Expressions Of COX-2,iNOS And HIF-1α And Their Correlations With The Clinicopathologic Characteristics In Osteosarcoma

Abnomal expression of oncoma related gene often is associated with the formation and growth of tumors, and manifests multifold metabolic abnormality correlated with the malignant behavior.The abnomal expression of cyclooxygenase-2(COX-2) genes in osteosarcoma which both predilected to teenagers and possess higher malignant potential not only causes variation of osteogenesis but also closely correlated with the angiogenesis and the aggressiveness of the tumor.Moreover, regional hypoxia becomes to a significant hall mark in the tumor microenviroenment, which is caused by the active proliferation of tumor accompanied with quickly consume to oxygen and chaotic proliferation of vessels. Hypoxia in tumor tissue may occur in 100-200μm away from a functional blood supply. In physiological condition, at the organs level hypoxia will be improved by reflex hyperventilation and increasing oxygen delivery from the atmosphere to the tissue, at the cellular level the adaptation involves increasing inversion from oxidative phosphorylation into anaerobic glycolysis and the glucose uptake and the expression of stress proteins. In pathological condition tumor cells adapting and improving hypoxia microenviroenment which facilitating tumor's growth relies on its self-regulations correlated with tumorous related genes. It has been confirmed that tumor cells improve hypoxia and maintain the stability of oxygen and metabolism to promote the infiltration and metastasis of tumor by inducing expression of hypoxia -Inducible factor-1 alpha(HIF1α) which is the centre transcription regulator in hypoxia reaction, cyclooxygenase-2 may be induced to express in tumorous hypoxia condition as well as iNOS (Inducible nitric oxidesynthase) which is a key induced enzyme of the important signaling transmit molecule—NO(nitric oxide).It is suggested that expression of COX-2 be closely correlated with the expression of iNOS and HIFla in multiple epithelial tumour such as endometrial cancer, lung cancer, gastric cancer, colon carcinoma, pancreatic carcinoma, breast cancer, prostatic carcinoma, cervix cancer, bladder cancer and so on, which has an distinguished influence in various degree upon tumorous vasoformation, promotion of the growth, infiltrating and metastasis of the tumor in many ways. Nevertheless, in mesenchymal tumors, especially in osteosarcoma, it is unknown whether do HIFla and iNOS the overexpress and Whether the overexpression of COX-2 is correlated with that of HIFla or iNOS. This topic is going to investigate the relationships among the expressions of COX-2, HIFla and iNOS in osteosarcoma, their associations with clinicopathologcal characteristics.Purpose:This study aims at investigation into the expressions of COX-2, HIFla and iNOS at protein level in 59 cases osteosarcoma by immunohistochemistry staining and their correlations with clinicopathologcal features, and try to reveal the potential mechanisms of the formation and progression in this tumor, and to provide theoretical foundations for the possibility of using the inhibitors in clinical therapeutics.Material and methods1 Materials: 59 cases typical HE sections of osteosarcoma without os osseum tissue and non decalcification were obtained from zhejiang university medical college affiliated second hospital department of pathology from the year of 2002 to 2005. the corresponding paraffin imbedding tissue blocks were extracted, the sections were consecutively prefomed at 4um in thickness. 59 cases of SaOS were grouped according to the clinicopathologic characteristics, the Enneking GTM clinical stage, the histological types and histological grading, followed-up data of postoperative patients were collected.2 Methods: the expressions of COX-2, iNOS and HIFla in osteosarcoma were examined by immunohistochemical staining (EnVision? two steps method), 11 cases of osteofibrous dysplasia were used as the negative control. The results were evaluated by the semi-quantitative standard ofIHC according to tumor cell positive ratio and coloring strength .Correlations of the expressions with osteosarcomatous clinicopathologic characteristics and between the tumor markers were evaluated by x2 analysis and spearman rank correlation analysis.the Survival data of postoperative were performed by Kaplan-meier Survival curve analysis Method. Significance of difference between the survival rates in various groups were tested by Log-Rank method. All experimental data were manipulated by the SPSS statistics and analyse software three times at least.Results:1 The positive expression rates of COX-2, iNOS and HIFla in osteosarcoma were 69.5%(41/59), 86.4(51/59) and 38.98%(23/59)respectivety, which were significantly higher than that