Compromised PKM2 activity has been observed to shift preferentially towards anabolic metabolism, resulting in macromolecular biosynthesis, instead of leading to oxidative metabolism for energy production, which promotes cancer cell proliferation and tumor growth. as carcinogen metabolism, cell division, differentiation, apoptosis, in pancreatic cancer development, has generated interest in dietary phytochemicals for potential cancer chemoprevention. A mechanistic link between diet and pancreatic cancer comes from its long-recognized interrelationship [4]. One such dietary agent, which could be used in combination with GCB for treatment of pancreatic cancer, is Betulinic acid (BA), a naturally occurring penta-cyclic-triterpene with a variety of biological activities including potent antitumor properties [5]. BA is contained Protosappanin B in the outer bark of various plants throughout the plant kingdom, including white-barked birch trees [6], with anti-inflammatory, anti-viral, and anti-neoplastic activities [7]. Anticancer activity of BA has been linked to its ability to directly trigger mitochondrial membrane permeabilization, a central event in the apoptotic process, raising the hope to bypass the resistance to conventional chemotherapeutics [6]. Thymoquinone (TQ), another potential anticancer-nutraceutical agent, is a bioactive compound derived from black seed (and in vivo. In vitro, curcumin was shown to inhibit the proliferation of Protosappanin B various pancreatic cancer cell lines, potentiating apoptosis induced by gemcitabine, and inhibiting constitutive NF-B activation in the cells. In vivo, tumors from nude mice injected with pancreatic cancer cells and treated with a combination of curcumin and gemcitabine showed a significant reduction in volume (P?=? 0.008 vs control; P?=? 0.036 vs gemcitabine alone) [42]. In our experiments, a combined interaction of betulinic acid (BA) or thymoquinone (TQ), the two neutraceuticals with gemcitabine (GCB), in GCB-sensitive-MIA PaCa-2 and GCB-resistant-PANC-1 – pancreatic cancer cell lines, showed synergism. The viability assay revealed a synergistic cytotoxic interaction between GCB and BA or GCB and TQ in both GCB-sensitive and -resistant cell lines, as determined by the DRI (Dose Reduction Index) of >1 obtained from calcusyn [43]. Mechanistically, GCB mediates its cytotoxic effects by inhibiting the repair synthesis step through its action as a ribonucleotide-reductase inhibitor, depleting the intracellular deoxy-nucleotide pools, and enhancing the potential of its own incorporation into newly synthesized DNA. Once incorporated into DNA, the analog causes termination of DNA synthesis and is resistant to removal by exo-nucleases, resulting in DNA strand breaks [44]. Betulinic acid (BA), Rabbit Polyclonal to AML1 a Protosappanin B nutraceutical, has been shown to induce apoptosis through mitochondrial pathways, where during this process both the mitochondrial outer and inner membranes are permeabilized, resulting in release of soluble proteins, such as cytochrome c, Smac or AIF, from the mitochondrial interspace into the cytosol [45]. BA also induces deathin several different cancer cell lines through multiple pathways, which include p53-independent induction of p21/Waf1,up-regulation of death receptors, inhibition of specificity protein(Sp) transcription factors [46]. Similarly, Thymoquinone (TQ) induces apoptosis in tumor cells by several mechanisms, including NF-kB suppression, Akt activation and extracellular signal-regulated kinase signaling pathways, and also by inhibiting tumor angiogenesis [47]. On the basis of ED50, ED75 and ED90 of drug combinations, isobolograms were generated and the synergy evaluated, using CalcuSyn. Betulinic acid (BA) or Thymoquinone (TQ) in combination with gemcitabine (GCB), at multiple drug concentrations, showed that in GCB-sensitive-MIA PaCa-2 and GCB-resistant-PANC-1 cells, the combination index (CI)was under 0.88 at Fa of 0.75 (75% reduction of cell growth) in MIA PaCa-2 cells. Whereas, in case of PANC-1, the combination index (CI) was under 0.76 at Fa of 0.5. The biological basis for this synergy was clear in differential changes in apoptosis with both the dietary molecules. Even though mechanistically our combinations are mutually non-exclusive in nature but both the dietary molecules showed a significant difference in quantum of cytotoxicity induction in comparison to stand alone GCB. However, the rate of apoptosis induction by BA in combination with GCB was more efficient in both the cell lines, than TQ in combination with GCB. Also, decrease in the trans-membrane mitochondrial potential () was observed when BA alone or in combination with GCB was provided to both the cell lines. No change, however, was observed on trans-membrane mitochondrial potential () when cancer cell lines were treated with GCB alone. PANC-1, which is resistant to GCB than MIA PaCa-2, did not decrease the trans-membrane mitochondrial potential () more efficiently in our experiment with BA in combination with GCB. It was surmised that targeting the glycolytic regulation may be an effective strategy for a shift from predominantly mitochondrial metabolism toward glycolysis. A shift toward glycolysis appears to confer a number of survival advantages for tumor cells. These include resistance to hypoxia, which is Protosappanin B prevalent in.