Current Eye Research, Early Online, 1–13, 2014 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2014.987870

RESEARCH REPORT

Synergistic Effect of Curcumin in Combination with Anticancer Agents in Human Retinoblastoma Cancer Cells Lines

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Seethalakshmi Sreenivasan1,2 and Subramanian Krishnakumar1 1

L&T Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, Nungambakkam, Chennai, Tamil Nadu, India and 2Birla Institute of Technology & Science (BITS) Scholar, Pilani, Rajasthan, India

ABSTRACT Purpose: Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of the herb Curcuma longa, is known to have anti-proliferative and anti-tumor properties. In this study, we evaluated the cytotoxic effect of curcumin alone and in combination with individual drugs like carboplatin, etoposide, or vincristine in a human retinoblastoma (RB) cancer cell line. Materials and methods: A drug–drug interaction was analyzed using the median effect/isobologram method and combination index values were used to characterize the interaction as synergistic or additive. We also performed the apoptosis and cell-cycle kinetics study with single drugs in combination with curcumin in a human RB cell lines (Y79 and Weri-Rb1). Results: Curcumin caused concentration-dependent decrease in cell proliferation, cell kinetics, and also induced apoptosis in both the RB cell lines. When combination of curcumin with individual drugs like carboplatin or etoposide or vincristine was treated on to RB cells, both cell viability and cell cycling were reduced and increased apoptosis was noted, in comparison with single drug treatment. These effects were significant in both the cell lines, indicating the ability of curcumin to increase the sensitivity of RB cells to chemotherapy drugs. Conclusion: Our in vitro findings showed that the combination of curcumin with single drug treatment showed marked synergistic inhibitory effect against RB cell lines. These results suggest that curcumin can be used as a modulator which may have a potential therapeutic value for the treatment of RB cancer patients. Keywords: Additive, chemotherapy drugs, combination index, curcumin, isobologram, retinoblastoma, synergistic

preserve eye vision and avoid enucleation.3 However, the efficiency of cancer chemotherapy is often limited due to severe adverse reactions and development of drug resistance. Cancer cells that exhibit multi-drug resistance phenomenon will decrease the intracellular accumulation of drug and enhanced its efflux due to the over-expression of multi-drug resistance proteins like P-glycoprotein (P-gp), multi-drug resistance associated protein (MRP), and lung resistance-related

INTRODUCTION Retinoblastoma (RB) is the most common primary intraocular tumor affecting one in 15,000 children. The disease is caused by the loss of function of both alleles of RB1 gene in the retina that initiates development of RB tumours.1,2 Chemotherapy drugs (carboplatin, etoposide, and vincristine) have become an important modality for the treatment of RB in an effort to

Received 28 March 2013; revised 23 October 2014; accepted 7 November 2014; published online 9 December 2014 Correspondence: Dr Subramanian Krishnakumar, Department of Ocular Pathology, Vision Research Foundation, Sankara Nethralaya, 18 College Road, Chennai 600 006, Tamil Nadu, India. Tel: +91 044 28271616. Fax: +91 044 28254180. E-mail: [email protected]

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protein (LRP).4 In our earlier study, we found that, prior to chemotherapy, the expression of P-gp and the LRP protein in RB tumors.5 It has been reported that in RB patients the use of cyclosporine reverses multidrug resistance and also increased the intracellular concentration of chemotherapeutic agents.6 However, the use of cyclosporine has some drawback due to its neurotoxicities to the RB cells which may contribute to the morbidity of chemotherapy in these patients.7,8 Extensive research in the last few years has demonstrated that phytochemicals are natural compounds that exert their inhibitory effects on carcinogenesis and tumor progression.9 Recent studies show that combining conventional or new cancer drugs with dietary compounds have been shown to have anti-cancer properties. Similarly, these natural compounds developed to have a synergistic effect with anti-cancer drugs, thereby making them to work better or require less toxic dosages.10 Curcumin(1,7-bis(4-hydroxy-3-methoxyphenyl)-1, 6-heptadiene-3,5-dione), commonly called diferuloylmethane, is a hydrophobic polyphenol derived from the turmeric of the herb Curcuma longa.11 Curcumin exhibits a wide range of pharmacological activities including antioxidant, anti-inflammatory, antimicrobial, and anti-carcinogenic activities.12 It has been shown to possess chemosensitization and modulation of MDR phenotype13,14 in MDR cell lines. The antitumor effect of curcumin has also been attributed in part to the suppression of cell proliferation, reduction of tumor load, and induction of apoptosis in various cancer cell lines both in vitro and in vivo model.15 The sensitizing effect of curcumin on cancer cell apoptosis has been reported for a variety of chemotherapeutic agents that are related to inactivation of NF-kB, modulation of p38 MAPK and AKT, and up-regulation of death receptor.16,17 Isobologram analysis has been widely used for calculating the combined effect of two chemotherapy drugs in vitro and becoming an increasingly essential and useful tool to combat the aggressive nature of cancer. Combining two anticancer drugs with different mechanisms of action may elicit a synergistic response, greater than the sum of the individual drug effects, which is much more effective than either drug alone.18,19 The aim of the present work was to study the effect of a combination of curcumin with chemotherapy agents (carboplatin, etoposide, or vincristine) on cell viability, apoptosis, and cell-cycle analyses in human RB cell lines. Combinations of curcumin and individual chemotherapy drugs were characterized using the median-effect/combination index isobologram method of multiple drug effect analysis. Combination index (CI) values were calculated for different dose-effect levels based on parameters derived from median effect plots of the curcumin, individual drug, and the combination of the both.

