AGE-RAGE Promotes Prostate Cancer Cells Proliferation Through Regulation Of Retinoblastoma Protein Via PI3K/Akt Signaling Pathway

BackgroundProstate cancer is one of the most common malignant tumors of the male genitourinary system. With the increasement of human average longevity and the improvement of diagnostic techniques, the incidence of prostate cancer is on the rise. According to the global cancer incidence and death statistics in 2008, prostate cancer accounts for the second most common cancer and ranks sixth in causes of death in males. In United States, prostate cancer is the most common malignant tumor and the second-deadliest cancer after lung cancer in males. In China, the westernization of life style, the aging population and the wildly used screening techniques contributed to the rising prostate cancer incidence, and prostate cancer has become the third most common malignant tumors of the male genitourinary system, although its incidence and mobidity rate in still far lower than that of western countries.People have attached great importance to the basic and clinical researches on prostate cancer. Nowadays, researches on the mechanism of prostate cancer development and progression almost focus on the following fields:the alteration of androgen receptor, the mutations of the oncogenes and tumor suppressor genes and the abnormal activation of relevant cell signaling pathways.Recent studies show that the interaction between receptor for advanced glycation end products(RAGE) and its ligand advanced glycation end products(AGE) is closely associated with the development and progression of prostate cancer.AGE is the product of the Maillard reaction between sugar and protein. The concentration of AGE in human body will increase significantly when under certain pathological state such as diabete, dialysis-associated kidney diseases and alzheimer’s disease, due to the rising production of AGE or the decreasement of AGE elimination. Many studies have suggested that high concentration of AGE in human body is closely associated with the development and progression of diabete, vascular diseases, aging, kidney diseases and tumors.RAGE, a membrane protein, is a member of the immunoglobulin superfamily of cell-surface molecules. RAGE is recognized firstly as a receptor for AGE and interact with AGE to mediate biological effects. The extracellular domain of RAGE, whose structure looks like immunoglobulin for consisting of a V-like domain and two C-like domain, functions as a specific domain to bind RAGE ligands. There are two N-linked glycosylation sites lie in the V-like domain. These structures are important’ to the molecular stability and the function of identifying ligands. It has been confirmed that the V-like domain of RAGE interacts with AGEs on prostate carcinoma cells. Many studies have shown that the interaction between RAGE and its ligands activated many kinds of signal transduction pathways in mononuclear macrophage, vascular endothelial cells, renal mesangial cells, nerve cells, vascular smooth muscle cells and a variety of tumor cells, and produce pathogenic effects.AGE-RAGE interaction plays an important role in prostate cancer development and progression. Recent studies showed that AGE treatment promoted prostate cancer cells growth and proliferation; the V domain of RAGE interacted with AGE on prostate carcinoma cells; knockdown of RAGE expression induced prostate cancer cells apoptosis and inhibited prostate tumor growth. However, the related molecular mechanisms behind AGE-RAGE interaction induced promotion of prostate cancer cells proliferation is remain unclear, and it worth studying.What’s the mechanisms behind AGE induced promotion of prostate cancer cell proliferation? Which proteins and signaling pathways involves this effect? These questions aroused our attention. Based on our previous findings and the research advance, we predict that the interaction between AGE and RAGE may promote prostate cancer cells by regulating the retinoblastoma protein.RB is the first tumor suppressor gene identified by human, it encodes a nuclear protein, retinoblastoma protein, which is 110 kd with the ability to bind DNA. RB plays an important role in the regulation of cell cycle, in particular, in the regulation of the process in which cell cycle progress from G1 phase to S phase. RB acts as a negative regulator in the cell cycle. There are two forms of RB, dephosphorylated RB protein owns biological activity, while phosphorylated RB protein lose its activity. Normally, dephosphorylated RB binds the E2F family of transcription factor and inhibit the activity of E2F, maintaining the cell cycle in G1 phase and inhibiting cell proliferation. E2F will be released from the RB-E2F complex when RB is phosphorylated, and E2F subsequently promotes transcription, which impels the cell cycle from G1 phase to S phase, and thus promotes prostate cancer cells proliferation. The phosphorylation of RB was mainly induced by the CDK-cyclin complex which consist of cyclin-dependent kinase and cyclin. The CDK-cyclin complex, in particular, CDK4-cyclinD, causes the phosphorylation of RB and promotes proliferation. Phosphorylated RB degrades by the ubiquitin-proteasome system.Recent studies show that the mutation of RB gene and the inactivation of RB protein exert significant influence on the development and progression of prostate cancer. Researchers have found that the loss of RB allele lead to the functional defection of the RB protein in most of the prostate cancer cases. The inactivation of RB up-regulates the expression of E2F and thus promotes the development, progression and metastasis of prostate cancer. Taneja and co-workers found that androgen stimulation enhanced the phosphorylation of RB and accelerated the degradation of RB protein by the ubiquitin-proteasome system, and thus promoted the growth and proliferation of LnCap cells.AGE interacts with RAGE in the cell