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Study on Effect and Mechanism of Aspirin on Murine S180Sarcoma

Author: ZhangXiaoYue
Tutor: WuLiCun
School: Jinan University
Course: Pathology and Pathophysiology
Keywords: aspirin angiogenesis lymphangiogenesis microvessel density vascularendothelia growth factor
CLC: R73-361
Type: Master's thesis
Year: 2013
Downloads: 33
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Abstract

ObjectiveAspirin, a safe, effective and extensively used NSAID has antipyretic,analgesic，anti-thrombotic and anti-inflammatory effects through inhibiting theactivity of cyclooxygenase (COX) enzymes. In recent years, aspirin was found tohave a potential in preventing and treating a tumor, which is a major area of concernin cancer research. Epidemiological and clinical studies suggest that by using aspirinas a chemopreventive agent, it can reduce cancer risk by as much as50%in somecancers. The molecular mechanisms of aspirin inhibiting tumor remains unclear. Thisstudy utilized murine S180sarcoma model and focused on the impact on tumorangiogenesis and lymphangiogenesis by evaluating the MVD, LVD, and theexpression of VEGF-A and VEGF-C in tumor tissues in order to investigate themolecular mechanisms that aspirin inhibits tumor growth.Methods1.The growth inhibition effects of aspirin in murine S180cells were detected byMTT assay.2. All tumor-bearing mice were randomly divided into5groups: control group,5FU group, high-dose aspirin group, low-dose aspirin group and combination groupwith10mice in each group. The tumor volume was detected and inhibition rate was caculated to observe the inhibition of tumor growth of aspirin.3. Tumor pathological changes were observed by light microscopy after routineHE staining.4. Immunohistochemistry were performed to detect the microvessel density(MVD), lymphatic vessel density (LVD), and the expression level of vascularendothelial growth factor A (VEGF-A) and VEGF-C.5. The expression of VEGF-A and VEGF-C was confirmed and quantified bywestern blotting.Results1. Effects of aspirin on proliferation of murine sarcoma180cellsAspirin significantly inhibited the growth of S180cells whether added before orafter cells adhered. When the pH was adjusted with sodium bicarbonate, theinhibition had a little decrease.2. Effects of aspirin on transplantable sarcoma180growth5-FU alone, aspirin either high-dose or low-dose alone, and combination of5-FU and aspirin resulted in inhibition of tumor growth compared to control group.The inhibitory rate was64.1%,33.5%,22.2%and70.1%, respectively (P<0.05foreach comparison). The inhibition effect was stronger in both in combination and5-FU groups (P<0.01). Although the inhibitory rate of high and low-doses of aspiringroup was lower, the two-grouped mice were in a good condition with an increasingbody weight after the experiment. This showed aspirin not only inhibited the growthof S180sarcoma, but also improved the body wasting caused by tumor.3. Pathologic and morphometric analysis of sarcoma180after treatmentHematoxylin and eosin staining showed that S180sarcoma cells are distributedas sheets and nests. Tumor cells vary in size and shape. High and low-dose aspirin,5FU and combination groups resulted in morphological changes of apoptotic tumorcells such as nuclear pyknosis and karyorrhexis, and microvessel density was significantly lower than control group. In high-dose aspirin,5FU and combinationgroups were accompanied by large patchy necrosis and along with frequentobservation of monocyte infiltration.4. Effcts of aspirin on the expression of VEGF-A and VEGF-C in sarcoma180By immunochemical staining, VEGF-A and VEGF-C were expressed in thecytoplasm of tumor cells. Positive cells for VEGF-A and VEGF-C are stained brown.The expression of VEGF-A and VEGF-C in control group was markedly higher thanthe other treatment groups. Treatment with5FU and aspirin resulted in reduction ofVEGF-A and VEGF-C expression. VEGF-A and VEGF-C expression decreasedsignificantly in both5FU alone and combination with other treatment groupsshowing a dose-dependency on high and low dose of aspirin. In each comparison,there was a significant difference (P<0.05).The effect of aspirin treatment on VEGF-A and VEGF-C protein expression wasalso assessed by western blotting analysis. VEGF-A and VEGF-C expression werereduced in high and low dose of aspirin,5-FU and combination groups. VEGF-A andVEGF-C expression decreased significantly in S180sarcoma treatment using only5FU or in combination with aspirin. It also showed dose-dependency on high andlow doses of aspirin groups.5. Effcts of aspirin on MVD and LVD in sarcoma180Positive cells for CD34are stained brown. Microvessels distribution was shown.Necrotic tumors cells were frequently present in the5-FU treated group and in thehigh-dose aspirin group. The5FU, the high and low dose of aspirin and thecombination group all have an inhibitory effect on MVD in comparison to thecontrol group (P<0.05for each comparison).D2-40positive tubular structures were stained brown.5-FU, aspirin andcombination groups having an inhibitory effect on LVD. High-dose aspirin showed anoticeable inhibited of LVD in comparison to the control group (P<0.05). The result revealed that LVD of the control group was the highest in the5groups and the lumenshowed obvious expansion.5FU, high and low dose of aspirin group had aninhibitory effect on LVD and VEGF-C. High-dose aspirin inhibited lymphatic vesseldensity obviously compared with the control group.ConclusionAspirin can inhibit tumor angiogenesis by reducing the production of VEGF-A,and inhibiting tumor lymphangiogenesis through the reduction of the VEGF-C in adose-dependent manner, which might explain why aspirin could inhibit tumorgrowth of murine S180sarcoma in vivo. We also found that aspirin synergisticallyenhanced the anti-tumor effect of5-FU.

Study on Effect and Mechanism of Aspirin on Murine S180Sarcoma

Abstract

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