MATERIALS AND METHODS Reagents Curcumin was purchased from Sigma Chemical Co. (St. Louis, MO) and dimethyl sulphoxide (DMSO) from Merck Chemicals (Darmstadt, Germany). RPMI 1640 was purchased from Gibco BRL (Grand Island, NY). Fetal bovine serum and antibiotic-antimycotic solution were purchased from Himedia Laboratories Pvt. Ltd. (Mumbai, India). All other chemicals and reagents were of the highest grade commercially available.

Cell lines and culture conditions Y79 and Weri-Rb1 RB cell lines were obtained from the Riken Cell Bank (Ibaraki, Japan). The cells were maintained in the RPMI-1640 medium supplemented with 20% fetal bovine serum, 50 ng/ml of streptomycin, and 1.25 ng/ml of Amphotericin B at 37  C in a humidified incubator with 95% air and 5% CO2.

Drugs Curcumin and etoposide were dissolved in DMSO and stored, protected from light, as a concentration solution at 20  C. Chemotherapy drugs (carboplatin and vincristine) were made in sterile distilled water and stored at 20  C. Each compound was diluted in the culture medium on the day of experiment with DMSO concentrations never exceeding 0.1%.

Cell Viability Assay RB cell lines were harvested at confluence and plated into 96-well tissue culture plates, at a density of 5  103 cells/well and allowed to incubate overnight. Next day the plating medium was replaced with 0.1 ml fresh culture medium containing the vehicle control, curcumin (mM), or a combination of curcumin and carboplatin (mg/ml) or etoposide (mg/ml) or vincristine (nM). Cells were incubated for 48 h and after incubation period 10 ml of MTT (5 mg/ml) was added and the plates were allowed to incubate further for 4 h at 37  C. Crystals were dissolved with 100 ml DMSO, after which the reading was taken spectrophotometrically at 540 nm using an ELISA reader (Bio-Tek Instruments, Winooski, VT). IC50 values were determined from the dose–response curve of percent growth inhibition against test concentrations. DMSO was used as the negative (vehicle). Each experiment was carried out in triplicate. Current Eye Research

Novel combination therapy for RB

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Isobologram Analysis of Interactions Between Curcumin and Individual Chemotherapy Drugs The nature of drug interaction between curcumin and chemotherapeutic agents was analyzed using the isobologram method with the software program Calcusyn (Biosoft, Cambridge, UK). In the isobologram method, the concentration of one agent producing a desired (e.g., 50% inhibitory) effect is plotted on the horizontal axis, and the concentration of another agent producing the same degree of effect is plotted on the vertical axis; a straight line joining these two points represents the zero interaction between the two agents. The combination index of these interactions was provided by the isobologram equation CI50 = d1/D1 + d2/D2, where d1 and d2 represent the doses of chemotherapeutic agents and curcumin were administered in simultaneous combination required to produce a fixed level of inhibition IC50, while D1 and D2 represent the respective concentrations of curcumin and chemotherapeutic agents, respectively, required to produce a fixed level of inhibition (IC50) when administered alone. If CI values is less than 1, the effect of combination is synergistic, whereas if CI = 1 or greater than 1, the effect is additive or antagonistic.20 The dose-reduction index (DRI) is a measure of how much the dose of each drug in a synergistic combination can be reduced and still produces an effect level comparable to that for each drug alone. The DRIs for two drugs are derived from combination index values such that DRI1 = D1/d1 and DRI2 = D2/d2.21

