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INTRODUCTION

Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated.

METHODS

4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status.

RESULTS

Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8, IFNγ, granzyme B, CD11c cells in comparison to 4NQO treated mice with a decrease in Ki67 and CD4FoxP3 cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP.

DISCUSSION

Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.

INTRODUCTION

METHODS

RESULTS

DISCUSSION

简介

在各种鼠肉瘤、黑色素瘤和癌的移植模型中，印度楝叶糖蛋白（NLGP）抑制肿瘤生长。然而，NLGP 在连续致癌步骤中的作用尚未得到探索。因此，我们用 4-硝基喹啉 1-氧化物（4NQO）诱导瑞士小鼠舌癌变，因为它与人的癌变过程非常相似。本研究在 4NQO 介导的舌癌变的起始-促进方案中，研究 NLGP 的干预作用与系统免疫改变和上皮-间充质转化（EMT）的关系。

方法

用 5mg/ml 的储备溶液 25μl 每天在舌头上涂 4NQO，连续涂 5 天（从第 7 天开始）。在用 4NQO 处理五次后（开始于第 7 天），一组小鼠用 NLGP（sc，25μg/只/周）治疗，另一组用 PBS 作为对照。在不同时间间隔处死小鼠，收集舌头进行组织学、免疫组织化学、流式细胞术和 RT-PCR 检测，研究不同免疫细胞和 EMT 标志物（E-钙粘蛋白、波形蛋白）的表型和分泌状态。

结果

连续 300 天局部给予 4NQO 可显著改变舌黏膜，包括乳头上的糜烂和恶性上皮细胞向下方结缔组织基质迁移，形成细胞巢（外生性-过度角化伴轻度发育不良）。治疗性 NLGP 治疗通过维持正常结构，延缓前瘤性变化，促进黏膜正常化。流式细胞术证据表明，与 4NQO 治疗组相比，NLGP 治疗组的 CD8、IFNγ、颗粒酶 B、CD11c 细胞上调，Ki67 和 CD4FoxP3 细胞减少。RT-PCR 显示，与 4NQO 治疗组相比，4NQO+NLGP 治疗组舌组织中 MMP9、IL-6、IL-2、CD31 表达减少，CCR5 表达上调。此外，4NQO 介导的变化与上皮细胞中 E-钙粘蛋白减少和波形蛋白表达同时上调有关，而 NLGP 部分逆转了这种变化。