Antitumor Activities and Apoptosis-regulated Mechanisms of Fermented Wheat Germ Extract in the Transplantation Tumor Model of Human HT-29 Cells in Nude Mice

Objective A subcutaneous transplantation tumor model of human HT-29 cells in nude mice was established to evaluate anticarcinogenic activities, and the apoptosis-regulated mechanism effect of aqueous extract of fermented wheat germ with Lactobacillus plantarum dy-1 （LFWGE）. Methods The HT-29 cells were transplanted via subcutaneous injection of 1×10^7cells into the right flank of each nude mouse. Then, nude mice were treated for 30 d with LFWGE （high-dose 2 g/kg/d; low-dose 1 g/kg/d） and for 7 d with 5-fluorouracil （5-FU, 25 mg/kg/d） by gavage and intraperitoneal injection, respectively. An inhibition of tumor growth was observed. Results Tumor volume and weights decreased significantly in both groups of nude mice treated with LFWGE. In addition, the cell apoptosis rate of the LFWGE group （2 g/kg/d, 60.2%＋4.4%; 1 g/kg/d, 58.6%＋6.9%） was significantly higher than that of the control group （11.5%＋1.6%） and 5-FU group （32.1%＋3.5%） as measured by the TUNEL assay. Moreover, the real-time fluorescent quantitative PCR and Western blot method further confirmed these enhancing apoptosis and growth inhibition effects. The involvement of LFWGE in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, Caspase-3, and CyclinD1. Conclusion The results showed that LFWGE could induce subcutaneous transplantation tumor apoptosis in nude mice and could be as a natural nutrient supplements or chemopreventive agent in the treatment of human colon cancer.

Antitumor Activities and Apoptosis-regulated Mechanisms of Fermented Barley Extract in the Transplantation Tumor Model of Human HT-29 Cells in Nude Mice

Objective A subcutaneous transplantation tumor model of human HT-29 cells was established in nude mice to study the anticarcinogenic activities and apoptosis-regulatory mechanistic effect of aqueous extract of fermented barley with Lactobacillus plantarum dy-1 （LFBE）. Methods HT-29 cells were transplanted via subcutaneous injection of 1 × 107cells into the right flank of each nude mouse. Then, nude mice were treated for 30 days with LFBE （high-dose 2 g·kg-1·d-1; low-dose 1 g·kg-1·d-1） and for 7 days with 5-fluorouracil （5-FU, 25 g·kg-1·d-1） by gavage and intraperitoneal injection, respectively. Results Tumor volume and weight decreased significantly in both groups of nude mice treated with LFBE. In addition, the cell apoptosis rate of the LFBE group was significantly higher than that of the control group and 5-FU groups as measured by the TUNEL assay. Moreover, the real-time fluorescent quantitative PCR and Western blot methods further confirmed these apoptosis-enhancing and growth-inhibiting effects. The involvement of LFBE in inducing apoptosis was confirmed by the expression of Bax, Bcl-2, caspase-3, and cyclin D1. Conclusion The results showed that LFBE could induce subcutaneous transplantation tumor apoptosis in nude mice and could be used as a natural nutrient supplement or chemopreventive agent in the treatment of human colon cancer.

Curcumin suppresses PPARδ expression and related genes in HT-29 cells

AIM:To investigate the effects of curcumin on the expression of peroxisome proliferator-activated receptorδ(PPARδ)and related genes in HT-29 cells. METHODS:HT-29 cells were treated with curcumin (0-80μmol/L)for 24 h.The effects of curcumin on the morphology of HT-29 cells were studied by Hoechst 33342 staining.The activity of caspase-3 was determined using DEVD-p NA as substrate.The levels of peroxisome PPARδ,14-3-3εand vascular endothelial growth factor(VEGF)in HT-29 cells were determined by Western blotting analysis and their mRNA expression was determined by real-time quantitative RT-PCR. RESULTS:Treatment with 10-80μmol/L curcumin induced typical features of apoptosis and activated the caspase-3 in HT-29 cells.The expression of PPARδ, 14-3-3εand VEGF was reduced and the activity of β-catenin/Tcf-4 signaling was inhibited by curcumin treatment. CONCLUSION:Curcumin can induce apoptosis of HT-29 cells and down-regulate the expression of PPARδ,14-3-3εand VEGF in HT-29.

