|Year : 2015 | Volume
| Issue : 2 | Page : 352-357
Enhancement of the cytotoxic effects of Cytarabine in synergism with Hesperidine and Silibinin in Acute Myeloid Leukemia: An in-vitro approach
Urja N Desai1, Krupa P Shah1, Sheefa H Mirza2, Darshil K Panchal3, Sonia K Parikh1, Rakesh M Rawal1
1 Departments of Cancer Biology and Medical Oncology, The Gujarat Cancer and Research Institute, Ahmedabad, Gujarat, India
2 Department of Life Sciences, Gujarat University, Ahmedabad, Gujarat, India
3 Department of Pharmacology, Lallubhai Motilal College of Pharmacy, Ahmedabad, Gujarat, India
|Date of Web Publication||7-Jul-2015|
Rakesh M Rawal
303, Medicinal Chemistry and Pharmacogenomics, Department of Cancer Biology, The Guajrat Cancer and Research Institute, New Civil Hospital Campus, Asarwa, Ahmedabad - 380 016, Gujarat
Source of Support: Gujarat State Biotechnology Mission (GSBTM), Conflict of Interest: None
Objectives: Acute Myeloid Leukemia (AML) therapy continues to be a daunting challenge. Cytosine Arabinoside (Ara-C) is widely used to treat hematological malignancy in humans, but often becomes ineffective because of increased resistance to the drug which may lead to a worse prognosis. Therefore new strategies are needed to understand the mechanism responsible for drug resistance and to develop new therapies to overcome it. Research evidence based on natural compounds used alone or in combination with current chemotherapeutic agents proved their efficacy to treat and prevent cancer. Hesperidin and Silibinin displayed anti-cancer activity against various types of cancers and cell lines and can be used in combination with Cytarabine with the aim to increase cytotoxicy profile and reduction in drug resistance.
Experimental Work: Primary cells obtained from AML patient's bone marrow were used to develop in-vitro model and further exposed to various concentration of Cytarabine (10 nM-5000 nM), Hesperidin (0.5 μM-100 μM) and Silibinin (0.5 μM-100 μM) alone and in combination with Cytarabine (Hesperidin-25 μM, Silibinin10 μM) to check cytotoxicity using MTT assay. Synergistic effect was evaluated by Combination Index method.
Result and Conclusion: In-vitro study of Hesperidin and Silibinin indicated their cytotoxicity at IC 50 value 50.12 μM and 16.2 μM, respectively. Combination Index study revealed Hesperidin and Silibinin both showed synergistic potential and decreased the IC 50 value of Cytarabine by ~5.9 and ~4.5 folds, respectively. Both natural compounds showed potential anti-leukemic activity hence may be used for AML therapy alone or in combination with other chemotherapeutic agents.
Keywords: Acute myeloid leukemia, combination index, cytarabine, drug resistance
|How to cite this article:|
Desai UN, Shah KP, Mirza SH, Panchal DK, Parikh SK, Rawal RM. Enhancement of the cytotoxic effects of Cytarabine in synergism with Hesperidine and Silibinin in Acute Myeloid Leukemia: An in-vitro approach. J Can Res Ther 2015;11:352-7
|How to cite this URL:|
Desai UN, Shah KP, Mirza SH, Panchal DK, Parikh SK, Rawal RM. Enhancement of the cytotoxic effects of Cytarabine in synergism with Hesperidine and Silibinin in Acute Myeloid Leukemia: An in-vitro approach. J Can Res Ther [serial online] 2015 [cited 2017 Sep 21];11:352-7. Available from: http://www.cancerjournal.net/text.asp?2015/11/2/352/157330
| > Introduction|| |
