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Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells Salvatore Genovese a, Francesco Epifano a, *, Francesca Preziuso a, Jill Slater b, Pratima Nangia-Makker c, Adhip P.N. Majumdar c, Serena Fiorito a “G. D’Annunzio” Chieti-Pescara, Via dei Vestini 31, 66100, Chieti, Italy Dipartimento di Farmacia, Universita Department of Biological Sciences, University of Michigan-Flint, Michigan, USA c VA Medical Center, Karmanos Cancer Institute, Department of Medicine, Wayne State University, Detroit, MI, USA a
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Article history: Received 25 October 2019 Accepted 11 November 2019 Available online xxx
Advanced colon cancer is extremely difficult to cure, underscoring the need to develop novel therapeutic agents. Prenylated curcumins that are semisynthetic curcumin derivatives with significant anti-cancer potential have been studied herein to assess their therapeutic potential for colon cancer and tested to this aim in vitro for their growth inhibitory properties against 5-fluorouracil þ oxaliplatin resistant human colon cancer CR-HT29 and HCT-116 cells. The resulting most active product, gercumin (mono-Ogeranylcurcumin), has been further tested for its synergistic effects with FOLFOX (a combination of 5fluorouracil and oxaliplatin) on the same cell lines. Activity of this combination on colonosphere formation was also investigated. Gercumin was able to suppress the growth of cancer cells with a potency similar to that of curcumin. A synergistic effect of this compound and FOLFOX was also observed. doses tested for synergy in the colonosphere assays did not show greater suppression of colonosphere formation than independent treatment with either reagent alone. Only one of the combinations was shown to be more effective at suppressing colonosphere formation [gercumin 5 mM þ FOLFOX (2x)]. Thus, the growth inhibitory effects of curcumin against human cancer cells can be modulated and enhanced by the introduction of hydrophobic chains, normally found in several natural compounds, like the geranyl one. Such compounds are also able to synergize with known chemotherapeutics. © 2019 Elsevier Inc. All rights reserved.
Keywords: Cancer chemotherapy Curcumin Geranylation Gercumin Colon cancer
1. Introduction Natural products traditionally have been and still represent the main source of bioactive compounds used both in the pharmaceutical industry and in traditional health care systems [1]. In particular, in vital indications such as anti-cancer and antibiotic drugs, most compounds currently used in Western and alternative and complementary medicines stem either directly from natural sources (e.g. morphine) or are derived from scaffolds provided by natural products (e.g. taxol). Structural modifications of a lead compound have in most cases pronounced effects on pharmacokinetic and/or pharmacodynamic properties. Explicative examples have been thoroughly reported in the literature. This is the case of nalorphine in which the addition of an allyl moiety to the skeleton of morphine provide a drug that antagonized the effects of the
* Corresponding author. E-mail address: [email protected] (F. Epifano).
same morphine, while ethylmorphine resulted in a complete lack of analgesic activity. Curcumin 1 is well recognized among the naturally occurring compound with the most promising therapeutic potential. It is a diferuloylmethane derivative isolated from the rhizome of turmeric, Curcuma longa L. (Zingiberaceae), and has been proposed for treatment of several acute and chronic syndromes like inflammatory, cancer, neurodegenerative, cardiovascular, and metabolic diseases [2]. The mechanism of action of curcumin involves interactions with several biological targets like transcription factors, protein kinases, several enzymes, genes, and different receptors [3,4]. Due to its plethora of effects, curcumin has several potential clinical applications. Thus, this natural compound and its semisynthetic derivatives are currently passing through different clinical trials [5]. Examples of chemical modification of curcumin skeleton with the aim of modifying selectivity, potency, and bioavailability have been recently reported in the literature. In particular the addition of a prenyl group in position 9 favorably affect the biological selectivity of the resulting curcuminoid [6,7].
https://doi.org/10.1016/j.bbrc.2019.11.068 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: S. Genovese et al., Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.068
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More recently Piacente, Bifulco, and coworkers investigated the effects of mono- and di-O-prenylation of curcumin on the inhibitory properties on histone deacetylase (HDAC) and membraneassociated prostaglanid E synthase-1 (mPEGS-1) and found that monoprenylated compounds 2 and 5 acted as effective inhibitory agents of this latter enzyme [8]. The one just mentioned represents the only report published in the literature about pharmacological properties of O-prenylated curcumin derivatives. Thus, investigations on such semisynthetic compounds is a field of research of current and growing interest. In the current manuscript we wish to report the in vitro growth inhibitory effects of four O-prenylated curcumin derivatives (e.g. mono- and di-3,3-dimethylallyloxy and mono- and digeranyloxy) 2e5 (Fig. 1) on chemo-resistant human colon cancer CR-HT29 cells and the activity of the most effective compound resulting from this preliminary assay on the same cells and also on colon cancer CR-HCT116 cells, when combined with FOLFOX (5-fluorouracil and oxaliplatin) using the Chou-Talalay method [9]. The effects of O-prenylated curcumins and FOLFOX alone or in combination on colonosphere formation have been also investigated.
plates. 24 Hours after plating, cells were treated with various testing agents and incubated for another 48 h. Following this incubation period, the reaction was terminated by adding 0.5-mg/ml stock of MTT to each plate. The reaction was allowed to proceed for 3e4 h at 37 C. The culture medium was then removed. The resulting formazan crystals were then dissolved by adding 1 N HCl þ isopropanol (1:24). The intensity of the resulting color change reflected the number of live cells; absorbance was measured at a wavelength of 570 nm. All values were compared with the corresponding controls. All assays were performed in four replicates.
2. Materials and methods
2.5. Colonosphere formation or disintegration
2.1. Chemicals
To examine the effects of gercumin 2 on the formation of colonospheres, the FOLFOX-resistant cells were suspended in serumfree stem cell medium containing DMEM/F12 (1:1) supplemented with B27 (Life Technologies, Gaithersburg, MD) and antibioticantimycotic. The cells (1000 per well) were then plated in a 24well plate (Corning Inc, Lowell, MA) and incubated overnight. They were subsequently treated with compound 2 and/or FOLFOX for 7 days. The number of colonospheres was evaluated by light microscopy. To examine its effects on the disintegration of colonospheres, cells were plated as described above and incubated for 7 days, allowing colonospheres to form. At this time colonospheres were incubated in the absence (controls) or presence of the testing agent for 7 days and evaluated for the number of colonospheres by light microscopy.
