Specific Targets for Pancreatic Cancer Therapy

描述(申请人提供)：胰腺肿瘤是美国第五大常见癌症，每年有28,000例新诊断病例，年死亡率大于99%。其病因在很大程度上尚不清楚，目前尚无根治性治疗方法。在胰腺癌中，几种重要的生存分子如EGFR、NF-kB、COX-2和Bclxl被高度激活。核因子-kB调控许多参与肿瘤进展、生存、血管生成和侵袭的基因，如Bclxl、COX-2、VEGF、MMP9和uPAR。抗凋亡蛋白Bclxl的高表达促进了细胞的存活，并在胰腺癌的发展过程中发挥了关键作用，导致了耐药性的产生。其在90%的胰腺癌中均有表达。环氧合酶-2(COX-2)酶在胰腺癌中的过度表达也被证明与肿瘤的生长、转移和化疗耐药有关。因此，抑制NF-kB、Bcl2/Bclxl和COX-2有望成为治疗胰腺癌的新策略。我们的合作研究人员设计了非肽、类药物、细胞通透性的有效小分子抑制剂(SMI)，它们结合在Bcl2和Bclxl的口袋上，阻断/破坏它们的抗凋亡功能。体外结合实验表明，这些SMI能有效抑制Bak BH3多肽与Bcl2和Bclxl的结合。我们还发现，金雀异黄素通过转录失活使核因子-kB失活，并下调了Bcl-xL、血管内皮生长因子、基质金属蛋白酶-9和uPAR。此外，塞来昔布对COX-2活性和Akt/NF-kB的抑制可能在杀伤胰腺癌细胞方面具有潜在的作用。我们发现人胰腺癌细胞系以高表达Bclxl、NFkB和COX-2而著称。因此，我们推测，SMI靶向Bcl2/Bclxl生存通路，金雀异黄素和塞来昔布抑制NF-kB诱导的Bcl2/BCI-xl/COX-2的生成，可能是治疗胰腺癌的新策略。本工作的重要意义在于，核因子-kB在转录水平上调节Bclxl的表达，因此金雀异黄素使NFkB失活和SMI处理将导致Bclxl的降低。此外，塞来昔布的加入可能通过抑制NF-kB以及COX-2的转录和酶活性而诱导胰腺癌细胞死亡。本研究的主要目的是：1)探讨参麦注射液促进人胰腺癌细胞株凋亡的分子机制；2)采用人胰腺癌移植瘤模型，研究参麦参碱对胰腺癌细胞的抗肿瘤活性；3)研究染料木素对Bclxl基因转录的抑制作用是否能在体内外协同参麦碱诱导的胰腺癌细胞凋亡；4)研究塞来昔布抑制环氧合酶-2对金雀异黄素和参麦黄素诱导的胰腺癌细胞Akt/NF-kB激活的影响。靶向Bcl2/Bclxl、NFkB和COX-2有望成为治疗胰腺癌的新途径。

DESCRIPTION (provided by applicant): Pancreatic neoplasia is the fifth most common cancer in the United States with >28,000 newly diagnosed cases per year with an annual mortality rate of greater than 99%. Its etiology is largely unknown and no curative treatment is presently available. In pancreatic cancer, several important survival molecules such as EGFR, NF-kB, COX-2, and Bcl-xL are highly activated. NF-kB regulates many genes involved in tumor progression, survival, angiogenesis and invasion such as Bcl-xL, COX-2, VEGF, MMP-9 and uPAR. High expression of anti-apoptotic Bcl-xL protein promotes cell survival and plays a key role in pancreatic cancer progression leading to drug resistance. Its expression was found in 90% of pancreatic cancer. The overexpression of the cyclooxygenase-2 (COX-2) enzyme in pancreatic cancer has also been shown to contribute to growth, metastasis, and chemoresistance. Therefore, inhibitions of NF-kB, Bcl-2/Bcl-xL, and COX-2 should serve as a novel treatment strategy for pancreatic cancer. Our co-investigator has designed non-peptide, drug-like, cell permeable potent small molecule inhibitors (SMI) that bind to the pocket of Bcl-2 and Bcl-xL and block/disrupt their anti-apoptotic function. These SMI were tested in an in vitro binding assay and found to be potent inhibitor of the binding of Bak BH3 peptide to Bcl-2 and Bcl-xL. We have also shown that genistein inactivates NF-kB and down regulates Bcl-xL, VEGF, MMP-9, and uPAR by transcriptional inactivation. Furthermore, the inhibition of COX-2 activity and Akt/NF-kB by celecoxib could be potentially useful in killing pancreatic cancer cells. We have found that human pancreatic cancer cell lines are notable for high expression of Bcl-XL, NF-kB, and COX-2. Therefore, we hypothesize that targeting the Bcl-2/Bcl-xL survival pathway by SMI and inactivating NF-kB-induced Bcl-2/BcI-xL/COX-2 generation by genistein and celecoxib could be novel therapeutic strategies for the treatment of pancreatic cancer. Of importance to this work is that NF-kB transcriptionally regulates the expression of Bcl-xL, hence inactivation of NF-kB by genistein and treatment with SMI will lead to decreased Bcl-xL. Moreover, the addition of celecoxib may be very useful for inducing pancreatic cancer cell death by inhibiting NF-kB as well as transcription and enzyme activity of COX-2. Our Specific Aims are: 1) To investigate the molecular mechanisms by which SMI promote apoptosis in human pancreatic cancer cell lines, 2) To determine anti-tumor activity of SMI using human pancreatic cancer xenograft model, 3) To determine whether transcriptional repression of Bcl-xL by genistein will synergize SMI-induced apoptosis in vitro and in vivo, and 4) To determine the effects of inhibition of COX-2 by celecoxib on Akt/NF-kB activation in genistein-treated and SMI-treated pancreatic cancer cells in vitro and in SCID mouse xenograft models. Targeting Bcl-2/Bcl-xL, NF-kB and COX-2 could be novel therapeutic treatment of pancreatic cancer.