Roy, Sudeshna
Citation
Roy, S. (2008, November 25). Synergy of intercalation and coordination binding to design novel DNA-targeting antineoplastic metallodrugs. Retrieved from
https://hdl.handle.net/1887/13281
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†
| 5
Comparative studies of DNA cleavage, conformational change and antiproliferative activity of platinum(II) and copper(II) compounds of Hpyramol †
Chapter
The single-step reaction of the redox-active ligand, Hpyramol [4- methyl-2-N-(2-pyridylmethyl)aminophenol] with K2PtCl4 yields the platinum compound of formula, [Pt(pyrimol)Cl]. This compound is formed by intramolecular oxidative dehydrogenation of the starting ligand yielding Hpyrimol. The compound has been structurally characterised using X-ray diffraction. This compound cleaves ΦX174 supercoiled phase DNA stoichiometrically and attacks at multiple sites without any specificity. The uptake studies of platinum and copper pyrimol compounds in ovarian carcinoma cell lines have been performed along with time- dependent cytotoxicity assays in vitro. The variation in incubation times has significant effect on the cytotoxicity values. Cyclic voltammetry studies have been carried out to support the proposed ligand-based radical mechanism. Conformational changes of right-handed B-DNA induced by these compounds have been recorded by circular dichroism spectroscopy.
5.1. Introduction
Medicinal bioinorganic chemistry research has taken several steps forward from the accidental discovery of cisplatin.1 A plethora of transition metal compounds, which have been synthesised and showed promising activity against cancer cell lines, keeps this research area fertile and challenging.2 Some of these second- or third-generation drugs, such as oxaliplatin, carboplatin, picoplatin (JM473), nedaplatin (JM118), or Pt(IV)-based satraplatin, tetraplatin and ormaplatin. overcome the limitation by a reduced toxicity, better bio-availability,3 or displaying improved antitumour activity.4
Genomic DNA is generally accepted as the main cellular target of platinum drugs and for cisplatin it has been demonstrated that the major antitumour activity originates from intrastrand crosslinks.1 The detailed investigation of cisplatin activation, biotransformation, accumulation, coordinative binding, kinking and unwinding of the DNA explains much of the antitumour activity of this drug.5 Therefore, DNA-targeting drugs6 remain in the limelight of anticancer research and loading the cancer cells selectively over healthy cells is getting more attention. The mono-substituted adduct of cisplatin with guanine has been shown to be more genotoxic than the bis-substituted adducts.7 This observation leads to search for monofunctional active platinum compounds. There are also some reports of ‘rule-breaker’ platinum compounds, which do not follow the basic SAR of active platinum compound (vide Chapter 1, section 1.3), are highly active against some tumour cells. The approach directs to design of the compounds with a different adduct to DNA. This can be coordinative (interstrand crosslinking), non- coordinative (intercalation, electrostatic, groove-binding or hydrogen-bonding) interactions8 or monofunctional binding.9 The monofunctional platinum compounds have gained attention recently by the bioinorganic research groups.
The unconventional approach for antitumour research can be successful by use of the well-known ‘chemical nucleases’. This class of compounds cleaves DNA in several pathways namely (a) nucleobase oxidation, (b) phosphate group hydrolysis and/or (c) deoxyribose unit oxidation. These molecular scissors can cut DNA either by single-strand or double-strand breaks. The cellular response against this damage is the activation of multi-faceted repair mechanism as base excision repair, double-strand break repair, crosslink repair and nucleotide excision repair. Most efficient chemical nucleases assimilate trace elements (copper, iron or zinc) while in action, or contain redox active
transition-metal compounds. The classical examples of this type of artificial nucleases are bleomycin10-12 and [Cu(phen)2]n+.13-16
Recently a quite unique ligand (Hpyramol L8)17 has been introduced to investigate DNA cleavage and antitumour activity upon metallation. This ligand is redox active, but devoid of any cleaving or antineoplastic property on its own. Upon coordination, L8, 4-methyl-2-N-(2-pyridylmethyl)aminophenol undergoes oxidative dehydrogenation to the imine (“pyrimol”) state. The intermediate form (oxidised) of the ligand, i.e., before metallation, is Hpyrimol, (L9), 4-methyl-2-N-(2- pyridylmethylene)aminophenol. The chemical structure of the ligand and changes after coordination to a metal are shown in Fig. 5.1. The free ligand, L8 when coordinated to redox-inactive zinc, cleaves double stranded DNA oxidatively, most probably by non- diffusible ligand-based radical mechanism.18 The copper analogue shows high to moderate cytotoxicity against ovarian carcinoma and murine leukaemia cell lines and catalytically cleaves DNA without any added reductant.19
N CH3
OH N H
Hpyramol L8
(a)
N CH3
N OH
Hpyrimol L9
(b)
N CH3
N
O M
M-pyrimol (c)
Figure 5.1. Structure of the starting ligand L8 (a) in free from, (b) oxidised form L9 and (c) coordinated to metal, M.
