Abstract
Currently used platinum drugs fail to provide long-term cure for ovarian cancer mainly because of acquired drug resistance. With the idea that the difference may translate into an altered spectrum of activity, monofunctional planaramineplatinum(II) complex tris(quinoline)monochloro-platinum chloride (coded as LH5) was synthesized and investigated for its activity against human ovarian A2780, cisplatin-resistant A2780 (A2780cisR) and ZD0473-resistnat A2780 (A2780ZD0473R) cancer cell lines alone and in combination with the phytochemicals capsaicin (Caps) and curcumin (Cur) as a function of concentration and sequence of administration. Cell viability was quantified using the MTT reduction assay, while combination was used as a quantitative measure of the combined drug action. LH5 is found to be more active than cisplatin (CS) against both resistant cell lines. Combination of LH5 with capsaicin showed synergism in all three cell lines, with the bolus being most synergistic. Lack of association between the levels of platinum accumulation and platinum–DNA with cytotoxicity can be seen to indicate that binding with DNA may not be the main determinant of activity of LH5. Greater activity of LH5 compared to cisplatin, especially against the resistant cell lines, indicates that the compound may have the potential for development as a novel anticancer drug and that its combination with phytochemicals can serve to further enhance drug efficacy.
Ovarian cancer is the leading cause of death from gynecological cancers in women in the Western world for which overall treatment success rate remains low (1). Although tumour generally responds well to combination of platinum with paclitaxel, the drugs fail to function when relapse occurs (2). Because of the lack of early symptoms, most patients would be at an advanced stage at the time of initial diagnosis and they die of recurrence of the tumour that is resistant to conventional treatment (3, 4). Hence, there is a need for new platinum-based drugs with novel mechanisms of action and use of different treatment regimens consisting of two or more drugs in combination to improve the therapeutic outcome.
Recently, it has been shown that some monofunctional cationic platinum complexes with only one site for binding with DNA have a greater efficacy than cisplatin (CS) and oxaliplatin in killing cancer cells and inhibiting transcription (5-7). In this study, we report the synthesis and activity of the monofunctional platinum(II) tris(quinoline)chloroplatinum(II) (coded as LH5) (Figure 1) against ovarian A2780, A2780cisR and A2780ZD0473R cancer cell lines. The interaction of LH5 with salmon sperm DNA and pBR322 plasmid DNAs have also been investigated. The cytotoxicity of cisplatin has also been determined to serve as reference.
The effect of combination of LH5 with the phytochemicals capsaicin (Caps) and curcumin (Cur) (Figure 1) was also investigated with the idea that the combination might be better able to overcome platinum resistance. Caps is a major pungent smelling compound found in red pepper and hot chilli that belongs to the plant genus Capsicum (Solanaceae) (8, 9). It has been reported to control obesity and possesses anti-carcinogenic and chemopreventive activities (10, 11). It can inhibit the growth of tumour cells and induce apoptosis in various cancer cells, including human leukemia HL-60 cell line (12), gastric adenocarcinoma cell line (AGS cells) (13) and the human breast cancer MCF-7 cell line (13). Caps is a chemopreventive able to inhibit multi-stage carcinogenesis (14) and angiogenesis in vitro and in vivo (15) and does so by blocking the translocation of nuclear factor kappa B (NF-κB), activator protein 1 (AP-1) and signal transducer and activator of transcription (STAT3) signaling pathway that are required for carcinogenesis (8, 15, 16).
Structures of LH5, capsaicin, curcumin and cisplatin.
Cur is a polyphenol from rhizome of the plant Curcuma longa (Figure 1). It is a key component of turmeric that has antioxidant, anti-toxic, anti-inflammatory, cancer chemo-preventive and potentially chemotherapeutic properties (3, 17). Recent studies have shown that diverse activities of Cur are mediated through multiple signalling pathways (18) and that the molecular targets of Cur include transcription factors, growth factors, cytokines, enzymes and other gene products (19). Cur has been shown to inhibit the Fanconi anaemia (FA)/Breast Cancer (BRCA) pathway and sensitize CAOV3 and SKOV3 ovarian cancer cells to cisplatin-induced apoptosis (20, 21). Because of expected differences in mechanism of action of LH5 and Cur, it is logical to think that combination of LH5 with Caps and Cur may exhibit sequence-dependent synergism. The structures of LH5, capsaicin and curcumin and cisplatin are given in Figure 1.
