The Effect Of IGF-1 And IGF-2 And Their Receptors On Growth Of Endometrial Carcinoma Cells

Endometrial carcinoma(EMC)is one of the three major malignant tumors in the female reproductive system,accounting for 7% of the female malignant tumors and,and 20-30% of a malignant tumor in female genital tract.The average age of EMC is 60 years old.The incidence of EMC in developed countries is on the rise,and about more than 40 thousand new cases of EMC in the United States.The incidence of EMC in China is also increasing year by year,which seriously threatens the health of women.The excessive irritation of estrogen to the endometrium lack of progestin antagonism(“Unopposed estrogen”theory)is now one of etiology and pathogenesis of EMC.While the specific pathogenesis of EMC is still not very clear to date.At present,the research on the molecular mechanism of EMC is being deepened,and its development mechanism and targeted therapy are widely concerned.It is observed in the clinic that patients with endometrial cancer are associated with obesity,diabetes,and hypertension,suggesting that we have a close relationship with glycolipid metabolism.Diabetes is a common chronic disease worldwide.Epidemiological studies have shown that diabetics have insulin resistance and a significant increase in the risk of cancer and cancer death.Epidemiologic study suggests that people with diabetes are at significantly higher risk for many forms of cancer and greater cancer mortality,predominantly insulin-resistant type 2diabetes mellitus(T2DM).The incidence and mortality of cancer and diabetes are increasing with age,although the two diseases have trends of attacking young adults.Meta-analyses have revealed T2 DM to be an independent risk factor for the development of several different types of cancer,such as EMC,breast cancer,colorectal cancer,etc.The presence of IR in T2 DM is closely related to endometrial cancer.Hyperinsulinemia caused by IR is a high riskfactor for endometrial cancer that is independent of estrogen.More and more epidemiological evidence suggests that the disease associated with IR,such as obesity,T2 DM,polycystic ovary syndrome,metabolic syndrome,etc,is also a high risk factor for EMC.IR and EMC are regulated by common factors at the molecular level,such as inflammatory mediators,fat factors,Kaohsiung hormones,and so on.Although the mechanisms that underlie the associations between T2 DM and EMC remain far from understood,the insulin-like growth factors(IGFs)have been proposed to be important factors.IGFs includes insulin-like growth factor 1(IGF-1)and insulin-like growth factor 2(IGF-2).IIGF-1 is a potent growth factor with a role in cancer pathogenesis,which has been linked in epidemiological studies to cancer.The IGF-1R signaling pathway initiates with binding of IGF1 to its cell-surface receptor IGF-1R to activate phosphatidylinositol-3 kinase(PI3K)/ Akt signaling pathway,to stimulate cell growth and proliferation,and to inhibit programmed cell death.In obese individuals,total IGF-1 levels has been shown to be normal or even low due to decreased concentrations of IGF binding proteins,though the free/active IGF-1 levels are generally higher than in the non-obese.IGF-1 signals some of the same pathways as insulin,including PI3 K,ERK,AKT,and mTOR,which as described above could increase cancer cell proliferation and impair apoptosis.IGF-1 can also increase normal cell cycling,leading to increased risk of mutation and malignant transformation.Most circulating IGF-1 is produced by the liver,though paracrine secretion of IGF-1 occurs at the growth plate,and perhaps other tissues.As with insulin,inhibition of IGF-1 signaling has anticancer effects.Knockdown of the IGF-1 receptor enhances chemotherapy sensitivity in some cancers.IGF-1 receptor antibody has been explored as a treatment,and recently shown to have in vitro efficacy against some cancers.Nevertheless,although previous studies have suggested that serum IGF-1 in insulin-resistant T2 D patients is likely associates with higher risk for developing endometrial carcinoma(EMC),the underlying mechanisms are not understood.This topicexplores the role of IGF-1,IGF-2 and their receptors in the pathogenesis of EMC,and provides a basis for the targeted therapy of EMC.Part 1 The expression of IGF-1 and IGF-2 related molecules in the serum and endometrial cells of T2 DM patients with EMCObjectives:To investigate the expression of IGF-1,IGF-2,and IGF1BP3 in the serum of T2 DM patients with and without EMC,as well as to detect the levels of IGF-1R,IGF-2R,and PI3 k phosphorylation in endometrial cells.To clarify the role of IGF-1 and IGF-2 in the development of endometrial carcinoma.Methods:A total of 162 T2 DM subjects(22 with diagnosis of EMC and140 without EMC as controls)were included in this study.First,we examined the serum levels of IGF-1,IGF-2,and IGF-1 binding protein 3(IGF1BP3)by ELISA between the T2 DM patients with and without EMC.The levels of IGF-1R,IGF-2R,and PI3 k phosphorylation in endometrial cells were detected by Western blot.Results:1 No difference were detected in serum IGF-1 and serum IGF-2 between two groups by ELISA(P>0.05).2 No difference were detected in serum serum IGF1BP3 between two groups by ELISA(P>0.05).3 It was found that the levels of IGF-1R phosphorylation in endometrial(EM)cells were no difference between T2 DM patients with or without EMC(P>0.05).However,the levels of phosphorylated IGF-2R as well phosphorylation of PI3 k,an IGF-1R downstream factor,were significantly higher in endometrial cells