| Oral squamous cell carcinoma (OSCC) is one of the most common malignancies of the head and neck. Epidemiological studies revealed that majority cases of OSCC are seen in patients over the age of 40 years, whereas the age distribution of OSCC tends to be younger, and has become one of the major threats to human health. For patients with early stage of OSCC, the primary therapeutic method is surgical treatment. For patients with moderate and advanced stage of OSCC, the primary therapeutic method is operation based comprehension treatment. Despite advances in understanding of the disease and improved therapeutic interventions, it remains has a poor prognosis. And the mechanisms involved in tumor initiation, development, angiogenesis of tumor microenvironment, cancer cell metastasis are still unclear.Arachidonic acid is an eicosanoic polyunsaturated fatty acid, belongs to ω-6 long-chain polyunsaturated fatty acids. It contains four carbon-carbon double bonds, and one carbon-oxygen bond, belongs to high unsaturated fatty acids. Researches showed that arachidonic acid is not only molecule that is rich in energy; it also can induce the expression of some genes. Arachidonic acid plays an important role in the development and progression of cancer. AA pathway is an important metabolic pathway in which phospholipase A2s (PLA2s), cyclooxygenases (COXs), lipoxygenases (LOXs) and cytochrome P450 (CYP) monooxygenases and respective metabolites lysophospholipids (LPLs), prostanoids, leukotriens (LTs), hydroxyl eicosanoitetraic acids and epoxy eicosanoitetraic acids are involved and plays a key role in normal and various pathophysiological functions. AA pathway metabolizing enzymes and their products orchestrate the inflammatory response and regulate multiple cellular processes, including cell proliferation, survival, angiogenesis, invasion and metastasis, which can promote carcinogenesis. Recent years, the reasearches about the tumor promotion of arachidonic acid mainly concentrate on the cyclooxygenase signal pathway.Cyclooxygenase (COX) is a key rate-limiting enzyme which can catalyze arachidonic acid (AA) into prostaglandins (PGs). As far as found there are two isoforms of cyclooxygenase:COX-1 and COX-2. The COX-1 isoform constitutively expressed in most of healthy tissue cells, which plays crucial roles in diverse physiological functions, such as platelet aggregation, inhibition of gastrointestinal acid secretion, regulation of glomerular function, and labor. COX-2, the inducible form of cyclooxygenase, cannot be detected in most of healthy tissue cell. However, the expression of COX-2 can be induced by a variety of inflammatory factor, cytokine, mitogen, tumor promoter. It had been repeatedly reported that the expression of COX-2 was significantly elevated in many tumors, and play an important role in the development of tumorigenesis, such as colorectal cancer, breast carcinoma, lung cancer, prostatic cancer, head and neck cancer and oral carcinoma. Epidemiological studies have revealed that in patients with arthritis who take aspirin as a long-term use has a lower incidence of colorectal cancer. Clinical observation indicated that tumor regression has been induced by indomethacin in patients with breast cancer and squamous cell cancer of the skin. The mechanism by which NSAIDs inhibit tumor cell growth is not clearly understood, whereas it can block the activity of COX, which can inhibit the production of prostaglandins, and may affect cell proliferation, apoptosis and immune response. It seems that NSAIDs are promising drugs which can inhibit the development of tumor. However, clinical observation revealed that long term use of NSAIDs can exert side effects of cardiovascular system and gastrointestinal tract system. These side effects hinder the widespread use of NSAIDs clinically. Now researches focus on the downstream signal pathway of COX-2, looking forward to the more safe and effective molecular target which can bypass the side effect exerted by inhibiting COX-2.Among the downstream productions of COX-2, the most relevant to the development of cancer is PGE2. The expression of PGE2 was significantly increased in many kinds of tumors, and played a very important role in the development and progression of tumor, such as colorectal cancer, prostate cancer, skin cancer and oral cancer. PGE2 can promote the development and progression of tumor from the following aspects:(1) it can promote the cascade reaction induced by some growth factor receptors. (2) It can inhibit cancer cell apoptosis by elevating the expression of Bcl-2. (3) It can promote the expression of angiogenesis factor, such as VEGF. (4) It can inhibit the production of immune regulating lymphatic factor; inhibit the proliferation of B lymphocyte and T lymphocyte; inhibit the production of tumor necrosis factor (TNF); inhibit the toxic effect of natural killer cell (NKC). So it can suppress the local immunity, which can facilitate cancer cell to escape the attack of immune system. (5) It can enhance the invasion ability of cancer cell, which can promote the invasion and metastasis of cancer cell. PGE2 exerts its physical and pathology functions mainly through four EP receptors, namely EP1, EP2, EP3, and