Expression Of Endostatin In Lung Adenocarcinoma, And Effects On The Lymphangiogenesis And Lymphatic Metastasis Of Lewis Lung Carcinoma Xenograft In Mice

Background and ObjectiveLung cancer is now the greatest health risks to human, particularly lung adenocarcinoma, not only with a high incidence and poor prognosis. Lymphatic and hematogenous metastasis is the leading cause of death, and lymphangiogenesis and angiogenesis play important roles in the development, growth and metastasis of lung carcinoma. Therefore, study about the mechanism and method to suppress lymphangiogenesis is therefore essential to the development of better treatment.However the usual treatments, including surgery, chemotherapy and radiotherapy for malignant tumors cannot prevent metastasis completely. Anti-angiogenic therapy has become an option for the treatment of malignant disease. However, tumor cells migrate and metastasize not only through vascular system, but through lymphatic system as well. In fact, lymphatic metastasis may occur more commonly than metastasis through vascular system.Endostatin is a 20-kDa internal fragment of the carboxyterminus of collagen XVIII, first produced in hemangioendothelioma by O'Reilly, capable of inhibiting endothelial cell proliferation and inducing endothelial cell apoptosis. Endostatin has been reported as one of the most potent endothelial cell inhibitors of angiogenesis and tumor growth. Studies have showed that endostatin also inhibits the proliferation and migration of lymphatic endothelial cells in vitro. However, it was apparently not successful in clinical trials because of problems with the technique for recombinant human endostatin production.Compared with endostatin reported previously, an additional nine-amino acid sequence was added at the N-terminal of the protein. These changes cut down the preparation cost and improve the stability of protein without decreasing its antiangiogenic efficacy.In the present study, we investigate the expression of endostatin and explore the relationship between endostatin and lymphangiogenesis,lymph node metastasis, and the clinical pathological features of lung adenocarcinoma. In addition,to investigate the effects of endostar on the lymphangiogenesis and lymphatic metastasis of lung carcinoma. We used Lewis lung carcinoma cells and C57BL/6 mice to establish the lung carcinoma animal model, and studied the effects of endostar on the growth, lymphangiogenesis and lymphatic metastasis of the tumor.Materials and methodsPart one Expression of Endostatin and MLVD in Lung AdenocarcinomaFormalin-fixed,paraffin-embedded tissue specimens from 60 patients with lung adenocarcinoma who underwent surgical resection at Second Hospital of Shandong University were studied. There was no death due to operation.Patients included in this study had not received any preoperative chemotherapy or radiotherapy. Of which:33 males and 27 females, aged 45 to 69 years old;poorly differentiated adenocarcinoma in 29 cases,31 cases in moderately and well-differe-ntiated adenocarcinoma; central lung cancer 32 cases, peripheral 28 cases;ⅠandⅡperiod are 25 cases,ⅢandⅣperiod are 35 cases.Consecutive 4-μm sections were cut from each study block.Sections were immunostained for endostatin and MLVD. According to staining intensity and density of the tumor cells, the expression of endostatin was evaluated semi-quantitatively as negative/low (<2 score), positive/high (>2 score) by two independent observers blinded to the mice's status. The number of D2-40 positive vessels was counted as follows:search the dense area of D2-40 positive vessels with low power (100X), switch to high power (400X) and select 5 visual fields to perform counting, and take the average value as MLVD. Their correlation with lymph node metastasis and clinicopathological features were evaluated.Part two Effects of Endostatin on the Growth and Lymphangiogenesis of Lewis Lung Carcinoma Xenograft in MiceLewis lung carcinoma cell line was grown in RPMI-1640. Forty Male C57BL/6 mice weighing 20-24g were purchased from Peking, China. For in vivo implantation, LLC cells were washed in Hanks' balanced salt solution(HBSS) and injected intravenously at 1×106 cells in the caudal vein of the mice. On day 8 post cell injection, the