| Abstract: | 自民國七十一年開始，肺癌一直是台灣女性和男性癌症的第一和第二大死亡原因。已知抽菸是導致肺癌的最重要原因，歐美國家的肺癌有90% 以上可用抽菸來解釋。但是台灣地區則有一半的肺癌無法以抽菸來解釋，尤其台灣女性肺癌患者有90% 以上是不抽菸者。因此可能有其他的環境因子參與不抽菸者肺癌的形成。而本研究所探討之環境因子包括環境致癌物之暴露和微生物的感染。
已知香菸中的多環芳香烴致癌物-benzo[a]pyrene (BaP) 會經過體內酵素代謝產生具高度活性的中間代謝物，然後攻擊DNA形成DNA鍵結物，造成抑癌或致癌基因的突變，終致腫瘤形成。因此首先了解肺癌患者的肺組織中DNA 鍵結物含量，是否較非癌症患者高？以確定DNA鍵結物是否可做為肺癌之危險生物因子？因此收集73位肺癌和33位非癌症患者之腫瘤周圍或非病灶的正常肺組織，以32P-postlabeling 分析DNA鍵結物之含量，結果發現肺癌患者DNA 鍵結物之含量 (49.58±33.39 adducts/108 nucleotides) 顯著高於非癌症患者 (18.00±15.33 adducts/108 nucleotides, P<0.001)。又發現抽菸和不抽菸之肺癌患者肺組織中之DNA 鍵結物含量並無差異 (抽菸者: 49.03±37.21, 非抽菸者: 49.28±30.73; P = 0.719)。而DNA鍵結物含量和患者本身之CYP1A1和GSTM1的基因多形性無關，但和CYP1A1的蛋白表現有關。這些結果顯示環境致癌物暴露所造成之肺組織DNA鍵結物形成之貢獻度可能不低於抽菸。由多變項統計分析的結果顯示，參與肺癌之危險因子中，以肺組織DNA鍵結物含量高於48.66 adducts/108 nucleotides者罹患肺癌之危險性是低者的25.19倍為最高 (P = 0.003)，另外僅有年齡因子達到統計意義，其餘因子如性別、抽菸習慣、CYP1A1和GSTM1的基因多形性都無法做為肺癌之危險因子。
過去研究大多認為女性對抽菸之感受性較高，為了了解不抽菸之婦女是否對環境致癌物之感受性也較高？因此各收集30位不抽菸之男、女性肺癌患者以及20位不抽菸之非癌症患者之肺組織，以32P-postlabeling和ELISA兩種方法分析DNA 鍵結物含量，以了解不抽菸之肺癌和非癌症患者間，是否同樣是肺癌患者對環境暴露有較高之感受性？並了解不抽菸之男、女性對環境致癌物暴露是否有不同之感受性？結果發現確實肺癌患者對環境暴露有較高之感受性。同時亦發現女性肺癌患者肺組織中之 DNA 鍵結物顯著高於不抽菸男性。且其DNA鍵結物之差異並非是CYP1A1 和GST-M1 基因多形性及蛋白表現不同所致，這結果顯示女性對環境致癌物暴露有較高之感受性。因此台灣婦女有較高之肺癌死亡率或許是由於他們對環境致癌物之感受性較高有關。
由於台灣女性肺癌患者有較高之DNA鍵結物含量，因此推測可能有較高之p53和K-ras突變頻率。但本研究室的初步結果卻發現52位不抽菸女性肺癌患者中僅有兩位有p53基因突變，其突變率僅有3.9%，而沒有任何一個女性肺癌患者有k-ras基因突變。在免疫組織化學分析的結果發現，肺腫瘤組織中的p53 及Rb 蛋白又大部分不表現，且p53及Rb 之調控基因如MDM2、p16及cyclinD1之蛋白表現又無法完全解釋p53及Rb蛋白的去活化。因此推測有其他外來之生物因子參與了肺癌的形成。
人類乳突瘤病毒 (human papillomarvirus 16/18；HPV 16/18) 所轉譯出來的E6及E7致癌蛋白，具有去活化p53及Rb蛋白的能力。因此大膽假設 HPV 可能參與肺腫瘤組織中 p53 及 Rb 的去活化作用。本實驗收集了141位肺癌和60位非癌症患者，以 巢疊式PCR (nested PCR)分析HPV 16/18 兩種高危險型和 HPV 6/11兩種低危險型之 HPV，以了解肺癌和非癌症患者之感染率是否不同？結果發現四種HPV在肺癌患者的感染率分別為 35.5%, 41.1% , 28.4% 和10.0%，其中HPV 6, 16, 18的感染率在肺癌和非癌症患者間具有統計上之差異。因此HPV 16, HPV 18和HPV 6三種HPV的感染可能和肺癌形成有關。若比較抽菸和性別時，則發現不抽菸之女性肺癌患者HPV 16/18的感染率竟高達60% 和73%，其感染率遠高於男性肺癌患者 (HPV 16: 24%, HPV 18: 26%)。而HPV 6的感染率卻以抽菸男性最高，達到38.1%，但不抽菸女性的HPV 6的感染率最低，僅有11.1%。這結果顯示不同性別和抽菸習慣之肺癌可能有不同型之HPV參與。因此HPV 16/18感染可能與不抽菸的台灣婦女肺癌形成有關。同時也發現HPV 6 及16 之DNA 感染與腫瘤期別有關，第一期之肺癌患者有最高之 HPV 6和HPV 16感染率，因此HPV可能參與肺癌早期之形成。
已知HPV 16/18的E6/E7蛋白參與p53和Rb蛋白的去活化，因此本研究進一步以in situ RT-PCR分析HPV 16/18 E6/E7 mRNA在肺腫瘤組織的表現，以了解是否參與p53和Rb蛋白的去活化而造成肺腫瘤形成。結果發現在兩張連續病理切片中，肺腫瘤組織中有E6/E7 mRNA表現的，有80% 測不到p53和Rb蛋白的表現，且HPV 16 E6/E7表現p53/Rb 去活化之肺癌患者之預後也明顯較差。因此 HPV 16/18 可能透過E6/E7致癌蛋白去活化p53和Rb蛋白之致癌路徑參與肺癌形成。總之，有較高的環境致癌物暴露而形成的DNA傷害，同時又有較高之HPV 16/18的感染，可能是不抽菸之台灣婦女為何有較高之肺癌死亡率的原因。
