The Activation of ERK1/2 and JNK MAPK Signaling by Insulin/IGF-1 Is Responsible for the Development of Colon Cancer with Type 2 Diabetes Mellitus

Abstract

Previous studies showed that type 2 diabetes mellitus (T2DM) is linked to increased risk of developing colon cancer. Insulin and insulin-like growth factor 1 (IGF-1) are increased in patients with T2DM. The increased insulin and IGF-1 may be responsible for the developing of colon cancer. In this study, we investigated the effects and mechanisms of insulin and IGF-1 in colon cancer development in vitro and in vivo. Insulin and IGF-1 alone or together elevated proliferation and reduced apoptosis in colon cancer MC38 cells. Meanwhile, insulin and IGF-1 promoted the phosphorylation of extracellular-signal regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Treatment with ERK1/2 or JNK inhibitor in the presence of insulin and IGF-1 significantly decreased B-cell lymphoma 2 (Bcl-2) and increased Bcl-2-associated X protein (Bax) expression and finally increased apoptosis and inhibited the proliferation. Accelerative colon tumor growth was found in a mouse model of T2DM with db/db mice which got high level of endogenous insulin and IGF-1. Furthermore, the inhibition of ERK1/2 or JNK suppressed the development of colon tumor in vivo. These results suggest that the activation of ERK1/2 and JNK signaling by insulin and IGF-1, at least in part, is responsible for the development of colon cancer with T2DM.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1. Insulin/IGF-1 promotes colon cancer cells proliferation and cell cycle progression in vitro.

MC38 cells were cultured with various concentrations of insulin and IGF-1 for 72 hours. Control groups were treated with PBS. (A) Cell morphology was observed. (B) Cells were harvested for proliferation analysis with CCK-8 assay. *P#P<0.05; ##P<0.01 between the indicated two groups, n = 3 per group. (C) Cell cycle analysis of insulin/IGF-1 treated cells. DNA content was measured by PI staining on flow cytometry. The percentages of cell cycle phases are shown in each panel. The data shown are representative of three separate experiments.

Fig 2. Insulin/IGF-1 inhibits colon cancer cells apoptosis in vitro.

MC38 cells were cultured with insulin and IGF-1 alone or both together for 72 hours. Control groups were treated with PBS. (A) Cells were harvested for apoptosis analysis by Annexin V and PI staining on flow cytometry. The early stage (Annexin V+/PI-) and late stage (Annexin V+/PI+) apoptotic events were gated. The data shown are representative of three separate experiments. Quantification of total percentage (B) and early/late stage percentage (B) of apoptotic cells after the treatments. *P

Fig 3. Insulin/IGF-1 activates ERK1/2 and JNK signaling of colon cancer cells in vitro.

MC38 cells were cultured with insulin and IGF-1 alone or both together for 72 hours and then collected for western blotting analysis. Control groups were treated with PBS. (A) Western blotting analysis of p-ERK1/2, ERK1/2, p-JNK, JNK, p-P38 and P38 protein expression in treated cells. GAPDH served as a loading control. The blots shown are representative of three separate experiments. Semi-quantitation for the expressions of (B) ERK1/2 and pERK1/2, (C) JNK and p-JNK, (D) P38 and p-P38 proteins. Fold changes were normalized by control groups. *P

Fig 4. Inhibition of ERK1/2 or JNK signaling abolishes the proliferative and anti-apoptotic effects of insulin/IGF-1 in vitro.

MC38 cells were cultured with insulin and IGF-1 alone or both together for 72 hours. ERK1/2 inhibitor PD98059 (B), JNK inhibitor SP600125 (C) or their vehicle DMSO added to the cultures when MC38 cells were treated with both insulin and IGF-1. (A) Cells were collected for proliferation analysis with CCK-8 assay at 24, 48 and 72 hour. **P

Fig 5. Establishment of type 2 diabetes model with db/db mice.

Male db/db mice were used as mouse type 2 diabetes models, while db/+ littermates as normal controls. Body weight (A), blood glucose (B), insulin (C) and IGF-1 (D) were determined before MC38 cells injection at 8th week. *P<0.05; ***P<0.001, n = 5 per group.

Fig 6. Endogenous insulin/IGF-1 accelerates colon tumor growth in a mouse type 2 diabetes model.

2 × 106 MC38 cells suspended in 0.1 ml of PBS were subcutaneously injected into the db/db and db/+ mice to initiate tumor growth in vivo. (A) Tumor size was measured every 3 days. *P<0.05; ***P<0.001. (B) The tumors were excised and weighted 3 weeks after cell injection. ***P< 0.001. A representative tumor mass from each group was shown in the inset (scale bar = 1cm). (C and E) Western blotting analysis of p-ERK1/2, ERK1/2, p-JNK, JNK, Cyclin D1, Bcl-2, Bax and Caspase3 protein expression in tumors. GAPDH served as a loading control. The blots shown are representative of three separate experiments. (D and F) Semi-quantitation for the expressions of ERK1/2 and pERK1/2, JNK and p-JNK, Cyclin D1, Bcl-2, Bax and Caspase3 protein. Fold changes were normalized by control groups. *P<0.05; **P<0.01 versus control, n = 3 per group.

Fig 7. Inhibition of ERK1/2 or JNK signaling suppresses the development of colon tumor in type 2 diabetes model.

2 × 106 MC38 cells suspended in 0.1 ml of PBS were subcutaneously injected into the db/db mice to initiate tumor growth in vivo. 10 mg/kg PD98059 or 30 mg/kg SP600125 was administered intraperitoneally every 3 days when tumor volume reached 100mm3. 1% DMSO was used as control treatment. (A) Tumor size was measured every 3 days. *P<0.05; ***P<0.001. (B) The tumors were excised and weighted 3 weeks after cell injection. ***P<0.001. A representative tumor mass from each group was shown in the inset (scale bar = 1cm). (C and E) Western blotting analysis of p-ERK1/2, ERK1/2, p-JNK, JNK, Cyclin D1, Bcl-2, Bax and Caspase3 protein expression in tumors. GAPDH served as a loading control. The blots shown are representative of three separate experiments. (D and F) Semi-quantitation for the expressions of ERK1/2 and pERK1/2, JNK and p-JNK, Cyclin D1, Bcl-2, Bax and Caspase3 protein. Fold changes were normalized by control groups. **P<0.01; ***P<0.001 versus control, n = 3 per group.