This fascinating result inspired to the coupling of L8 with platinum, to yield a potent monofunctional drug targeting the cellular DNA. So, the platinum-pyrimol complex has now been synthesised and characterised by X-ray diffraction studies, and detailed investigations are reported in this chapter to correlate the cleaving property with its in vitro cytotoxicity. As DNA being the supposed target, the interaction with calf thymus DNA was studied by circular dichroism. Both the copper and the platinum compounds were tested for the growth-inhibition of cultured tumour cells and the total drug uptake was also measured. High-resolution gel experiments were done to test the sequence specificity of the compounds by 32P radio-labelled oligonucleotide. As the
ligand itself shows redox activity, the formed platinum compound has also been tested for its electrochemical properties by cyclic voltammetry.
5.2. Experimental 5.2.1. Materials
The deuterated solvents used for NMR experiments were purchased from Sigma- Aldrich B.V. (The Netherlands). The supercoiled plasmid DNA used for cleavage studies has been purchased from Invitrogen Life Technology as ΦX174 phage DNA (0.25 µg/µL). K2PtCl4 was generously provided as a loan by Johnson-Matthey (Reading, U.K.).
The ligand L8 was synthesised in a single-step reaction as reported in literature.17 The solvents used for synthesis were purchased from Biosolve, The Netherlands (AR grade) and used without further purifications.
5.2.2. Synthesis of [Pt(pyrimol)Cl] (C7)
An ethanolic solution of L8 (51.616 mg, 0.2409 mmol) was added dropwise to the aqueous solution of K2PtCl4 (100 mg, 0.2409 mmol) in dark and stirred at 40 °C for 24 h.
The reaction mixture was filtered while warm and the filtrate was evaporated to 5 mL under reduced pressure. A deep green precipitate was obtained by cooling this concentrated filtrate at 4 °C overnight. The precipitate was washed with cold ethanol (3×2 mL) and finally with diethyl ether (3×5 mL) and dried under suction in dark. Yield: 65.98 mg (62%). Elemental analysis for C13H11N2OClPt expt.(calcd.): C: 35.25 (35.34), H: 2.42 (2.50); N: 6.10 (6.34).
This solid product was characterised as [Pt(pyrimol)Cl], C7, by 1H, 13C, 195Pt, 2D- COSY and 1H-13C HETCOR spectroscopy, but the spectra were not shown. 195Pt NMR (dmso-d6 and dmf) showed a single peak at -2326 ppm and -2322 ppm, respectively, which corresponds to a PtN2OCl environment. Single crystals suitable for X-ray crystallography were obtained from a concentrated dmso solution from an NMR tube, which was kept in the dark.
5.2.3. Synthesis of [Cu(pyrimol)Cl] (C8)
This compound was reproduced following the reported method.19 CuCl2·2H2O (60 mg, 0.35 mmol) was dissolved in 10 mL of CH3OH/H2O (7:3 v/v). The resulting solution was added dropwise to a solution of L8 (106 mg, 0.50 mmol) in 15 mL of methanol.
After 12 h, brown crystals, suitable for X-ray crystallography, were collected by filtration.
The yield was 54 mg (47%). Elemental analysis for C13H11ClCuN2O·H2O expt.(calcd.):
C: 47.58 (47.57); H: 4.35 (3.99); N: 8.60 (8.53). The crystal structure was reported before.19
5.2.4. X-ray crystal structure determination of C7
Single crystals suitable for diffraction were obtained directly from an NMR tube containing a concentrated dmso-d6 solution of C7 at ambient temperature in the dark. X- ray intensities were measured on a Nonius Kappa CCD diffractometer with rotating anode (graphite monochromator, λ = 0.71073 Å) at a temperature of 150 K. Data were integrated with EvalCCD20 using an accurate description of the experimental setup for the prediction of the reflection contours. Absorption correction and scaling was performed with the program SADABS21. The structure was solved applying automated Patterson methods (DIRDIF-9922). Refinement was performed with SHELXL-9723 against F2 of all reflections. Non-hydrogen atoms were refined with anisotropic displacement parameters.
All hydrogen atoms were located in difference-Fourier maps refined with a riding model.
Geometry calculations and checking for higher symmetry was performed with the PLATONprogram.24 Further details of the molecular structure of the C7 are given in the Tables 5.1 and 5.2, respectively.
5.2.5. NMR experiments
The one-dimensional (1H and 13C) and two-dimensional COSY and HETCOR spectra were recorded on a 600 MHz Bruker DPX600 spectrometer at ambient temperature (24 °C) in dmso-d6. The 195Pt spectra were recorded on a 300 MHz Bruker spectrometer with a 5 mm multi-nucleus probe at 24 °C in dmso-d6 and non-deuterated dmf solvents. For 195Pt NMR, Na2PtCl6 was used as external reference with δ = 0 ppm.
5.2.6. Biological studies
(a) Cells and culture conditions
The experimental details have been described in detail in Chapter 2 (vide section 2.2.5) and are not repeated in this chapter.
(b) Cytotoxicity assays (i) MTT assay
The experimental details have been provided in Chapter 2 (vide section 2.2.6) and are not repeated in this chapter. The stock solution of C7 was made in dmf to check any
Table 5.1. Crystal data and structure refinement for the compound C7.
