Indian Journal of Experimental Biology Vol. 44, March 2006, pp 216-220 Inhibitory effect of cinnamoyl compounds against human malignant cell line M A Indap a, S Radhika a, Leena Motiwale b & K V K Rao b * a Chemotherapy Group and b Chemical Carcinogenesis Group, Khanolkar Shodhika, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410 208, India Received 14 July 2005; revised 7 December 2005 In the present study, anti-proliferative effects of dietary polyphenolic compounds have been observed and demonstrated the strong anticancer efficacy of curcumin (CMN), an active constituent of dietary spice (turmeric) using human leukemia cancer cell line. CMN inhibited the proliferation of K562 leukemic cells by induction of apoptosis. The current study demonstrated synergy with combination of drug therapy, and suggested that combination of ferulic acid and cisplatin synergistically inhibited cellular proliferation. Cytotoxic synergy was observed independent of the sequence of addition of two drugs to cultured cells. The synergized growth inhibitory effect with cisplatin was probably associated with G2-M arrest in cell cycle progression. These findings suggested that among the cinnamoyl compounds, CMN was most potent and FER appeared to be a better modulating agent on human malignant cell line. Keywords: Antitumor, Caffeic acid, Cinnamoyl compounds, Curcumin, Ferulic acid, Gallic acid, Platinum drugs. *Correspondent author: Phone: 91-22-27405023 Fax: 91-22-27412894 E-mail: krao@actrec.res.in kvkrao1@yahoo.com Microconstituents derived from fruits, vegetables and dietary plants have been shown to exert antitumor activity against variety of rodent tumors 1. The mechanism by which they exert antitumor effects has not been fully elucidated, although a number of potential targets have been implicated. Some interesting biological targets are nuclear factor kappa B (NF-kB) 2, angiogenesis and EGFR 3 which are involved in diverse cellular processes. We have been working on polyphenolic compounds occurring abundantly in food products like grapes, peanuts and various herbs. Polyphenolics are generally believed to be antioxidant in nature 4. The mechanism by which these antioxidants exert tumor suppressing activity is likely to involve modulation of cell signaling pathways resulting in inhibition of cell proliferation which leads to induction of apoptosis, the biological end point 5. Cinnamoyl compounds of many plants have been studied (1) Ferulic acid (FER): It is a major constituent of rice bran or germ. Its anti-poliferative effects on K562 and lymphocytes derived from ALL and CML are reported 6. It is less studied hydroxycinnamate; (2) Caffeic acid (CAF): It is found to be widely present in many members of plant kingdom. Caffeic acid and its esters inhibited cell growth of colon adenocarcinoma HT-29 and HCT-116 and exhibited differential toxicity to cancer cells 7 ; (3) Curcumin (CMN): One of its richest sources is the traditional herbaceous plant Curcuma longa (turmeric) and related Curcuma species of the family Zingiberaceca. The yellow color turmeric is being used as a spice, food preservative, coloring agent and an additive in cosmetic and drug preparations. Curcumin possesses antioxidant, antiinflammatory and anti-carcinogenic properties 8. CMN treatment causes induction of apoptosis in cancer cells and reduces the expression of vital factors such as C- Jun, C-myc, NF-kappaB, AP-1 etc 3 ; (4) Gallic acid (GAL): It is a trihydroxybenzoic acid, naturally occurring plant phenol, and showed selective cytotoxicity against tumor cells with higher sensitivity than normal cells 9. Although not a cinnamoyl compound, its hydroxy and carboxic acid groups are important for anticancer activity. In order to investigate the effects of these compounds and their combined use with chemotherapeutic drugs on cellular functions including cell viability, survival and cell cycle, we used human erythroleukemic cell line K562. In the present report, we present our findings of the studies carried out on some interesting dietary constituents emphasizing more on cinnamates.
