Peiminine inhibits the proliferation of colon cancer cells by inducing G0/G1 phase arrest / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨贝母素乙对结肠癌HCT116细胞增殖的抑制作用及其分子机制。方法：利用不同浓度的贝母素乙处理人结肠癌细胞HCT116和正常结肠上皮细胞CCD841 CON，通过CCK-8法和结晶紫染色法检测贝母素乙对HCT116和CCD841 CON细胞增殖活力的影响，流式细胞术和WB法检测贝母素乙对HCT116细胞周期及其细胞周期相关蛋白表达的影响。构建HCT116移植瘤裸鼠模型和AOM/DSS结肠癌小鼠模型，观察贝母素乙对小鼠模型肿瘤生长和OS的影响，免疫组化法和WB法检测对移植瘤或肿瘤组织中细胞周期相关蛋白CDK4、CDK6和cyclin D1表达的影响。结果：贝母素乙可显著抑制结肠癌HCT116细胞的增殖能力（P<0.01），诱导HCT116细胞周期G0/G1期阻滞（P<0.01），降低CDK4、CDK6和cyclin D1的蛋白表达水平（均P<0.01）。荷瘤小鼠实验结果显示，贝母素乙（0.75 mg/kg）显著抑制HCT116细胞移植瘤的生长并延长荷瘤裸鼠的OS（P<0.05或P<0.01），降低AOM/DSS模型小鼠的体质量、延长OS、减少癌变肠组织的肿瘤个数和肿瘤体积，下调肿瘤组织中CDK4、CDK6和cyclin D1的蛋白表达（P<0.01或P<0.05）。结论：贝母素乙通过下调CDK4、CDK6和cyclin D1的表达水平，引起细胞周期G0/G1期阻滞，从而抑制结肠癌HCT116细胞的增殖。

Baitouweng Tang Suppresses Colorectal Cancer HCT116 Cells by Regulating Hedgehog Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effect of Baitouweng Tang （BTWT） on the apoptosis of human colorectal cancer HCT116 cells and decipher the underlying mechanism based on the Hedgehog （Hh） signaling pathway. MethodHCT116 cells were treated with BTWT （25， 50， 100， 200， 500， 750， and 1 000 mg·L-1） for 24 h， and then the cell proliferation was detected by methyl thiazolyl tetrazolium （MTT） colorimetry. Five groups were designed for the treatment of HCT116 cells， including a blank control group， BTWT groups （125， 250， and 500 mg·L-1）， and a positive control （5-fluorouracil， 5-FU， 40 mmol·L-1） group. The cell morphology was observed under an inverted microscope. The migration of the cells was detected by scratch test， and the apoptosis by Hoechest 33324/propidium iodide （PI） staining and flow cytometry. Western blot was employed to determine the protein levels of sonic hedgehog （SHh）， GLI family zinc finger protein 1 （Gli1）， smoothened （Smo）， suppressor of fused （SuFu）， cellular-myelocytomatosis viral oncogene （c-Myc）， and the apoptosis-related proteins B-cell lymphoma-2 （Bcl-2） and Bcl-2-associated X protein （Bax）. The quantitative real-time reverse transcription PCR （Real-time PCR） was employed to determine the mRNA levels of Bax， Bcl-2， SHh， Gli1， Smo， SuFu， and c-Myc. ResultCompared with the blank control group， BTWT changed the cell morphology （making the cell become round with dense nucleus）， inhibited the proliferation of HCT116 cells in a dose-dependent manner， decreased the ability of migration （P<0.05， P<0.01）， and increased apoptotic cells. Compared with the blank control group， BTWT （500 mg·L-1） treatment for 24 h up-regulated the protein and mRNA levels of Bax （P<0.05， P<0.01） and down-regulated the protein and mRNA levels of Bcl-2 in HCT116 cells （P<0.05， P<0.01）. Moreover， the treatment down-regulated the mRNA and protein levels of SHh， Gli1， Smo， and c-Myc （P<0.05， P<0.01） and up-regulated the mRNA and protein levels of SuFu （P<0.05， P<0.01）. ConclusionBTWT inhibited the proliferation and migration and induced the apoptosis of colorectal cancer HCT116 cells by down-regulating the Hh signaling pathway.

