Colon cancer-associated B2 Escherichia coli colonize gut mucosa and promote cell proliferation

AIM: To provide further insight into the characterization of mucosa-associated Escherichia coli (E. coli) isolated from the colonic mucosa of cancer patients.

Correlation of dynamic contrast-enhanced magnetic resonance imaging parameters before colon cancer surgery with the angiogenesis and cell proliferation in tumor lesions

Objective: To discuss the correlation of dynamic contrast-enhanced magnetic resonance imaging parameters before colon cancer surgery with the angiogenesis and cell proliferation in tumor lesions. Methods: A total of 186 patients with colon cancer who were treated in the hospital between January 2015 and January 2017 were collected as colon cancer group;100 patients with multiple polyposis of colon who received colonoscopy in the hospital during the same period were selected as colon polyp group. The differences in DCE-MRI parameters as well as the expression of angiogenesis and cell proliferation genes were compared between the two groups, and Pearson test was used to evaluate the correlation of preoperative DCE-MRI parameters with angiogenesis and cell proliferation gene expression in patients with colon cancer. Results: Preoperative DCE-MRI parameters Ktrans and Kep levels in colon cancer group were significantly higher those in colon polyp group;Cyr61, HIF-α, VEGF, MMP-9, CXCR7, EZH2, SphK1 and IFT57 mRNA expression in lesion tissue of colon cancer group were significantly higher than those of colon polyp group while Kiss-1 mRNA was lower than that of colon polyp group. Pearson test showed that the DCE-MRI parameters Ktrans and Kep levels before colon cancer surgery were directly correlated with the expression of angiogenesis and cell proliferation genes in lesion tissues. Conclusion: Preoperative DCE-MRI parameters can accurately reflect the severity of colon cancer.

Stem cell characteristics of early colon cancer tissue and their relationship with cell proliferation and invasion

Objective:To study the stem cell characteristics of early colon cancer tissue and their relationship with cell proliferation and invasion.Methods: Colon cancer tissues and adjacent tissues surgically removed in Dongguan People's Hospital between January 2010 and October 2017 were selected as the clinical samples of this study, the protein was extracted to determine the protein expression of tumor stem cell genes USP22, Nanog, Lgr5 and CD44, and RNA was extracted to determine the mRNA expression of cell proliferation genes and cell invasion genes.Results:USP22, Nanog, Lgr5 and CD44 protein expression as well as Rab5A, TBX2, MDM2, TGF-β1, Smad2/3, Vimentin, Rac1 and VEGF mRNA expression in colon cancer tissues were significantly higher than those in adjacent tissues while Bad, Bax and Fas mRNA expression were significantly lower than those in adjacent tissues;USP22, Nanog, Lgr5 and CD44 protein expression in colon cancer tissues were positively correlated with Rab5A, TBX2, MDM2, TGF-β1, Smad2/3, Vimentin, Rac1 and VEGF mRNA expression, and negatively correlated with Bad, Bax and Fas mRNA expression.Conclusion: The activation of stem cell characteristics in early colon cancer can promote the proliferation and invasion of cancer cells.

Effect of nutritional support + intravenous chemotherapy on anti-tumor immunity and cancer cell proliferation in patients with colon cancer complicated by incomplete intestinal obstruction