Curcumin was purchased from Merck Sigma Aldrich (USA) and purified by crystallization (MeOH/H2O) before use. Compounds 2e5 were chemically synthesized as described previously [8] and their purity (>98.1%) assessed by HPLC and 1H NMR. Analytical data for curcuminoids 2e5 were in full agreement for those already reported for the same compounds [8]. 2.2. Cell cultures Chemo-resistant (CR) human colon cancer cell lines, CR-HT29 and CR-HCT116, were generated as previously described [10e13]. Cells were maintained in Dulbecco’s modified Eagle medium (DMEM; 4.5 g/L D-glucose) supplemented with 10% FBS, 1% antibiotic/antimycotic, and 50 mM 5-fluorouracil (5-FU) þ 1.25 mM oxaliplatin (FOLFOX) in tissue culture flasks in a humidified incubator at 37 C in an atmosphere of 95% air and 5% carbon dioxide. The medium was changed twice per week, and cells were passaged using 0.05% trypsin/EDTA. 2.3. Growth inhibition assay Inhibition of cell growth in response to curcumin analogues and/or FOLFOX (5FU þ oxaliplatin) was assessed by 3-(4,5dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay as described previously [10]. Briefly, cells were dispersed by trypsin-EDTA treatment, resuspended in DMEM containing 10% FBS, and seeded at a density of 5000 cells/well into 96-well culture
Fig. 1. Structure of prenylated curcuminoids: 1 R1 ¼ R2 ¼ H, 2 R1 ¼ geranyl R2 ¼ H, 3, R1 ¼ R2 ¼ geranyl 4 R1 ¼ 3,3-dimethylallyl R2 ¼ H, 5 R1 ¼ R2 ¼ 3,3-dimethylallyl.
2.4. Analysis of the interaction between gercumin 2 and FOLFOX The software program Calcusyn (Biosoft, Ferguson, MO) was employed to determine the nature of interaction between compound 2 and FOLFOX using the Chou-Talalay methodology [9]. This method utilizes a multiple drug-effect equation derived from an enzyme kinetics model in which the output is represented as combination indexes (CI) and/or isobologram analysis.
3. Results and discussion Curcumin and curcuminoids 2e5 were first screened for their in vitro growth inhibitory capacities on CR-HT29 cancer cell line using the MTT test in the concentration range 2.5e40 mM. Results are shown in Fig. 2. Of all the curcuminoids, only gercumin 2 had the greatest effect displaying a potency comparable or somewhat better respect to the parent compound 1. Thus, all further analyses, aimed at assessing the effects of a combination of gercumin 2 and FOLFOX on the
Fig. 2. Growth inhibitory effects of curcumin 1 and prenyloxycurcuminoids 2e5.
Please cite this article as: S. Genovese et al., Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.068
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growth of CR-HT29 and also CR-HCT-116 colon cancer cells, were focused on this latter sample. Experiments were carried out using the Chou-Talalay method [9] and the program Calcusyn was employed to determine the nature of interaction between gercumin and FOLFOX. The employed methodology utilizes a multiple drug-effect equation derived from an enzyme kinetics model in which the output is represented as combination indexes (CI) and/or isobologram analysis. Based on CIs values, the extent of synergism/ antagonism is determined. Calcusyn software defines synergy as CI value less than 1. CI values between 0.9 and 0.85 suggest moderate synergy, whereas those in the range of 0.7 to 0.3 are indicative of clear synergistic interactions between the drugs. CI values in the range of 0.9e1.10 suggest a near additive effect. The ChoueTalalay equation has been also utilized to perform isobologram analysis as represented by the isobole for ED75. According to Calcusyn glossary an “isobologram is a graph indicating the equipotent combinations of various doses that can be used to illustrate additivity, synergism, or antagonism. For a given effect level such as fraction of affected (FA) cells of 0.5, the required doses for (ED50)1 and (ED50)2 are drawn on the x-axis and y-axis, respectively. If the combination data point for FA ¼ 0.5 falls on the diagonal, an additive effect is indicated whereas when it is on the lower left, synergism is indicated. On the other hand, if the line falls on the upper right, antagonism is suggested.”. Dose effect curves, isobolograms, and CIs tables about the effects of combinations at various doses of gercumin and FOLFOX on growth of chemo-resistant colon cancer CR-HT29 and CR-HCT-116 cells as derived by Calcusyn calculations are shown in Figs. 3 and 4 and Tables 1e2. Similar patterns of results have been obtained for both cancer cell lines with the only exceptions of data recorded in isobolograms in Fig. 3, for which synergy between compound 3 and FOLFOX is suggested for all EDs, and Fig. 4 for which synergy is suggested only for ED50 and ED75. We then assessed the effects of the combination mono-O-geranylcurcumin 2 (10 mM) with FOLFOX (5-fluorouracil 50 mM þ oxaliplatin 1.25 mM) on colonosphere formation and disintegration. Results are reported in Tables 3 and 4 respectively. Curcumin is well recognized among the most promising biologically active natural products able to exert a plethora of beneficial effects on human health as well as to trigger a wide array of cell targets [2e5]. Its complexity in pharmacological activities and mechanisms of action recently prompted studies to better characterize its pharmacophore and to synthesize and assay structural analogues in order to depict a detailed structure-activity relationship [6,7].To this end the most of attention has been dedicated to the diacryloylmethane moiety while few data have been reported
3
in the literature about structural modifications in the aromatic rings of curcumin. Thus, many other efforts to this concern need to be addressed in the next future. In 2011 Majumdar and coworkers showed that difluorocurcumin was able to efficiently inhibit the growth of chemo-resistant colon cancer cells accompanied by induction of apoptosis and colonosphere disintegration when used in combination with FOLFOX [14]. In 2013 Minassi, Appendino, and co-workers studied for the first time the effects of aromatic Cprenylation of curcumin on its pharmacological activity [6]. Earlier, they demonstrated that an increase in lipophylicity was detrimental for the capacity to act as a Michael acceptor towards thiol containing biofactors (e.g. NF-kB and Nrf-2) but enhanced the effects of the parent compound as an anti-HIV agent. A year later, they demonstrated further how prenylation combined with the introduction of a pyrazole ring instead of the two carbonyls largely increased the activity and selectivity of the obtained curcuminoid towards 5-lipoxygenase [7]. Piacente, Bifulco, and co-workers turned their attention in 2015 on mono-O- and symmetrical di-Oprenylated (3,3-dimethylallyl, geranyl, and farnesyl) curcumin analogues [8]. In addition, they extensively investigated the effects of these compounds on HDAC and mPEGS-1 enzymes by means of in silico and in vitro analysis. They also provided a detailed structureactivity relationship of the synthesized O-prenylcurcuminoid and showed how the prenylation pattern is a determinant for the selectivity towards HDAC or mPGES-1 but did not perform any kind of assay on cultured cells. In the present manuscript, we investigated for the first time the effects of four O-prenylated curcumins 2e5 on chemotherapyresistant human colon cancer cells in vitro. Results of the MTT, assay revealed that the addition of only one geranyloxy chain in position 8 of the curcumin skeleton greatly reduced cell viability in a dose dependent manner and with a profile similar to that recorded for the parent unprenylated sample in the concentration range 2.5 mMe40 mM. Only at the highest dose tested (40 mM), all compounds were found to be strongly cytotoxic reducing cell viability down to about 20% of the control. On the contrary in the range 2.5 mMe20 mM compounds 3, 4, and 5 essentially showed the same effects with an average reduction of cell viability less than 20%. Thus, the best activity displayed by gercumin 2 as colon cancer cells growth inhibitory agent was the rationale for selectin to perform further tests with this compound as a potential in vitro anti-cancer agent against two chemo-resistant human colon cancer cells together with FOLFOX, the backbone for colon cancer for chemotherapy [15]. Results obtained by the Chou-Talalay method and Calcusyn analysis showed that the application of individual
Fig. 3. (a) Calcusyn analysis of gercumin þ FOLFOX synergy in CR-HT29 cells (Combined R ¼ 0.95, MONO R ¼ 0.92, FOLFOX R ¼ 0.72), (b) isobologram of Calcusyn analysis of gercumin þ FOLFOX synergy in CR-HT29 cells.