Materials and Methods
Materials. Chemicals used were potassium tetrachloroplatinate (II) (K2[PtCl4]) (Sigma Chemical Company, St. Louise, MO, USA); N,N-dimethylformamide (DMF) [C3H7NO]; imidazo(1,2-α)pyridine [C7H6N2] (Sigma Aldrich Chemical Company, Milwaukee, MO, USA); HCl (Ajax chemicals, Auburn NSW, Australia); ethanol (Merck Ltd., Kilsyth, Australia).
Method. 0.5 mmol of K2PtCl4 (0.210 g) was dissolved in mQ water (10 ml) and treated with concentrated hydrochloric acid (0.25 ml). The temperature of the solution was increased to 50°C. Five millimoles of quinoline (0.60 ml) were added dropwise over 1 h to the solution of potassium tetrachloroplatinate. The reaction mixture, protected from light, was stirred at room temperature for 2 weeks. Fifteen ml of 0.25 M hydrochloric acid was added to the mixture and stirring was continued for another 2 weeks at room temperature in dark. After 24 h, the mixture was centrifuged at 5,500 rpm for 10 min to collect precipitate of LH5. The crude product was washed with ice cold ethanol and purified by precipitation from 0.05 M HCl. The yield of the product was 42% (139 mg) as a pale yellow powder.
Characterization. C, H and N were determined using a Carlo Erba 1106 automatic analyzer available at the Australian National University. Pt was determined by graphite furnace atomic absorption spectroscopy (AAS). As LH5 could not be obtained in crystalline form, infrared (IR), mass spectrum (MS) and proton nuclear magnetic resonance (1H NMR) spectra were used to aid in its structural characterization. The IR spectrum was obtained using a Varian FT-IR spectrometer (Bruker IFS66 spectrometer; Agilent Technologies, Mulgrave, Victoria, Australia). To obtain the mass spectrum, solution of LH5 made in 90% methanol and 10% DMF was sprayed into a Finnigan LCQ mass spectrometer. To obtain 1H NMR spectrum, the compound was dissolved in deuterated dimethyl sulfoxide (DMSO) and prepared in 5 mm high precision Wilmad NMR tube and a Bruker DPX400 spectrometer was used with frequency of 400.2 MHz. In 1H NMR, s, d and q denote respectively singlet, doublet and quartet. Numbering scheme, adopted for quinoline ligand, is given in Figure 2.
Numbering scheme, adopted for quinoline ligand, present in LH5.
LH5: As a pale yellow powder (139 mg, 42%); 1H NMR (400 MHz, [D6] DMSO: δ (ppm)=8.4 (d, due to C2H), 8.2 (d, due to C6H), 7.95 (q, due to C7H), 7.6 (q, due to C9H), 7.5 (q, due to C8H), 7.1 (q, due to C5H); 2.49 (s, due to DMSO); IR (KBr): 2361, 1503, 1124, 812 and 775 cm−1; MS (ESI) m/z (%):617 (100)=[Pt(C9H7N)3Cl2 – Cl]; 618 (80)=[Pt(C9H7N)3Cl2 – Cl + H]; 616 (78)=[Pt(C9H7N)3Cl2 – Cl – H]; 453 (82)=[Pt(C9H7N)3Cl2 – (C9H7N) – 2Cl]; 545 (75)=[Pt(C9H7N)3Cl2 – (C9H7N) + (-NH2)2 – 6H]; 545 (75)=[Pt(C9H7N)3Cl2 – (C9H7N) + (-NH2)2 – 6H]; 530 (68)=[Pt(C9H7N)3Cl2 – (C9H7N) + 6H]; Anal. calcd for C27H21Cl2N3Pt: C 46.6, H 3.2, N 6.0, Pt 29.8, found: C 45.9±0.4, H 3.4±0.4, N 5.7±0.4, Pt 29.8±1.0
Molar conductivity. Molar conductivity values for solutions of LH5 and CS were determined at concentrations ranging from 60 μM to 200 μM. First, to obtain 1 mM solutions, CS was dissolved in 1:4 mixture of DMF and mQ water and LH5 in 2:1 mixture of DMSO and mQ water; these were then further diluted with mQ water. The molar conductivity values were plotted against concentration to obtain limiting molar conductivity values (Λo).
