| Abstract: | 由衛生署的統計資料中得知，近十幾年來肺癌為我國惡性腫瘤死因之首，尤其是女性癌症死因第一位。根據流行病學的調查，罹患肺癌的危險因子包括抽煙習慣、空氣污染、職業暴露等因子。據調查台灣地區的婦女抽煙比例只有3 ﹪，華裔非吸煙婦女比歐美各國非吸煙婦女具有較高的肺癌發生率，因此華裔婦女中可能存在與抽煙無關之肺癌危險因子。近年來暴露中國式烹調油煙霧與肺癌發生的探討受到人們的關注。已有報告指出吸入食物烹調煙霧及食用油煙霧明顯影響人的肺功能，另外食用油加熱後所產生的煙霧對細菌具有致突變性，食用油煙霧可能具有遺傳毒性。食用油煙霧的成分，大部分為脂肪酸、醛類、酮類或B[a]P。本實驗室發現當肺癌細胞株CL3暴露食用油煙萃取物後，細胞生長被抑制。已知許多環境污染物不只造成生物損傷而且改變基因表現，例如柴油燃燒廢氣顆粒、空氣中懸浮微粒，會促使細胞株分泌cytokines。所以本研究欲以RT-PCR方法檢測，肺癌細胞株CL3暴露食用油煙萃取物或B[a]P後，對cytochrome P4501A1、1B1、2E1及cytokines和生長因子基因表現之影響。
CL3細胞暴露食用油煙霧後細胞cytochrome P4501A1和1B1等的基因表現程度於4小時內短暫增加為對照組的2至4倍；cytochrome P4502E1基因表現程度於24小時後增加為2倍；Tumor growth factor β1（TGFβ1）基因表現程度於24小時後增為對照組的4倍。cytokines包括IL-2、IL-4、IL-6、IL-8、IL-13、interferon-γ及tumor necrosis factor-α及TGFβ2等基因表現程度並無明顯改變。細胞暴露於B[a]P也有相似的情形，B[a]P會增加CYP1A1、CYP1B1和TGFβ1 mRNA表現約3-4倍，而IL-2、IL-4、IL-6、IL-8、IL-13、interferon-γ及tumor necrosis factor-α及TGFβ2等基因表現程度並無明顯改變。
接著我們以ELISA定量培養液中之TGFβ1，證實CL3細胞經食油煙霧或B[a]P處理後，不僅會改變TGFβ1 mRNA程度，也會刺激細胞分泌TGFβ1。我們又發現處理食用油煙霧或B[a]P，會誘導細胞產生氧化緊迫之作用，由此可知細胞處理食用油煙霧或B[a]P誘導細胞產生氧化緊迫，或許與TGFβ1 mRNA表現增加有關。
已知B[a]P經由活化多環芳香烴類受器（aryl hydrocarbon receptor，AhR），增加細胞CYP1A1基因表現及其酵素活性，我們利用三種對B[a]P敏感性不同的肺癌細胞株CL3、A549和H1355細胞來探討B[a]P誘導TGFβ1基因表現是否與AhR訊息傳遞路徑相同。CL3和H1355細胞經B[a]P處理後細胞生長受抑制，但A549細胞生長速率並未受影響。比較B[a]P對三種細胞株之CYP1A1和TGFβ1基因表現的影響，發現10μM B[a]P處理24小時後，皆會使CL3、A549、H1355細胞CYP1A1基因表現增加，為對照組的2、1.8、5倍，比較CYP1A1酵素活性也分別增加為對照組的7.4、2.5、56.3倍，B[a]P在H1355細胞中誘導CYP1A1酵素活性的能力最高，但是檢測三種細胞之TGFβ1基因表現，只有CL3細胞的TGFβ1基因表現有增加情形。再觀察B[a]P在三種細胞株中誘導氧化緊迫之情形，只有在CL3細胞中發現細胞有氧化緊迫的情形，A549和H1355細胞則無氧化緊迫的現象出現。綜合以上的結果發現，B[a]P誘導CL3細胞之TGFβ1基因表現增加，可能與氧化緊迫有關，但是與AhR所調控之路徑可能無關。
由本研究結果得知，人類肺癌細胞株暴露食用油煙萃取物後能增加cytochrome P450與TGFβ1基因表現，刺激細胞分泌TGFβ1且產生氧化緊迫；處理B[a]P也可看到相同的影響。進一步探討B[a]P增加TGFβ1基因表現之機轉時發現，B[a]P增加CL3之TGFβ1基因表現可能與氧化緊迫有關，但是與AhR所調控之路徑可能無關。
Lung cancer is the most common cause of cancer death worldwide around 1990. Lung cancer mortality among Chinese females was above the world average. Cigarette smoking has been associated with the development of lung cancer. However, the prevalence of cigarette smoking is low among Chinese females. The high mortality among Chinese females was unexplained by the habit of cigarette smoking. Thus, other risk factors may contribute the lung cancer incidence among Chinese females. Exposure to air pollutants, such as cooking oil fumes (COF), has been considered as an important risk factor for lung cancer. It was recently demonstrated that many environmental pollutants not only inhibited cell growth but also altered gene expression in lung cells. For example, diesel exhaust particle and air pollution particles induced cytokines production in human lung epithelial cells. Benzo[a]pyrene (B[a]P), a pulmonary carcinogen, was detected in COF and air particulates. Therefore, in the present