中草藥成分Solamargine、CH1027和CH1128對人類肺癌細胞的作用機轉之研究

Characterization of the Action Mechanism of Herbal Extracts Solamargine, CH1027 and CH1128 on Human Lung Cancer Cells

梁家華(Chia-Hua Liang)

指導教授 ： 郭國華 ； 黃裕勝

高雄醫學大學/醫學院/醫學研究所/博士(2005年)

摘要

肺癌是國人第一大死亡原因，基本上肺癌可分成小細胞癌(small cell lung cancer, SCLC)和非小細胞癌(non-small cell lung cancer, NSCLC)兩大類。小細胞肺癌患者對目前所使用之抗癌藥物及放射治療的感受性高，但是其復發率高，且復發後易對化療藥物產生抗藥性；而非小細胞肺癌患者的治療情形則不甚理想，尤其是其抗藥性問題是目前臨床治療的最大困擾。因此判斷治療藥物對肺癌的敏感性或是抗藥性的分子機轉，將有助於改善化學治療的效果。另外，許多的文獻報導均證實，由化學抗癌藥物及放射的治療所誘導的細胞死亡過程是經由細胞設定性死亡(apoptosis)機轉。因此開發尋求一經由誘導細胞設定性凋亡而殺死癌細胞的新抗癌藥物，從而改進、研發更有效的臨床治療，將是癌症研究人員努力的目標。
以中草藥抗癌新藥來治療、抑制癌細胞之生長已被使用數個世紀，且有逐年攀升的趨勢。為了由中草藥中來篩選並研究判斷其有效之化合成分，其結構成分的特性作用分析將是研究的重點。近年來許多研究者證實從茄科植物中固醇類生物鹼所分離出的成份Solamargine (SM)，於抗癌活性方面具有很強的作用。因此本論文擬以研究從茄科植物中所萃取出的SM，以及由中草藥中所萃取出之CH1027和CH1128來誘導肺癌細胞進行設定式凋亡為指標，並進行一系列的死亡訊息機制之探討。
腫瘤壞死因子(tumor necrosis factors, TNFs)：TNF-?捋PTNF-?牷A此種激素具有調節細胞之增生、分化和直接對抗腫瘤引起細胞凋亡的活性作用。此兩種細胞激素具有相似的生物活性作用，其所結合的腫瘤壞死因子受器(tumor necrosis factor receptor, TNFR)又可分為兩型：TNFR-I與TNFR-II。之前的文獻中指出，肺癌細胞中腫瘤壞死因子受器族群的表現明顯的較正常肺細胞降低。人類A549肺腺癌細胞為TNF-???n非感受性之肺癌細胞，且對多種抗癌藥物，例如Cisplatin、Paclitaxel、Doxorubicin，則有顯著抗藥性。因此推論肺癌細胞中TNFR的表現明顯的較正常肺細胞降低的情形，是否與癌細胞無法進行細胞凋亡而具抗藥性有關。論文的第二章節我們以Solamargine (SM)來誘導人類肺癌細胞(A549、H441、H520、H661和H69)進行設定式凋亡為指標，探討腫瘤壞死因子受器族群所扮演的角色，進行死亡訊息機制探討。結果顯示SM作用於肺癌細胞後，可觀察到細胞核內染色質濃染、DNA的片段化、偵測到sub-G1 peak之DNA螢光強度增強，以及細胞設定性死亡訊息下游之caspase-3、-8和-9的活性增加。實驗亦發現肺癌細胞中的TNFR-I和TNFR-II的表現在受到SM的調控後均明顯的增加，且經由啟動TNFR的表現後，會誘導TNF-?悕MTNF-?狺孝畢X能力增加，進而促使TRADD和FADD的表現，並誘導肺癌細胞凋亡。另外許多研究中指出，過度表現原致癌基因Bcl-2和Bcl-xL，與TNF的表現降低所造成之的抗藥性相關連。Bcl-2和Bcl-xL的過度表現可抑制細胞進行設定性死亡作用，且對多種臨床上所使用之肺癌抗癌藥物具有顯著抗藥性。於我們的實驗結果中發現肺癌細胞經SM作用後，會促使細胞色素c (cytochrome c)從粒線體釋放至細胞質中，且抑制凋亡蛋白Bcl-2和Bcl-xL蛋白質之表現被降低，促進細胞凋亡的蛋白Bax則明顯的增加。另一方面我們亦測試是否對於化學抗癌藥物Cisplatin不敏感的細胞，經由SM的作用後可提升其敏感性，且以SM配合Cisplatin來評估A549細胞之抗藥性程度及藥物合併治療之作用效果。結果顯示SM與Cisplatin的毒殺加成效果(synergistic effect)是經由細胞死亡訊息下游之caspase-3、-8和-9的活性增加，進而促使細胞凋亡。綜合而論，在對於TNFs和Cisplatin具抵抗性的肺癌細胞中，SM或許可發展為一良好的抗癌藥物。