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。

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 effects of curcumin on the biological behavior of colorectal cancer cells through the JAK/STAT3 and RAS/MAPK/NF-κB pathways / Los efectos de la curcumina en el comportamiento biológico de las células del cáncer colorrectal mediante las vías JAK/STAT3 y RAS/MAPK/NF-KB

Abstract The purpose of this work was to investigate the effects of curcumin on the biological behavior of colorectal cancer cells through the JAK/STAT3 and RAS/MAPK/NF-κB pathways. Human colorectal cancer HCT116 cells were cultured and divided into a control group and low, medium and high-dose curcumin groups (n =5). HCT116 colorectal cancer cells became long-growing cells after incubation and culture at 37°C. The control group was treated with 15μL phosphate-buffered saline, and the low-dose, medium-dose and high-dose curcumin groups were treated with 20, 40 and 80μmol/L curcumin, respectively. All groups were treated with relevant drug intervention, digested and centrifuged for 48h, washed twice with a PBS solution, centrifuged at 1000 rpm for 3 min, and the cells precipitated. The proliferation, apoptosis and growth cycle of cells in each group were observed, and the expressions of the JAK/STAT3 and RAS/MAPK/NF-κB pathways and related proteins in each group were studied. Compared with the curcumin low-dose and medium-dose groups, the proliferation ability of the curcumin high-dose group was significantly decreased (P<0.05). When the low-dose and medium-dose curcumin groups were compared with the high-dose curcumin group, the apoptosis ability was significantly increased (P<0.05). When the low-dose and medium-dose curcumin groups were compared, the growth ratio of the G0/G1 phase in the high-dose curcumin group was significantly increased, and the percentage of the S phase was significantly decreased (P<0.05). Compared with the curcumin low-dose and medium-dose groups, the expression of JAK-STAT3 and RAS/MAPK/NF-κB pathway in the curcumin high-dose group was significantly decreased (P<0.05). The protein expressions of STAT3, RAS, P-P38 and P65 in the curcumin high-dose group were significantly lower than those in the curcumin low-dose and medium-dose groups (P<0.05). Curcumin can inhibit the expression of JAK/STAT3 and RAS/MAPK/NF-κB pathways, block the growth cycle, and inhibit the proliferation and induce apoptosis of colorectal cancer cells, providing a new idea for the clinical treatment of colorectal cancer.

Resumen El objetivo del presente trabajo fue investigar los efectos de la curcumina en el comportamiento biológico de las células del cáncer colorrectal mediante el estudio de las vías JAK/STAT3 y RAS/MAPK/NF-KB. Las células del cáncer colorrectal humano HCT116 se cultivaron y dividieron en un grupo control y en grupos con dosis baja, media y alta (n = 5) de curcumina. Las células de cáncer colorrectal HCT116 se convirtieron en células de crecimiento prolongado después de la incubación y cultivo a 37°C. El grupo de control se trató con 15 μL de solución tampón fosfato salina (PBS) y los grupos de curcumina de dosis baja, media y alta se trataron con 20, 40 y 80 μmol/L de curcumina, respectivamente. Todos los grupos fueron tratados con la intervención farmacológica pertinente, digeridos y centrifugados durante 48 horas, lavados dos veces con solución de PBS, centrifugados a 1000 rpm durante 3 minutos, y las células precipitadas. Se observaron la proliferación, la apoptosis y el ciclo de crecimiento de las células de cada grupo, y fueron estudiados las expresiones de las vías JAK/STAT3, RAS/MAPK/NF-KB y proteínas relacionadas en cada grupo. Comparado con los grupos de la dosis baja y media de la curcumina, disminuyó obviamente la capacidad de proliferación del grupo de la dosis alta de la curcumina (P<0,05). Comparado con los grupos de la dosis baja y media de la curcumina, aumentó de modo significativo la capacidad de la apoptosis del grupo de la dosis alta de la curcumina (P<0,05). Comparado con los grupos de la curcumina de dosis baja y media, aumentó obviamente la proporción del crecimiento de la fase G0/G1 en el grupo de la curcumina de dosis alta y el porcentaje de la fase S disminuyó considerablemente (P<0,05). Las expresiones proteicas STAT3, RAS, P-P38 y P65 en el grupo de la dosis alta de la curcumina fueron evidentemente más bajas que las de los grupos de la dosis baja y media de la curcumina (P<0.05). La curcumina puede inhibir la expresión de las vías JAK/STAT3 y RAS/MAPK/NF-KB, bloquear el ciclo del crecimiento y luego inhibir la proliferación e inducir apoptosis de las células del cáncer colorrectal, lo que brinda una nueva idea para el tratamiento clínico del cáncer colorrectal.

