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@#[摘 要] 目的： 基于Hedgehog信号通路探讨石斛提取物毛兰素（erianin，ER）抑制结直肠癌HT29细胞上皮间质转化（EMT）和血管生成的作用机制。方法: 将HT29细胞分为空白对照组、ER-L（25 μg/mL）组、ER-M（50 μg/mL）组、ER-H（75 μg/mL）组、ER-H（75 μg/mL）+PM（Hedgehog通路激活剂，1.5 μmol/L）组。MTT法检测细胞增殖活力，克隆形成实验检测细胞克隆形成能力，划痕实验和Transwell实验检测细胞迁移与侵袭能力，血管拟态形成实验检测血管生成能力，WB法检测与EMT进程、Hedgehog信号通路和拟态血管生成相关蛋白质的表达。结果: HT29细胞增殖活性随着ER质量浓度的升高而逐渐降低（P<0.05）；与空白对照组比较，ER各组细胞克隆形成率、迁移与侵袭能力、血管形成能力、间质标志蛋白（N-cadherin、vimentin）、血管生成相关蛋白（VEGF、VE-cadherin）及Hedgehog通路相关蛋白（SHH、GLI1、SMO、c-Myc）表达均显著下降（均P<0.05），上皮标志蛋白（E-cadherin）、Hedgehog通路中融合蛋白抑制剂（SUFU）蛋白表达均显著上升（均P<0.05）；PM处理在一定程度上逆转了ER对于HT29细胞增殖、EMT和血管生成的抑制作用（均P<0.05）。结论: ER可以抑制结直肠癌HT29细胞的增殖、迁移与侵袭、EMT和血管生成，其机制可能与抑制Hedgehog信号通路激活有关。
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Objective:To establish a colon cancer cell line which overexpressing LINC01018 stably and study its biological characteristics.Methods:The expression of LINC01018 in HCoEpiC and HT-29 cells were detected by real time fluorescence quantitative polymerase chain reaction (qRT-PCR). HT-29 cells were infected with LINC01018 overexpression lentivirus to screen and establish HT-29 cell lines which overexpressing LINC01018 stably. The effect of LINC01018 on the proliferation, invasion and migration of HT-29 cells were detected by cell counting kit-8 (CCK-8) assay and Transwell assay separately. The expression of CDK6 and matrix metalloproteinase-2 (MMP-2) in HT-29 cells was detected by Western blot.Results:The expression of LINC01018 in HT-29 cells was significantly lower than that in the human colonic epithelial cells (HCoEpiC). HT-29-L18 cell lines which overexpressing LINC01018 stably was screened successfully. Overexpression of LINC01018 significantly inhibited the cell proliferation, invasion and migration, and reduced the protein expression of CDK6 and MMP-2 in HT-29 cells.Conclusions:The expression of LINC01018 was decreased abnormally in colon cancer cells. Up-regulation of LINC01018 expression can inhibit the proliferation, invasion and migration of colon cancer cells, which may be related to CDK6 and MMP-2.
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AIM: To study the inhibitory effect and mechanism of Jolkinolide B (JB) on the proliferation and metastasis of colon cancer HT-29 cells. METHODS: HT-29 cells were treated with different concentrations of JB, the cell proliferation rate was detected by MTT method, the cell clone formation rate was detected by plate cloning experiment, the cell cycle change was detected by flow cytometry, the cell migration ability was analyzed by wound healing assay, the cell invasion ability was studied by Transwell assay, E-cadherin, N-cadherin, vimentin, Snail1, Snail2, matrix metalloproteinase (MMP)-2 and MMP-9 protein expression levels were detected by immunofluorescence and Western blotting, and p-PI3K, PI3K, p-Akt, Akt, NF-κB P65and p-NF-κB P65 protein expression levels were detected by Western blotting. HT-29 cells were treated with 100 μg/L IGF-1 and 100 μg/L IGF-1+20 μmol/L JB, respectively. The expression of PI3K/Akt/NF-κB pathway related proteins was detected by Western blotting. RESULTS: JB inhibited the proliferation of HT-29 cells in a concentration-dependent manner, with an IC
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RESUMEN Objetivos: Determinar el efecto citotóxico y genotóxico in vitro del extracto crudo y etanólico del rizoma de Curcuma longa L. Materiales y métodos: El efecto citotóxico fue evaluado utilizando líneas celulares DU-145, HT-29, 3T3 BALB/c. Se hallaron los porcentajes de crecimiento en 48 horas y se determinó la concentración inhibitoria 50 (CI50). El efecto genotóxico en el ADN genómico humano se determinó mediante el método Tomasevich. Resultados: El extracto crudo produjo una CI50 de 12,98 ± 0,21 μg/mL para la línea celular tumoral HT-29, que es inferior a DU-145 con una CI50 de 36,77 ± 9,12 μg/mL; el extracto etanólico presentó una CI50 de 13,24 ± 0,77 y 20,54 ± 2,58 µg/mL para ambas líneas celulares, respectivamente; el compuesto estándar curcumina presentó una CI50 de 3,96 ± 0,60 y 13,94 ± 