be concluded that MSG possibly confers a pro-proliferative effect on CRC cells.

O câncer colorretal (CCR) é uma doença com alta incidência mundial. Desde 2018, é a segunda principal causa de mortes por câncer no mundo. Na Arábia Saudita, a incidência dessa doença vem aumentando na população mais jovem. Tanto fatores genéticos quanto de estilo de vida podem ter contribuído para o aumento da sua incidência e patogênese. O glutamato monossódico (MSG) é um intensificador de sabor de alimentos que pode ser encontrado em muitos alimentos comerciais e às vezes pode ser usado como um substituto do sal de cozinha. O MSG tem sido investigado por sua possível genotoxicidade, produzindo resultados controversos. Neste estudo, foram estudados o efeito do MSG na viabilidade celular e seu efeito na expressão dos genes APC, BECN1 e TP53 em linhas de células de câncer de cólon SW620 e SW480. TP53 é um gene supressor de tumor que atua modificando erros de DNA e/ou induzindo apoptose de células danificadas, estando os genes APC e BECN1 envolvidos no CRC e sendo importantes no crescimento celular e metástase. A viabilidade das células cancerosas foi analisada por meio do ensaio MTT, e os resultados mostraram um aumento significativo no número de células viáveis após 24 h de tratamento com MSG em diferentes concentrações (0,5; 1,0; 10; 50 e 100mM). Além disso, os resultados da expressão gênica mostraram um aumento significativo nos níveis de expressão de APC e BECN1 sob condições especificadas em ambas as linhagens celulares. Por outro lado, TP53 mostrou uma diminuição significativa na expressão em células SW620. Assim, pode-se concluir que, possivelmente, o MSG confere um efeito pró-proliferativo às células CRC.

Alterations in APC, BECN1, and TP53 gene expression levels in colon cancer cells caused by monosodium glutamate

Abstract Colorectal cancer (CRC) is a disease with high incidence worldwide. As of 2018, it is the second leading cause of cancer deaths in the world. In Saudi Arabia, the incidence of this disease has been increasing in the younger population. Both genetic and lifestyle factors may have contributed to its increased incidence and pathogenesis. Monosodium glutamate (MSG) is a food flavor enhancer that can be found in many commercial foods, and it can sometimes be used as a substitute to table salt. MSG has been investigated for its possible genotoxicity, yielding controversial results. In the present study, the effect of MSG on cell viability and its effect on expression of APC, BECN1, and TP53 genes in SW620 and SW480 colon cancer cell lines were studied. TP53 is a tumor suppressor gene that functions in modifying DNA errors and/or inducing apoptosis of damaged cells, and both APC and BECN1 genes are involved in CRC and are of importance in cellular growth and metastasis. Cancer cell viability was analyzed using MTT assay, and the results showed a significant increase in the number of viable cells after 24 h of treatment with MSG with different concentrations (0.5, 1.0, 10, 50, and 100mM). Moreover, gene expression results showed a significant increase in the expression levels of APC and BECN1 under specified conditions in both cell lines; conversely, TP53 showed a significant decrease in expression in SW620 cells. Thus, it can be concluded that MSG possibly confers a pro-proliferative effect on CRC cells.

Resumo O câncer colorretal (CCR) é uma doença com alta incidência mundial. Desde 2018, é a segunda principal causa de mortes por câncer no mundo. Na Arábia Saudita, a incidência dessa doença vem aumentando na população mais jovem. Tanto fatores genéticos quanto de estilo de vida podem ter contribuído para o aumento da sua incidência e patogênese. O glutamato monossódico (MSG) é um intensificador de sabor de alimentos que pode ser encontrado em muitos alimentos comerciais e às vezes pode ser usado como um substituto do sal de cozinha. O MSG tem sido investigado por sua possível genotoxicidade, produzindo resultados controversos. Neste estudo, foram estudados o efeito do MSG na viabilidade celular e seu efeito na expressão dos genes APC, BECN1 e TP53 em linhas de células de câncer de cólon SW620 e SW480. TP53 é um gene supressor de tumor que atua modificando erros de DNA e/ou induzindo apoptose de células danificadas, estando os genes APC e BECN1 envolvidos no CRC e sendo importantes no crescimento celular e metástase. A viabilidade das células cancerosas foi analisada por meio do ensaio MTT, e os resultados mostraram um aumento significativo no número de células viáveis após 24 h de tratamento com MSG em diferentes concentrações (0,5; 1,0; 10; 50 e 100mM). Além disso, os resultados da expressão gênica mostraram um aumento significativo nos níveis de expressão de APC e BECN1 sob condições especificadas em ambas as linhagens celulares. Por outro lado, TP53 mostrou uma diminuição significativa na expressão em células SW620. Assim, pode-se concluir que, possivelmente, o MSG confere um efeito pró-proliferativo às células CRC.

