以干擾性核糖核酸剔除人類肺癌細胞CL1-0之穀胱甘肽還原酶基因所得選殖細胞株之cathepsin S蛋白酶表現以及其生物效應之研究

Cathepsin S expression and its effects in human lung adenocarcinoma CL1 cells after glutathione reductase (GR) siRNA transfection

林俞村(Yu-Cun Lin)

指導教授 ： 黃海美

國立清華大學/生命科學院/生物科技研究所/碩士(2011年)

摘要

肺腺癌原始細胞株CL1-0 (台大醫院楊泮池的病患樣品)，培養在24-孔盤，每孔含一塗佈Matrige/基底膜基質的小內插杯中，72小時後，穿透過膜的細胞繼續培養為CL1-1子細胞，再連續4次重複相似操作，取”穿透過的細胞” 繼續培養，一共獲得5種肺腺癌子細胞株CL1-X(1~5)。前人的實驗結果獲知：5種子細胞株之侵入能力是CL1-0原始細胞株的四至六倍，然而許多年來，它們的遺傳不穩定性而造成入侵能力不同的特性，並不十分了解。
最近，我們實驗室的數據顯示CL1-3細胞株之內源性與外源性半胱胺酸組織蛋白酶S (CTSS)的活性分別各為CL1-0的5~7倍與2~3倍；此外，與CL1-0原始細胞株之抗氧化相關分子相較，本研究顯示較高侵入能力且高CTSS的CL1-3細胞株只有原始細胞株的榖光甘肽還原酶(GR)含量的52.4%、榖光甘肽(GSH)含量的82.5%、榖光甘肽轉移酶(GST)含量的122.7%、榖光甘肽過氧化酶(Gpx)含量的113.6%。為了解CL1-3細胞是否因為由原始細胞株CL1-0經過 “穿越基底膜”之選殖”增加侵入能力”方式，而導致GR活性降低以及CTSS活性增高，本研究應用干擾性核醣核酸技術降低CL1-0細胞內的GR活性到近似CL1-3細胞中GR的含量(程度)，並觀察所得選殖細胞株之CTSS活性及其後續影響。
病毒載體含有GR之干擾性核醣核酸基因序列(編號二、三、五，購自中央央研究院國家型干擾性和醣核酸設施)以及抵抗嘌呤黴素基因序列，將之轉染至CL1-0細胞株內；病毒載體僅含有抵抗嘌呤黴素基因序列，轉染至CL1-0細胞株內則作為控制組實驗。前者所得抗嘌呤黴素細胞株，分別以酵素活性分析，並挑選出低活性之GR和高活性之CTSS的穩定性單一細胞株。在轉殖基因實驗1中，從含有嘌呤黴素之培養液挑選出來68株細胞，其中48株細胞(>70%)之GR活性低於33.1 ± 4.5 U/mg protein (接近於CL1-3細胞之背景值)。48株細胞中的19株細胞(28%)之GR活性約為原始細胞CL1-0中GR含量(33.1 ± 4.5 U/mg protein)的0.53倍 (接近於CL1-3細胞中GR含量)。將GR活性較低的10株 (<原始細胞CL1-0中GR含量之0.4倍)繼續測試其CTSS活性，與對照組比較後，發現只有一株細胞#3-16具有1.64倍的CTSS活性。在轉殖基因實驗2中，含有GR干擾性核醣核酸基因序列(編號五) ，以及抵抗嘌呤黴素基因序列之載體轉殖至CL1-0原始細胞株後，共挑選出抗嘌呤黴素之77株細胞，其中38株細胞(49.4%)之CTSS超過原始細胞CL1-0之活性(10.7 ± 0.4 RFU)，約為後者之1.1至2.0倍。38株細胞內有6細胞株 所含之CTSS活性超過原始細胞~1.5倍，各細胞株之GR活性，也比較接近於低GR活性的CL1-3細胞株。
從實驗1中選出細胞株3-13、3-16、5-04，實驗2中選出細胞株5-29來進行傷口癒合實驗(wound healing assay)及細胞穿透實驗(Boyden chamber assay)，分析各選出的細胞株的移動能力，與對照原始CL1-0細胞株相較之下，細胞株3-13(1.64倍之CTSS和0.22倍之GR活性)具有1.5倍的移動能力；細胞株5-29 (2倍之CTSS和0.38倍之GR活性)具有16倍的轉移能力，結果顯示: 低GR活性及高CTSS活性的細胞株具有較好的移動能力。因此，本研究證實: 經由干擾性核醣核酸技術降低原始CL1-0細胞株之GR活性，獲得一些比原始細胞低GR活性的細胞株，且可獲得具有高CTSS活性(1.5~2.0倍)，及高移動能力之細胞株。
在本研究的第二部分，選定NEM、PMSF及E-64這三種半胱胺酸蛋白酶抑制劑來抑制CL1-3細胞內CTSS的活性，經in vitro實驗發現: E-64之IC50 為5 nM， NEM之IC50 為800 μM。 至於PMSF抑制劑，對於CTSS的抑制程度與不加藥的控制組相比，並沒有統計上的差異；此外，in vivo處理細胞實驗結果則顯示: E-64之IC50 為10 μM，因此推論E-64具有良好的抑制CL1-3細胞內CTSS活性效果。
雖然前述選殖的細胞株3-13之GR活性是CL1-0細胞的0.22倍，但暴露於25~50 μM過氧化氫H2O2四小時後，相較於CL1-0細胞其存活率僅有些許不同, 對H2O2敏感程度大一些；然而，CL1-3細胞之GR活性為CL1-0之0.5倍，暴露於25~50 μM過氧化氫四小時或40 μM過氧化氫1~4小時之後，相較於相同處理之CL1-0細胞，兩者之存活率有顯著性的差異(p<<0.05)。CL1-3細胞的CTSS是CL1-0的6~7倍，除了其GR是CL1-0的0.5倍, 較高的6~7倍的CTSS活性是否增加了CL1-3細胞對於過氧化氫的敏感性，則有待進一步來研究。

