探討抗阿黴素之非小細胞肺癌細胞株NCI-H23在乙型轉型因子調控下訊息傳遞路徑的改變

化學療法是今日控制轉移性腫瘤生長最常使用的方法之一，然而也因此發生了腫瘤細胞產生抗藥性的問題，已經有報導指出抗藥性的產生，往往加速了腫瘤細胞更快的生長導致病情惡化。因此，本篇論文研究利用臨床上普遍使用的抗癌藥物Adriamycin(ADM)，對於人類非小細胞肺腺癌(Non-small Cell Lung Carcinoma, NSCLC)細胞株NCI-H23所產生的抗藥性而引發細胞不受TGF-β的生長抑制調控加以探討。以NCI-H23細胞株做為本研究計劃的實驗用細胞，經過ADM的處理進行長期的培養，最後得到一株具有抗藥能力(0.1 μM ADM)的細胞株(NCI-H23/ADR)。在生長速率的實驗中，發現NCI-H23母株受到TGF-β1誘導，使得細胞倍數生長時間由36小時遲緩至96小時，但在抗藥株中並不受到調控。利用RNA萃取技術，收集原母細胞株及抗藥性細胞株的total RNA，並以RT-PCR的技術觀察MDR-1及TGF-β相關基因表現情形，也未發現兩株細胞TGF-β訊息傳遞系統的基因表現有所差異。但經由Luciferase assay則發現NCI-H23母株及抗藥株細胞受到TGF-β1時，相較於貂肺上皮細胞株(Mv1Lu cell line)，Smad蛋白並沒有完全的被活化來啟動目標基因，在流式細胞儀分析細胞週期的G1 arrest及細胞凋亡的結果中，NCI-H23細胞亦皆完全不受TGF-β1的影響，兩株細胞皆沒有明顯的細胞週期G1 arrest和細胞凋亡現象。最後以西方點墨法(Western blotting)分析和細胞生長有關的蛋白在TGF-β誘導下的表現，包括MAPK成員，如ERK、p38以及JNK等激酶活性，還有AKT/PKB、Smad2和p53蛋白的表現程度，在NCI-H23母株及抗藥株細胞中的表現並沒有顯著的差異。但在PKC活化的表現上，可以看到NCI-H23母株細胞在TGF-β誘導下，48小時後提高了PKC的活性，相對地，在抗藥株細胞中，一直遲至96小時才觀察到PKC的活化表現。另外，在STAT3的活化表現上，僅僅發生在TGF-β誘導了NCI-H23母株細胞上，而在抗藥株細胞方面則不受到TGF-β所調控，因此，NCI-H23母株細胞生長週期的延遲可能與STAT3在NCI-H23母株細胞中漸增的活化有所關聯。本篇研究的結果顯示，當NCI-H23細胞獲得抗Adriamycin的抗藥性之後，便失去了受TGF-β所調控的生長抑制，而抗藥性的獲得並不會造成NCI-H23細胞株的TGF-β接受器蛋白表現有所差異。NCI-H23母株細胞受到TGF-β1的生長抑制作用並非由細胞週期的G1 arrest或是造成細胞凋亡所引起，而是延遲了細胞的生長週期。其間TGF-β的傳遞也並非經由Smad蛋白所引導的訊息傳遞系統，而可能透過JAK/STAT路徑的傳導，NCI-H23母株經過TGF-β1處理後，STAT3漸增的活化可能就是導致細胞生長遲滯的原因。

關鍵字

乙型轉型因子

並列摘要

Chemotherapy is a common therapy used for controlling metastatic tumor growth on cancer treatment today. However, it also often causes tumor cells resistant to the treatment. The displaying drug-resistance has been reported to be strongly associated with tumorgenesis. Therefore, this research is going to focus on how adriamycin-resistant cells prevent from TGFβ-mediated cell growth inhibition by altering its downstream signaling pathways in a non-small cell lung carcinoma cells (NSCLC), NCI-H23 cells. NCI-H23 Adriamycin —resistant (H23/ADR) cells are more insensitive to TGFβ-mediated cell growth inhibition than their parental (H23P) cells after exposure of 5 ng/ml TGFβ1 treatment. The doubling times of H23/ADR are about 36 hrs when compared to 96 hrs for H23P cells. RT-PCR to quantify the TGFβ receptors and their ligands showed no any distinct differences between these two cell lines except for TGFβ2 ligands mRNA level were not detectable in the H23/ADR. By Western blotting analysis using anti-phospho-Samd2 antibody or by a luciferase reporter gene assay, the results all indicated Smad-mediated signaling pathways were defect in both cell lines, suggesting that TGFβ-mediated cell growth inhibition in H23P cells is Smad-independent. The suppression of the cell growth on H23P cells did not cause cell cycle G1 arrest, but also not induced cell apoptosis, in stead, increasing their cell cycle doubling time. The increased cell cycle doubling time was not associated with activations of MAPK kinase members (ERK, p38 and JNK), AKT/PKB, Smad2, and protein level of p53 since the activities of these cell recycle regulators did not demonstrate distinguished differences in both cells except for PKC activation. The increasing PKC activation was observed in H23P cells (48 hrs post-treatment) much earlier than that in the H23/ADR cells (96 hrs post-treatment). Furthermore, the increasing activity of Stat3 in NCI-H23 cells was only seen in H23P cells, but not in H23/ADR cells, suggesting this pathway may be associated with the cell growth inhibition of H23P cells. In future, further experiments are needed to confirm its activity and explore how H23/ADR cells alter this signaling pathway to escape from TGFβ-mediated growth block.

並列關鍵字

TGF-β

參考文獻

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探討抗阿黴素之非小細胞肺癌細胞株NCI-H23在乙型轉型因子調控下訊息傳遞路徑的改變

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