本研究主要探討Pipoxolan對於抑制人類前骨髓性白血病細胞株HL-60的增生以及其相關機制。Pipoxolan是臨床上普遍被使用的ㄧ種藥物，作用於平滑肌的舒緩與解痙攣，尤其是腸胃道、膽道、泌尿道引起的疼痛，或是經痛、偏頭痛都有明顯的改善作用。
在我們的實驗研究中發現，藉由Trypan Blue assay可以得知Pipoxolan會隨著濃度的增加，相對抑制人類前骨隨性白血病細胞株HL-60的增生。此外，Pipoxolan會誘導細胞凋亡，藉由流式細胞儀的分析中得知隨著給予不同濃度的Pipoxolan (6.25,12.5,25及 50μg/ml)處理24小時後，sub-G1(apoptosis)的表現量顯著的增加，DNA斷裂情形也被觀察到(6.25,12.5, 25μg/ml)，且poly (ADP) ribose polymerase (PARP)也有裂解情形。Pipoxolan會抑制細胞週期的進行，並使細胞週期停滯於G0/G1期。同時藉由DAPI螢光染劑的實驗中可發現，Pipoxolan會誘導凋亡小體的形成，當以濃度6.25μg/ml處理自2小時到48小時可見隨時間增加凋亡小體的表現量也增加。
進ㄧ步分析蛋白質的表現量發現，Pipoxolan可以誘導p53的表現，並且活化p21以致作用在Cyclin D1使細胞週期停滯於G0/G1期。並且經由減少抗凋亡蛋白質Bcl-2的表現；增加誘導凋亡蛋白質Bax的表現，使得位於粒腺體中的cytochrome c 釋放到細胞質中，同時也會增加細胞內ROS的表現量，大量的ROS被表現後作用於p53及粒腺體上促使cytochrome c 更多釋放於細胞質中，活化caspase-9、caspase-3走向凋亡路徑。
因此我們可以確立，Pipoxolan可誘導HL-60產生細胞凋亡，並且藉由抑制細胞週期停滯使之無法進行增生、分裂，也會誘導細胞凋亡之內在路徑的活化並且增加細胞內ROS的含量，使血癌細胞HL-60走向凋亡。
The object of this study is to investigate the anti-proliferative effect and the mechanisms of Pipoxolam toward HL-60 cells. Pipoxolan, a well-known smooth muscle relaxant and an anti-analgesic, alleviates spasm of the stomach, intestine, bile duct and urinary tract. It could also relieve the menstrual pain and migraine headache.
In the present study, we reported that Pipoxolan could reduce human promyelocytic leukemia cells (HL-60) proliferation in a dose dependent manner by Trypan Blue assay. It could also induce apoptosis in HL-60 characterized by flow cytometry array showing sub-G1 significant increase following 24 h exposure to various dosage (6.25,12.5,25 and 50μg/ml) of Pipoxolan. DNA fragmentation (6.25,12.5 and 25μg/ml) and poly (ADP) ribose polymerase (PARP) cleavage were significantly enhanced and simultaneously arrested the cell cycle in G0/G1 phase. DAPI staining was significantly when conducted with 6.25μg/ml pipoxolan for 2~48h incubation of HL-60 cells.
Further molecular analysis showed that Pipoxolan caused p53- mediated apoptosis by enhanceing p21 then decreasing cyclin D1 to arrest the cell cycle in G0/G1 phase. It decreased anti-apoptosis protein Bcl-2 and induced Pro-apoptosis protein Bax expressions, causing cytochrome c release, cleavage of procaspase -9 and -3, and hydrolysis of PARP. Intracellular reactive oxygen species (ROS) seem to play key role since high levels of ROS were produced early (<15min) of the experiment.
These findings suggest that Pipoxolan-induced cell cycle arrest in HL-60 cells by apoptosis. And ROS could probably be involved the initiation of the apoptosis, and caused mitochondrial dysfunction pathway.