METTL3-modified RNASEH1-AS1 regulates the malignant biological behaviors of colorectal cancer SW480 cells through the BUD13/ANXA2/Wnt/β-catenin axis / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探究甲基转移酶样蛋白3（METTL3）修饰的RNASEH1-AS1通过BUD13/膜联蛋白A2/ Wnt/β-连环蛋白（BUD13/ANXA2/Wnt/β-catenin）轴调控结直肠癌细胞SW480恶性生物学行为的分子机制。方法：选取2022年6月至2022年11月间在复旦大学附属中山医院厦门医院手术治疗的24例CRC患者，收集患者的CRC组织和对应的癌旁组织，用qPCR法检测其中METTL3、RNASEH1-AS1、BUD13、ANXA2、β-catenin、GSK-3β mRNA的表达，用Pearson法分析CRC组织中RNASEH1-AS1表达分别与METTL3和BUD13表达的相关性。常规培养结直肠癌细胞SW480，分为sh-NC组、sh-RNASEH1-AS1组、NC组、sh-METTL组、si-NC组、si-BUD13组、sh-RNASEH1-AS1+pc-NC组、sh-RNASEH1-AS1+pc-ANXA2组、sh-METTL+pc-NC组、sh-METTL+pc-ASEH1-AS1组，用Lipofectamine® 2000转染试剂将sh-NC、sh-RNASEH1-AS1、sh-NC、sh-METTL、si-NC、si-BUD13、sh-RNASEH1-AS1+pc-NC、sh-RNASEH1-AS1+pc-ANXA2、sh-METTL+pc-NC、sh-METTL+pc-ASEH1-AS1分别转染SW480细胞，用qPCR法检测后SW480细胞中METTL3、RNASEH1-AS1、BUD13、ANXA2的表达，细胞克隆形成实验检测转染后各组SW480细胞的增殖能力，FCM术检测转染后各组SW480细胞的凋亡情况，细胞划痕实验检测转染后各组SW480细胞的迁移能力，WB法检测转染后各组SW480细胞中ANXA2、β-catenin、p-β-catenin、GSK-3β、p-GSK-3β、c-Myc、cyclinD1蛋白表达，RNA免疫沉淀（RIP）法检测RNASEH1-AS1与BUD1、BUD1与ANXA2的靶向关系。结果：数据库CRC数据分析和中国人CRC组织和癌旁组织的qPCR法检测结果均显示，与癌旁组织相比CRC组织中METTL3、RNASEH1-AS1、BUD13、ANXA2、β-catenin、GSK-3β均呈明显高表达（均P<0.01），且RNASEH1-AS1表达与METTL3（r=0.698，P<0.01）、BUD13（r=0.784，P<0.01）的表达呈正相关。在结直肠癌各细胞中METTL3、RNASEH1-AS1、BUD13、ANXA2 mRNA均呈高表达（均P<0.05）；敲减RNASEH1-AS1或METTL3后SW480细胞中RNASEH1-AS1、BUD13、ANXA2表达显著降低（均P<0.05），而过表达RNASEH1-AS1后上述分子表达明显上调（均P<0.05）；敲减RNASEH1-AS1或METTL3可抑制SW480的增殖、迁移和p-β-catenin、p-GSK-3β、c-Myc、cyclinD1蛋白的表达，促进其凋亡（均P<0.05），而过表达RNASEH1-AS1则可促进SW480增殖、迁移和p-β-catenin、p-GSK-3β、c-Myc、cyclinD1蛋白的表达和抑制其凋亡（均P<0.05）；RNASEH1-AS1通过招募BUD13靶向促进ANXA2的表达（均P<0.05）；过表达ANXA2可部分逆转敲减RNASEH1-AS1对SW480细胞的影响（均P<0.05）。结论：METTL3修饰的RNASEH1-AS1通过BUD13/ANXA2/Wnt/β-catenin轴调控SW480细胞的恶性生物学行为。

Specific activation of G protein-coupled estrogen receptor regulates colorectal cancer cell migration through reactive oxygen species pathways / 中国肿瘤生物治疗杂志

@# [摘 要] 目的：探讨特异性激活G蛋白偶联雌激素受体（GPER）对结直肠癌（CRC）细胞迁移的作用及其可能的机制。方法：体外培养CRC细胞RKO和SW480，使用0.5或1.0 μmol/L的GPER特异性激活剂（G-1）处理CRC细胞，采用CCK-8法、划痕实验和Transwell实验分别检测G-1对CRC细胞增殖、迁移的影响。用qPCR和WB法分别检测G-1及G-1+活性氧（ROS）清除剂N-乙酰半胱氨酸（NAC）处理对RKO细胞E-钙黏素（E-Cad）、纤连蛋白（FN）mRNA和蛋白表达的影响，用流式细胞术检测RKO细胞中ROS的水平。结果：经G-1处理后RKO和SW480细胞的迁移能力均明显减弱（均P<0.05），能显著上调细胞中E-cad mRNA及蛋白表达、下调FN mRNA及蛋白表达（均P<0.05）。G-1处理能刺激RKO细胞ROS水平上升，在NAC的作用下，由G-1引起的E-cad、FN蛋白表达变化被部分逆转。结论：特异性激活GPER通过上调ROS水平抑制EMT进程，进而抑制CRC细胞的迁移。