2,79 μg/mL, respectivamente. El extracto crudo a concentraciones de 50 y 100 mg/mL fragmentó entre el 40% a 95% de ADN genómico humano; mientras que, a 200 mg/mL, la fragmentación fue mayor al 95%. El extracto etanólico a todas las concentraciones no fragmentó el ADN. La curcumina a 200 mg/mL fragmentó menos del 5% de ADN genómico humano. Conclusiones: Los extractos crudo y etanólico de Curcuma longa L. demuestran efecto citotóxico in vitro diferencial para la línea celular tumoral humana DU-145 y HT29 semejante al compuesto estándar curcumina. El extracto crudo de Curcuma longa L. presenta una potente actividad genotóxica in vitro frente al ADN genómico humano, esta actividad está ausente en el extracto etanólico.
ABSTRACT Objectives: To determine the in vitro cytotoxic and genotoxic effect of the crude and ethanolic extract from the Curcuma longa L. rhizome. Materials and methods: The cytotoxic effect was evaluated using DU-145, HT-29, 3T3 BALB/c cell lines. The growth percentages in 48 hours; and the half maximal inhibitory concentration (IC50) were determined. The genotoxic effect on human genomic DNA was determined using the Tomasevich method. Results: Crude extract produced an IC50 of 12.98 ± 0.21 μg/mL for the HT-29 tumor cell line, which is lower than the value obtained for DU-145, with an IC50 of 36.77 ± 9.12 μg/mL. The ethanolic extract presented an IC50 of 13.24 ± 0.77 and 20.54 ± 2.58 μg/mL for both cell lines, respectively; the curcumin standard compound presented an IC50 of 3.96 ± 0.60 and 13.94 ± 2.79 μg/mL, respectively. Crude extract concentrations of 50 and 100 mg/mL fragmented between 40% to 95% of human genomic DNA; while at 200 mg/mL, fragmentation was greater than 95%. The ethanolic extract at all concentrations did not fragment the DNA. Curcumin at 200 mg/mL fragmented less than 5% of human genomic DNA. Conclusions: The crude and ethanolic extracts of Curcuma longa L. demonstrate different in vitro cytotoxic effects for the human tumor cell lines DU-145 and HT-29; similar to the standard curcumin compound. The crude extract of Curcuma longa L. shows a potent genotoxic in vitro activity against human genomic DNA; this type of effect is not produced by the ethanolic extract.
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In Vitro Techniques , Curcuma , Rhizome , Cell Line, Tumor , Complex Mixtures , Cell Line , HT29 Cells , Inhibitory Concentration 50 , BALB 3T3 CellsABSTRACT
@#[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.
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OBJECTIVE：To investigate the effects and its mechanism of pseudostrychnine on the apoptosis of human colon can - cer HT- 29 cells. METHODS ：Human colon cancer HT- 29 cells were randomly divided into blank group ，pseudostrychnine low-dose，medium-dose and high-dose groups （125，250，500 μmol/L）. They were cultured with culture medium without medicine or relevant concentration of pseudostrychnine for 48 h. The cell apoptosis and mitochondrial transmembrane potential were detected by flow cytometry. Western blotting assay was employed to detect the protein expression of P 53，Caspase-3，Caspase-9，DNA re - pair enzyme from rabbit （c-PARP）and Bcl- 2. RESULTS ：Compared with blank group ，apoptotic rate of cells was increased signifi - cantly in pseudostrychnine low-dose ，medium-dose and high-dose groups ，while mitochondrial transmembrane potential was de - creased significantly （P＜0.01），in concentration-dependent manner. The protein expression levels of P 53，Caspase-3，Caspase-9 and c-PARP were increased in pseudostrychnine medium-dose and high-dose groups ，compared with blank group ；while those of Bcl-2 were decreased significantly in pseudostrychnine low-dose ，medium-dose and high-dose groups （P＜0.05 or P＜0.01）. CON - CLUSIONS：Pseudostrychnine may change mitochondrial membrane potential by up-regulating the protein expression of P 53 and down-regulating the protein expression of Bcl- 2，and activate the expression of Caspase- 3，c-PARP and Caspase- 9，so as to acti - vate endogenous mitochondrial pathway and promote the apoptosis of HT- 29 cells.
