3133 - 3-Bromopyruvate in Combination with Radiation Inhibits Pancreatic Tumor Growth by Suppressing Hexokinase II (HK2) Enzyme of Aerobic Glycolysis

Purpose/Objective(s): Pancreatic cancer (PC) is the fourth-most-deadly cancer in the United States. The majority of patients with locally advanced PC undergo chemoradiation therapy (CRT). However, current treatments are inadequate, and novel strategies are desperately required. 3-Bromopyruvate (3-BP) is a promising drug against pancreatic cancer which exerts potent anticancer effects by inhibiting hexokinase II enzyme (HK2) of the glycolytic pathway of cancer cells while not affecting the normal cells. 3-BP in combination with radiation is a promising modality to treat locally advanced PC.

Materials/Methods: Two human pancreatic cancer cell lines mia-paca2 and Panc1 were used. The cells were treated with 3BP for 12 hours and cell viability was measured using MTS and cellular ATP. Apoptosis and ROS production were measured using Annexin -V and DHE, respectively. Western blots were performed using antibodies against phospho-S552 beta catenin, c-Myc, MCT-1, ß-actin and IHC staining were performed using specific HK2 antibodies. The mRNA of HK2, MCT-1 and ß-actin were quantified by real time qRT-PCR. The interaction of ß-catenin and HK2 was detected by co-IP. The mouse tumor xenograft was created by subcutaneously injecting Panc-1 cells in nude mice and tumor was treated with 3-BP and RT. The tumor tissues were also electron micrographed for detecting mitochondrial damage. The TCGA data analysis of pancreatic cancer patients were performed using CBIOPORTAL genomic database.

Results: We investigated the mechanism of 3-BP in MIA PaCa-2 and Panc-1 cells, and we observed inhibition of Hexokinase II (HK2) enzyme of aerobic glycolytic pathway when treated with 100 uM of 3-BP. We also observed inhibition in phosphorylation of ß-catenin at S552, responsible for translocating ß-catenin from cytoplasm to the nucleus and transcribing other downstream genes including c-Myc for tumor growth. We also observed inhibition in the MCT1 expression which is responsible for transporting 3-BP into the cell which also exports lactate out of tumor cells making TME more acidic. The Co-IP data showed inhibition in HKII interaction with ß-catenin. Notably, 3-BP with RT significantly enhanced the radiation-induced G2/M cell cycle arrest (RT only 25.1% vs 3 BP+RT at 37.8%). We also observed significant ROS production in MIA PaCa-2 and Panc-1 cells treated with combination of 3-BP and RT. The combination of 3 BP with RT inhibited tumor growth in nude mice. We treated tumor bearing animlas with 10 mg/kg of 3-BP and 2 Gy RT twice a week for 4 weeks. We observed significant inhibition in tumor growth in animals treated with combination as compared to individual treatment of 3-BP or RT alone. There was substantial damage to mitochondria in treated tumor tissues. The TCGA data analysis showed alteration of HK2 gene in pancreatic cancer patients also linked to worst prognosis than the unaltered group.

Conclusion: 3-BP in combination with RT exhibited significant inhibition of pancreatic tumor growth in animals with no apparent toxicity.