Influence of paeonol on expression of COX-2 and p27 in HT-29 cells

AIM： To investigate the effect of paeonol on controlling the proliferation of colorectal cancer cell line HT-29 and to discuss its possible mechanism. METHODS： The inhibitory effect of paeonol on proliferation of HT-29 cells was detected by M-I-I- assay. The results of apoptosis were measured by flow cytometry. Immunocytochemical staining was performed to detect the expression of cyclooxygenase-2 （COX-2） and protein p27 in HT-29 cells treated with paeonol at different concentrations. Reverse transcription-polymerase chain reaction （RT- PCR） was used for mRNA analysis. RESULTS： From the data of both MTT and flow cytometry, we observed that cell proliferation was inhibited by different concentrations of paeonol. By immunocytochemical staining, we found that HT-29 cells treated with paeonol （0.024-1.504 mmol/L） reflected reduced expression of COX-2 and increased expression of p27 in a dose-dependent manner. RT-PCR showed that paeonol down-regulated COX-2 and up-regulated p27 in a dose- and time-dependent manner in HT-29 cells. CONCLUSION： One of the apoptotic mechanisms of paeonol is down-regulation of COX-2. p27 is upregulated simultaneously and plays an important part in controlling cell proliferation and is a crucial factor in the Fas/FasL apoptosis pathway.

Pien Tze Huang(片仔癀)-Induced Apoptosis in Human Colon Cancer HT-29 Cells Is Associated with Regulation of the Bcl-2 Family and Activation of Caspase 3

Objective：To investigate the cellular effects of Pien Tze Huang（片仔癀,PZH） in the HT-29 human colon carcinoma cell line.Methods：The viability of HT-29 cells was determined by MTT assay.A fluorescence-activated cell sorting（FACS） analysis with annexin-V/propidium iodide（PI） and JC-1 staining were performed to determine cell apoptosis and the loss of mitochondrial membrane potential,respectively.Activation of caspase 3 was evaluated by a colorimetric assay.The mRNA expression levels of Bcl-2 and Bax were measured by reverse transcription polymerase chain reaction（RT-PCR）.Results：PZH,in a dose- and time-dependent manner,reduced viability and induced apoptosis of HT-29 cells.Moreover,PZH treatment resulted in the collapse of the mitochondrial membrane potential,activation of caspase 3,and an increase in the Bax/Bcl-2 ratio.Conclusion：PZH inhibits the growth of HT-29 cells by inducing cancer cell apoptosis via regulation of the Bcl-2 family and activation of caspase 3,which may,in part,explain its anticancer activity.

Curcumin induces apoptosis through the mitochondria-mediated apoptotic pathway in HT-29 cells

Objective： To investigate the effects of curcumin on release of cytochrome c and expressions of Bcl-2, Bax, Bad, Bcl-xL, caspase-3, poly ADP-ribose polymerase （PARP）, and survivin of HT-29 cells. Methods： HT-29 cells were treated with curcumin （0-80 μmol/L） for 24 h. The release of cytochrome c from the mitochondria and the apoptosis-related proteins Bax, Bcl-2, Bcl-xL, Bad, caspase-3, PARP, and survivin were determined by Western blot analysis and their mRNA expressions by reverse transcriptase-polymerase chain reaction （RT-PCR）. Results： Curcumin significantly induced the growth inhibition and apoptosis of HT-29 cells. A decrease in expressions of Bcl-2, Bcl-xL and survivin was observed after exposure to 10-80 μmol/L curcumin, while the levels of Bax and Bad increased in the curcumin-treated cells. Curcumin also induced the release of cyto- chrome c, the activation ofcaspase-3, and the cleavage of PARP in a dose-dependent manner. Conclusion： These data suggest that curcumin induced the HT-29 cell apoptosis possibly via the mitoehondria-mediated pathway.

