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 [ 1 , 2 ] . therefore , exploring new approaches for better maintenance of this disease is of great interest . 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 . over the years , several natural bioactive compounds or phytochemicals have been isolated , identified , and their potential anticarcinogenic properties have been evaluated . there is a growing body of evidence that chemotherapeutic combination strategies would be more efficient in reducing drug toxicity , inhibiting tumor development and progression than with either agent alone . research suggests that increased consumption of curcumin isolated from curcuma longa or turmeric , contributes to lower risk of cancer in asian ( indian ) populations as compared to industrialized western nations [ 4 , 5 ] . also recently , the anticancer properties of garcinol , a bioactive compound derived from the rind of the fruit , garcinia indica , kokum or mangosteen has been revealed . the anticarcinogenic properties of this compound in several cancers such as breast , oral , and colon cancer have been tested . very recently , we and others have showed that garcinol reduces pancreatic cancer cell viability and increases apoptosis via downregulation of nuclear factor nf-b [ 6 , 7 ] . asian ( indian ) populations consume turmeric and kokum as a spice in their diet regularly . it is possible that the lower incidence of pancreatic cancer in india ( according to 2008 statistics , 2 cases per 100,000 persons per year ) as compared to the western nations ( in 2008 , an estimated 37,680 new cases of pancreatic cancer ( 18,770 in men and 18,910 in women ) were diagnosed in the united states ) [ 3 , 8 ] might be attributed in part from interactions between the bioactive components in diet including that of turmeric and kokum . 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 garcinia indica and curcumin derived from curcuma longa will work in synergism and inhibit the growth of paca cells . therefore , garcinol , curcumin , and their combination were tested in paca cell lines bxpc-3 and panc-1 , carrying wildtype and mutated k - ras , respectively . our results indicate that the combination of garcinol and curcumin significantly inhibited cell viability ( p < 0.05 ) and caused induction of apoptosis via upregulation of caspase-3 and -9 activity ( p < 0.05 ) in both cell lines . these data suggest a potential for the combination therapy for improvement of efficacy in paca treatment and hence warrants further investigation . 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 ( dmem , cellgro manassas , va ; panc-1 cells ) , respectively and supplemented with 10% fetal bovine serum , 100 units / ml penicillin , and 10  mg / ml streptomycin in a humidified incubator containing 5% co2 in air at 37c . garcinol ( 95% ( tlc ) , biomol international , usa ) and curcumin ( 94% ( curcuminoid content ) , 80% ( curcumin ) sigma aldrich , usa ) were dissolved in dmso to make 20  mm stock solution . cells were seeded at a density of 3  10 cells / well in 96-well microtiter culture plates . after overnight incubation , the medium was removed and replaced with fresh medium containing different concentrations of garcinol ( 040  m ) diluted from a 20  mm stock and/or curcumin ( 050  m ) . after 72  hrs , mts ( 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h - tetrazolium , inner salt ) solution was added to each well and incubated further for one hour . color development was measured spectrophotometrically at 595  nm on a plate reader ( bio - tek instruments ) and quantified as per the manufacturer 's protocol ( promega , usa ) . cell viability has been expressed as a percentage , for each treatment group relative to control in the absence of garcinol or curcumin . 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 , 5  10 cells were seeded into 6-well plates containing 1 - 2  ml medium . after 2436  hrs , garcinol and/or curcumin was added and incubated for another 48  hrs . cells were harvested by trypsinization , washed with pbs , and subsequently incubated for 30 minutes with dapi at room temperature in dark for 30 minutes . prior to microscopic analysis , the cells were stained with prolong gold antifade reagent and visualized under fluorescence microscope ( nikon eclipse , 80i ) with an excitation maximum at 358  nm and an emission maximum at 461  nm . 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 . 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 . 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 : ( d)1/(dx)1 + ( d)2/(dx)2 + (d)1(d)2/(dx)1(dx)2 , where ( dx)1 and ( dx)2 are the doses for x% inhibition by drug 1 and drug 2 alone . these values are obtained from the median - effect equation . ( d)1 and ( d)2 are the doses in combination that inhibit cell growth by x% . a more detailed description of degrees of synergism and antagonism is adapted from chou and hayball ( calcusyn , windows software for dose effect analysis . 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 k - ras mutation status . panc-1 is a poorly differentiated paca cell line with a mutated k - ras , whereas bxpc-3 is a moderately differentiated paca cell line with a wild type k - ras . we observed that both curcumin and garcinol significantly ( p < 0.05 ) reduced cell viability in both cell lines in a dose - dependent manner . however , garcinol exhibited a more potent effect ( ic50 = ~7  m ) in panc-1 cells which was comparable to the efficacy of curcumin ( ic50 = ~10  m ) in bxpc-3 cells as seen in figure 1 . cells with mutant oncogenes such as k - ras continue to grow even when they are not receiving any growth signals . 