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Objective: To compare the anti-proliferative effect of sodium thiosulfate on human colorectal cancer cells (HT-29) and normal small intestine cells (IEC6). Methods: Cells (HT-29 and IEC6) were treated with different concentrations of sodium thiosulfate ranging from 0.5 mM to 80 mM for 24 h. Cell viability was measured via crystal violet and MTT assays. HT-29 cells were further treated in the presence and absence of mitochondrial electron transport chain (ETC) inhibitors, KATP channel opener and closer and H2S inhibitors for 24 h followed by sodium thiosulfate in order to study their respective roles in the anti-proliferative activity of sodium thiosulfate. Results: The IC50 values of sodium thiosulfate on HT-29 cells were 40.93 mM and 42.45 mM by crystal violet and MTT assay whereas, in the case of IEC6 cells, the values were 45.17 mM and 47.22 mM. The inhibition of endogenous H2S enzymes and KATP channel induced no change in the anti-proliferative capacity of sodium thiosulfate. However, the anti-proliferative activity of sodium thiosulfate was enhanced in the presence of mitochondrial ETC inhibitors. Conclusions: HT-29 cell growth is effectively attenuated by sodium thiosulfate and the anti-proliferative activity of sodium thiosulfate is enhanced in the presence of mitochondrial ETC inhibitors.
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OBJECTIVE：To investigate the inhibitory effects of total alkaloids of Gelsemium elegans （TAG） on the proliferation and angiogenesis of human colon cancer cells. METHODS ：Human colon cancer cell line HT- 29 and HUVEC were cultured in vitro . After the intervention of low- ，medium-，high-dose TAG （40，80，120 μg/mL），the morphology of the two cells was observed by fluorescence inversion microscope. The survival rate of HT- 29 cells and HUVEC was detected by CCK- 8 assay. Flow cytometry was used to detect HT- 29 cell cycle. The migration rate ，invasion rate and tube number of HUVEC were observed by scratching test ，Transwell invasion experiment and tube formation experiment. RESULTS ：Compared with blank group ，HT-29 cells and HUVEC were decreased to different extents in TAG groups ；dead cells were observed ，and the survival rate of both decreased significantly （P＜0.05 or P＜0.01）. The proportion of HT- 29 cells at G 2/M phase in TAG groups as well as those at G 0/G1 phase in medium-dose group were increased significantly ；the proportion of HT- 29 cells at S phase in TAG groups as well as those at G 0/G1 phase in high-dose group were decreased significantly （P＜0.05 or P＜0.01）. Survival rate ，migration rate and invasion rate of HUVEC were decreased significantly in TAG groups ，and tube number was also decreased significantly at each time point during 4-24 h（P＜0.01）. CONCLUSIONS ：TAG have inhibitory effect on the proliferation of human colon cancer HT- 29 cells and HUVEC，can change HT- 29 cell cycle ，inhibit the migration ，invasion and tube formation of HUVEC.
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Objective: To compare the anti-proliferative effect of sodium thiosulfate on human colorectal cancer cells (HT-29) and normal small intestine cells (IEC6). Methods: Cells (HT-29 and IEC6) were treated with different concentrations of sodium thiosulfate ranging from 0.5 mM to 80 mM for 24 h. Cell viability was measured via crystal violet and MTT assays. HT-29 cells were further treated in the presence and absence of mitochondrial electron transport chain (ETC) inhibitors, K
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Objective:To investigate the effect of oxymatrine (OMT) on the proliferation and migration of human colon cancer cell line HT-29 under Type Ⅱ diabetes environment by co-culturing HT-29 with insulin to simulate hyperinsulinemia. Method:The effect of OMT (2, 4, 8 mmol·L-1) on insulin-induced proliferation of HT-29 was detected by methyl thiazolyl tetrazolium (MTT) assay and cloning assay. The morphology change and cell migration were evaluated under microscope and by wound healing assay. The Annexin V/propidium iodide(PI) assay was used to detect the change of insulin-induced HT-29 cell cycle and apoptosis. Western blot was performed to validate the expression of cell cycle-related protein and cell migration protein. Result:Insulin significantly increased growth of HT-29 (P-1 showed a significant inhibitory effect in this model (P0/G1 phase (P1, CDK4 and the up-regulation of p27 by OMT might involve the growth inhibition mechanism. Furthermore, OMT reduced the migration of insulin-induced HT-29 according to wound healing assay(PPPConclusion:OMT can inhibit the proliferation and migration of insulin-induced HT-29 cells. The changes of cell cycle and migration related proteins may be correlated with the mechanism.