Lung cancer has been the leading and second cause of cancer death of women and men, respectively, in Taiwan since 1982 and is responsible for about 20% of overall cancer death. Similar trend was also observed in western countries and Japan. Prevention of lung cancer incidence is an important study topic in human health not only in Taiwan, but also worldwide. Cigarette smoking has been implied to be the most important etiological factor of lung cancer and up to 90% of lung cancer in the western countries could be explained by cigarette smoking. Intriguingly, in Taiwan, only around 50% of lung cancer incidence could be related to cigarette smoking, particularly, less than 10% of Taiwanese female lung cancer patients are smokers. Thus, it is conceivable that environmental factors other than cigarette smoking may be associated with lung cancer development in Taiwan and the aim of this study, therefore, is to evaluate possible involved environmental factors, including exposure to environmental carcinogen and microbial infections.
It has been well known that benzo[a]pyrene (BaP), one polycyclic aromatic hydrocarbon, is converted into a active metabolite, which will attack DNA to form DNA adduct, through biotransformation. The DNA adduct has been shown to result in mutation on tumor suppressor genes or oncogenes and ultimately, lead to formation of tumor. To prove the feasibility of DNA adduct levels in lung tissues being a risk biomarker of lung cancer, we have to firstly evaluate if DNA adduct levels in lung tissues of lung cancer patients are higher than that of normal controls. In this study, 73 lung cancer patients and 33 non-cancer control were recruited and corresponding DNA adduct levels were analyzed by 32P-postlabeling. Our data showed that DNA adduct levels in lung cancer patients’ non-tumor tissues (49.58±33.39 adducts/108nucleotides) were significantly higher than that of non-cancer controls (18.00±15.33 adducts/108nucleotides, P<0.001). And DNA adduct levels in smoking lung cancer patients (49.03±37.21 adducts/ 108nucleotides) were similar to that of non-smoking ones (49.28±30.73 adducts/108nucleotides, P=0.719). The DNA adduct levels were irrelevant to genetic polymorphisms of CYP1A1 and GSTM1, but related to the CYP1A1 protein expression. The data showed that exposure to environmental carcinogens may play an important role in lung cancer formation other than cigarette smoking. Multivariate logistic regression analysis showed that individuals with higher DNA adduct levels (>48.66 adducts/108nucleotides) had an approximate 25-fold risk of lung cancer compared to that of those with low adduct levels (48.66 adducts/108nucleotides). The other factor such as gender, smoking behavior, or genetic polymorphism of CYP1A1 and GSTM1 all could not be risk biomarkers for lung cancer.
