| 摘要: | 檳榔嚼塊 (betel quid, BQ)與檳榔子 (areca nut, AN) 已經被公認為致癌物，引起各種口腔疾病，包含口腔鱗狀細胞上皮癌 ( oral squamous cell carcinoma, OSCC )。已知檳榔子萃取液 ( AN extract, ANE )與檳榔素 (arecoline檳榔子中主要的檳榔鹼)，已知細胞毒性。我們發現檳榔嚼塊的二種組成：紅灰與荖花均引起細胞皺縮，類似細胞凋亡的產生，而其另一種組成檳榔子本身的萃取液與其經 10K濃縮管部份純化分子量大於 10 kDa的分子 (ANE > 10 kDa)，卻造成 OSCC 有細胞膨脹且類似細胞壞死 ( necrosis-like cell death, NCD )的現象。與初級培養的口腔纖維母細胞相較之下 ( IC50 = 82.5 g/ml )，有兩株 OSCC細胞株對ANE > 10 kDa的處理更為敏感 ( IC50 = 38.3與27.03 g/ml )。經葡聚醣凝膠 G-100型膠體層析的檳榔子萃取液，造成類似細胞壞死的物質之分子量大於 67 kDa ( LLS-1)。但進一步純化ANE > 10 kDa與LLS-1均未成央C加入熱休克蛋白 90型的抑制劑 (Hsp 90 inhibitor)，膠達納黴素 (geldanamycin)，無法抑制由檳榔子萃取液所引起的 NCD。而加入抗氧化劑 N-acteyl-L-cystein (NAC)可以保護檳榔素造成的細胞死亡；但是對檳榔子萃取液與 LLS-1的細胞毒性則不具此保護效果。在免疫螢光與活性測試的實驗結果均顯示，檳榔子萃取液與檳榔素均有活化第三型凋亡蛋白酵素(caspase-3)的能力，而 LLS-1則無法活化此酵素。此外，檳榔子萃取液與檳榔素均可刺激細胞外訊號調節激酶 ( extracellular-regulated kinase, ERK ) 、p70 S6激酶 S6 kinase, S6K-Thr421, Ser424與 p70 S6K-Thr389(可以反應 mTOR的活性 ) 的磷酸化。而檳榔素與檳榔子萃取液均可誘導 p53的表現，且其中以檳榔素的效果較大。就細胞外觀上的變化而言，檳榔素造成細胞死亡的型態有細胞皺縮與凋亡小體的產生；而檳榔子萃取液與 LLS-1則使得細胞呈現圓形膨脹，並且伴隨著細胞核明顯的濃縮。這些結果顯示在檳榔子的成分中，檳榔素與 LLS-1導致不同的細胞死亡型態。
Betel quid (BQ) and areca nut (AN) are both recognized as carcinogens causing various oral diseases including oral squamous cell carcinoma (OSCC). Extract of AN (ANE) and arecoline, a major alkaloid of AN, are known to be cytotoxic. We have found that two components of BQ caused cell shrinkage －the apoptosis-like cell death (ACD). ANE and its ingredients with molecular weights hights than 10 kDa (ANE > 10 kDa) induced cell swelling and necrosis-like cell death (NCD). Two OSCC cell lines showed higher sensitivity to ANE > 10 kDa as compared to that of the primary culture of oral fibroblasts. Gel filtration of ANE by Sephadex G-100 indicated the molecular weight of NCD-inducing ingredient is higher than 67 kDa (LLS-1). Further purification of the necrotic activity from neither ANE > 10 kDa nor LLS-1 was not successful. An Hsp90 inhibitor, geldanamycin, did not inhibit ANE-induced NCD. An antioxidant, N-acteyl-L-cystein (NAC), protected arecoline-induced ACD but not those induced by ANE and LLS-1. Our results also showed that arecoline and ANE, but not LLS-1, were able to activate caspase-3 as revealed by immunofluorescence and caspase-3 assay kit. In terms of morphological changes, arecoline caused cell shrinkage and apoptotic bodies, while ANE and LLS-1 brought about the swollen cells and nuclear condensation. Both ANE and arecoline stimulated the phosphorylation of ERK1/2, p70 S6 kinase Thr421, Ser424 and p70 S6 kinase-Thr389 (reflecting the activity of mTOR), The also stimulated the expression of p53, and arecoline was a stronger stimulator. These results indicate that among the components of ANE, arecoline and LLS-1 may different pattern of cell death via different mechanism. |