GB7 acetate,a galbulimima alkaloid from Galbulimima belgraveana,possesses anticancer effects in colorectal cancer cells / 药物分析学报

GB7 acetate is a galbulimima alkaloid obtained from Galbulimima belgraveana.However,information regarding its structure,biological activities,and related mechanisms is not entirely available.A series of spectroscopic analyses,structural degradation,interconversion,and crystallography were performed to identify the structure of GB7 acetate.The MTT assay was applied to measure cell proliferation on human colorectal cancer HCT 116 cells.The expressions of the related proteins were measured by Western blotting.Transmission electron microscopy(TEM),acridine orange(AO)and monodansylcadaverine(MDC)staining were used to detect the presence of autophagic vesicles and autolysosomes.A transwell assay was performed to demonstrate metastatic capabilities.Oxygen consumption rate(OCR)and extracellular acidification rate(ECAR)assays were performed to determine the mitochondrial oxidative phosphorylation(OXPHOS)and glycolysis activity of HCT 116 cells.The data showed that GB7 acetate suppressed the proliferation and colony-forming ability of HCT 116 cells.Pretreatment with GB7 acetate significantly induced the formation of autophagic vesicles and autolysosomes.GB7 acetate upregulated the expressions of LC3 and Thr172 phosphorylated adenosine 5'-monophosphate(AMP)-activated pro-tein kinase α(p-AMPKα),which are key elements of autophagy.In addition,GB7 acetate suppressed the metastatic capabilities of HCT 116 cells.Additionally,the production of matrix metallo-proteinase-2(MMP-2)and MMP-9 was reduced,whereas the expression of E-cadherin(E-cad)was upregulated.Furthermore,GB7 acetate significantly reduced mitochondrial OXPHOS and glycolysis.In conclusion,the structure of the novel Galbulimima alkaloid GB7 acetate was identified.GB7 acetate was shown to have anti-proliferative,pro-autophagic,anti-metastatic,and anti-metabolite capabilities in HCT 116 cells.This study might provide new insights into cancer treatment efficacy and cancer chemoprevention.

Predominant low Hematopoietic Cell Transplantation Specific Comorbidity Index (HCT-CI) score in a Mexican referral center: a selection bias caused by limited-resources?

Abstract Introduction: To date, many studies have validated the Hematopoietic Cell Transplantation Specific Comorbidity Index (HCT-CI) scoring system in allogeneic hematopoietic stem cell transplantation (allo-HSCT), but studies from developing countries remain scarce. Objective: The aim of this study was to evaluate and categorize Mexican patients using the HCT-CI at a referral center. Methods: One hundred and nineteen consecutive patients undergoing allo-HSCT at the National Institute of Medical Sciences and Nutrition in Mexico City were included. Patients were classified according to the HCT-CI scores. Results: The median age was 31 years and most were males (56%). Most patients had hematological malignancies (73%) and a low HCT-CI score (72%). The non-relapse mortality and survival were predicted according to the score. Conclusions: This is one of the few studies to evaluate the HCT-CI in adults with HLA-matched donors in a developing country and our findings suggest that the high percentage of patients with a low HCT-CI scores, contrary to international reports, could be explained by different comorbidities and demographics, but mainly due to stricter filters applied to HSCT candidates and consequently, a potential selection bias caused by limited resources.

