Prognostic significance of CDKN2A expression in colon adenocarcinoma: insights from TCGA analysis

Background:The cyclin-dependent kinase inhibitor 2a(CDKN2A)gene,identified as the multiple tumor suppressor gene,functions as a regulatory gene implicated in cancer pathogenesis.Its significance lies in its pivotal involvement in the genesis of various tumors;notwithstanding,the precise connection between CDKN2A and c olon adenocarcinoma(COAD)remains undisclosed.Methods:The objective of this research was to assess the predictive importance of CDKN2A in COAD by analyzing data from The Cancer Genome Atlas database.Logistic regression,signed rank test,Wilcoxon test,and Kruskal-Wallis test were used to examine CDKN2A expression levels and clinicopathological features.Univariate and multivariate Cox r egression analyses and Kaplan-Meier analysis found prognostic variables.Additionally,gene set enrichment analysis identified key CDKN2A expression pathways.The study additionally examined CDKN2A expression with tumor immune infiltration using The Cancer Genome Atlas data and single sample gene set enrichment analysis.Results:The results of this investigation indicated a substantial connection between higher CDKN2A expression and negative outcomes in terms of overall survival and disease-related survival among COAD patients.Gene set enrichment analysis indicated a tight link between CDKN2A and both the cell cycle and hedgehog signaling pathways.Subsequent evaluation employing single sample gene set enrichment analysis demonstrated a positive link between CDKN2A expression with infiltration by iDCs,whereas a negative correlation was detected with infiltration by helper T cells.Conclusion:In conclusion,the present study gives strong data supporting the predictive value of CDKN2A and its possible usefulness as a biomarker for COAD.Additionally,our results show a reasonable link between CDKN2A expression and immune influx in COAD,putting light on the role of CDKN2A in the control of the tumor microenvironment.Nevertheless,additional studies are needed to confirm the underlying mechanisms of these relationships and to discover the therapeutic possibilities of targeting CDKN2A in the treatment of COAD.

Growth inhibitory effect of wild-type Kras2 gene on a colonic adenocarcinoma cell line

AIM： To observe the growth inhibitory effect of wild-type Kras2 gene on a colonic adenocarcinoma cell line Caco-2. METHODS： Recombinant plasmid pCI-neo-Kras2 with wild type Kras2 open reading frame was constructed. The Caco-2 cells were transfected with either pCI-neo or pCI-neo-Kras2 using Upofectamine 2000. The expression of wild type Kras2 was examined by Northern blot analysis. And the expression of wild type Kras2 protein was examined by Western blot analysis. The effects of wild-type Kras2 on cell proliferation were analyzed by monotetrazolium （MTT） assay, meanwhile analyses of cell cycle and spontaneous apoptosis rate were carried out by flow cytometry （FCM）. RESULTS： The plasmid of pCI-neo-Kras2 was successfully established. The growth rate of cells transfected with pCI-neo-Kras2 was significantly lower than the control cells transfected with the empty pCI- neo vector （P 〈 0.05）. Cell cycle analysis revealed arrest of the pCI-neo-Kras2 transfected cells in G0/G1 phases, decreased DNA synthesis and decreased fractions of cells in S phase. The proliferative index of cells transfected with pCI-neo-Kras2 was decreased compared with the control cells （49.78% vs 64.21%）, while the apoptotic rate of Caco-2 cells with stable Kras2 expression increased （0.30% vs 0.02%）. CONCLUSION： The wild-type Kras2 gene effectively inhibits the growth of the colonic adenocarcinoma cell line Caco-2.

Molecular analysis and anticancer properties of two identified isolates,Fusarium solani and Emericella nidulans isolated from Wady El-Natron soil in Egypt against Caco-2(ATCC) cell line

Objective:To characterize,identify and investigate the anticancer properties of two new soil fungal isolates,Emericella nidulansand Fusarium solani isolated from Wady El-Natron in Egypt against colon cancer Caco-2(ATCCj cell line.Methods:Soil sample was cultured and two strains were chosen for morphological and phenotypical characterization.Partial sequences of the 18s rRNA gene and the internal transcribed spacer region ITS of the two isolates were amplified by PCR.Phylogenetic tree construction and analysis of the resulted multiple sequences from the two fugal isolates were also carried out.In vitro anticancer activity of the two strains was done against colon Caco-2 cancer cell line.Reverse transcription — PCR was carried out to detect level of expression of p53 in Caco-2 cell line.Results:HF.I displayed morphological and genotypic characteristics most similar to that of Fusarium solani while HF.2 was most similar to Emericella nidulans with high similarity of 99%and 97%respectively.The multiple sequence alignment of the two fungal isolates showed that,the maximum identical conserved domains in the 18s rRNA genes were identified with the nucleotide regions of Slst to 399th base pairs,88th to 525th base pairs respectively.While those in the ITS genes were identified with the nucleotide regions of 88th to 463rd and Slst to 274th.The two isolates showed IC<sub>50 value with（6.24±5.21） and（9.84±0.36） μ g/mL) concentrations respectively at 28h.Reverse transcription- PCR indicated that these cells showed high level of expression for p53 mRNA.Conclusions:The morphology and molecular analysis identified HF.1 and HF.2 to be Fusarium solani and Emericella nidulans;new isolates of anticancer producing fungi from Wady El-Natroon city in Egypt.Treatment with the two isolates caused P53 expression in Caco-2 cell line.These two isolates can be used as an anticancer agents.