Biological Activity
Interaction with DNA. Interaction of cisplatin and LH5 with salmon sperm DNA and pBR322 plasmid DNA (without and with BamH1 digestion) were investigated using agarose gel electrophoresis in which the amount of DNA was kept constant, while concentrations of the compounds varied. DNA bands were viewed under short wave UV light and a Kodak Gel Logic 100 imaging system (GL 100) was used for taking gel pictures. Images were analyzed using Kodak molecular imaging software (Kodak MI software; https://www.google.com.au/#q=kodak+molecular+imaging+softwar)
Salmon sperm DNA (ssDNA). To prepare stock solutions of ssDNA, 10-15 mg of ssDNA was added to 10 ml of 0.05 M Trizma buffer at pH 8 to give DNA concentrations ranging from 1 mg ml−1 to 1.5 mg ml−1 and stored at −18°C until used. Four μl aliquots of ssDNA (at 1.5 mg ml−1) were added to solutions of varying amounts of LH5 and cisplatin and the total volume was made up to 20 μl by adding mQ water so that the concentrations of compounds ranged from 1.87 to 60 μM. Sixteen μl of mQ water was added with 4 μl of ssDNA to prepare a DNA blank. Incubation was carried out in shaking water bath at 37°C for 2 h following which 16 μl aliquots of drug–DNA mixtures were loaded onto 1% agarose gel and electrophoresis was run in TAE buffer for 50 min at 120 Vcm−1 at room temperature.
pBR322 plasmid DNA. pBR322 plasmid DNA was supplied in a 10 mM Tris-HCl, pH 7.5, 1 mM EDTA buffer at a concentration of 0.5 μg/μl. Exactly 1 μl of supplied pBR322 plasmid DNA in solution was added to solutions of LH5 and cisplatin at different concentrations ranging from: 1.87 to 60 μM. The total volume was made up to 20 μl by adding mQ water. The DNA blank was prepared by adding 19 μl of mQ water to 1 μl of pBR322 plasmid DNA. The samples were incubated for 5 h on a shaking water bath at 37°C in the dark, at the end of which the reaction was quenched by rapid cooling to 0°C for 30 min. The samples were thawed and mixed with 2 μl of marker dye (0.25% bromophenol blue and 40% of sucrose). Seventeen μl aliquots of drug-DNA mixtures containing 1 μl of DNA were loaded onto the 1% agarose gel made in TAE buffer (22) that contained ethidium bromide (1 mg ml−1). Electrophoresis was carried out at room temperature for 1 h and 30 min at 150 V cm−1.
BamH1 digestion. In this series of experiment, an identical set of drug–DNA mixtures, as that described previously, was first incubated for 5 h in a shaking water bath at 37°C and then subjected to BamH1(10 units ul−1) digestion. To each 20 μl of incubated drug-DNA mixture were added 3 μl of 10 x digestion buffer SB followed by 0.2 μl BamH1 (2 units).The mixtures were left in a shaking water bath at 37°C for 1 h, at the end of which the reaction was terminated by rapid cooling. Electrophoresis was carried out for 1 h and 30 min at 150 V cm-1 and the gel was subsequently stained with ethidium bromide, visualized under UV light and photographed as described previously.