Curcumin Uptake in RB cells The ability of RB cells to absorb curcumin was quantified using a flow cytometer. Cells were treated for 48 h with different concentrations of curcumin or combination of curcumin and carboplatin or etoposide or vincristine in RB cells. After the incubation period, the cells were washed thrice with ice cold PBS. Since curcumin exhibits a green fluorescent signal, the cells were analyzed using the FACScalibur flow cytometer (Becton Dickinson; Franklin Lakes, NJ). Fluorescence was detected and quantified using CellQuest Software (Becton Dickinson; Franklin Lakes, NJ). Quantification results are presented as percent increase of mean fluorescence intensity of the curcumin treated samples, compared with untreated cells in triplicate experiments.22,23

Cell-Cycle Distribution Analysis RB cell lines were treated with curcumin (5 and 10 mM) and different concentrations of carboplatin or !

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etoposide or vincristine in combination for 48 h, then harvested by centrifugation, washed twice with icecold PBS, and fixed by 70% ethanol at 20  C overnight. The fixed cells were then washed twice with ice-cold PBS and treated with 10 mg/ml RNAse for 30 min at 37  C. Cells were stained with propidium iodide buffer (0.1 mM EDTA, 0.1% Triton-X 100, 50 mg/ml propidium iodide, PBS pH 7.4) for 15 min in the dark at room temperature. Cell-cycle distribution was analyzed on a BD FACSCalibur flow cytometer (Becton Dickinson; Franklin Lakes, NJ). Data for 10,000 cells per sample were collected and analyzed using Cell Quest software programs (BD Biosciences, San Jose, CA).

Annexin V/Propidium Staining Assay RB cell lines (1  105cells/cm2) were plated in a 12well plate for overnight at 37  C. After overnight incubation, the cells were treated with different concentrations of curcumin and carboplatin or etoposide or vincristine in combination for 48 h. AnnexinVfluos staining was performed using an AnnexinVfluos apoptosis detection kit (Roche, Indianapolis, IN) in accordance with the instructions of the manufacture. In brief, treated cells were centrifuged, resuspended in 100 ml of Annexin-V-Fluos reagent, and incubated for 10–15 min at RT in the dark. After the incubation period, flow cytometry analysis was immediately performed. Data acquisition and analysis were performed by a FACSCalibur flow cytometer using Cell Quest software (Becton Dickinson; Franklin Lakes, NJ). Cells showing Annexin V negative and PI negative were considered viable cells whereas cells that were Annexin V positive and PI negative or Annexin V positive and PI positive were considered early stage or late-stage apoptotic cells, respectively.

Measurement of Caspase 3 Activity RB cell lines (1  106cells/cm2) were plated in 12 well plates and treated with curcumin and carboplatin or etoposide or vincristine in combination for 48 h. Cells were washed with ice-cold PBS and resuspended in a buffer containing 5 mmol/L Tris (pH 8), 20 mmol/L ethylenediamine tetraacetate (EDTA), and 0.5% Triton-X 100 on ice for 30 min. After incubation, lysates were centrifuged for 5 min at 13,000 rpm and the clear supernatant was taken for caspase activity. Reactions were carried out with 50 mg of protein, 20 mmol/L HEPES (pH 7), 10% glycerol, 2 mmol/L dithiothreitol, and 200 mmol/L N-acetyl-Asp-Glu-ValAsp (DEVD)-pNA substrate (caspase 3). Reaction mixtures were placed into a flat-bottomed mitrotiter plate and read with a 405 nm filter using a microtiter plate reader. Caspase activities were detected by

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measuring the proteolytic cleavage of the colored substrates.

Statistical Analysis Data were presented as mean values ± standard deviation (SD). Statistical comparisons between groups were performed by one-way analysis variance (ANOVA) followed by posthoc Tukey’s test. Value of *p50.05 was assumed as statistically significant.