Scutellarin induces apoptosis of colorectal cancer HCT116 cells via Hippo signaling pathway / 中国药理学与毒理学杂志

OBJECTIVE To investigate the effect of scutellarin on the apoptosis of human colorectal cancer cells via the Hippo signaling pathway in vitro. METHODS MTT colorimetric method was used to detect the influence of scutellar?in on the survival rate of HCT116 cells. And the effect of scutellarin at various concentrations on cell morphology was observed by microscopy. Cell scratch experiment was used to detect the influence of scutellarin on the migration of HCT116 cells. Hoechst33342/PI double staining method was used to detect the effect of scutellarin on the apoptosis of HCT116 cells. Western blotting method was used to assess the action of scutellarin on the expressions of Hippo signal?ing pathway-related proteins Mst1, Lats1, YAP1, p-YAP(Ser127), TAZ, and its downstream effector proteins c-Myc and cyclin D1, as well as apoptosis-related proteins Bcl-2 and Bax in HCT116 cells. RESULTS Scutellarin significantly affected the morphology of HCT116 cells and reduced the survival rate of HCT116 cells. Hoechst33342/PI double stain?ing showed that scutellarin effectively induced the apoptosis of HCT116 cells. Western blotting analysis showed that the expression levels of Hippo signaling pathway-related proteins Mst1, Lats1, YAP1, TAZ and its downstream effector pro?teins c-Myc, cyclin D1 were down-regulated in a concentration-dependent manner by scutellarin, and the expression of p-YAP (ser127) was up-regulated. Moreover, scutellarin substantially lessened the expression level of apoptosis-related protein Bcl-2, and promoted the protein level of Bax. CONCLUSION Scutellarin may inhibit the proliferation and migra?tion of HCT116 cells, while induce its apoptosis, potentially by activation of Hippo signaling pathway.

Effects of Shikonin on Autophagy and Apoptosis of Human Colon Cancer HCT 116 Cells / 中国药房

OBJECTIVE：To study the effects of shikonin on autophagy and apoptosis of human colon cancer HCT 116 cells. METHODS：After treating HCT 116 cells for 48 h with shikonin at 0（blank control ）10，20，40 μg/mL，MTT method was used to detect inhibitory rate of cell proliferation. Flow cytometry was used to detect cell apoptosis rate. RT-qPCR assay and Western blotting were respectively used to detect mRNA and protein expressions of microtubule associated protein light chain 3（LC3）and autophagy-related protein Beclin- 1 and p 62. RESULTS ：Compared with blank control ，after treated with 10，20，40 μ g/mL shikonin for 48 h，proliferation inhibitory rate and apoptosis rate of HCT 116 cells were increased significantly （P＜0.05 or P＜ 0.01）. After treated with 10，20，40 μg/mL shikonin for 48 h，mRNA and protein expressions of LC 3，Beclin-1 and p 62 in HCT116 cells were increased to different extents ；except that mRNA expression of LC 3 was not increased significantly after treated with 10 μg/mL shikonin，the difference were statistically significant in other indexes ，compared with blank control （P＜0.05 or P＜0.01）. CONCLUSIONS ：Shikonin can induce the apoptosis of human colon cancer HCT 116 cells and activate its autophagy pathway.

Efficacy of Jianpi Xiaoai Prescription in Inhibition of Metastasis of Colon Cancer HCT116 Cells： An Exploration Based on LncRNA HOTAIR/JAK2/STAT3 Signaling Pathway / 中国实验方剂学杂志

Objective:To observe the effect of Jianpi Xiaoai prescription on long non-coding RNA Hox transcript antisense intergenic RNA （lncRNA HOTAIR）/Janus kinase 2 （JAK2） /signal transducer and activator of transcription 3 （STAT3） signaling pathway and to explore the potential mechanism of Jianpi Xiaoai prescription in suppressing the metastasis of colon cancer. Method:The expression of lncRNA HOTAIR in different cells was analyzed. Following the treatment of HCT116 cells with 10%，15%，and 20% Jianpi Xiaoai prescription -containing serum， the invasive ability of Jianpi Xiaoai prescription on HCT116 cells was assessed by transwell assay. The mRNA expression levels of lncRNA HOTAIR，JAK2，and STAT3 were measured by Real-time polymerase chain reaction （Real-time PCR）， and the protein expression levels of JAK2， phosphorylated STAT3 （p-STAT3） and STAT3 by Western blot. Result:The highest expression of lncRNA HOTAIR was detected in HCT116 cells. Compared with the blank group， each Jianpi Xiaoai prescription group exhibited a decreased number of invasive cells （<italic>P</italic><0.05， <italic>P</italic><0.01）. The relative JAK2 mRNA expression in the middle-dose Jianpi Xiaoai prescription group was down-regulated （<italic>P</italic><0.05）， and the relative lncRNA HOTAIR mRNA expression in the middle- and high-dose Jianpi Xiaoai prescription groups and the relative JAK2 mRNA expression in the high-dose Jianpi Xiaoai prescription group were remarkably down-regulated （<italic>P</italic><0.01）. Compared with the blank group，the relative p-STAT3 protein expression was down-regulated in the middle-dose Jianpi Xiaoai prescription group （<italic>P</italic><0.05）， and the relative JAK2 protein expression in the middle- and high-dose Jianpi Xiaoai prescription groups and the relative p-STAT3 protein expression in the high-dose Jianpi Xiaoai prescription group were remarkably down-regulated （<italic>P</italic><0.01）. Conclusion:Jianpi Xiaoai prescription effectively inhibits the metastasis of colon cancer cells， which may be related to the inhibition of lncRNA HOTAIR/JAK2/STAT3 signaling pathway.