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@# Objective: To investigate the effect and mechanism of polysaccharide of atractylodes macrocephala (PAM) on the growth of colon cancer cells in mice bearing in-situ colon cancer transplantation tumor. Methods: 1×107 colon cancer HT-29 cells labeled with luciferase were injected into colon serosa of the mice to establish the in-situ colon cancer transplantation tumor model. When the tumor volume reached 230 mm3, the mice were given 30 mg/kg PAM (PAM group) or equal volume of normal saline (Control group) by gavage for 10 consecutive days. The effect of PAM on the growth of colon cancer cells in mice was tested by in vivo tumor imaging technology. The expressions of MHCII and IL-12 in granulocytes, dendritic cells and macrophages, the activation of lymphocytes, and IFN-γ expression in CD4+ and CD8+ cells of tumor tissues were detected by Flow cytometry. Results: PAM significantly inhibited the growth of colon cancer cells in mice bearing in-situ colon cancer transplantation tumor (P<0.01). PAM activated immune cells though increasing the expression levels of MHCII and IL-12 in dendritic cells and macrophages (both P<0.01). PAM significantly increased the frequency of CD8+ cells, NK cells, CD44+/NK cells and CD44+/CD4+ cells in tumor tissues and the number of CD8+ cells and NK cells per unit volume (all P<0.01). PAM significantly increased the IFN-γ secretion of CD4+ and CD8+ cells (both P<0.01), too. Conclusion: PAM inhibits the growth of colon cancer by activating immune cells in tumor tissues of mice bearing in-situ colon cancer transplantation tumor.
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@# Objective: To investigate the therapeutic effect of dendritic cell-induced killer cells (DC-CIK) combined with 5-fluorouracil (5-FU) and loaded with CD133+ HT-29 cell lysate or RNA on mice bearing colon cancer HT-29 cell transplanted tumor, and to explore the underlying mechanism. Methods: Colon cancer xenograft model was established in BALB /c nude mice by using human colon cancer HT-29 cells at logarithmic growth phase; Antigen-free DC-CIK, 5-FU+DC-CIK, R+DC-CIK (loaded with total RNA of CD133+ cells), L+DC-CIK (loaded with CD133+ cell lysate), 5-FU and normal saline were respectively injected into transplanted mice, and the treatment efficacies on the growth of transplanted tumor in each group after three treatment cycles were observed, and the tumor growth curve was drawn. The nude mice were sacrificed by cervical dislocation and the tumor volume and body weight were measured. qPCR was used to detect the expression of AKT mRNA in transplanted tumor tissue, and WB was used to detect the expression of phosphorylated AKT protein. Results: After treatment, the body mass of nude mice in R+DC-CIK group, L+DC-CIK group and 5-FU+DC-CIK group increased steadily, while the body mass of nude mice in DC-CIK group and 5-FU group decreased gradually; the tumor growth speed of nude mice in R+DC-CIK group, 5-FU+DC-CIK group and L+DC-CIK group was significantly slower than that of the control group (P<0.05). Compared with 5-FU and DC-CIK alone, the combined treatment with loaded lysate/RNA had more sig nificant effect on mRNA and protein expressions of AKT(P<0.05). Conclusion: The effect of DC-CIKwithdifferentloadingoritscombinationwith5-FUisbetterthanthatofchemotherapy alone. One of the mechanisms is related to the down-regulation ofAKT level.