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RESULTS Cytotoxic Effect of Curcumin and Chemotherapy Drugs Alone and in Combination in RB Cell Lines The anti-proliferative effect of curcumin and/or individual chemotherapy drugs on RB cells was determined by MTT assay. After 48 h of treatment, growth inhibition (GI50) values for each agent in Y79 and Weri-Rb1 cells, respectively, were as follows: curcumin, 30 mM and 25 mM24; carboplatin, 30 mg/ml and 35 mg/ml; etoposide, 4.5 mg/ml and 5 mg/ml; vincristine, 1 nM and 50 nM (Figure 1). These percentage

inhibition data indicate that all the compounds possess anti-proliferative property with varying concentrations of the particular drug in both the RB cell lines and the effect was found to be greater when the cells were treated with combination of curcumin and the chemotherapeutic drugs in the RB cell lines (Figure 2A and B).

Interaction of Combined Treatment of Curcumin with Individual Chemotherapy Agents in RB Cell Lines In order to determine the extent of interaction between curcumin/chemotherapy drugs, these data were subjected to Calcusyn software (Biosoft, Cambridge, UK) to study the synergism/antagnosim effect and also to evaluate the benefits of combined treatment compared with individual treatment. In the combination experiments, carboplatin concentrations ranged from 5 to 30 mg/ml, etoposide, 0.1–5 mg/ml, and vincristine 0.1–50 nM were tested with curcumin (5 mM and 10 mM) in Y79 and WeriRb1 cells. In the Weri-Rb1 cell line, the combination of 20 and 30 mg carboplatin, 1 and 5 nM vincristine with 10 mM curcumin, 5 nM vincristine with 5 mM curcumin, and

FIGURE 1 Effects of individual treatment of carboplatin, etoposide, and vincristine on RB cell survival. After exposure of Y79 and Weri-Rb1 cells to the indicated concentration of chemotherapy drug for 48 h, the cell viability was determined by MTT assay. Each bar represents the mean ± SD (n = 3). *p50.05 significant difference compared with the untreated RB cell lines. Current Eye Research

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Novel combination therapy for RB

FIGURE 2 Combination of curcumin and chemotherapy drugs (carboplatin, etoposide, and vincristine) on growth of RB cell survival. RB cell lines (Y79 and Weri-Rb1) were treated for 48 h with different concentrations of chemotherapy drugs in combination with 5 and 10 mM concentration of curcumin. The cell viability was determined by the MTT assay and the difference in cell growth after exposure to individual and combination of curcumin and chemotherapy drug was determined. The combination of curcumin with carboplatin, etoposide, and vincristine in both the RB cell lines is represented in (A) and (B). The data are mean ± S.D. values from three independent experiments.

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TABLE 1 Combination index values and dose reduction index for combined treatments of curcumin and chemotherapy drug in Weri cell line. DRI

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Chemotherapy drug 5 mg carboplatin 10 mg carboplatin 20 mg carboplatin 30 mg carboplatin 5 mg carboplatin 10 mg carboplatin 20 mg carboplatin 30 mg carboplatin 0.1 mg etoposide 0.5 mg etoposide 1 mg etoposide 5 mg etoposide 0.1 mg etoposide 0.5 mg etoposide 1 mg etoposide 5 mg etoposide 5 nM vincristine 10 nM vincristine 20 nM vincristine 50 nM vincristine 5 nM vincristine 10 nM vincristine 20 nM vincristine 50 nM vincristine

Curcumin (mM)

Viable cells (%)

CI

Degree of additve/syn

Curcumin

Chemotherapy drug

5 5 5 5 10 10 10 10 5 5 5 5 10 10 10 10 5 5 5 5 10 10 10 10

78.0 ± 2.6 52.7 ± 1.5 46.7 ± 3.2 39.3 ± 0.6 73.3 ± 2.5 64.7 ± 4.5 51.0 ± 2.0 40.3 ± 1.5 75.0 ± 2.0 66.0 ± 2.6 51.7 ± 1.5 37.0 ± 1.0 67.0 ± 1.0 50.7 ± 1.5 37.3 ± 2.1 35.3 ± 2.1 63.0 ± 2.0 52.0 ± 1.0 36.7 ± 1.5 33.0 ± 2.0 69.7 ± 1.5 50.0 ± 2.0 33.2 ± 21.3 28.7 ± 1.1

0.75 0.50 0.66 0.67 0.85 0.9 0.97 1.08 2.0 0.9 1.55 0.4 0.6 0.6 0.35 0.75 0.9 1.3 0.75 0.42 1.4 1.1 0.53 0.6