Expression of Runt-related Transcription Factor 3 in Human Colon Cancer Cell Line HCT-116 Resistant to 5-Fluorouracil and the Mechanism of Drug Resistance / 中国医学科学院学报

Objective To establish a human colon cancer cell line HCT-116/5-FU resistant to 5-fluorouracil(5-FU)and explore the relationship between runt-related transcription factor 3(RUNX3)and drug resistance of colorectal cancer.Methods The human colon cancer cell line HCT-116/5-FU with resistance to 5-FU was established by low concentration gradient increment combined with high-dose intermittent shock.CCK-8 method was used to determine the half maximal inhibitory concentration(IC

Quinacrine causes apoptosis in human cancer cell lines through caspase-mediated pathway and regulation of small-GTPase

Quinacrine (QC), an FDA-approved anti-malarial drug, has shown to have anticancer activities. Due to its‘shotgun’ nature, QC has become an inevitable candidate for combination chemotherapy. There is lack of studyof the molecular interplay between colorectal cancer (CRC) microenvironment and its metastasis. In this study,we focused on the differential anti-cancerous effect of QC on two different human cancer cell lines, HCT 116and INT 407. Results suggest that cytotoxicity increased in both the cell lines with an increase in QCconcentration. The expression patterns of small-GTPases and caspases were altered significantly in QC-treatedcells compared to non-treated cells. HSP70 and p53 showed comparable differences in the expression pattern.The wound-healing assay showed an increase in the denuded zone, with an increase in the concentration ofQC. The formation of apoptotic nuclei increased with a rise in the concentration of QC in both the cell lines.The decrease and increase in caspase 9 and caspase 3 expression respectively were studied, confirmingapoptosis by the extrinsic pathway

Effects of picropodophyllin on proliferation and cell cycle of human colorectal cancer HCT-15 cells and its mechanism / 解剖学报

Objective To investigate the effect of picropodophyllin (PPP) on proliferation and cell cycle of human colorectal cancer cell line HCT-15 and to clarify the related mechanism. Methods Different concentrations of PPP were used to treat HCT-15 cells, the proliferative activity of HCT-15 cells was detected by cell courting kit-8(CCK-8). Morphological changes of HCT-15 cells were observed under inverted microscope. Flow cytometry was used to detect the changes of HCT-15 cell cycle and Western blotting was used to detect the expression of proliferating cell nuclear antigen (PCNA) and cell cyclinD1. Results The result of CCK-8 assay showed that PPP inhibited the proliferation of HCT-15 cells in a dose-time-dependent manner. Under the inverted microscope, it was found that the HCT-15 cells lost their original shape and became round, the refractive index decreased, and the number of living cells decreased significantly. Flow cytometry showed that the proportion of G