Expression of phosphoglycerate-mutase 1 in colorectal cancer tissues and its effects on the prognosis and malignant biological behaviors of cancer cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨结直肠癌（CRC）组织中磷酸甘油酸变位酶1（PGAM1）的表达及其与患者预后的关系，研究PGAM1对CRC细胞增殖、迁移和侵袭的影响。方法：选择2003年3月至2008年11月间在天津医科大学肿瘤医院手术切除的30例CRC患者的肿瘤组织标本及临床资料，采用免疫组织化学染色法检测CRC组织中PGAM1蛋白的表达，分析PGAM1表达与患者临床病理特征的关系，Kaplan-Meier生存分析法比较PGAM1高表达与低表达患者的OS、PFS来评价PGAM1表达与患者预后的关系。利用RNA干扰技术分别将si-PGAM1及si-NC质粒转染至HCT-116和SW480细胞，WB法检测转染细胞中PGAM1蛋白的表达水平，CCK-8、Transwell实验分别检测敲低PGAM1对CRC细胞增殖、迁移和侵袭的影响。结果：30例CRC组织中PGAM1阳性染色定位于CRC细胞的细胞质，其中33.3%（10/30例）呈高表达。虽然PGAM1高表达与CRC患者年龄、性别、组织学类型、肿瘤大小、淋巴结转移、远处转移及临床TNM分期无关（均P>0.05），但是PGAM1高表达与低表达患者相比其OS、PFS显著缩短。在CRC细胞中敲低PGAM1后，细胞的增殖、迁移和侵袭能力均显著降低（均P<0.05）。结论：CRC组织中PGAM1呈高表达，PGAM1高表达的患者预后较差；敲低PGAM1后细胞的增殖、迁移及侵袭能力均显著降低，提示PGAM1可能是CRC患者预后的生物标志物。

Expression of low-density lipoprotein receptor-associated protein 11 in colorectal cancer tissues and its effects on proliferation and apoptosis of SW480 cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨低密度脂蛋白受体相关蛋白11（LRP11）在结直肠癌（CRC）组织中的表达及其对结肠癌SW480细胞增殖与凋亡的影响。方法：利用生物信息学方法分析TCGA数据库中LRP11在CRC组织中的表达水平。用慢病毒感染技术分别将sh-LRP11及sh-NC质粒转染至SW480细胞，采用qPCR、WB法检测感染后各组细胞中LRP11的mRNA和蛋白的表达，CCK-8法、流式细胞术分别检测细胞的增殖活力、凋亡率及细胞周期分布情况，WB法检测SW480细胞中cyclin D1、BAX、Bcl-2、β-catenin、活化β-catenin等蛋白的表达水平。结果：TCGA数据库数据分析显示，LRP11 mRNA在CRC组织中的表达水平显著高于正常组织（P<0.05）。与sh-NC组比较，sh-LRP11组SW480细胞的增殖活力明显降低、细胞凋亡率显著升高（均P<0.01），细胞中BAX表达显著升高、Bcl-2表达显著降低（均P<0.01）；G0/G1期细胞增多、S期细胞明显减少（均P<0.01），cyclin D1的蛋白表达显著降低（P<0.01）；Wnt/β-catenin信号通路中β-catenin和活化β-catenin的蛋白表达均显著下降（均P<0.01）。结论：LRP11 mRNA在CRC组织中呈高表达，干扰LRP11表达可抑制结肠癌SW480细胞增殖并促进其凋亡，为CRC提供了一种潜在的治疗靶点。

Expression of TRAP1 in colon cancer tissues and its relationship with clinicopathological features and patient prognosis and the possible molecular mechanisms / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨肿瘤坏死因子受体相关蛋白1（TRAP1）在结肠癌组织和细胞中的表达及其与临床病理特征和患者预后的关系和相关分子机制。方法：通过TCGA和GEO数据全面分析TRAP1在结肠癌中的表达及其与临床病理特征和患者预后的关系，选取2020年10月至2021年03月间在山西医科大学第一医院手术切除的10例结肠癌组织及相应癌旁组织标本，用IHC染色法检测中国人结肠癌组织中TRAP1的表达进行验证，运行R包（survival和survminer）进行Kaplan-Meier生存分析；在线分析TRAP1蛋白的信号肽及穿膜结构域，通过基因富集分析软件进行GO分析和KEGG分析。培养结肠癌SW480和SW620细胞，将si-NC和si-TRAP1转染结肠癌细胞，实验分为空白对照组、si-NC组和si-TRAP1组，采用qPCR法检测转染后各组结肠癌细胞中TRAP1的表达，FCM检测转染后各组细胞的细胞周期和凋亡情况。结果：与癌旁组织比较，TRAP1在结肠癌组织中呈高表达（P<0.01），TRAP1表达水平与淋巴结转移有关联（P<0.05），TRAP1高表达组结肠癌患者5年OS率较低（P<0.05）。TRAP1蛋白属于细胞质蛋白，功能富集结果显示TRAP1及其相关分子与细胞周期、核糖体生物发生等信号通路有关（均P<0.01），TRAP1高表达组的结肠癌代谢重编程基因簇和线粒体蛋白输入基因簇水平升高（均P<0.01）。敲减TRAP1后，结肠癌细胞周期阻滞于G1期，细胞凋亡水平显著升高（均P<0.01）。结论：TRAP1在结肠癌组织中呈高表达，且与患者淋巴结转移和低OS率相关联，敲减TRAP1可阻滞结肠癌细胞周期并促进其凋亡。

