| Objective: Tumor and diabetes are the two kinds of severe diseases harming the health and life of human at present and the morbidity and mortality of them are increasing year by year. Of late years, more and more clinical and epidemiological data discovered that the patients with diabetes easily suffer from colorectal cancer. The risk suffering from colorectal cancer by the patients with diabetes is significantly higher than the non-diabetes patients, and it was suggested that diabetes is the independent risk factor for colorectal cancer. The morbidity, relapse rate and mortality of colorectal cancer in the diabetes patients are notably higher than that in the non-diabetes patients. The relationship between diabetes and colorectal cancer has become a highly interested heat point. Studying and discovering the pathological physiology relationship between diabetes and colorectal cancer and its molecular mechanisms has become an important question for exploration needed very in this scientific territory.The tumor immunological studies have discovered that the tumor development closely related not only to the immunofunction of body, in a sense, but also even more to the immunosuppression mediated by tumor cells. That the tumor cells secrete immunosuppressive substances to suppress the immunofunction of body, i.e. the immunosuppression of tumor cells, is the important mechanism with which the tumor cells escape the immunosurveillance of body to develop. The immunosuppressive substances secreted by tumor cells not only possibly could play a immunosuppression in the sites apart from the tumor in the body, but also even more can form the deeply immunosuppressive area, i.e. called"collapsar", severely suppressing not only the immunofunction of the immunocytes in situ within the area, but also that of the function-normal and even activated immunocytes entered into the area to form"silence"cells, making all immunocytes in the area to loss the ability of effectively killing the tumor cells, and thus this apparently should be the important mechanism that in the body with completely normal immunofunctions the tumor yet could escape the immunosurveillance to develop.One of the important pathological physiology characteristics of diabetes is the abnormal changes of the concentrations of glucose and insulin in blood of the patients. Just according to the abnormal changes of the concentrations of glucose and insulin in blood of the patients,the modern medicine divided the natural course of diabetes into four phases, and there are the different concentrations of glucose and insulin in blood corresponding to the different phases. It was not reported at present that the patients with diabetes easily suffer from colorectal cancer would be related either to the different concentrations of glucose and insulin in blood in the different phases of diabetes or not , very needing to study on it. Up to now, it has been reported that there are about twenty kinds of immunosuppressive substances which could be secreted by tumor cells. Among these, those reported more frequently, having more intensive immunosuppression and just to be secreted by the colorectal cancer cell line Colon26 tumor cells are the vascular endothelial growth factor(VEGF), the transforming growth factorβ1(TGF-β1)and interleukin-6(IL-6). Thus, in this study using the different concentrations of glucose and insulin mimicing the different phases of diabetes to affect Colon26 tumor cells, the effect on the secretion of VEGF, TGF-β1 and IL-6 from Colon26 tumor cells was detected and the molecular mechanism leading to the effect was explored primarily, so that from the aspect of affecting the secretion of immunosuppressive substances from colorectal cancer cells, for the investigating the relationship between diabetes and colorectal cancer and discovering why the diabetes patients easily suffer from colorectal cancer, to supply new studying way and experimental evidences.Methods: Colon26 tumor cells were cultured conventionally in vitro. According to the final concentrations of glucose and insulin added into the cell medium (RPMI1640 complete medium,CM), the Colon26 tumor cells cultured were divided into 12 groups: A group(control), CM; B group (normal glucose, nG), 5mmol/L glucose; C