Abstract A28: Mechanistic study involving the combined antiproliferative effect of Etoricoxib- cyclooxygenase-2 inhibitor and cholecystokinin-2 receptor antagonist in human pancreatic cancer cells
Authors: Sikka Manisha 
Published: November 2016

Introduction: Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer death in United States. Due to poor prognosis and increasing incidences, over 80% of the patient are given palliative chemotherapy with combination drugs. Despite decades of efforts, the survival rate still remains 3-6%. The most common cause of pancreatic cancer are genetic alterations, cigarette smoking, obesity and lack of exercise. Patients with Type-2 Diabetes Mellitus are also on higher risk of pancreatic cancer. Although numerous chemotherapeutic drugs have been tested in this malignancy, survival of advanced pancreatic cancer has not improved over the past several decades. Because of the lack of effective treatment options available for this disease, identification of novel targets and approaches has been made. Recent studies have shown an association between the gastrointestinal hormone gastrin, the expression of Cholecystokinin -B/ gastrin receptor and an increased generation of COX-2 in colon-carcinoma cells and progression of gastric cancer, suggesting that gastrin has direct stimulating potencies. However, it remains unknown whether the combination of Cholecystokinin-2 (CCK-2) receptor antagonist plus COX-2 inhibitor exerts synergistic antitumor effects on human gastric cancer. Therefore, for a better treatment and understanding the underlying mechanism that leads to pancreatic adenocarcinoma initiation, chemoresistance and progression, we further investigated the combined effect of Cyclooxygenase (COX-2) inhibitor and Cholecystokinin receptor-2 (CCK-2) antagonist on pancreatic cancer cells. Thus it is essential to recognize more effective targets/ receptors for treatment of pancreatic cancer.

Materials & Methods: Antiproliferative effects were carried out using Cell Proliferation Kinetics, Trypan blue staining technique, Morphological analysis and MTT Assay. Apoptotic activity was confirmed by DNA Fragmentation, Western Blot analysis and Cell cycle analysis

Result and Discussion: The present study demonstrate that COX-2 enzyme is a modulator of carcinogenesis, apoptosis and angiogenesis in pancreatic cancer and is such rational target for drug development. Similarly Cholecystokinin antagonists is also a potential target for developing novel strategies for pancreatic cancer and further defines the pathway affected by CCK-BR. To study the combinatorial effect of COX-2 inhibitor and Cholecystokinin antagonist we successfully purified Etoricoxib using HPLC and further characterized by spectroscopic techniques using FT-IR, NMR and Mass spectroscopy. The anti-proliferative activity of Etoricoxib was checked on various cell lines and it induced proliferation arrest in Miapaca-2, Panc-I, BXPC-3 and ASPC-1 both in time and concentration dependent manner. The IC50 values were found to be 280 μM (Miapaca-2), 400μM (Panc-1), 300μM (BXPC-3) and 450 μM (ASPC-1) cells. The IC50 of procured Cholecystokinin antagonists YM-022, LY 288513 and L-365-260 was found out to be 0.002μM, 0.001μM and 0.01μM on Panc-1 respectively. Similarly, combinatorial studies was carried out on Panc-1, BXPC-3, ASPC-1 and Miapaca-2 respectively using cytotoxicity and apoptotic techniques. It is thus, a probable insight in stating that it has lesser side effects and thus have selectivity for cancer cell toxicity.

Conclusion: The combined effect of Cyclooxygenase-2 inhibitor and Cholecystokinin-2 Receptor antagonists was calculated using Chaou Tatlay method. A combination index was calculated. As a class NSAIDs possess analgesic, antipyretic, anti-inflammatory, and there is persuasive evidence that COX-2 inhibitor suppress cancer cell proliferation owing to their role in apoptosis, compelling evidence suggest that COX-2 over-expression promotes whereas COX-2 inhibition prevents tumor initiation and promotions. NSAIDs and COX-2 selective inhibitors may have different effects on cancer may be stage dependent therefore a better understanding of the critical COX related mechanisms of carcinogenesis, proliferation, apoptosis, inflammation, angiogenesis and tumor invasion will help to define the potential of COX-2 blockage in cancer therapy. Previous studies have shown that NSAIDs and specific COX-2 inhibitors are able to enhance the effect of certain cystostaic agents, a specific COX-2 inhibitor Celecoxib enhances the cystostaic effect of doxorubicin on tumor cell lines (Wijnggaarden J.V. et.al. 2007). Results in our lab showed that combination of doxorubicin and etoricoxib synergistically inhibited the proliferation of Hela and Miapaca cell lines. These results has indicated that the combination therapy of COX-2 selective inhibitors may be the potential chemotherapeutic strategy for cancer prevention. In the present study, human pancreatic cancer cell lines, in which the CCK-2 receptor and COX-2 were expressed, was applied to examine whether blockade of the CCK-2 receptor and COX-2 exerts synergistic anti-tumor effects on human pancreatic cancer in vitro. The detailed studies are under process. In conclusion, both Etoricoxib and YM-022 had growth inhibitory and apoptosis inductive effects on the pancreatic cancer cells through down regulation of Bcl-2 with simultaneously up-regulation of BAX expression which suggest that COX-2 inhibition is new molecular targets for effective therapy against pancreatic cancer.

Citation Format: Manisha Sikka. Mechanistic study involving the combined antiproliferative effect of Etoricoxib- cyclooxygenase-2 inhibitor and cholecystokinin-2 receptor antagonist in human pancreatic cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr A28.