Cell cycle arrest effect of compound YSY-01A, a new proteasome inhibitor, on HT-29 cells in vitro

Compound YSY-01A, a recently synthesized proteasome inhibitor, has shown potent growth-inhibitory effect on tumor cells in previous researches. However, the mechanism of its inhibitory effects, especially on cell cycle, remains largely unclear. This study aims to evaluate the correlation between cell cycle arrest effect of YSY-01A and its anti-cancer effect, and to probe the possible molecular mechanisms for its effects on human colorectal adenocarcinoma cells HT-29. The results suggested that YSY-01A significantly （P0.05） inhibited cellular proliferation of HT-29 cells in a time and concentration-dependent manner. Furthermore, YSY-01A suppressed the G 2 /M transition of HT-29 cells, whereas the mitotic inhibitor paclitaxel induced M phase accumulation. Further investigation revealed that YSY-01A significantly （P0.05） up-regulated the expression levels of a series of cell cycle related protein, such as cyclin B1, Wee1, p-cdc2 （Tyr15）, p53, p21, and p27. The HT-29 cells only exhibited typical cytotoxic symptom when YSY-01A concentration reached 0.5 μM （P0.05）, which was above the dose we used in the mechanism research. In conclusion, YSY-01A showed remarkable anti-cancer activity on HT-29 cells, and its molecular mechanisms are related to G 2 /M cell cycle transition arrest.

Extracellular vesicles derived from mesenchymal stem cells mediate extracellular matrix remodeling in osteoarthritis through the transport of microRNA-29a

BACKGROUND Knee osteoarthritis(KOA)is a common orthopedic condition with an uncertain etiology,possibly involving genetics and biomechanics.Factors like changes in chondrocyte microenvironment,oxidative stress,inflammation,and immune responses affect KOA development.Early-stage treatment options primarily target symptom relief.Mesenchymal stem cells(MSCs)show promise for treatment,despite challenges.Recent research highlights microRNAs(miRNAs)within MSC-released extracellular vesicles that can potentially promote cartilage regeneration and hinder KOA progression.This suggests exosomes(Exos)as a promising avenue for future treatment.While these findings emphasize the need for effective KOA progression management,further safety and efficacy validation for Exos is essential.AIM To explore miR-29a’s role in KOA,we’ll create miR-29a-loaded vesicles,testing for early treatment in rat models.METHODS Extraction of bone marrow MSC-derived extracellular vesicles,preparation of engineered vesicles loaded with miR-29a using ultrasonication,and identification using quantitative reverse transcription polymerase chain reaction;after establi-shing a rat model of KOA,rats were randomly divided into three groups:Blank control group injected with saline,normal extracellular vesicle group injected with normal extracellular vesicle suspension,and engineered extrace-llular vesicle group injected with engineered extracellular vesicle suspension.The three groups evaluation,histological detection,and immunohistochemical detection to compare and evaluate the progress of various forms of arthritis.RESULTS General behavioral observation results showed that the extracellular vesicle group and engineered extracellular vesicle group had better performance in all four indicators of pain,gait,joint mobility,and swelling compared to the blank control group.Additionally,the engineered extracellular vesicle group had better pain relief at 4 wk and better knee joint mobility at 8 wk compared to the normal extracellular vesicle group.Imaging examination results showed that the blank control group had the fastest progression of arthritis,the normal extracellular vesicle group had a relatively slower progression,and the engineered extracellular vesicle group had the slowest progression.Gross histological observation results showed that the blank control group had the most obvious signs of arthritis,the normal extracellular vesicle group showed signs of arthritis,and the engineered extracellular vesicle group showed no significant signs of arthritis.Using the Pelletier gross score evaluation,the engineered extracellular vesicle group had the slowest progression of arthritis.Results from two types of staining showed that the articular cartilage of rats in the normal extracellular vesicle and engineered extracellular vesicle groups was significantly better than that of the blank control group,and the engineered extracellular vesicle group had the best cartilage cell and joint surface condition.Immunohistochemical detection of type II collagen and proteoglycan showed that the extracellular matrix of cartilage cells in the normal extracellular vesicle and engineered extracellular vesicle groups was better than that of the blank control group.Compared to the normal extracellular vesicle group,the engineered extracellular vesicle group had a better regulatory effect on the extracellular matrix of cartilage cells.CONCLUSION Engineered Exos loaded with miR-29a can exert anti-inflammatory effects and maintain extracellular matrix stability,thereby protecting articular cartilage,and slowing the progression of KOA.

