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Curcumin Derivative FLLL-31 Sensitizes Pancreatic Cancer Cells to TRAIL Induced Cell Death through Death Receptor Up-regulation
Kaustav Majumder^*, Steven Skube, Amanda O. Salzwedel, Nivedita Arora, Bhuwan Giri, Shrey Modi, Bharti Garg, Sulagna Banerjee, Ashok Saluja, Vikas Dudeja
Surgery, University of Minnesota, Minneapolis, MN

Introduction: TRAIL (Tumor Necrosis Factor-Related Apoptosis Inducing Ligand) is emerging as a promising anti-cancer therapy by virtue of its strong anti-tumor activity in wide range of cancer cells and minimal toxicity to normal cells and tissues. TRAIL acts via Death Receptors (DR4 and DR5) which form a DISC (Death inducing signaling complex) that further activates downstream caspases leading to cell death via apoptosis. Unfortunately, pancreatic cancer is resistant to TRAIL. Curcumin, a bioactive compound derived from turmeric has been shown to sensitize cancer cells to multiple chemotherapeutic agents. The clinical use of curcumin has been hampered by poor bioavailability. FLLL-31 is a diketone analog of curcumin with markedly increased bioavailability and increased translational potential. The aim of the current study was to evaluate whether FLLL-31 sensitizes pancreatic cancer cells to TRAIL induced cell death.
Methods: Highly aggressive metastatic pancreatic cancer cell lines (S2013, S2VP10) were treated with FLLL-31 (0-5µM), TRAIL (0-40ng/ml) and a combination of FLLL-31 and TRAIL. The effect on viability and parameters of apoptosis (Caspase 3, 8 and 9 activation) was measured. Protein was extracted from cell lysates and assessed for levels of anti and pro apoptotic proteins via Western Blotting. FACS (Fluorescence activated cell sorting) was done to evaluate surface receptor levels of Death Receptor 4 (DR4) and Death Receptor 5 (DR5).
Results: While TRAIL alone was ineffective in inducing cell death in pancreatic cancer cells, combination of TRAIL and FLLL-31 induced marked decreased in viability (Table 1). Combination of TRAIL and FLLL-31 induce significantly more caspase 3, 8 and 9 activation when compared to TRAIL or FLLL-31 alone suggesting that TRAIL and FLLL-31 synergize in inducing apoptosis in pancreatic cancer cells. Western blotting for protein showed increased cleavage of Caspase 3, Caspase 8, Caspase 9 and PARP (Poly ADP-Ribose Polymerase) in cells treated with combination of FLLL-31 and TRAIL. However the levels of anti-apoptotic proteins (Survivin, Bcl-xl, Bcl-2, XIAP, cIAP1 and cIAP2) showed no significant changes with combination. FACS showed a decrease in the number of DR5 surface receptors in the TRAIL treated cells as compared to control in both cell lines. However the levels of DR5 were significantly up-regulated in the combination group (Table 2).
Conclusion: FLLL-31 sensitizes pancreatic cancer cells to TRAIL induced apoptosis and cell death by preventing down-regulation of DR5. The increased availability of death receptors in the presence of TRAIL enhances pro-apoptotic action of TRAIL in resistant cells. Combination of FLLL-31 and TRAIL has immense potential to emerge as novel therapeutic strategy against pancreatic cancer.
Table 1

S2VP10
	Viability (24h)	Casapse 3 (24h)	Caspase 8 (12h)	Caspase 9 (12h)
Control	100 ± 0	100 ± 0	100 ± 0	100 ± 0
TRAIL (20 ng/mL)	103 ± 1	150 ± 36	114 ± 12	109 ± 7
FLLL-31 (5uM)	55 ± 6	242 ± 2	129 ± 29	162 ± 1
TRAIL (20ng/mL) and FLLL-31 (5uM)	23 ± 2*	3890 ± 910*	842 ± 252*	295 ± 57*
S2013
	Viability (24h)	Caspase 3 (24h)	Caspase 8 (12h)	Caspase 9 (12h)
Control	100 ± 0	100 ± 0	100 ± 0	100 ± 0
TRAIL (20ng/mL)	100 ± 3	231 ± 74	143 ± 5	146 ± 11
FLLL-31 (5uM)	62 ± 10	357 ± 77	116 ± 7	111 ± 11
TRAIL (20ng/mL) and FLLL-31 (5uM)	23 ± 5*	3812 ± 546*	758 ± 380*	723 ± 448*

Table 2

Surface Death Receptor 5 /DR5 levels (FACS)
	S2VP10	S2013
Control	100 ±0	100 ±0
TRAIL (20 ng/mL)	46.38 ±19	62 ±13
FLLL-31 (5uM)	126.5 ±6	130.4 ±31
TRAIL (20 ng/mL) and FLLL-31 (5uM)	100.2 ±15	101 ±8.8

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