miR-145-5p affects the proliferation of colorectal cancer cells and cytotoxicity of NK cells by targeting regulation of RAD18 expression / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探索RAD18影响结直肠癌细胞增殖及调节NK细胞对结直肠细胞的杀伤作用及其可能的机制。方法：采用生物信息学技术分析结直肠癌组织中RAD18和miR-145-5p的表达及两者之间的调控关系、分析RAD18富集通路。采用qPCR法验证RAD18和miR-145-5p在结直肠癌细胞中的表达，双荧光素酶报告基因实验验证miR-145-5p与RAD18的调控关系。按转染物的不同将SW480、HCT-15细胞分为将si-RAD18组、si-NC组，另向SW480细胞分别转染inhibitor-NC+si-NC、miR-145-5p inhibitor+si-NC或miR-145-5p inhibitor+si-RAD18，采用CCK-8法、克隆形成实验分别检测敲降miR-145-5p和/或RAD18对细胞增殖、克隆形成的影响；将各组细胞分别与经IL-2激活的NK92细胞共培养，采用乳酸脱氢酶释放法、ELISA和免疫荧光染色法分别检测NK细胞的细胞毒性、细胞因子分泌及细胞表面穿孔素和颗粒酶B表达的影响。结果：RAD18在结直肠癌组织和细胞中呈高表达（均P<0.01）。敲降RAD18可以抑制结直肠癌细胞增殖能力（P<0.05）和促进NK细胞活力、细胞毒性、IFN-γ、TNF-α、GM-CSF分泌及穿孔素和颗粒酶B的表达（均P<0.05）。双荧光素酶报告实验验证了RAD18-3’UTR与miR-145-5p的结合关系，miR-145-5p在结直肠癌组织和细胞中低表达（P<0.05或P<0.01）。miR-145-5p可以靶向下调RAD18的表达（P<0.05），过表达RAD18可以逆转miR-145-5p过表达对NK细胞杀伤效应的促进作用（均P<0.05）。结论：miR-145-5p可靶向下调RAD18的表达，miR-145-5p/RAD18轴能够影响结直肠癌细胞的增殖和NK细胞对其的细胞毒作用。

The anti-colorectal cancer effect of biphenyl-furocoumarin derivative BFD-6b / 西安交通大学学报(医学版)

【Objective】 To investigate the inhibitory effect of BFD-6b on colorectal cancer cell growth and its preliminary mechanism. 【Methods】 The inhibitory effect of BFD-6b on the growth of various tumor cells （SW480， MCF-7， T47D， SMMC-7721， BEL-7402， 97H， SK-HEP-1， H460， H1299， A549， MS751， and HELA） was investigated by MTT assay； the effect of BFD-6b on apoptosis and cell cycle was detected by flow cytometry； the effect of BFD-6b on the expressions of cell cycle-related proteins and apoptosis-related proteins was examined by Western blotting. 【Results】 BFD-6b inhibited the proliferation of different cancer cells such as SW480， MCF-7， H460， H1299， A549， and HELA. Among all of them， SW480 cells were most sensitive to BFD-6b， and the IC50 value was 7.09 μmol/L. Furthermore， BFD-6b could inhibit the growth of another three colorectal cell （SW620， Lovo， and HCT116 cells）； the IC50 values were 7.23， 8.08 and 8.96 μmol/L， respectively. BFD-6b could downregulate the amount of cyclinD1， cyclinE， and cell cycle-dependent kinase CDC2， thereby arresting the cell cycle at G1 phase. Also， BFD-6b downregulated the expressions of anti-apoptotic proteins Mcl-1 and Bcl-2， and upregulated the expressions of pro-apoptotic proteins BAK and BAX， thus inducing apoptosis of SW480 cells. 【Conclusion】 BFD-6b arrests SW480 cells at G1 phase by downregulating the expressions of cyclinD1， cyclinE and CDC2， and induces SW480 cell apoptosis by downregulating the expressions of Mcl-1 and Bcl-2 and upregulating the expressions of BAK and BAX， thereby inhibiting the proliferation of colorectal cancer SW480 cells.