in T2 DM patients with EMC(P<0.05).Conclusions:1 The levels of phosphorylated IGF-2R and PI3 k were significantly higher in endometrial cells in T2 DM patients with EMC,these results suggest that the presence of a complex regulation of PI3 k signaling by IGF-1R and IGF-2R in EM cells in T2DM2 The activation of pIGF-2R signals existed in the endometrial cells ofT2 DM patients with EMC.It is speculated that IGF-2R plays an important role in the development of EMC in T2 DM patients.Part 2 The effect of IGF-1 and IGF-2 on the growth of endometrial carcinoma cells and the expression of PI3 K signaling pathwayObjectives: To investigate the effect of exogenous IGF-1 or IGF-2 on the expression of phosphorylated IGF-1R,IGF-2R and the effect on growth of HEC-1A cells,as to explore the potential mechanism of IGF-1,IGF-2 and their receptors in the pathogenesis of EMC.Methods: The human endometrial carcinoma cell line HEC-1A was cultured.The activation of IGF-1R,IGF-2R and PI3 k in cultured HEC-1A cells treated with IGF-1 or IGF-2 was detected after 1 and 2 days by Western blot.The cell growth was measured by MTT assay after adding different growth factors.Results:1 IGF-1 or IGF-2 was given to the cultured HEC-1A cells and the activation of IGF-1R,IGF-2R and PI3 k was examined.We found that IGF-1R was significantly and seemingly activated by either IGF-1 or IGF-2(P<0.05).However,IGF-2R was only significantly activated by IGF-2,but not by IGF-1(P<0.05).Interestingly,PI3 k and Cyclin D1(CCND1)were only activated by IGF-1,but not by IGF-2(P<0.05).2 Next,we examined the effects of IGF-1 or IGF-2 on EMC cell growth in an MTT assay.We found that IGF-1,but not IGF-2,increased EMC cell growth(P<0.05).Conclusions:1 IGF-1 stimulation increased the expression of pIGF-1R,p-PI3 K and CCND1 protein,but had no effect on the expression of pIGF-2R protein.IGF-2 stimulation up-regulated the expression of pIGF-1R and pIGF-2R protein,but had no effect on the expression of p-PI3 K and CCND1 protein.2 IGF-1 activated the downstream PI3 K signaling pathway by combining with IGF-1R to increase the expression of CCND1 and promote the growth of endometrial cancer cells.3 IGF-2,as a bait ligand for IGF-1R,made IGF-1R selectively phosphorylation,but it did not activate PI3 K and CCND1 downstream of IGF-1R,and did not promote the growth of endometrial cancer cells.4 IGF-1 promoted the growth of endometrial cancer cells,while IGF-2has no effect on the growth of endometrial cancer cells.Part 3 Effect of overexpression of IGF-2R on the growth of endometrial carcinoma cells and the PI3 K pathway in IGF-1 regulated endometriumObjectives:Objective to study the role of IGF-1 and IGF-2 in HEC-1A overexpressing IGF-2R cells,the expression of various factors and the effect on HEC-1A cell growth,and further explore the mechanism of IGF-2R in the pathogenesis of EMC.Methods:The IGF-2R construct were designed and synthesized and then transfected into HEC-1A cells.IGF-2R expression was detected by RT-PCR and Western blot.The growth of IGF-2R overexpressed HEC-1A cells treated with IGF-1 and IGF-2 was measured by MTT assay.In addition,the effect of IGF-2R on the expression of IGF-1-related genes and activation of some molecular in signaling pathway were also observed.Results:1 The results of fluorescence quantitative PCR showed that the expression of mRNA in IGF-2R was significantly increased after IGF-2R construct transfected to HEC-1A cells 48 h,while the expression of IGF-2R mRNA was not significantly changed after the cells were transfected into the missense sequence.At the same time,the protein expression of IGF-2R detected by Western blot was also significantly increased,which was consistent with the results of fluorescence quantitative RT-PCR,which confirmed the overexpression of IGF-2R.While the expression of IGF-2R protein was not significantly changed after the cells were transfected into the missense sequence.2 The activation of IGF-1R by IGF-1 and IGF-2 was not significantly different between control SCR and IGF-2R-overexpressing HEC-1A cells.Overexpression of IGF-2R further increased the activation of IGF-2R by IGF-1 and IGF-2.Moreover,the activation of PI3 k and CCND1 were both further increased in IGF-2R-overexpressing HEC-1A cells upon IGF-1 and IGF-2 stimulation.These data suggest that high IGF-2R may increase the sensitivity of IGF-1R to IGF-1 to increase cell growth.3 MTT assay showed that overexpression of IGF-2R further increased EMC cell growth upon IGF-1 and IGF-2 stimulation.These data support our hypothesis,and demonstrate that high IGF-2R in T2 DM may increase the sensitivity of IGF-1R to IGF-1 to increase EM cell growth.Conclusions:1 After IGF-1 and IGF-2 stimulation,no matter whether there was IGF-2R overexpression,the expression of p IGF-1R,pIGF-2R,pPI3 K and CCND1 protein in HEC-1A cells increased,and the growth of HEC-1A cells increased significantly.2 Overexpression of IGF-2R up-regulated the expression of p IGF-2R,pPI3 K and CCND1 protein,but had no effect on the expression of pIGF-1R protein.And the overexpression of IGF-2R significantly increased the growth rate of HEC-1A cells.3 IGF-2 and IGF-1 competitively combined to IGF-1R.High level IGF-2R reduced free IGF-2,enhanced IGF-1R sensitivity to IGF-1,thereby promoted downstream signaling pathway activation and endometrial cancer cell growth.4 IGF-2R may be involved in the PI3 K signaling pathway regulated by IGF-1 and receptor IGF-1R and the growth of CCND1 and endometrial cancer cells by enhancing the sensitivity of IGF-1R to IGF-1.