| Abstract: | 在台灣、印度、東南亞國家和南非等地嚼食檳榔是非常普遍的。檳榔是否具致癌性為近年來受爭議之話題。但嚼食檳榔對形成口腔癌的促癌性則很少有人討論。本實驗中，以MTT試驗表現紅灰檳榔萃取液對JB6細胞確實有細胞毒性。將JB6細胞浸在紅灰檳榔萃取液經過7天會看到JB6細胞有著癌化般特質的細胞形態改變。以低濃度(1、5、10ug/ml)的紅灰檳榔萃取液浸泡JB6細胞30天時，能測到H2O2的產生同時也測到MPO活性與ODC活性的增加。分析細胞週期發現經紅灰檳榔萃取液處理過48小時的JB6細胞，G1期會減少而相對的S期會增加。然後連續以濃度0.5 mg/ml的紅灰檳榔萃取液作為促癌劑(promotor)浸泡JB6細胞15天後，可以看到JB6細胞形成了typeⅢ的細胞集。而由此結果證明紅灰檳榔萃取液確實具有促癌性。
由前實驗結果進一步探討紅灰檳榔萃取液是否會使JB6細胞產生癌化轉型作用。我們使用了3種不同濃度的紅灰檳榔萃取液，結果不論是以高濃度(0.1mg/ml)的LPB浸泡JB6細胞5天後，或是JB6細胞被低濃度(0.1、0.5 mg/ml)的LPB作用15天後，都能造成轉型的JB6細胞集的形成。而這些轉型細胞集能生長在soft agar中，與正常細胞相比更能快速生長以及在培養皿中會重疊多層生長的癌化細胞特性。同時這些轉型的JB6細胞能夠藉著繼代培養到第50代，這也是正常細胞無法做到的。而在繼代培養的第50代轉型JB6細胞中測到C-Fos蛋白，C-Jun蛋白與C-Myc蛋白與正常JB6細胞相較有2倍的增加，而這些第50代的細胞，它們細胞週期的S期則明顯延長，以上結果顯示LPB作用形成的轉型JB6細胞已具有癌化形成。
之後對其各種不同世代(癌化程度)進行細胞週期蛋白表現分析，藉此實驗證實轉形JB6細胞的增生作用，是經由受到G1早期表現蛋白cyclin D-dependent kinase的活化來誘發Rb磷酸化(pRb, retinoblastoma tumor suppressor protin)，進而促使釋出轉錄因子E2F-1，使細胞進入S期，進行細胞增生作用。本實驗以西方點墨法(Western blot)、免疫沉澱法(IP, Immuno-precipition) 和流式細胞儀(Flow cytometry)來分析不同世代數之轉形JB6細胞中細胞週期與細胞週期相關蛋白之變化。結果顯示pRb明顯增加趨勢，而屬於cdk抑制劑 (CKIs , cdk inhibitors) 的p21和p16蛋白則呈現下降趨勢。另外，在第20th、30th、40和50th 代數中，其cyclinD1-cdk4和cyclinE-cdk2複合體有明顯增加趨勢。由此結果推測轉形JB6細胞是經由cyclin-cdk複合體活化導致Rb磷酸化，使細胞進入S期，進行細胞增生作用。而在細胞週期分析顯示在第50th 代中G1期明顯下降，由76.92 %降至52.26 %，S期則呈增加趨勢，由7.68 %增至16.36 %。
因此，這些結果證實LPB (紅灰檳榔萃取液)確實對老鼠上皮細胞(JB6 cell)具有促癌性，進一步也證實LPB作用形成的轉型JB6細胞已具有癌化特性，其增生作用，是經由cyclin D-dependent kinase的活化來誘發Rb磷酸化，釋出E2F-1，細胞進入S期所致。
Betel quid chewing is very general behavior in Taiwan, India, southeastern Asian and South Africa. In this study, microculture tetrazolium test (MTT) showed that the extract of lime-piper betel quid (LPB) (1.0-20 mg/ml) was toxic to JB6 cells. Cells exposed to LPB (0.1, 0.5, 1.0 mg/ml) for 7 days resulted in a change in cytomorphology with charasteristics of the carcinogenesis. With a long-term treatment (~30 days) of low doses of LPB (1, 5, 10 mg/ml), JB6 cells exhibited increases in the H2O2 production, myeloperoxidase (MPO) and ornithine decarboxylase (ODC) activities. Cell cycle analysis showed a decrease in the G1 phase and an accumulation in the S phase 48 h after LPB treatment. When treating with 0.5 mg/ml LPB for 15 days as a promoter, type III foci were formed in the JB6 culture. These results demonstrated that the tumor promotional effect of LPB in JB6 cells.
Betel quid chewing is a general oral habit in Taiwan, India, southeastern Asian and South Africa with or without the additive of tobacco, alcohol or lime. In this study, the tumour-promoting neoplastic transformation effect of Lime-Piper betel quid (LPB) was examined on JB6 cells. The treatment of LPB at a high dose (1.0 mg/ml) for over 5 days or at lower doses (0.1, 0.5 mg/ml) for over 15 days induced the formation of transformed foci. The transformed cells showed the characteristics of colony formation in soft agar, a higher growth rate and multilayer on culture dish. A two-fold induction of the protein levels of c-fos and c-jun proto-oncogenes was observed in the cells from the 50th passage (Cl1/p50, Cl2/p50 and Cl3/p50), suggesting that LPB-transformed cells were oncogenic. In addition, the LPB-transformed cells possessed an elevated level of c-Myc and an increased cell population distributed in the S phase of cell cycle. These results demonstrated the promotion effect of LPB, and indicate that it could be a tumor promoter.
In order to understand the mechanism of the increased cell proliferation in lime-piper betel quid-transformed JB6 cells, we analyzed the cell cycle distribution and the levels of cell cycle related proteins in the different passages of transformed JB6 cells. The data of Western blot and immunoprecipitation showed that LPB-transformed JB6 cells (from the 20th, 30th, 40th, and 50th passages) had increases in the formations of cyclinA/Cdk2, cyclinD1/Cdk4 and cyclinE/Cdk2 complexes. At the meanwhile, the levels of phospho-Rb were also increased, and those of Cdk inhibitors (CKIs) p21 and p16 were decreased. These results suggested that the increased cell proliferation in the transformed JB6 cells was mediated by the activation of cyclin-cdk complexes and the inhibition of CKIs that led to Rb phosphorylation and subsequent cell cycle progression. Cell cycle analysis showed a decrease in the G1 phase and an accumulation in the S phase of the 50th passage cells. This study demonstrated that the increased proliferation of LPB-transformed JB6 cells was mediated via the activation of cyclin-Cdk complexes and the subsequent RB phosphorylation. |
