Expression Of Platelet Type 12-lipoxygenase, Integrin αv MRNA And Their Correlation With Angiogenesis In Esophageal Squamous Cell Carcinoma

Background and Objective Esophageal squamous cell carcinoma (ESCC) is one of the most aggressive malignancies in China. The poor prognosis of ESCC is due mainly to its potency to local invasion and distant metastasis. It is critical to understand the detailed information on the metastasis mechanism for the improvement of prognosis in such patients.Arachidonic acid (AA) is a main constituent of cell membrane phospholipid that can be catalyzed by three distinct enzyme pathways, including cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P₄50 epoxygenase (EPO). Their metabolisms lead to the generation of biologically active metabolites that may be involved in carcinogenesis by modulating mitogenic signaling and regulating cellular proliferation. Furthermore, it has been shown that the LOXs in particular are key regulators of cell survival and apoptosis. Substantial data have implicated a role for the platelet-type 12-lipoxygenase (P-12-LOX) metabolite, 12(S)-hydroxyeicosatetraenoic acid (12S-HETE), in a variety of tumor functions, including cell cycle progression, apoptosis, angiogenesis and metastasis. However, compared with the clear-cut concept of COX-2 in cancer development, the underlying mechanism by which P-12-LOX contributes to these processes remains obscure. In the present study, we focus our attention on this specific enzyme to investigate its potential role in ESCC. Tumor cell proliferation and metastasis are angiogenesis-dependent. Integrinsare a family of heterodimeric membrane glycoproteins that consist of various a and P subunits, which provide the physical interaction with extracellular matrix (ECM) necessary for tumor cell adhesion and angiogenesis and induce signal events essentialfor cell survival and differentiation. Of the wide spectrum of integrin subunits, ctvintegrin, which can combine with different p subunits such as fb and P5, has been in particular implicated in neovascularization, but its exact role in the process of angiogenesis induced by P-12-LOX is not fully clear.The intensity of angiogenesis, as assessed by counting the microvessles, may act as a prognostic factor for many solid tumors. In contrast to the panendothelial markers (e.g. CD31, CD34 and vWF), CD 105 (Endoglin) is preferentially expressed in the angiogenic vessel, and thus it may be a more ideal index to evaluate neovacularization.In this study, we investigated the mRNA expression of P-12-LOX and integrin a v subunit, and measured microvessle density (MVD) by means of CD 105 immuno-stainning in cancerous and adjacent normal tissue samples of ESCC. Furthermore, we analyzed the possible associations of these parameters with main clinicopathologic data of ESCC.Materials and method Esophageal carcinoma and adjacent normal tissue specimens were obtained from 44 patients who underwent radical resection for esophageal cancer. The mRNA levels of P-12-LOX and integrin otv were measured by reverse transcription-polymerase chain reaction (RT-PCR), and the expression levels of each sample were corrected by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA levels. The S-P immunohistochemical technique was used to detect the expression of CD 105 in esophageal carcinoma, and microvessel density (MVD) was counted in the field of X 200 magnification. Comparisons between groups were performed using independent-samples / test and x2 test, respectively. The correlation between two continuous variables was determined using the Pearson rank correlation (Y ) test. P value < 0.05 was considered significant.Result 1. Of 44 pairs of matched cancer and noncancerous tissues, the relativelevels of normalized P-12-LOX mRNA expression was significantly higher in tumor samples compared to its adjacent normal tissues (0.78+0.22 vs. 0.43 ±0.17, P < 0.01). 2. Correlation with clinicopathological data demonstrated that greater expression of P-12-LOX mRNA was found among patients with adventitious invasion and positive lymph node metastasis than those without adventitious invasion (0.83+0.19 vs. 0.67 ±0.24, P = 0.019) and negative lymph node metastasis (0.89+0.19 vs. 0.70±0.21, P <0.05), respectively. No significant differences were found between the levels of P-12-LOX mRNA expression and gender, age, tumor size or differentiation status. 3. Of 44 pairs of ESCC cases, the positive rate of av mRNA expression in tumor tissue samples was significantly higher than that of adjacent normal tissue samples (34.09% vs. 13.64%, P = 0.044). 4. No significant correlations were found between av mRNA expression status and any clinicopathological data (P > 0.05). 5. Of 44 pairs of ESCC samples, the MVD score in tumor tissues was significantly greater than that in adjacent noncancerous tissues (44.70+21.01 vs. 32.35 + 17.17, P < 0.01) from the same patient. 6. The MVD score of esophageal cancer tissues was related to the size of tumor. Tumors exceeding or equal to 3cm showed more number of microvessles than those in tumors less than 3cm in diameter (46.06± 18.97 vs. 34.47± 15.32, P = 0.047). 7. MVD score of esophageal cancer tissues was related to the differentiation status. MVD score was significantly higher in tumors with poor differentiation than that in tumors with well or moderate differentiation (51.10+21.42 vs. 39.51 ±16.17, P = 0.010). 8. MVD score of esophageal cancer tissues was related to adventitious invasion and lymph node metastasis, respectively. MVD score was significantly higher in adventitious invasion group than that in tumors without adventitious invasion group (49.61 ±21.70 vs. 34.19+ 15.32, P = 0.021). MVD score was significantly higher in positive lymph node metastasis group than that in negative lymph node metastasis group (54.07 ±21.93 vs. 37.59+17.54, P = 0.008). 9. No significant correlations were found between MVD score and gender or age of patients. 10. MVD score was significantly correlated with P-12-LOX mRNA expression level (r = 0.570, P < 0.01). 11. No significant differences of the levels of P-12-LOX mRNAexpression were found between positive av mRNA expression group and negative av mRNA expression group (0.85±0.22 vs. 0.75 + 0.21, P = 0.112), whereas there was a significant difference of MVD score between positive avmRNA expression group and negative avmRNAexpression group (55.51+20.08 vs. 39.11 ± 19.52, P = 0.012).Conclusion 1. In esophageal squamous cell carcinoma, Platelet type 12-lipoxygenase mRNA expression is higher than that in adjacent normal tissues. P-12-LOX is involved in the processes of the tumor cell invasion and metastasis. 2. P-12-LOX mRNA expression is correlated with MVD score. 3. The expression of integrin avmRNA is up-regulated in ESCC. MVD score is higher in positive integrin avmRNA expression group compared to negative avmRNA expression group. 4. There are relationships between MVD score and tumor size, the extent of differentiation, adventitious invasion and lymph node metastasis.