Cytotoxicity Assay
The cell kill due to drugs alone and in combination were determined using the MTT (3-(4,5–Di-methyl-2-thiazole)-2,5-diphenyl-2H-tetrazolium bromide) reduction assay (23, 24). Briefly, 4,000 to 5,500 cells (maintained in logarithmic growth phase in complete medium consisting of RPMI 1640, 10% heat-inactivated foetal bovine serum (FBS), 20 mM HEPES, 0.112% sodium bicarbonate and 2 mM glutamine without antibiotics) were seeded into flat-bottomed 96-well culture plates in 10% FBS/RPMI 1640 culture medium and allowed to attach overnight. For single-treatments, drugs were added at least three to five different concentrations to triplicate wells and left in an incubator (at 37°C, with 5% carbon dioxide in air, at pH 7.4) for 72 h. Solutions of drugs made in 10% FCS/RPMI-1640 medium (0.16 to 20 μM for CS and LH5, 0.16 to 200 μM for Caps and Cur), 100 μl of drugs were added to equal volumes of cell culture in triplicate wells, then left to incubate under normal growth conditions for 72 h at 37°C in a humidified atmosphere. For combinations studies, cells were treated with increasing concentrations of drugs at constant ratios of their half maximal inhibitory concentration (IC50) values, i.e. drug concentrations required for 50% cell kill, using the sequences: 0/0 h, 0/4 h and 4/0 h, where 0/0 h meant that both drugs were added at the same time, 0/4 h meant that LH5 was added first followed by the addition of phytochemical (capsaicin/curcumin) 4 h later and 4/0 h meant the converse. The concentration ranges were: LH5: 0.23-3.63 μM, 0.29-4.63 μM and 0.29-4.56 μM; capsaicin: 3.6-57.7 μM, 3.12-49.9 μM and 4.13-66.02 μM; curcumin: 1.10-17.63 μM, 1.34-21.41 μM and 1.35-21.52 μM for A2780, A2780cisR and A2780ZD0473R cell lines, respectively. At the completion of the 72 h incubation period, the MTT reduction assay was performed as described previously (23, 24).
Median effect analysis (25, 26), based on the pooled data from 3 to 5 individual experiments each comprising at least three data points for each drug alone and in combination, was carried out to calculate combination index (CI) as a quantitative measure of combined action using Calcusyn software (V2) (Biosoft, Cambridge, UK).
where D1 and D2 in the numerator stand for the concentrations of compounds 1 and 2 in combination needed to achieve x% inhibition, whereas D1x and D2x in the denominator represent the same when they are present alone. In the following equation, Dx denotes dose of drug, Dm is the median-effect dose, which is equivalent to IC50, fa is the fraction of cells affected (i.e. killed) by the dose, fu is the fraction of cells remaining unaffected so that fu=1-fa and m is the exponent defining the shape of the dose effect curve.
CI of <1,=1 and > 1 indicates, respectively, synergism, additivity and antagonism in the combined drug action. The linear correlation coefficient ‘r’ was used as a measure of goodness of fit for the pooled data (where r=1 is a perfect fit). For the cell culture system, r should be greater than 0.95.
Cellular platinum accumulation and platinum (Pt)–DNA binding. Exponentially growing A2780 and A2780cisR cells in 10 ml of 10 % FCS/RPMI 1640 culture medium (cell density: 1×106 cells ml−1) were incubated with solutions of LH5 and cisplatin (at 10 μM for LH5 and 50 μM for cisplatin) for 24 h at 37°C. Two sets of cell culture dishes were prepared, one set for DNA binding studies and the other for cellular accumulation of platinum. Cells were collected by scraper and the cell suspensions were transferred into 10 ml centrifuge tubes. The cell suspensions were spun at 3,500 rpm at 4°C for 2 min to obtain the cell pellet. The supernatant was drained out and the cell pellets were washed with 4 ml PBS kept in 4°C. The mixtures were centrifuged again at 3,500 rpm at 4°C for 2 min by using CS-15R Centrifuge (Beckman, Gladesville, NSW, Australia) to obtain the cell pellet. After discarding the supernatant, 500 μl of the PBS was added to re-suspend the cells and the suspensions were transferred to corresponding labelled of 1.5 ml centrifugal eppendorf tubes. Surviving cell concentration was determined by TC10TM Automated Cell Counter (Bio-Rad, Gladesville, NSW, Australia) using tryphan blue. The samples in the tubes were spun for 2 min at 10,000 rpm at 4°C to obtain the pellet and stored at −20°C until assayed for platinum contents. At least three independent experiments were performed for both cellular accumulation and DNA binding studies. Total platinum contents in cell pellets were determined by graphite furnace AAS.
Platinum-DNA binding. Following incubation of cells with drugs, high molecular weight DNA was isolated from cell pellet using JETQUICK Blood DNA Spin Kit/50 according to the modified protocol of Bowtell et al. (27) DNA content was determined by UV spectrophotometry (260 nm). Pt levels were determined by graphite furnace AAS. A260/A280 ratios were found to be between 1.75 and 1.8 for all samples ensuring its high purity of the DNA (27) and the DNA concentration was calculated according to the following equation: Concentration=Absorbance at 260 nm × 50 ng/μl.
