氧化氮供給劑及抗癌藥物Adriamycin對路易士肺癌細胞之細胞-遺傳毒性的研究

氧化氮 (Nitric oxide；NO) 普遍存在於我們周圍的環境中，為一個具有生理功能的活躍分子；而其雖具有許多生物功能，但相同的亦會引發一些疾病。伴隨著Furchgott、Ignarro和Murad榮獲1998年諾貝爾醫學獎的旋風， NO所擁有之獨特性質再次受到世人的注目。延續這股熱潮，本研究亦想了解其對於路易士肺癌細胞 ﹝Lewis lung carcinoma (LLC) cells ﹞ 之細胞-遺傳上的毒性。我們以路易士肺癌細胞 (LLC) 標本為主，中國頰鼠肺細胞 ﹝V79 Chinese hamster lung (V79) cells﹞ 為對照細胞，分別加入氧化氮供給劑 (NO dornors)：sodium nitroprusside (SNP)、3- morpholinosydnonimine (SIN-1)、S-nitrosoglutathione (GSNO)和S-nitroso-N-acetyl—DL-penicillamine (SNAP) 和抗癌藥物adriamycin (ADM) 後，再利用微小核法觀察其遺傳毒性、以MTT分析法評估其細胞毒性、以流式細胞儀分析法偵測其對細胞週期的改變與細胞凋亡之情形以及利用CPP32分析法來更進一步確認這些藥物與細胞凋亡的關聯性。另一方面，也以MTT分析法來觀測鈣離子抑制劑verapamil (VPM) 加強SNP或ADM作用之情形，藉此我們希望可以提供更具前瞻性的化學療法。實驗結果發現： (1).在LLC細胞中，四種100mM NO供給劑或0.3mM ADM均會呈現時間相關性地增加MN (Micronuclei) 出現的頻率，降低細胞存活率和NDI (Nuclear division index) 值。 (2).對V79細胞而言，100mM SNP或0.3mM ADM均會呈現時間相關性地增加MN出現的頻率，降低細胞存活率和NDI值。此外，SNP和ADM在V79細胞之遺傳毒性均大於在LLC細胞。 (3).四種NO供給劑呈濃度相關性地降低LLC細胞之細胞存活率。四種NO供給劑以SNP (ED50：100mM) 之細胞毒性最強，但遠不及ADM。 (4).在LLC細胞中，100mM SNP會加強0.3mM ADM的細胞毒性；相同地，30mM和100mM VPM會加強0.3mM ADM之細胞毒性。 (5).在V79細胞中，30mM和100mM SNP會加強0.3mM ADM之細胞毒性。 (6).在分析細胞凋亡指標方面，我們發現四種NO供給劑於100mM的濃度時，無論是在LLC細胞或是V79細胞均不會出現sub-G1 細胞，顯示其在LLC細胞或V79細胞均不引發細胞凋亡。此外四種NO供給劑在LLC細胞在存在下會減少其細胞體積，但增加其細胞顆粒度。 (7).相對地，0.3mM ADM不僅會引發LLC細胞的凋亡，而且會造成LLC細胞和V79細胞大量累積在G2/M phase。 (8).CPP32活性分析法結果顯示只有0.3mM ADM 均會經由活化CPP32活性而造成細胞凋亡。 總而言之，在我們的研究中發現：四種100mM NO供給劑或0.3mM ADM在V79細胞和LLC細胞均會增加MN的頻率，降低細胞存活率和NDI值；但在光學顯微鏡下並不會顯著改變LLC和V79細胞之型態。由於四種 NO供給劑必須相當高的濃度下 (ED50：100mM - 1mM) 才會對LLC細胞有細胞的毒性，因此在臨床上100mM NO供給劑並不適合當抗癌藥。另一方面，對LLC細胞而言100mM SNP能加強0.3mM ADM對LLC細胞之毒殺性；但對V79細胞而言30mM SNP便可加強0.3mM ADM對V79細胞之毒性，顯示SNP不適合和0.3mM ADM聯合使用。VPM ³ 30mM亦可加強ADM在LLC細胞之細胞毒性，但因未做V79細胞之對照實驗，所以其相關性尚待證實。而實驗中亦顯示0.3mM ADM 會經由活化CPP32活性引發細胞的凋亡，而四種100mM之NO供給劑似乎與此途徑沒有關聯。最後，由於0.3mM ADM無論在LLC細胞或V79細胞之細胞凋亡和微小核數據無法相互佐證，所以初步推論微小核之形成和細胞凋亡並無相關性。因此對於尋求更具潛力的化學療法而言，無疑地仍待我們努力做進一步的探討。

Nitric oxide (NO) is a ubiquitous, physiologically active molecule involved in many biological functions and diseases. The unique attributes and importance of NO were recently recognized by the Nobel Prize Committee, with their decision to award the 1998 Prize in Medicine to Drs. Furchgott, Ignarro, and Murad. In this study, we attempted to investigate the genotoxic and cytotoxic effects of different nitric oxide donors, sodium nitroprusside (SNP), 3-morpholinosydnonimine (SIN-1), S-nitroso -glutathione (GSNO), and S-nitroso-N-acetyl-DL -penicillamine (SNAP), and anticancer drug, adriamycin (ADM), respectively, both in Lewis lung carcinoma (LLC) cells and V79 Chinese hamster lung (V79) cells. We used micronucleus assay to evaluate the genotoxic effects, MTT assay to evaluate the cytotoxic effects, flow cytometry to evaluate cell cycle and apoptosis, and also used assay the CPP32 activity to confirm apoptosis. In addition, we also used MTT assay to evaluate the effect of Ca2+-channel blocker (verapamil；VPM) on SNP, or ADM-induced cytotoxicity in LLC cells, we hope the results will afford useful references to improve the potency of cancer chemotherapy. We demonstrated that: (1). All of four 100 mM NO donors or 0.3 mM ADM significantly increased MN (Micronuclei) frequency, but decreased both cell viability and NDI (Nuclear division index) in a time-dependent manner in LLC cells. (2). 