membrane, while RB protein locates in the nucleus. How does the interaction between AGE and RAGE regulate RB?Previous studies have shown that the interaction between RAGE and its ligands could activate PI3K/Akt signaling pathway; proteins involved in cell cycle regulation, such as RB, may be regulated by PI3K/Akt signaling pathway. PI3K/Akt signaling pathway plays an important role in the proliferation and apotosis of cancer cells, the abnormal activation of this pathway is closely associated with the development and progression of cancer. Therefore, we predict that AGE-RAGE interaction may regulate RB by activating PI3K/Akt signaling pathway.Phosphatidyl Inositol 3-kinase(PI3K) and its downstream molecule Akt(or PKB) comprise the PI3K/Akt signaling pathway which is closely associated with cancer development and progression and attracts tremendous attention. This pathway regulates cancer cells survival and proliferation, and also plays an important role in cancer invasion and metastasis. Akt, whose molecular mass is about 57 kd, is the serine/threonine protein kinase, and the core protein in PI3K/Akt signaling pathway. Normally, PI3K/Akt signaling pathway is activated by receptors in cell membrane. The activation of PI3K leads to the phosphorylation of phosphatidylinosital biphosphate(PIP2) and generates phoSphatidylinositol-3-phosphate(PIP3).which interacts with the PH domain of Akt and induce the transposition of Akt from cytoplasm to cell membrane. The phosphorylation of two sites is necessary for the fully activation of PI3K/Akt signaling pathway:the phosphorylation of Thr308 induced by PDK1 and the phosphorylation of Ser473 induced by PDK2. The activation of PI3K/Akt pathway leads to the phosphorylation of some substrates and thus regulates cell growth, proliferation and metabolism.The abnormal activation of PI3K/Akt signaling pathway plays an important role in prostate cancer cell survival and proliferation. The over-expression of Akt is very common in prostate cancer. In 50%-80% of the aggressive prostate cancer cases, the inactivation of PTEN leaded to the over-expression of PIP3 and induces the abnormal activation of Akt, and then promoted the survival of cancer cells. The abnormal activation of PI3K/Akt signaling pathway may enhance the invasion of prostate cancer cells and the progression of tumor. The blockade of this pathway can significantly inhibit the proliferation of prostate cancer cells and maintain the cell cycle of cancer cells in G1 phase.Cancer is a kind of cell cycle disease, and cancer cells is characterized by the ability to infinite proliferation. Our study explore the mechanisms behind the promotion of cell proliferation of prostate cancer induced by AGE. This study will reveal the role of AGE/RAGE axis in prostate cancer cell proliferation and the related mechanisms, and will deepen and expand our understanding of the biological function of AGE/RAGE axis. And will offer theoretical and experimental evidence for targeting AGE and/or RAGE to diagnose and treat prostate cancer.MethodsBased on the above research advance, in our study, we stumilated prostate cancer cells with AGE-BSA in dose-response and time-course maner, then, the effect of AGE stimulation on proliferation of the PC-3 cells was evaluated by CCK-8. After confirming the effect of AGE on prostate cancer cells proliferation, we tried to explored the related mechanisms. The total quantity of RAGE, RB and P-RB in prostate cancer cells were assessed by immunoblot analysis after AGE treatment. Meanwhile, The total quantity of P-Akt were also evaluated to confirm whether the PI3K/Akt signaling pathway was activated by AGE treatment. Then, expression of RAGE and RB were knockdowned by specific siRNA, in order to assesse the function of RAGE and RB in AGE induced cancer cells proliferation. Finally, to explored the role of PI3K/Akt pathway activation in cell proliferation promotion caused by AGE, Ly294002 was used to block the PI3K-dependent Akt phosphorylation and thus block the PI3K/Akt signaling pathway.ResultsDepending on all above studies and experiments, we have got the following results:1. AGE stimulation promoted prostate cancer proliferation.2. AGE induced prostate cancer cells proliferation via RAGE ligation.3.AGE-RAGE interaction enhanced prostate cancer cells proliferation through phosphorylation of retinoblastoma, increasing the total amount of phospho-RB and decreasing the total amount of RB.4. AGE-RAGE interaction activated PI3K/Akt signaling pathway.5.Based on the current research advance, our study revealed the possible mechanism behind AGE induced prostate cancer cell proliferation promotion:the interaction between AGE and RAGE activates PI3K/Akt signaling pathway, and then enhances the phosphorylation of RB and increases the total quantity of phospho-RB. Phospho-RB releases the transcription factor E2F, then, E2F promotes transcription and drives the cell cycle from G1 phase to S phase, and thus promotes prostate cancer cells proliferation.ConclusionsIn this study, we evaluated the effect of AGE on prostate cancer proliferation and explored the rated mechanisms. We find that, in prostate cancer cells, the interaction between AGE and RAGE activates PI3K/Akt signaling pathway, and then enhances the phosphorylation of RB and increases the total amount of phospho-RB. Phospho-RB releases the transcription factor E2F, then, E2F promotes transcription and drives the cell cycle from G1 phase to S phase, and thus promotes prostate cancer cells proliferation. These results reveal the role of AGE/RAGE axis in prostate cancer cell proliferation and the related mechanisms, and will deepen and expand our understanding of the biological function of AGE/RAGE axis. Our findings offer theoretical and experimental evidence for targeting AGE and/or RAGE to diagnose and treat prostate cancer, and provide new thinking and method for the diagnosis and treatment of prostate cancer.