Intervention Of QHBJ Prescription With The Biological Effects Of COX-2/5-LOX Metabolic Pathway Of Arachidonic Acid On Ovarian Carcinogenesis Induced By DMBA In Rats

Ovarian cancer is the leading course of death in women with gynecological malignancy. Majority of the patients extensive extraovarian spread at the time of diagnosis.The5-years survival rate of advanced disease is only about25%. Pelvic infection or pelvic inflammation is a high risk factor to ovarian cancer. The finding of epithelial ovarian cancer pathogenesis will undoubtedly provide a novel potential target for diagnosis or treatment of the disease.Nonresolving inflammation is not a primary cause of some cancers, but it contributes significantly to their pathogenesis. Epidemical and clinical studies have confirmed that nonresolving inflammation contributes to cancer progression. Tumor associated macrophages (TAMs) which represent a prominent component of the mononuclear leukocyte population that infiltrate in the tumor micro-environment play an indispensable role in tumorigenesis growth, invasion,and metastasis by expression cyclooxygenase-2(COX-2) and5-lipoxygenase(5-LOX).Arachidonic acid (AA) metabolites may play a modulator role in several immunological and inflammatory diseases. Numerous studies investigated the modulation of eicosanoid synthesis through the inhibition of various metabolism pathways of AA. The literature to date suggests that the production of AA metabolites through lipoxygenase(LOX) and cyclooxygenase(COX) pathways plays a important role in cancer growth, promotion, and metastasis. Both cyclooxygenase-2(COX-2) and5-lipoxygenase (5-LOX) as inflammatory factors are usually produced by macrophages and monocytes which are hypoxia or injured. The metabolites of AA by COX-2and5-LOX, prostaglandin E2(PGE2) and leukotriene B4(LTB4) might modulate tumor growth by the activation of vascular endothelial growth factor (VEGF) which can direct stimulus tumor angiogenesis, an important process in tumor progression. Cancer cells in solid tumors require access to blood vessels for growth and metastasis. Therefore, it is expected that to reduce TAMS infiltration in tumor tissues, and furthermore to inhibit AA metabolic COX-2/5-LOX pathway may be clinically useful for the treatment of tumors.QHBJ decoction was originated from Wenbingtiaobian, a classical prescriptions written by Wu Jutong in Qing Dynasty. The original prescribe was consists of artemisa apiacea6g, turtle shell15g, rehmannia12g, rhizoma anemarrhenae6g, tree peony root-bark9g. Chinese scholars in the clinical used for "cancer hot" is very satisfied with the treatment effect of QHBJ decoction, meanwhile, it also adjust immune function of patients with tumor. In our previous study, we have found QHBJ decoction exhibited anti-inflammatory effects and improve tumor microenvironment driven by protein HIF-1α, it also exhibited sensitization of chemotherapy and anti-proliferative effect against carcinomas including the human lung adenocarcinoma and S180sarcoma. However, to our knowledge, whether QHBJ decoction impact against tumors through the inhibition of AA metabolic pathway by COX-2/5-LOX that contribute to nonresolving inflammation has not been reported.Based on above, we investigated the expression of COX-2and5-LOX in human ovarian epithelial carcinoma by immunohistochemistry methods and analyzed the relationship between COX-2,5-LOX expression level and ovarian cancer clinical parameters toexplore the correlation of COX-2and5-LOX in contributing to development and progression of ovarian cancer. Furthermore, we observed the chemopreventive effect of QHBJ decoction on dimethylbenzanthracine induced ovarian carcinogenesis in rats which is comparable with COX-2inhibitors and5-LOX path inhibitors (5-LOX pathways downstream metabolites leukotriene receptor blockers). Moreover, expression of the tumor-associated macrophage phenotypes CD68was examined by IHC methods and expression COX-2,5-LOX and VEGF was assessed by western blot in rat ovarian tumor tissues. The levels of inflammatory mediators PGE2/LTB4in blood plasma were determined by radio-immunity assay. We attempt to find