Formula C13H11ClN2OPt
Formula weight 441.76
Crystal colour dark red
Crystal size [mm3] 0.21 × 0.03 × 0.03
Temp [K] 150(2)
λ [Å] 0.71073
Crystal system triclinic Space group P 1 (no. 2)
a [Å] 7.1447(3)
b [Å] 8.5058(3)
c [Å] 10.2432(3)
α [°] 83.230(2)
β [°] 76.509(1)
γ [°] 87.059(2)
V [Å3] 600.91(4)
Z 2
Dx [g/cm3] 2.442
μ [mm-1] 11.884
Abs. corr. multi-scan
Abs. corr. range 0.36 – 0.70 (sin θ/λ)max [Å-1] 0.65 Reflections (collected / unique) 9030 / 2743
Parameters / restraints 164 / 0 R1/wR2 [I>2σ(I)] 0.0246 / 0.0492 R1/wR2 [all refl.] 0.0355 / 0.0518
S 1.050
ρmin/max [e/Å3] -1.05 / 0.94
Table 5.2. Selected bond lengths and bond angles for compound C7.
Bond lengths (Å) Bond angles (°)
Pt(1) – N(1) 2.009 (3) Cl(1) – Pt(1) – O(1) 94.88 (9) Pt(1) – N(2) 1.946 (4) Cl(1) – Pt(1) – N(1) 99.15 (12) Pt(1) – Cl(1) 2.301 (12) Cl(1) – Pt(1) – N(2) 177.03 (11)
Pt(1) – O(1) 2.016 (3) N(1) – Pt(1) – N(2) 81.73 (15) O(1) – Pt(1) – N(2) 84.22 (14)
solvent effect. For comparison in activity, L8 and cisplatin were tested in the same batch and the stocks in both cases were made in dmf. The copper compound, C8 is highly soluble in water, so the aqueous solution was used for in vitro cytotoxicity. In all cell plates, dmf was used as the blank solvent. In addition, for comparison purpose with the reported literature value (IC50 of cisplatin), the activity of cisplatin was also estimated using typical aqueous stock solution.
(ii) SRB assay
The cytotoxicity of compounds C7, C8 and L8 was evaluated using the microculture sulforhodamine B (SRB) test25 against seven human tumour cell lines at Pharmachemie (Haarlem, The Netherlands) commercially.
(c) Cellular uptake experiments
Cells of the A2780 and A2780R cell lines were plated in sterile 6-well plates in 5 mL of Dulbecco’s Modified Eagle’s Medium (DMEM) at a density of 1×105 cells per well and incubated for 48 h. Cisplatin, C7 and C8 were added to the plates to reach a final concentration of 200 nM and incubated for 0.5 h, 2 h and 24 h. After incubation the cells were washed twice with 5 mL PBS per well and triton (lysis buffer) was added 350 µL per well, to lyse the cells. After transferring to eppendorf vials 10% SDS (100 µL per well) and Proteinase K (20 µg/mL and 1 mL per well) were added. The viscous pellets were subsequently incubated at 55 °C for 30 min and 1.5 mL 20% HNO3 was added.
After shaking for 45 min the samples were measured by Varian Vista-MPX charge- coupled simultaneous ICP-OES (Inductively Coupled Plasma-Optical Emission Spectroscopy) for estimation of the % Pt and the % Cu taken up by the cancer cells intracellularly. To compare the accumulation of platinum species inside the cisplatin
sensitive, A2780 cells, time-dependent uptake studies were also performed for C7 up to 24 h with each 2 h intervals.
(d) DNA cleavage studies
A typical reaction mixture, containing supercoiled plasmid DNA and C7 in a 10 mM phosphate buffer at pH 7.2, was incubated at 37 °C for 2 h without additives and external reductant. After the incubation period, the reaction was quenched by keeping the samples at -20 °C followed by the addition of loading buffer (bromophenol blue, xylene cyanol, and 25% ficoll). This solution was then loaded on a 0.8% agarose gel containing ethidium bromide (2.54 μM final concentration in the gel as well as in the buffer). The gels were run at a constant voltage of 80 V for 60-90 minutes in TBE buffer containing ethidium bromide. After washing with distilled water, the gels were visualised under a UV transilluminator and the bands were documented and quantified using a BioRad Gel Doc 1000 apparatus interfaced with a computer.
(e) High resolution PAGE (with radio-labelled oligonucleotides)
For the ultra-high resolution analysis of the cleavage process, a double stranded oligonucleotide has been used. The synthetic procedure to prepare this type of oligonucleotides has a coupling efficiency of 97-99%. Therefore, in each step, a short side product is produced, whose intensity on the electrophoresis gel is associated with the coupling efficiency (99% efficiency implies that 1% side product is present, 97% gives 3% side product and so on). Consequently, the electrophoresis reveals a ladder-like pattern, with a weak band at each nucleotide (nt) position. This is ideally suited for the detailed analysis of the cleavage mechanism. A 54 nt oligonucleotide FNA1 (DNA sequence 5'-ATCCTGGTGGAGCTAAGCGGGATCGAACCGCTGACCTCTTGCTTG CATAGCAA-3') was radiolabelled with [γ-32P]ATP (MP Biomedicals) using T4 polynucleotide kinase (Fermentas) as indicated by the supplier, and purified over S200 spin columns (GE healthcare), effectively removing all products up to 15 nt. Duplex DNA was obtained by annealing the 10 μM [32P]-labelled FNA1 with an equimolar amount of oligonucleotide FNA2 (DNA sequence 5'-AGCGCTTGCTATGCAAGCAAG AGGTCAGCGGTTCGATCCCGCTTAGCTCCACCA-3') in 250 mM NaCl solution at 80 °C for 2 min, followed by slow cooling to ambient temperature. On either side, the probe has 5 nt of single-stranded region at the 5' end. For copper-induced cleavage experiments, the FNA1/2 duplex DNA was incubated at a concentration of 100 μM bp
with 20 μM C8 at 37 °C. At indicated time intervals (0-60 min), samples were taken and quenched with an equal volume of stop solution (90% v/v formamide and 50 mM EDTA) at 80 °C.19 DNA was incubated at a concentration of 100 μM bp with 50 μM and 200 μM of C7 at 37 °C at indicated time intervals (0-150 min). Samples were separated by high resolution denaturing PAGE (15%) and visualised by phosphor imaging (Bio-Rad).