Expression of TAGLN in colorectal cancer tissues and its effect on malignant biological behaviors of SW480 cells / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the expression of transgelin (TAGLN) in colorectal cancer (CRC) tissues and its effect on the proliferation, migration and invasion of CRC SW480 cells. Methods: Surgically resected CRC tissues and corresponding para-cancerous tissues of 97 CRC patients from May 2015 to August 2016 in the Affiliated Tumor Hospital of Zhengzhou University were collected; In addition, CRC cell lines SW620, SW480, HCT116 and normal colorectal mucosal cell line FHC were also collected for this study. Immunohistochemical staining was used to detect the expression of TAGLN in CRC tissues, and the correlation between TAGLN and patients’ clinicopathological features was analyzed. Quantitative Real-time quantitative polymerase chain reaction (qPCR) and Western blotting (WB) were used to detect the mRNA and protein expressions of TAGLN in CRC cell lines. si-TAGLN and si-Ctrl were respectively transfected into SW480 cells by liposome transfection method. The effects of silencing TAGLN on the proliferation, migration and invasion of SW480 cells were detected by CCK-8, Wound-healing assay and Transwell assay, respectively; and the expression of EMT-related proteins E-cadherin, N-cadherin and vimentin were detected by WB. Results: The positive expression rate of TAGLN in CRC tissues was significantly higher than that in para-cancerous tissues (P<0.01), and TAGLN expression was correlated with TNM stage, degree of tumor differentiation and lymph node metastasis in CRC patients (P<0.05 or P<0.01). The mRNA and protein expression levels of TAGLN in SW480 cells were significantly higher than those in FHC cells (all P<0.01). After TAGLN silence, the proliferation, invasion and migration ability of SW480 cells were significantly reduced (all P<0.01), the expression level of E-cadherin in SW480 cells was increased, while the expression levels of N-cadherin and vimentin were decreased (all P<0.01). Conclusion: TAGLN is highly expressed in CRC tissues and cells. Silencing TAGLN can inhibit the proliferation, invasion and migration of CRC cells, suggesting that TAGLN plays an important role in the occurrence and development of CRC.

Capsaicin Induces Apoptosis of Colon Cancer SW480 Cells Through TRPV1 / 中国实验方剂学杂志

Objective:To investigate the effects of capsaicin on colon cancer SW480 cells and the underlying molecular mechanism through the transient receptor potential vanilloid 1（TRPV1）. Method:Capsaicin groups with different concentrations and a blank group were set up. The cell viability was detected by cell counting kit-8 （CCK-8） after SW480 cells were treated with capsaicin（50，100，200，300，400，500，600，800，1 000 μmol·L^-1） for 12，24，and 48 h to select the concentration of capsaicin which can effectively inhibit proliferation. The cell cycle and apoptosis were detected by flow cytometry after SW480 cells were treated with capsaicin （200，400，800 μmol·L^-1） for 24 h. The protein expression levels of TRPV1，p53，p-p53，B-cell lymphoma-2（Bcl-2），Bcl-2-associated X protein（Bax），cleaved cysteinyl aspartate-specific protease-3（cleaved Caspase-3），cleaved Caspase-8，and cleaved poly adenosine diphosphate ribose polymerase （PARP） were detected by Western blot after SW480 cells were treated with capsaicin （200，400 μmol·L^-1） for 24 h．In addition，the apoptosis was detected after SW480 cells were treated with TRPV1 microRNA（mRNA） and capsaicin（200 μmol·L^-1）. Western blot analysis was used to detect the protein expression levels of the above proteins. Result:As compared with the blank group，capsaicin（≥200 μmol·L^-1）significantly inhibited the cell viability of SW480 cells（<italic>P</italic><0.01） in dose- and time-dependent manners. The cell cycle was arrested in G₂/M phase by 200 and 400 μmol·L^-1 capsaicin treatment，and arrested in G₁ phase by 800 μmol·L^-1 capsaicin treatment （<italic>P</italic><0.05）. Flow cytometry showed that capsaicin （200， 400， 800 μmol·L^-1） significantly promoted apoptosis of SW480 cells simultaneously（<italic>P</italic><0.05，<italic>P</italic><0.01）. Western blot showed that capsaicin （200，400 μmol·L^-1） significantly up-regulated the protein levels of apoptosis-related proteins（p53，p-p53，Bax，cleaved Caspase-3，cleaved Caspase-8，and cleaved PARP） （<italic>P</italic><0.05，<italic>P</italic><0.01），and significantly down-regulated Bcl-2（<italic>P</italic><0.01）. In addition，siRNA-mediated knockdown of TRPV1 significantly attenuated capsaicin-induced apoptosis and the protein levels of apoptosis-related proteins in SW480 cells（<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:Capsaicin can inhibit cell proliferation，arrest cell cycle，and induce apoptosis of SW480 cells，and the possible mechanism may be related to TRPV1 activation.