Effect of DUOX2 on sensitivity of colorectal cancer cells to 5-fluorouracil / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of double oxidase 2 (DUOX2) on the sensitivity of colorectal cancer (CRC) cells to 5-fluorouracil (5-FU). Methods: CRC cell lines DLD-1, SW480, HCT116, SW620 and normal intestinal epithelial cell line NCM460 were selected, and the expression of DUOX2 in these cell lines were detected by qPCR. DUOX2 expression in HT-29 and HCT116 cells was stably knocked down by lentivirus infection technique. The knockdown efficiency was detected by qPCR and WB. Cells in sh-Control and sh-DUOX2 groups were treated with 5-FU at different concentrations (0, 5, 10, 20, 40, 80, 120 μg/ml). The effects of 5-FU on cell proliferation, apoptosis and cell cycle were detected by CCK-8 method and flow cytometry. HT29 cell transplanted xenograft model in nude mice was constructed to observe the effect of DUOX2 gene on the treatment efficacy of 5-FU. Results: the expression level of DUOX2 mRNA in CRC cells was significantly higher than that in NCM460 cells (P<0.05 or P<0.01). Compared with sh-Control group, the mRNA and protein expressions of DUOX2 in sh-DUOX2 group were significantly decreased (all P<0.01); the sensitivity of cells to 5-FU was enhanced, the apoptosis rate and the ratio of cells at G0/G1 phase were significantly increased (all P<0.01), and the ratio of cells at G2 and S phase was significantly decreased (all P<0.01). There was no significant difference in tumor volume and mass between sh-Control group and sh-DUOX2 group without 5-FU treatment (all P>0.05), but the volume and mass of transplanted tumor in sh-DUOX2+5-FU group after 5-FU treatment was significantly lower than that in sh-Control+5-FU group (all P<0.01). Conclusion: The sensitivity of CRC cells to 5-FU can be significantly enhanced by knocking down DUOX2 gene.

Effect and mechanism of down-regulating REV7 expression on the radiosensitivity of human colon cancer cell HCT116 / 中华放射肿瘤学杂志

Objective@#To evaluate the effect and mechanism of down-regulating the expression of REV7 on the radiosensitivity of human colon cancer cell HCT116.@*Methods@#HCT116 cells were cultured and the expression of REV7 was down-regulated by RNA interference technique. HCT116 cells were divided into the blank group, negative control transfected with negative RNA oligo group and REV7 expression down-regulation transfected with REV7 RNA oligo group, respectively. The cell proliferation was determined by colony formation assay. The expression levels of the proteins of relevant genes were detected by Western blot. The level of cell apoptosis and non-homologous end joining was evaluated.@*Results@#The colony formation rate was significantly reduced in THE REV7 siRNA group after 6Gy irradiation (P<0.05). The down-regulating efficiency rate of REV7 gene was>60% in the REV7 siRNA group. The expression levels of γH2AX and Caspase9 were significantly up-regulated, whereas those of KU80 and XRCC4 were remarkably down-regulated in the REV7 siRNA group (all P<0.05).@*Conclusions@#The radiosensitivity of human colon cancer cell HCT116 can be increased by down-regulating the expression of REV7. The underlying mechanism may be related to the lower incidence rate of non-homologous end joining.

Effect of Polyphyllin Ⅰ on Transcriptional Factor FOXQ1 and Epithelial-mesenchymal Transition in Colorectal Cancer Cell HCT116 / 中国实验方剂学杂志

Objective:To observe the effect of polyphyllin Ⅰ on the expressions of forkhead box Q1（FOXQ1）and epithelial-mesenchymal transition (EMT)-related factors, in order to explore the possible mechanism of polyphyllin Ⅰ in inhibiting the metastasis of colon cancer. Method:After the treatment with 1.25，2.50 μmol·L-1 polyphyllin Ⅰ on HCT116 cells, Western blot and Real-time PCR were used to detect the expressions of FOXQ1，E-cadherin，Vimentin protein and mRNA. Result:Compared with the blank group, relative expressions of FOXQ1 protein and mRNA in low-concentration polyphyllin Ⅰ group were decreased, while relative expression of E-cadherin mRNA was increased, the differences were not statistically significant, and relative expressions of Vimentin protein and mRNA in low-concentration polyphyllin Ⅰ group were decreased (P<0.05)， and relative expression of E-cadherin protein in low-concentration polyphyllin Ⅰ group was increased (P<0.01). Compared with the blank group, relative expressions of FOXQ1, Vimentin protein and mRNA in high-concentration polyphyllin Ⅰ group were decreased，while relative expressions of E-cadherin protein and mRNA were increased (P<0.05, P<0.01). Compared with low-concentration polyphyllin Ⅰ group, relative expressions of Vimentin protein and mRNA in high-concentration polyphyllin Ⅰ group were decreased, but the difference was not statistically significant, relative expressions of E-cadherin protein and mRNA in high-concentration polyphyllin Ⅰ group were increased, whereas relative expressions of FOXQ1 protein and mRNA were decreased (P<0.05, P<0.01). Conclusion:Mechanism of polyphyllin Ⅰ inhibiting the metastasis of colon cancer may be related to the decrease of FOXQ1 and Vimentin expressions, and the up-regulation of E-cadherin.