由中草藥中所萃取之CH1027是一新的固醇類化合物，固醇類的化合物可直接進入細胞，和DNA有高度的親合力，並調控基因的表現。然而目前對於CH1027所引起的細胞毒殺作用機制仍不是很清楚。本論文的第三章節我們將CH1027與四種臨床抗癌藥物Cisplatin、Methotrexate、5-fluorouracil和Cyclosphamide作用於H441、A549、H520、H661和H69五株肺癌細胞，並比較其對肺癌細胞的治療效果。結果發現相較於此四種抗癌藥物，CH1027對肺癌細胞具強烈的毒殺效果。CH1027作用於肺癌細胞後，可觀察到細胞型態的變化及細胞核內染色質的濃染，於電泳的結果觀察到DNA的階梯狀表現，偵測到sub-G1 peak之DNA螢光強度增強。這些結果顯示，CH1027所引起的細胞毒殺作用是經由誘導細胞進行設定性凋亡。由實驗結果亦發現CH1027投與肺癌細胞後，可經由啟動TNFR-I和TNFR-II的表現，進而誘發TNF-???n和TNF-?猁熊畢X能力增加，使細胞凋亡下游作用因子TRADD、FADD、caspase-8和-3的表現增強，進而促使細胞進行設定式凋亡。於這一章節，我們期望透過CH1027對於肺癌細胞的研究依據，能提供研發成為一具有開發潛力之中草藥抗癌治療藥物。
大部分的肺癌是屬於肺癌分類中的非小細胞肺癌，且非小細胞肺癌對於許多化學抗癌藥劑具有抗藥性。於本論文的第三章節證實，CH1027經由啟動調節TNF之活化途徑，進而誘導細胞凋亡。在第四章節中，我們將探討是否原致癌基因Bcl-2家族蛋白成員的表現亦參與CH1027所誘導細胞的設定性凋亡，且可藉由Bcl-2家族蛋白的參與而增加抗癌藥物Cisplatin的作用。由實驗結果中發現，將低濃度的CH1027和Cisplatin合併作用於四株非小細胞肺癌(H441、A549、H520和H661細胞)後，CH1027可增強Cisplatin之藥物毒殺作用。且將CH1027和Cisplatin合併作用於四株非小細胞肺癌比此兩種藥物單獨作用時，抑制細胞凋亡蛋白Bcl-2和Bcl-xL之表現顯著的被降低，而促進凋亡蛋白Bax的表現、caspase-3、-8和-9之蛋白質活性、腫瘤抑制基因p53的表現則顯著的增加。因此，綜合第三和第四章節的實驗結果，我們證實CH1027所引起人類非小細胞肺癌之設定性凋亡，與調控TNF/TNFR之訊息途徑、Bcl-2家族蛋白之作用和死亡訊息機制下游caspase之變化相關，且此兩個途徑之活化對於CH1027和Cisplatin之藥物合併治療之作用效果亦息息相關。透過這些實驗與理論依據我們建議CH1027可作為一有效的抗肺癌藥物。
於前幾章節中我們發現從中草藥中所分離出的成份Solamargine (SM)和CH1027可藉由調節TNFR途徑和Bcl-2家族蛋白的參與，進而誘導肺癌細胞凋亡，抑制人類腫瘤細胞的生長。在第五章節，我們亦發現從中草藥中所萃取的CH1027和CH1128作用於A549細胞後，可以調節降低致癌基因HER2/neu之表現，解決因為HER2/neu基因之放大及蛋白質過度表現所產生抗藥性問題之發生，進而增強A549細胞對化療藥物之敏感性。將CH1027和CH1128作用於A549細胞後，我們由反轉錄聚合酶鏈反應(RT-PCR)測得核糖核酸mRNA的表現中發現，HER2/neu基因表現和拓樸異構酶II?? (topoisomerase II??)基因表現具協同調節(coregulation)的作用，以低濃度的CH1027/CH1128和取對細胞低毒性的拓樸異構酶II之抑制劑Epirubicin合併作用於A549細胞後，可得到加成的細胞毒殺作用。因此我們推斷A549細胞經CH1027/CH1128作用後，HER2/neu基因和拓樸異構酶II?n?扆穧]均被降低表現，與所觀察到CH1027/CH1128和Epirubicin合併作用之細胞毒殺加成作用相關。綜合以上的數據結果，以CH1027/CH1128和Epirubicin合併使用來治療肺癌，將可提供合併化療藥物治療的一良好選擇。
本論文證實中草藥萃取物SM、CH1027和CH1128於肺癌細胞之作用機制，並探討經SM和CH1027作用後，肺癌細胞中TNFR和Bcl-2的表現情形，以及經CH1027和CH1128作用後，肺癌細胞中HER2/neu的表現情形，以評估藥物單獨或合併使用對抗藥性肺癌的治療潛力。並期望透過這些研究，能在肺癌預防及治療上提供一新的策略與方向。