Please cite this article as: S. Genovese et al., Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.068
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Fig. 4. (a) Calcusyn analysis of gercumin þ FOLFOX synergy in CR-HCT-116 cells (Combined R ¼ 0.81, MONO R ¼ 0.77, FOLFOX R ¼ 0.94), (b) Isobologram of Calcusyn analysis of gercumin þ FOLFOX synergy in CR-HCT-116 cells.
Table 1 Synergistic effects of gercumin with FOLFOX on growth of chemo-resistant colon cancer CR-HT29 cells. 2 (mM)
5-FU þ Ox (mM)
FA 2
FOLFOX
2 þ FOLFOX
CI
1 2.5 5 10 20 30
5/0.125 12.5/0.313 25/0.625 50/1.25 100/2.5 150/3.75
0.01 ± 0.0005 0.01 ± 0.0004 0.01 ± 0.0006 0.15 ± 0.003 0.42 ± 0.008 0.65 ± 0.011
0.19 ± 0.005 0.01 ± 0.0004 0.15 ± 0.004 0.15 ± 0.002 0.56 ± 0.012 0.93 ± 0.013
0.11 ± 0.002 0.22 ± 0.006 0.25 ± 0.006 0.85 ± 0.011 0.99 ± 0.022 0.99 ± 0.021
0.439 0.613 1.09 0.306 0.088 0.133
Table 2 Synergistic effects of gercumin with FOLFOX on growth of chemo-resistant colon cancer CR-HCT-116 cells. 2 (mM) 5-FU þ Ox (mM) FA
1 2.5 5 10 20 30
5/0.125 12.5/0.313 25/0.625 50/1.25 100/2.5 150/3.75
2
FOLFOX
2 þ FOLFOX
CI
0.01 ± 0.0003 0.01 ± 0.0002 0.01 ± 0.0005 0.01 ± 0.0002 0.07 ± 0.0009 0.43 ± 0.0014
0.01 ± 0.0001 0.01 ± 0.0003 0.02 ± 0.0003 0.29 ± 0.002 0.56 ± 0.006 0.81 ± 0.004
0.25 ± 0.003 0.18 ± 0.0009 0.12 ± 0.0007 0.42 ± 0.002 0.79 ± 0.002 0.86 ± 0.004
0.110 0.351 0.93 0.711 0.577 0.666
Table 3 Effects of gercumin ± FOLFOX on colonosphere formation. Chemoresistant colon cancer cells were plated in stem cell media at a density of 1000 cells/well in a 24well plate. Following overnight incubation, cells were treated with gercumin ± FOLFOX and incubated for another 7 d. Colonospheres were counted using light microscopy. CR-HT29
Control 2 FOLFOX Combination
CR-HCT116
∅ 50e100 mm
∅ > 100 mm
∅ 50e100 mm
∅ > 100 mm
26 ± 2 58 ± 9 60 ± 13 70 ± 12
1 ± 0.6 3 ± 0.3 1 ± 0.7 0±0
33 ± 3 29 ± 3 31 ± 4 47 ± 12
2±1 5 ± 0.3 2±1 6±3
samples (e.g. compound 2 or FOLFOX alone) to either CR-HT29 or CR-HCT116 cells had little effect at low doses (10 mM for 2 and 100 mM [5-FU] and 2.5 mM [oxalilplatin] for FOLFOX). However, the combination of gercumin 2 and FOLFOX displayed a strong synergism of growth inhibitory action in both cancer cell lines. As highlighted in Tables 1 and 2 the percentages of cells affected largely increased when compound gercumin 2 and FOLFOX were
Table 4 Effects of gercumin ± FOLFOX on colonosphere disintegration. Chemoresistant colon cancer cells were plated in stem cell media at a density of 1000 cells/well in a 24well plate. Following overnight incubation, cells were treated with gercumin ± FOLFOX and incubated for another 7 d. Colonospheres were counted using light microscopy. CR-HT29
Control 2 FOLFOX Combination
CR-HCT116
∅ 50e100 mm
∅ > 100 mm
∅ 50e100 mm
∅ > 100 mm
28 ± 1 31 ± 4 34 ± 3 28 ± 3
11 ± 3 6±2 3±1 2±1
23 ± 2 21 ± 1 21 ± 2 25 ± 1
14 ± 2 9±2 6±2 5±1
either applied together or alone in a dose-dependent manner. This synergism of action was seen to be more pronounced in CRHT29 cells, where the growth inhibitory effect found to be 6 to 20 times over the individual compound or FOLFOX in the concentration range 2.5e10 mM for gercumin and 50 mM (5-FU)/1.25 mM (Ox) for FOLFOX. Isobologram analysis confirmed such a synergism at all EDs values (50, 75, and 90) for CR-HT29 cells as depicted in Fig. 3. Data reported herein seem to indicate that gercumin 2 is able to overcome the intrinsic resistance of CR-HT29 and CR-HCT 116 colon cancer cells to FOLFOX. On the basis of the data, reported earlier, a valid hypothesis of synergism of action can be formulated. Piacente, Bifulco, and coworkers put in evidence how compound 2 is able to inhibit the activity of m-PGES-1 thus abolishing the biosynthesis of PGE2 [8].The inhibition of production of this latter endogenous metabolite in turn is known to largely decrease the levels of resistance of colon cancer cells to both components of FOLFOX. This effect is accomplished by acting on several metabolic ways including targeting COX-2, that has been shown to be tightly linked to a decrease of resistance to 5-fluorouracil in colon cancer cells [15,16], down-regulation of the orphan nuclear receptor NR4A2, that has been found to be among the main determinants to confer increase in chemoresistance to FOLFOX in patients affected by colorectal carcinoma, induced by an abolished PGE2 biosynthesis [17], attenuation of PI3K signaling pathway, the activation of which in turn is known to positively regulate cancer cell proliferation and survival [18]. Several other modes of action have also been recently reported [19]. However, until now only effective target of gercumin, namely m-PGES-1 [8],has been recognized to be involved in colon cancer pathogenesis and identified as a valid target alternative to COX-2 to reduce colon cancer incidence in humans [20,21]. Prenylation of curcumin skeleton is currently attracting much attention of medicinal chemists and pharmacologists. This
Please cite this article as: S. Genovese et al., Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.068
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structural modification indeed has been seen to favorably modify pharmacological properties of the parent compound. However, until now the few data reported in the literature focused only on assays carried out on isolated enzymes. Herein, we report for the first time, the growth inhibitory potential of gercumin 2 together with FOLFOX in FOLFOX-resistant chemo-resistant CR-HT29 and CR-HCT116 human colon cancer cells. Such a synergism deserves to be investigated in several other cancer cells and studies to accomplish this goal are now underway in our laboratories.