Results and Discussion
Chemistry. Monofunctional platinum tris(quinoline) chloroplatinum (II) (coded as LH5) was synthesized according to modified Kauffman method (28). Cisplatin, used as a reference, was synthesized according to Dhara's method (29). Synthesis and characterization are fully described in the experimental section. Structures of LH5, capsaicin, curcumin and cisplatin are shown in Figure 1.
The half-maximal inhibitory concentration (IC50) values and resistance factors (RF) of LH5, cisplatin (used as a reference compound), capsaicin and curcumin against the three cell lines: A2780, A2780cisR and A2780ZD0473R.
Molar conductivity. Whereas non-polar and non-ionic substances may cross the cell membrane by both passive diffusion and carrier mediated transport (30), polar molecules and ions usually cross the cell membrane by carrier-mediated transports (30, 31). Cisplatin, being administered by intravenous route, will remain largely intact in the extracellular fluid that contains high chloride concentration. Hence, it is expected to cross the cell membrane by both passive diffusion and carrier-mediated transport (30). It was also suggested that cisplatin can also cross the cell membrane by pinocytosis (32). Unlike cisplatin, which can cross the cell membrane by both passive diffusion and carrier-mediated transport, monofunctional platinum LH5, being ionic in nature, is expected to cross the cell membrane by carrier-mediated transport only.
Thus, limiting molar conductivity value (Λ0) for LH5 was found to be 272 Ω−1 cm2 mol−1 compared to 136 for cisplatin, reflecting the difference in nature of the compounds (ionic for LH5 and polar covalent for cisplatin) (33). However, the smaller difference can be seen to the bulky nature of the cation in LH5.
Drugs administered alone. Table I lists the IC50 values and the resistant factors (RF) of LH5, cisplatin (used as a reference), capsaicin and curcumin applying to the ovarian cancer cell lines: A2780 (cisplatin-sensitive; the parent cell line), A2780cisR (cisplatin-resistant) and A2780ZDO473R (picoplatin-resistant) cell lines. IC50 represents the drug concentration required for 50% cell kill and RF is defined as the ratio of the IC50 value in the resistant cell line to that in the parent cell line.Whereas the activity of cisplatin decreases markedly from the parent A2780 cell line to the resistant A2780cisR and A2780ZD0473R cell lines, that of LH5 in A2780cisR cell line remains the same as that in the parent A2780 cell line and shows less pronounced decrease (from 2.27 to 2.85 μM for LH5 and from 1.0 to 8.2 μM for cisplatin) than cisplatin in A2780ZD0473R cell line.
Dose-effect parameters applying to combinations of LH5 with capsaicin and curcumin in the A2780, A2780cisR and 2780ZD0473R cell lines; 0/0 h meant that both the compounds were administered at the same time, 0/4 h meant that LH5 was administered first followed by the phytochemical 4 h later and 4/0 h meant the converse.
The pronounced cytotoxicity of LH5 against all the three ovarian cancer cell lines clearly indicates that it must differ from cisplatin in the mechanisms of antitumour action and the ability to overcome drug resistance. As LH5 contains three bulky quinoline ligands bound to platinum(II) compared to two ammonia ligands present in cisplatin, it is expected to undergo distinctly different non-covalent interactions in terms of hydrogen bonding and stacking interactions with nucleobases in the DNA and these may translate into significant differences in protein recognition and, hence, the ability to induce cell death or the escape from it. Irrespective of the mechanisms involved, the results indicate that LH5 has been better able to overcome resistance operating in the ovarian cancer cell lines. In line with these results, it has been reported that monofunctional cationic compounds containing bulky planaramine ligand can kill cancer cells with a greater efficacy than cisplatin and oxaliplatin (5, 33). Between the two phytochemicals, curcumin is found to be more active than capsaicin against all the three cell lines. Like LH5, the two phytochemicals have lower RF values than cisplatin, indicating the mechanisms of resistance operating in the resistant cell lines do not apply to the phytochemicals, as well as LH5.