關鍵字

中草藥成分 ； 肺癌 ； 腫瘤壞死因子 ； 細胞設定性死亡 ； 抗藥性

並列摘要

Lung cancer is the primary cancer causing death in Taiwan. Lung cancer can be divided into two major groups, small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). SCLC in the early stages of the disease is highly sensitive to chemotherapy and radiotherapy, although resistant relapses occur in the majority of patients. Moreover, drug-resistance to anticancer drugs is a common clinical problem in the treatment of NSCLC. Defining the molecular determinants of sensitivity or resistance to chemotherapy in lung cancer would have important implications for treatment. In addition, many reports have demonstrated that the cancer cells die through apoptotic mechanisms induced by chemotherapeutic drugs and irradiation. Therefore, the development of new treatments for cancer via apoptosis is the way that researchers have spent much effort to achieve their goal.
The use of herbs in the treatment of malignant diseases has dramatically risen in recent years. In order to identify active ingredients from Chinese herbs, structural characterization of components is needed. Recently, a purified compound from Solanum incanum herb, Solamargine (SM), a steroidal glycoalkaloid, was demonstrated to inhibit the growth of human tumor cells. Therefore, we performed a series of studies to investigate if SM, CH1027 or CH1128, purified compounds from herbs, play an apoptotic role in lung cancer cells.
Tumor necrosis factors (TNFs), TNF-?? and TNF-??, have been shown to modulate proliferation, differentiation and apoptotic cell death in a number of different cell lines. Both TNFs exert very similar biological activities, and bind with nearly identical affinities to TNFR-I and TNFR-II distinct transmembrane receptors. Previous studies demonstrated lung normal cells strongly express TNFRs, however, there is a loss or down-regulation of TNFR expression in lung cancer cells. The human lung cancer cell line A549 is known to be resistant to the cyototxic effects of both TNF-?? and chemotherapy agents, e.g. Cisplatin, Paclitaxel, and Doxorubicin. Since inherent and acquired drug resistance is commonly present in human lung cancers, the down-regulation of TNFR in lung cancers may be an avenue for drug resistance. In the second chapter of this study, we present mechanisms of how SM may trigger TNFR gene expression in lung cancer cells (A549, H441, H520, H661 and H69), which may lead to apoptosis. These results indicate that SM induced apoptotic death: SM-treated cells exhibited of chromatin condensation, DNA fragmentation, sub-G1 peak in the DNA histogram and increased caspase-3, -8 and -9 activities in lung cancer cells. SM elevated TNFR-I and TNFR-II expression to improve the susceptibility to TNF-?? and TNF-??, thereby increasing the expression of TRADD and FADD, and consequently inducing complete cell death of lung cancer cells. In addition, several mechanisms implicated in TNF resistance, one of which is to alter expression of oncogenes (e.g. Bcl-2) that subsequently activate anti-apoptotic pathways may also contribute to the resistance phenotype. Overexpression of anti-apoptotic protein Bcl-2 and Bcl-xL protein may increase apoptotic resistance and correlate with the multidrug resistance of lung cancers. Release of cytochrome c, decreased expression of Bcl-2 and Bcl-xL and an increase in levels of anti-apoptosis-related Bax were observed in SM-treated lung cancer cells. We also tested whether Cisplatin-insensitive tumor cells retain sensitivity for SM-induced apoptosis. Combination studies showed synergistic interactions for SM with Cisplatin through caspase-3, -8 and -9 activities, regardless of drug-resistance cell phenotypes. In conclusion, SM may elicit an anticancer potential against TNFs- and Cisplatin-resistant lung cancer cells.
