Pancreatic cancer (PaCa) is a major health concern due to its aggressiveness and early metastasis. Current treatments for PaCa are limited by development of resistance against therapy. As an alternative strategy, we assessed the combinatorial effect of dietary compounds, garcinol and curcumin, on human PaCa cells (BxPC-3 and Panc-1). A significant (
Cancer is a major health concern across the globe today with pancreatic cancer (PaCa) being the fifth major cause of deaths due to cancer in the United States. Development and progression of this chronic disease involves deregulation and activation of multiple signaling pathways at different stages of carcinogenesis. This complexity associated with the disease causes limitations in designing high-efficacy therapeutic strategies. Although immense work has been done in the prevention and treatment of PaCa, the results are not satisfactory and need improvement. For example, gemcitabine is a standard cytotoxic chemotherapeutic agent used for treatment in PaCa. However, this drug provides limited survival advantage along with several side effects and development of chemoresistance [
Epidemiological studies have consistently shown that consumption of a healthy diet including fruits, vegetables, and whole grains is strongly associated with reduced risk of cancer and other diseases [
When a combination of two or more compounds exhibit a more potent therapeutic effect than that of individual compounds at equal concentrations, the effect is described to be a synergistic one. In this study, we tested the hypothesis that the bioactive compounds garcinol from
Human pancreatic carcinoma cell lines BxPC-3 and Panc-1 were obtained from American Type Culture Collection (Manassas, VA, USA). The cell lines were maintained in continuous exponential growth by twice a week passage in RPMI-1640 medium (Cellgro Manassas, VA; BxPC-3 cells) and Dulbecco modified Eagle’s medium (
Garcinol (≥95% (TLC), Biomol International, USA) and curcumin (≥94% (curcuminoid content), ≥80% (Curcumin) Sigma Aldrich, USA) were dissolved in
Cells were seeded at a density of
The cell apoptosis ELISA Detection Kit (Roche, Palo Alto, CA, USA) was used to detect apoptosis according to the manufacturer’s protocol. Briefly, after treatment of BxPC-3 and Panc-1 cells with garcinol or curcumin for 48 hrs, the cytoplasmic histone/DNA fragments from cells were extracted and bound to immobilized antihistone antibody. Subsequently, a peroxidase-conjugated anti-DNA antibody was used for the detection of immobilized histone/DNA fragments. After addition of the peroxidase substrate, the absorbance by the samples was determined at 405 nm with an ULTRA Multifunctional Microplate Reader (BIO-TEK Instruments).
Morphological changes characteristic of apoptosis were determined by DAPI (4′, 6-diamidino-2-phenylindole) staining as per manufacturer’s protocol (Invitrogen, USA). Briefly,
Caspase-3 and -9 activities were measured in whole-cell lysates prepared from garcinol and/or curcumin-treated samples using a commercially available assay kit (R&D Assay System, Minneapolis, MN, USA) according to the manufacturer’s instructions.
Cells were plated as described above and allowed to attach overnight. The culture medium was replaced with fresh medium containing curcumin and garcinol individually or in combination in different ratios of 1 : 2.5, 1 : 5, and 1 : 10 for 72 hrs, and the effect on cell growth was examined by the MTS assay method as described above and then analyzed using the Calcusyn (Biosoft) software program, which utilizes the T.C. Chou method of determining synergy and antagonism.
