上皮生長因子與其訊號傳遞抑制劑對人結直腸癌細胞程式化凋亡作用的調控

摘要上皮生長因子 (epidermal growth factor; EGF) 可與細胞表面的上皮生長因子接受器結合，調控正常與腫瘤腸細胞的增殖作用。本研究之目的乃在探討上皮生長因子及其訊號傳遞抑制劑，對於人類結直腸癌細胞之程式化凋亡作用及其相關蛋白的影響。將人類結直腸癌細胞 (SW480) 培養於含有90% Leibovitz’s L-15/10%胎牛血清，使其生長至90%滿後，轉換成不含血清的基本培養液培養24小時。之後分別加入0.06% dimethyl sulfoxide (DMSO)、225 ng/mL EGF、225 ng/mL EGF + 2.5 ?g/mL EGF antibody或225 ng/mL EGF + 215 ng/mL tyrphostin 51 (上皮生長因子受器酪胺酸激酉每抑制劑) 至細胞培養液。由於tyrphostin 51需溶解於DMSO，所以其餘各組需同時加入等量的DMSO，以校正此因子。持續培養48小時後，收集細胞及培養液，每一組皆為七重複試驗 (n=7)。結果顯示添加EGF組細胞內EGF含量為DMSO組之418%；而EGF + EGF antibody 組和EGF + tyrphostin 51組皆明顯高於EGF組 (p < 0.05)；EGF組培養液內EGF含量明顯高於DMSO組 (p < 0.05)；而EGF組、EGF + EGF antibody組與EGF + tyrphostin 51組無明顯差異。活化型上皮生長因子受器 I 的表現於EGF組為DMSO組之183%；而EGF + EGF antibody組與EGF + tyrphostin 51組則分別為EGF組之36%與79%。觀察細胞程式化凋亡的結果，發現於12小時，EGF組與DMSO組之apoptotic cells數目無明顯差別。EGF + EGF antibody組與EGF + tyrphostin 51組之apoptotic cells數目在12小時，明顯比EGF組少；而EGF + EGF antibody組與EGF + tyrphostin 51組比較在12小時，並無明顯差別。此外，p53蛋白的表現於EGF組為DMSO組之102%,而EGF + EGF antibody組與EGF + tyrphostin 51組分別為EGF組之97%與83%；p21蛋白的表現於EGF組為DMSO組之115%，而EGF + EGF antibody組與EGF + tyrphostin 51組分別為EGF組之5%與51%。Caspase-3酵素的表現於EGF組為DMSO組之139%，而EGF + EGF antibody與EGF + tyrphostin 51組分別為EGF組之124%與133%。因此，在高濃度EGF (225 ng/mL) 下，會促進活化型上皮生長因子受器的表現，進而誘導caspase-3表現，若再添加EGF訊號傳遞抑制劑，則會降低活化型上皮生長因子受器、p53與p21蛋白的表現，並抑制apoptosis的進行，但caspase-3的表現反而會增加。

關鍵字

上皮生長因子； p21 ； p53 ； caspase-3 ；人類結直腸癌細胞；上皮生長因子抗體； tyrphostin 51

並列摘要

ABSTRACT Epidermal growth factor (EGF) has been considered to regulate the proliferation of normal and tumor intestinal cells through the EGF receptor (EGFR) on the cell surface. The study was to investigate the effects of EGF and its signal transduction inhibitors on apoptosis and its related proteins in human colorectal cancer cells. Human colorectal adenocarcinoma cells (SW480) were grown in 90% Leibovitz’s L-15 medium with 10% fetal bovine serum until 90% confluency, and switched to serum-free medium for 24 h. Cells (n = 7/group) were treated with 0.06% dimethyl sulfoxide (DMSO), 225 ng/mL EGF in 0.06% DMSO, 225 ng/mL EGF + 2.5 ?g/mL EGF antibody in 0.06% DMSO, or 225 ng/ml EGF + 215 ng/mL tyrphostin 51 (a tyrosine kinase blocker of EGFR) in 0.06% DMSO. Cells and media were collected after 48-h incubation. The results showed that cellular EGF contents in the EGF group were 418% of the DMSO group. Whereas, EGF contents in SW 480 cells were higher in the EGF + EGF antibody and EGF + tyrphostin 51 groups than those in the EGF groups (p < 0.05). The level of EGF in III the medium of the EGF group was higher than that of the DMSO group (p < 0.05). However, there was no significant difference among the EGF, EGF + EGF antibody and EGF + tyrphostin 51 groups. The expression of phosphorylated EGFR in the EGF group was 183% of the DMSO group. Phosphorylated EGFR in the EGF + EGF antibody and EGF + tyrphostin 51 groups was 36% and 79% of the EGF group, respectively. In addition, the expression of p53 protein in the EGF group was 102% of the DMSO group, and that in the EGF + EGF antibody and EGF + tyrphostin 51 groups was 97% and 83% of the EGF group, respectively. The expression of p21 protein in the EGF group was 115% of the DMSO group, and that in the EGF + EGF antibody and EGF + tyrphostin 51 groups was 5% and 51% of the EGF group, respectively. However, the expression of caspase-3 enzyme in the EGF group was 139% of the DMSO group, and that in the EGF + EGF antibody and EGF + tyrphostin 51 groups was 124% and 133% of the EGF group, respectively. Therefore, high concentration of EGF (225 ng/mL) could stimulate activated EGFR expression, and further induce caspase-3 expression. After the addition of signal transduction inhibitor IV of EGF, the expression of activated EGFR, p53 and p21 proteins was decreased, and apoptosis was inhibited, but the expression of caspase-3 was elevated.

並列關鍵字

Key words: epidermal growth factor ； p21 ； p53 ； caspase-3 ； human colorectal adenocarcinoma cells ； EGF antibody ； tyrphostin 51

參考文獻

Barnes, DW (1982) Epidermal growth factor inhibits growth of A431 human epidermoid carcinoma in serum-free cell culture. J Cell Biol 93: 1-4.

Boldin, MP, Goncharrov, T, Golstev, YV and Wallach, D (1996) Involvement of MACH, a novel MORT1/FADD-interacting protease, in FAS/APO-1- and TNF receptor-induced cell death. Cell 85: 803-815.

Brabyn, CJ and Kleine, LP (1995) EGF causes hyperproliferation and apoptosis in T51B cells: involvement of high and low affinity EGFR binding sites. Cell Signal 7: 139-150.

Brugarolas, J, Chandrasekaran, C and Gordon, JI (1995) Radiation-induced cell cycle arrest compromised by p21 deficiency. Nature 377: 552-557.

Buckbinder, L, Talbott, R and Velasco-Miguel, S (1995) Induction of the growth inhibitor IGF-binding protein 3 by p53. Nature 377: 646-649.

國際替代計量

上皮生長因子與其訊號傳遞抑制劑對人結直腸癌細胞程式化凋亡作用的調控

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