(f) Circular dichroism
The experimental details have been provided in Chapter 2 (vide section 2.2.7) and are not repeated here. A typical sample containing DNA and metal compounds (C7 and C8) in phosphate buffer was incubated at 37 °C and the spectra were collected at the given time intervals. As reference samples cisplatin and L8 were also followed for similar time- and concentration-dependent experiments.
(g) Cyclic voltammetry
The electrochemical studies were performed for L8 and on the platinum compound C7, using CV. Two samples were dissolved in dmf and (Bu4N)PF6 was used as supporting electrolyte of 0.1 M concentration. A three-electrode system consisting of a platinum wire (working electrode), platinum plate (counter electrode) and Ag/AgCl (reference electrode) was used. During all measurements an inert gas blanket (Ar) was used at room temperature. The concentration of the freshly prepared sample solution was 1×10-3 M and the voltage scan rate was maintained at 200 mVs-1 for all measurements.
5.3. Results and Discussion 5.3.1. Syntheses
The chemical synthesis of C7 is straightforward and successfully completed in a single step. K2PtCl4 and L8 are reacted in equimolar ratio to yield the compound as shiny crystalline green powder. The spectroscopic and elemental analyses prove the formation of compound [Pt(pyrimol)Cl]. The assignment of 1H NMR in (CD3)2SO at 600 MHz δ:
2.10 (s, 3H, CH3), 6.50 (d, 1H, C–Harom), 6.88 (d, 1H, C–Harom), 7.20 (s, 1H, C-Harom), 7.63 (dd, 1H, 5-py-H), 7.65 (d, 1H, 3-py-H), 8.12 (t, 1H, 4-py-H), 8.55 (d, 1H, 6-py-H);
8.77 (s, 1H,CH). The 195Pt NMR shows a single peak (at -2326 ppm) which is in the region common for PtN3O and PtN3Cl environments. There is no evidence of any fold- back signal, so the single peak can be attributed to a PtN2OCl environment indicating the formation of compound C7. The ESI-MS of dmso solution shows the major peak at m/z =
484.32, which corresponds to the species, [Pt(pyrimol)(dmso)]+ after the solvolysis of labile chloride ligand by dmso.
5.3.2. Crystal structure
Dark red needles of C7 have been obtained from slow evaporation of a concentrated dmso solution in an NMR tube at ambient temperature, while kept in the dark for around four weeks. The crystal structure shows that the starting ligand L8 has been dehydrogenated to the Hpyrimol form, L9, and the metal is coordinated through the O,N,N-binding motif after loss of H+, which results in an almost square-planar geometry with cis angles between 81.74(15) and 99.14(11)° (Fig. 5.2). The molecular structure is quite similar to that of the reported Cu-analogue, (C8), [Cu(pyrimol)Cl] but the packing in the crystal is completely different due to the co-crystallised water molecules in C8.19 L8 undergoes similar metal-assisted oxidative dehydrogenation to form planar pyrimol via Hpyrimol as intermediate.
Figure 5.2. Displacement ellipsoid plot of C7 (50% probability level) and the used atomic numbering.
The planar molecules of C7 are stacked on top of each other in an anti-parallel fashion in the direction of the crystallographic a axis (Fig. 5.3) with intermolecular Pt···Pt distances of 5.0418(3) and 4.7540(3) Å. The corresponding interplanar distances are 3.43 and 3.31 Å, respectively. Besides this molecular stacking no other strong intermolecular
interactions could be detected, which is explained by the fact that no strong hydrogen bond donors are present. Consequently the long dimension of the needle shaped crystals corresponds to the crystallographic a axis. In the Cu compound there is also anti-parallel molecular stacking in the a direction with Cu···Cu distances of 5.1647(3) and 6.1325(3) Å and interplanar distances of both 3.34 Å, but here the stacks are linked with each other by intermolecular hydrogen bonds involving the co-crystallised water molecules. The crystallographic details and some selected bond parameters for C7 are presented in Tables 5.1 and 5.2, respectively.
Figure 5.3. Packing diagram of C7 in the crystal. Hydrogen atoms are omitted for clarity.
Viewed along the crystallographic c axis. Symmetry operations i: -x, 1-y, 1-z; ii: 1-x, 1-y, 1-z. The colour magenta is denoted for Pt, green for chloride, red for oxygen and blue for nitrogen atom.
5.3.3. Cytotoxicity assays (a) MTT assay
The in vitro cytotoxicity tests were performed against human ovarian carcinoma and mouse leukaemia cancer cell lines and the IC50 values (in µM) are summarised in Table 5.3.