Mechanism of ginsenoside CK inhibiting proliferation of human colon cancer SW480 cells / 中草药

Objective: To study the effects of ginsenoside CK on proliferation and apoptosis of human colon cancer cell line SW480, and further explore the mechanism. Methods: Cell viability was measured by CCK-8 assay. Cell cycle, apoptosis, reactive oxygen species (ROS) levels and changes in mitochondrial membrane potential were measured by flow cytometry. Hoechst staining further detected apoptosis. Western blotting was used to detect the release of cytochrome C and the expression of apoptosis-related proteins such as Bcl-2, Bax and cleaved Caspase-3. Results: Ginsenoside CK had a significant inhibitory effect on the proliferation of human colon cancer cell line SW480. Ginsenoside CK induced SW480 cells arrest in G0/G1 phase, promoted early apoptotic cells, significantly increased intracellular ROS levels and reduced the MMP level. Ginsenoside CK promoted the expression of Bax and cleaved-Caspase-3 and inhibited the expression of Bcl-2. In addition, ginsenoside CK released a large amount of cytochrome C in SW480 cells. Conclusion: Ginsenoside CK has a significant inhibitory effect on the proliferation of human colon cancer cell line SW480. The mechanism may be through the promotion of mitochondrial superoxide elevation, resulting in a significant increase in intracellular ROS levels and a significant decrease in MMP level, further leading to the release of cytochrome C, the up-regulated expression of Bax, the down-regulated expression of Bcl-2, and ultimately leading to apoptosis of cells.

Clostridium difficile toxin B-induced apoptosis of colon cancer cells and related mechanisms / 中华微生物学和免疫学杂志

Objective To analyze the effects of Clostridium difficile toxin B (TcdB) on the prolif-eration and apoptosis of colon cancer cell line SW480 and the possible mechanisms related to cell apoptosis. Methods SW480 cells were treated with different concentrations of TcdB. Cell proliferation was detected by MTT assay. Cell apoptosis and mitochondrial membrane potential were measured with flow cytometry. Re-sults TcdB significantly inhibited the proliferation of SW480 cells in a time-concentration dependent man-ner and the inhibition rate reached 46. 36％ at 48 h. Flow cytometry results showed that TcdB could induce the apoptosis of SW480 cells in a time-concentration dependent manner and a 20. 83％ apoptosis rate was in-duced by 800 ng/ml of TcdB at48 h. Conclusions TcdB could inhibit the proliferation and induce the ap-optosis of colon cancer SW480 cells, and the possible mechanisms might be relate to the initiation of mito-chondrial apoptosis pathway.

Sevoflurane inhibits proliferation and invasion of colon cancer SW480 cells and standard regimen for advanced colorectal cancer / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect and mechanism of sevoflurane on the proliferation and invasion of colon cancer SW480 cells and the growth of transplanted tumor in nude mice by regulating the phosphorylation of PI3K. Methods: Colon cancer SW480 cells were treated with sevoflurane and randomly divided into control group, 0.5% sevoflurane group, 1.0% sevoflurane group and 2.0% sevoflurane group for subsequent experiments. The proliferation ability of SW480 cells was detected by Clone formation assay, mRNA expression levels of MDM2 and survivin in cells were detected by RT-PCR, invasion ability of cells was detected by Transwell assay, and protein expression levels of MDM2, survivin, VEGF, PI3K, p-PI3K, AKT and p-AKT were detected by Western blotting. PI3K activator 740Y-P was added for verification. SW480 cell transplanted tumor model was constructed on nude mice, and the tumor mass was weighed. The positive expression rates of MDM2 and VEGF in the transplanted tumor tissues were detected by Immunohistochemistry. Results: As compared with the control group and the low-dose group, the clone formation rate of SW480 cells and the number of invaded cells in the 1.0% and 2.0% sevoflurane groups were significantly decreased (all P<0.01), the mRNA and protein levels of MDM2 in the cells were significantly increased (all P<0.01), while the mRNA and protein levels of survivin were significantly decreased (all P<0.01); and the protein levels of VEGF, p-PI3K/PI3K and p-AKT/AKT were significantly decreased (all P<0.01). 740Y-P could reverse the effect of sevoflurane on the proliferation, invasion and expression of proteins associated with the PI3K/AKT signaling pathway in SW480 cells. The mass of transplanted tumor in 2.0% sevoflurane group was significantly decreased (P<0.01), and the positive MDM2 expression rate in tumor tissues was significantly increased (P<0.01), while the positive VEGF expression rate was significantly decreased (P<0.01). Conclusion: Sevoflurane inhibits the proliferation and invasion of colon cancer SW480 cells and the growth of xenografts in nude mice possibly by inhibiting PI3K phosphorylation.