Drugs in combination. Combinations of LH5 with capsaicin and curcumin were administered to human ovarian A2780, A2780cisR and A2780ZD0473R cancer cell lines as a function of the sequence of administration and concentrations. Combination indices (CIs), median-effect dose (ED50), dose required for 75% cell kill (ED75) and that for 90% cell kill (ED90), shape (sigmoidicity), conformity (linear correlation coefficient r) are given in Table II. Figure 3 provides a bar graph of CI values at ED50.
The results show that, as applied to the combinations of LH5 with capsaicin administered to A2780 and A2780cisR cell lines, all three sequences of administration resulted in synergistic outcomes in A2780ZD0473R; however, it was the bolus administration that produced most significant synergistic outcomes. Generally a greater variation in combination index values with the sequence of administration was observed in A2780 and A2780cisR cell lines than in the A2780ZDO473R cell line.
As applied to the combinations of LH5 with curcumin also, the bolus administration was most synergistic in all the three cell lines.
The above findings indicate that the two compounds in combination need to administered at the same time to maximise their ability to overcome the mechanisms of resistance operating in the cell lines. The findings are similar to those observed for the combinations of other monofuntional platinums and phytochemicals where the bolus administration was found to be most synergistic as well (7); however, they are different from those observed for the combinations of bifunctional platinums cisplatin and oxaliplatin with phytochemicals (curcumin and quercetin) where pre-treatment with the phytochemicals produced most synergistic outcomes (34). The results indicate that the mechanisms underpinning synergistic outcome from combination of capsaicin and curcumin with monofunctional platinum LH5 are different than those for their combination with bifunctional platinums cisplatin and oxaliplatin (34).
Like bifunctional platinums, monofunctional compounds with bulky planaramine ligands can also bind covalently to N7 position of guanine (although forming only monofunctionl adducts), but are expected to undergo stacking interaction and hydrogen bonding with DNA (5). But why the greatest synergism at the median effect dose was found to result from concurrent administration of LH5 with capsaicin and curcumin than administration using 0/4 h and 4/0 h sequences remains an open question. Possible mechanisms involved in the combined drug are considered in the following paragraphs.
Combination indices (CIs) applying combinations of LH5 with capsaicin (Caps) and curcumin (Cur) at the median effect dose (ED50) in (a) A2780, (b) A2780cisR and (c) A2780ZD0473R cell lines, calculated based on the pooled data from three to five individual experiments; 0/0 h means that both the compounds were administered at the same time, 0/4 h means that LH5 was administered first followed by the phytochemical 4 h later and 4/0 h means the converse.
Further on mechanisms of combined drug action. The results of the present study and the reported results (5-7) can be seen to indicate that the combined drug action from the combinations of monofunctional platinum and phytochemicals is a function of the nature of the phytochemical, that of the monofunctional platinum and the status of the cell. Thus, although capsaicin was found to be less cytotoxic than curcumin against both the resistant A2780cisR and A2780ZD0473R cell lines, in combination with LH5 it produced more synergistic outcomes than curcumin at the median effect dose. This is not unexpected when we note that, although both the phytochemicals act as antioxidants and inducers of cell death (35), they differ in the mechanisms involved as described below.
For example, capsaicin was found to inhibit growth and induce apoptosis of various tumour cells, including those of leukaemia HL-60 (12), gastric adenocarcinoma (13), breast MCF-7 (13) and glioblastoma U87MG, believed to be mediated via p-38 MAPK signalling and mitochondrial dysfunction, reactive oxygen species generation and caspase 3 activation (36). Treatment of cancer cells with capsaicin was also reported to inhibit the activation of NF-κB by blocking the degradation of IκBα and, thus, preventing the nuclear translocation of the p65 subunit (which is essential for NF-κB activation) (16). Capsaicin was also reported to block the activation of STAT3 (induced by interleukin-6 (IL-6)) (37). JAK1 and c-Src (implicated in STAT3 activation) had no effect on extracellular signal-regulated kinases (Erk1/2). It was also reported to down-regulate the expression of the STAT3-regulated gene products, such as cyclin D1, Bcl-2, Bcl-xL, survivin and vascular endothelial growth factor (37). Finally, whereas capsaicin induces the accumulation of cells in G1 phase, monofunctional platinums were found to inhibit proliferation of tumour cells (by arresting cell cycle progression) at the G2 phase, thus preventing their mitotic entry and cause apoptosis through a p53-dependent pathway (33, 38). Thus, it was not unexpected to find that combinations of LH5 with capsaicin were most synergistic. In this context, it is also important to note that the bulky quinolone ligand present in LH5 can undergo stacking interaction with base pairs of DNA and can form hydrogen bond with the DNA. The processes associated with the combination of LH5 and capsaicin are illustrated in the Figure 4 (13, 31, 38).