Objective:To study the effect of nutritional support + intravenous chemotherapy on anti-tumor immunity and cancer cell proliferation in patients with colon cancer complicated by incomplete intestinal obstruction.Methods: Patients with colon cancer complicated by incomplete intestinal obstruction who were treated in Midi Branch, Pangang Group General Hospital between March 2015 and October 2017 were selected and randomly divided into the nutrition group who accepted nutritional support + FOLFOX4 intravenous chemotherapy and the control group who accepted FOLFOX4 intravenous chemotherapy alone, and they underwent surgery after two cycles of chemotherapy. The contents of immune cells in peripheral blood and the contents of immune cytokines in serum were determined before chemotherapy and two cycles after chemotherapy;the expression levels of proliferation genes in colon cancer lesions were determined after surgical resection.Results:Compared with those of same group before chemotherapy, peripheral blood Treg, Th9, Th17 and Th22 contents as well as serum IL-4, IL-9, IL-10, TGF-β1, IL-17 and IL-22 contents of nutrition group were decreased significantly after chemotherapywhereas peripheral blood Treg, Th9, Th17 and Th22 contents as well as serum IL-4, IL-9, IL-10, TGF-β1, IL-17 and IL-22 contents of control group did not change significantly after chemotherapy, and compared with those after chemotherapy between groups, peripheral blood Treg, Th9, Th17 and Th22 contents as well as serum IL-4, IL-9, IL-10, TGF-β1, IL-17 and IL-22 contents of nutrition group were significantly lower than those of control group, and CyclinD1, Bcl-2, USP22, VEGF and N-cadherin mRNA expression were not different from those of control group.Conclusion:Nutritional support + intravenous chemotherapy can improve the anti-tumor immune response without affecting the proliferation of cancer cells in the lesion of patients with colon cancer complicated by incomplete intestinal obstruction.

Luteolin inhibits the colon cancer HT-29 cell proliferation, migration and epithelial-mesenchymal transition: an experimental study

Objective: To study the regulating effect of luteolin on colon cancer HT-29 cell proliferation, migration and epithelial-mesenchymal transition. Methods: Colon cancer HT-29 cells were cultured and randomly divided into two groups, control group were treated with serum-free medium without drugs and LUT group were treated with serum-free medium containing luteolin. After 24 h of treatment, cells were collected to extract RNA, and then fluorescent quantitative PCR method was used to determine the mRNA expression of proliferation genes, migration genes and epithelial-mesenchymal transition genes. Results: After 24 h of luteolin treatment, Lrig1, TSPYL5, Bim, SOX15 and DLC1 mRNA expression in LUT group were significantly higher than those in control group while RPS15a, Bad, TRPV5, TRPV6, PLD2, IBP, SphK1, FAK, Vimentin and N-cadherin mRNA expression were significantly lower than those in control group. Conclusion: Luteolin has inhibiting effect on colon cancer HT-29 cell proliferation, migration and epithelial-mesenchymal transition.

Sm-like 5 knockdown inhibits proliferation and promotes apoptosis of colon cancer cells by upregulating p53,CDKN1A and TNFRSF10B

BACKGROUND The role of Sm-like 5(LSM5)in colon cancer has not been determined.In this study,we investigated the role of LSM5 in progression of colon cancer and the potential underlying mechanism involved.AIM To determine the role of LSM5 in the progression of colon cancer and the potential underlying mechanism involved.METHODS The Gene Expression Profiling Interactive Analysis database and the Human Protein Atlas website were used for LSM5 expression analysis and prognosis analysis.Real-time quantitative polymerase chain reaction and Western blotting were utilized to detect the expression of mRNAs and proteins.A lentivirus targeting LSM5 was constructed and transfected into colon cancer cells to silence LSM5 expression.Proliferation and apoptosis assays were also conducted to evaluate the growth of the colon cancer cells.Human GeneChip assay and bioinformatics analysis were performed to identify the potential underlying mechanism of LSM5 in colon cancer.RESULTS LSM5 was highly expressed in tumor tissue and colon cancer cells.A high expression level of LSM5 was related to poor prognosis in patients with colon cancer.Knockdown of LSM5 suppressed proliferation and promoted apoptosis in colon cancer cells.Silencing of LSM5 also facilitates the expression of p53,cyclin-dependent kinase inhibitor 1A(CDKN1A)and tumor necrosis factor receptor superfamily 10B(TNFRSF10B).The inhibitory effect of LSM5 knockdown on the growth of colon cancer cells was associated with the upregulation of p53,CDKN1A and TNFRSF10B.CONCLUSION LSM5 knockdown inhibited the proliferation and facilitated the apoptosis of colon cancer cells by upregulating p53,CDKN1A and TNFRSF10B.