The combination index was determined at a 25%, 50%, and 75% toxicity level for each cell line and at each drug ratio. The median-effect equation and combination index (CI) analysis was used to calculate the interaction between treatment modalities. This analysis determines if the effect of the combination is antagonistic, additive, or synergistic. A CI value of one indicates that the effect of one drug is additive to the second, a CI value of greater than one indicates antagonism between the two agents, and a CI value of less than one indicates synergism between the agents. The equation used to calculate CI is as follows:
BxPC-3 and Panc-1 cells were tested for their effects on cell viability under the influence of curcumin or garcinol. Both cell lines are p53 mutated with differences in their
Percentage of metabolically viable cells was reduced in a dose-responsive manner on 48 hr treatment with garcinol (upper panel) or curcumin (lower panel) in both PaCa cell lines. (a) BxPC-3 and (b) Panc-1 as analyzed using MTS assay. *
Garcinol and curcumin hold structural resemblance, but our results suggest that their therapeutic mechanistic targets might be different. Overall, this indicates that garcinol might play an important role in targeting the
In order to confirm whether the reduction in cell viability was in part due to apoptotic induction, we performed the ELISA assays and saw that both garcinol (upper panel) and curcumin (lower panel) induced apoptosis in a dose-dependent manner in both PaCa cell lines, BxPC-3 (Figure
Garcinol-(upper panel) or curcumin-(lower panel) treated cytosolic extracts were used to evaluate induction of apoptosis in PaCa cells. (a) BxPC-3 and (b) Panc-1 using ELISA-Histone DNA Enrichment Assay. Results demonstrate a significant dose-dependent increase in apoptotic cells in individual treatment with either agent for 48 hours. Enrichment factor was measured using subtraction of background signal. *
Both BxPC-3 (Figure
Apoptotic morphological changes such as abnormal nuclear morphology, reduction in cell number with apoptotic body formation, and cell shrinkage induced by combination treatment with curcumin and garcinol in different ratios for 48 hours were observed using DAPI stain in both PaCa cell lines: BxPC-3 (left panel) and Panc-1 (right panel). (1 : 4 ratio is 2.5
Caspases are a family of cysteine proteases that play a very important role in apoptosis. Caspase-9 is an initiator caspase that causes the cleavage of procaspase to active form, and Caspase-3 is an executioner caspase that cleaves other protein substrates triggering the process of apoptosis. We measured caspase-3 and 9 activities using a colorimetric assay and observed a significant 2 to 3 fold induction of active caspase-3 (
Garcinol and curcumin significantly increased Caspase-3 (a and b) and Caspase-9 (c and d) activity by ~2 to 3 folds in both PaCa cell lines: BxPC-3 (left panel) and Panc-1 (right panel) relative to untreated control after 48 hour treatment. Caspase activity was measured in garcinol- and/or curcumin-treated whole cell extracts using colorimetric assay. *
In order to determine the extent of synergism between these two agents, we tested the combination effect of garcinol and curcumin in different ratios. Cell viability reduced significantly (
(a) combination effect of curcumin and garcinol on Panc-1 cell viability was determined using MTS assay. Combinatorial treatment significantly reduced cell viability more effectively than monotherapy on 48 hr treatment. *
Figure
Similarly when the ratio of garcinol and curcumin was reversed, and the cells were treated with 1 : 10, 1 : 5, and 1 : 2.5 of garcinol : curcumin, the CI values at ED50 were 0.756, 0.747, and 0.921, respectively. The
Another parameter describing the sigmoidicity of the dose effect curve is the
Collectively, the above results suggest that dietary interactions can be a beneficial option for the control of PaCa. This disease is a major health problem because of its increasing incidence worldwide. Given the limited therapeutic options and current unmatched clinical needs for the treatment of the patient, there is an urgent need for the development of novel agents that can influence the survival rates and quality of life for the patients. Relatively few studies have reported that the additive and synergistic effects of phytochemicals in fruits and vegetables are responsible for their potent anticancer activities. One school of thought is that this synergistic or additive effect of various bioactive compounds could be due to targeting of multiple signaling pathways. Our data clearly exhibited that garcinol in combination with curcumin had potent synergistic effect on cell viability and apoptosis.
Dietary modification is a practical approach where we can combine nontoxic phytochemicals from fruits and vegetables, and this approach may also enhance the chemotherapeutic efficacy of malignant cells with minimal toxicity to normal cells. This study demonstrates a synergistic effect between curcumin and garcinol in pancreatic cancer cells. Our findings have important implications for combination of different dietary agents for cancer therapy. However, further studies are needed to elucidate the underlying mechanisms of combinatorial approach in pancreatic cancer for enhancing efficacy and simultaneously lowering cytotoxicity to normal cells.