the intervention effect of QHBJ decoction on nonresolving inflammation malignant transformation. At the same time, we analyzed the infrared spectrum(IR) similarity in Qinghao, Qinghao-Biejia Combination, and Qinghao-Biejia Prescriptions.Results:1Expression of cyclooxygenase-2(COX-2) and5-lipoxygenase (5-LOX) in human epithelial ovarian carcinomas1.1Expression and Localization of COX-2,5-LOX in different ovarian tissuesThe immunohistochemistry method was applied to determine the expression and localization of COX-2and5-LOX in epithelial ovarian neoplasm(48malignant,30borderline and20benign) and10normal ovarian tissues. COX-2was found expressed mainly in the cytoplasm, while5-LOX was expressed in the nucleus,nuclear membrane and cytoplasm.No apparent expression of COX-2and5-LOX was found in normal and benign ovarian tissues, but they were overexpressed in ovarian carcinomars and borderline ovarian tumors. The positive rates of COX-2expression in ovarian carcinomas were77.1%, and in borderline ovarian tumors were73.3%, which were significantly higher than those in benign ovarian tumors and normal ovarian tissues (P<0.001). The positive rates of5-LOXexpression in ovarian carcinomas and borderline ovarian tumors were81.3%and76.7%respectively, which was also significantly higher than that in benign and normal groups (P<0.001).1.2Correlations between expression of COX-2,5-LOX and pathological parameters in ovarian carcinomasNo significantly correlation was found between the expression of COX-2and the histology types. However, the expression of COX-2in low differentiated grade was significantly higher than those in high and middle differentiated grade(P<0.01). In FIGO grades, the expression of COX-2in Ⅲ-Ⅳ stages was different from the Ⅰ-Ⅱ stages (P<0.05), But the expression of5-LOX was not different between different histological types. The positive expression of5-LOX in low differentiated grade was higher than that in high and middle differentiated grade (P<0.001). The Ⅲ-Ⅳ stages of ovarian cancer showed stronger expression of5-LOX than the Ⅰ-Ⅱ stages (P<0.01).In summary, the positive rates of COX-2and5-LOX were correlated with histopathology- cal grade and clinical stage. There was a positive correlation between the expression of COX-2and5-LOX in epithelial ovarian carcinoma.2. Intervention of QHBJ decoction on the biological effects of COX-2/5-LOX metabolic pathway of Arachidonic acid on chemical induced ovarian carcinogenesis in rats2.1General observationRats that induced by DMBA were randomly divided into six groups:Control, meloxicam(Mel), montelukast sodium(Mon), and QHBJ decoction(750mg·kg-1,1500mg·kg-1,3000mg·kg-1). Several rats in meloxicam group appeared intestinal ulcer and hematochezia after administrated for9weeks, while other groups didn’t showen. The mean body weights of meloxicam group was significantly lower than that of control group after administrated for16weeks (P<0.01), but the liver weight was significantly higher than the latter (P<0.05). Both the mean body weights and the mean weights of liver did not show statistical significances among other treated groups after administrated for16weeks. At36weeks after the experiment, the body weights of control group and QHBJ low dose group were both significantly declined, but no statistically difference was found between them. The body weights of QHBJ middle and high dose group were significantly higher than that of control group (P<0.01). At the end of the study, the mean liver weights of all groups were comparable.2.2QHBJ decoction decrease the incidence rate of ovarian malignant tumorThe ovarian tumors were observed successively at22weeks since the experiment in control group and Mon group. In QHBJ low dose group and Mel group, the ovarian tumors were found at26weeks after the experiment, and that were found at28weeks after the experiment in QHBJ middle and high dose groups.At the end of the study, the incidences rate of ovarian malignant tumor was84.61%in control group,53.84.