Tetramethylpyrazine inhibits proliferation of colon cancer cells in vitro

BACKGROUND Colon cancer is one of the most common malignancies worldwide,and chemotherapy is a widely used strategy in colon cancer clinical therapy.However,chemotherapy resistance is a major cause of disease recurrence and progression in colon cancer,and thus novel drugs for treatment are urgently needed.Tetramethylpyrazine(TMP),a component of the traditional Chinese medicine Chuanxiong Hort,has been proven to exhibit a beneficial effect in tumors.AIM To investigate the potential anticancer activity of TMP in colon cancer and its underlying mechanisms.METHODS Colon cancer cells were incubated with different concentrations of TMP.Cell viability was evaluated by crystal violet staining assay and cell counting kit-8 assay,and cell apoptosis and cell cycle were assessed by flow cytometry.RESULTS TMP significantly inhibited the proliferation of colon cancer cells in a dose-and time-dependent manner.In addition,flow cytometry revealed that TMP induced cell cycle arrest at the G0/G1 phase.TMP treatment caused early stage apoptosis in SW480 cells,whereas it caused late stage apoptosis in HCT116 cells.CONCLUSION Our studies demonstrated that TMP inhibits the proliferation of colon cancer cells in a dose-and time-dependent manner by inducing apoptosis and arresting the cell cycle at the G0/G1 phase.Our findings suggest that TMP might serve as a potential novel therapeutic drug in the treatment of human colon cancer.

Effects of MIF on proliferation,migration,and STAT1 pathway of colon cancer cells

Objective This study aimed to investigate how macrophage migration inhibitory factor(MIF)regulates the interaction of signal transducer and activator of transcription 1(STAT1)with CD74,and affects colon cancer proliferation and invasion.Methods After transfecting MIF small interfering RNA into the SW480 cell line,the expression of STAT1 and CD74 mRNA was detected by qRT-PCR and western blotting.Transwell and MTT assays were performed to detect the colon cancer cell invasion and proliferation ability.Co-immunoprecipitation was used to detect the interaction between CD74 and STAT1 proteins in the treated and control groups.Results The cellular biological assays(MTT and Transwell)showed that the proliferation and invasion ability of colon cancer cells decreased after MIF knockdown;the results showed significant statistical difference(P<0.05).The results of the co-immunoprecipitation assay suggested that MIF knockdown in colon cancer cells could inhibit the binding of CD74 and STAT1 proteins;statistical difference was observed between the two groups(P<0.05).Conclusion MIF can increase the proliferation and invasion of colon cancer cells by promoting the combination of CD74 and STAT1.

Exosome-Transmitted miR-224-5p Promotes Colorectal Cancer Cell Proliferation via Targeting ULK2 in p53-Dependent Manner

Objective To investigate the role and molecular mechanism of exosomal miR-224-5p in colorectal cancer(CRC).Methods The miR-224-5p expression in CRC patient tissues and cell-derived exosomes was measured by laser capture microdissection and qRT-PCR,respectively.Dual-luciferase reporter gene assay was used to determine the target gene of miR-224-5p.The protein expressions of p53 and unc-51 like kinase 2(ULK2)in CRC cells were detected by western blot.Flow cytometry was used to detect cell cycle and apoptosis.Cell proliferation was measured by CCK8 and EdU assay.Results The miR-224-5p expression was upregulated in CRC tissues and increased progressively with the rise of CRC stage.CRC cells secreted extracellular miR-224-5p mainly in an exosome-dependent manner,and then miR-224-5p could be transferred to surrounding tumor cells to regulate cell proliferation in the form of autocrine or paracrine.Moreover,ULK2 was characterized as a direct target of miR-224-5p and was downregulated in CRC tissues.Interestingly,ULK2 inhibited CRC cell proliferation in a p53-dependent manner.Furthermore,exosome-derived miR-224-5p partially reversed the proliferation regulation of ULK2 on CRC cells.Conclusion Our findings demonstrate that exosome-transmitted miR-224-5p promotes p53-dependent cell proliferation by targeting ULK2 in CRC,which may offer promising targets for CRC prevention and therapy.