As a thumb rule, RF (resistant factor) values between 0.5 and 2.0 indicate similar cytotoxic activities in both cisplatin sensitive and resistant cell lines, whereas compounds having RF values greater than 2 are partially cross-resistant with cisplatin. The IC50 value
Table 5.3. IC50 values (µM) of samples against human ovarian carcinoma and mouse leukaemia cancer cell lines (cisplatin sensitive and resistant) after 48 h and 120 h of incubation.
Cell lines Compounds
Time
(h) A2780 A2780R RFa L1210(0) L1210(2) RF
Cisplatin (water) 2.9 13.1 4.5 4.0 24.2 6.1
Cisplatin (dmf) 8.6 17.2 2.0 5.6 14.6 2.6
L8 (dmf) 15.0 13.8 0.9 >>50 >50 ---
C7 (dmf) 22.4 43.6 1.9 8.1 27.5 3.4
C8 (water)
48
3.4 8.3 2.4 3.6 10.3 2.9 Cisplatin (water) 1.1 21.9 19.9 3.1 39.7 12.8
C7 (dmf) 38.8 120.9 3.1 42.2 121.6 2.9
C8 (water)
120
63.9 158.8 2.5 62.8 153.8 2.4
aThe Resistance Factor (RF) is defined as the relative ratio of IC50 values in resistant and sensitive variety of similar cell lines e.g., A2780R/A2780 or L1210(2)/L1210(0), respectively.
IC50 value is defined as the amount of compound needed to inhibit 50% of cell growth.
is inversely related to the activity, which means that low IC50 values for a compound imply high activity in the cancer cell lines. In the ovarian carcinoma cell line (both cisplatin sensitive and resistant) the IC50 values for C7 are relatively high (22.4 and 43.6 µM, respectively), which indicates its low activity when compared to leukaemia cell line (8.1 and 27.5 µM, respectively). The IC50 values in cisplatin resistant cell lines are higher than in cisplatin sensitive cell lines, which is common and as expected for the two types of cell lines. In leukaemia cell lines C7 overcomes cisplatin resistance to some extent only, as evident from the RF value of 3.4. Therefore, the compound C7 is active towards leukaemia cell line and moderately active towards ovarian carcinoma cell lines.
The Cu(II) analogue, C8 [Cu(pyrimol)Cl] shows a very high antiproliferative property, as in all cases the IC50 values are similar or even lower than the IC50 values for cisplatin itself; moreover this compound almost completely overcomes cross-resistance to
cisplatin in both types of cancer cell lines, as evident from the RF values of 2.4 and 2.9, respectively. L8 is active in ovarian carcinoma cell lines, but exhibits almost no activity in leukaemia cell lines (as IC50 values are higher than 50 µM). Therefore, it is evident that on coordination and oxidation with platinum or copper the cytotoxicity profile has changed. It is evident from the table that though dmf is known as non-coordinating (innocent) solvent, and dmf in medium is used as blank, the IC50 values calculated for cisplatin (in dmf) are quite different from the IC50 values for cisplatin in aqueous solution.
Therefore, dmf as a solvent for in vitro cytotoxicity assay has more detrimental effect than water or dmso. In the literature some reports are known of the detrimental effect of dmf on cells, but the detailed comparison has not been reported yet.
The effect of different times of incubation on the activity towards cell lines has also been investigated. The activity of C7 remains almost unchanged, but there is significant change in the cytotoxicity profile of C8. When the incubation time is shorter, C8 exhibits activity as high as cisplatin, though longer incubation times repress the activity. The results are shown in Table 5.3 (48 h and 120 h incubation times, respectively). Upon increasing the incubation time, the loss of activity in resistant cell lines, A2780R and L1210(2), is more significant in the cisplatin sensitive cell lines, A2780 and L1210(0). The prolonged incubation time reduces the activity of C8 up to 18- 20-folds in cell lines tested. The difference in the absolute values of IC50 can be explained by either an increased efflux, or an increased cellular assimilation of biologically relevant metal, copper, in C8. In the case of cisplatin and C7, the results can be explained also by an enhanced efflux and several side reactions.
(b) SRB assay
Seven human tumour cell lines with different origin were used to test the activity of compounds C7 and C8, in comparison with cisplatin. For cisplatin and C8 stock solutions were made in MilliQ water and for C7 in dmso (following the NCI protocol dmso was used and not dmf). The results are summarised in Table 5.4. The activity profile clearly shows dependence on the time of incubation. For all cell lines, the activity of cisplatin is higher with prolonged incubation.
The activity exhibited by cisplatin after 120 h of incubation is 6-12-fold higher than after 48 h of incubation. This can be explained by higher accumulation over a longer time period and a reduced efflux. In case of C7 there is no smooth trend of change with incubation time. The activity in the A498 cell line tremendously increases after 120 h by
6-fold (from 51.5 µM to 8.8 µM). In H226 and MCF7 the activity of C7 is enhanced also by 1-fold. For other cell lines no prominent effect of incubation time is seen. The significant change in activity is noticed in case of C8 as well. In the A498, H226 and MCF7 cell lines the activity of C8 is increased by 7, 2.1 and 2-folds, respectively. In the other three cell lines, IGROV, M19 and WIDR, the activity of C8 increases, but to a smaller extent. Completely contrasting behaviour is observed in the EVSA-T cell line, where the activity of C8 is repressed by almost 8-fold upon longer incubation time.