INDAP et al.: INHIBITORY EFFECT OF CINNAMOYL AGAINST MALIGNANT CELLS 217 Materials and Methods Chemicals Ferulic acid and gallic acid were purchased from s.d. Fine Chemicals, Mumbai. Caffeic acid and curcumin were purchased from Merck, Mumbai. Cisplatin (CDDP) and carboplatin (CBDC) were purchased locally. Growth medium, fetal bovine serum (FBS), antibiotics were obtained from Sigma Chemical Co., St. Louis, M. O. All other chemicals and reagents were purchased from Gibco Laboratories, Grand Island (USA). Cell lines and cell cultures Human erythroleukemic cell line K562 was procured from National centre for cell sciences, Pune. Cells were maintained at 37 C in IMDM medium (Gibco BRL) containing 10% FBS besides penicillin G (100μg/ml) and streptomycin (100μg/ml) under a humidified atmosphere of 5% CO 2 in air. Assessment of cell viability Cells (1 10 5 cells/ml) were seeded into pertidishes (35mm) and incubated with different concentrations of compounds for 24-72 h. Cell viability was measured by trypan blue assay and also by 3-(4,5- Dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) colorimetric dye assay 10. Measurement of GSH and GST activity Total GSH was measured from cells growing in culture as described previously 10. Total GST activity was determined using 1-chloro-2,4-dinitrobenzene as a substrate 10. Reactions were performed with cytosolic extracts and the conversion of 1-chloro-2,4- dinitrobenzene by GST was measured with spectrofluorometer. The data are expressed as amount of dinitrophenyl glutathione formed/min/mg of protein at 37 C using extinction coefficient 9.6 mm/cm. Cell cycle analysis Analysis of cell cycle effects was performed as follows. Cells were collected in PBS, fixed in 70% ethanol (in PBS) at 4 o C and were incubated for 5 min in citrate buffer (750mM, Na 2 HPO 4; 750µM, sodium citrate; ph 7.8) to permeabilize the cells. The cells were resuspended in PBS (1ml) containing 20μg/ml of propidium iodide (Sigma) plus 5 units of Rnase A (Sigma) and incubated for 30 min at room temperature. Flow cytometric analyses were carried out in a fluorescence activated sorter with a 360 nm Argon- Iron laser. Analysis of cell cycle and measurement of subdiploid (<2N) DNA content of propidium iodide stained nuclei was performed as published previously 10. Cyclodextrin complexes Preparation and characterization of beta cyclodextrin inclusion complexes of the compounds under investigation was carried out as reported earlier 11. Results and Discussion The main polyphenolic dietary sources for human are fruits and beverages (fruit juice, tea, coffee, coco, beer and wine) and to a lesser extent vegetables, legumes and cereals. Recently, various purified polyphenolic compounds were shown to display beneficial effects in vitro or in vivo, including antioxidant, chemopreventive and antitumoral properties 5. However, mechanisms by which these polyphenolics inhibit tumor cell growth and their therapeutic potential are poorly understood. The present study was, therefore, undertaken to define the anti-proliferative doses and the mechanism of action of a few selected polyphenolics namely ferulic acid, caffeic acid, curcumin and gallic acid. The phenolics were tested for their effect on the growth of human erythroleukemic cell line K562. As shown in Figure 1, FER CAF, CMN, GAL induced a dose dependent decrease of leukemia cell member, compared with cells incubated in medium alone (control group). The data also indicated that leukemia cells differ in their sensitivity to these compounds. Inhibitory doses of FER were relatively higher compared with CAF, CMN and GAL (Fig. 1). It was clear from the data that CMN was the most potent compound. Data clearly showed that phenolics mediated inhibition of leukemia cell proliferation was dose and time dependent (Fig. 2). Besides K562, CMN also inhibited the growth of estrogen responsive MCF7 mammary carcinoma cell line (data not shown). CMN can, therefore, be considered as a potent inhibitory agent for human malignant cell lines taking into consideration the early reported work 8. Besides growth inhibition, we checked whether a phenolics mediated decrease of leukemic cell numbers was related to cell cycle arrest. Data showed FERmediated cell cycle modification in K562 cell line (Table 1). K562 cells showed clear accumulation in G2/M phase, correlations with increasing concentration and consequently decreased numbers of cells in Go/G1 phase were noticeable.