關鍵字

穀胱甘肽還原酶 ； 半胱胺酸組織蛋白酶 ； 人類肺癌細胞CL1 ； 干擾性核糖核酸

並列摘要

CL1-X (1~5) cells as a lung-cancer metastasis cell model were established through Matrigel-coated Transwell-membrane in cell culture insert (in each well of 24-well micro-titer dish) after 72-h selection and 5 consecutive procedures from CL1-0 human lung adenocarcinoma in the laboratory of Dr. P. C. Yang from National Taiwan University Hospital. Their invasive abilities through basement membrane matrix showed a 4- to 6- fold increase over that of the parental cells. Nevertheless, not all characters for their genetic instabilities were known for many years.
Recently, data from our laboratory indicated that endogenous and exogenous cathepsin S (CTSS) protease activity in CL1-3 increased 5~7-fold and 2~3-fold, respectively, in comparison with those in CL1-0 parental cells. Measurement on several anti-oxidative molecules resulted in about one-half glutathione reductase (GR) activity (P<<0.01), less glutathione (GSH) (P<0.01), more GSH transferase (GST) (P<0.03), slightly more Glutathione peroxidase (GpX) activity (P=0.06-0.09) in CL1-3 cells having relatively more invasive (higher CTSS) abilities. To understand the causes of decreased GR and increased CTSS activity in CL1-3 cells after invasive ability selection from parental CL1-0 cells, gene manipulation and GR-siRNA transfection in CL1-0 cells to produce similar phenomenon as CL1-3 cells was conducted in this study.
GR-siRNA (#2, #3 and #5 different sequence, from Academia Sinica) and puromycin gene containing lentivirus vectors were transfected into CL1-0 cells. Clones containing low GR activity and high CTSS activity were selected. In Exp. I, among 68 clones picked from puromycin containing medium, 48 clones (>70%) had GR activity lower than 18.1 U/mg protein (close to background of CL1-3 cells). Among them, 19 clones (28%) showed lower than 0.53-fold of parental GR activity. Ten (relative lower GR, < 0.4-fold) of these 19 clones were chosen to further examine their CTSS activities. Only one clone, #3-16 (GR-siRNA #3 transfected) from all selected clones showed one-fifth GR activity (very low) and 1.64-fold endogenous CTSS activity in comparison with that in CL1-0-mock cells. In Exp. II, vectors containing GR-siRNA#5 and puromycin gene were transfected into CL1-0 cells. Among 77 clones picked from puromycin containing medium, 38 clones (49.4%) had 1.1~2.0-fold of parental CTSS activity. Six clones among these high CTSS clones contained more than 1.5-fold of parental CTSS activity and less than one-half (0.16~0.38-fold) of parental GR activity (close to background of CL1-3 cells).
Cell migration ability examination by means of wound healing assays and Boyden chamber assays was determined in clones 3-13, 3-16, 5-04 from Exp. I, clone 5-29 from Exp. II and parental CL1-0 cells, respectively. The results indicated that those clones from GR-knock down manipulation and high-CTSS selection have better cell migration abilities. Clone 3-13 [1.64-fold CTSS, 0.22-fold GR] and clone 5-29 [2.0-fold CTSS, 0.38-fold GR] showed 1.5-fold and 16-fold of parental cell migration ability. Therefore, clones containing higher CTSS activity (1.5~2.0-fold) and cell migration abilities than parental cells had been obtained from genetic manipulation to lower down GR activity in parental CL1-0 cells.
In the second part of this study, CTSS inhibitors, L-trans-epoxysuccinyl- L-leucylamido (4-guanidino) butane (E-64), N-Ethylmaleimide (NEM) and phenylmethanesulfonyl fluoride (PMSF) were applied in CL1-3 cells. E-64 reduced 50% CTSS activity at 5 nM in vitro and 10 μM in vivo. In addition, NEM reduced 50% CTSS activity at 800 μM in vitro. However, there was no significant inhibition on CTSS activity after PMSF treatment. Therefore, E-64 was a considerable potent inhibitor.
Although clone 3-13 had 0.22-fold of parental GR activity, clone 3-13 showed slightly SRB viability difference from parental CL1-0 cells after H2O2 exposure for 4 h. In contrast, CL1-3 cells, containing 0.5-fold GR activity of parental CL1-0 cells, showed significant different sensitivity to H2O2 (4 h at 25~50 μM dose or 1~4 h at 40 μM), from CL1-0 cells. CL1-3 cells have 6~7-fold of parental CTSS activity. Whether higher CTSS activity in CL1-3 cells further increased H2O2 sensitivity (in comparison with clone 3-13 cells) remained to be further investigated.

並列關鍵字

glutathione reductase ； cysteine cathepsin S ； human lung adenocarcinoma CL1 cells ； siRNA

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