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@# Objective: To investigate the effect of astragalus polysaccharides (APS) on proliferation, invasion, apoptosis and drugresistance of cisplatin-resistant colorectal cancer (CRC) HT-29/DDP cells through regulating miR-20a/TGFBR2 axis, and to explore the possible mechanism. Methods: Human CRC HT-29 cells and HT-29/DDP cells were used as non-drug resistant and resistant cell models, respectively; HT-29/DDP cells were randomly divided into four groups, including untreated (HT-29/DDP) group, APS treatment group, miR-20a mimics + APS group, and si-TGFBR2 + APS group. qPCR and Western blotting were applied to detect the expressions of miR-20a and TGFBR2 in HT-29/DDP cells treated with different concentrations ofAPS (0, 0.5, 1.0, 1.5 and 2.0 mg/ml). Subsequently, dual luciferase reporter gene assay was used to verify whether TGFBR2 was a target gene of miR-20a. In addition, CCK-8, Transwell andAnnexin V-FITC/PI double staining were applied to examine the effect ofAPS on proliferation, invasion and apoptosis of HT29/DDP cells. Furthermore, subcutaneous HT-29/DDP cell xenograft model was established on nude mice, and the effect ofAPS on the growth of transplanted tumor was observed. Results: APS significantly inhibited the proliferation of HT-29/DDP cells in a dose-dependent manner (P<0.01). Meanwhile, the expression of miR-20a was down-regulated in HT-29/DDP cells treated with APS, while the expression of TGFBR2 was significantly up-regulated (all P<0.01). Additionally, dual luciferase reporter gene assay result showed that TGFBR2 was a direct target of miR-20a in HT-29/DDP cells and its expression was suppressed. Furthermore, APS could enhance the drug sensitivity of HT-29/DDP cells through downregulating the inhibitory effect of miR-20a on TGFBR2 expression, thereby suppressed proliferation and invasion, and induced apoptosis of HT-29/DDP cells in vitro and in vivo. It was also found that this effect was related with the suppression of PCNA and Bcl-2 proteins and promotion of Bax and Caspase-3 proteins. Conclusion: APS reverses the resistance of HT-29/DDPcells to cisplatin by down-regulating the inhibitory effect of miR-20a on TGFBR2 expression.
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Drug Metabolizing Enzyme (DME) has been a target of natural chemopreventive agents to inhibit, retard and reverse theprocess of carcinogenesis. Pterostilbene, an analog to resveratrol has been reported to possess various pharmacologicalbenefits including chemoprevention. In our study, benzo[a]pyrene-induced HT-29 colorectal cell line was used as theDME model. The activity of phase I enzyme CYP1A as determined by the 7-ethoxyresorufin O-deethylation (EROD) assaywas found to be inhibited significantly by pterostilbene at 50 µM, 75 µM and 100 µM (p ≤ 0.01, p ≤ 0.05, p ≤ 0.01respectively) compared to the benzo[a]pyrene treated group. Meanwhile, pterostilbene induced glutathione-S-transferase(GST) activity significantly (p ≤ 0.01) at 50 µM as compared to the untreated. In addition, However, the protein expressionof CYP1A1 and GST in pterostilbene treated group was not significantly affected compared to untreated. On the otherhand, pterostilbene at 25 and 75 µM were able to increase the protein expression of transcription factor Nrf2 significantly(p ≤ 0.01). Results indicated that pterostilbene could reduce metabolic activation of procarcinogens and increase thedetoxification process which can be potentially developed as chemopreventive agent.
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Objective: To identify the bioactive extracts from Alternanthera sessilis and investigate its cytotoxicity potential against colon cancer cells, HT-29. Methods: This study examined the effects of three parts (aerial, leaf, stem) of whole plant on HT-29 colon cancer cell lines. Three different extracts from the plant parts were prepared by maceration technique using 80％ ethanol. The anticancer activities were determined using MTT, clonogenic, cell motility and AOPI assay. The chemical composition profiling was analyzed by GC-MS. Results: Among three plant part extracts, leaf extract greatly suppressed the growth of colon cancer cells in time and dosage-dependent manner, followed by aerial and stem. The cytotoxicity results were rationalized with clonogenic, cell motility and AO/PI assay, where extract showed the most active activity compared to aerial and stem extracts. GC-MS analysis of leaf extract showed there were various recognized anti-cancer, anti-oxidant and anti-inflammatory compounds. Conclusions: Amid the screened extracts, the leaf extract exhibits the credible cytotoxic, anti-proliferative and apoptotic activity and hence, our findings call for additional research to conclude the active compounds and their mechanisms determining the apoptotic activity.
