Molecular mechanism of bitter melon juice efficacy against pancreatic cancer.

描述(申请人提供)：胰腺癌(PANC)是一种侵袭性疾病；PANC患者确诊后的中位生存期为6个月，总体5年生存率为3-5%。吉西他滨是治疗PANC的一线化疗药物，可以有效消除大量PANC细胞，但保留了导致癌症复发和侵袭性的肿瘤干细胞(CSC)群体。总而言之，很明显，迫切需要更多的策略来有效降低PANC的发生率，以CSC为目标来控制PANC的复发和相关的死亡率。值得注意的是，PANC是一种具有多种突变组合的复杂疾病，因此有必要确定具有多个靶点的药物来控制PANC的生长和CSC相关的PANC复发。我们已发表的和初步的研究表明，苦瓜汁(BMJ)显著降低了人PANC细胞系的存活率，并诱导了强烈的凋亡性死亡，这与AMPK的激活有关，因为AMPK抑制剂和siRNA都逆转了BMJ诱导的细胞凋亡。此外，BMJ还能抑制PANC细胞的糖酵解和氧化磷酸化速率。综上所述，这些结果表明BMJ在PANC细胞中发生了代谢转变。BMJ还强烈地抑制PANC CSCs的球体形成，表明它也针对CSC的抗PANC效果。最值得注意的是，BMJ仅以5 mg/只/鼠/天的剂量灌胃，对裸鼠MIA-Paca-2异种移植瘤的生长有60%的抑制作用，没有任何明显的副作用或毒性。异种移植的免疫组织化学分析显示，BMJ还在体内抑制增殖和CSC生物标志物，诱导细胞凋亡，并激活AMPK。综上所述，我们假设BMJ通过营养应激、AMPK激活和抑制与代谢和增殖相关的信号分子导致PANC细胞代谢改变，从而导致强烈的生长抑制和细胞凋亡。此外，BMJ以Notch/Hedgehog信号为靶点，有效地消除PANC CSC种群；从而产生强大的抗PANC活性。提出的具体目标是：i)进一步明确BMJ靶向代谢并影响AMPK介导的PANC细胞生长抑制和凋亡的机制；ii)进一步明确BMJ靶向Notch和Hedgehog通路并有效抑制PANC中CSC群体的机制；以及iii)在PDX1-CRE、LSL-KrasG12D转基因小鼠模型中进一步建立特定目标I和II中定义的BMJ分子机制。值得一提的是，苦瓜作为蔬菜和果汁被广泛食用，特别是在亚洲国家；苦瓜被认为具有多种有益健康的特性，如抗糖尿病、消炎等。 已经在几个临床试验中测试了它的抗糖尿病效果，并具有足够的人体安全性 数据。因此，我们预计，拟议研究的积极结果将为启动临床试验以确定BMJ对人类胰腺癌的活性提供令人信服的理由。

DESCRIPTION (provided by applicant): Pancreatic cancer (PanC) is an aggressive disease; median life of PanC patients post-diagnosis is <6 months and overall 5-year survival rate is 3-5%. Gemcitabine is the frontline chemotherapy for PanC that effectively eliminates bulk PanC cells; however spares cancer stem cell (CSC) population which causes cancer relapse as well as aggressiveness. Together, it is clear that additional strategies are urgently warranted to effectively lower PanC incidence, target CSC to control PanC relapse, and associated mortality. Noteworthy, PanC is a complex disease with multiple combinations of mutations, and therefore it is necessary to identify the agents with multiple targets to control both PanC growth and CSC-associated PanC relapse. Our published and preliminary studies show that bitter melon (Momordica charantia) juice (BMJ) significantly decreases the viability and induces strong apoptotic death of human PanC cell lines, which was associated with a robust AMPK activation, as both AMPK inhibitor and siRNA reversed BMJ-caused apoptosis. Furthermore, BMJ inhibited glycolysis and oxidative phosphorylation rate in PanC cells. Together these results suggested a 'metabolic shift' by BMJ in PanC cells. BMJ also strongly inhibited the sphere formation by PanC CSCs suggesting that it also targets CSC for its anti-PanC efficacy. Most notably, BMJ feeding by oral gavage at only 5mg dose/mouse/day resulted in 60% inhibition in MIA PaCa-2 xenograft growth in nude mice without any noticeable side effects or toxicity. Immunohistochemical analysis of xenografts showed that BMJ also inhibits proliferation and CSC biomarkers, induces apoptosis, and activates AMPK in vivo. Together, based on these and other findings, we hypothesize that BMJ causes metabolic shift in PanC cells through nutrient stress, AMPK activation and inhibiting signaling molecules related to metabolism and proliferation, which leads to strong growth inhibition and apoptosis specifically in PanC cells. Additionally, BMJ targets Notch/ Hedgehog signaling to effectively eliminate PanC CSC population; resulting in strong activity against PanC. The specific aims proposed are: I) To further define the mechanisms by which BMJ targets metabolism and affects AMPK-mediated growth inhibition and apoptosis in PanC cells; II) To further define the mechanisms by which BMJ targets Notch and Hedgehog pathways and effectively inhibits CSC population in PanC; and III) To further establish BMJ molecular mechanisms defined in specific aims I and II in PDX1-Cre; LSL-KRASG12D transgenic mouse model. It is important to highlight here that bitter melon is widely consumed as vegetable as well as juice especially in Asian countries; and has been attributed with multiple health beneficial properties such as anti-diabetic, anti-inflammatory, etc. Most notably, bitter melon has been tested in several clinical trials for its anti-diabetic effects and has plenty of human safety data. We, therefore, anticipate that the positive outcomes from the proposed studies will provide compelling rationale for initiating clinical trials to establish BMJ activity against human pancreatic cancer.