in control group (x2=15.07, PO.01), (x2=25.13, PO.01), (%2=4.74, 0.050.05). the over expression of HIFla in osteosarcoma were related with the age, histological classification (x2 =8.61 % 12.24, P<0.01) and not with its clinical stage and metastasis, (x2 = 0.54, 0.09, P>0.05). the over expression of iNOS in osteosarcoma were not related with the age, histological classification, clinical stage and metastasis (x2 = 0.95% 0.34% 0.03% 0.42, P>0.05).3 The overexpression of COX-2 % HIFla and iNOS in osteosarcoma were closely related with the histological classifications, in COX-2 positive group, total positive rate 16.66% (1/6 )of intraosseous well-differentiated and parostea osteosarcoma was significantly lower than that of the other groups (71.7%, 33/46) which showed significant difference(x2=7.113, P=0.008);In HIFla positive group , total positive rate of conventional, telangiectatic, periosteal and high-grade surface osteosacoma was 41.3%(19/46) which was significantly higher than that of the other groups 0 with significant difference (x2=3.91, P=0.048);Likewise, in iNOS positive group, total positive rate of intraosseous well-differentiated and parostea osteosarcoma was 33.33%(2/6) which is significantly lower than that of the other groups (91.3%, 42/46) with significant difference (x2=13.70, P=0.000);the over-expression of iNOS in osteosarcoma were related with the histological classification, with significantly difference between the classifications.4 The coexpression of iNOS and COX-2 was 67.8% (40/59);positively expressed iNOS without COX-2 expression was 18.6% (11/59);positively expressed COX-2 and without iNOS expression was 11.86% (7/59);No expression of both iNOS and COX-2 was 1.69% (1/59). The positive expression rate of iNOS in the group with positive expression of COX-2 was 97.56% (40/41), the positive expression rate of iNOS in the group with no expression of COX-2 was 61.11% (11/18), which shows a compacted positive correlation between them (x2=6.75, r=0.636;P<0.01).The expression of both HIF-la and COX-2 was 38.98% (23/59);positively expressed COX-2 without HIF-la expression was 30.50%(18/59);positively expressed HIF-la with negatively expressed COX-2 was 0;No expression of both HIF-la and COX-2 was 30.50% (18/59). the positive expression rate of HIF-la in the group of positive expression of COX-2 was 56.10% (23/41), the positive expression rate of HIF-la in the group of negative expression of COX-2 was 0, which shows a compacted positive correlation between them (x2= 16.06, r=0.610;P<0.01).The expression of both HIF-la and iNOS was 38.98% (23/59);positively expressed iNOS without HIF-laexpression was 47.46% (28/59);the rate of positively expressed HIF-la and negatively expressed iNOS was 0;No expression of both HIF-la and iNOS was 13.56% (8/59). the positive expression rate of HIF-la in the group of positive expression of iNOS was 45.10% (23/51), the positive expression rate of HIF-la in the group of negative expression of iNOS was 0, which shows a compacted positive correlation between them (x2=26.04, r=0.460;P<0.01).5 Cumulative Survival curve of postoperative patients was drawn by Kaplan-meier Method. Significance of difference of interclass survival rate were tested by Log-Rank Method. Expressions of COX-2 and HIFla protein in osteosarcoma demonstrate negative correlation with the postoperative survival time, expression of iNOS protein in osteosarcoma demonstrate noncorrelation with the postoperative survival time, The patients with positive expression of COX-2 had a lower postoperative accumulative survival rate compared with those without expression, which showed significant difference (x2 =3.825, P=0.05). The patients with positive expression of HIFla had a significantly lower postoperative accumulative survival rate compared with those without expression which showed significant difference (x2= 10.53, P<0.01). The patients with positive expression of iNOS had a slightly lower postoperative accumulative survival rate compared with those withoutexpression which showed nonsignificant difference (x2= 1.325, P>0.05). conclusion:The expressions of COX-2, iNOS and HIFla in osteosarcoma were strongly positive in 41 of 59 (69.5%), 51 of 59 ( 86.4) and 23of 59 (38.99%) cases, respectively. The combined expression of COX-2> iNOS and HIFla may play a cooperative role in influenceing biological behavior of osteosarcoma, they may closely correlated with the formation and development of osteosarcoma. The patients with positive expression of COX-2 and HIFla had a lower postoperative accumulative survival rate, the expression of COX-2 and HIFla may be useful prognostic marker of steosarcoma. The inhibitors of COX-2, iNOS may be a potential suppressant of the tumor, the presumption provides a theoretical foundation for the further empirical study.