甘草查尔酮A抑制口腔鳞癌细胞转移的关键分子鉴定

Oral squamous cell carcinoma metastasis is the primary cause of death in patients. Currently, it still lacks the specific drugs which inhibit metastasis of squamous cell carcinoma clinically. Licorice, a traditional Chinese medicine, has thousands of years of history in medicine, and licochalcone A is one of the main medicinal ingredient. Our previous in vitro and in vivo studies have already proved that licochalcone A significantly inhibited the migration and invasion of human squamous cell, and the epithelial-mesenchymal transformation, yet had no significant effects on the proliferation, apoptosis and cell cycle of normal oral mucosa keratinocyte. Based on these, licochalcone A may be a candidate drug for the inhibition of squamous cell carcinoma metastasis. However, the molecular targets of licochalcone A inhibiting squamous cell carcinoma metastasis remains unclear. To clarify the mechanism, we intend to use affinity purification, mass spectrometry, dissociation constant determination and fluorescence colocalization to screen and confirm the key molecules of squamous cell and licochalcone A interactions. Then siRNA silencing and lentiviral over-expression are going to be used to study the effect of the molecules on squamous cell migration, invasion and epithelial-mesenchymal transition and other effects. At last, the effects of interaction of licochalcone A and the molecular on the squamous cell carcinoma metastasis will be tested in vitro and in vivo. We are not only going to propose a new understanding of squamous cell metastasis mechanism, but also provide theoretical foundation for clarifying the drug targets of licochalcone A inhibiting squamous cell carcinoma metastasis.

口腔鳞状细胞癌转移是导致患者死亡的主要原因。目前，临床上仍缺乏特异性抑制鳞癌转移的药物。甘草在我国已有数千年的医药史，甘草查尔酮A是其主要的药效成分之一。我们前期的体内外实验证明：甘草查尔酮A可显著抑制人鳞癌细胞的迁移、侵袭能力及上皮-间充质转化，但对正常的口腔粘膜角化细胞的增殖、凋亡及细胞周期无明显影响。鉴此，甘草查尔酮A可考虑为抑制鳞癌转移的候选药物。然而，甘草查尔酮A抑制鳞癌转移的分子靶不清。我们拟首先用亲和纯化、质谱鉴定、解离常数测定及荧光共定位等手段，筛选并确定鳞癌细胞中与甘草查尔酮 A相互作用的关键首触分子；其次采用siRNA沉默及慢病毒过表达等手段，研究该分子对鳞癌细胞迁移、侵袭及上皮-间质转化等的影响；最后采用体内、外实验探明甘草查尔酮 A与该分子相互作用对鳞癌细胞转移的影响。即为鳞癌细胞转移机制提出新认识，又为阐明甘草查尔酮 A抑制鳞癌细胞转移的药物作用靶点提供依据。

口腔鳞状细胞癌转移是导致患者死亡的主要原因。目前，临床上仍缺乏特异性抑制鳞癌转移的药物。甘草在我国已有数千年的医药史，甘草查尔酮A是其主要的药效成分之一。我们前期的体内外实验证明：甘草查尔酮A可显著抑制人鳞癌细胞的迁移、侵袭能力及上皮-间充质转化，但对正常的口腔粘膜角化细胞的增殖、凋亡及细胞周期无明显影响。鉴此，甘草查尔酮A可考虑为抑制鳞癌转移的候选药物。本项目探索甘草查尔酮A对人口腔鳞癌细胞自噬活动的影响以及MAP4K3-mTOR信号级联在甘草查而酮A诱导的人口腔鳞癌细胞自噬与凋亡中的作用。结果显示：甘草查尔酮 A可剂量及时间依赖地促进SCC-25鳞癌细胞自噬活性，表现为促进其溶酶体及自噬泡的形成、促进其表达自噬调节因子beclin1及LC3，且显著增高LC3-II/LC3-I的比率（P<0.05）。未经刺激组SCC-25系细胞基本不表达LC3蛋白，而经50微摩尔甘草查尔酮A刺激后，LC3蛋白表达显著增强（P<0.01），且主要表达在胞浆。甘草查尔酮 A可剂量及时间依赖性地降低SCC-25细胞MAP4K3、p-mTOR及p-p70S6蛋白的表达水平，促进其LC3-II、beclin1、caspase-3及caspase-9蛋白表达水平，并抑制其bcl-2蛋白表达水平（P<0.05）。采用慢病毒过表达MAP4K3后，p-mTOR、caspase-3及caspase-9蛋白表达水平显著增高，LC3-II、beclin1及bcl-2蛋白表达水平则显著降低（P<0.05）。经甘草查尔酮A刺激后，SCC-25细胞早期凋亡数与晚期凋亡数均较对照组显著增多，该效应可被MAP4K3过表达处理显著增强（P<0.05）。结论：甘草查尔酮A可时间剂量依赖地抑制MAP4K3-mTOR信号级联，并促进人口腔鳞癌细胞的自噬与凋亡，当过表达MAP4K3活化mTOR信号级联后，鳞癌细胞自噬活动受到抑制，但其凋亡活动则显著增强。甘草查尔酮 A可作为抑制口腔鳞癌转移的重要药物。