IGFBP-rP1 Is Associated With Colorectal Cancer And Type Ⅱ Diabetes Mellitus

Metabolic syndrome represents a cluster of related cardiovascular risk factors that include central obesity, insulin resistance, atherogenic lipid profile, and hypertension. Insulin resistance is a central feature of metabolic syndrome, showing a strong association with most components of the syndrome. Thus metabolic syndrome is also named as insulin resistance syndrome. In the last decades, the incidence of metabolic syndrome rose rapidly. In China, the incidence of metabolic syndrome was 17.14% in Shanghai in 2003. The prevalence of the metabolic syndrome is high among U.S. adults. The unadjusted prevalence of the metabolic syndrome was 26.7% and the age-adjusted prevalence was 27.0% in the National Health and Nutrition Examination Survey 1999-2000. Metabolic syndrome has been a common disease which severely impairs the health of human.The exact molecular mechanism of metabolic syndrome is not clear so far. In the recent years lots of researches have been carried out to clarify the relationship between metabolic syndrome and cardiovascular diseases. The relationship between metabolic syndrome and cancer has been validated. The incidence and mortality of cancers, such as colorectal cancer, endometrial cancer and breast cancer, are increased by overweight and obesity. Patients with type II diabetes mellitus (DM2) are apt to suffer cancers. It was also reported that the incidence of cancers was increased in those patients with type II diabetes mellitus who received insulin treatment, and the incidence was positively correlated to the time of insulin treatment. It has been clear that hyperinsulinemia is the key of metabolic syndrome and cancer. However, the exact mechanism between metabolic syndrome and cancer has not been clarified.Our laboratory focused on the research in molecular pathology of colorectal cancer. We have constructed three cDNA libraries using SSH (suppression subtractive hybridization): adenocarcinoma VS adenoma (T-A), adenocarcinoma VS normal mucosa (T-N), adenoma VS normal mucosa (A-N) in 1999, and have screened a series of differentially expressed genes. Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is screened from the cDNA library of adenocarcinoma- normal mucosa, which was overexpressed in colorectal adenocarcinoma tissue.IGFBP-rP1 belongs to the IGFBP superfamily and is a member of insulin-like growth factor axis. As a 31KD secreted protein, IGFBP-rP1 is widely detectable in serum, urine, amniotic fluid, and cerebrospinal fluid. It also has been found in various types of tissue and organs, including brain, prostate, bladder, liver, lung, kidney, skeletal muscle and colon. It has been found that IGFBP-rP1 is significantly correlated with carcinogenesis. IGFBP-rP1 has been proposed as a potential tumor suppressor gene, because the down-regulation of IGFBP-rP1 expression is a common phenomenon in most human tumors, such as breast carcinoma, prostate carcinoma, meningioma and liver carcinoma. Overexpression of IGFBP-rP1 can inhibit the proliferation of immortalized or malignant human cells. Moreover, IGFBP-rP1 may be involved in the regulation of cell growth, differentiation and apoptosis. However, it is interesting to find that the expression of IGFBP-rP1 is upregulated in colorectal cancers. The fasting glucose level was significantly correlated with the staining of IGFBP-rP1 in cancer tissues. Colorectal cancer patients with type II diabetes mellitus demonstrated relatively high expression of IGFBP-rP1. These results indicated that IGFBP-rP1 may be an important factor both in colorectal cancer and type II diabetes mellitus. Yamanaka et al also found that IGFBP-rP1 is a high-affinity insulin binding protein. IGFBP-rP1 blocks insulin binding to the insulin receptor and thereby inhibits the earliest steps in insulin action, such as autophosphorylation of the insulin receptor beta subunit and phosphorylation of IRS-1, indicating that IGFBP-rP1 is a functional insulin-binding protein. Therefore, we hope to explore the relationship among IGFBP-rP1, colorectal cancer and type II diabetes mellitus, which may be helpful to clinical diagnoses and therapy. We also expect to explore the role of IGFBP-rP1 in colorectal cancer cells, which may contribute to understand the role of IGFBP-rP1 in colorectal cancer.Colorectal carcinoma (CRC) is one of the most common gastrointestinal malignancies that severely threaten the health of human. The incidence of colorectal cancer rose rapidly in the world in recent years. In western countries, colorectal cancer is the second cause of cancer-related death, only next to lung cancer. In China, the incidence of colorectal carcinoma ranks from the third to fourth among all the malignant cancers. Further the incidence and mortality are still increasing, due to the change of life style and diet elements. Therefore, it is of great significance to study the underlying mechanisms of colorectal cancer for its prevention and treatment. However, it is still difficult to diagnose colorectal cancer at the early stage. Enzyme-Linked Immunosorbnent Assay (ELISA) showed