Acute myeloid leukemia (AML) is characterized by an arrest in differentiation and uncontrolled proliferation of myeloid precursors in the bone marrow whose standard treatment approach has been predominantly based on Cytarabine and Anthracyclines.  1-β3-D-arabinofuranosyl cytosine or Cytarabine is the most effective antimetabolite used for the treatment of AML since more than 40 years. Initial remission rate was typically around 70% but patients tend to relapse early, where less than 25% can expect to have a sustained long-term survival. , The clinical importance of Ara-C has stimulated extensive laboratory investigations aimed to understand biochemical correlation of drug action and mechanisms of drug resistance to provide ideas however, response to its treatment and development of resistance is still a major drawback in AML.  Accumulating research evidence suggests that many dietary agents/medicinal plants can be used alone or in combination with traditional chemotherapeutic agents to prevent the occurrence of cancer, their metastatic spread, or even to treat cancer. , Therefore, the situation mandates development of new approaches to improve the outcome for patients with relapsed and refractory leukemia.  Phytochemicals were used for cure of so many diseases from the ancient times. Many of the natural products such as Taxol and Vincristine are still in use for treatment of various cancers. Wide range of naturally occurring phytochemicals like Hesperidin, Silymarin, Curcumin were used in cancer chemoprevention. , One of the approaches to increase the therapeutic efficacy of therapy for AML is to combine Ara-C with natural compounds like Hesperidin and Silibinin which may sensitize the cells to the cytotoxic activity of the drugs however there is no published preclinical study of this method. Silymarin and Hesperidin act by anti-oxidative, anti-lipid peroxidative, anti-fibrotic, anti-inflammatory, membrane stabilizing, immunomodulatory and liver regenerating mechanisms in experimental liver diseases. Furthermore, Silymarin and Hesperidin has been extensively studied, both in-vivo and in-vitro, for its cancer chemopreventive potential against various cancers. ,, Therefore our aim of the study was to investigate effect of Hesperidin and Silibinin alone and their synergism with Cytarbine in treatment of AML patients.
| > Material and methods|| |
Sample collection and processing
Bone marrow samples were collected with prior consent of patients. The diagnosis of leukemia was made according to the FAB criteria.  Mono-nuclear cells (MNC) were separated by Ficoll Histopaque-1077 (Sigma) density gradient centrifugation method  and further cultured in RPMI-1640 medium (Hi-Media) with 10% heat inactivated FBS (Cellclone), Antibiotic (Hi-Media) at 37 ° C under 5% CO 2 .
In-vitro cytotoxicity assay
CD34 + cells were isolated from MNC by IMCS using anti-CD34 antibodies followed by manufacturer's protocol (Miltenyi Biotec Inc.). These isolated CD34 + cells were treated with various concentrations of Cytarabine (10nM-5000 nM), Hesperidin (0.5-100 μM) and Silibinin (0.5-100 μM) alone as well as in combination with Cytarabine (Hesperidin-25 μM, Silibinin 10 μM) for 24 hours. (Cytarabine- Biobin, Hesperidin-Sigma, Silybinin-Microlabs Ltd.)
Cells were seeded in 96-well plates at 1 × 10 4 cells/well in 200 μl of medium. They were cultured for 24h to allowed to settle down. Then different concentrations of drugs were added alone and in combination in duplicate. After adding drugs plate was incubated for 24h and then 10 μl of MTT [3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl-tetrazolium bromide] (5 mg/mL, Hi-Media) was added to each well and the plates were incubated at 37°C for 4 h. To each well 200 μl of Dimethyl Sulfoxide (DMSO) was added, and the plates were agitated on a plate shaker for 10 min. Optical density at 570 nm was read with an ELISA reader (Multiskan Spectrum Microplate Reader, Thermo Scientific). 
Cell viability assay
Cell viability assay was done by Trypan Blue Exclusion dye in Automated Cell Counter TC10 (BioRad) according to manufacturer's instructions and counter checked by manual method using Neubauer's Chamber. 
Statistical Analysis of Combination Index and IC 50 were done using GraphPad Prism-5 (free Trail Version 5.04) software.
