Pristimerin Causes G1 Arrest, Induces Apoptosis, and Enhances the Chemosensitivity to Gemcitabine in Pancreatic Cancer Cells

and Enhances the Chemosensitivity to Gemcitabine in Pancreatic Cancer Cells. PLoS ONE 7(8): e43826. doi:10.1371/journal.pone.0043826 Pristimerin Causes G1 Arrest, Induces Apoptosis, and Enhances the Chemosensitivity to Gemcitabine in Pancreatic Cancer Cells Yongwei Wang. 0 Yinan Zhou. 0 Haoxin Zhou 0 Guang Jia 0 Ji Liu 0 Bing Han 0 Zhuoxin Cheng 0 Hongchi Jiang 0 Shangha Pan 0 Bei Sun 0 Sharmila Shankar, University of Kansas Medical Center, United States of America 0 Department of Pancreatic and Biliary Surgery, The First Affiliated Hospital of Harbin Medical University , Harbin , People's Republic of China Despite rapid advances in chemotherapy and surgical resection strategies, pancreatic cancer remains the fourth leading cause of cancer related deaths in the United States with a 5-year survival rate of less than 5%. Therefore, novel therapeutic agents for the prevention and treatment of pancreatic cancer are urgently needed. The aim of this study was to investigate the effect of pristimerin, a quinonemethide triterpenoid compound isolated from Celastraceae and Hippocrateaceae, on inhibition of cell proliferation and induction of apoptosis in three pancreatic cancer cells, BxPC-3, PANC-1 and AsPC-1, in both monotherapy and in combination with gemcitabine. Treatment with pristimerin decreased the cell proliferation of all three pancreatic cancer cells in a dose- and time-dependent manner. Treatment of pancreatic cancer cells with pristimerin also resulted in G1-phase arrest which was strongly associated with a marked decrease in the level of cyclins (D1 and E) and cyclin-dependent kinases (cdk2, cdk4 and cdk6 ) with concomitant induction of WAF1/p21 and KIP1/p27. Pristimerin treatment also resulted in apoptotic cell death, cleavage of caspase-3, modulation in the expressions of Bcl-2 family proteins, inhibition of the translocation and DNA-binding activity of NF-kB. In addition, pristimerin potentiated the growth inhibition and apoptosis inducing effects of gemcitabine in all three pancreatic cancer cells, at least in part, by inhibiting constitutive as well as gemcitabine-induced activation of NF-kB in both its DNA-binding activity and transcriptional activity. Taken together, these data provide the first evidence that pristimerin has strong potential for development as a novel agent against pancreatic cancer. - Funding: This work was supported in part by grants from the New Century Support Foundation for Elitist of Chinese Ministry of Education (NCET-07-0248), the Scientific Foundation for Prominent Youth of Heilongjiang Province, China (JC200717), the Scientific and Technological Project of Heilongjiang Province, China (GC09C407-2), the research Special Fund For public welfare industry of health (201202007) and the National Natural Scientific Foundation of China (81170431, 81101799). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Pancreatic cancer is one of the most aggressive and lethal human cancers, which has steadily increased in incidence over the past decades, and is currently the fourth leading cause of cancer death in the United States. The collective 1-year survival from the time of diagnosis at any stage is only 26%, while a long-term overall survival (OS) falls down to ,5% [1]. In 2010, it was estimated 43,140 Americans were diagnosed with pancreatic cancer and 36,800 died of the disease [2]. The main reason for such poor prognosis of pancreatic cancer is mostly due to the lack of early diagnosis, the highly aggressive biological behavior of the tumor and the poor response to most therapies including chemotherapy, radiotherapy, and immunotherapy [3]. Currently, Gemcitabine (29-deoxy-29,29-difluorocytidine) is known to be the mainstay in the treatment for advanced and metastatic pancreatic cancer. However, gemcitabine treatment results in a response rate of less than 20% and is associated with multiple adverse events and chemoresistance [4,5]. Therefore, developing alternative chemopreventive and chemotherapeutic strategies are urgently needed for the treatment of this deadly disease. Nuclear factor-kB (NF-kB),which plays a critical regulatory role in the expression of genes involved in inflammation, cell proliferation, invasion, angiogenesis, metastasis, suppression of apoptosis, is constitutively activated in a variety of cancer cells including pancreatic cancer cells [6,7,8]. In addition, Multiple lines of evidence suggest that NF-kB plays a pivotal role in the growth and chemoresistance of pancreatic cancer. First, NF-kB is constitutively activated in pancreatic cancer cells, but not in normal pancreatic tissues or nontumorigenic cell lines [7,9]. Second, NF-kB could promote pancreatic cancer growth due to its ability to inhibit cell apoptosis, induce mitogenic gene products (cMyc and cyclin D1), increase expression of proangiogenic factor including vascular endothelial growth factor(VEGF), and promote the migration and invasion of pancreatic cancer cells [10,11,12,13,14]. Finally, NF-kB plays a pivotal role in mediating chemoresistance in pancreatic cancer [15]. Together, this evidence implicates the role of NF-kB in pancreatic cancer and suggests agents that can block NF-kB activation have potential to combat the growth of pancreatic cancer. Pristimerin (Fig. 1), a naturally occurring quinonemethide triterpenoid compound isolated from Celastraceae and Hippocrateaceae, has recently attracted considerable interest due to its potential chemopreventive or chemotherapeutic properties. It has anti-inflammatory, antioxidant, antimalarial, and insecticidal activities and has been shown to possess growth inhibitory effect on a series of human cancer cell lines such as breast and lung cancer, prostate cancer, cervical cancer and multiple myeloma tumors [16,17,18,19,20]. However, the potential efficacy of pristimerin against pancreatic cancer remains unknown. In the present study, we used three human pancreatic cancer cell lines, BxPC-3, PANC-1 and AsPC-1, to evaluate the potential of pristimerin as an effective chemopreventive and chemotherapeutic agent against pancreatic cancer. We demonstrate that pristimerin strongly suppresses the growth of all three pancreatic cancer cell lines by inducing G1-phase cell cycle arrest and apoptosis, and sensitizes them to gemcitabine-induced cell death. Furthermore, Our results demonstrate that the effects and molecular mechanisms of action of pristimerin in pancreatic cancer cells are associated with inhibition of NF-kB activation. Pristimerin Induces Cell Growth Inhibition in Pancratic Cancer Cells To determine the effect of pristimerin on cell growth, human pancreatic cancer cells (BxPC-3, PANC-1 and AsPC-1) were treated with the varying concentrations (01000 nM) of pristimerin for 24 h, 48 h and 72 (...truncated)