EP4. Understanding the role and mechanisms of action of the EP receptors potentially offers new targets for the prevention and therapy of OSCC.EP receptors belong to the G protein-coupled receptor superfamily. The EP receptors have seven transmembrane segments and each receptor is coupled to different Ga subunits of heterotrimeric G proteins. EP2 and EP4 are coupled to Gas, commonly referred to as Gs. EP1 is linked to Gq, while EP3 is linked to Gi. Different EP receptor is linked to different Ga subunits and activated different downstream signal pathway, so the biological functions activated by EP receptors are different. The signal pathways activated by EP receptors are context specific and likely depend on the level of PGE2 synthesis, the different expression levels of the different EP receptors, as well as the levels of expression of other interacting receptors. Different EP receptor can play different biology action, and also the same EP receptor may play different function in different types of cancer. EP1 activation stimulates the release of intracellular calcium via a mechanism involving G proteins. EP2 and EP4 activate adenylate cyclase via stimulatory G protein. EP3 induces Ca2+ mobilization or inhibits adenylate cyclase via inhibitory G-binding proteins. Current studies are focused on identifying which of the G protein-coupled EP receptors mediate the tumor promotion/progression activities of PGE2 and the signaling pathways involved. It had been repeatedly reported that EP receptors play an important role in tumorigenesis and progression. For example, PGE2 upregulates β1-integrin expression and cell migration in HCC cells by activating the EP1/PKC/NF-κB/FoXC2 signaling pathway. In breast cancer, the activation of EP2 and EP4 receptor can suppress the function of NK cell, and promote the invasion and metastasis of breast cancer cell. The identification of EP receptors makes it possible that inhibit the development of tumor through regulating the activity of EP receptors. This also can bypass the side effects of gastrointestinal tract and cardiovascular exerted by long-term use of NSAIDs. Nonetheless, the expression profile of EP receptors and the roles mediated by EP receptors in OSCC are still unclear. Therefore, the present study was designed to elucidate the expression pattern of EP receptors in OSCC, and lay the foundation of the role of EP receptors in the development of OSCC.Chapter I the expression of EPs mRNA in oral squamous cell cancer detected by Real-time PCRObjective:To examine the expression of EP1, EP2, EP3, and EP4 mRNA in oral squamous cell cancer tissues. Methods:This study included analysis of 40 oral squamous cell carcinoma specimens excised from patients undergoing operative treatment in the Department of Oral and Maxillofacial Surgery, Nanfang Hospital, from May 2013 to May 2015. None of the patients received radiotherapy, chemotherapy, or any treatment before operation in the present study. They had no family history of cancer or other complications. All samples were examined by hematoxylin-eosin (HE) staining, and diagnosis was confirmed on its basis by three independent pathologists. A total of 80 specimens, including 40 cases of OSCC tissues and 40 cases of paired para-carcinoma tissues which are confirmed carcinoma cell negative by intraoperative frozen section, were obtained at the time of surgery. The samples were deposited in RNA-later liquor and flash frozen in liquid nitrogen. The experiment was dispart into two groups. (1) Experimental group:40 oral squamous cancer tissues. (2) Control group:40 paired paracarcinoma tissues. The expression of EPs mRNA was examined by real-time PCR. Results were normalized against ACTB in each sample and calculated relative to the control group. Fold change in the mRNA expression level was calculated as 2-ΔΔCt. Experiments were performed in triplicate. Results:Real-time quantitative PCR showed that the mRNA expression of EP1 and EP2 was significantly up-regulated in OSCC tissues compared to para-carcinoma tissues. The average mRNA expression of EP1 and EP2 in OSCC tissues was 1.442 ± 0.4245,1.207 ± 0.4259 respectively. By pared t test, both P value< 0.05. The difference was statistically significant. Real-time quantitative PCR showed that the mRNA expression of EP3 receptor was significantly down-regulated in OSCC tissues compared to para-carcinoma tissues. The average mRNA expression of EP3 receptor in OSCC tissues was 0.4455 ± 0.3292. By pared t test, P value<0.05. The difference was statistically significant. Real-time quantitative PCR showed that the mRNA expression of EP4 receptor was up-regulated in OSCC tissues compared to para-carcinoma tissues, however, it failed to reach statistically difference. The average mRNA expression of EP4 receptor in OSCC tissues was 1.147±0.4965. By pared t test, P value> 0.05. The difference was not statistically significant. Conclusion:compared to paracarcinoma tissue, the expressions of EP1, EP2 mRNA were up-regulated in OSCC and the expression of EP3 mRNA was down-regulated in OSCC. The disordered expression of EP mRNA may play an important role in the development of oral squamous cell cancer.Chapter II the expression of EPs protein in