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.068.
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References [1] D.J. Newman, G.M. Cragg J., Natural products as sources of new drugs over the last 25 years, Nat. Prod 70 (2007) 461e477, https://doi.org/10.1021/ np068054v. [2] A. Noorafshan, S. Ashkani-Esfahani, A review of therapeutic effects of curcumin, Curr. Pharm, DES 19 (2013) 2032e2046. PMID 23116311. [3] A. Shehzad, y.s. Lee, Molecular mechanisms of curcumin action: signal transduction, Biofactors 39 (2013) 27e36, https://doi.org/10.1002/biof.1065. [4] S. Shishodia, Molecular mechanisms of curcumin action: gene expression, Biofactors 39 (2013) 37e55, https://doi.org/10.1002/biof.1041. [5] X. Fan, C. Zhang, D.B. Liu, J. Yan, H.P. Liang, The clinical applications of curcumin: current state and the future, Curr. Pharm. Des.19 (2013) 2011e2031. PMID 23116310. ~ oz, G. Appendino, Dis[6] A. Minassi, G. S anchez-Duffhues, J.A. Collado, E. Mun secting the pharmacophore of curcumin. Which structural element is critical for which action? J. Nat. Prod. 76 (2013) https://doi.org/10.1021/np400148e, 105e1112. ~ oz, G.B. Appendino, A. Minassi, S. Pace, A. Rossi, [7] A. Koeberle, E. Mun C. Weinigel, D. Barz, L. Sautebin, D. Caprioglio, J.A. Collado, O. Werz, SAR studies on curcumin’s pro-inflammatory targets: discovery of prenylated pyrazolocurcuminoids as potent and selective novel inhibitors of 5lipoxygenase, J. Med. Chem. 57 (2014) 5638e5648, https://doi.org/10.1021/ jm500308c. [8] M. Iranshahi, M.G. Chini, M. Masullo, A. Sahebkar, A. Javidnia, M.C. Yazdi, C. Pergola, A. Koeberle, O. Werz, C. Pizza, S. Terracciano, S. Piacente, G. Bifulco, Can small chemical modifications of natural Pan-inhibitors modulate the
[15]
[16]
[17]
[18]
[19]
[20]
[21]
5
biological selectivity? The case of curcumin prenylated derivatives acting as HDAC or mPGES-1 inhibitors,, J. Nat. Prod. 78 (2015) 2867e2879, https:// doi.org/10.1021/acs.jnatprod.5b00700. T.C. Chou, Drug combination studies and their synergy quantification using the Chou-Talalay method, Cancer Res. 70 (2010) 440e446, https://doi.org/ 10.1158/0008-5472.CAN-09-1947. J. Nautiyal, S.S. Kanwar, Y. Yu, A.P.N. Majumdar, Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells, J. Mol. Signal. 6 (2011) 7, https://doi.org/10.1186/1750-2187-6-7. C.H. Kohne, Successes and limitations of targeted cancer therapy in colon cancer, Prog. Tumor Res. 41 (2014) 36e50, https://doi.org/10.1159/ 000356436. A. Yu, S.S. Kanwar, B.B. Patel, J. Nautiyal, F.H. Sarkar, Elimination of colon cancer stem-like cells by the combination of curcumin and FOLFOX, Transl. Oncol 2 (2009) 321e328, https://doi.org/10.1593/tlo.09193. Y. Yu, F.H. Sarkar, A.P.N. Majumdar, Down-regulation of miR-21 induces differentiation of chemoresistant colon cancer cells and enhances susceptibility to therapeutic regimens, Transl. Oncol 6 (2013) 180e186. PMID 23544170. S.S. Kanwar, Y. Yu, I. Nautyial, B.B. Patel, S. Padhye, F.H. Sarkar, A.P.N. Majumdar, Difluorinated-curcumin (CDF): a novel curcumin analog is a potent inhibitor of colon cancer stem-like cells, Pharm. Res. 28 (2011) 827e838, https://doi.org/10.1007/s11095-010-0336-y. C.H. Choi, T.B. Lee, Y.A. Lee, S. Choi, K.J. Kim, Up-regulation of cyclooxygenase2-derived prostaglandin E2 in colon cancer cells resistant to 5-fluorouracil, J. Korean Surg. Soc. 81 (2011) 115e121, https://doi.org/10.4174/ jkss.2011.81.2.115. D.F. Romagnolo, A.J. Papoutsis, O. Selmin, Nutritional targeting of cyclooxygenase-2 for colon cancer prevention, Infl. Allergy Drug Targets 9 (2010) 181e191, https://doi.org/10.2174/187152810792231922. Y. Han, H. Cai, L. Ma, Y. Ding, Y. Tan, Y. Liu, T. Su, Y. Yu, WChang, H. Zhang, C. Fu, G. Cao, Nuclear orphan receptor NR4A2 confers chemoresistance and predicts unfavorable prognosis of colorectal carcinoma patients who received postoperative chemotherapy, Eur. J. Cancer 49 (2013) 3420e3430, https:// doi.org/10.1016/j.ejca.2013.06.001. D. Tougeron, D. Sha, S. Manthravadi, F.A. Sinicrope, Aspirin, Colorectal Cancer, Back to the Future,Clin, Can. Res. 20 (2014) 1087e1094, https://doi.org/ 10.1158/1078-0432.CCR-13-2563. X. Zuo, Y. Shurelqi, Eicosanoid profiling in colon cancer: emergence of a pattern, Prostaglandins Other Lipid Mediat, 104-105, https://doi.org/10.1016/ j.prostaglandins.2012.08.004, 2013, 139-143. Y. Sasaki, Y. Nakatani, S. Hara, Role of microsomal prostaglandin E synthase-1 (mPGES-1)-derived prostaglandin E2 in colon carcinogenesis, Prostaglandins Other Lipid Mediat. 121 (2015) 42e45, https://doi.org/10.1016/ j.prostaglandins.2015.06.006. K. Larsson, P.J. Jakobsson, Inhibition of microsomal prostaglandin E synthase-1 as targeted therapy in cancer treatment, Prostaglandins Other Lipid Mediat. 120 (2015) 161e165, https://doi.org/10.1016/j.prostaglandins.2015.06.002.
