Abstract
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.