Moderate synergistic Synergistic Synergistic Synergistic Slight synergistic Slight synergistic Additive Additive Strong antagonistic Slight synergistic Strong antagonistic Synergistic Synergistic Synergistic Synergistic Slight synergistic Slight synergistic Moderate antagonistic Slight synergistic Synergistic Moderate antagonistic Additive Synergistic Synergistic

2 5 6 8 1.5 2 2.5 NA NA 4 NA 8 2 2.5 4 4 4 NA 8 8 NA NA 4 5

4 3.5 2 6 4 1.5 1.75 NA NA 2 NA 2 10 10 10 2 20 NA 6 3 NA NA 6 3.5

Chemotherapy drug was dosed in combination with curcumin at several different concentrations. Combination index values were calculated using the Calcusyn software (Biosoft, Cambridge, UK) and values 51 indicate synergism; CI = 1, additivity; and CI41, antagonism. The dose reduction index for each curcumin and chemotherapy drug is a measure of the extent, i.e., fold that the dose of each drug in a synergistic combination may be reduced at a given effect level compared with the doses for each drug alone. NA, not applicable to antagonism. Synergy/antagonism as a function of CI values (1.45–3, strong antagonism; 1.2–1.45, moderate antagonism; 0.9–1.1, additive; 0.75–0.9, slight synergistic; 0.7–0.75, moderate synergistic; 0.3–0.7, synergistic; 0.1–0.3, strong synergistic). Carboplatin and etopside = mg/ml concentration.

combination of 0.1–1 mg etoposide with 5 mM curcumin exerted an antagonistic or additive effect. Although there was no strong synergistic effect, the degree of synergy (high to slight synergistic) effect was seen in almost all other combination of carboplatin or etoposide or vincristine with curcumin in WeriRb 1 cell line (Table 1). In Y79 cells, the combination of 5 and 10 mg carboplatin with 5 mM curcumin yielded CI = 1.25–1.5 (having an antagonistic effect), similarly combination of 0.1–1 nM vincristine with 5 mM curcumin yielded CI = 1.1–1.25, pointing to an additive effect and combination of 0.5 and 1 mg etoposide with 5 mM curcumin showed an antagonistic effect. All other combinations of carboplatin or etoposide or vincristine with curcumin (5 and 10 mM) showed CI50.9, indicating synergism between these drugs (Table 2). The DRI quantitating the advantage synergism provides for dose reduction when two drugs are used in combination. The DRI value of 10 for etoposide (0.1 mg of etoposide + 5 mM curcumin) indicates that this drug would need a 10-fold higher concentration to produce the same inhibition effect in the absence of curcumin. In other words, the addition of 5 mM

curcumin allowed for a 10-fold reduction in the concentration of etoposide achieving the same inhibition effect. DRI values are listed for all doses of chemotherapy drug and curcumin used in isobologram experiments that identified synergism. Thus, the combination of curcumin and chemotherapeutic agent together was more effective than either treatment alone in some of the combination observed (Figure 3).

Combination Effects of Curcumin with Individual Chemotherapy Agents on the Curcumin Uptake We quantified curcumin uptake by flow cytometry as curcumin is known to fluorescence in the green band. The flow cytometry results showed that the RB cells (Y79 & Weri-Rb1) treated with curcumin exhibit increased fluorescence than the control cells and significant increase was observed with certain combination of 5 mM and 10 mM curcumin with a concentration of carboplatin or etoposide or vincristine (Figure 4). Current Eye Research

Novel combination therapy for RB

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TABLE 2 Combination index values and dose reduction index for combined treatments of curcumin and chemotherapy drug in Y79 cell line. DRI

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Chemotherapy drug 5 mg carboplatin 10 mg carboplatin 20 mg carboplatin 30 mg carboplatin 5 mg carboplatin 10 mg carboplatin 20 mg carboplatin 30 mg carboplatin 0.1 mg etoposide 0.5 mg etoposide 1 mg etoposide 5 mg etoposide 0.1 mg etoposide 0.5 mg etoposide 1 mg etoposide 5 mg etoposide 0.1 nM vincristine 0.5 nM vincristine 1 nM vincristine 5 nM vincristine 0.1 nM vincristine 0.5 nM vincristine 1 nM vincristine 5 nM vincristine

