| Abstract: | 泌乳激素(Prolactin)為一多功能的內分泌(endocrine)荷爾蒙，除了具有促進乳房成長及發育、乳腺分化及乳汁分泌等功能，還被發現具有三百多種作用，其中包括調節細胞功能，如繁殖、分化、血管新生、抗細胞凋亡及發炎反應，作用如同細胞激素(cytokine)。在前人NB2 T細胞的研究上，泌乳激素可提高抗細胞凋亡蛋白質，如Bcl-2及Bcl-xL，以抵抗荷爾蒙缺乏或類固醇所引起的細胞凋亡。另外泌乳激素可以提升鳥胺酸去羧化（Ornithine decarboxylase, ODC, EC 4.1.1.17）的活性。鳥胺酸去羧化催化鳥胺酸（L-ornithine）成為腐胺（putrescine）及二氧化碳，為製造多胺酸（polyamines）生合成的第一個，也是速率決定步驟之酵素，具有調控細胞發育、分化、轉形、生長和凋亡等功能。本實驗室之前的研究發現，過度表現的鳥胺酸去羧化可以對抗胺甲葉酸所引起的細胞凋亡現象，其機制為減少藥物導致的活性氧分子、增加Bcl-2的表現、穩定粒線體膜電位、抑制cytochrome c放及劊蛋白活化。接著我們進一步研究是否泌乳激素也可藉由提高鳥胺酸去羧化的活性，達到抗細胞凋亡的效果，同時分析鳥胺酸去羧化與抗細胞凋亡蛋白質Bcl-2的關係。依據我們實驗的結果，泌乳激素呈現隨劑量增加而增加抗胺甲葉酸所引起人類前骨髓白血球癌母細胞株（human promyelocytic leukemia, HL-60 cells）的細胞凋亡作用，包括增加細胞的存活、減少凋亡小體的出現、減少去氧核醣核酸的片斷化及流式細胞儀分析Sub-G1的比例。經由泌乳激素的刺激，人類前骨髓白血球癌母細胞株細胞的鳥胺酸去羧化活性於三小時達到高峰，隨後逐漸減少，的活性與其劑量呈正相關。泌乳激素提高鳥胺酸去羧化活性的現象可被PKC-delta抑制劑rottlerin完全抑制。然而鳥胺酸去羧化的傳訊核醣核酸及蛋白質的表現卻不受泌乳激素的影響。由此可推論泌乳激素是藉由PKCdelta抑制劑rottlerin完全抑制。然而鳥胺酸去羧化的傳訊核醣核酸及蛋白質的表現卻不受泌乳激素的影響。由此可推論泌乳激素是藉由PKC 作用造成蛋白質轉譯後調升的活性。鳥胺酸去羧化與Bcl-2的關係上，我們發現在泌乳激素刺激後的一個小時即可發現抗細胞凋亡蛋白Bcl-2的增加，此一增加不受鳥胺酸去羧化不可逆抑制劑DFMO (DL-alpha-difluoromethylornithine)的影響，刺激四小時後Bcl-2更明顯地增加，此一後續的增加表現則會被DFMO抑制，而Bcl-xL則沒有變化。無論使用Bcl-2抑制劑HA14-1或轉殖基因以過度表現Bcl-2，都無法改變細胞鳥胺酸去羧化的活性及蛋白質表現。然而過度表現的鳥胺酸去羧化則可以增加Bcl-2的表現，且 DFMO可以減少Bcl-2表現及鳥胺酸去羧化對其的作用。因此泌乳激素以兩種不同的機制先增加Bcl-2，接著再調升鳥胺酸去羧化的活性，活化的鳥胺酸去羧化再進一步使Bcl-2更增加。而Bcl-2不影響鳥胺酸去羧化。泌乳激素抗細胞凋亡的作用會受到DFMO的影響而降低，如果再外加鳥胺酸去羧化的產物，即腐胺，則此一影響可被回復。顯然鳥胺酸去羧化活性也是泌乳激素可以對抗細胞凋亡的機制之一。同時使用DFMO及HA14-1則可完全抵銷泌乳激素抗細胞凋亡的作用，似乎調升Bcl-2及鳥胺酸去羧化活性為泌乳激素抗細胞凋亡主要的兩個機制。最後我們的結論是泌乳激素增加鳥胺酸去羧化活性為其抗胺甲葉酸所引發細胞凋亡之另一機制，被提升的鳥胺酸去羧化活性進一步增加抗細胞凋亡蛋白Bcl-2，以發揮其最大保護作用。
Prolactin is a versatile hormone, produced in the anterior pituitary gland, and affects the growth, differentiation and secretion of mammary glands. In addition, it has more than 300 separate functions, including regulating cellular functions, such as proliferation, differentiation, angiogenesis and protection against apoptosis and inflammation, as a cytokine. In the previous studies on NB2 T cells, prolactin induces Bcl-2 and Bcl-xL expression to prevent apoptosis under the conditions of lactogren deprivation or following exposure to glucocorticoids. Prolactin also induces the activity of ornithine decarboxylase (EC 4. 1. 1. 