Please cite this article as: S. Genovese et al., Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.068
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Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells Salvatore Genovese a, Francesco Epifano a, *, Francesca Preziuso a, Jill Slater b, Pratima Nangia-Makker c, Adhip P.N. Majumdar c, Serena Fiorito a “G. D’Annunzio” Chieti-Pescara, Via dei Vestini 31, 66100, Chieti, Italy Dipartimento di Farmacia, Universita Department of Biological Sciences, University of Michigan-Flint, Michigan, USA c VA Medical Center, Karmanos Cancer Institute, Department of Medicine, Wayne State University, Detroit, MI, USA a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 October 2019 Accepted 11 November 2019 Available online xxx
Advanced colon cancer is extremely difficult to cure, underscoring the need to develop novel therapeutic agents. Prenylated curcumins that are semisynthetic curcumin derivatives with significant anti-cancer potential have been studied herein to assess their therapeutic potential for colon cancer and tested to this aim in vitro for their growth inhibitory properties against 5-fluorouracil þ oxaliplatin resistant human colon cancer CR-HT29 and HCT-116 cells. The resulting most active product, gercumin (mono-Ogeranylcurcumin), has been further tested for its synergistic effects with FOLFOX (a combination of 5fluorouracil and oxaliplatin) on the same cell lines. Activity of this combination on colonosphere formation was also investigated. Gercumin was able to suppress the growth of cancer cells with a potency similar to that of curcumin. A synergistic effect of this compound and FOLFOX was also observed. doses tested for synergy in the colonosphere assays did not show greater suppression of colonosphere formation than independent treatment with either reagent alone. Only one of the combinations was shown to be more effective at suppressing colonosphere formation [gercumin 5 mM þ FOLFOX (2x)]. Thus, the growth inhibitory effects of curcumin against human cancer cells can be modulated and enhanced by the introduction of hydrophobic chains, normally found in several natural compounds, like the geranyl one. Such compounds are also able to synergize with known chemotherapeutics. © 2019 Elsevier Inc. All rights reserved.
Keywords: Cancer chemotherapy Curcumin Geranylation Gercumin Colon cancer
1. Introduction Natural products traditionally have been and still represent the main source of bioactive compounds used both in the pharmaceutical industry and in traditional health care systems [1]. In particular, in vital indications such as anti-cancer and antibiotic drugs, most compounds currently used in Western and alternative and complementary medicines stem either directly from natural sources (e.g. morphine) or are derived from scaffolds provided by natural products (e.g. taxol). Structural modifications of a lead compound have in most cases pronounced effects on pharmacokinetic and/or pharmacodynamic properties. Explicative examples have been thoroughly reported in the literature. This is the case of nalorphine in which the addition of an allyl moiety to the skeleton of morphine provide a drug that antagonized the effects of the
* Corresponding author. E-mail address: [email protected] (F. Epifano).
same morphine, while ethylmorphine resulted in a complete lack of analgesic activity. Curcumin 1 is well recognized among the naturally occurring compound with the most promising therapeutic potential. It is a diferuloylmethane derivative isolated from the rhizome of turmeric, Curcuma longa L. (Zingiberaceae), and has been proposed for treatment of several acute and chronic syndromes like inflammatory, cancer, neurodegenerative, cardiovascular, and metabolic diseases [2]. The mechanism of action of curcumin involves interactions with several biological targets like transcription factors, protein kinases, several enzymes, genes, and different receptors [3,4]. Due to its plethora of effects, curcumin has several potential clinical applications. Thus, this natural compound and its semisynthetic derivatives are currently passing through different clinical trials [5]. Examples of chemical modification of curcumin skeleton with the aim of modifying selectivity, potency, and bioavailability have been recently reported in the literature. In particular the addition of a prenyl group in position 9 favorably affect the biological selectivity of the resulting curcuminoid [6,7].
https://doi.org/10.1016/j.bbrc.2019.11.068 0006-291X/© 2019 Elsevier Inc. All rights reserved.
Please cite this article as: S. Genovese et al., Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.068
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More recently Piacente, Bifulco, and coworkers investigated the effects of mono- and di-O-prenylation of curcumin on the inhibitory properties on histone deacetylase (HDAC) and membraneassociated prostaglanid E synthase-1 (mPEGS-1) and found that monoprenylated compounds 2 and 5 acted as effective inhibitory agents of this latter enzyme [8]. The one just mentioned represents the only report published in the literature about pharmacological properties of O-prenylated curcumin derivatives. Thus, investigations on such semisynthetic compounds is a field of research of current and growing interest. In the current manuscript we wish to report the in vitro growth inhibitory effects of four O-prenylated curcumin derivatives (e.g. mono- and di-3,3-dimethylallyloxy and mono- and digeranyloxy) 2e5 (Fig. 1) on chemo-resistant human colon cancer CR-HT29 cells and the activity of the most effective compound resulting from this preliminary assay on the same cells and also on colon cancer CR-HCT116 cells, when combined with FOLFOX (5-fluorouracil and oxaliplatin) using the Chou-Talalay method [9]. The effects of O-prenylated curcumins and FOLFOX alone or in combination on colonosphere formation have been also investigated.
plates. 24 Hours after plating, cells were treated with various testing agents and incubated for another 48 h. Following this incubation period, the reaction was terminated by adding 0.5-mg/ml stock of MTT to each plate. The reaction was allowed to proceed for 3e4 h at 37 C. The culture medium was then removed. The resulting formazan crystals were then dissolved by adding 1 N HCl þ isopropanol (1:24). The intensity of the resulting color change reflected the number of live cells; absorbance was measured at a wavelength of 570 nm. All values were compared with the corresponding controls. All assays were performed in four replicates.
2. Materials and methods
2.5. Colonosphere formation or disintegration
2.1. Chemicals
To examine the effects of gercumin 2 on the formation of colonospheres, the FOLFOX-resistant cells were suspended in serumfree stem cell medium containing DMEM/F12 (1:1) supplemented with B27 (Life Technologies, Gaithersburg, MD) and antibioticantimycotic. The cells (1000 per well) were then plated in a 24well plate (Corning Inc, Lowell, MA) and incubated overnight. They were subsequently treated with compound 2 and/or FOLFOX for 7 days. The number of colonospheres was evaluated by light microscopy. To examine its effects on the disintegration of colonospheres, cells were plated as described above and incubated for 7 days, allowing colonospheres to form. At this time colonospheres were incubated in the absence (controls) or presence of the testing agent for 7 days and evaluated for the number of colonospheres by light microscopy.