Differentiation and immunosuppressive function of CD19^(+)CD24^(hi)CD27^(+) regulatory B cells are regulated through the miR-29a-3p/NFAT5 pathway

Background: Regulatory B cells(Bregs) is an indispensable element in inducing immune tolerance after liver transplantation. As one of the microRNAs(miRNAs), mi R-29a-3p also inhibits translation by degrading the target mRNA, and yet the relationship between Bregs and mi R-29a-3p has not yet been fully explored. This study aimed to investigate the impact of miR-29a-3p on the regulation of differentiation and immunosuppressive functions of memory Bregs(m Bregs) and ultimately provide potentially effective therapies in inducing immune tolerance after liver transplantation. Methods: Flow cytometry was employed to determine the levels of Bregs in peripheral blood mononuclear cells. TaqMan low-density array miRNA assays were used to identify the expression of different miRNAs, electroporation transfection was used to induce mi R-29a-3p overexpression and knockdown, and dual luciferase reporter assay was used to verify the target gene of miR-29a-3p. Results: In patients experiencing acute rejection after liver transplantation, the proportions and immunosuppressive function of m Bregs in the circulating blood were significantly impaired. mi R-29a-3p was found to be a regulator of m Bregs differentiation. Inhibition of miR-29a-3p, which targeted nuclear factor of activated T cells 5(NFAT5), resulted in a conspicuous boost in the differentiation and immunosuppressive function of m Bregs. The inhibition of mi R-29a-3p in CD19~+ B cells was capable of raising the expression levels of NFAT5, thereby promoting B cells to differentiate into m Bregs. In addition, the observed enhancement of differentiation and immunosuppressive function of m Bregs upon mi R-29a-3p inhibition was abolished by the knockdown of NFAT5 in B cells. Conclusions: mi R-29a-3p was found to be a crucial regulator for m Bregs differentiation and immunosuppressive function. Silencing mi R-29a-3p could be a potentially effective therapeutic strategy for inducing immune tolerance after liver transplantation.

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

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

LIGHT sensitizes IFNγ-mediated apoptosis of HT-29 human carcinoma cells through both death receptor and mitochondria pathways

LIGHT [homologous to lymphotoxins, shows inducible expression, and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator (HVEM/TR2)] is a new member of TNF superfamily. The HT-29 colon cancer cell line is the most sensitive to LIGHT-induced, IFNγ-mediated apoptosis among the cell lines we have examined so far. Besides downregulation of Bcl-XL, upregulation of Bak, and activation of both PARP [poly (ADP-ribose)polymerase] and DFF45 (DNA fragmentation factor), LIGHT-induced, IFNγ-mediated apoptosis of HT-29 cells involves extensive caspase activation. Caspase-8 and caspase-9 activation, as shown by their cleavages appeared as early as 24 h after treatment, whereas caspase-3 and caspase-7 activation, as shown by their cleavages occurred after 72 h of LIGHT treatment. Caspase-3 inhibitor Z-DEVD-FMK (benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone)and a broad range caspase inhibitor Z-VAD-FMK (benzyloxycarbonyl-Val-Ala-Asp fluoromethylketone) were able to block LIGHT-induced, IFNγ-mediated apoptosis of HT-29 cells. The activity of caspase-3, which is one of the major executioner caspases, was found to be inhibited by both Z-DEVD-MFK and Z-VAD-FMK. These results suggest that LIGHT-induced, IFNγ-mediated apoptosis of HT-29 cells is caspase-dependent, and LIGHT signaling is mediated through both death receptor and mitochondria pathways.

Anticancer potential of Alternanthera sessilis extract on HT-29 human colon cancer cells

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.