that serum IGFBP-rP1 was correlated with colorectal cancer. And it may be an independent indicator of colorectal cancer, which may be contribute to diagnose colorectal cancer.As we all know, insulin resistance is the basis of type II diabetes mellitus. IGFBP-rP1 is a member of insulin-like growth binding protein family, and it can block insulin binding to the insulin receptor and thereby inhibits the earliest steps in insulin action. Thus, insulin resistance can be induced by IGFBP-rP1. We found that serum IGFBP-rP1 was correlated with type II diabetes mellitus. The concentration of serum IGFBP-rP1 in type II diabetes mellitus patients was more than 400 times compared with that in non- diabetes mellitus patients. We also found that insulin could induce expression of IGFBP-rP1 in colorectal cancer cells. All these results indicated that IGFBP-rP1 is an independent indicator of type II diabetes mellitus.The above research of IGFBP-rP1 solved our puzzle that IGFBP-rP1 exactly is a key factor both in colorectal cancer and type II diabetes mellitus. However, high IGFBP-rP1 concentration was found only in 1 of 5 colorectal cancers that had high serum glucose concentration. This result indicated that IGFBP-rP1 plays a more important role in colorectal cancer than in type II diabetes mellitus. The previous studies in our lab also found that the expression level of IGFBP-rP1 in colorectal cancer tissue was significantly upregulated. Thus, we hope to explore the exact role of IGFBP-rP1 in colorectal cancer.We selected two colorectal cancer cell lines without endogenous IGFBP-rP1 expression and with different inherited characteristics, RKO and CW2, as our cell models. We constructed pcDNA3.1 (IGFBP-rP1) expression vectors and performed the transfection experiment. Expression of IGFBP-rP1 was detected on the level of mRNA and protein in the transfectants by reverse transcription polymerase chain reaction (RT-PCR) and Western blot. Cell function assays was conducted subsequently.We performed cell migration assay and matrigel invasion assay to observe the movement of colorectal cancer cells between pre- and post- IGFBP-rP1 transfection. The width of scratched lines was measured as a marker of migration assay. No significant difference on the change of width between IGFBP-rP1 transfectans and controls cells was detected. And in matrigel invasion assay there was no significant difference either between IGFBP-rP1-transduced and control vector transduced cells. Neither the IGFBP-rP1-transfectants nor the pcDNA 3.1-transduced cells migrated through the matrigel.Growth proliferation assay using MTT showed that IGFBP-rP1 could suppress the growth of colorectal cancer cells. On day 5, IGFBP-rP1 transduced CW2 cells showed a 28.6% reduction rate in cell number compared with pcDNA 3.1-transduced CW2 cells (p = 0.008). A higher pronounced reduction rate (63.5%) in cell number existed in IGFBP-rP1-transduced RKO cells versus pcDNA 3.1-transduced RKO cells on day 5 (p = 0.001). At the same time by flow cytometry after propidium iodine staining, we observed more IGFBP-rP1-transduced cells in the G1 phase than control cells (76.1% in RKO transfectants versus 61% in control cells, 14.1% in CW2 transfectants versus 0.83% in control cells, p < 0.005). Furthermore, IGFBP-rP1-transfectants were less in the S phase than those in control cells.Senescence-associatedβ-galactosidase (SA-β-gal) staining showed that IGFBP-rP1 transfectants exhibited at least 2-fold higher SA-β-galactosidase activity than control vector transduced cells. Cells with SA-β-galactosidase staining increased from 0 to 8.7% and 26.7% to 53.3% in RKO and CW2 cells (p < 0.05). Expression of pRB protein was significantly downregulated in IGFBP-rP1 transfectants compared with control cells by Western blot (p < 0.005). Both p21 and p16 expression were about 1.5-folder higher in IGFBP-rP1 transfectants than that in control cells (p21 p < 0.01, p16 p< 0.05). p53 expression increased 7.4% in IGFBP-rP1-transduced RKO cells versus vector transduced RKO cells and 2-folder in IGFBP-rP1-transduced CW2 cells versus control cells (p < 0.05). These results suggested that IGFBP-rP1 should be an important molecule that triggers senescence.IGFBP-rP1 could inhibit growth of colorectal cancer cells through senescence-like pathway. However, we found the expression level of IGFBP-rP1 in colorectal cancer tissue was significantly upregulated and serum IGFBP-rP1 was positively correlated with colorectal cancer. This paradoxical phenomenon made us puzzled. We believe that factors in the circumstance of cancer make the expression of IGFBP-rP1. Much more work should be carried out to clarify the mechanism. From the above researches of both IGFBP-rP1's biological function and the relationship among IGFBP-rP1, colorectal cancer and type II diabetes mellitus, we conclude that:1,IGFBP-rP1, which might be an indicator of colorectal cancer and type II diabetes mellitus, may be an important factor to bridge colorectal cancer and type II diabetes mellitus.2,IGFBP-rP1 could inhibit cell growth and induce cellular senescence in CRC cells through two distinct pathways, p53 and pRB pathway.