Curcumin was purchased from Merck Sigma Aldrich (USA) and purified by crystallization (MeOH/H2O) before use. Compounds 2e5 were chemically synthesized as described previously [8] and their purity (>98.1%) assessed by HPLC and 1H NMR. Analytical data for curcuminoids 2e5 were in full agreement for those already reported for the same compounds [8]. 2.2. Cell cultures Chemo-resistant (CR) human colon cancer cell lines, CR-HT29 and CR-HCT116, were generated as previously described [10e13]. Cells were maintained in Dulbecco’s modified Eagle medium (DMEM; 4.5 g/L D-glucose) supplemented with 10% FBS, 1% antibiotic/antimycotic, and 50 mM 5-fluorouracil (5-FU) þ 1.25 mM oxaliplatin (FOLFOX) in tissue culture flasks in a humidified incubator at 37 C in an atmosphere of 95% air and 5% carbon dioxide. The medium was changed twice per week, and cells were passaged using 0.05% trypsin/EDTA. 2.3. Growth inhibition assay Inhibition of cell growth in response to curcumin analogues and/or FOLFOX (5FU þ oxaliplatin) was assessed by 3-(4,5dimethylthiazol-2yl)-2, 5-diphenyltetrazolium bromide (MTT) assay as described previously [10]. Briefly, cells were dispersed by trypsin-EDTA treatment, resuspended in DMEM containing 10% FBS, and seeded at a density of 5000 cells/well into 96-well culture
Fig. 1. Structure of prenylated curcuminoids: 1 R1 ¼ R2 ¼ H, 2 R1 ¼ geranyl R2 ¼ H, 3, R1 ¼ R2 ¼ geranyl 4 R1 ¼ 3,3-dimethylallyl R2 ¼ H, 5 R1 ¼ R2 ¼ 3,3-dimethylallyl.
2.4. Analysis of the interaction between gercumin 2 and FOLFOX The software program Calcusyn (Biosoft, Ferguson, MO) was employed to determine the nature of interaction between compound 2 and FOLFOX using the Chou-Talalay methodology [9]. This method utilizes a multiple drug-effect equation derived from an enzyme kinetics model in which the output is represented as combination indexes (CI) and/or isobologram analysis.
3. Results and discussion Curcumin and curcuminoids 2e5 were first screened for their in vitro growth inhibitory capacities on CR-HT29 cancer cell line using the MTT test in the concentration range 2.5e40 mM. Results are shown in Fig. 2. Of all the curcuminoids, only gercumin 2 had the greatest effect displaying a potency comparable or somewhat better respect to the parent compound 1. Thus, all further analyses, aimed at assessing the effects of a combination of gercumin 2 and FOLFOX on the
Fig. 2. Growth inhibitory effects of curcumin 1 and prenyloxycurcuminoids 2e5.
Please cite this article as: S. Genovese et al., Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.068
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growth of CR-HT29 and also CR-HCT-116 colon cancer cells, were focused on this latter sample. Experiments were carried out using the Chou-Talalay method [9] and the program Calcusyn was employed to determine the nature of interaction between gercumin and FOLFOX. The employed methodology utilizes a multiple drug-effect equation derived from an enzyme kinetics model in which the output is represented as combination indexes (CI) and/or isobologram analysis. Based on CIs values, the extent of synergism/ antagonism is determined. Calcusyn software defines synergy as CI value less than 1. CI values between 0.9 and 0.85 suggest moderate synergy, whereas those in the range of 0.7 to 0.3 are indicative of clear synergistic interactions between the drugs. CI values in the range of 0.9e1.10 suggest a near additive effect. The ChoueTalalay equation has been also utilized to perform isobologram analysis as represented by the isobole for ED75. According to Calcusyn glossary an “isobologram is a graph indicating the equipotent combinations of various doses that can be used to illustrate additivity, synergism, or antagonism. For a given effect level such as fraction of affected (FA) cells of 0.5, the required doses for (ED50)1 and (ED50)2 are drawn on the x-axis and y-axis, respectively. If the combination data point for FA ¼ 0.5 falls on the diagonal, an additive effect is indicated whereas when it is on the lower left, synergism is indicated. On the other hand, if the line falls on the upper right, antagonism is suggested.”. Dose effect curves, isobolograms, and CIs tables about the effects of combinations at various doses of gercumin and FOLFOX on growth of chemo-resistant colon cancer CR-HT29 and CR-HCT-116 cells as derived by Calcusyn calculations are shown in Figs. 3 and 4 and Tables 1e2. Similar patterns of results have been obtained for both cancer cell lines with the only exceptions of data recorded in isobolograms in Fig. 3, for which synergy between compound 3 and FOLFOX is suggested for all EDs, and Fig. 4 for which synergy is suggested only for ED50 and ED75. We then assessed the effects of the combination mono-O-geranylcurcumin 2 (10 mM) with FOLFOX (5-fluorouracil 50 mM þ oxaliplatin 1.25 mM) on colonosphere formation and disintegration. Results are reported in Tables 3 and 4 respectively. Curcumin is well recognized among the most promising biologically active natural products able to exert a plethora of beneficial effects on human health as well as to trigger a wide array of cell targets [2e5]. Its complexity in pharmacological activities and mechanisms of action recently prompted studies to better characterize its pharmacophore and to synthesize and assay structural analogues in order to depict a detailed structure-activity relationship [6,7].To this end the most of attention has been dedicated to the diacryloylmethane moiety while few data have been reported
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in the literature about structural modifications in the aromatic rings of curcumin. Thus, many other efforts to this concern need to be addressed in the next future. In 2011 Majumdar and coworkers showed that difluorocurcumin was able to efficiently inhibit the growth of chemo-resistant colon cancer cells accompanied by induction of apoptosis and colonosphere disintegration when used in combination with FOLFOX [14]. In 2013 Minassi, Appendino, and co-workers studied for the first time the effects of aromatic Cprenylation of curcumin on its pharmacological activity [6]. Earlier, they demonstrated that an increase in lipophylicity was detrimental for the capacity to act as a Michael acceptor towards thiol containing biofactors (e.g. NF-kB and Nrf-2) but enhanced the effects of the parent compound as an anti-HIV agent. A year later, they demonstrated further how prenylation combined with the introduction of a pyrazole ring instead of the two carbonyls largely increased the activity and selectivity of the obtained curcuminoid towards 5-lipoxygenase [7]. Piacente, Bifulco, and co-workers turned their attention in 2015 on mono-O- and symmetrical di-Oprenylated (3,3-dimethylallyl, geranyl, and farnesyl) curcumin analogues [8]. In addition, they extensively investigated the effects of these compounds on HDAC and mPEGS-1 enzymes by means of in silico and in vitro analysis. They also provided a detailed structureactivity relationship of the synthesized O-prenylcurcuminoid and showed how the prenylation pattern is a determinant for the selectivity towards HDAC or mPGES-1 but did not perform any kind of assay on cultured cells. In the present manuscript, we investigated for the first time the effects of four O-prenylated curcumins 2e5 on chemotherapyresistant human colon cancer cells in vitro. Results of the MTT, assay revealed that the addition of only one geranyloxy chain in position 8 of the curcumin skeleton greatly reduced cell viability in a dose dependent manner and with a profile similar to that recorded for the parent unprenylated sample in the concentration range 2.5 mMe40 mM. Only at the highest dose tested (40 mM), all compounds were found to be strongly cytotoxic reducing cell viability down to about 20% of the control. On the contrary in the range 2.5 mMe20 mM compounds 3, 4, and 5 essentially showed the same effects with an average reduction of cell viability less than 20%. Thus, the best activity displayed by gercumin 2 as colon cancer cells growth inhibitory agent was the rationale for selectin to perform further tests with this compound as a potential in vitro anti-cancer agent against two chemo-resistant human colon cancer cells together with FOLFOX, the backbone for colon cancer for chemotherapy [15]. Results obtained by the Chou-Talalay method and Calcusyn analysis showed that the application of individual
Fig. 3. (a) Calcusyn analysis of gercumin þ FOLFOX synergy in CR-HT29 cells (Combined R ¼ 0.95, MONO R ¼ 0.92, FOLFOX R ¼ 0.72), (b) isobologram of Calcusyn analysis of gercumin þ FOLFOX synergy in CR-HT29 cells.