miR-93 suppresses proliferation and colony formation of human colon cancer stem cells

AIM： TO identify differentially expressed microRNAs （miRNAs） in human colon cancer stem cells （SW1116csc） and study their function in SW1116csc proliferation. METHODS： SW1116csc were isolated from the human colon cancer cell line, SW1116 and cultured in serum- free medium. A miRNA microarray was used to detect differential expression profiles of rniRNAs in SW1116csc and SW1116 cells. Real-time quantitative polymerase chain reaction （PCR） was performed to verify the dif- ferential expression of candidate miRNAs obtained from the microarray. Target mRNAs of differentially expressed miRNAs were predicted with target predic- tion tools, miRNA expression plasmids were transfected into SW1116csc using Lipofectamine 2000 reagent. Cell proliferation curves were generated with trypan blue staining, and the colony formation rate of transfected cells was measured with the soft agar colony formation assay. Expression of target mRNAs and proteins from differentially expressed miRNAs were detected using reverse transcription （RT）-PCR and western blotting.RESULTS： Compared with expression in SW1116 cells, 35 miRNAs （including hsa-miR-192, hsa-miR-29b, hsa-miR-215, hsa-miR-194, hsa-miR-33a and hsa- miR-32） were upregulated more than 1.5-fold, and 11 miRNAs （including hsa-miR-93, hsa-miR-1231, hsa- miRPlus-F1080, hsa-miR-524-3p, hsa-miR-886-3p and hsa-miR-561） were downregulated in SW1116csc. The miRNA microarray results were further validated with quantitative RT-PCR. miR-93 was downregulated, and its predicted mRNA targets included BAMBI, CCND2, CDKNIA, HDACS, KIF23, MAP3K9, MAP3K11, MYCN, PPARD, TLE4 and ZDHHCl. Overexpressed miR-93 sig- nificantly inhibited cell proliferation and colony forma- tion by SW1116csc. Furthermore, miR-93 negatively regulated the mRNA and protein levels of HDAC8 and TLE4. CONCLUSION： Some miRNAs were differentially ex- pressed during differentiation of SW1116csc into SW1116 cells, miR-93 may inhibit SW1116csc proliferation and colony formation.

EFFECT OF TNF-a AND IFN-g ON THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE AND PROLIFERATION INHIBITION OF HUMAN COLON CANCER CELL LINE

Objective: To study the expression of the inducible nitric oxide synthase (iNOS) gene and the effects of tumor necrosis factor-α(TNF-a) and interferon-γ(IFN-g)on proliferation of the continuous cultured human colon cancer cell line CCL229. Methods: Using the molecular and biochemical techniques and electron microscopy to analyze the expression of iNOS, production of NO and growth characteristics of human colon cancer cells. Results: cytokine treatment can induce expression of the iNOS gene and production of nitric oxide was significantly higher after treatment of CCL229 cells with TNF-αor IFN-γ. Treatment with either cytokine or a combination of both significantly increased levels of Malondialdehyde (MDA) over control. Furthermore, cytokine treatment increased the proliferation inhibition rate as assessed in vitro and decreased the cell proliferation index on flow cytometry. Electron microscopy showed that cells treated with cytokines had fewer pseudopodia or cell processes than control cells and that cytokine treated cells had dilatation of the mitochondria and endoplasmic reticulum and dilated vesicular or tubular cisternae. Conclusion: Our findings indicate that TNF-α and IFN-γ induce the expression of iNOS gene in CCL229 cells, which increases the production of nitric oxide, inhibits proliferation, causes lipid peroxidation, and results in ultrastructural changes.