Curcumin (mM)

Viable cells (%)

CI

Degree of additve/syn

Curcumin

Chemotherapy drug

5 5 5 5 10 10 10 10 5 5 5 5 10 10 10 10 5 5 5 5 10 10 10 10

75.0 ± 2.0 53.7 ± 3.1 45.7 ± 3.1 38.7 ± 1.5 61.0 ± 2.94 47.3 ± 2.05 37.7 ± 1.25 43.0 ± 1.63 53.3 ± 3.5 47.7 ± 2.5 51.3 ± 1.5 33.0 ± 3.6 47.7 ± 2.52 36.0 ± 2.65 38.0 ± 2.65 32.7 ± 2.08 54.7 ± 4.5 37.7 ± 0.6 25.3 ± 2.5 20.7 ± 0.0 42.0 ± 2.6 26.7 ± 1.5 32.0 ± 2.6 26.0 ± 1.0

1.5 1.25 0.7 0.87 0.65 0.55 0.48 0.30 0.14 1.0 2.0 0.35 2.0 0.5 0.52 0.5 1.25 1.1 1.1 0.56 0.34 0.25 0.4 0.7

Strong antagonistic Moderate antagonistic Synergistic Slight synergistic Synergistic Synergistic Synergistic Synergistic Strong synergistic Additive Strong antagonistic Synergistic Strong antagonistic Synergistic Synergistic Synergistic Moderate antagonistic Additive Additive Synergistic Synergistic Strong synergistic Synergistic Synergistic

NA NA 7 8 2.5 3 4 3 6 NA NA 10 NA 4 4 4 NA NA NA 15 3.5 5 5 5

NA NA 1 1.1 4 3 2 1 10 NA NA 4 NA 40 10 4 NA NA NA 2 25 20 5 2

Chemotherapy drug was dosed in combination with curcumin at several different concentrations. Combination index values were calculated using the Calcusyn software (Biosoft, Cambridge, UK) and values 51 indicate synergism; CI = 1, additivity; and CI41, antagonism. The dose reduction index for each curcumin and chemotherapy drug is a measure of the extent, i.e., fold that the dose of each drug in a synergistic combination may be reduced at a given effect level compared with the doses for each drug alone. NA, not applicable to antagonism. Synergy/antagonism as a function of CI values (1.45–3, strong antagonism; 1.2–1.45, moderate antagonism; 0.9–1.1, additive; 0.75–0.9, slight synergistic; 0.7–0.75, moderate synergistic; 0.3–0.7, synergistic; 0.1–0.3, strong synergistic). Carboplatin and etopside = mg/ml concentration.

Combination Effects of Curcumin with Chemotherapy Agents on Induction of Apoptosis Annexin V/PI staining was performed to assess the induction of apoptosis with combination treatment of curcumin with individual chemotherapy drugs. The average percentage of apoptotic cells was increased significantly after exposure to all chemotherapy drugs and curcumin treatment applied alone when compared with the control. However, the combination of curcumin with chemotherapy drugs produced greatest increase in apoptotic cells when compared with the individual agents when applied alone (Figure 5).

Effect of Treatment with Curcumin and Chemotherapy Drugs, Separately and in Combination, on Cell-Cycle Distribution in RB Cell Lines We examined the effect of curcumin (5 mM and 10 mM) and different concentrations of chemotherapy drugs either alone or in combination in the RB cells, to see !

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whether the growth inhibitory effect of these compounds could be the result of a disturbance of cellcycle distribution. The analysis of DNA content in RB cells revealed that curcumin significantly increased the percentage of the cells in the S phase and decreased in the percentage of cells in the G0/G1 phase in RB cells. However, the effect of combination treatment demonstrated a significant decrease in the G0/G1 phase compared with the individual treatment (Figure 6).

Effect of Combined Treatment with Curcumin and Chemotherapy Agents on Caspase 3 Activity Caspase 3 activity was measured to confirm the induction of apoptosis in RB cell lines. RB cells were incubated for 48 h with curcumin in the presence and absence of carboplatin or etoposide or vincristine. The activity of caspase 3 was increased after treatment of RB cell lines with chemotherapy agents and curcumin alone. This was significantly enhanced upon combination of chemotherapy drugs and curcumin treatment in RB cell lines (Figure 7).