17; ODC). ODC is the first and rate-limiting enzyme in the synthesis of polyamines. ODC decarboxylates L-ornithine to form putrescine. Polyamines (putrescine, spermidine and spermine) and ODC play an important role in diverse biological functions including cell development, differentiation, transformation, growth and apoptosis. Our previous studies showed overexpression of ODC prevents MTX-induced apoptosis. Overexpression of ODC can reduce reactive oxygen species (ROS), increase Bcl-2 expression, stabilize mitochondrial membrane potential, inhibit cytochrome c release and prevent caspase activation. Here, we further investigate whether prolactin can prevent MTX-induced apoptosis through inducing ODC activity or not, and the relationship between ODC and Bcl-2. Prolactin prevents MTX-induced apoptosis in a dose dependent manner, in the experiments of cell viability and DNA gel electrophoresis. Decreased apoptotic bodies and sub-G1 ratio by flow cytometry are also observed. After being stimulated by prolactin, ODC activity is increased dose-dependently to maximum at 3 h in HL-60 cells, and then it declines. Prolactin-induced ODC activity can be blocked completely by rottlerin, a PCK inhibitor. However, mRNA and protein expression of ODC don’t change after prolactin treatment. It indicates prolactin induces ODC activity by post-translation regulation through PKC. In the studies of the relationship between ODC and Bcl-2, we found prolactin induces Bcl-2 expression in the first hour. This effect of prolactin on Bcl-2 in the first hour is not inhibited by DL-alpha-difluoromethylornithine (DFMO, an irreversible inhibitor of ODC). After being stimulated by prolactin for 4 hours, the expression of Bcl-2 is further enhanced. DFMO block this enhancement at the time when ODC activity is up-regulated by prolactin. Bcl-xL doesn’t change during prolactin treatment. There are no changes in ODC activity and protein expression in cells treated by HA14-1 or Bcl-2-overexpressing cells, compared with controlled cells. Overexpression of ODC increases the expression of Bcl-2. DFMO decreases the expression of Bcl-2 and inhibits the effect of ODC overexpression on Bcl-2. According to our results, it is demonstrated clearly that there are two different ways of prolactin to induce Bcl-2, and then ODC activity. Induced ODC activity further enhances the expression of Bcl-2. Bcl-2 doesn’t affect ODC. The anti-apoptotic effect of prolactin is diminished by DFMO and recovered by putrescine. Obviously, prolactin-induced ODC activity is one of the mechanisms of anti-apoptosis. HA14-1, together with DFMO, blocks the anti-apoptotic effects of prolactin completely. In conclusion, increasing ODC activity is another way of prolactin to prevent MTX-induced apoptosis. Prolactin-induced ODC activity further enhances the expression of Bcl-2 to bring the anti-apoptotic function to full play. |