%in Mel group,69.23%in Mon group, and in QHBJ low. middle, high dose groups were76.92%,46.15%,30.76%respectively. The incidences rate of ovarian malignant tumor and ovarian model weights in QHBJ middle dose group, high dose group and Mel group were significantly lower than that in control group (P<0.05). Also, the incidences in QHBJ high dose group was statistical significance compared with Mon group (P<0.05) 2.3Histopathological observation in ovarian carcinomaThe biological feature of the ovarian tumor in rat induced by DMBA was quite comparable with the ovarian tumor in woman. The induced adenocarcinomas in control group were showed poor and moderate differentiated, and the tumor cells wereexcessively proliferated and showed distinct heterotype. The induced adenocarcinomas in QHBJ high dose group were well and moderately differentiated. While in Mel and Mon group, the ovarian tumor tissues were showed poor and moderate differentiated.2.4Inhibition of inflammatory mediators PGE2, LTB4production in blood plasmaThe levels of PGE2and LTB4in blood plasma at the administration16weeks were determined by ELISA assay according to the manufacturers instruction. In the QHBJ high dose group and middle dose group, the content of PGE2and LTB4in blood plasma was lower than that of the control group singnificantly(P<0.05). But the levels of PGE2production in QHBJ high and middle dose group did not show statistical significances compared with Mel group (P>0.05), and the levels of LTB4was not statistically significant compared with Mon group (P>0.05)2.5Suppression of TAMs infiltration in ovarian tumor tissuesThe CD68protein was used to mark TAMs in ovarian tumor tissues by immunohistochemistry. Numbers for TAMs in QHBJ middle and high dose group were significantly lower than those in control group (P<0.05, P<0.01). The numbers of TAMs in QHBJ high dose group did not show statistical different compared with Mel group. But the numbers of TAMs in QHBJ high dose group were significantly lower than that of Mon group (P<0.05)2.6Down-regulation of the expression of COX-2,5-LOX, VEGF in ovarian carcinomaImmunohistochemistry method was used to determine the expression of COX-2,5-LOX and VEGF in ovarian cancer tissue. The positive expression was confirmed by the presence of brown stained in the cytoplasm or nucleus. The expressions of these three proteins in control group were all observed positively. Compared with control group, the expression of COX-2,5-LOX and VEGF in QHBJ middle and high dose groups were slighter significantly. Western-blot was exploited to detect the expression of COX-2,5-LOX and VEGF protein in tumor tissue. The results showed that the expression of COX-2,5-LOX and VEGF in QHBJ middle and high dose groups were significantly decreased than that in control group. Compared with the Mel and Mon groups, the levels of COX-2,5-LOX and VEGF in QHBJ high dose group were also significantly down-regulated.Conclusion:1. COX-2and5-LOX protein were over expressed in epithelial ovarian carcinoma. The positive rates of COX-2and5-LOX expression were correlated with histopathological grade and clinical stage. There was a positive correlation between the expression of COX-2and5-LOX in epithelial ovarian carcinoma. Therefore, both of them play important roles in tumor genesis and malignant progress, which may be helpful diagnostic parameters and prognostic indexes. Hence, it may be an attractive target for chemopreventive strategy in the treatment of ovarian carcinoma.2. QHBJ decoction significantly reduced the incidence rate of DMBA-induced ovarian cancer in rats. In addition, QHBJ decoction treatment suppressed the infiltration of TAMs in ovarian tumor and significantly down-regulated the expression of COX-2,5-LOX, as well as the downstream inflammatory mediators PGE2, LTB4production, further more decreased expression VEGF protein in ovarian tumor tissues. Taken together, these finding suggest that anti-inflammatory properties of QHBJ decoction through the COX/LOX pathways of arachidonic acid metabolism which maybe play an important action in control of the nonresolving inflammation malignant transformation.3. It is demonstrated that Qinghao contains smaller polarity volatile compositions and possess’leading function in Qinghao-Biejia Prescriptions by analysis of IR calculated similarity.