Clostridium difficile toxin B-induced apoptosis of colon cancer cells and related mechanisms / 中华微生物学和免疫学杂志

Objective@#To analyze the effects of Clostridium difficile toxin B (TcdB) on the proliferation and apoptosis of colon cancer cell line SW480 and the possible mechanisms related to cell apoptosis.@*Methods@#SW480 cells were treated with different concentrations of TcdB. Cell proliferation was detected by MTT assay. Cell apoptosis and mitochondrial membrane potential were measured with flow cytometry.@*Results@#TcdB significantly inhibited the proliferation of SW480 cells in a time-concentration dependent manner and the inhibition rate reached 46.36% at 48 h. Flow cytometry results showed that TcdB could induce the apoptosis of SW480 cells in a time-concentration dependent manner and a 20.83% apoptosis rate was induced by 800 ng/ml of TcdB at 48 h.@*Conclusions@#TcdB could inhibit the proliferation and induce the apoptosis of colon cancer SW480 cells, and the possible mechanisms might be relate to the initiation of mitochondrial apoptosis pathway.

Potential using of microRNA-34A in combination with paclitaxel in colorectal cancer cells

Background: MicroRNAs are small noncoding RNAs which modulate gene expression at different levels. It has been shown that downregulation of miR-34a occurs in varieties of cancers including colorectal cancer (CRC). In this study, we investigated the potential tumor inhibitory effects of miR-34a alone or in combination with paclitaxel in CRC cells. Materials and Methods: SW480 cells were transduced with lentiviral overexpressed miR-34a. First, using 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay, the effect of miR-34a induction alone or in combination with paclitaxel on the cell viability and cell proliferation were estimated. Then, the expression level of target genes was measured using quantitative reverse transcription-polymerase chain reaction analysis. Eventually, the role of miR-34a and paclitaxel on cell cycle were determined with flow cytometry. Results: Gene expression analysis showed that miR-34a downregulates the expression of BCL2 and SIRT1 genes at mRNA level. Furthermore, miR-34a has a potential to reduce cell viability and cell cycle arrest at G1 phase. Combination of paclitaxel with overexpression of miR-34a significantly decreased cell viability compared to cell treated with miR-34a or paclitaxel alone. Interestingly, a combination of miR-34a and paclitaxel arrested cell cycle at two phases. Conclusion: Our results suggested that combination therapy of miR-34a and paclitaxel could be considered as the potential treatment of CRC.

Investigation of apoptotic effect of juglone on CCL-228-SW 480 colon cancer cell line

Background: Colon cancer is a major cause of morbidity and mortality in the world. Juglone is a natural compound which has been isolated from Juglans mandshurica Maxim, and it has various pharmacological effects such as antiviral, antibacterial, and anticancer. In our study, we aimed to investigate the effect of juglone on CCL-228-SW 480 colon carcinoma cell line in monolayer and spheroid culture medium. Materials and Methods: The CCL-228-SW 480 cell lines were cultured in both monolayer and spheroid cultures. Cells were treated with juglone at 24, 48, and 72 h of incubation. ID50 inhibition was determined on the dose for juglone and after it was found 20 μM was applied to the cells to examine the effect of juglone on CCL-228-SW 480 colon carcinoma cell line. After Juglone was applied the BrdU marking index, Transferase dUTP Nick ends Labeling (TUNEL) assay, active caspase-3 assay, apoptosis-inducing factor (AIF) assay were determined by immunohistochemistry in both the monolayer and spheroid cultures. Results: The control group had a healthy pattern of S-phase fraction, and many of the CCL-228-SW 480 cells nuclei were observed to be positive for BrdU. Terminal Deoxynucleotidyl TUNEL-positive cells, active Caspase-3, and AIF were detected after treatment with juglone in both the monolayer and spheroid cultures. Conclusions: The dead cell count was higher in the CCL-228-SW 480 cell lines with juglone applied than in the controls. Juglone significantly inhibits the proliferation and induces the apoptosis of CCL-228-SW 480 cells in vitro

Noscapine inhibits migration of colon cancer SW480 cells by down-regulating Wnt3a/β-catenin signaling pathway and cadherin 17 / 中国肿瘤生物治疗杂志

@# Objective: To explore the effects of noscapine (Nos) on the expression of cadherin 17 (CDH17) in colon cancer SW480 cells and the mechanism of Nos on cell migration. Methods: SW480 cells were divided into the control group, empty vector (si-EV) group, CDH17 interference (si-CDH17) group, Nos treatment group, and CDH17 interference+Nos treatment (si-CDH17+Nos) group. Small interfering RNA (siRNA) was used to knockdown CDH17, and the selected concentration of Nos was (55.30±2.21) µg/ml (IC50). The mRNA expression of CDH17 was detected by qPCR; the apoptosis and migration abilities of SW480 cells were observed by Hoechst33258 staining and Transwell assay; the contents of VEGF, MMP2 and MMP9 in SW480 cells were measured by ELISA, and the protein expressions of CDH17, Wnt3a and β-catenin were determined by WB. Results: Compared with the control group, mRNA and protein expressions of CDH17 obviously decreased, cell apoptosis and migration significantly reduced, while the contents of VEGF, MMP2 and MMP9 as well as the protein expressions of Wnt3a and β-catenin significantly decreased in Nos treatment group, siCDH17 group and si-CDH17+Nos treatment group (all P<0.01).The effect of si-CDH17+Nos treatment was more significant than that of si-CDH17 (P<0.01). Conclusion: Nos induces apoptosis and inhibits the migration of human colon cancer SW480 cells, which may be related to the down-regulation of CDH17 expression and inhibition of the Wnt3a/β-catenin signaling pathway.