group(middle glucose, mG), 10mmol/L glucose ; D group(high glucose, hG), 25mmol/L glucose; E group (normal insulin, nI), 5μIU/mL insulin; F group(middle insulin, mI), 25μIU/mL insulin; G group(high insulin, hI), 125μIU/mL insulin; H group (nGnI), 5mmol/L glucose and 5μIU/mL insulin; K group (nGhI), 5mmol/L glucose and 125μIU/mL insulin; L group(hGhI), 25mmol/L glucose and 125μIU/mL insulin; M group(hGnI), 25mmol/L glucose and 5μIU/mL insulin; N group(high glucose and low insulin, hGloI), 25mmol/L glucose and 1.25μIU/mL insulin. All groups were triple holes, placed in 37℃5 % CO2 saturated humidity to be cultured for 48h. After culture completion the cells and the supernatants were collected respectively. The concentrations (pg/mL) of TGF-β1, VEGF and IL-6 in the supernatant were detected by ELISA respectively. The total RNA of the cells collected was extracted. The cDNA of VEGF, TGF-β1, IL-6 and interior referenceβ-actin were amplificated by RT-PCR respectively. The agarose gel electrophoresis of amplificated products was performed and analyzed by scaning in the gel imaging system, and the mRNA relative expressive values of TGF-β1, VEGF and IL-6 were calculated respectively and the expressive intensity was shown as the relative expressive coefficient RC. The all experiments were triplicated and the results were shown as x±s. The statistical analysis was performed using the t test and the sigle factor analysis of variance, and the significant size of test was P<0.05.Results:1 The effect of glucose at different concentrations on the secretion of VEGF from Colon26 tumor cellsThe concentrations of VEGF in the supernatants of B (nG) group, C (mG) group and D (hG) group (9.4167±0.4646,9.4667±0.1528 and 9.6167±0.2887 respectively) had not significant difference compared with that of A (control) group (9.6167±0.2887) (all P>0.05), indicating that the three concentrations [(5, 10 and 25) mmol/L] of glucose tested did not affect the secretion of VEGF from Colon26 tumor cells.2 The effect of insulin at different concentrations on the secretion of VEGF from Colon26 tumor cellsThe concentration of VEGF in the supernatant of G (hI) group (27.7667±0.2517) was significantly higher than that of A (control) group (9.5583±0.2691), E (nI) group (9.5833±0.0764) and F (mI) group (9.6167±0.1258) ( all P<0.01), but there was no significant difference among the three (i.e. A, E and F) groups (P>0.05), indicating only the high concentration (125μIU/mL) of insulin enabaling to increase the secretion of VEGF from Colon26 tumor cells.3 The effect of the combination of glucose and insulin at different concentrations on the secretion of VEGF from Colon26 tumor cellsThe concentrations of VEGF in the supernatants of K(nGhI) group and L(hGhI) group(27.8333±0.3512 and 27.6000±0.6557respectively)were significantly higher than that of A group(9.5583±0.2691)(all P<0.01) and there was no difference between K group and L group(P>0.05), and the concentrations of VEGF in the supernatants of H(nGnI) group, M(hGnI) group and N(hGloI) group(9.7167±0.00289,9.8667±0.0577 and 9.90±0.1732, respectively) had no differences compared with A group(all P>0.05), indicating that the combination of glucose and insulin at different concentrations could not affect the pattern of the effect by oneself on the secretion of VEGF from Colon26 tumor cells, yet showing that only that adding high concentration(125μIU/mL)of insulin(K group and L group) could increase the secretion of VEGF from Colon26 tumor cells, and that the combination of both had neither synergistic effect nor antagonistic effect.4 The effect of glucose and insulin at different concentrations on the VEGF genetic transcription of Colon26 tumor cellsIn G, K and L groups in which in the cell medium the final concentration of insulin all was high(125μIU/mL), their electrophoresis strip of the amplificated product of VEGF mRNA had become thickening and bright compared with A group, and their VEGF mRNA expression level (0.8802±0.1900,0.8605±0.1800 and 0.8569±0.2200 respectively) compared with A group(0.4653±0.0200) had significant differences(all P<0.01). In other groups compared with A group either the electrophoresisi strips or the VEGF mRNA expression levels had not significant differences(all P>0.05). These findings indicated that the high concentration(125μIU/mL)of insulin could increase the VEGF genetic transcription and suggested that this should be the molecular mechanism that the high concentration of insulin could increase the secretion of VEGF from Colon26 tumor cells as above-mentioned.5 The effect of glucose at different concentrations on the secretion of TGF-β1 from Colon26 tumor cellsThe concentration of TGF-β1 in the supernatant of D(hG) group(863. 