水解磷脂酸對人類子宮頸癌細胞及卵巢癌細胞的生物效應探討

Abstract

Lysophosphatidic acid (LPA)是人體內結構最簡單的磷脂質，在人體中會表現LPA的細胞包含活化的血小板、脂肪細胞、血管內皮細胞，事實上正常微量的LPA對人體內的血管具保護作用、與神經痛覺傳導、胚胎著床、免疫發炎反、傷口癒合應均有關聯。而上述生理作用中的LPA來源主要為活化的血小板，也因此心血管疾病也被研究發現有LPA的異常表現情況。此外，在基礎的研究上則發現卵巢癌細胞與子宮頸癌細胞在受到刺激或平時都會產生並釋放LPA。 LPA在人類腫瘤的角色探索起源於對卵巢癌病人腹水中的growth factors 及cytokines，LPA具有引發卵巢癌細胞胞內鈣離子濃度變化的特性。LPA在婦科癌症(卵巢癌、子宮頸癌、子宮內膜癌)病人血清中會顯著上升，被認為具有發展為診斷標記的潛力。也由此使LPA在卵巢癌中的角色逐漸被研究探討，目前為止卵巢癌是所有人類癌症被認為與LPA最具關聯性的類型，而其在卵巢癌的作用機轉及生物效應也被研究得最為深入。進年來，LPA在其他人類腫瘤如乳癌、大腸癌、子宮頸癌、前列腺癌的研究也日漸受到重視，被認為是癌症治療極佳的分子標地之一。 臨床的研究發現LPA在卵巢癌病人的腹水及其他婦科腫瘤病人血清中的濃度是有意義升高的，其專一性受體LPA1/Edg2, LPA2/Edg4 及 LPA3/Edg7的表現模式在正常細胞與腫瘤細胞有很顯著的差別；正常細胞被發現僅表現LPA1/Edg2。而腫瘤細胞則具有LPA2/Edg4 and LPA3/Edg7的表現。LPA主要會透過其專一性的受體LPA1/Edg2, LPA2/Edg4 及 LPA3/Edg7與G-protein- coupled receptors經由不同的訊息傳遞路徑，調控癌細胞的增生、抗凋亡、轉移侵襲能力、血管新生能力以及抗藥性等生理功能。治療性的基礎研究也發現調控LPA的代謝、專一性的受體的表現或阻斷其訊息傳遞路徑都對卵巢癌治療有很好的效果。 本論文分為三個部分。第一部分，主要探討LPA在卵巢癌細胞調控IL-6表現的機轉，我們的研究結果顯示LPA會藉由PTX敏感性G型蛋白質受體活化PI3K/Akt/NF-kB的訊息傳遞途徑增強卵巢癌細胞表現IL-6。我們的研究結果顯示LPA能透過Gi/PI3K/NFkB的訊息傳遞途徑增強或誘發卵巢癌細胞表現IL-6，此一結果為卵巢癌病人血清或腹水中IL-6的高表現量提出一個來源的解釋。第二部分，主要探討抗血小板活化藥物YC-1應用於LPA活化之細胞生物效應的可行性。本研究中我們以LPA會誘發細胞內鈣離子濃度變化的特性，測試能抑制細胞內鈣離子濃度變化的藥物 YC-1對於LPA誘發作用的影響。結果顯示YC-1能透過抑制LPA誘發的β-integrin的表現而影響卵巢癌細胞附著，另一方面YC-1也能抑制LPA對懸浮狀態巢癌細胞的抗凋亡作用以及能抑制血管內皮細胞的VEGFR2的活化而影響LPA調節之促血管新生作用。此一結果顯示YC-1應用於LPA活化之細胞生物效應的可行性。第三部分，主要探討LPA在子宮頸癌細胞的生物效應。我們的研究結果顯示LPA對於子宮頸癌細胞具有促血管新生、促細胞移行侵襲及增加對放射線治療的抗性等作用。我們也發現LPA在子宮頸癌細胞的促血管新生效應與對放射線治療的抗性等效應，分別與LPA會誘發子宮頸癌細胞產生IL-8及IL-6有關。此部分之結果在現有文獻中尚未有類似結果發表。 綜合我們的研究結果與國際上的相關文獻的知識，我們認為作用於LPA專一性的受體或作用於LPA活化之訊息傳遞路徑或調控LPA的代謝等方式將是以LPA為分子治療標的的發展方向。此外我們也期望藉由本研究結果闡明LPA在婦癌細胞惡性化過程中可能具有的生物效應與作用機轉進而尋求可能的治療方式，以釐清將LPA作為婦癌分子治療標的可行性。