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 k - ras pathway . in bxpc-3 cells ( figure 1(a ) ) ic50 for garcinol treatment ( upper panel ) was observed at approximately 15  m as compared to 10  m curcumin treatment for 48 hours . in addition , in panc-1 cells ( figure 1(b ) ) ic50 values for garcinol ( upper panel ) and curcumin treatment were 7  m and 25  m , respectively . we and others have consistently reported no toxicity at treatment dose of either curcumin   or garcinol [ 6 , 7 ] on normal cells . hence , these two agents in combination could be a useful strategy for the treatment of this aggressive disease .   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 2(a ) ) and panc-1 ( figure 2(b ) ) . p53 is a key tumor suppressor protein that limits cell proliferation by inducing cell cycle arrest and apoptosis in response to cellular stress . hence , the role of p53-independent pathways such as retinoblastoma protein ( rb ) or p73 in induction of apoptosis needs further investigation . we determined morphological changes associated with apoptosis , such as formation of apoptotic bodies and reduction in cell number using the dapi stain as in figures 2(c ) and 2(d ) in both paca cell lines upon treatment . both bxpc-3 ( figure 3 left panel ) and panc-1 ( figure 3 right panel ) were subjected to different concentrations of curcumin and garcinol individually , and reduction in cell number along with formation of apoptotic bodies was observed as a dose - dependent effect . figure 3 depicts the structural changes when curcumin and garcinol were given in combination in different ratios ( 1  :  4 ratio is 2.5  m  : 10  m concentration and 1  :  10 ratio is 2  m  : 20  m respective concentrations ) . combination treatment had a more pronounced effect on induction of apoptosis than their respective individual treatments . 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 ( p < 0.05 ) ( figures 4(a ) and 4(b ) ) and caspase-9 ( p < 0.05 ) ( figures 4(c ) and 4(d ) ) in both panc-1 and bxpc-3 cells upon treatment with garcinol and curcumin . similar or higher induction was observed in combination treatment with lower doses in different ratios . caspases are usually associated with the activity of tumor suppressor genes such as p53 , p73 , or rb . since both the cell lines exhibit p53 mutation , activation of caspases-3 and -9 might be due to the involvement of another tumor suppressor gene , which needs to be confirmed by further analysis .   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 ( p < 0.05 ) upon treatment in panc-1 cells , and the effects were more pronounced in the combinatorial approach as compared to individual doses ( figure 5(a ) ) . using the isobologram analysis method , we determined the combination index values ( ci ) for different ratios of treatment in panc-1 cells . ci = 1 indicates additive effect , < 1 indicates synergistic effect , and > 1 suggests antagonistic effect . the ci values for ed50 ( effective dose for 50% inhibition ) when curcumin and garcinol were administered in the ratios of 1  :  10 , 1  :  5 , and 1  :  2.5 were 0.201 , 0.422 , and 0.659 , respectively . dm can be defined as the median - effect dose or concentration signifying the potency of the treatment . we observed that when panc-1 cells were treated with only curcumin and garcinol , the dm values were 26  m and 17.65  m , respectively . however , when curcumin and garcinol were administered in the ratios of 1  :  10 , 1  :  5 and 1  :  2.5 , the dm values were 4.57 , 8.57 , and 10.79  m , respectively . based on the ci ed50 values and the dm values , it can be concluded that there was 2 to 5 fold lower concentration requirement in combination treatment than individual treatments in order to show similar effect . 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 dm values recorded for the same ratios were 1.25 , 2.32 , and 5.11  m , respectively . dm is an important parameter which helps in establishing dose reduction leading to lowered toxicity in the host , thus retaining overall therapeutic efficacy . an m = 1 , > 1 , and < 1 correlates with a hyperbolic , sigmoidal , and negative sigmoidal shape , respectively . we observed m > 1 value in all our samples except when curcumin  :  garcinol were in the ratio of 1  :  10 suggesting sigmoidicity of the curve . we report an r value close to 1 ( 0.971.00 ) in all our samples . collectively , the above results suggest that dietary interactions can be a beneficial option for the control of paca . 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 . 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 .
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 ( p < 0.05 ) dose - dependent reduction in cell viability and increase in apoptosis were observed in both cell lines as compared to untreated controls . a combination index ( ci ) 
 value  <  1 , for a two - way comparison of curcumin and garcinol , suggests synergism . 
 the potency ( dm ) of the combination of garcinol and curcumin was 2 to 10 fold that of the individual agents . 
 this indicates that curcumin and garcinol in combination exhibit a high level of synergism , with enhanced bioactivity , thereby reducing the required effective dose required for each individually . 
 this combinatorial strategy may hold promise in future development of therapies against paca .