Identification And Characterization Of Anti-Pancreatic Cancer Natural Compounds From Chinese Medicinal Herbs

Pancreatic cancer is an uncommon tumor worldwide, it accounts only1～7%of all malignant tumor. Pancreatic cancer has now become a common cause of cancer mortality, because the mortality rate of this form of cancer approaches100%. According to data published in2010, the incidence of pancreatic cancer in North America, Europe, Australia, and New Zealand is around100per1000000inhabitants, with male rates are1.5folds higher than females rates. Pancreatic cancer is the fourth most frequent cause of cancer death in males after cancer of lung, prostate and colo-rectum; in females it is also the fourth frequent cause of cancer death after cancer of the lung, breast and colo-rectum.Pancreatic cancer is difficult to diagnose at early stage, and can’t be removed surgically at the time of diagnosis. Due to its inaccessible location, proximity to other vital organs, and inherently aggressive pattern of growth, it has extremely poor survival rate. Surgical operation remains the only choice for long term survival of patients. However, difficulty in achieving early diagnosis and aggressive nature of this type of cancer limited the surgical operation to only about10～15%patients. Therefore, the majority of pancreatic cancer patients are treated with chemotherapy. Currently, gemcitabine is the best chemotherapeutic drug for the treatment of pancreatic cancer. However, the objective tumor response remains considerably low (less than20%). Several other cytotoxic and chemotherapy agents such as cisplatin, fluoroucil, docetaxel, erlotinib, oxaliplatin and irinotecan have been tested as single agent or in combination with gemcitabin for the treatment of advanced pancreatic cancer. However, most of these studies failed to ameliorate overall patient survival compared to gemcitabine. Therefore, there is an urgent need to identify new compounds that can effectively kill pancreatic cancer cells. With the development in high-throughput screening technique in the early century, great progress has been achieved in drug discovery world wide; many leading drugs against certain drug targets have been identified.Aim of our study is to find tumor-specific killer. A compound is regarded as a tumor-specific killer, if it kills only the PANC-1cells but not the mice splenocytes or the Fischer rat thyroid epithelial cells (FRT). Cell proliferation Assays were used in primary screening and MTT analysis was done to confirm the results. Primary screening was done the Traditional Chinese Medicine (TCM) fraction library which is generated from the500most common used TCM and contains40,000fractions by our lab previously. Once a positive fraction is determined, counter current chromatography prep-HPLC, liquid-liquid extraction will be used in the following active-directed single compound isolation. Mass Spectrometry (MS), nuclear magnetic resonance (NMR) and infrared spectrum (IR) methods were used in deciphering chemical structures of active compounds. We have finished screening of the planned40,000fractions from500herbs and identified262positive fractions from128herbs, the positive rates of herbs and fractions are25.6%and0.66%, respectively. Further analysis determined that only21positive fractions fit the criteria of PANC-1-specific killer, positive rate is only0.053%. We have isolated2compounds (Casticin and Isoalantolactone) with PANC-1inhibition activities. Further mechanistic studies revealed that these two compounds share some similarity in PANC-1inhibition mechanisms:both inhibited proliferation of PANC-1through cell cycle arrest and apoptosis induction mechanisma; both compounds increased the expression of Bax, cleaved Caspase-3and decreased the expression of Bcl-2protein in a dose-dependent manner. There are still some differences in PANC-1inhibition mechanisms between the2compounds in that: Induction of apoptosis by isoalantolactone is associated with increased generation of reactive oxygen species (ROS), cardiolipin oxidation, reduced mitochondrial membrane potential, release of cytochrome c and cell cycle arrest at S phase. While the growth inhibitory effect of casticin was associated with cell cycle arrest at G2/M phase. Furthermore, our in vivo toxicity study demonstrated that isoalantolactone did not induce any acute or chronic toxicity in liver and kidneys of mice at dose of100mg/kg body weight. Therefore, casticin and isoalantolactone may be safe chemotherapeutic candidates in the treatment of human pancreatic carcinoma.