The second phytochemical, curcumin, is a polyphenol and a key component of turmeric that has anti-oxidant, anti-toxic, anti-inflammatory, cancer chemopreventive and chemotherapeutic properties (3, 17). It has been suggested that both platinum resistance and enhancement of platinum action due to its combination with phytochemicals may be related to the multidrug resistance of the NF-κB gene. Whereas resistance to platinum drugs may be associated with aberrant activation of NF-κB, the synergistic combination of LH5 and curcumin is thought to have dampened its expression. In addition to NF-kB, other mediators are also believed to be involved in platinum based chemotherapy (39). For example, curcumin has been found to sensitize ovarian cancer cells (CAOV3 and SKOV3) to cisplatin-induced apoptosis (20, 21) by inhibiting the FA/BRCA pathway and the production of IL-6 (3). Like capsaicin, curcumin may also be a promising candidate for combination with monofunctional platinum.
Cellular accumulation of platinum and platinum-DNA binding. Tables III and IV give the total intracellular platinum levels and level of platinum DNA binding from cisplatin and LH5 in the A2780 and A2780cisR cells after their interaction with solutions of compounds (50 μM in the case of cisplatin and 10 μM in the case of LH5) for 24 h at 37°C.
The designed monofunctional platinum compound, LH5, was found to have higher platinum accumulation in both A2780 and A2780cisR cell lines than cisplatin.
As noted earlier, whereas cisplatin is expected to cross the cell membrane by both passive diffusion and carrier-mediated transport (40), LH5 (being ionic in nature) can cross the cell membrane only by carrier-mediated transport so that their total flux of transport into the cell can be lower (33). However, LH5 is found to result in higher Pt accumulation than cisplatin in both cell lines. It is possible that the presence of bulky hydrophobic quinoline ligand in LH5 facilitates its uptake through the cytoplasmic membrane and it is also possible that LH5 is better able to bind more efficiently to the carrier molecules.
It can be seen that the higher platinum accumulation from LH5 than cisplatin in the resistant A2780cisR cell line is in line with greater activity of the compound against the cell line. In contrast, higher platinum accumulation from LH5 in the parent A2780 cell line did not translate into higher activity (as cisplatin is more active than LH5 against the cell line). The results are clearly related to the nature of LH5 and cisplatin, as well as characteristics of the cell lines. As noted earlier, positively charged monofunctional platinum complex can cross the cell membrane more efficiently than cisplatin even though it must be carried across by carrier molecules, such as organic cationic transporters (41).
Electrophoretograms applying to the interaction of a: salmon sperm DNA (ssDNA); b: pBR322 plasmid DNA without BamH1 digestion; c: pBR322 plasmid DNA followed by BamH1 digestion, with increasing concentrations of cisplatin and LH5. Lanes: SS, Untreated salmon sperm DNA; P, untreated pBR322 plasmid DNA; B, untreated and digested pBR322 DNA by BamH1 digestion; 1, 1.87 μM; 2, 3.75 μM; 3, 7.5 μM; 4, 15 μM; 5, 30 μM; 6, 60 μM.
Platinum accumulations in A2780 and A2780cisR cells after their interaction with solutions of CS and LH5 (50 μM in the case of CS and 10 μM in the case of LH5) for 24 h at 37°C.
Since cell kill due to platinum drugs is considered to be a consequence of their binding with DNA (42), platinum–DNA binding level is deemed to be more significant than cellular accumulation of platinum, although uncertainty remains whether for monofunctional platinums this is true or not. Indeed platinum drugs may undergo deactivation due to binding with cellular platinophiles, such as glutathione and metallothionein before binding with DNA (43, 44).