Analysis of interactions
Attempts have been made during the past century to quantitatively measure the dose-effect relationships of each drug alone and its combinations and to determine whether or not a given drug combination would gain a synergistic effect. Drug synergism assessed by Chou and Talalay method also known as combination index method. ,,
The CI method is a quantitative representation of pharmacological interaction between two drugs. Briefly, CI method used to check drug interaction between Silibinin/Hesperidin with Cytarabine and for that variable ratios of drug concentrations were used in different combinations for experiment. Cell growth inhibition was determined using MTT assay, as previously described. CI value of 1 indicates an additive effect, whereas a CI < 1 or > 1 indicates synergism or antagonism, respectively. The involved drugs were assumed mutually non-exclusive meaning that they have totally independent modes of action. The CI values were calculated at x% cell growth inhibition, as:
CI = (D) 1 /(Dx) 1 + (D) 2 /(Dx) 2 + (D1)(D2)/(Dx) 1 (Dx) 2
where, (DX 1 ) and (DX 2 ) are the doses of D 1 (drug # 1, for example, Cytarabine) and D 2 (drug # 2, for example, Silymarin/Hesperidin) alone that gives x% inhibition, whereas (D 1 ) and (D 2 ) are the doses of D 1 and D 2 in combination that also inhibits x% (isoeffective). The (DX 1 ) and (DX 2 ) can be readily calculated from the Median-effect equation of Chou.
D x = D m [fa/(1-fa) ] 1/m
A mean CI was calculated from data points with fraction affected (Fa) > 0.5. (Fa) < 0.5 would imply lower growth inhibition and a large fraction of the cell population would still grow. Fa < 0.5 was therefore considered not relevant.
The median effect equation
The median effect equation states that:
fa/fu = (D/Dm) m - ----------(1)
where D is the dose, fa and fu are the fractions of the system affected and unaffected, respectively, by the dose D, D m is the dose required to produce the median effect (analogous to the more familiar IC 50 , ED 50 , or LD 50 values), and m is a Hill-type coefficient signifying the sigmoidicity of the dose-effect curve, that is, m = I for hyperbolic (first order or Michaelis-Menten) systems.
The median effect plot
The median effect equation (equation 1) may be linearized by taking the logarithms of both sides, that is
log (fa/fu) = m log (D) - m log (Dm) or
log [(fa) -1 - 1] -1 = m log (D) - m log (Dm) or
log [(fu) -1 - 1] = m log (D) - m log (Dm)
Median effect plot is used for determining pharmacological median doses for lethality (LD 50 ), toxicity (TD 50 ), effect of agonist drugs (ED 50 ), and effect of antagonist drugs (IC 50 ). Thus, the median-effect principle of the mass-action law encompasses a wide range of applications. The plot gives the slope, m, and the intercept of the dose-effect plot with the median-effect axis [i. e. when fa = fu, fa/fu = 1 and hence y = log (fa/fu) =0] which gives log (Dm) and consequently the Dm value. Any cause-consequence relationship that gives a straight line for this plot will provide the two basic parameters, m and Dm, and thus, an apparent equation that describes such a system.
| > Result|| |
In vitro drug induced cell proliferation inhibition (Single Drug)
Primary AML cells were simultaneously exposed to Cytarabine (10-5000 nM), Hesperidin (0.5-100 μM) and Silibinin (0.5-100 μM) to find out IC 50 value of single drug by MTT assay. Result shows IC 50 value of Cytarabine, Hesperidin and Silibinin was 1.12 μM, 50.12 μM and 16.2 μM respectively at 24 hrs. All of these compounds showed growth inhibitory effect in dose dependent manner. Drug effect and Dose-response values are depicted in [Figure 1] and [Table 1].
|Figure 1: Median Effect plot and Dose Responses of Single Drug. (a) IC50 value of Cytarabine, (b) IC50 value of Hesperidin, (c) IC50 value of Silibinin|
Click here to view
|Table 1: IC50 value of Cytarabine, Hesperidin and Silibinin (single and combine)|
Click here to view
In vitro drug induced cell proliferation inhibition (Combination Drug)
To find out the IC 50 value in combination treatment Cytarabine (100nM-1μM) was given at different doses in combination with fixed dose of Silibinin (10 μM) and Hesperidin (25 μM) and incubated for 24 hours to plated cells. Result showed antiproliferative effect in dose dependent manner. Dose response effect is presented by Median effect plot in [Figure 2] and [Table 1].
|Figure 2: Median Effect plot and Dose responses of curve of Cytarabine in combination with (a) Hesperidin (25 μM) and (b) Silibinin (10 μM)|
Click here to view
IC 50 values obtained from median effect plot of combination treatment for Cytarabine + Hesperidin (25 μM) and Cytarabine + Silibinin (10 μM) shows that IC 50 value of Cytarabine reduced ~5.9 and ~4.5 folds in combination with Hesperidin and Silibinin, respectively.