218 INDIAN J EXP BIOL, MARCH 2006 Defects in G2-M arrest checkpoint may allow a damaged cell to enter mitosis and undergo apoptosis and efforts to enhance this effect may increase the cytotoxicity of chemotherapy 12. Many reported herbal derivatives capable of G2-M arrest may be mediating their effects through microtubules, which induce mitotic arrest beyond the late G2 checkpoint. Discovery of agents with synergy in combination with chemotherapy and associated effects on cdk1 function, suggests that modulation of checkpoint regulators may contribute to cytotoxicity in clinical trials. Cellular glutathine (GSH) has been shown to be crucial for regulation of cell proliferation, cell cycle progression and apoptosis 10. We, therefore, analyzed changes of GSH levels of K562 cells by using standard procedure. GSH level was depleted by 72 h exposure of K562 cells to FER (data not shown). Depletion of GSH by FER may explain why exposure to FER leads to cell cycle arrest and cytotoxicity. Data showed CMN mediated cell cycle modification in K562 cell line (Table 2). CMN induced apoptosis (33%) in K562 cells. Genotoxic damage induces cell cycle arrest and/or apoptosis. Apoptosis or programmed cell death is an active physiological mode of cell death. A number of anticancer drugs are genotoxic and their damaging effects upon cells are mediated by this mechanism 13. CMN has been reported to Table 1 Effect of FER on cell cycle progression of K562 cells Group Fig. 1 Dose dependent effect of phenolic compounds. K562 cells were incubated for 72h with various concentration of compounds and the IC 50 of each compound was determined from the dose response curve. Each value represents mean ± SD from 3 independent experiments. Concentration (μg/ml) G0/G1 S G2/M M1 (% apoptosis) Control - 32.56 67.44 0.00 1.56 FER 50 29.89 70.11 0.00 3.12 100 26.97 67.59 5.45 3.29 250 22.09 67.06 10.84 5.12 500 18.3 65.72 15.98 9.79 1000 7.3 61.84 30.86 * 14.41 *Significant at P<0.01, FER vs control Table 2 Cell cycle analysis of K562 cells after exposure to CMN Group Concentration (μg/ml) G0/G1 S G2/M M1 (% apoptosis) Control - 37.93 55.73 6.34 1.09 CMN 0.5 38.32 53.54 8.14 1.00 1.0 34.02 56.56 9.42 1.05 2.5 34.43 63.82 1.75 4.29 5.0 47.84 52.16 0.0 19.99 10.0 12.02 84.92 3.06 33.2 * *Significant at P<0.01, CMN vs control
INDAP et al.: INHIBITORY EFFECT OF CINNAMOYL AGAINST MALIGNANT CELLS 219 induce apoptosis through mitochondrial pathway involving caspase-8, cytochrome C release and caspase- 3 activation in human neoplastic cells 8. Increase in systemic toxicity and drug resistance, the major drawbacks of cancer chemotherapeutic agents have led to a new challenge in the field of cancer research. To overcome this problem, extensive research has been directed toward reducing systemic toxicity and increasing drug activity in cancer therapy. In this regard, combination chemotherapy has received more attention for the purpose of finding compounds with a known mechanism of action that could increase the therapeutic index of clinical anticancer drugs 14. Some dietary supplements possess anticancer activity apart from their other biological efficacy, which is an added advantage in combination chemotherapy. Combined effect of FER/CDDP and FER/CBDC showed effectiveness on K562 cell line (Figs 3A, B, 4). These results implied that FER could potentiate the activity of platinum complexes. The biological mechanism responsible for increased therapeutic gain might deserve further investigation. CMN showed significant antitumor activity that was comparable to the activity of CDDP (Figs 1-3). Further study was conducted to observe the enhancement of the antitumor effect of CMN by preparing its beta cyclodextrin inclusion complex 11. The antiproli- Fig. 3 Effect of polyphenolics and cisplatin alone and in combination on K562 cell growth (A). Also shown are the co-operative efforts of FER with CDDP (B). Cells were cultured and treated with DMSO, cisplatin, polyphenolics alone or their combinations and the % viability was determined at the end of 72h of treatment by tyrypan blue dye exclusion test. Experiments were conducted in duplicates and repeated thrice. The values represent mean ± SD. Fig. 2 Time bound effect on cell survival. Human erythroleukemic K562 cells were treated with phytochemical at various IC 50 concentrations. % Viability was determined on Days 1, 2 and 3 by trypan blue dye exclusion. Data represents the average (± SD) of triplicate determinations.