Effect of soy saponin on the growth of human colon cancer cells

AIM:To investigate the effect of extracted soybean saponins on the growth of human colon cancer cells.METHODS:WiDr human colon cancer cells were treated with 150,300,600 or 1200 ppm of soy saponin to determine the effect on cell growth,cell morphology,alkaline phosphatase(AP) and protein kinase C(PKC) activities,and P53 protein,c-Fos and c-Jun gene expression.RESULTS:Soy saponin decreased the number of viable cells in a dose-dependent manner and suppressed 12-Otetradecanol-phorbol-13-acetate-stimulated PKC activity(P < 0.05).Cells treated with saponins developed cytoplasmic vesicles and the cell membrane became rougher and more irregular in a dose-dependent manner,and eventually disassembled.At 600 and 1200 ppm,the activity of AP was increased(P < 0.05).However,the apoptosis markers such as c-Jun and c-Fos were not significantly affected by saponin.CONCLUSION:Soy saponin may be effective in preventing colon cancer by affecting cell morphology,cell proliferation enzymes,and cell growth.

Interaction of 14-3-3σ with KCMF1 suppresses the proliferation and colony formation of human colon cancer stem cells

AIM: To investigate the biological function of 14-3-3σ protein and to look for proteins that interact with 14-3-3σ protein in colon cancer stem cells. METHODS: Reverse transcription polymerase chain reaction was performed to amplify the 14-3-3σ gene from the mRNA of colon cancer stem cells. The gene was then cloned into the pGEM-T vector. After being sequenced, the target gene 14-3-3σ was cut from the pGEM-T vector and cloned into the pGBKT7 yeast expression plasmid. Then, the bait plasmid pGBKT7-14-3-3σ was transformed into the yeast strain AH109. After the expression of the pGBKT7-14-3-3σ fusion protein in the AH109 yeast strain was accomplished, a yeast two-hybrid screening assay was performed by mating AH109 with Y187 that contained a HeLa cDNA library plasmid. The interaction between the 14-3-3σ protein and the proteins obtained from positive colonies was further confirmed by repeating the yeast two-hybridscreen. After extracting and sequencing the plasmids from the positive colonies, we performed a bioinformatics analysis. A coimmunoprecipitation assay was performed to confirm the interaction between 14-3-3σ and the proteins obtained from the positive colonies. Finally, we constructed 14-3-3σ and potassium channel modulatory factor 1 (KCMF1) siRNA expression plasmids and transfected them into colon cancer stem cells. RESULTS: The bait plasmid pGBKT7-14-3-3σ was constructed successfully, and the 14-3-3σ protein had no toxic or autonomous activation effect on the yeast. Nineteen true-positive colonies were selected and sequenced, and their full-length sequences were obtained. We searched for homologous DNA sequences for these sequences from GenBank. Among the positive colonies, four coding genes with known functions were obtained, including KCMF1 , quinone oxidore-ductase (NQO2 ), hydroxyisobutyrate dehydrogenase (HIBADH ) and 14-3-3σ . For the subsequent coimmu-noprecipitation assay, the plasmids PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH were successfully constructed, and the sequences were further confirmed by DNA sequencing. The Fugene 6 reagent was used to transfect the plasmids, and fluorescence-activated cell sorting analysis showed the transfection efficiency was 97.8% after 48 h. The HEK 293FT cells showed the stable expression of the PCDEF-Flag-14-3-3σ, PCDEF-Myc-KCMF1, PCDEF-Myc-NQO2 and PCDEF-Myc-HIBADH plasmids. After anti-Myc antibody immunoprecipitation with Myc-KCMF1, Myc-NQO2 and Myc-HIBADH from cell lysates, the presence of Flag-14-3-3σ protein in the immuno-precipitated complex was determined by western blot analysis. The knock-down expression of the 14-3-3σ and KCMF1 proteins significantly inhibited cell proliferation and colony formation of SW1116csc. CONCLUSION: Genes of the proteins that interactedwith 14-3-3σ were successfully screened from a HeLa cDNA library. KCMF1 and 14-3-3σ protein may affect the proliferation and colony formation of human colon cancer stem cells.