Table IV shows the levels of Pt–DNA binding (expressed as nmol Pt per mg DNA) and platinum-DNA binding ratio (expressed as Pt(nmol)/DNA (nmol)) applying to designed monofunctional compound, LH5, and cisplatin.
Pt–DNA binding level, as well as platinum-DNA binding ratio from cisplatin, are found to be higher than LH5 in both the parent and resistant cell lines, indicating that binding with DNA may not be the only or the critical determinant of activity of the compounds.
Pt–DNA binding in A2780 and A2780cisR cells after their interaction with solutions of CS and LH5 (50 μM in the case of CS and 10 μM in the case of LH5) for 24 h at 37°C.
Notwithstanding the complication resulting from difference in incubation time for cytotoxicity measurement and determination of Pt–DNA binding level (whereas cytotoxicity measurement was carried out after 72 h of incubation, Pt–DNA binding level was determined after 24 h of incubation), discordance between order of cytotoxicity and that of Pt–DNA binding level can be seen to indicate that binding with DNA may not be the critical determinant or the only critical determinant of activity of the designed monofunctional platinums. As discussed earlier, proteomic studies to determine key proteins associated with platinum resistance in ovarian cancer may provide a clearer picture.
Interaction with DNA. Figure 5 gives the electrophoretograms applying to the interaction of (a) ssDNA, (b) pBR322 plasmid DNA without BamH1 digestion and (c) pBR322 plasmid DNA followed by BamH1 digestion, with increasing concentrations of cisplatin and LH5 ranging from 1.87 to 60 μM (lanes 1 to 6) for 5 h at 37°C. Lane P applied to the untreated pBR322 plasmid DNA that served as control.
In the case of ssDNA, only a single band was observed in both untreated and treated ssDNA. There was a decrease in intensity of the band at higher concentrations of LH5, indicating that the compound was able to induce DNA damage at higher concentrations. In contrast, no noticeable change in band intensity in the case of cisplatin indicates that cisplatin was unable to cause any damage to ssDNA in the studied range of concentration. As noted earlier, LH5 can only form monofunctional adducts with DNA as against predominantly intrastrand 1,2–Pt(GG) and 1,2–Pt(AG) adducts formed by cisplatin (although cisplatin is also known to form monofunctional adducts and less frequent but more cytotoxic interstrand adducts with DNA) (45) that cause significant change in DNA conformation at and close to the binding site (46). However, this was not reflected as any observable change in mobility of the linear ssDNA band. Damage to ssDNA at higher concentrations of LH5 is attributable to non-covalent interactions involving quinoline ligand, such as stacking interaction and hydrogen bonding.
When pBR322 plasmid DNA was interacted with LH5, no noticeable change in either mobility or intensity of DNA bands was observed with the increase in concentration of the compound, thus indicating that LH5 failed to cause any observable change in DNA conformation or to induce DNA damage.
In contrast, when pBR322 plasmid DNA was interacted with cisplatin, two bands corresponding to forms I and II were observed at all concentrations of the compound ranging from 0 to 60 μM. However, there was a decrease in separation between the bands, an increase in mobility and a decrease in intensity of the bands with the increase in concentration of cisplatin. The results are indicative of conformational change in the DNA and the occurrence of DNA damage. As noted earlier, cisplatin forms mainly intrastrand bifunctional 1,2–Pt(GG) and 1,2–Pt(AG) adducts with duplex DNA that cause significant bending of the DNA strand at and close to the binding site, thus bringing about changes in conformation, especially to the supercoiled form I DNA (45). Such conformational changes are not caused by monofunctional LH5 cation.
Conclusion
Synthesized monofunctional platinum, coded as LH5, is found to be significantly more active than cisplatin against the resistant A2780cisR and A2780ZDO473R cell lines, indicating that the compound has been able to overcome mechanisms of platinum resistance. The observed synergism from combinations of LH5 with capsaicin and curcumin can be seen to indicate enhanced ability of the combinations to induce cell death and provides support to the idea that the synergistic combinations of targeted therapy and tumour-active phytochemicals may provide effective and affordable means of overcoming platinum resistance in ovarian cancer.
- Received March 28, 2016.
- Revision received May 1, 2016.
- Accepted May 10, 2016.
- Copyright© 2016 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