12±20.26) was significantly higher than that of A group (625.14±12.02) (P<0.01), and that of B (nG) group and C (mG) group (635.51±16.97 and 632.52±17.88 respectively) compared with A group had not significant differences(all P>0.05). These findings indicated that the high concentration (25mmol/L) of glucose could increase significantly the secretion of TGF-β1 from Colon26 tumor cells.6 The effect of insulin at different concentrations on the secretion of TGF-β1 from Colon26 tumor cellsThe concentrations of TGF-β1 in the supernatants of E(nI), F(mI) and G (hI) groups(622.18±10.53, 614.85±14.92 and 617.12±16.12, respectively) compared with A group(625.14±12.02) had not significant differences(all P>0.05), indicating that the three concentrations [(5, 25 and 125)μIU/mL ] of insulin tested had not affect the secretion of TGF-β1 from Colon26 tumor cells.7 The effect of the combination of glucose and insulin at different concentrations on the secretion of TGF-β1 from Colon26 tumor cellsThe concentrations of TGF-β1 in the supernatants of L(hGhI),M(hGnI)and N(hGloI)groups in which the high concentration(25mmol/L)of glucose had be added into the cell medium were 982.78±19.02, 882.54±17.08 and 871.46±16.21 and significantly higher than that of A group (625.14±12.02) (all P<0.01). The concentrations of TGF-β1 in the supernatants of H(nGnI)group and K(nGhI)group in which the high concentration of glucose had not be added into the cell medium were 626.18±15.98 and 630.18±18.95 respectively and compared with A group had not significant differences (all P>0.05). These findings indicated that the combination of glucose and insulin did not change the effect patterns by oneself, yet showing that only the high concentration of glucose could increase significantly the secretion of TGF-β1 from Colon26 tumor cells, and that the combination of both had neither synergistic effect nor antagonistic effect.8 The effect of glucose and insulin at different concentrations on the TGF-β1 genetic transcription of Colon26 tumor cellsThe expression level of TGF-β1 mRNA in D(hG) group(0.768±0.017), L(hGhI) group(0.889±0.028), M(hGnI) group(0.764±0.027) and N (hGloI) group (0.752±0.035) in which the high concentration (25mmol/L) of glucose was added into the cell medium were significantly higher than that of A group(0.540±0.012()all P<0.01) and that in B(nG) group(0.545±0.033),C(mG) group(0.558±0.035),E(nI) group(0.566±0.039), F(mI) group(0.549±0.030), G(hI) group(0.531±0.022), H(nGnI) group(0.550±0.037) and K(nGhI) group(0.537±0.025) in which the high concentration of glucose did not be added into the cell medium were equivalent to that in A group(all P>0.05), indicating that only the high concentration(25mmol/L) of glucose could increase the TGF-β1 mRNA transcription of Colon26 tumor cells and suggesting that the molecular mechanism with which the high concentration of glucose could increase the secretion of TGF-β1 from Colon26 tumor cells as above-mentioned should be due to the increasing the TGF-β1 mRNA transcription at the genetic level.9 The effect of glucose at different concentration on the secretion of IL-6 from Colon26 tumor cellsThe concentration of IL-6 in the supernatant of D(hG) group(27.70±1.53) was significantly higher than that of A group(18.33±2.52)(P <0.01), but that of B(nG) group(19.33±2.31) and C(mG) group(22.6±0.58) compared with A group had not significant differences(all P>0.05), indicating that the high concentration(25mmol/L)of glucose could increase significantly the secretion of IL-6 from Colon26 tumor cells.10 The effect of insulin at different concentration on the secretion of IL-6 from Colon26 tumor cellsThe concentrations of IL-6 in the supernatants of E(nI) group, F(mI) group and G(hI) group(19.67±1.53, 21.33±4.04 and 21.67±2.08, respectively) compared with that of A group(18.33±2.52) had not significant differences (all P>0.05), indicating that the three kinds of concentrations [(5, 25 and 125 )μIU/mL] of insulin tested had not the effect on the secretion of IL-6 from Colon26 tumor cells.11 The effect of the combination of glucose and insulin at different concentration on the secretion of IL-6 from Colon26 tumor cellsThe concentrations of IL-6 in the supernatants of L group , M group and N group (27.67±2.52, 27.33±2.08 and 27.33±1.53, respectively) in which the high concentration (25mmol/L) of glucose was added into the cell medium were significantly higher than that of A group (18.33±2.52)( all P <0. 