Down-regulation of p110β Expression Increases Chemosensitivity of Colon Cancer Cell Lines to Oxaliplatin

This study examined the synergetic effect of class ⅠA Phosphoinositide 3-kinases catalytic subunit p110β knockdown in conjunction with oxaliplatin treatment on colon cancer cells. Down-regulation of p110β by siRNA interference and oxaliplatin treatment were applied in colon cancer cell lines HT29, SW620 and HCT116. MTT assay was used to measure the inhibitory effect of p110β knockdown on the proliferation of colon cancer cell lines. SubG1 assay and Annexin-Ⅴ FITC/PI double-labeling cytometry were applied to detect cell apoptosis. And cell cycle was evaluated by using PI staining and flow cytometry. The expression of caspase 3, cleaved PARP, p-Akt, T-Akt and p110β was determined by western blotting. The results suggested that down-regulation of p110β expression by siRNA obviously reduced cell number via accumulation in G0-G1 phase of the cell cycle in the absence of notablely increased apoptosis in colon cancer cell lines HT29 and SW620 (S phase arrest in HCT116). Moreover, inhibition of p110β expression increased oxaliplatin-induced cell apoptosis and cell cycle arrest in HT29, HCT116 and SW620 cell lines. In addition, increases of cleaved caspase-3 and cleaved PARP induced by oxaliplatin treatment were determined by immunoblotting in p110β knockdown group compared with normal control group and wildtype group. It is concluded that down-regulated expression of p110β could inhibit colon cancer cells proliferation and result in increased chemosensitivity of colorectal cancer cells to oxaliplatin through augmentation of oxaliplatin-induced cell apoptosis and cell cycle arrest.

Epigenetic modification regulates both expression of tumor-associated genes and cell cycle progressing in human colon cancer cell lines: Colo-320 and SW1116

The aim of this study is to assess the effects of DNA methylation and historic acetylation, alone or in combination, on the expression of several tumor-associated genes and cell cycle progression in two established human colon cancer cell lines: Colo-320 and SW1116. Treatments with 5-aza-2'-deoxycytidine (5-aza-dC) and trichostatin A, alone or in combination, were applied respectively. The methylation status of the CDKN2A promoter was determined by methyla-tion-specific PCR, and the acetylated status of the histones associated with the p21WAF1 and CDKN2A genes was examined by chromatin immunoprecipitation. The expression of the CDKN2A, p21WAF1, p53, p73, APC, c-myc, c-Ki-ras and survivin genes was detected by real-time RT-PCR and RT-PCR. The cell cycle profile was established by flow cytometry. We found that along with the demethylation of the CDKN2A gene promoter in both cell lines induced by 5-aza-dC alone or in combination with TSA, the expression of both CDKN2A and APC genes increased. The treatment of TSA or sodium butyrate up-regulated the transcription of p21WAF1 significantly by inducing the acetylation of histones H4 and H3, but failed to alter the acetylation level of CDKN2A-associated histones. No changes in transcription of p53, p73, c-myc, c-Ki-ras and survivin genes were observed. In addition, TSA or sodium butyrate was shown to arrest cells at the G1 phase. However, 5-aza-dC was not able to affect the cell cycle progression. In conclusion, regulation by epigenetic modification of the transcription of tumor-associated genes and the cell cycle progression in both human colon cancer cell lines Colo-320 and SW1116 is gene-specific.

