羧胺三唑与小剂量地塞米松合用对A549肺癌移植瘤生长抑制作用的研究

目的观察羧胺三唑（CAI）对A549肺癌移植瘤生长及肿瘤环境中肿瘤坏死因予-α含量和血管生成的影响，研究小剂量地塞米松（DEX）是否能通过降低肿瘤环境中肿瘤坏死因子-α含量和抑制血管生成增强CAI对移植瘤生长的抑制作用。方法向裸鼠腋下接种A549肺癌细胞，建立A549肺癌移植瘤模型，接种第2天随机分5组，并开始给药，分别是PEG400溶剂对照组（每天一次灌胃给药，0．1ml／10g体重）、CAI组（每天一次灌胃给药，20mg／kg）、DEX组（每周两次背部皮下注射给药，1mg／kg）、CAI与DEX合用组（CAI，每大一次灌胃给药，20mg／kg；DEX，每周两次背部皮下注射给药，1mg／kg）及英夫利昔单抗组（每周两次腹腔注射给药，10mg／kg）。40d后剥离瘤组织，拍照并称重；将部分新鲜肿瘤组织速冻后制作冰冻切片，进行CD31荧光染色；另一部分新鲜瘤组织制各匀浆后用ELISA方法对其TNF—α含量进行检测。结果CAI、DEX和TNF—α抗体（英夫利昔单抗）对移植瘤生长均有抑制作用，而CAI与DEX的联合应用抑癌作用最强，PEG、CAI、DEX、COM和TAB组瘤重分别为（0．23±0．04）、（0．21±0．02）、（0．17±0．04）、（0．12±0．03）和（0．17±0．04）g。CD31血管染色显示，CAI和DEX均能减少肿瘤组织内的血管数目，两者合用在极大程度上抑制了血管的生成；CAI能够降低肿瘤组织内TNF-α含量，与DEX合用使TNF-α含量降至更低水平，PEG、CAI、DEX、COM和TAB组肿瘤组织匀浆中TNF-α的含量分别为（933．9±286．8）、（636．1±157．4）、（684．9±170．1）、（364．7±50．1）和（606．8±190．3）pg／mg蛋白。结论CAI不仅能够直接影响肿瘤细胞的增殖和凋亡，还能通过调控肿瘤环境中的TNF-α抑制血管的生成，从而减缓肿瘤的生长，这可能是CAI发挥抗肿瘤作用一个新的机制。小剂量DEX与CAI联合用药对移植瘤生长的抑制作用明显增强，优化了CAI的抗肿瘤作用，可能成为一种新的联合用药方案。

Carboxyamidotriazole and low dose dexamethasone significantly inhibit the growth of A549 xenograft tumors

Objective To investigate the effect of carboxyamidotriazole （CAI） on the A549 xenograft tumor growth as well as the tumor necrosis factor-u （TNF-α） level and angiogenesis in tumor microenvironment; to investigate whether the antitumor effect of CAI could be enhanced by low dose dexamethasone （DEX） via further decreasing the TNF-α level and inhibiting angiogenesis. Methods A549 xenograft tumor model was established by injecting A549 cells into the subcutaneous tissue of the right axillary fossa of nude mice. The tumor bearing mice were divided into 5 groups and treated with CAI and/or DEX （PEG400 vehicle group： intragastric administration daily by 0.1 ml/10g bodyweight; CAI group： intragastric administration daily by 20 mg/kg; DEX group： subcutaneous administration twice a week for 1 mg/kg; combination group （COM）： CAI, intragastric administration daily by 20 mg/kg, DEX, subcutaneous injection twice a week for 1 mg/kg; TNF-α antibody group （TAB）： peritoneal injection twice a week for 10 mg/kg） on the next day. After 40 days, the tumors were isolated and weighed. The tissues were immediately frozen for preparation of frozen sections or tissue homogenates. The frozen sections were stained for CD31 immuno-fluorescent test. TNF-α concentration in the tissue homogenates was determined by a specific ELISA kit. The involvement of TNF-α in this xenograft model was confirmed by specific TNF-α antagonist infliximab. Results CAI, DEX and infliximab could inhibit the growth of A549 xenograft tumors, and the combination of CAI and DEX was the most effective among the groups. The tumor weights of PEG, CAI, DEX, COM and TAB group were （0.23 ± 0.04）, （0.21 ± 0.02）, （0.17 ± 0.04）, （0.12 ± 0.03） and （0.17 ± 0.04）g, respectively. Both CAI and DEX could decrease the number of blood vessels in tumor tissues, and the number decreased to a lower level in the combination group. CAI could inhibit the TNF-α production in tumor tissues, which could also be enhanced by DEX. TNF-α concentrations in tumor tissues of PEG, CAI, DEX, COM and TAB group were （933.9 ± 286.8）, （636.1 ±157.4）, （684.9 ± 170.1）, （364.7 ± 50.1） and （606.8 ± 190.3）pg/mg total protein, respectively. Conclusion CAI inhibits tumor growth not only by directly acting on tumor cells, but also by suppressing TNF-α production and angiogenesis in the tumor microenvironment, which may be a new antitumor mechanism of CAI. As those actions of CAI could be augmented by low dose DEX, the combination of CAI and low dose DEX may be further developed and applied clinically.