S. Sreenivasan & S. Krishnakumar

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FIGURE 3 The isobologram as described in Materials and methods was used to study the interaction of curcumin with carboplatin, etoposide, or vincrisine treatment in both the RB cell lines (Y79 and Weri-Rb1). Isobolograms were plotted by combining the IC50 values of curcumin with IC50 values of carboplatin, etoposide, or vincristine. The data are mean value from three individual experiments, values below the additive line consider as synergism and those above the additive line are taken as antagonism. Current Eye Research

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FIGURE 4 Effect of chemotherapeutic agents on curcumin uptake: RB cells were treated with different concentration of curcumin (5 and 10 mM) in the presence and absence of chemotherapy agents and analyzed by flow cytometry. Fold changes were compared with the control cells. *p50.05 significant difference compared with the concentration of curcumin (5 and 10 mM) without chemotherapy agents.

DISCUSSION The major drawback involved in chemotherapy treatment is the dose-limiting toxicity and induction of drug resistance. Several molecular mechanisms are involved in MDR.4 Various natural compounds have been identified which overcome drug resistance to chemotherapy drugs and thereby increase drug accumulation in cancer cells.25,26 Curcumin, a natural polyphenolic compound, has been reported to have strong anticancer effects against a broad range of human cancer cells like breast, prostate, colon, hepatocellular carcinoma, T-cell leukemia, B-cell lymphoma, mantle cell lymphoma, basal cell carcinoma, melanoma, renal carcinoma, and neuroblastoma.27 Although curcumin exhibits limited bioavailability, even low levels of physiologically achievable concentrations are sufficient for its chemopreventive and chemotherapeutic activity. Many approaches have been made on formulations of curcumin, including its complexation with phospholipid or nanoparticle-based encapsulation that exhibit greater bioavailability, stability, and anti-tumour activities than native curcumin.28 Moreover, various clinical trial and preclinical models have documented !

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minimal toxicity on administrating curcumin to human subjects.29 Our previous works showed that curcumin modulated the expression of LRP and similarly we also showed through in vitro and in silico studies the inhibitory effect of curcumin on MRP1 in RB cell line.24,30 Similar results were found by Das et al.31 where they have shown that curcumin is effective enough to modulate the expression and function of various MDR proteins.31 Thus, in the present study, we investigated the chemosensitizing effect of curcumin in combination with carboplatin or etoposide or vincristine on cell growth, uptake study, apoptosis, and cell kinetics on the RB cell lines. Our result found that curcumin inhibits RB cells (Y79 and Weri-Rb1) proliferation in vitro with the IC50 value of 30 mM and 25 mM, respectively. We used RB cells to study the interaction between curcumin and chemotherapy drugs using the isobolographic method. It has been studied that isobolographic analysis has been widely used to evaluate the interaction between two anticancer agents and provides qualitative and quantitative measure of nature and extent of drug interaction. Thus, in this study, a range of drug ratios was chosen to provide a deeper insight for each drug–drug combination. Combination index

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FIGURE 5 (A) and (B) Induction of apoptosis by annexin V staining in Y79 and Weri-Rb1 RB cells after 48 h treatment with curcumin alone or in combination with chemotherapy drugs (carboplatin, vincristine, and etoposide). Each bar represents the mean ± SD (n = 3). *p50.05 significant difference compared with the chemotherapeutic agents.

were calculated to characterize the degree of interaction between curcumin and chemotherapeutic agents as synergistic, additive, or antagonistic. Using this method, we found that several synergistic combinations of chemotherapeutic agents with curcumin fall in the range of CI = 0.1–0.9 in Y79 and with a CI = 0.3–0.9 in Weri-Rb1 cell lines. Curcumin has been shown to augment the effects of various chemotherapeutic agents like vincristine and doxorubicin, thereby enhancing the cellular accumulation of these drugs onto cancer cells.32 For example, Leila et al.33

demonstrated that curcumin in conjugation with the doxorubicin showed synergistic effect in H9c2 cardiomyoblast cells. Similarly, the combination of curcumin with paclitaxel augments the anticancer effects of paclitaxel against Hela cells via a mechanism involving down-regulation of NF-kB activation and phosphorylation of AKT pathways.34 Our study also found that curcumin enhanced the cytotoxicity of RB cells in combination with chemotherapy drugs (carboplatin or etoposide or vincristine) when compared with the individual treatment in a synergism manner Current Eye Research

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FIGURE 6 Effect of curcumin in combination with carboplatin or etoposide or vincristine on the cell cycle in RB cells. The cells were treated with drugs for 48 h and changes in cell-cycle distribution were calculated by flow cytometry in Y79 and Weri-Rb1 RB cells after 48 h treatment. Each bar represents the percentage of cells in G0/G1, S, and G2/M phases from the mean ± SD value (n = 3).