An effective vaccine against colon cancer in mice:Use of recombinant adenovirus interleukin-12 transduced dendritic cells

AIM： To investigate the effect of a vaccine with recombinant adenovirus interleukin-12 （AdVIL-12） transduced dendritic cells （DCs） against colon cancer in mice. METHODS： DCs and AdVIL-12 were incubated together at different time intervals and at different doses. Supernatant was collected and tested for IL-12 by enzyme-linked immunosorbent assay （ELISA）. In order to determine whether tumor cell lysate-pulsed （TP） AdVIL-12/DCs enhance therapeutic potential in the established tumor model, CT26 colon tumor cells were implanted subcutaneously （s.c.） in the midflank of naive BALB/c mice. Tumor-bearing mice were injected with a vaccination of CT26 TP AdVIL-12/DCs on d 3 and 10. As a protective colon tumor model, naive BALB/c mice were immunized s.c. in their abdomens with CT26 TP AdVIL-12/DCs twice at seven day intervals. After the immunization on d 7, the mice were challenged with a lethal dose of CT26 tumor cells and survival times were evaluated. Subsequently, cytotoxic T lymphocyte （CTL） activity and interferon gamma （IFNy） secretion was evaluated in the immunized mice, and assayed CTL ex vivo. RESULTS： Murine DCs were retrovirally transduced with AdVIL-12 efficiency, and the AdVIL-12 transduced DCs secreted a high level of IL-12 （AdVIL-12/DCs, 615.27 ± 42.3 pg/mL vs DCs, 46.32 ± 7.29 pg/mL, P 〈 0.05）. Vaccination with CT26 TP AdVIL-12/DCs could enhance anti-tumor immunity against CT26 colon tumor in murine therapeutic models （tumor volume on d 19：CT26 TP AdVIL-12/DCs 107 ± 42 mm^3 vs CT26 TP DCs 383± 65 mm^3, P 〈 0.05） and protective models. Moreover, the CT26 TP AdVIL-12/DC vaccination enhances tumor-specific CTL activity, producing high levels of IFN7 in immunized mice. Ex vivo primed T cells with AdVIL-12/DCs were able to induce more effective CTL activity than in primed T cells with CT26 TP/DCs （E：T = 100：1, 69.49% ± 6.11% specific lysis vs 37.44% ＋ 4.32% specific lysis, P 〈 0.05）.CONCLUSION： Vaccination with recombinant AdVIL-12 transduced DC pulsed tumor cell lysate enhance antitumor immunity specific to colon cancer in mice.

Ca2+/calmodulin-dependent protein kinase II regulates colon cancer proliferation and migration via ERK1/2 and p38 pathways

AIM To investigate the role of calmodulin-dependent protein kinase Ⅱ(Ca MKⅡ) in colon cancer growth,migration and invasion.METHODS Ca MKⅡ expression in colon cancer and paracancerous tissues was evaluated via immunochemistry. Transcriptional and posttranscriptional levels of Ca MKⅡin tissue samples and MMP2,MMP9 and TIMP-1 expression in the human colon cancer cell line HCT116 were assessed by q RTPCR and western blot. Cell proliferation was detected with the MTT assay. Cancer cell migration and invasion were investigated with the Transwell culture system and woundhealing assay.RESULTS We first demonstrated that CaMK Ⅱ was ove rexpressed in human colon cancers and was associated with cancer differentiation. In the human colon cancer cell line HCT116,the Ca MKII-specific inhibitor KN93,but not its inactive analogue KN92,decreased cancer cell proliferation. Furthermore,KN93 also significantly prohibited HCT116 cell migration and invasion. The specific inhibition of ERK1/2 or p38 decreased the proliferation and migration of colon cancer cells.CONCLUSION Our findings highlight Ca MKⅡ as a potential critical mediator in human colon tumor development and metastasis.

Therapeutic implications of colon cancer stem cells

Colorectal cancer is the second most common cause of cancer-related death in many industrialized countries and is characterized by a heterogenic pool of cells with distinct differentiation patterns. Recently, the concept that cancer might arise from a rare population of cells with stem cell-like properties has received support with regard to several solid tumors, including colorectal cancer. According to the cancer stem cell hypothesis, cancer can be considered a disease in which mutations either convert normal stem cells into aberrant counterparts or cause a more differentiated cell to revert toward a stem cell-like behaviour; either way these cells are thought to be responsible for tumor generation and propagation. The statement that only a subset of cells drives tumor formation has major implications for the development of new targeted therapeutic strategies aimed at eradicating the tumor stem cell population. This review will focus on the biology of normal and malignant colonic stem cells, which might contribute to our understanding of the mechanisms responsible for tumor development and resistance to therapy.