oral squamous cell cancer detected by immumohistochemical stainingObjective:To examine the expression of EP1, EP2, EP3, and EP4 protein in oral squamous cell cancer tissue. Methods:This study included analysis of 40 oral squamous cell carcinoma specimens excised from patients undergoing operative treatment in the Department of Oral and Maxillofacial Surgery, Nanfang Hospital, from May 2013 to May 2015. None of the patients received radiotherapy, chemotherapy, or any treatment before operation in the present study. They had no family history of cancer or other complications. All samples were examined by hematoxylin-eosin (HE) staining, and diagnosis was confirmed on its basis by three independent pathologists. A total of 80 specimens, including 40 cases of OSCC tissues and 40 cases of paired para-carcinoma tissues which are confirmed carcinoma cell negative by intraoperative frozen section, were obtained at the time of surgery. The samples were deposited in formaldehyde solution immediately after dissection and stored at room temperature. The experiment was dispart into two groups. (1) Experimental group:40 oral squamous cancer tissues. (2) Control group:40 paired paracarcinoma tissues. The expression of EPs protein was examined by IHC. Negative controls were obtained by using PBS instead of the primary antibody, and sections of colorectal cancer were used as positive controls. The IHC stains were semi quantified by staining intensity and the percentage of positive cells that stained yellow or brown. Results:IHC showed that EP1 receptor protein presents moderately positive expression in most of OSCC tissues. However, in most of para-carcinoma tissues, EP1 receptor protein presents weakly positive expression. By Wilcoxon rank sum test, T=55, P<0.05, the difference was statistically difference. The expression of EP1 protein in OSCC was significantly up-regulated.IHC showed that EP2 receptor protein presents moderately positive expression in most of OSCC tissues. However, in most of para-carcinoma tissues, EP2 receptor protein presents weakly positive expression. By Wilcoxon rank sum test,T=91, P<0.05, the difference was statistically difference. The expression of EP2 protein in OSCC was significantly up-regulated. IHC showed that EP3 receptor protein presents weakly positive expression in most of OSCC tissues. However, in most of para-carcinoma tissues, EP3 receptor protein presents moderately positive expression. By Wilcoxon rank sum test,T=-114, P<0.05, the difference was statistically difference. The expression of EP3 protein in OSCC was significantly down-regulated. IHC showed that EP4 receptor protein presents moderately positive expression in most of OSCC tissues and para-carcinoma tissues. By Wilcoxon rank sum test,T=14, P>0.05, the difference was not statistically difference. Conclusion:compared to paracarcinoma tissue, the expressions of EP1, EP2 protein were up-regulated in OSCC and the expression of EP3 protein was down-regulated in OSCC. The disordered expression of EP protein may play an important role in the development of oral squamous cell cancer.Chapter III the expression of arachidonic acid in oral squamous cell cancer detected by ELISAObjective:To examine the expression of arachidonic acid in oral squamous cell cancer tissue. Methods:This study included analysis of 40 oral squamous cell carcinoma specimens excised from patients undergoing operative treatment in the Department of Oral and Maxillofacial Surgery, Nanfang Hospital, from May 2013 to May 2015. None of the patients received radiotherapy, chemotherapy, or any treatment before operation in the present study. They had no family history of cancer or other complications. All samples were examined by hematoxylin-eosin (HE) staining, and diagnosis was confirmed on its basis by three independent pathologists. 40 cases of OSCC tissues and 40 cases of paired para-carcinoma tissues which are confirmed carcinoma cell negative by intraoperative frozen section were obtained at the time of surgery. The samples were stored in -80℃ immediately after dissection. The experiment was dispart into two groups. (1) Experimental group:40 oral squamous cancer tissues. (2) Control group:40 paired paracarcinoma tissues. The expression of arachidonic acid was examined by ELISA. Results:Compared to paracarcinoma tissue, the expression of arachidonic acid in oral squamous cell cancer tissue was significantly elevated. The average mass fraction of arachidonic acid in 40 carcinoma tissues was 477.4±39.84 pg/g, The average mass fraction of arachidonic acid in 40 para-carcinoma tissues was 38.4±4.92 pg/g, by paired t test, P<0.05. The difference is statistically significant. Conclusion:In oral squamous cell carcinoma tissues, the up-regulated expression of arachidonic acid may play an important role in the development of oral squamous cell carcinoma.In summary, compared to paracarcinoma tissue, the expressions of EP1, EP2 were significantly up-regulated in OSCC and the expression of EP3 was significantly down-regulated in OSCC. The expression of AA in OSCC was significantly up-regulated. The disordered expression of EPs and arachidonic acid may play an important role in the development of OSCC. |
PDF Full Text Request