Combination index method
Median effect curves
CI was found for Hesperidin and Silibinin in two different combinations with Cytarabine. Cells were exposed for 24 hours to respective drug combination. Median effect curves for different combinations with Hesperidin and Silibinin were depicted in [Figure 3]a and b and [Figure 4]a and b respectively. CI values of Hesperidin and Silibinin with Cytarabine shown in [Table 2] suggestive of synergy between drug and natural compounds.
|Figure 3: Median Effect Curve of (a) Cytarabine: Hesperidin (1:125), (b) Cytarabine: Hesperidin (1:250)|
Click here to view
|Figure 4: Median Effect Curve of (a) Cytarabine: Silibinin (1:50), (b) Cytarabine: Silibinin (1:100)|
Click here to view
|Table 2: Combination index values of Hesperidin and Silibinin with Cytarabine|
Click here to view
Fraction affected versus combination index plots
Fraction affected (Fa) values (indicating the fraction of cells inhibited after drug exposure) were obtained after exposure of the cells to a series of drug concentrations. To indicate the effects at different Fa values, combination index (CI) values were calculated for each Fa and plotted in FA-CI plot as seen in [Figure 5] and [Figure 6].
Mean CI was calculated from data points with fraction affected (Fa) > 0.5. (Fa) < 0.5 would imply lower growth inhibition and a large fraction of the cell population would still grow. Fa < 0.5 was therefore considered not relevant.
Synergistic effects have been found in all the experimental combinations. Among all the groups, combination of Cytarabine: Silibinin (1:50) showed the best potential of synergism while combinations with Hesperidin showed comparable CI value.
| > Discussion|| |
Drug combination is most widely used in treating the most dreadful diseases, such as cancer and AIDS. The main aims are to achieve synergistic therapeutic effect, dose and toxicity reduction, and to minimize or delay the induction of drug resistance.  Normal and leukemic stem cells are clonogenic and it is thus possible to study the effects of chemotherapeutic drugs on these cells in-vitro. Such tests could possibly provide a means to predict the response to chemotherapy in acute leukemia and eventually be used to select the most efficient drug combination in individual patients. Another development would be to use such in-vitro tests to establish the efficacy of new drugs before clinical trials are started. There are a few reports demonstrating the feasibility of in-vitro approach in leukemia. ,,,
Natural compounds are known to exhibit anti-cancer properties and induce apoptosis in a variety of cancer. , Silibinin (Silymarin), a major flavonolignan obtained from milk thistle extract, have showed its anti-cancer effects against variety of cancer such as skin cancer, prostate cancer, human colorectal LoVo cells and leukemic K562 cell lines .,, It has also been observed that Silibinin at lower doses inhibit extracellular signal-regulated kinases (ERK1/2) and in higher doses leads to apoptosis through MAPK/JNK pathway and also induces growth arrest at the G1 and G2 checkpoints.  Silibinin-Doxorubicin combinations were previously investigated in breast, prostate and lung cancer using range 10-100 μM and showed synergistic effect. 
Hesperidin (5, 7, 3'- trihydroxy-4'- methoxy-flavanone7- rhamnoglucoside) is a flavanone glycoside abundantly found in sweet orange and lemon peel. Hesperetin, aglycone moiety of Hesperidin, significantly inhibits cell proliferation in a dose-dependent manner and resulted in significant cell cycle arrest in the G1 Phase in breast carcinoma MCF-7 cells.  Hesperidin induces cell growth arrest and apoptosis in a large variety of cells including colon and pancreatic cancer cells through caspase-3 activation. 