CH1027, herbal and molluscicidal medicine is a new steroidal alkaloid glycoside, that increases the molecule’s affinity to particular DNA regions and alters gene expression. However, the mechanism of cytotoxicity of CH1027 is unknown. In the third chapter of this study, we show that CH1027 displayed a greater cytotoxicity than the conventional chemotherapeutic agents (Cisplatin, Methotrexate, 5-fluorouracil, and Cyclosphamide) in human lung cancer cells (H441, A549, H520, H661, and H69). The appearance in CH1027-treated lung cancer cells of morphological changes such as chromatin condensation, DNA ladders, and increased sub-G1 populations indicate that CH1027 induces cell death by apoptosis. CH1027 elevated the expression of TNFRs thereby improving lung cancer cells susceptibility to TNF-?? and TNF-???z?nleading to cell apoptosis. We also determined the mechanism of CH1027 in lung cancer: levels of TRADD and FADD signal cascade of TNFR-I, TNFR-II, caspase-8 and -3 effectors of apoptosis were all increased after CH1027 treatment lung cancer cells. In this chapter, the study demonstrates the mechanism of action CH1027, and sheds light on the investigation of herbs as new chemotherapeutic agents for clinical cancer therapy.
NSCLC is the most prevalent form of lung cancer and displays resistance to anticancer treatments. The third chapter demonstrates that CH1027-induced apoptosis involves the TNF-mediated pathway in human lung cancer cells. In the forth chapter, we examine to what extent Bcl-2 family members contribute to CH1027-induced apoptosis, and augment the effect of the anticancer drug Cisplatin. We found short incubation periods with low concentrations of CH1027 enhanced Cisplatin the cytotoxicity of NSCLC (H441, A549, H520, and H661) cells. A combined treatment of CH1027 and Cisplatin resulted in a marked decrease in expression of Bcl-2 and Bcl-xL, increased expression of Bax, caspase-3, -8, -9, and accumulation of p53, compared with single agents in NSCLC cells. In conclusion, we have demonstrated that CH1027 acts through both receptor and Bcl-2 mediated caspase-dependent cell death pathways in NSCLC cells, which may play an important role in mediating the augmented effect of CH1027 and Cisplatin. Based on these findings we suggest that CH1027 may be a good candidate for additional evaluation as a cancer therapeutic agent for human lung cancer.
We have previously shown that Solamargine (SM) and CH1027, mediate the expression of TNFRs and Bcl-2 family members, and sensitize human lung cancer cell lines to apoptosis. In the fifth chapter, we show that CH1027/CH1128 downregulates the expression of HER-2/neu, overexpression of which is correlated to drug resistance, and enhances chemotherapy-induced apoptosis in NSCLC A549 cells. We also found that after treatment with CH1027/CH1128, expression of HER-2/neu mRNA coregulated expression of topoisomerase II?? mRNA as determined by RT-PCR. The combinatory use of low concentrations of CH1027/CH1128 with low-toxic it’s of the topoisomerase II inhibitor Epirubicin accelerated apoptotic cell death in A549 cells. Thus, down-regulation of HER-2/neu and topoisomerase II?? expression by CH1027/CH1128 with Epirubicin may in part explain its cytotoxic synerginistic effect in A549 cells. Taken together, our data suggest that the combinatory use of CH1027/CH1128 and Epirubicin may be useful for lung cancer therapy.
This dissertation investigated that the mechanism of action of purified SM, CH1027 and CH1128 in lung cancer cells. The expression of TNFR and Bcl-2 family members after SM and CH1027 treatment in lung cancer cells was analyzed. After treatment with CH1027 and CH1128, determine the expression of HER2/neu to evaluate the potential of drugs’ treatments and drug-resistance of human lung cancers. By these studies, new strategies may provide in the lung cancer prevention and therapy.

並列關鍵字

Solamargine ； Lung cancers ； TNF ； Apoptosis ； Drug resistance

參考文獻

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