Tumors have long been viewed as a population in which all cells have the equal propensity to form new tumors,the so called conventional stochastic model.The cutting-edge theory on tumor origin and progression,tends to...Tumors have long been viewed as a population in which all cells have the equal propensity to form new tumors,the so called conventional stochastic model.The cutting-edge theory on tumor origin and progression,tends to consider cancer as a stem cell disease.Stem cells are actively involved in the onset and maintenance of colon cancer.This review is intended to examine the state of the art on colon cancer stem cells(CSCs),with regard to the recent achievements of basic research and to the corresponding translational consequences.Specific prominence is given to the hypothesized origin of CSCs and to the methods for their identification.The growing understanding of CSC biology is driving the optimization of novel anti-cancer targeted drugs.展开更多
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 per...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 construct展开更多
Recent clinical and research development supports the use of 5-fluorouracil in combination with oxaliplatin for the treatment of patients with advanced colorectal cancer (CRC). 5-Fluorouracil (5-FU), which is as an an...Recent clinical and research development supports the use of 5-fluorouracil in combination with oxaliplatin for the treatment of patients with advanced colorectal cancer (CRC). 5-Fluorouracil (5-FU), which is as an anti-metabolite, is a widely used cytostatic drug. Although the rate of response, quality of life and overall survival differs between CRC patients, the above combination remains a widely used chemotherapeutic regimen. In some cases, a cancer stem cell (CSC) population may resist the majority of chemotherapeutic models. This study investigated if monotherapy is more efficacious than 5-fluorouracil and oxaliplatin combined for the treatment of CRC, using a CRC cell line and a CSC-like line. Cell viability was evaluated by cellular-based assays, and quantitative polymerase chain reaction (q-PCR) assays were performed to assess the expression of specific genes (TYMS,?DNMT1,?NANOG, DHFR, SHMT1, ERCC1, DPYD) correlated with 5-FU and oxaliplatin resistance. We observed that 5-fluorouracil was more effective in both CRC and CSCs. This finding proved the hypothesis that, in some cases, monotherapy may be more successful in CRC treatment than a drug combination that may be cytotoxic and inflict adverse side effects.展开更多
文摘Tumors have long been viewed as a population in which all cells have the equal propensity to form new tumors,the so called conventional stochastic model.The cutting-edge theory on tumor origin and progression,tends to consider cancer as a stem cell disease.Stem cells are actively involved in the onset and maintenance of colon cancer.This review is intended to examine the state of the art on colon cancer stem cells(CSCs),with regard to the recent achievements of basic research and to the corresponding translational consequences.Specific prominence is given to the hypothesized origin of CSCs and to the methods for their identification.The growing understanding of CSC biology is driving the optimization of novel anti-cancer targeted drugs.
基金Supported by The Medical Guidance Projects of Shanghai Science Committee,No.10411961800National Natural Science Foundation of China,No.81101617
文摘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 construct
文摘Recent clinical and research development supports the use of 5-fluorouracil in combination with oxaliplatin for the treatment of patients with advanced colorectal cancer (CRC). 5-Fluorouracil (5-FU), which is as an anti-metabolite, is a widely used cytostatic drug. Although the rate of response, quality of life and overall survival differs between CRC patients, the above combination remains a widely used chemotherapeutic regimen. In some cases, a cancer stem cell (CSC) population may resist the majority of chemotherapeutic models. This study investigated if monotherapy is more efficacious than 5-fluorouracil and oxaliplatin combined for the treatment of CRC, using a CRC cell line and a CSC-like line. Cell viability was evaluated by cellular-based assays, and quantitative polymerase chain reaction (q-PCR) assays were performed to assess the expression of specific genes (TYMS,?DNMT1,?NANOG, DHFR, SHMT1, ERCC1, DPYD) correlated with 5-FU and oxaliplatin resistance. We observed that 5-fluorouracil was more effective in both CRC and CSCs. This finding proved the hypothesis that, in some cases, monotherapy may be more successful in CRC treatment than a drug combination that may be cytotoxic and inflict adverse side effects.