Please cite this article as: S. Genovese et al., Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.068
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Fig. 4. (a) Calcusyn analysis of gercumin þ FOLFOX synergy in CR-HCT-116 cells (Combined R ¼ 0.81, MONO R ¼ 0.77, FOLFOX R ¼ 0.94), (b) Isobologram of Calcusyn analysis of gercumin þ FOLFOX synergy in CR-HCT-116 cells.
Table 1 Synergistic effects of gercumin with FOLFOX on growth of chemo-resistant colon cancer CR-HT29 cells. 2 (mM)
5-FU þ Ox (mM)
FA 2
FOLFOX
2 þ FOLFOX
CI
1 2.5 5 10 20 30
5/0.125 12.5/0.313 25/0.625 50/1.25 100/2.5 150/3.75
0.01 ± 0.0005 0.01 ± 0.0004 0.01 ± 0.0006 0.15 ± 0.003 0.42 ± 0.008 0.65 ± 0.011
0.19 ± 0.005 0.01 ± 0.0004 0.15 ± 0.004 0.15 ± 0.002 0.56 ± 0.012 0.93 ± 0.013
0.11 ± 0.002 0.22 ± 0.006 0.25 ± 0.006 0.85 ± 0.011 0.99 ± 0.022 0.99 ± 0.021
0.439 0.613 1.09 0.306 0.088 0.133
Table 2 Synergistic effects of gercumin with FOLFOX on growth of chemo-resistant colon cancer CR-HCT-116 cells. 2 (mM) 5-FU þ Ox (mM) FA
1 2.5 5 10 20 30
5/0.125 12.5/0.313 25/0.625 50/1.25 100/2.5 150/3.75
2
FOLFOX
2 þ FOLFOX
CI
0.01 ± 0.0003 0.01 ± 0.0002 0.01 ± 0.0005 0.01 ± 0.0002 0.07 ± 0.0009 0.43 ± 0.0014
0.01 ± 0.0001 0.01 ± 0.0003 0.02 ± 0.0003 0.29 ± 0.002 0.56 ± 0.006 0.81 ± 0.004
0.25 ± 0.003 0.18 ± 0.0009 0.12 ± 0.0007 0.42 ± 0.002 0.79 ± 0.002 0.86 ± 0.004
0.110 0.351 0.93 0.711 0.577 0.666
Table 3 Effects of gercumin ± FOLFOX on colonosphere formation. Chemoresistant colon cancer cells were plated in stem cell media at a density of 1000 cells/well in a 24well plate. Following overnight incubation, cells were treated with gercumin ± FOLFOX and incubated for another 7 d. Colonospheres were counted using light microscopy. CR-HT29
Control 2 FOLFOX Combination
CR-HCT116
∅ 50e100 mm
∅ > 100 mm
∅ 50e100 mm
∅ > 100 mm
26 ± 2 58 ± 9 60 ± 13 70 ± 12
1 ± 0.6 3 ± 0.3 1 ± 0.7 0±0
33 ± 3 29 ± 3 31 ± 4 47 ± 12
2±1 5 ± 0.3 2±1 6±3
samples (e.g. compound 2 or FOLFOX alone) to either CR-HT29 or CR-HCT116 cells had little effect at low doses (10 mM for 2 and 100 mM [5-FU] and 2.5 mM [oxalilplatin] for FOLFOX). However, the combination of gercumin 2 and FOLFOX displayed a strong synergism of growth inhibitory action in both cancer cell lines. As highlighted in Tables 1 and 2 the percentages of cells affected largely increased when compound gercumin 2 and FOLFOX were
Table 4 Effects of gercumin ± FOLFOX on colonosphere disintegration. Chemoresistant colon cancer cells were plated in stem cell media at a density of 1000 cells/well in a 24well plate. Following overnight incubation, cells were treated with gercumin ± FOLFOX and incubated for another 7 d. Colonospheres were counted using light microscopy. CR-HT29
Control 2 FOLFOX Combination
CR-HCT116
∅ 50e100 mm
∅ > 100 mm
∅ 50e100 mm
∅ > 100 mm
28 ± 1 31 ± 4 34 ± 3 28 ± 3
11 ± 3 6±2 3±1 2±1
23 ± 2 21 ± 1 21 ± 2 25 ± 1
14 ± 2 9±2 6±2 5±1
either applied together or alone in a dose-dependent manner. This synergism of action was seen to be more pronounced in CRHT29 cells, where the growth inhibitory effect found to be 6 to 20 times over the individual compound or FOLFOX in the concentration range 2.5e10 mM for gercumin and 50 mM (5-FU)/1.25 mM (Ox) for FOLFOX. Isobologram analysis confirmed such a synergism at all EDs values (50, 75, and 90) for CR-HT29 cells as depicted in Fig. 3. Data reported herein seem to indicate that gercumin 2 is able to overcome the intrinsic resistance of CR-HT29 and CR-HCT 116 colon cancer cells to FOLFOX. On the basis of the data, reported earlier, a valid hypothesis of synergism of action can be formulated. Piacente, Bifulco, and coworkers put in evidence how compound 2 is able to inhibit the activity of m-PGES-1 thus abolishing the biosynthesis of PGE2 [8].The inhibition of production of this latter endogenous metabolite in turn is known to largely decrease the levels of resistance of colon cancer cells to both components of FOLFOX. This effect is accomplished by acting on several metabolic ways including targeting COX-2, that has been shown to be tightly linked to a decrease of resistance to 5-fluorouracil in colon cancer cells [15,16], down-regulation of the orphan nuclear receptor NR4A2, that has been found to be among the main determinants to confer increase in chemoresistance to FOLFOX in patients affected by colorectal carcinoma, induced by an abolished PGE2 biosynthesis [17], attenuation of PI3K signaling pathway, the activation of which in turn is known to positively regulate cancer cell proliferation and survival [18]. Several other modes of action have also been recently reported [19]. However, until now only effective target of gercumin, namely m-PGES-1 [8],has been recognized to be involved in colon cancer pathogenesis and identified as a valid target alternative to COX-2 to reduce colon cancer incidence in humans [20,21]. Prenylation of curcumin skeleton is currently attracting much attention of medicinal chemists and pharmacologists. This
Please cite this article as: S. Genovese et al., Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.068
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structural modification indeed has been seen to favorably modify pharmacological properties of the parent compound. However, until now the few data reported in the literature focused only on assays carried out on isolated enzymes. Herein, we report for the first time, the growth inhibitory potential of gercumin 2 together with FOLFOX in FOLFOX-resistant chemo-resistant CR-HT29 and CR-HCT116 human colon cancer cells. Such a synergism deserves to be investigated in several other cancer cells and studies to accomplish this goal are now underway in our laboratories.