According to the National Comprehensive Cancer Network (NCCN) guidelines currently available therapy used in clinical practice for AML involved intermediate dose of Cytarabine in combination with Daunorubicin/Idarubicin or high dose of Cytarabine. Jim Glare et al., observed that higher dose of Cytarabine was as effective with intermediate dose but at higher doses there is an increase in cytotoxicity.  Therefore an alternative therapy or combination will be required which produced synergistic or additive effect and reduce the dose of Cytarabine.
In the present study the use of natural compounds like Silibinin and Hesperidin showed 50% cell inhibition at 16.2 μM and 50.12 μM respectively when given alone. While Hesperidin and Silibinin in a fixed dose with Cytarabine in a different concentration, reduces the IC 50 value of Cytarabine by ~5.9 and ~4.5 folds, respectively. This may provide better combination treatment strategy and reduce the cytotoxicity profile. Drug interaction analysis by Combination Index method revealed Silibinin and Hesperidin both showed synergism with Cytarabine upto 1:50 to 1:250 ratios. Silibinin at higher dose ratio (1:100) showed synergism but it was less in comparison to low dose ratio (1:50). This finding was also observed by Colombo et al., in which Silibinin in combination with Doxorubicin was used on colon carcinoma LoVo cell lines.  This may be due to cell cycle delays and/or deregulations of protein expression produced by Silibinin which may delay other cytotoxic agent action and hence accounts for less synergy.  While in the case of Hesperidin comparable synergistic effect was observed at both the ratios.
| > Conclusion|| |
Concluding our finding suggests that Silibinin and Hesperidin revealed synergistic potential with Cytarabine and have reduced cytotoxicity. Hence, both of them may use as chemotherapeutic agents either alone or in combination which emerges as better alternative treatment modality for AML. However, validation using in-vivo models is needed to further establishing its underlying mechanism of activity.
| > References|| |
Fathi AT, Grant S, Karp JE. Exploiting cellular pathways to develop new treatment strategies for AML. Cancer Treat Rev 2010;36:142-50.
Gale RP. Advances in the treatment of acute myelogenous leukemia. New Engl J Med 1979;300:1189-99.
Astrom M, Bodin L, Nilsson I, Tidefelt U. Treatment, long-term outcome and prognostic variables in 214 unselected AML patients in Sweden. Br J Cancer 2000;82:1387-92.
Mahlknecht U, Dransfeld CL, Bulut N, Kramer M, Thiede C, Ehninger G, et al
. SNP analyses in cytarabine metabolizing enzymes in AML patients and their impact on treatment response and patient survival: Identification of CDA SNP C-451T as an independent prognostic parameter for survival. Leukemia 2009;23:1929-32.
Prasain JK, Barnes S. Metabolism and bioavailability of flavonoids in chemoprevention: Current analytical strategies and future prospectus. Mol Pharm 2007;4:846-64.
Nishino H, Satomi Y, Tokuda H, Masuda M. Cancer control by phytochemicals. Curr Pharm Des 2007;13:3394-9.
Shiozawa K, Nakanishi T, Tan M, Fang HB, Wang WC, Edelman MJ, et al
. Preclinical studies of vorinostat (suberoylanilide hydroxamic acid) combined with cytosine arabinoside and etoposide for treatment of acute leukemias. Clin Cancer Res 2009;15:1698-707.
Tsuda H, Ohshima Y, Nomoto H, Fujita K, Matsuda E, Iigo M, et al
. Cancer prevention by natural compounds. Drug Metab Pharmacokinet 2004;19:245-63.
Aggarwal BB, Van Kuiken ME, Iyer LH, Harikumar KB, Sung B. Molecular targets of nutraceuticals derived from dietary spices: Potential role in suppression of inflammation and tumorgenesis. Exp Biol Med (Maywood) 2009;234:825-49.
Meiyanto E, Hermawan A, Anindyajati. Natural products for cancer-targeted therapy: Citrus flavonoids as potent chemopreventive agents. Asian Pac J Cancer Prev 2012;13:427-36.
Post-White J, Ladas EJ, Kelly KM. Advances in the use of milk thistle (Silybummarianum). Integr Cancer Ther 2007;6:104-9.
Ramasamy K, Agarwal R. Multitargeted therapy of cancer by silymarin. Cancer Lett 2008;269:352-62.
Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA, Gralnick HR, et al
. Proposed revised criteria for the classification of acute myeloid leukemia. A report of the French-American-British Cooperative Group. Ann Intern Med 1985;103:620-5.
Boyum A. Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 1968;97:77-89.
Roy M, Chakraborty S, Siddiqi M, Bhattacharya RK. Induction of apoptosis in tumor cells by natural phenolic compounds. Asian Pac J Cancer Prev 2002;3:61-7.
Chou TC, Talalay P. Analysis of combined drug effects: A new look at a very old problem. Trends Pharmacol Sci 1983;4:450-4.
Chou TC, Talalay P. Quantitative analysis of dose effect relationships: The combined effects of multiple drugs or enzyme inhibitors. Adv Enzyme Regul 1984;22:27-55.
Chou TC. Theoretical basis, experimental design, and computerized simulation of synergism and antagonism in drug combination studies. Pharmacol Rev 2006;58:621-81.
Chou TC. Drug combination Studies and their synergy quantification using the Chou-Talalay method. Cancer Res 2010;70:440-6.
McCulloch EA, Curtis JE, Messner HA, Senn JS, Germanson TP. The contribution of blast cell properties to outcome variation in acute myeloblastic leukemia (AML). Blood 1982;59:601-8.
Park CH, Amare M, Savin MA, Goodwin JW, Newcomb MM, Hoogstraten B. Prediction of chemotherapy response in human leukemia using an in vitro
chemotherapy sensitivity test on the leukemic colony-forming cells. Blood 1980;55:595-601.
Preisler HD. Prediction of response to chemotherapy in acute myelocytic leukemia. Blood 1980;56:361-7.
Rustum YM, Preisler HD. Correlation between leukemic cell retention of 1-β-D-arabinofuranosylcytosine-5′- triphosphate and response to therapy. Cancer Res 1979;39:42-9.
Deep G, Agarwal R. Chemopreventive efficacy of silymarin in skin and prostate Cancer. Integr Cancer Ther 2007;6:130-45.
Kaur M, Velmurugan B, Tyagi A, Deep G, Katiyar S, Agarwal C, et al
. Silibinin suppresses growth and induces apoptotic death of human colorectal carcinoma LoVo cells in culture and tumor xenograft. Mol Cancer Ther 2009;8:2366-74.
Zhong X, Zhu Y, Lu Q, Zhang J, Ge Z, Zheng S. Silymarin causes caspases activation and apoptosis in K562 leukemia cells through inactivation of Akt pathway. Toxicology 2006;227:211-6.
Singh RP, Agarwal R. A Cancer chemopreventive agent silibinin, targets mitogenic and survival signaling in prostate cancer. Mutat Res 2004;555:21-32.
Flaig TW, Su LJ, Harrison G, Agarwal R, Glodé LM. Silibinin synergizes with mitoxantrone to inhibit cell growth and induce apoptosis in human prostate cancer cells. Int J Cancer 2007;120:2028-33.
Choi EJ. Hesperetin induced G1-phase cell cycle arrest in human breast cancer MCF-7 Cells: Involvement of CDK4 and p21. Nutr Cancer 2007;59:115-9.
Park HJ, Kim MJ, Ha E, Chung JH. Apoptotic effect of hesperidin through caspase3 activation in human colon cancer cells, SNU-C4. Phytomedicine 2008;15:147-51.
Glare J. High-dose cytarabine no more effective in AML than intermediate doses, with more toxicity. New Engl J Med 2011;364:1027-38.
Colombo V, Lupi M, Falcetta F, Forestieri D, D′Incalci M, Ubezio P. Chemotherapeutic activity of silibinin combined with doxorubicin or paclitaxel in sensitive and multidrug-resistant colon cancer cells. Cancer Chemother Pharmacol 2011;67:369-79.
Gallo D, Giacomelli S, Ferlini C, Raspaglio G, Apollonio P, Prislei S, et al.
Antitumour activity of the silybin-phosphatidylcholine complex, IdB 1016, against human ovarian cancer. Eur J Cancer 2003;39:2403-10.
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2]