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Objective To study the inhibitory effects of Chinese yam combined with tegafu on colon cancer HT-29 cells in vivo and in vitro.Methods The changes of cell morphology were observed by the method of adopting 2×2 factorial designs,after HT-29 colon cancer cells were treated with physiological saline containing dimethyl sulfoxide(blank control), tegafur(36 mg/L)and Chinese yam(125 mg/L).MTT method was used to detect the proliferation of tumor cells,and flow cytometry was used to detect the expression of tumor stem cells(CD133+cells).The nude mouse model of colon cancer HT-29 cells was established. The treatment was given in the above method, and the tumor inhibition rate was calculated. The VEGF positive rate of the tumor was detected by immunohistochemistry. Results The inhibited proliferation of colon cancer HT-29 cells was significantly higher in the synergistic group than that in Chinese yam group and tegafur group,and the inhibitory rates were higher in the three treatment groups than that of blank control group.The proportion of CD133+cells was significantly lower in HT-29 cells in the synergistic group compared with that of the Chinese yam group and the tegafur group,and which was lower in these three groups than that of the blank control group.After the treatment in three treatment groups,the tumor quality and VEGF positive rate were significantly lower than those of the blank control group,and which was lower in the tegafur+Chinese yam group than that of Chinese yam group and the tegafur group. Conclusion Chinese yam combined with tegafu can inhibit the proliferation of colon cancer HT-29 cells and their stem cells.
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Objective:To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus (O. 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)-2H-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 with 4′,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-GConclusions:Combining the above results, it is thought that the survival of HT-29 cells is suppressed by ethylacetate fraction from O. japonicus through mitochondrial regulation-induced caspase cascade activation, induction of apoptosis and cell cycle arrest.
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Objective: To identify the bioactive extracts from Alternanthera sessilis and investigate its cytotoxicity potential against colon cancer cells, HT-29. Methods: This study examined the effects of three parts (aerial, leaf, stem) of whole plant on HT-29 colon cancer cell lines. Three different extracts from the plant parts were prepared by maceration technique using 80% ethanol. The anticancer activities were determined using MTT, clonogenic, cell motility and AOPI assay. The chemical composition profiling was analyzed by GC-MS. Results: Among three plant part extracts, leaf extract greatly suppressed the growth of colon cancer cells in time and dosage-dependent manner, followed by aerial and stem. The cytotoxicity results were rationalized with clonogenic, cell motility and AO/PI assay, where extract showed the most active activity compared to aerial and stem extracts. GC-MS analysis of leaf extract showed there were various recognized anti-cancer, anti-oxidant and anti-inflammatory compounds. Conclusions: Amid the screened extracts, the leaf extract exhibits the credible cytotoxic, anti-proliferative and apoptotic activity and hence, our findings call for additional research to conclude the active compounds and their mechanisms determining the apoptotic activity.
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Objective: To investigate the anti-colon cancer effects of ethylacetate fraction from Orostachys japonicus (O. 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)-2H-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 with 4′,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
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OBJECTIVE:To study the effect and its mechanism of Fuzheng Jiedu Quyu formula(short forFuzheng formula) combined with oxaliplatin (L-OHP) on human colon cancer HT-29 cell proliferation and apoptosis. METHODS:HT-29 cells were divided into blank control group(without drugs),Fuzheng formula group(1000 mg/L),L-OHP group(31.25 mg/L)and combi-nation group (1000 mg/L Fuzheng formula+31.25 mg/L L-OHP). After cultured with corresponding drug for 48 h,MTT method was used to detect the cell proliferation;the changes of cellular morphology were observed by invert microscope;flow cytometry was used to detect the cell cycle and apoptosis rate. Proapoptotic gene Bax,apoptotic gene Bcl-2 mRNA expressions were deter-mined by real-time fluorescence quantitative polymerase chain reaction method;Bax,Bcl-2 protein expressions were assayed by Western blot. RESULTS:Compared with blank control group,cell proliferation was inhibited in L-OHP group and combination group;cell proportion was increased in S stage,G2/M stage and decreased in G0/G1 stage(P<0.05). Cell apoptosis rate in L-OHP group,Fuzheng formula group and combination group was increased;Bax mRNA and protein expression were up-regulated,Bcl-2 mRNA expression was downregulated(P<0.05);and combination group changed more obviously than the single drug groups(P<0.05). CONCLUSIONS:Fuzheng formula combined with L-OHP can inhibit HT-29 cell proliferation and promote its apoptosis, showing better effects than either of the two drugs alone. The mechanism may be associated with up-regulation of Bax gene and pro-tein expressions and down-regulation of Bcl-2 gene expressions in cells.