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Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.11.068.
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References [1] D.J. Newman, G.M. Cragg J., Natural products as sources of new drugs over the last 25 years, Nat. Prod 70 (2007) 461e477, https://doi.org/10.1021/ np068054v. [2] A. Noorafshan, S. Ashkani-Esfahani, A review of therapeutic effects of curcumin, Curr. Pharm, DES 19 (2013) 2032e2046. PMID 23116311. [3] A. Shehzad, y.s. Lee, Molecular mechanisms of curcumin action: signal transduction, Biofactors 39 (2013) 27e36, https://doi.org/10.1002/biof.1065. [4] S. Shishodia, Molecular mechanisms of curcumin action: gene expression, Biofactors 39 (2013) 37e55, https://doi.org/10.1002/biof.1041. [5] X. Fan, C. Zhang, D.B. Liu, J. Yan, H.P. Liang, The clinical applications of curcumin: current state and the future, Curr. Pharm. Des.19 (2013) 2011e2031. PMID 23116310. ~ oz, G. Appendino, Dis[6] A. Minassi, G. S anchez-Duffhues, J.A. Collado, E. Mun secting the pharmacophore of curcumin. Which structural element is critical for which action? J. Nat. Prod. 76 (2013) https://doi.org/10.1021/np400148e, 105e1112. ~ oz, G.B. Appendino, A. Minassi, S. Pace, A. Rossi, [7] A. Koeberle, E. Mun C. Weinigel, D. Barz, L. Sautebin, D. Caprioglio, J.A. Collado, O. Werz, SAR studies on curcumin’s pro-inflammatory targets: discovery of prenylated pyrazolocurcuminoids as potent and selective novel inhibitors of 5lipoxygenase, J. Med. Chem. 57 (2014) 5638e5648, https://doi.org/10.1021/ jm500308c. [8] M. Iranshahi, M.G. Chini, M. Masullo, A. Sahebkar, A. Javidnia, M.C. Yazdi, C. Pergola, A. Koeberle, O. Werz, C. Pizza, S. Terracciano, S. Piacente, G. Bifulco, Can small chemical modifications of natural Pan-inhibitors modulate the
[15]
[16]
[17]
[18]
[19]
[20]
[21]
5
biological selectivity? The case of curcumin prenylated derivatives acting as HDAC or mPGES-1 inhibitors,, J. Nat. Prod. 78 (2015) 2867e2879, https:// doi.org/10.1021/acs.jnatprod.5b00700. T.C. Chou, Drug combination studies and their synergy quantification using the Chou-Talalay method, Cancer Res. 70 (2010) 440e446, https://doi.org/ 10.1158/0008-5472.CAN-09-1947. J. Nautiyal, S.S. Kanwar, Y. Yu, A.P.N. Majumdar, Combination of dasatinib and curcumin eliminates chemo-resistant colon cancer cells, J. Mol. Signal. 6 (2011) 7, https://doi.org/10.1186/1750-2187-6-7. C.H. Kohne, Successes and limitations of targeted cancer therapy in colon cancer, Prog. Tumor Res. 41 (2014) 36e50, https://doi.org/10.1159/ 000356436. A. Yu, S.S. Kanwar, B.B. Patel, J. Nautiyal, F.H. Sarkar, Elimination of colon cancer stem-like cells by the combination of curcumin and FOLFOX, Transl. Oncol 2 (2009) 321e328, https://doi.org/10.1593/tlo.09193. Y. Yu, F.H. Sarkar, A.P.N. Majumdar, Down-regulation of miR-21 induces differentiation of chemoresistant colon cancer cells and enhances susceptibility to therapeutic regimens, Transl. Oncol 6 (2013) 180e186. PMID 23544170. S.S. Kanwar, Y. Yu, I. Nautyial, B.B. Patel, S. Padhye, F.H. Sarkar, A.P.N. Majumdar, Difluorinated-curcumin (CDF): a novel curcumin analog is a potent inhibitor of colon cancer stem-like cells, Pharm. Res. 28 (2011) 827e838, https://doi.org/10.1007/s11095-010-0336-y. C.H. Choi, T.B. Lee, Y.A. Lee, S. Choi, K.J. Kim, Up-regulation of cyclooxygenase2-derived prostaglandin E2 in colon cancer cells resistant to 5-fluorouracil, J. Korean Surg. Soc. 81 (2011) 115e121, https://doi.org/10.4174/ jkss.2011.81.2.115. D.F. Romagnolo, A.J. Papoutsis, O. Selmin, Nutritional targeting of cyclooxygenase-2 for colon cancer prevention, Infl. Allergy Drug Targets 9 (2010) 181e191, https://doi.org/10.2174/187152810792231922. Y. Han, H. Cai, L. Ma, Y. Ding, Y. Tan, Y. Liu, T. Su, Y. Yu, WChang, H. Zhang, C. Fu, G. Cao, Nuclear orphan receptor NR4A2 confers chemoresistance and predicts unfavorable prognosis of colorectal carcinoma patients who received postoperative chemotherapy, Eur. J. Cancer 49 (2013) 3420e3430, https:// doi.org/10.1016/j.ejca.2013.06.001. D. Tougeron, D. Sha, S. Manthravadi, F.A. Sinicrope, Aspirin, Colorectal Cancer, Back to the Future,Clin, Can. Res. 20 (2014) 1087e1094, https://doi.org/ 10.1158/1078-0432.CCR-13-2563. X. Zuo, Y. Shurelqi, Eicosanoid profiling in colon cancer: emergence of a pattern, Prostaglandins Other Lipid Mediat, 104-105, https://doi.org/10.1016/ j.prostaglandins.2012.08.004, 2013, 139-143. Y. Sasaki, Y. Nakatani, S. Hara, Role of microsomal prostaglandin E synthase-1 (mPGES-1)-derived prostaglandin E2 in colon carcinogenesis, Prostaglandins Other Lipid Mediat. 121 (2015) 42e45, https://doi.org/10.1016/ j.prostaglandins.2015.06.006. K. Larsson, P.J. Jakobsson, Inhibition of microsomal prostaglandin E synthase-1 as targeted therapy in cancer treatment, Prostaglandins Other Lipid Mediat. 120 (2015) 161e165, https://doi.org/10.1016/j.prostaglandins.2015.06.002.
Please cite this article as: S. Genovese et al., Gercumin synergizes the action of 5-fluorouracil and oxaliplatin against chemoresistant human cancer colon cells, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.11.068