Therefore, the changes in activity profile are strongly dependent on the time of incubation and eventually on uptake and efflux, on deactivation and interaction with cellular nucleophiles.
Table 5.4. IC50 values (µM) of samples against seven human tumour cell lines after 48 h and 120 h of incubation.
Cell-lines Samples
Time
(h) A498 EVSA-T H226 IGROV M19 MCF7 WIDR
Cisplatin 8.2 17.2 10.4 3.0 9.7 16.3 21.1
C7 51.5 26.2 69.5 24.8 59.3 31.5 50.9
C8
48
33.4 1.3 29.4 18.6 2.2 21.4 36.2
Cisplatin 6.2 2.5 2.1 0.74 2.3 2.7 1.8
C7 8.8 38.4 56.6 45.2 54.4 27.4 62.7
C8
120
4.3 10.6 11.5 17.2 1.5 10.6 29.9
5.3.4. Uptake of metal compounds in cancer cells
In order to explain significant difference in the cytotoxicities of C7 and C8, uptake studies have been performed in A2780 and A2780R cell lines.26 The active concentration of cisplatin (reference compound), C7 and C8 added to the cells was 200 nM and the incubation times were 0.5 h, 2 h and 24 h for each of the compounds. The amount of platinum or copper which has been taken up by both cisplatin sensitive and resistant ovarian carcinoma cells is graphically presented in Figs. 5.4(a) and 5.4(b), respectively. In the A2780 cell line, both cisplatin and C7, exhibit time-dependent
increased accumulation whereas C8 is accumulated at its highest concentration after 2 h incubation. The amount of platinum accumulated inside the A2780 cells in the case of C7 is very low when compared to cisplatin can reason out the lower cytotoxicity of C7.
Excretion of some copper is seen after 24 h incubation, though the high influx of the copper compound inside the cells from the moment of incubation explains the high activity.
0 20 40 60 80 100
120 cisplatin
Pt-pyrimol Cu-pyrimol
Total Pt/Cu (nm)
0.5 h 2 h 24 h
Incubation time (a)
0 20 40 60 80 100
cisplatin Pt-pyrimol Cu-pyrimol
Total Pt/Cu (nm)
Incubation time
0.5 h 2 h 24 h
(b)
Figure 5.4. Amount of platinum and copper accumulated (in nM) inside (a) A2780 and (b) A2780R cell lines with time.
In resistant cell lines the same trend has been followed by platinum compounds, but maximum accumulation of copper is achieved only after 24 h. As the resistant cell lines are obtained after multiple exposures of cell lines to cisplatin over a certain time period, the uptake of platinum compounds is expected to be lower than its sensitive
counterpart. As copper is more abundant in physiological environment, the cellular uptake for the copper compound would be expected to be faster than the toxic metal compounds, cisplatin and C7. The uptake of copper is expected to depend on its speciation but the uptake of C8 cannot be compared with that of generic copper. The amounts of copper and platinum detected in the cells are in comparable range (33 and 42 nM for C7 and C8, respectively), whereas for cisplatin the amount of platinum is somewhat lower (24 nM). In the resistant cell lines after 24 h accumulation of copper is two times lower than platinum, but the activity of C8 is five fold higher than C7.
In order to quantitate the platinum accumulated inside the A2780 cells with certain time interval (2 h) a separate experiment has been performed for C7 using a concentration of 50 µM. The maximum accumulation was observed after 18 h of incubation, but the time-scale of cancer cell-cycle is around 24 h. The changes in the amount of platinum accumulated inside the cells do not follow a specific trend, which may be interesting to study in more detail. In Fig. 5.5, the concentration of platinum inside the cell versus the incubation time has been plotted to find a possible correlation of the uptake-accumulation and cytotoxicity in vitro for C7. After the initial higher accumulation of platinum in cells, the efflux process most probably is getting activated.
So, the amount of total platinum inside the cells gradually decreases. After 10 h of incubation the uptake and accumulation is starting to reach the highest concentration at 18 h. After that time the platinum compound appears to be excreted from the cells and there is only some residual platinum after 24 h. Therefore, after a full cell cycle a significant amount of platinum is present, however, it does not correlate with the in vitro cytotoxicity data.27 This behaviour can be explained by removal or deactivation by platinophiles inside the cells.28-31
5.3.5. DNA cleavage studies (a) Agarose gel electrophoresis
The DNA-cleavage property of C7 has been investigated in the presence of ΦX174 supercoiled phage DNA by agarose gel mobility shift. The compound (20-200 µM) has been incubated with DNA (20 µM in base pairs) at 37 °C for 2 h in phosphate buffer (pH = 7.2) with or without additives (H2O2, dmso, TEMPO, NaN3, etc.) and reductant (ascorbic acid or mercaptopropionic acid). With increasing concentration of C7, form II is increased, which is a clear evidence of non-catalytic stoichiometric cleavage.
The added reductant (lane 7, ascorbic acid of 100 µM) or the radical initiator (lane 9,
4 8 12 16 20 24 0
50 100 150 200 250 300
Total platinum (ppb)
Time of incubation (h)
Figure 5.5. Time–dependent concentration of C7 in ovarian carcinoma A2780 cell line.