Upregulation of 25-hydroxyvitamin D_3-1α-hydroxylase by butyrate in Caco-2 cells

AIM： To investigate the possible involvement of 25-hydroxyvitamin D3-1cx-hydroxylase [1α-25（OH）2D3] in butyrate-induced differentiation in human intestinal cell line Caco-2 cells. METHODS： Caco-2 cells were incubated either with 3 mmol/L butyrate and 1 umol/L 25（OH）2D3 or with 1 umol/L 1α-25（OH）2D3 for various time intervals ranging from 0 to 72 h. Additionally, cells were co-incubated with butyrate and either 25（OH）2D3 or 1α-25（OH）2D3. 1α-25（OH）2D3 mRNA was determined semi-quantitatively using the fluorescent dye PicoGreen. Immunoblotting was used for the detection of 1α-25（OH）2D3 protein. Finally, enzymatic activity was measured by ELISA. RESULTS： Both butyrate and 1α-25（OH）2D3 stimulated differentiation of Caco-2 cells after a 48 h incubation period, while 25（OH）2D3 had no impact on cell differentiation. Synergistic effects on differentiation were observed when cells were co-incubated with butyrate and vitamin D metabolite. Butyrate transiently upregulated 1α-25（OH）2D3 mRNA followed by a timely delayed protein upregulation. Coincidently, enzymatic activity was enhanced significantly. The induction of the enzyme allowed for comparable differentiating effects of both vitamin D metabolites. CONCLUSION： Our experimental data provide a further mechanism for the involvement of the vitamin D signaling pathway in colonic epithelial cell differentiation by butyrate. The enhancement of 1α-25（OH）2D3 followed by antiproliferative effects of the vitamin D prohormone in the Caco-2 cell line suggest that 25（OH）2D3 in combination with butyrate may offer a new therapeutic approach forthe treatment of colon cancer.

共轭亚油酸混合物对人结肠癌细胞(Caco-2)的抑制作用

研究不同浓度、不同作用时间的共轭亚油酸混合物对人结肠癌细胞Caco-2增殖的影响。采用体外细胞培养方法,通过MTT试验,观察不同浓度、不同作用时间的共轭亚油酸混合物对Caco-2细胞的增殖抑制作用。结果表明:共轭亚油酸混合物抑制了Caco-2细胞的增殖,且对Caco-2细胞增殖的抑制作用与浓度和作用时间密切相关。共轭亚油酸混合物对人结肠癌细胞Caco-2的增殖有抑制作用。

c9,t11-CLA对人结肠癌细胞(Caco-2)的抑制作用

采用体外细胞培养方法,通过MTT实验,研究了c9,t11-CLA(Conjugated Linoleic acid,CLA)在不同浓度,(0,25,50,100,200μmol/L)、不同作用时间(1,2,3,4 d)下,对人结肠癌细胞Caco-2增殖的影响。结果表明,c9,t11-CLA抑制了Caco-2细胞的增殖,且对Caco-2细胞增殖的抑制作用与浓度和作用时间密切相关。

东亚钳蝎毒素对人结肠癌细胞Caco-2的增殖抑制作用研究

为研究东亚钳蝎毒素对人结肠癌细胞Caco-2增殖的影响,以不同浓度的东亚钳蝎毒素(10、20、40μg/m L)干预体外培养的Caco-2细胞,分别于24、48 h后,用四甲基偶氮唑盐(MTT)比色法,观察毒素对Caco-2细胞的增殖抑制作用,运用淋巴细胞转化试验和乳酸脱氢酶(LDH)释放试验检测蝎毒素对Caco-2细胞的作用途径。结果表明:东亚钳蝎毒素不仅能抑制Caco-2细胞的增殖而且能促进淋巴细胞转化,且毒素对Caco-2细胞增殖的抑制作用具有浓度和时间的依赖关系,随浓度加大和时间延长,对Caco-2细胞增殖的抑制作用增强。