220 INDIAN J EXP BIOL, MARCH 2006 GSH by FER led to an increase in the sensitivity of the K562 cells to platinum complexes. Similarly, G2/M cell cycle arrest of K562 cells was also contributory in enhancing the tumor cell killing affected by cisplatin. It could be concluded that CMN was capable of demonstrating the most pronounced effect on cell viability. Additional work on water soluble derivatives of CMN and its various cyclodextrin complexes must be undertaken to determine in what manner CMN will be more effective in vivo. Fig. 4 Antiproliferative activity of FER and carboplatin (CBDC) against K562. Cells were cultured in 60 mm dishes and treated with increasing concentration of CBDC with or without FER for 72 h. After incubation period cells were stained with trypan blue and counted. Values represent the mean ± SD of three independent experiments. Fig. 5 Time course effect of β-cyclodextrin complex of CMN on K562 cells. Cells were treated with the complex (B-CMN) for different time intervals. At the end of each time point the number viable cells were determined by trypan blue. ferative property of CMN complex was clearly visible on K562 cancer cells (Fig. 5). In conclusion, the present study showed that curcumin, an anti-inflammatory antioxidant compound consumed widely as a dietary supplement, possessed strong anticancer activity against human cancer cell lines in vitro. Among the cinnamoyl compounds, FER appeared to be a better modulating agent and CMN was found to be the most potent on human malignant cell lines. For further studies, we propose to include cinnamaldehyde 15, which may contribute to the development of new directions in anticancer therapies. Depletion of References 1 Orsolic N, Kosalec I & Basic I, Synergistic antitumor effect of polyphenolic components of water soluble derivatives of propolis against Ehrlich ascites tumor, Biol Pharm Bull, 28 (2005) 694. 2 Aggarwal B B, Takada Y, Shishodia S, Gutierrez A M, Oommen O V, Ichikawa H, Baba Y & Kumar A, Nuclear transcription factor NF-kappa B: role in biology and medicine, Indian J Exp Biol, 42 (2004) 341. 3 O Reilly M S, Targeting multiple biological pathways as a strategy to improve the treatment of cancer, Clin Cancer Res, 8 (2002) 3309. 4 Bravo L, Polyphenols: Chemistry, dietary sources, metabolism and nutritional significance, Nature Rev, 56 (1998) 317. 5 Galati G, Teng S, Moridani M Y, Chan T S & O Brien P J, Cancer chemoprevention and apoptosis mechanism induced by dietary polyphenolics, Drug Metabol Drug Interact, 17 (2000) 311. 6 Nair S C & Panikkar K R, Antitumor principles from Ixora javanica. Cancer Lett, 49 (1990) 121. 7 Lee Y J, Liao P H, Chen W K & Yang C Y, Preferential cytotoxicity of caffeic and phenethyl ester analogues on oral cancer cells, Cancer Lett, 153 (2000) 51. 8 Moragda L, Jaszewski R & Majumdar A P, Curcumin induced modulation of cell cycle and apoptosis in gastric and colon cancer cells, Anticancer Res, 21 (2001) 873. 9 Isuzugawa K, Ogihara Y & Inoue M, Different generations of inhibitors against gallic acid induced apoptosis produce different sensitivities to gallic acid, Biol Pharm Bull, 24 (2001) 249. 10 Indap M A, Bhosale S C, Miranda M & Rao S G A, Induction of quercetin mediated apoptosis in human erythroleukemic K562 cells, Indian Drugs, 35 (1998) 128. 11 Indap M A, Bhosale S C, Tayade P T & Vavia P R, Evaluation of toxicity and antitumor effects of a hydroxypropyl beta-cyclodextrin inclusion complex of quercetin, Indian J Pharm Sci, 64 (2002) 349. 12 DiPaola R S, To arrest or not to G 2 -M cell cycle arrest, Clin Cancer Res, 8 (2002) 3311. 13 Reed J C, Apoptosis-targeted therapies for cancer, Cancer Cell, 3 (2003) 17. 14 Merlin J L, Concepts of synergism and antagonism, Anticancer Res, 14 (1994) 2315. 15 Dornish J M, Pettersen E O & Oftebro R, Synergistic cell inactivation of human NHIK 3025 cells by cinnamaldehyde in combination with cis-diamminedichloroplatinum(ii), Cancer Res, 48 (1988) 938.