Figure 5.6. Agarose gel electrophoresis of C7 in presence of ΦX174 supercoiled phage DNA at 37 °C in a phosphate buffer at pH 7.2. Lane 1, DNA blank (20 µM in base pairs), Lane 2, 20 µM + 10 µM C7, Lane 3, 20 µM + 20 µM C7, Lane 4, 20 µM + 50 µM C7, Lane 5, 20 µM + 100 µM C7, Lane 6, 20 µM + 200 µM C7, Lane 7, 20 µM + 10 µM C7+ 20 µM Ascorbic acid, Lane 8, 20 µM + 20 µM Ascorbic acid, Lane 9, 20 µM + 10 µM C7+20 µM TEMPO, Lane 10, 20 µM + 20 µM TEMPO.
TEMPO of 20 µM) does not affect the cleavage activity of C7. With 200 µM of C7, the DNA is completely digested and a faint smearing is observed. The effect of C7 on plasmid DNA is shown in Fig. 5.6. C7 thus behaves differently than C8 (cleaves DNA oxidatively and catalytically).19
(b) Radiolabelled oligonucleotide cleavage
The most important observation of the high resolution gel experiment is the
‘smear’, suggesting that degradation occurs in a less specific manner than expected on the basis of a single cleavage event. For a more detailed analysis the same experiment on a 5' radiolabelled double-stranded oligonucleotide was performed, which was known to
produce a ladder of bands inherent to the fact that each coupling step occurs with a 98- 99% efficiency. The samples were then run on a 15% denaturing polyacrylamide gel, to allow identification of DNA fragments at single nucleotide resolution. Excitingly, while the control lane clearly showed individual bands, on progressive degradation these simply became fainter (more prominent for 200 µM from 90 min incubation), eventually producing a continuous 'smear' (Fig. 5.7, for C7). This smearing is most likely the result of the cleavage process and is not due to excessive background, as highlighted by the intensity of the smear below the lowest nucleotide band [15 nucleotide (nt), vide bottom part of Fig. 5.7]. The platinum compound C7 therefore attacks the DNA at multiple sites without any specificity. The copper compound C8 in fact degrades the DNA in a very non-specific manner, perhaps attacking the base and/or the sugar in multiple positions (Fig. 5.7, for C8).
Figure 5.7. High-resolution analysis of the cleavage process using 15% denaturing PAA gel. A 54 bp DNA fragment was incubated with C7 for 0-150 minutes (left) and with C8 for 0-60 minutes (right). DNA size marker in nucleotides (nt) is shown on the left. Time (min) is indicated above the lanes. The probe was uniquely radiolabelled at the 5' end of the top strand.
The relative efficiency of cleavage activity towards plasmid DNA by C7 and C8 was investigated by transformation to E. coli competent cells. DNA was linearised with a restriction enzyme (BamHI) and then incubated with C7 and C8. DNA was purified to
Compound C7 Compound C8
remove the excess complexes, dissolved in water, and concentration is determined exactly by the nano-drop spectrophotometer. Exactly 5 ng (set to precision by mechanical methods) of the digestion mixture was then religated by T4 DNA ligase and transformed to fresh competent cells of E. coli, to determine the number of colony forming units (CFU). Comparing uncleaved pUC DNA with the control experiment (digested, not treated and then religated) shows that the ligation efficiency is close to 95%. Each experiment was done in triplicate and appeared reproducible. From this graphical-plot (Fig. 5.8), it can be clearly seen that C7 cuts 10 times more efficiently than C8. This experiment is somewhat indecisive for the exact mode of digestion, but clearly is very informative about the comparison of two metal analogues, C7 (Pt) and C8 (Cu).
Figure 5.8. Relative efficiency of cleavage activity of complex C7 and C8 on pUC DNA as (a) CFU per ng DNA (b) and relative CFU.
5.3.6. Circular dichroism
The conformational changes induced by C7, C8, and L8 on CT DNA were investigated by CD spectroscopy in the presence of calf-thymus DNA. Typical samples contains DNA (100 µM per nucleotide phosphate) in the presence or absence of a metal compound, or a free ligand (1 mM) in 10 mM phosphate buffer (pH = 7.2); the ionic strength is kept constant at 50 mM.
Therefore, the R value is kept constant at 0.1 (10 metals per base) C7, C8, cisplatin, and L8. The samples were incubated at physiological temperature of 37 °C and spectra were recorded at selected time intervals (10 min. interval up to 100 min. and then 1 h interval up to 12 h). As a reference compound, cisplatin was used under identical experimental conditions. The changes in the native right-handed B-form DNA are shown in Fig. 5.9.
The right-handed B-DNA exhibits dramatic changes upon addition of C7 and C8 [Figs. 5.9(a)-(d)]. The change starts immediately after addition of the compound to the DNA solution and changes were recorded up to 12 h of incubation. C7 induces a
Figure 5.9. Time-dependent conformational changes of right-handed DNA upon addition of C7, C8, L8, and cisplatin at pH=7.2 at 37 °C with R=0.1.
