III the medium of the EGF group was higher than that of the DMSO group (p < 0.05). However, there was no significant differen

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

 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/m L 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 + tyrpho stin 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 a poptotic 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 1 15% ，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。

Regulations of Apoptosis by Epidermal Growth Fa ctor and Its Signal Transduction Inhibitors in Huma

n Colorectal Cancer Cells



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 inve stigate the effects of EGF and its signal transduction inhibitors on apoptosis and its related proteins in human colorectal cance r 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% dimet hyl 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 2  25 ng/ml EGF + 215 ng/mL tyrphostin 51 (a tyrosine kinase blocker of EGFR) in 0.06% DMSO. Cells and media were collect ed after 48-h incubation. The results showed that cellular EGF contents in the EGF group were 418% of the DMSO group. W hereas, EGF contents in SW 480 cells were higher in the EGF + EGF antibody and EGF + tyrphostin 51 groups than those in t he 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 differen

ce among the EGF, EGF + EGF antibody and EGF + tyrphostin 51 groups. The expression of phosphorylated EGFR in the E

GF 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 t

he 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 anti

body 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 grou

ps was 124% and 133% of the EGF group, respectively. Therefore, high concentration of EGF (225 ng/mL) could stimulate a

ctivated 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 expressi

on of caspase-3 was elevated.