study we investigated whether COF and B[a]P alter gene expression of cytokines, growth factors, and cytochrome P450 in human lung cancer cells CL3. CL3 cells were exposed to COF or B[a]P for 24 hrs and then the mRNA levels were measured with the RT-PCR method. When CL3 cells were treated with 200μg/ml COF, the mRNA level of TGF-β1 was increased to 4 fold of controls. The mRNA level of cytochrome P4502E1 (CYP2E1) was increased to 2 fold 24 hr after treatment. The mRNA levels of cytochrome P4501A1 (CYP1A1) and cytochrome P4501B1 (CYP1B1) were transiently increased to 2-4 fold of control, but returned to the basal levels at 24 hr. However, the mRNA levels of IL-2、IL-4、IL-6、IL-8、IL-13、IFN-γ、TNF-α and TGF-β2 were similar between control and treated cells. Similarly, treatment with 10μM B[a]P for 24hr also increased the mRNA levels of TGF-β1、CYP1A1 and CYP1B1 to approximately 3-4 folds of controls in CL3 cells. The mRNA levels of IL-2、IL-4、IL-6、IL-8、IL-13、IFN-γ、TNF-α and TGF-β2 were not changed after treatment with 10μM B[a]P. In summary, treatment with COF and B[a]P increased TGF-β1 mRNA levels in human lung CL3 cells. CL3 cells treated with COF or B[a]P also increased TGF-β1 secretion（measured by ELISA assay） in the dose- and time-dependent manner. It suggests that TGF-β1 may involve in the mechanism of COF and B[a]P-induced lung carcinogenesis. We further found that COF and B[a]P increased oxidative stress in CL3 cells. It implies that COF and B[a]P-induced TGF-β1 mRNA expression may relate to oxidative stress generation.
B[a]P induced gene expression of cytochrome P4501A1 gene expression and enzyme activity through activation of aryl hydrocarbon receptor（AhR）. We used three lung cancer cell lines CL3、A549 and H1355, with differential sensitivity to B[a]P, to study whether B[a]P-induced TGF-β1 gene expression is AhR-dependent. After treatment with 10μM B[a]P, growth of H1355 cells was significantly inhibited, but not A549 cells. In consistent with growth inhibition, CYP1A1 gene and enzyme inducibility by B[a]P was higher in H1355 cells than in CL3 and A549 cells. However, treatment with 10μM B[a]P increase TGF-β1 mRNA levels only in CL3 cells, but not in H1355 and A549 cells. When we examined the generation of oxidative stress by B[a]P treatment in these cell lines, oxidative stress was increased only in CL3 cells, but not in H1355 and A549 cells. These results suggest that TGF-β1 induction by B[a]P is AhR-independent, but may depend on the generation of oxidative stress. In summary, COF and B[a]P induced TGF-β1 production and oxidative stress generation in human lung CL3 cells. Therefore, TGF-β1 and oxidative stress may play a role in COF and B[a]P-related lung carcinogenesis. |