decrease in intensity for both the positive (272 nm) and negative band (248 nm). There is also an evolution of two new ellipticities at 230 and 310 nm, respectively. The decrease in positive band intensity indicates destabilisation of base stacking and a decrease in the -ve band intensity points to loss in right-handed helicity, respectively.8 For C8, similar behaviour is observed. The change starts almost immediately and continues up to 12 h of incubation. The two new peaks also start appearing at 230 and 310 nm, respectively. The conformational change induced by this compound indicates the transition of B-form to an intermediate form to Z-form.32
The decrease in the positive band can be easily correlated to the cleavage activity of the compounds. Therefore the degradation of native DNA is indicated clearly. In a recent report the reduction in the intensity of the positive band is reported to be due to interstrand crosslinks to DNA (similar as transplatin) formed by trans-platinum compounds.33 Assumably, the single chloride might be solvolysed forming a positively charged species which then can eventually assist the electrostatic attraction of the metal compound with the DNA strand, followed by intercalation of the planar species between the base pairs. Therefore the intercalation and coordination is probable for both C7 and C8. The decrease in the negative band intensity can be well explained by unwinding of the DNA.33
The CD spectral changes of DNA induced by L8 are shown in Figs. 5.9(e) and 5.9(f). The concentration of DNA used is 50 µM and the ligand concentration is 500 µM with the same R value 0.1 as in the other samples. A small time-dependent change is observed in the hyperchromicity of the positive maxima (278 nm) with a blue shift of 2 nm. The reduction in intensity of the negative band with an 1 nm red shift is well marked.
The appearance of the new ellipticity at 230 nm is consistent for both C7 and C8 interactions. In spite of the changes in intensity the base-stacking and right handed helicity are retained overall. This behaviour can be explained by interaction of the ligand with DNA (intercalation) and the mode of interaction is different. The starting ligand L8 does not exhibit any cleavage property and the CD spectra clearly support that fact.
The reference compound cisplatin can be considered as a classical example of a metallodrug which binds to DNA by coordination.34 The CD spectra in presence of cisplatin [Figs. 5.9(g) and 5.9(h)] remain mostly unperturbed. Initially, the positive band intensity decreases slightly and there is a 2 nm red shift after 12 h of incubation. The base stacking remains stabilised overall though the helicity of the right-handed B-DNA is
disturbed. The reduced intensity in the negative minima (246 nm) can be easily explained by the unwinding of the helix, resulting from intrastrand and interstrand crosslinkings.
5.3.7. Cyclic voltammetry
The starting ligand Hpyramol exhibits unique redox chemistry when coordinated with a divalent transition metal.18 The two metal compounds reported in this chapter C7 and C8 have two redox active metal centres, platinum and copper, respectively. The starting ligand L8 and the platinum compound C7 have been investigated by electrochemical studies. It has been proven that M-pyrimol compound (M= copper or zinc) exhibits biological activity due to short-lived highly reactive diffusible phenoxyl radical.35-37 The redox property of the platinum compound and the free ligand has been recorded by cyclic voltammetry in dmf solution under an argon blanket at room temperature. The relevant data are shown in Table 5.5 and detailed electrochemical data for compound C8 has already been reported in a recent article.
Table 5.5. Electrochemical data for platinum compound C7 and the ligand L8.
Samples Ea (V) Ec (V)
C7, Pt-pyrimol 0.14, 1.17, 1.34 -0.17, -0.41, -0.73, -0.86, -1.54 L8, Hpyramol 0.35, 0.64, 1.21 -0.26, -0.36, -0.74, -1.45
The voltammograms of C7 and L8 have been recorded for 1 mM solutions against Ag/Ag+ electrode at room temperature. C7 shows a single-step oxidation and on reverse scan no reversible cathodic peak is observed. Unlike the copper analogue C8 the only irreversible oxidation peak can be assigned to the Hpyramol to pyrimol conversion [Fig.
5.10(a)]. The oxidation peak related to phenoxyl radical has not been observed in this compound. The existence of any radical in compound C7 by EPR spectroscopy (powder and solution state at room and low temperature) has not been observed; therefore, it can be assumed that diffusion, or decomposition of the radical in dmf solution takes place. On the other hand, L8 shows two one-step oxidation peaks at 0.35 and 0.64 V [Fig. 5.10(b)]
corresponding to ligand dehydrogenation and phenoxyl radical generation, respectively.
Therefore the diffusion of the radical is not that rapid as in the metal compounds.
Figure 5.10. Cyclic voltammogram of (a) compound C7 and (b) the free ligand L8 in dmf at room temperature showing redox process.
5.4. Conclusions
The monofunctional platinum compound of formula [Pt(pyrimol)Cl] (C7) has been synthesised and characterised. The crystal structure shows an almost planar structure with a tripodal (N,N,O) binding motif of the Hpyrimol ligand. The compound is formed after intramolecular ligand dehydrogenation followed by phenolic hydrogen-abstraction.
This neutral Pt(II) compound along with the copper analogue C8 has been studied for in vitro cytotoxicity. The cancer cell-growth inhibition ability appears to be strongly time- dependent. Both these compounds cleave DNA oxidatively by multiple attacks, presumably by a ligand-based phenoxyl radical active species. However, the definite site of metallation on DNA strand remains unclear. The interaction of these compounds with calf thymus DNA has been studied by circular dichroism and denaturation of DNA along with unwinding of the helix is observed. In the literature it is known that the monofunctional cationic complexes are not active against cancer cell [only exception is cis-diamminepyridinechloridoplatinum(II)]. The compound C7 exhibits quite high activity in ovarian cancer cell line whereas C8 exhibits higher efficiency after longer incubation time. Thus these two compounds indicate different cellular processing and it is worthwhile to investigate their mode of action in details.
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