Expression of lncRNA SNHG16 in colorectal cancer tissues and cells and its regulationon expression of GPAM in colon cancer cells / 中国肿瘤生物治疗杂志

@# Objective: To investigate the expression of long non-coding RNASNHG16 (lncRNASNHG16) in colorectal cancer (CRC) tissues and cells, and to explore the mechanism of its regulation on the expression of mitochondrial glycerol-3-phosphate acyltransferase (GPAM) via sponging miR-128-3p. Methods: Sixty pairs of colorectal cancerous tissues and para-cancerous tissues that resected from CRC patients, who underwent surgery in the Department of Anorectal Surgery, Gansu Provincial People’s Hospital during Jan. 2014 and Jan. 2017, were collected for this study; In addition, CRC cell lines (SW480, SW620, HCT116, Caco-2,DLD-1, HT29) and colonic epithelial cell line CCD841 were also collected for the study. The expression of SNHG16 in collected tissues and cell lines was determined by Real-time quantitative PCR (qPCR), and its correlation to the clinicopathological features of CRC patients was also analyzed. SW480 cells were transfected with miR-128-3p mimic, miR-128-3p inhibitor, and si-SNHG16, respectively, and then the mRNA expressions of miR-128-3p and SNHG16 were detected by qPCR, the protein expression of GPAM was determined by Western blotting, and the cell proliferation, apoptosis and invasion were detected by CCK-8 assay, colony formation assay, cell apoptosis assay and Transwell chamber assay, respectively. The binding between SNHG16 and miR-128-3p was validated with dual luciferase reporter gene assay and RNA Immunoprecipitation assay. For in vivo experiment, mouse model of SW480 cell exnograft was constructed, and the effect of SNHG16 knockdown on the growth of exnograft was observed. Results: SNHG16 was found to highly expressed in human CRC tissues and cell lines (all P<0.01), and SNHG16 expression level was associated with lymph node metastasis, Duke's stage and patients’survival (all P<0.01). Knockdown of SNHG16 significantly inhibited CRC cell proliferation and invasion, and induced apoptosis (all P<0.01); After SNHG16 knockdown, the volume of exnograft was obviously reduced (P<0.05). Dual luciferase reporter gene assay and RNA Immunoprecipitation assay validated the interaction between miR-128-3p and SNHG16, and they were negatively correlated with each other in CRC patients (P<0.01). The SNHG16 regulated the expression of its down-stream gene GPAM via endogenously sponging miR-128-3p. Conclusion: SNHG16 regulates GPAM expression in CRC cells by sponging miR-128-3p, and SNHG16 and miR-128-3p may serve as potential targets for the diagnosis and treatment of CRC.

Effect of shRNA interfering BAMBI on malignant biological behaviors of human colon cancer SW480 cell / 中国肿瘤生物治疗杂志

@# Objective: To explore the effect of shRNA interfering BAMBI (bone morphogenetic protein and activin membrane bound inhibitor) on the proliferation, apoptosis, invasion and migration of human colon cancer SW480 cells and the possible mechanisms. Methods: After successful transfection with sh-BAMBI in SW480 cells, the mRNA and protein epxressions of BAMBI were detected by qRT-PCR and Western blotting, respectively. Cell proliferation was measured by MTT; apoptosis was tested by Hoechst33258 staining; cell invasion was detected by transwell assay; and cell migration was measured by wound healing assay. The expressions of TGF-β/ Smad/2 signaling pathway related proteins were detected by Western blotting. Results: The mRNA and protein levels of BAMBI in shBAMBI group were lower than those of control group (P<0.05). Compared with control group, cell proliferation in sh-BAMBI group was obviously decreased (P<0.05), while apoptosis was obviously increased (P<0.01); in the meanwhile, cell invasion and migration in sh-BAMBI group were significantly reduced (P<0.05). In addition, the protein level of TGF-β and the ratio of p-Smad/2/ Smad/2 in shBAMBI group were significantly higher than those in control group (P<0.05). Conclusion: Interference of BAMBI by shRNA inhibits proliferation, invasion and migration but induces apoptosis of human colon cancer SW480 cells and activates TGF-β/Smad/2 pathway.