新砷化合物对小鼠C26移植性肿瘤细胞体内生长抑制作用的研究

目的 探讨新砷化合物 (AsI3 - 3Tu)对C2 6移植性肿瘤细胞的抑制作用及其对肿瘤组织环氧合酶 - 2(COX - 2 )及微血管密度 (MVD)的影响。 方法 将Balb/c小白鼠随机分为对照组、5 Fu组和AsI3 - 3Tu组 ,于接种C2 6移植性肿瘤细胞后第 6天开始腹腔注射给药 ,隔天 1次 ,共给药 8次 ,观察抑瘤率 ,并采用免疫组织化学的方法研究AsI3 - 3Tu对肿瘤组织COX - 2及MVD的影响。 结果 AsI3 - 3Tu对C2 6移植性肿瘤有明显的抑制作用 ,抑瘤率为 5 5 .0 7%。免疫组化结果显示 ,AsI3 - 3Tu对肿瘤组织的COX - 2表达有明显的抑制作用 ,同时MVD明显降低。 结论 AsI3 - 3Tu对C2 6移植性肿瘤有抑制作用 ,其作用途径可能是通过抑制COX - 2的表达而抑制肿瘤血管的形成 。

The combination of sitagliptin and bee honey extract potentiates the anti-proliferative properties of 5-fluorouracil on Caco-2 cell line without detectable inflammatory events

Background:Colon cancer originates in the colon,specifically the large intestine.It carries a poor prognosis and high mortality rate due to late diagnosis and migration.Objective:Here our objective was to evaluate the anticancer effects of sitagliptin(Sita)in colon cancer using Caco-2 cells.Additionally,we examined the role of bee honey extract in modulating cancer cell division and necrotic events commonly observed during drug treatments.Methods:We monitored cell viability rate to evaluate the effect of bee honey extract compared to 5-fluorouracil(5-Fu),Sita,and their combinations.To gain further foresights into the implicated molecular interaction,we assessed the expression outline of Raf-1 and MEK,as proliferation effectors.Additionally,we examined the expression outline of p53 and Caspase 3,which are associated with programmed cell death(PCD),through western blot analysis.Results:We identified the Raf-1 expression pattern as a likely target for the drug combination and bee honey extract(HE),which effectively controlled colon cancer cell proliferation.Our study demonstrates that honey extract,either alone or in combination with drugs,can induce PCD by restoring the p53 and CASP-3 proteins.This was accompanied by a synergistic effect on the production of apoptotic cytokines,particularly interlukine-6(IL-6)and IL-8,in cancer cells.Moreover,the treatment modulated the levels of pro-inflammatory cytokines,including IL-1𝛼and IL-1𝛽and anti-inflammatory cytokines,including IL-4 and IL-10.Conclusion:Our findings shed light on honey extract and its combinations with 5-Fu and Sita in stimulating PCD and modulating cytokine production in Caco-2 cells.

Interaction between enteric epithelial cells and Peyer's patch lymphocytes in response to Shigella lipopolysaccharide: Effect on nitric oxide and IL-6 release

AIM： TO investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer＇s patch on the release of nitric oxide （NO） and IL-6 in response to Shigella lipopolysaccharide （LPS）. METHODS： Human colonic epithelial cells （Caco-2） were mixed cocultured with lymphocytes of Peyer＇s patch from wild-type （C57 mice） and inducible NO synthase knockout mice, and challenged with Shigella F2a-12 LPS. Release of NO and raiL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay （ELISA）, respectively. RESULTS： In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer＇s patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer＇s patch from the wild-type mice but not from iNOS knockout mice. LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6 production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice. CONCLUSION： Lymphocytes of Peyer＇s patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer＇s patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.

Phytochemical profiling and anticancer potential of Cymbopogon citratus extract

Objective:To evaluate the anticancer potential of Cymbopogon citratus extract.Methods:GC-MS analysis was used to identify phytocomponents in the methanolic extract of Cymbopogon citratus.A fractionation method was employed to isolate and assess the bioactivity of different fractions and their cytotoxic activities against cancer cell lines HCT116,LoVo,Caco-2,and HT-29 were investigated.A dual staining method with acridine orange and ethidium bromide was used to assess the effect of the extract on cell apoptosis.Additionally,the expression levels of Bax and TP53 were quantified using real-time PCR in Caco-2 cells treated with the ethyl acetate fraction of Cymbopogon citratus extract.A protein array was employed to profile key pro-and anti-apoptotic proteins in Caco-2 cells.Moreover,molecular docking studies were conducted to investigate the interactions between key compounds of Cymbopogon citratus extract and specific apoptosis-related protein domains(PDB IDs:7wql and 4bkx).Results:A significant growth inhibition was observed in Caco-2 cells treated with Cymbopogon citratus extract.Among the seven fractions of the plant extract,the ethyl acetate fraction showed the highest cytotoxicity against Caco-2 cells with an IC50 value of(6.16±0.01)μg/mL.The immunofluorescence assay showed that the ethyl acetate fraction could induce apoptosis of Caco-2 cells.Moreover,the fraction upregulated the gene expressions of Bax and TP53 in a dose-dependent manner.The docking analysis demonstrated the interaction of five compounds isolated from the ethyl acetate fraction with key proteins in Caco-2 cells,indicating their anticancer properties.Conclusions:Cymbopogon citratus extract shows anticancer activity against Caco-2 cells by inducing apoptosis.It may be a promising candidate for the treatment of colon cancer,which needs further investigation.