01) and that of H group and K group (20.33±1.53 and 22.33±2.08, respectively) in which the high concentration of glucose did not be added into the cell medium compared with that of A group had not significant differences (all P>0.05). These findings indicated that the combinations of glucose and insulin at different concentrations did not change the pattern of effect by oneself, yet showing that the high concentration of glucose could increase the secretion of IL-6 from Colon26 tumor cells, and that the combination of both had neither synergistic effect nor antagonistic effect.12 The effect of glucose and insulin at different concentrations on the IL-6 genetic transcription of Colon26 tumor cellsThe IL-6 mRNA expression in D(hG) group(0.560±0.011) was significantly higher than that in A group(0.406±0.012 )(P <0.05), that in B (nG)group and C(mG) group(0.413±0.031 and 0.426±0.001 respectively) compared with A group had not significant differences(all P >0. 05), that in E(nI)group, F(mI)group and G(hI)group(0.416±0.002, 0.422±0.013 and 0.426±0.016 respectively) compared with A group had not significant differences(all P >0. 05), that in the L , M and N groups in which the high concentration(25mmo1/L)of glucose was added into the cell medium ( 0.560±0.011, 0.560±0.011 and 0.563±0.002, respectively) were significantly higher than that in A group(all P <0.05), and that in the H and K groups in which the high concentration of glucose was not added into the cell medium (0.420±0.001 and 0.426±0.002 respectively) compared with A group had not significant differences(all P >0.05). These findings indicated that the high concentration(25 mmo1/L)of glucose can significantly increase the IL-6 mRNA transcription of Colon26 tumor cells and suggested that the molecular mechanism with which the high concentration of glucose could increase the secretion of IL-6 from Colon26 tumor cells as above-mentioned should be due to the increasing the IL-6 mRNA transcription at the genetic level.Conclusion:1 The high concentration(25mmol/L of glucose can increase significantly the secretion of TGF-β1 and IL-6 from Colon26 tumor cells, the molecular mechanism of which should be due to that the genetic transcription of TGF-β1 and IL-6 within Colon26 tumor cell had be promoted.2 The different concentrations ([5-25)mmol/L] of glucose had not the effect on the secretion and genetic transcription of VEGF of Colon26 tumor cells.3 The high concentration(125μIU/mL)of insulin can increase significantly the secretion of VEGF from Colon26 tumor cells, the molecular mechanism of which should be due to that the genetic transcription of VEGF within Colon26 tumor cell had be promoted.4 The different concentrations [(1.25-125)μIU/mL] of insulin had not the effect on the secretion and genetic transcription of the TGF-β1 and IL-6 of Colon26 tumor cells.5 The combination of glucose and insulin at different concentrations did not change the pattern of effect by oneself on the secretion and genetic transcription of VEGF, TGF-β1 and IL-6 of Colon26 tumor cells.6 The effect of the combination of glucose and insulin at different concentrations on the secretion and genetic transcription of VEGF, TGF-β1 and IL-6 of Colon26 tumor cells was neither synergistic nor antagonistic.7 That the different concentrations of glucose and insulin in the natural course of diabetes could increase significantly the secretion of some immunosuppressive substances from the colorectal cancer cells should be one of the internal molecular mechanism with which the diabetes patients easily suffer from the colorectal cancer. |
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