| 摘要: | 不受控制的細胞分裂為惡性腫瘤發展的根本原因，在各種人類癌症中皆發現細胞中的細胞週期素 (cyclins) 過度表達或是細胞週期素依賴性激?抑制物 (CKIs) 的缺失以及腫瘤抑制因子的突變與缺失，導致細胞內的檢查點監控機制發生故障，癌細胞因而獲得抵抗凋亡的能力，而細胞無止盡的增生最終將發展成腫瘤。膀胱癌屬於人類泌尿系統中常見的腫瘤之一，對於初期膀胱癌之治療方案，一般採用手術後輔以膀胱灌注法之方式，將藥物直接灌注至病患膀胱中，透過藥物毒性徹底殺死癌細胞，以避免腫瘤復發。灌注藥物期間，病患膀胱內的壓力也會提高，有學者指出此種壓力刺激可能具有抑制細胞增生的效果，且配合藥物刺激有助於提升誘導膀胱癌細胞凋亡的效率，但仍不清楚此種機械刺激在細胞中所扮演的角色。因此，本研究將在符合生理情況下，探討靜水壓刺激對於膀胱癌細胞之影響。
本研究以靜水壓生物反應器系統對BFTC905膀胱癌細胞株施以靜水壓刺激，透過流式細胞儀分析與西方墨點分析證實，以10 kPa之靜水壓刺激5小時期間內並不會誘導細胞凋亡，也不會誘導p53的表現，然而處於G1期之細胞量比例卻明顯增加了，說明靜水壓刺激確實會誘導G1停滯。根據實驗結果，我們發現不論有無施加靜水壓刺激，皆不影響cyclin D (D1、D3)、CDK4與CDK6的表現，這可能是由於INK4蛋白質的缺失所致。在加壓2.5小時內，處於G2/M之細胞量比例降低，大部分細胞停留於S期，且Cdc2於Tyr15的磷酸化程度增加，但是持續加壓至5小時後，大部分細胞卻停滯於G1期，而且不僅cyclin E1與CDK2的表現下降，甚至cyclin A2與cyclin B1的表現也都降低了，加上組蛋白H3於Ser10皆維持低磷酸化的狀態，這些證據表示靜水壓刺激可能會先延遲S/G2的進展，同時誘導細胞細胞週期停滯於G1期。此外不同於p21，在BFTC905細胞株中p27的表達明顯受到抑制，然而經過靜水壓刺激後p27卻大幅地上升，而p21則僅有些微的增加，接著我們也發現Rb於Ser807/811的低磷酸化，這些證據說明了靜水壓刺激將透過p53-非依賴性路徑提高p21與p27的表現，抑制cyclin E1-CDK2並且間接影響了Rb於Ser807/811的磷酸化程度以誘導G1停滯。
Uncontrolled cell division is the cause of malignant cell development. To date, there are large amount of literatures that investigate the cause of cancer and try to develop applicable strategies for cancer therapy. It has been well addressed that the mutation of tumor suppressor, overexpression of cyclins or loss of CKIs are common in a variety of human cancers. Those mutations, overexpressions or deletions will result in the malfunction of regulatory checkpoints mechanism of the cell, or make the cancer cells obtain abilities to resist apoptosis. At the end, unlimited cell proliferation will eventually lead tumorigenesis.
Bladder cancer is a common cancer in human urinary system. In addition to the primary surgical treatment, patients will be treated by adjuvant intravesical therapy that utilize chemotherapy agents such as mytomicin C to kill the remaining cancer cells after the surgery to prevent tumor recurrence. However not only the cytotoxic stress for cells but also the pressure effect were present in the bladder during the intravesical therapy. It is suggested that hydrostatic pressure stimulation may have the ability to inhibit cancer cell proliferation, because the drugs induced apoptosis will be enhanced by applying the hydrostatic pressure. However, the cellular mechnism of this mechanical stress still remain unclear.
This study applied the hydrostatic pressure bioreactor system to provide the hydrostatic pressure (10 kPa) on BFTC905 cells, a cell line of bladder cancer, and investigated how this mechanical stress effects the bladder cancer cells under physiological conditions. Results from flow cytometry showed most of cells stay in G1 phase, and rare cells go into apoptosis after 5 h hydrostatic pressure application. And we also found undetectable p53 expression after treated hydrostatic pressure by Western blot analysis no matter we applied hydrostatic pressure or not in BFTC905 cells. Our data shown the expression of cyclin D (D1, D3) and CDK4, CDK6 were the same as control cells after treatment, and these would be the results of loss of INK4 proteins in BFTC905 cells. However, under hydrostatic pressure stimulation, we found most of cells stayed in S phase and cells in G2/M were declined, also the phosphorylation state of Cdc2 on Tyr15 was increased within 2.5 h. But after treatment for 5 h, most of cells stayed in G1 phase and cyclin E1 and CDK2 were decreased. Not only that, but the data also shown both of cyclin A2 and cyclin B1 were down-regulated after treatement for 5 h, and Ser10 of histone H3 remained in the hypophosphorylation state. These evidences suggested hydrostatic pressure stimulation might cause cell cycle delay in G2/M transition and induce G1 arrest, simultaneously.
Futhermore, unlike p21, p27 levels were strongly inhibited in BFTC905 cells in the originated status. However, p27 levels were significantly increased by hydrostatic pressure application whether p21 levels just slightly increase. Also Ser807/811 of Rb became hypophosphorylation after treatment within 2.5 h or 5 h. These finding concluded that hydrostatic pressure stimulation induce G1 arrest by elavated p21 and p27 levels in a p53-independent manner, results cyclin E1 and CDK2 degradation, and hypophophorylation of Ser807/811 of Rb. |
