Enhanced chemosensitivity to Gemcitabine and Oxaliplatin by cancer chemopreventive agent Thymoquinone in pancreatic cancer

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Purpose: Current standard adjuvant therapies for pancreatic cancer consist of administering cytotoxic chemotherapeutic agents or irradiation treatment alone, or in combination. Unfortunately the effect remains modest due to high degree of inherent and acquired chemoresistance. We recently reported the potential benefit of Thymoquinone (TQ), present in the volatile oil of the seed Nigella sativa seeds (also called black seeds) for prevention of prostate cancer (Kaseb A et.al., Cancer Research67: 7782-7788, 2007). The purpose of this study was to test our hypothesis for enhanced chemosensitivity in pancreatic cancer and explore molecular mechanism associated with better therapeutic outcome for future use in the clinic.
 Method: Pancreatic tumor cells (HPAC, BxPC-3, Panc-1 and MDA Panc-28, COLO 357, L3.6pl,) cells were treated with TQ (0-50µM) for 48hrs to examine antiproliferative and proapoptotic effects. Their response to chemosensitivity towards Gemcitabine and Oxaliplatin was also assessed in vitro using MTT and quantifying cytoplasmic histone-DNA fragments upon pretreatment with TQ (5-50µM; 48 hrs) followed by cytotoxic drugs for 24hrs. The molecular mechanism for enhanced chemosensitivity was investigated by performing Western immunobloting targeting proapoptotic as well as, antiapoptotic signaling moieties by either single regimen treatment and under conditions of pretreatment with TQ for 48hrs. Additionally, gel shift assay was performed to elucidate whether activation of the basal NF-κB transcription factor is responsible in suppressing apoptosis and its abrogation by TQ leads to sensitization and cell killing by the investigated cytotoxic chemotherapeutic agents.
 Results: With exception of Panc-1, most pancreatic cancer cells were found sensitive to TQ alone as deduced from reduced viability and undergoing apoptosis in the range between 30-50µM. Panc-1 cells were seen sensitive to TQ concentration as low as 5µM. TQ pretreatment also significantly (p<0.01) enhanced the anti-proliferative and apoptotic effect of Gemcitabine as well as, Oxaliplatin in these cells relative to untreated control and single drug regimen. Additionally, constitutively active NF-κB and several of its downstream effector genes related to metastasis and invasion were downregulated by TQ, suggestive of an important molecular target in reversing chemoresistance as well as, invasion and metastasis of pancreatic cancer.
 Conclusion: This is first report suggestive of chemosensitization of human pancreatic cancer cells by TQ. Based on our current research findings in vivo animal studies are currently under progress so as to obtain better clue for its efficacy for future phase-1clinical trial.