Effect of Jianpi Yangzheng Recipe on Apoptosis and EMT of HCT116 Cells in Colorectal Cancer by Inhibiting Aerobic Glycolysis Regulated by PKM2 / 中国实验方剂学杂志

Objective:To investigate the mechanism of Jianpi Yangzheng recipe in inhibiting aerobic glycolysis by down-regulating the expression of pyruvate kinase isoenzyme M2 (PKM2) protein, in order to promote apoptosis and inhibite epithelial-mesenchymal transition（EMT）in HCT116 cells of colorectal cancer. Method:The effect of different concentrations of Jianpi Yangzheng recipe on HCT116 cell proliferation was detected by methylthiazolyldiphenyl-tetrazolium bromide（MTT）colorimetry. Flow cytometry was used to detect the effect of different concentrations of Jianpi Yangzheng recipe（2.0, 4.0, 8.0 g·L-1） on HCT116 cell apoptosis. The effect of Jianpi Yangzheng recipe（2.0, 4.0, 8.0 g·L-1） on the migration and invasion ability of HCT116 cells was observed by cell scratch and cell invasion assay (Transwell). The effect of different concentrations of Jianpi Yangzheng recipe（2.0, 4.0, 8.0 g·L-1） on glycolysis metabolism of HCT116 cells were detected by lactic acid (LD) test kit and glucose assay kit, respectively. Western blot was used to detect the expressions of apoptosis-related proteins, like B lymphocyte tumor-2 gene (Bcl-2), Bcl-2 related X protein (Bax) and EMT-related proteins, like epithelial cadherin (E-cadherin)，neurogenic cadherin（N-cadherin）, Vimentin, and PKM2, the key protein of glycolysis, in each group. Result:MTT assay showed that, compared with the blank group, HCT116 cells were treated with Jianpi Yangzheng recipe for 48 h. With the increase of drug concentration, the inhibitory effect of Jianpi Yangzheng recipe on the proliferation of HCT116 cells was also increased; and when the concentration was 4.0 g·L-1, the inhibition rate of HCT116 cells was about 53.87%. Therefore, 2.0，4.0，8.0 g·L-1 were selected as low, medium and high-dose groups for the study. The cell flow cytometry results indicated that, compared with the blank group, the low, medium and high-dose groups all significantly induced the apoptosis of HCT116 cells (P<0.05), and the effect in inducing apoptosis was more obvious with the increase of drug concentration (P<0.05). Cell scratch and Transwell showed that, compared with the blank group, all the groups had an inhibitory effect on migration and invasion of HCT116 cells (P<0.05), and the effect was more significant with the increase of drug concentration (P<0.05). The determination of lactic acid and glucose indicated that compared with the blank group, with the increase of drug concentration, the amount of lactic acid produced by cells in each group gradually decreased (P<0.05), while the glucose dosage also gradually decreased (P<0.05). Western blot showed that, compared with the blank group, the protein expressions of E-cadherin and Bax were up-regulated in groups with different concentrations, whereas the protein expressions of N-cadherin, Vimentin, Bcl-2 and PKM2 were down-regulated (P<0.05). Conclusion:Jianpi Yangzheng recipe can effectively induce the apoptosis of HCT116 cells and inhibit EMT in colorectal cancer. The possible mechanism may be related to the inhibition of aerobic glycolysis pathway of HCT116 cells by down-regulating PKM2 protein expression.