Gene transfer and expression of enhanced green fluorescent protein in variant HT-29c cells

AIM: To study the expression of enhanced green fluorescent protein (EGFP) gene in retrovirally transduced variant HT29 cells.METHODS: The retroviral vector prkat EGFP/neo was constructed and transfected into the 293T cell using a standard calcium phosphate precipitation method. HT-29c cells (selected from HT-29 cells) were transduced by a retroviral vector encoding the GEFP gene. The fluorescence intensity of colorectal carcinoma HT-29c cells after transduced with the EGFP bearing retrovirus was visualized using fluorescence microscope and fluorescence activated cell sorter (FACS) analysis. Multiple biological behaviors of transduced cells such as the proliferating potential and the expression of various antigens were comparatively analyzed between untransduced and transduced cells in vitro. EGFP expression of the fresh tumor tissue was assessed in vivo.RESULTS: After transduced, HT-29c cells displayed a stable and long-term EGFP expression under the nonselective conditionsin vitro. After cells were successively cultured to passage 50 in vitro, EGFP expression was still at a high level. Their biological behaviors, such as expression of tumor antigens, proliferation rate and aggregation capability were not different compared to untransduced parental cells in vitro. In subcutaneous tumors, EGFP was stable and highly expressed.CONCLUSION: An EGFP expressing retroviral vector was used to transduce HT-29c cells. The transduced cells show a stable and long-term EGFP expression in vitro and in vivo.These cells with EGFP are a valuable tool forin vivo research of tumor metastatic spread.

甘草次酸对大肠癌HT-29细胞化疗增敏作用机制研究

目的:研究甘草次酸增强奥沙利铂对大肠癌HT-29细胞化疗敏感性的作用机制。方法:将大肠癌HT-29细胞分为对照组、甘草次酸组(甘草次酸浓度为100μmol/L)、联合组(100μmol/L甘草次酸+25 mg/L奥沙利铂)和阳性对照组(奥沙利铂25 mg/L)。采用四氮唑蓝法检测各组HT-29细胞增殖率;通过Annexin V/PI双标流式细胞术检测各组HT-29细胞凋亡比例;通过蛋白质印迹法检测各组HT-29细胞中核转录因子κB(nuclear factor kappa B,NF-κB)、B淋巴细胞瘤2(B-cell lymphoma 2,Bcl-2)、激活型胱天蛋白酶3(caspase-3,CASP-3)蛋白表达。结果:与对照组相比,联合组和阳性对照组HT-29细胞增殖率降低(P<0.05),联合组和阳性对照组凋亡细胞比例均升高(P<0.05);甘草次酸组、联合组和阳性对照组NF-κB、Bcl-2蛋白相对表达量均降低(P<0.05),而激活型CASP-3蛋白表达升高(P<0.05)。结论:甘草次酸可能通过调控NF-κB信号通路增强奥沙利铂对大肠癌HT-29细胞的化疗敏感性。

Ferrostatin-1 protects HT-22 cells from oxidative toxicity

Ferroptosis is a type of programmed cell death dependent on iron.It is different from other forms of cell death such as apoptosis,classic necrosis and autophagy.Ferroptosis is involved in many neurodegenerative diseases.The role of ferroptosis in glutamate-induced neuronal toxicity is not fully understood.To test its toxicity,glutamate(1.25–20 mM)was applied to HT-22 cells for 12 to 48 hours.The optimal experimental conditions occurred at 12 hours after incubation with 5 mM glutamate.Cells were cultured with 3–12μM ferrostatin-1,an inhibitor of ferroptosis,for 12 hours before exposure to glutamate.The cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.Autophagy was determined by monodansylcadaverine staining and apoptosis by caspase 3 activity.Damage to cell structures was observed under light and by transmission electron microscopy.The release of lactate dehydrogenase was detected by the commercial kit.Reactive oxygen species were measured by flow cytometry.Glutathione peroxidase activity,superoxide dismutase activity and malondialdehyde level were detected by the appropriate commercial kit.Prostaglandin peroxidase synthase 2 and glutathione peroxidase 4 gene expression was detected by real-time quantitative polymerase chain reaction.Glutathione peroxidase 4 and nuclear factor erythroid-derived-like 2 protein expression was detected by western blot analysis.Results showed that ferrostatin-1 can significantly counter the effects of glutamate on HT-22 cells,improving the survival rate,reducing the release of lactate dehydrogenase and reducing the damage to mitochondrial ultrastructure.However,it did not affect the caspase-3 expression and monodansylcadaverine-positive staining in glutamate-injured HT-22 cells.Ferrostatin-1 reduced the levels of reactive oxygen species and malondialdehyde and enhanced superoxide dismutase activity.It decreased gene expression of prostaglandin peroxidase synthase 2 and increased gene expression of glutathione peroxidase 4 and protein expressions of glutathione peroxidase 4 and nuclear factor(erythroid-derived)-like 2 in glutamate-injured HT-22 cells.Treatment of cultured cells with the apoptosis inhibitor Z-Val-Ala-Asp(OMe)-fluoromethyl ketone(2–8μM),autophagy inhibitor 3-methyladenine(100–400μM)or necrosis inhibitor necrostatin-1(10–40μM)had no effect on glutamate induced cell damage.However,the iron chelator deferoxamine mesylate salt inhibited glutamate induced cell death.Thus,the results suggested that ferroptosis is caused by glutamate-induced toxicity and that ferrostatin-1 protects HT-22 cells from glutamate-induced oxidative toxicity by inhibiting the oxidative stress.

