[Growth inhibition of combined pathway inhibitors on KRAS mutated non-small cell lung cancer cell line].

OBJECTIVE: To investigate the effect of the selective PI3K inhibitor and MEK inhibitor on KRAS and PTEN co-mutated non-small cell lung cancer cell line NCI-H157 and the relevant mechanisms. METHODS: NCI-H157 was cultured routinely and treated with different concentrations of the two inhibitors. Cell proliferation was detected by MTT cell cycle assay. Based on the MTT results the cells were divided into four groups: the control group, PI3K inhibitor group (GDC-0941, 0.5 and 5.0 µmol/L), combination group I (0.5 µmol/L AZD6244 + 0.5 µmol/L GDC-0941) and combination group II (5.0 µmol/L AZD6244 + 5.0 µmol/L GDC-0941). Colony formation assay was performed to detect colony formation efficiency. The cell cycle and apoptosis were analyzed by flow cytometry. The expression of protein related to apoptosis was tested with Western blot. RESULTS: Cell growth was inhibited by the two inhibitors. Combination groups led to stronger cell proliferation inhibition: combination group Ishowed synergistic effect of their actions and combination group II showed an additive effect; in both groups, there were decreased colony number [(77.2 ± 1.54)/well vs (61.50 ± 2.12)/well, P < 0.01] and [(51.00 ± 4.00)/ well vs (22.50 ± 3.53)/well, P < 0.01]; and enhanced apoptotic ratios [(18.30 ± 0.82)% vs (21.32 ± 0.56)%, P < 0.01] and [(27.14 ± 1.58)% vs (42.45 ± 4.42)%, P < 0.01]. In addition, compared to the PI3K inhibitor alone group, the NCI-H157 cells in the combination groups showed increased G0/G1 phase and decreased S phase (P < 0.01). Western blotting showed that the combination groups demonstrated significantly decreased expression of cyclin D1 and cyclin B1, increased p21 and cleaved PARP and decreased bcl-2/bax ratio, compared to the PI3K inhibitor only group. CONCLUSION: The combined inhibition of PI3K (AZD6244) and MEK (GDC-0941) has synergistic effects on the proliferation of NCI-H157 cells, but such effects appear to be in a dose-dependent manner.

[Effect of forced E-cadherin expression on adhesion and proliferation of human breast carcinoma cells].

OBJECTIVE: To investigate the role that E-cadherin (E-cad) plays on cell adhesion and proliferation of human breast carcinoma. METHODS: E-cad expression vector was transfected into an E-cad-negative human breast carcinoma MDA-MB-231 cells. G418 was used to screen positive clones. E-cad, ß-catenin (ß-cat) and cyclin D1 expressions of these clones were confirmed by Western blot. Their cell-cell and cell-matrix adhesion abilities were detected. E-cad/ß-catenin interaction was confirmed by immunoprecipitation. Cell proliferation was evaluated by MTT. Cell apoptosis was analyzed by flow cytometry. Direct two-step immunocytochemistry was used to detect the localization of ß-cat. RESULT: E-cad(+) cell strains Ecad-231-7 and Ecad-231-9 were established. When cultured in ultra-low-binding dishes Ecad-231 cells grow in suspension while Ecad-231-7 and Ecad-231-9 cells grow in large clamps. When co-cultured with HCT116 cells, the average adhesion rates at 30 min are 39.0%, 60.0% and 59.5% for MDA-MB-231, Ecad-231-7 and Ecad-231-9 respectively. The average detachment rates by EDTA for 5 min are 37.4%, 4.2% and 7.4% respectively. So E-cad expression enhanced hemotypic and heterotypic cell-cell adhesion and cell-matrix adhesion. Forced exogenously expressed E-cad could combine with endogenous ß-cat, whereas down stream cyclin D1 expression was significantly decreased, as evidenced by Western blot. The rates of cell apoptosis of MDA-MB-231, Ecad-231-7 and Ecad-231-9 were 1.8%, 2.0% and 2.1%. Expression of E-cad had no obvious effect on the apoptosis of tumor cells with regular culture. ß-cat increased in the cytoplasma. CONCLUSIONS: Two monoclonal tumor cell strains (Ecad-231-7 and Ecad-231-9) stably expressing E-cad were successfully established. E-cad could enhance adhesion and inhibit proliferation of human breast carcinoma cells through a pathway involving ß-cat and cyclin D1.

Expression of CD133 correlates with differentiation of human colon cancer cells.

CD133 has been identified as a cancer stem cell marker in colon and several other cancers, but its function is still unknown. We examined the CD133 expression in 44 human cancer cell lines, and found five of the 8 positive lines were from colon cancer. The CD133 positive subpopulation of colon cancer cells showed more vigorous growth and lower differentiation. Induction of differentiation reduced the CD133-positive population. Knockdown of CD133 expression in colon cancer cells could not induce cellular differentiation. Care must be taken if CD133 is used as the only marker of cancer stem cells in colon cancer, especially in established cell lines. CD133 negatively correlates with cell differentiation, but it is not a regulator of differentiation.