100 mM SNP or 0.3 mM ADM also significantly increased MN frequency, and decreased both cell viability and NDI in a time-dependent manner in V79 cells. However, both SNP and ADM-induced more genotoxicity in V79 cells than in LLC cells. (3). All of four NO donors significantly decreased cell viability in a concentration-dependent manner in LLC cells. SNP appears the best cytotoxic effect (ED50 : 100 mM) among the four NO donors but markedly weaker than 0.3 mM ADM. (4). In this study, 100 mM SNP potentiated ADM-induced cytotoxicity, 30 mM and 100 mM VPM also increased ADM-induced cytotoxicity in LLC cells, respectively. (5). 30 mM and 100 mM SNP also increased 0.3 mM ADM-induced cytotoxicity in V79 cells (6). The data of hyperploids showed that all of 100mM NO donors neither induced sub-G1 cells in LLC nor in V79 cells, the results point out all of 100 mM NO donors didn’t induced apoptosis in both LLC and V79 cells. However, the four NO donors decreased cellular size and increased cellular granules in LLC cells. (7). By contrast, 0.3 mM ADM induced apoptosis in LLC cells and induced G2/M phase block in both LLC cells and V79 cells. (8). The results of CPP32 assay showed that only 0.3 mM ADM increased the activity of CPP32 in LLC cells. In conclusion, the results indicated that all of four 100 mM NO donors and 0.3 mM ADM significantly increased MN frequency, and decreased cell viability and NDI both in LLC and V79 cells. However, they did not markedly change cell morphology of both LLC and V79 cells. Because all of four NO donors induced cytogenotoxic effects must in high concentrations in both LLC and V79 cells, so we supposed 100 mM NO donors could unsuitably be anticancer drugs in clinical therapeutics. On the other hand, 100 mM SNP increased 0.3 mM ADM-induced cytoxicity in LLC cells. However, SNP at lower concentration (30 mM) potentiated ADM-induced cytoxicity in V79 cells. Obviously, SNP can not combine with 0.3 mM ADM to treat cancer. VPM ³30 mM also increased ADM-induced cytotoxicity in LLC cells, but the value of potentially cytotoxic effect of VPM with ADM must do further study in V79 cells. In addition 0.3 mM ADM induced apoptosis via induced the CPP32 activity in LLC cells, but not NO donors. At last, the data of ADM-induced MN didn’t correlate with the data of ADM-induced apoptosis in both LLC cells and V79 cells; the results suggest that there is no relationship between apoptosis and MN. In this study, we afforded rarely new information in providing the better chemotherapy in cancer. Obviously, we have to design a further plan about this study.