Bioactive lysophospholipid, lysophosphatidic acid (LPA), is consistently raised in the ascites of patients with ovarian cancer and cervical cancer. In this study, we elucidate the role of LPA in the pathogenesis of ovarian cancer and cervical cancer. In the first chapter, we investigated how LPA affects IL-6 production in ovarian cancer cells. Experimental results indicated that LPA stimulates IL-6 expression in all ovarian cancer cell lines tested, but not in normal ovarian surface epithelial (NOSE) cells, owing to the lack of LPA specific Edg4 and/or Edg7 receptors in NOSE cells. This work demonstrated that LPA transcriptionally activates IL-6 expression, which can be totally blocked by pertussis toxin, indicating that Gi-mediated signaling is critically involved in inducing IL-6 by LPA. Pharmacological and genetic inhibition assays revealed that Gi-mediated PI3K activation phosphorylated downstream Akt and subsequently induced NF-kB activation causes the induction of IL-6 by LPA in SK-OV-3 cells. In sum, dada presented here demonstrate that LPA is an important inducer of IL-6 and LPA regulated IL-6 expression via a Gi/ PI3K-Akt/ NF-kB pathway in ovarian cancer cells. In the second chapter, we investigated the biological effect on the clear cell type of ovarian cancer cell line ES-2, furthermore, we used ES-2 clear cells for testing the therapeutic effect of YC-1(3-(53-Hydroxymethyl- 23furyl)-1-benzyl indazole) which is a Ca(2+) influx inhibitor on the LPA mediated biological effects in vitro and in vivo. Our results indicated that LPA specific Edg receptors 2, 4 and 7 were all expressed in ES-2 cells with different level. LPA could not only enhance ES-2 cells adhesion to type I collagen but also enhance the VEGF and IL-6 expression. LPA could inhibit LPA enhanced cell adhesion through down regulated LPA induced ß1-integrin, and the rescue effect of LPA on ES-2 cells anoikis. We further tested the therapeutic effect of YC-1 on ES-2 tumorigenesis in nude mice, the data indicated that i.p. administration significant attenuated the ascites formation, and sustain the survival days in nude mice. And we found that YC-1 can not reduce the LPA enhanced VEGF expression in the ES-2 cells condition medium, but YC-1 could inhibit the condition medium modulated HUVECs monolayer permeability, HUVECs tube formation on matrigel and angiogenesis on chicken embryo chorioallantoic membrane assays (CAM), and the mechanism may due to YC-1 downregulated VEGF or condition medium caused VEGF receptor 2(KDR) phosphorylation. In the third chapter, we investigate the biological role of LPA in cervical cancer, we found that the LPA specific receptors, including Edg2, Egd4 and Edg7, were differentially expressed in cervical cancer cell lines. Physiological achievement level of LPA treatment shows survival promoting effects in cervical cancer cell lines under the serum-starved condition. LPA also stimulated significant elevation of IL-6 and IL-8 in cervical cancer HeLa and SiHa cells. By using neutralizing antibodies to block LPA induced IL-6 and IL-8 in the condition medium, we found that LPA induced IL-8 modulated the tube formation, proliferation and permeability on HUVECs, and also the angiogenesis in the chorioallantoic membrane (CAM) assay system. By using IL-6 neutrolizing antibodies to block IL-6, we found that the rescue effect of LPA on radiation-reduced colony formation was alleviated. Further more, LPA treatment also enhanced the cell migration in wound-healing assay, invasiveness in Matrigel coated Boyden chamber assay, and in the in vivo invasiveness/ intravasation capability assay in chicken embryo. In sum, our study indicated that LPA is a multiple function activating factor in the human cervical carcinoma as well as in ovarian cancer, LPA may provide a new targeting treatment of cervical cancer and ovarian cancer. Furthermore, anti-platelet drug, YC-1, may be a potent drug for molecular targeting on LPA mediated tumorigenesis and angiogenesis.