mice were randomly assigned into two groups:control group and test group, and each group contained twenty mice. Control group mice received caudal vein injections of 0.2 ml of 0.9% sodium chloride once daily for 15 days, test group mice received 500μg endostar. Six weeks after LLC cell injection mice were sacrificed, and a complete autopsy, including lymph node dissection, was performed. Tumor incidence (percentage of mice with at least one tumor on lung surface), tumor multiplicity (number of tumors>2 mm in longest diameter per mouse) and tumor size (sum of tumor diameters divided by tumor number) were recorded.Tumor tissue samples were fixed with 10% buffered formalin, and embedded in paraffin after routine dehydration. Consecutive 5μm sections were cut from each block and were immunostained for VEGF-C and D2-40.VEGF-C expression was also detected by reverse transcription PCR.Take 200mg tissues and add 1 ml Trizol solution to completely homogenate.Furth extract total RNA according to the instructions of Trizol kit, and determine the RNA concentration and purity. Primer sequence for C-met:5'-AAGACCGTGTGCGAATCGA-3'(upper stream)and 5'-ACACAGCGGCATACTTCTTCAC-3'(down stream). The internal reference P-actin was used to detected reverse transcription rate.PCR product was treated by 1.5%agarose gel electrophoresis.After electrophoresis,automatic gel imaging system was used to take images and analyze.Results1, Expression of Endostatin and impact on microlymphatic vessel density in Lung AdenocarcinomaEndostatin-positive substances that were in the shape of brownish-yellow fine particles were mainly located in the cytoplasm of cancer cells.The positive-expression rate of endostatin in tumor tissue (61.7%,37/60).D2-40 was mainly expressed in interstitial vascular endothelial cells around cancer nests, and cancer cell infiltration can be seen frequently. MLVD was significantly lower in endostatin positive expression group (7.68±1.89) than that in negative expression group (9.17±1.95) (P<0.01), and MLVD decreased with the increase of the endostatin expression level. In addition, there was a close relationship between the expression level of endostatin and the stage of TNM, lymph node metastasis,tumor size and tumor differentiation,but no significant relationship with gender and age.2, Effects of Endostatin on the Growth and Lymphangiogenesis of Lewis Lung Carcinoma Xenograft in MiceNo differences in tumor incidence were detected, and the incidence rate was 90%(18/20)in both group. Tumor multiplicity of control group and test group were 5.61±1.24 and 4.67±1.14 respectively, control group was significantly higher than treatment group (P=0.023). Tumor size in the test group mice were smaller than those in the control mice (P=0.007).Two lymph node metastases were observed in test group mice, and eight in the corresponding control group. Lymphatic metastases were more frequent in control group than in the test group(.P= 0.026). A decrease in the number of D2-40 positive vessels was detected in the area surrounding the tumor in test group mice. The MLVD was 5.67±1.57 per field in the test group, which was markedly lower than in the control mice,7.78±1.56 (P<0.01).Expression of VEGF-C:VEGF-C positive substances appear as brownish-yellow fine particles mainly located in the cytoplasm of cancer cells. In addition, it was expressed in various degrees on cell membrane, appearing pale-yellow to brownish-yellow. Expression of VEGF-C in control group was significantly higher than that in the test group (P=0.019). VEGF-C gene in control group was significantly higher than that in the test group (P<0.01).Conclusions1, The expression of endostatin in lung adenocarcinoma has closely relationship with lymphangiogenesis and lymph node metastasis.2, There was a close relationship between the expression level of endostatin and the stage of TNM, lymph node metastasis,tumor size and tumor differentiation,but no significant relationship with gender and age.3, Endostar significantly inhibits the growth, lymphangiogenesis and lymphatic metastasis of Lewis lung carcinoma xenografts.4, Inhibitory effect on lymphangiogenesis and lymphatic metastasis of endostar is due to its ability to regulate the expression of VEGF-C of tumors in part.