Proteome of human colon cancer stem cells:A comparative analysis

AIM： To isolate and identify the biological characteristics of human colon cancer stem cells （SW1116 cells） and further study their proteome. METHODS： SW1116 cells were isolated and cultured with a serum-free medium （SFM）. Sphere formation was assayed to observe the formation of colon cancer stem cell spheres. SW1116 cells were inoculated into a serum-containing medium for observing their differentiation characteristics. Proliferation curve and cross-resistance of SWl116 cells to different drugs were detected by MTT. Percentage of SP cells in SW1116 cells was detected with Hoechst33342 staining. Telomerase activity in SW1116cells was checked by polymerase chain reaction （PCR）-enzyme linked immunosorbent assay. Expressions of stem cell relevant genes and proteins were detected by reverse transcription-PCR and Western blot, respectively. Total protein was isolated from SW1116 cells by two-dimensional gel electrophoresis （2-DE） and differentially expressed proteins were identified by tandem mass spectrometry （MALDI-TOF/TOF）. RESULTS： The isolated SW1116 cells presented as spheroid and suspension growths in SFM with a strong self-renewal, proliferation, differentiation and drug-resistance ability. The percentage of SP cells in SW1116 cells was 38.9%. The SW1116 cells co-expressed the CD133 and CD29 proteins. The telomerase activity in SW1116 cells was increased. The expressions of different stem cell relevant genes and proteins were detected. The proteomic analysis showed that the 26 protein spots were differently expressed in SW1116 cells and 10 protein spots were identified as ubiquitin fusion- degradation l-like protein, nuclear chloride channel protein, tubulin 13, Raichu404X, stratifin, F-actin cap- ping protein α-1 subunit, eukaryotic translation elongation factor 1 delta isoform 2, hypothetical protein, glyceraldehyde-3-phosphate dehydrogenase and guanine nucleotide binding protein 13 polypeptide 2-like 1, respectively. CONCLUSION： SW1116 cells are biologically characterized by self-renewal, proliferation and differentiation, and the differently expressed proteins in SW1116 cells may be essential for isolating cancer stem cells.

Upregulated CD133 expression in tumorigenesis of colon cancer cells

AIM: To analyze the upregulated CD133 expression in tumorigenesis of primary colon cancer cells. METHODS: Upregulated CD133 expression in tumorigenesis of colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) was analyzed by flow cytometry. Human colon cancer tissue samples were stained with anti-human CD133. SW620 cells were sorted according to the CD133 expression level measured by fluorescence-activated cell sorting. Spheroids of colorectal cancer cells were cultured with the hanging drop. Expression of CD133 and Lgr5 in spheroids of colorectal cancer cells and monolayer culture was detected by RT-qPCR. Spheroids of colorectal cancer cells were analyzed using anti-human CD133 with immunohistochemical staining. RESULTS: CD133 antigen was expressed in colorectal cancer cell lines (Lovo, Colo205, Caco-2, HCT116 and SW620) as well as in primary and metastatic human colon cancer tissues. However, the CD133 was differently expressed in these cell lines and tissues. The expression levels of CD133 and Lgr5 were significantly higher in spheroids of parental, CD133hi and CD133-cells than in their monolayer culture at the mRNA level (P < 0.05). Immunohistochemical staining of spheroids of CD133-cells showed that CD133 was highly expressed in colorectal cancer cell lines. CONCLUSION: Upregulated CD133 expression plays a role in tumorigenesis colorectal cancer cells, which may promote the expression of other critical genes that can drive tumorigenesis.