From previous reports, women have been mostly believed to be more susceptible to cigarette smoking. To evaluate whether non-smoking female have higher susceptibility to the environmental carcinogen exposure, DNA adduct levels of non-smoking lung cancer patients, including 30 female and 30 male, and 20 non-smoking non cancer controls were analyzed by 32P-postlabeling and ELISA. Our data suggested that lung cancer patients were indeed more susceptible to the environmental carcinogen exposure than non-cancer controls and DNA adduct levels of female lung cancer patients were actually higher than that of male. Furthermore, this difference in DNA adduct levels was not resulted from genetic polymorphism or protein expression of CYP1A1 and GSTM1. Therefore, high susceptibility to DNA damage of females may be partial responsible for the high mortality rate of lung cancer in non-smoking Taiwanese women.
Based on the higher DNA adduct levels in female lung cancer patients in Taiwan, it was suspected that mutation frequencies of p53 and k-ras gene were higher in such patients. From our preliminary data, it was showed that only 2 of 52 female have mutations occurred in p53 gene, which yielding a mutation frequency of 3.9% and there was no K-ras mutation in tumor tissues of female lung cancer patients being detected. However, by immunohistochemistry, p53 and Rb protein expression could not been detected in most lung cancer patients. Meanwhile, the inactivation of the P53 and Rb protein could not be explained by their regulated gene such as MDM2, p16 and cyclinD1. From all these, we, therefore suspected that other biological factors may be responsible for the inactivation of p53 and Rb and therefore involved in lung tumorigenesis in Taiwan.
Since the p53 and Rb protein could be inactivated by the E6 and E7 oncoproteins encoded by human papillomavirus 16/18 (HPV 16/18) we hypothesized that the HPV may be involved in P53 and Rb protein inactivation in lung tumor tissues. In this study, 141 lung cancer patient and 60 non-cancer control were recruited to evaluate the infection rate of low-risk (HPV 6/11) and high-risk HPV (HPV 16/18) by nested PCR and in situ hybridization, to determine the difference in the infection rate of HPV between lung cancer patients and non-cancer controls. The infection rate of HPV6, 11, 16 and 18 for lung cancer patients was 28.4%, 10.0%, 35.5% and 41.1%, respectively, and the difference in prevalence rates of 6, 16 and 18 between these two study groups were statistically different. Therefore, the infection of HPV 6, 16 and 18 may be involved in lung cancer development. When stratified by gender and smoking behavior, HPV 16/18 infection rate of non-smoking female lung cancer patients (60.0% and 73.0% for HPV 16 and 18, respectively) were significantly higher than that of man (24.0% and 26.0% for HPV 16 and 18, respectively). But for HPV6, the highest infection rate of HPV6 was in smoking male (38.1%) and the lowest was in non-smoking female (11.1%). The result suggested that the different type of HPV might be involved in lung cancer with different gender and smoking behavior while the infection of HPV 16/18 may be involved in female lung cancer formation in Taiwan. It was also found that the infection rates of HPV 6 and HPV 16 were correlated with tumor stage, with the highest frequency being for stage I patients, therefore, HPV infection may contribute in the early stage in lung cancer development. To understand whether the inactivation of P53 and Rb in lung tumor tissues were correlated with HPV16/18 E6/E7, the HPV16/18 E6/E7 mRNA expression in lung tumor tissue were detected by in situ RT-PCR. In the serial tissue sections, the p53 and Rb protein were not detected in 80% of the lung cancer patients with HPV16/18 E6/E7 mRNA positive expression and these patients have the poor prognosis. Because of these, inactivation of p53 and Rb protein by HPV16/18 E6/E7 may be linked in lung tumorigenesis.
In conclusion, the concurrence of high DNA damage susceptibility to environmental carcinogen exposure and high HPV16/18 infection may contribute to the higher mortality rate of non-smoking female lung cancer in Taiwan. |