谷氨酰胺对结直肠癌HT-29细胞放射敏感性的影响及机制探讨

目的观察不同浓度谷氨酰胺(glutamine,Gln)对结直肠癌HT-29细胞放射敏感性的影响,并探讨其可能机制。方法体外培养HT-29细胞,将对数生长期的HT-29细胞按照Gln不同浓度设置对照组(2 mmol/L,培养基基础浓度)、实验组Ⅰ(4 mmol/L)、实验组Ⅱ(6 mmol/L)、实验组Ⅲ(8 mmol/L),预处理2 h后给予Co 60源放射。通过CCK-8法检测未放射与放射后不同浓度Gln对细胞活力的影响;克隆形成实验检测放射后不同浓度Gln对细胞放射敏感性的影响;活性氧(ROS)试剂盒检测放射后24 h细胞ROS水平;流式细胞仪检测放射后48 h细胞凋亡率;蛋白印迹法测定放射后各组细胞Nrf2、HO-1、cleaved-Caspase3蛋白的表达水平。结果未放射的HT-29细胞在Gln干预24 h后实验组Ⅱ、实验组Ⅲ细胞活力明显高于对照组、实验组Ⅰ(P<0.05)。HT-29细胞放射24 h后,各实验组细胞活力明显高于对照组(P<0.05);放射后14 d,各实验组细胞克隆形成数均高于对照组(P<0.05);放射后24 h,各实验组ROS水平均低于对照组(P<0.05);放射后48 h,各实验组细胞凋亡率均低于对照组(P<0.05)。实验组ⅠNrf2表达高于对照组(P<0.05);实验组Ⅱ、ⅢNrf2、HO-1表达均高于对照组和实验组Ⅰ(P<0.05),cleaved-Caspase3表达均低于对照组和实验组Ⅰ(P<0.05);实验组Ⅲcleaved-Caspase3表达低于实验组Ⅱ(P<0.05)。结论谷氨酰胺能显著降低HT-29细胞的放射敏感性,其机制与减轻氧化应激损伤和减少细胞凋亡有关,这可能不利于结直肠癌患者放疗。

Correlation between metastatic potential and variants from colorectal tumor cell line HT-29

AIM: To evaluate the relationship between uPA, PAI-1,CEA, PI3K and metastatic potential in three colorectal tumor cell lines.METHODS: Metastatic model in nude rats was established by variants HT-29c and HT-29d cell lines and the metastatic potential of two tumor cell variants was compared.Urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) were determined using ELISA in colorectal carcinoma WiDr, HT29 and HT-29d cell lines with different metastatic potentials.Expression of carcinoembryonic antigen (CEA) and phosphoinositide 3-kinase (PI3-Kinase) was analyzed using immunohistochemistry (IHC) in these cell lines in vitro and in vivo. CEA expression was compared using fluorescence activated cell sorter (FACS)in vitro.RESULTS: The number of HT-29d cells arrested in liver dramatically decreased within the initial 24 hours after injection. The taking rate of liver metastases in the variant HT-29d increased as compared with parental HT-29 cells (70 % versus 50 %) and a variant HT-29b cells (70 % versus 60 %), and extensive organs were synchronously involved in metastases. The uPA concentration of variant HT-29d cell line was significantly higher than that of the non-metastatic WiDr and the low metastatic HT-29 cell lines. The variant HT-29d cells produced stronger PI3-kinase expression as compared with the non-metastatic WiDr cells and the low metastatic HT-29 cellsin vivo.CONCLUSION: The selected variant HT-29d cell exhibited an enhanced metastatic potential. The level of uPA and PAI-1 is positively correlated with the metastatic capacity of tumor cells. The expression of PI3-kinase correlates with tumor development and metastasis.

