(A) TRYPAN BLUE CELL VIABILITY ASSAY Fisetin inhibits growth, induces G1 arrest and apoptosis of human gastric carcinoma AGS and SNU-1 cells involving disruption of mitochondrial membrane potential Akash Sabarwal1,2, Rajesh Agarwal2, Rana P Singh1,3 1 School of Life Sciences, Central University of Gujarat, Gandhinagar, Gujarat, India.2Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, CO, USA. 3Cancer Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, India Email: akash.sabarwal@gmail.com INTRODUCTION SOURCES Fruits and vegetables - Strawberries, blueberries, grapes, apples, onion, yellow cypress Medicinal Plants- Acacia greggii, Butea frondosa, Gleditsia triacanthos Fisetin Globally, Gastric Cancer (GC) is the third leading cause of cancer death. Almost one million new cases of GC were estimated to have occurred in 2013 worldwide, out of which 63,000 new cases and 59,000 deaths occurred in India alone. Existing strategies available for the treatment of GC are surgery, chemotherapy, radiation therapy and chemo-radiation which have several side effects. Chemoprevention denotes the use of agents to inhibit, reverse or retard tumorogenesis. Several phytochemicals derived from edible and non-edible plants have been reported to obstruct at a precise stage of the carcinogenic process. Fisetin is a flavonol, a structurally distinct chemical substance that belongs to the flavonoid group of polyphenols. Fisetin exhibits anti-inflammatory, anti-carcinogenic and antiviral activities. Fisetin has shown promising results against lung, colon, prostate and cervical cancer types however yet not reported against gastric cancer. Herein, we assessed the anticancer potency and associated molecular alterations by fisetin in human gastric cancer AGS and SNU-1 cells. PROPERTIES Chemical formula – C15H10O6.xH2O Molecular weight- 286.24 g/mol Stock prepration- 100 mM by dissolving in DMSO Total cells x104 AGS (24h) % dead cells 0 25 50 75 100 $ Fisetin (μM) # * Figure 1: Effect of Fisetin on cell growth and proliferation. (A) Trypan blue cell viability assay of AGS and SNU-1 cells after 24 and 48 h. (B) BrdU cell proliferation assay of AGS cells after 24 and 48 h. (C) MTT cell viability assay of AGS cells after 24 and 48 h. The cell growth data shown were mean ± SEM of three independent plates. p<0.001 (*), p<0.01($), p< 0.05(#) 0 25 50 75 100 AGS (48h) 0 25 50 75 100 SNU-1 (24h) SNU-1 (48h) 0 25 50 75 100 0 25 50 75 100 (A) TRYPAN BLUE CELL VIABILITY ASSAY (B) BrdU CELL PROLIFERATION ASSAY (C) MTT ASSAY CELL CYCLE ANALYSIS AGS cells SNU-1 cells CDK4 CDK2 Cyclin E β-Actin C 25 50 75 C 25 50 75 24 hr 48 hr Cyclin D 1 p21 p27 P53 ser15 P53 Total MDM2 CDK 4 CDK 2 Cyclin D1 p53 ser 15 β -Actin Figure 2: FACS analysis cell cycle time kinetics using Saponin-PI staining after synchronization of AGS and SNU-1 cells Figure 3: Western blot of cell cycle regulatory proteins of AGS and SNU-1 cells Fisetin (μM) P53 inhibition experiments APOPTOSIS (A) AGS cells (A) Fisetin (μM) SNU-1 cells (D) (C) SNU-1 cells AGS cells p53 H2AX Ser-139 H2AX Ser 139 β-Actin C F-50 P-75 P+F cCaspase 3 cCaspase 3 F- Fisetin (μM) P- Pifthrin (μM) β-Actin BCl2 (B) SNU-1 cells Bax Bax (C) (B) AGS cells SNU-1 cells cPARP β-Actin cPARP H2AX Ser-139 β-Actin cPARP PCNA Fisetin (μM) C 25 50 75 C 25 50 75 24 hr 48 hr β-Actin β-Actin Fisetin (μM) Fisetin (μM) C 25 50 75 C 25 50 75 24 hr 48 hr Fisetin (μM) C 25 50 75 C 25 50 75 24 hr 48 hr Figure 4: Effect of Fisetin on Apoptosis of AGS and SNU-1 cells. (A) Annexin V Fitc for % apoptotic cell death, (B) JC-1 staining for mitochondrial Membrane depolarization, (C) Western blot analysis of apoptosis regulatory proteins Figure 5: AGS cells were pre-treated with Pifithrin-alfa (A) Trypan blue exclusion assay, (B) Cell cycle analysis, (C) AnnexinV FITC, (D) Before performing these experiments effective dose of pifithrin alfa which can inhibit p53 was standardized using western blot analysis CONCLUSION Fisetin decreased the cell proliferation and cell viability of gastric cancer AGS and SNU-1 cells at 24 and 48 hours. Fisetin induced G1 phase cell cycle arrest and apoptosis in both AGS and SNU-1. Phase of cell cycle was further confirmed by starvation-driven cell synchronization and release assay. Mechanism of induction of G1phase arrest included decrease in protein levels of Cyclin D1, Cyclin E and CDK 2/4. Fisetin also increased the cleavage of PARP and H2AX phosphorylation in time dependent manner. Fisetin has caused mitochondrial mediated apoptosis since decrease in mitochondrial membrane depolarization was observed. Increase in phosphorylation of H2AX and p53 indicated involvement of DNA damage mechanisms which might be responsible for G1 phase arrest and apoptosis in AGS and SNU-1 cells. P53 inhibitor pifithrin alfa is not able to reverse the cell death and apoptosis instead increased the drug effect suggesting protecting role of p53 which further indicate involvement of an p53 independent pathway for cell cycle arrest and apoptosis. Taken together, these data provide evidence that fisetin possesses anticancer potential against human gastric carcinoma AGS and SNU-1 cells and it could be developed as a novel agent for the management of gastric cancer. ACKNOWLEDGEMENT : UGC fellowship and Central instrumentation facility of CUG is highly acknowledged.