中国主要水果抑制肝癌HepG2细胞和结肠腺癌Caco-2细胞增殖活性评价

分别采用Folin-Ciocalteu法测定了水果提取物的总酚含量,采用亚甲基蓝法测定了其抗人肝癌细胞Hep G2和人结肠腺癌细胞Caco-2增殖的活性,分析了总酚含量与抗Hep G2和Caco-2细胞增殖活性之间的相关性。结果显示,25种水果中李子的总酚含量最高(1686.08±96.94μmol GAE/100鲜果),西瓜(83.54±2.10μmol GAE/100 g鲜果)和哈密瓜(79.35±0.76μmol GAE/100 g鲜果)的总酚含量最低;在可测出抗增殖EC50值的水果中,李子(18.99±0.06 mg/m L)和番石榴(20.94±0.09 mg/m L)抗Hep G2细胞增殖的活性最强,梨的活性最弱(389.63±10.82 mg/m L)。李子抗Caco-2细胞增殖的活性最强(8.73±0.11 mg/m L),火龙果的活性最弱(388.07±7.04 mg/m L)。水果的抗Hep G2和Caco-2细胞增殖活性与其总酚含量相关性显著(R2=0.4147,p<0.01;R2=0.4071,p<0.05),该相关性表明水果中的多酚具有良好的抗肿瘤细胞增值的活性。

乙酰水杨酸铜对HCT-116细胞生长的抑制及诱导凋亡作用

目的研究乙酰水杨酸铜对HTC—116细胞生长的抑制及诱导凋亡作用,探讨其抗肿瘤作用的机制。方法采用MTT法检测乙酰水杨酸铜对HTC-116细胞的毒性作用；荧光显微镜照相及流式细胞术检测乙酰水杨酸铜诱导HTC-116细胞凋亡的作用。结果乙酰水杨酸铜处理HTC-116细胞48h、72h的IC50值分别为6．10μmol/L和4．48μmol/L；10μmol/L乙酰水杨酸铜处理HTC-116细胞24h镜下可见核分裂及凋亡小体形成,处理48h流式细胞术可检测出明显的亚二倍体峰,凋亡率达34．1%。细胞凋亡发生呈剂量和时间依赖性。结论乙酰水杨酸铜可显著抑制HTC-116细胞生长并诱导其凋亡。

东亚钳蝎毒素对Caco-2人结肠癌细胞的体外药理学研究

研究东亚钳蝎毒素对人结肠癌细胞Caco-2增殖的影响。以不同浓度的东亚钳蝎（Buthus martensii Karsch）毒素（10、20、40滋g/mL）干预体外培养的Caco-2细胞，分别于24 h、48 h后，用四甲基偶氮唑盐（MTT）比色法，观察毒素对Caco-2细胞的增殖抑制作用。运用淋巴细胞转化实验和乳酸脱氢酶（LDH）释放实验检测蝎毒素对Caco-2细胞的作用途径。结果表明：东亚钳蝎毒不仅能抑制Caco-2细胞的增殖而且能促进淋巴细胞转化，毒素对Caco-2细胞增殖的抑制作用与浓度和作用时间密切相关。

人参稀有皂苷Compound K混合胶束的处方优化及体外评价

目的:优化人参稀有皂苷Compound K(CK)混合胶束的处方,考察其体外释放度和表观渗透系数(Papp)。方法:以大豆磷脂和维生素E聚乙二醇1000琥珀酸酯(TPGS)为辅料,采用溶剂挥发法制备CK混合胶束。以载药量、包封率、粒径为指标,采用正交试验优化投药量、大豆磷脂-TPGS比例、水化体积。考察最优处方所制胶束的形态、粒径、载药量、包封率、溶解度、体外释放度和在结肠腺癌Caco-2细胞模型中的Papp。结果:CK混合胶束的最优处方投药量为1.0 mg,大豆磷脂-TPGS比例为1∶1,水化体积为10 ml。所制混合胶束呈球形或类球形,平均粒径为(110±2.69)nm,载药量为(4.32±0.19)%,包封率为(92.23±2.76)%,溶解度为(469.21±0.024)μg/ml,150 h时累积释放度为(66.19±0.027)%(n=3)。CK原料药和CK混合胶束的Papp分别为26.20、3.78(n=6)。结论:优化的制备工艺可行,成功制得CK混合胶束。