Proinflammatory effects and molecular mechanisms of interleukin-17 in intestinal epithelial cell line HT-29

AIM:To evaluate the proinflammatory effects and molecular mechanisms of interleukin(IL)-17 in intestinal epithelial cell line HT-29.METHODS:HT-29 cells were cultured with IL-17,tumor necrosis factor(TNF)-α,or the combination of both IL-17 and TNF-α.Real-time PCR and Western blot were used to measure the gene expression levels of neutrophil chemokines CXCL1,CXCL2,CXCL5,CXCL6,IL-8and TH-17 cell chemokine CCL20,the phosphorylation levels of p38 and TNF-α,and the expression level of IL-8,after using the p38 inhibitor in HT-29 cells.The stable Act1 knockdown HT-29 cell line was established to further test the phosphorylation changes of p38,after using IL-17 and TNF-α.RESULTS:After HT-29 cells were cultured with IL-17and TNF-α,the expression levels of neutrophil chemokines(CXCL1,CXCL2,CXCL5,CXCL6,IL-8)and Th17chemokine(CCL20)significantly improved(24.96±2.53,28.47±2.87,38.08±2.72,33.47±2.41,31.7±2.38,44.37±2.73,respectively),and the differences were all statistically significant(P<0.01).Western blot results showed that IL-17 obviously enhanced the phosphorylation level of p38,which was induced by TNF-α.Compared with the control group,the expression level of IL-8 significantly declined(9.47±1.36 vs 3.06±0.67,P<0.01)when TH-29 cells were cultured with IL-17and TNF-α.p38 inhibition assay showed that the p38pathway played an essential role in the inflammatory response induced by IL-17.p38 phosphorylation levels could not be changed after using IL-17 and TNF-αin the stable Act1 knockdown HT-29 cell line.CONCLUSION:IL-17 significantly promoted the gene expression levels of TNF-α-induced neutrophil chemokines and Th17 cell chemokine.It is obvious that IL-17and TNF-αhave synergistic effects on p38.

Potential Mechanisms Involved in Ceramide-induced Apoptosis in Human Colon Cancer HT29 Cells

Objective To investigate the potential mechanisms of cell death after the treatment with ceramide. Methods MTT assay, DNA ladder, reporter assay, FACS and Western blot assay were employed to investigate the potential mechanisms of cell death after the treatment with C2-ceramide. Results A short-time treatment with C2-ceramide induced cell death, which was associated with p38 MAP kinase activation, but had no links with typical caspase activation or PARP degradation. Rather than caspase inhibitor, Inhibitor of p38 MAP kinase blocked cell death induced by a short-time treatment with ceramide （〈12 h）. However, inhibition of p38 MAP kinase could not block cell death induced by a prolonged treatment with ceramide （〉12 h）. Moreover, incubation of cells with ceramide for a long time （〉12 h） increased subG1, but reduced S phase accompanied by caspase-dependent and caspase-independent changes including NFr, B activation. Conclusion Ceramide-induced cell apoptosis involves both caspase-dependent and -independent signaling pathway. Caspase-independent cell death occurring in a relatively early stage, which is mediated via p38 MAP kinase, can progress into a stage involving both caspase-dependent and -independent mechanisms accompanied by cell signaling of MAPKs and NFκB.

Additional comments on extracellular vesicles derived from mesenchymal stem cells mediate extracellular matrix remodeling in osteoarthritis

Recently,we read an article published by the Yang et al.The results of this study indicated that engineered exosomes loaded with microRNA-29a(miR-29a)alleviate knee inflammation and maintain extracellular matrix stability in Sprague Dawley rats.The study’s results provide useful information for treating knee osteoarthritis(KOA).This letter,shares our perspectives on treating KOA using engineered exosomes for miR-29a.