FIGURE 7 Effect of curcumin in combination with carboplatin or etoposide or vincristine on caspase 3 activity in RB cells. RB cells were plated on 12-well plates with curcumin and CEV combination for 48 h. Aliquots of cell lysate were added to the reaction buffer containing DEVD-pNA as a substrate and the amount of activity was measured by the proteolytic cleavage of the colored substrate measured calorimetrically. Data represent means ± of at-least three independent experiments. *p50.05 significant difference compared with the chemotherapeutic agents.

(Table 1 and 2). We also observed additive and antagonistic response in certain combination treatment. However, the mechanism of this antagonistic effect is unknown, it is well established that the !

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degree of synergy or additive effect is mainly based on between two drug concentration and ratios. The results shown in Figure 4 demonstrate that curcumin absorption was enhanced in certain

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12 S. Sreenivasan & S. Krishnakumar concentrations of curcumin (5 and 10 mM) with a combination of carboplatin or vincristine or etoposide. A similar effect was seen in SK-BR-3 breast cancer cell line where docosahexaenoic acid enhanced the cellular uptake of curcumin in SK-BR-3 breast cancer cell line both by Altenburg et al.35. When viewed with the data-describing curcumin uptake, the flow cytometry does not distinguish intracellular curcumin from the curcumin bound to the cell surface. To study the possible mechanism involved in the anticancer activity of curcumin and combination, we evaluated the cell-cycle kinetics on RB cell lines. A combination of curcumin and chemotherapy agents induced cell-cycle arrest in the S-phase in both the RB cell lines. Similar results observed by Andjelkovic et al.36 were a combination treatment of curcumin and sulfinosine induced a more pronounced cell-cycle arrest in the S and G2/M phase in NCI-H4660/R cells.36 Several studies have demonstrated that anticancer drugs like cisplatin, etoposide, and camptothecin induce tumor cell death via induction of apoptosis.37 Although various chemotherapeutic agents are in clinical use, the need for identifying a better drug induces apoptosis in various cancer cells in an efficient manner. Curcumin, a dietary supplement, has been reported to induce apoptosis in various cancer cell lines.38 In our study, we investigated the synergistic effect of curcumin and chemotherapeutic agent combination on the growth suppression of RB cells by annexin staining and caspase 3 activities. Our study showed that combination treatment of curcumin and chemotherapy drugs produced a higher percentage of apoptosis than that caused by either curcumin or chemotherapy drug alone by annexin staining. Similarly, we observed a significant increase in the activity of caspase 3 in combination treatment when compared with a single drug treatment in RB cells. Our results are similar to the findings of LevAri39 & Hosseinzadeh et al.33, where combination treatment of curcumin with celecoxib showed synergistic pro-apoptotic effects in osteoarthritis synovial adherent and cardiomyoblast cells. Recently, in RB cancer cells, a similar sequencedependent effect was reported by Mahida et al.40 on the interaction of FDA approved drugs with the natural antioxidant resveratrol, who reported that the class of anti-cancer drugs used for the therapy showed an antagonistic effect when combined with resveratrol in RB cancer cell lines. In conclusion, our study showed the anti-proliferative efficacy of curcumin in both the RB cell lines and the combination treatment showed both additive and synergistic interaction between the curcumin and the clinically used chemotherapeutic agents. These interesting results need to be further evaluated by performing in vivo studies for enhancing the efficacy and lowering the toxicity without affecting the normal

cells. Thus, combination therapy might potentially offer a greater therapeutic effect for the treatment of RB cancer.

DECLARATION OF INTEREST The authors declare that they have no potential conflict of interest. The present work was supported by grant from Indian Council of Medical Research (5/13/25/04/NCD-III).

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Synergistic Effect of Curcumin in Combination with Anticancer Agents in Human Retinoblastoma Cancer Cell Lines.

Curcumin (diferuloylmethane), a phenolic compound obtained from the rhizome of the herb Curcuma longa, is known to have anti-proliferative and anti-tu...
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