[Expression and function of VAP-33 in murine dendritic cell sarcoma].

OBJECTIVE: To elucidate the expression and function of VAP-33 gene in dendritic cell sarcoma (DCS) cell line. METHODS: The expression of VAP-33 in DCS cells was investigated by mass spectrum with immunoprecipitation membrane protein. DCS cells were treated with antigens in different dosages (150, 850, and 1500 microl) for 24, 48 and 72 h respectively. Cell morphology and phagocytosis activity of DCS cells were measured. Indirect immunofluorescence, confocal microscopy and Western blotting were used to study the distribution and expression changes of VAP-33. Moreover, DCS cells were treated with 0.5 mol/L insulin for 20 min first and followed by Western blotting to detect changes of VAP-33 and glucose transfer protein 4 (GLUT-4) in the total cellular protein, cytoplasmic protein and membrane protein. Confocal microscopy was used to document the expression and distribution changes of VAP-33 and GLUT-4 in DCS cells. RESULTS: VAP-33 expression was obtained at the cell membrane and in the cytoplasm of DCS cells. Upon antigen stimulation, DCS cells showed more active phagocytosis and morphologically became more elongated with branched protrusions. The expression of VAP-33 was decreased by the antigen stimulation. Upon the insulin stimulation, the expression of VAP-33 and GLUT-4 were increased and co-localized. CONCLUSIONS: VAP-33 expression in DCS originated from the dendritic cells (DC) seemed relating to the vesicle transportation during antigen processing in DC. Additionally, VAP-33 and GLUT-4 also take part in the glucose transportation in the cells.

[Establishment of green-fluorescent protein expressing tumor metastasis models].

OBJECTIVE: To establish a green-fluorescent protein (GFP) labeled tumor metastasis model and to evaluate its biological characteristics. METHODS: Human gastric carcinoma cell MGC-803 and murine cervical carcinoma cell U14 were transfected with the plasmid pEGFP-N1 and the efficiency of transfection was assessed 24 h later. Limited dilution was employed to screen and establish monoclonal cell strains, MGC-803-GFP and U14-GFP. The two fluorescent tumor cell stains were transplanted into BALB/c-nu mice and C57BL/6J mice respectively. The latency period of tumor mass appearance and the growth curve in vivo were documented. The tumor growth and metastasis were evaluated in vivo by the Viviperception Fluorescence Imagining System (VFIS). Expressions of CD44 and E-cadherin in tumor tissue were monitored by immunohistochemistry. RESULTS: The efficiency of pEGFP-N1 transfection of MGC-803 cells and U14 cells were 30% and 60%, respectively. Monoclonal GFP(+) cell strains-MGC-803-GFP and U14-GFP were established. The latency periods of tumor formation of MGC-803-GFP and U14-GFP were 3-5 days and 2-4 days, respectively. Their tumorigenicity rates were 100% in both. The tumor growth of MGC-803-GFP was well defined by the VFIS. Only one mouse was shown to harbor lymphatic metastasis by VFIS, 60 days after transplantation. The metastasis process of U14-GFP was depicted through VFIS on 27, 37 and 52 days post-transplantation. The incidence of pulmonary metastasis and lymphatic metastasis of U14-GFP was 67% and 100% respectively when the tumor volume was >or=5 cm3. CD44 was positive and E-cadherin was negative in both tumors by immunohistochemistry. CONCLUSIONS: Successfully established two monoclonal tumor cell strains stably expressing GFP: MGC-803-GFP and U14-GFP. Transplantation of these cells into mice can establish tumor metastasis models which could be used for future visualized tumor research in vivo.

[Effect of inhibitor of differentiation-1 on murine dendritic cell sarcoma cells].

OBJECTIVE: To investigate the effect of down-expression of inhibitor of differentiation-1 (Id-1) on the differentiation of dendritic cell sarcoma (DCS) cells in vitro. METHODS: Down-regulation of the expression of Id-1 in DCS cells was performed by RNAi, and confirmed by protein and mRNA quantitative analyses. Cellular differentiation and biological behavior including malignant phenotypes of the cells were evaluated. All experiments included negative (no treatment group and no-target siRNA) and positive (induction-differentiation drug sodium butyrate) controls. RESULTS: When the expression of Id-1 was down regulated, the DCS cells showed more mature morphology including cell enlargement, longer cellular extensions, more branches, and decreased nuclear/plasma ratio. Differentiation marker expression (Id-2 and CD86) was also increased. RNAi treated cells at 24 and 48 hours, showed increase percentage of cells at G0/G1 phase and less cells at S phase (P < 0.01). Importantly, the abilities of cell proliferation, colony formation and invasiveness were significantly decreased (P < 0.01), as evidenced by MTT, colony formation and transwell assays respectively. CONCLUSION: RNAi inhibition of Id-1 protein can induce differentiation of malignant solid tumor cells along with reversion of their malignant phenotype.