Effect and mechanism of down-regulating REV7 expression on the radiosensitivity of human colon cancer cell HCT116 / 中华放射肿瘤学杂志

Objective To evaluate the effect and mechanism of down-regulating the expression of REV7 on the radiosensitivity of human colon cancer cell HCT116.Methods HCT116 cells were cultured and the expression of REV7 was down-regulated by RNA interference technique.HCT116 cells were divided into the blank group,negative control transfected with negative RNA oligo group and REV7 expression down-regulation transfected with REV7 RNA oligo group,respectively.The cell proliferation was determined by colony formation assay.The expression levels of the proteins of relevant genes were detected by Western blot.The level of cell apoptosis and non-homologous end joining was evaluated.Results The colony formation rate was significantly reduced in THE REV7 siRNA group after 6Gy irradiation (P＜0.05).The down-regulating efficiency rate of REV7 gene was ＞ 60％ in the REV7 siRNA group.The expression levels of γ H2AX and Caspase9 were significantly up-regulated,whereas those of KU80 and XRCC4 were remarkably down-regulated in the REV7 siRNA group (all P＜0.05).Conclusions The radiosensitivity of human colon cancer cell HCT116 can be increased by down-regulating the expression of REV7.The underlying mechanism may be related to the lower incidence rate of non-homologous end joining.

Overexpression of miR-31 regulates TLR4/NF-κB signaling pathway and apoptotic protein in colitis model mice / 中国应用生理学杂志

To investigate the effects of miR-31 on TLR4/NF-κB signaling pathway and apoptosis-related proteins in dextran sulfate sodium (DSS) induced mouse colon colitis. Methods: ① Mouse model of colon colitis: 1% DSS was used to induce mouse ulcerative colitis (UC). Fourteen FVB non-transgenic mice were randomly divided into control group (n= 6), DSS group (n= 8), and 16 FVB miR-31 transgenic mice were randomly divided into miR-31 overexpression group (n= 8), miR-31 overexpression +DSS group (n= 8). DSS was dissolved in water and administered to mice by drinking water. The DSS group and miR-31+DSS group drank 1% DSS water in the first week, normal sterilized water in the second week, and 1% DSS water in the third week, after 5 weeks, the modeling was completed, then the colon tissues of the mice were collected. Western blot and IHC were used to detect the expressions of NF-κB p65, TLR4, Bax and Bcl-2 proteins in mouse colon tissue, TUNEL was used to detect apoptosis of mouse colon tissues. ② Cell culture experiments: Transfection of miR-31mimic and inhibitor by lipofectamine resulted in overexpression or knockdown of miR-31 in human colon epithelial cell line HCT 116 cells, each group was repeated three times and cells were collected 48 h later, Western blot was used to detect the expressions of NF-κB p65 and TLR4 protein. ① In animal experiments, compared with the control group, the expression levels of NF-κB p65, TLR4 protein and apoptotic cell index in the DSS group and miR-31 overexpression group in mouse colon tissue were significantly increased (P＜0.05 or P＜0.01), and the Bcl-2 / Bax ratio was significantly reduced (P＜0.05 or P＜0.01); and compared with the DSS group, the expression levels of NF-κB p65, TLR4 protein and apoptotic cell index in the miR-31+DSS group were significantly increased (P＜0.01), while the Bcl-2/Bax ratio was significantly decreased (P＜0.01). ② In cell experiments, compared with the control group, the expression levels of NF-κB p65 and TLR4 protein in the over-expressed miR-31 group of HCT 116 cells were significantly increased (P＜0.05 or P＜0.01), the expressions of NF-κB p65 and TLR4 protein in miR-31 knockdown group were decreased (P＜0.05). miR-31 promotes the development of colitis by promoting TLR4/NF-κB signaling pathway and mediating apoptosis of intestinal epithelial cells.

Downregulation of DBN1 is related to vincristine resistance in colon cancer cells

Objective: This study was aimed to investigate the relationship between the expression of drebrin (DBN1) gene and resistance in colon cancer to reveal the mechanism of tumor drug resistance and provide a basis for the reversal of this drug resistance in tumor cells. Materials and Methods: The human colon carcinoma cell line HCT-8 was used, and vincristine (VCR)-resistant colon cancer cell line HCT-8/V was established by gradually increasing the concentration of VCR. Polymerase chain reaction (PCR) primers were designed for DBN1 gene. The DBN1 differential expression in colon cancer sensitive and resistant cell lines was detected by fluorescence quantitative PCR. Western blot analysis was used to study DBN1 expression in the resistant cells further. Results: VCR resistance of HCT-8/V cell line was established. Quantitative PCR and Western blot results showed that DBN1 expression in the resistant cell line was significantly lower, the difference being statistically significant (P < 0.05). Conclusion: Low DBN1 gene expression may be associated with colon cancer cell resistance to VCR.