Apoptosis is a highly conserved process of cell death, which is strictly regulated and makes the cell undergo its own death. 1,2 In multicellular organisms, it is an important mechanism that destroys unnecessary or superfluous cells during growth or neutralizes potentially harmful cells with DNA disruption, thus preventing carcinogenesis. 3,4 Different extrinsic and intrinsic triggers such as DNA-damage, mitochondrial dysfunction, production of reactive oxygen species and activation of caspases can activate apoptosis. 5 Reactive oxygen species (ROS) are usually small, short-lived and highly reactive molecules. 6 They may be free radicals derived from oxygen such as superoxide anion and radical hydroxyl or non-radical molecules like hydrogen peroxide (H2O2) which cause apoptosis. 7,8 Lactate dehydrogenase (LDH) belongs to family of NAD + dependent enzymes and is responsible for the interconversion of pyruvate and lactate under normal conditions. 9 Extracellular release of cytosolic LDH indicates increased permeability of cell and is a marker of cell death. 10 Mitochondria are energy producing organelles of the cell which have various other functions too like biogenesis of intermediates for various biochemical processes, 11 contribution in biochemical synthesis of hormones, 12 thermogenesis 13 and induction of apoptosis through intrinsic pathway after release of mitochondrial cytochrome c. 14,15 They also have transiently stored calcium to facilitate in maintaining cellular homeostasis. 16 Mitochondria have their own DNA (mtDNA) which is maternally inherited. 17 Triorganotin(IV) compounds are well known in targeting mitochondria by three mechanisms through ion (Cl - /OH -) exchange across membranes, hydrolysis of ATP and swelling of mitochondria. 18 Mitochondria become abnormally swollen with disorganized cristae after treatment with an agent targeting mitochondria in cells. Mitochondrial dysfunction leads to reduction in mitochondrial membrane potential and increased permeability leads to release of cytochrome c and hence apoptosis. 14,15,19 Caspases are a family of protease enzymes playing essential role in apoptosis and inflammation. 20 They all work together and maintain homeostasis in body by regulating apoptosis. 21 Caspase-9 is an important apoptosis marker and present on CASP9 gene. Caspase-9 is initiator in mitochondrial apoptotic pathway, which is activated when cytochrome c is released from mitochondria. Afterwards caspase-9 performs its initiation action by activating "effectors-caspases" such as caspase-3 and caspase-7 which eventually cause apoptosis. 22 Caspase 3 is mainly known for initiating the apoptosis in the cells of multicellular organisms. 23 Tin metal complexes have been shown to induce caspase dependent apoptosis in cancer cells. 24 Metal complexes have largely been employed in medicinal chemistry as bioactive molecules which are effective carbonic anhydrase inhibitors, 25 antimicrobial, 26 and anticancer 27 agents.
Various metals can be used to achieve libraries of these metallodrugs with structural diversities.
Cisplatin, a platinum-based lead molecule in chemotherapy, inaugurated the era of metal-based therapeutics. In recent times, other metal complexes containing Mn, Zn, Ni, Cd, Co, Cu, and Zn have successfully been investigated to obtain therapeutics. [28][29][30] The organic motifs of these metallodrugs are also vital to define and among a large choice of ligands sulfonamide, imidazole, 2'2-bipyridine and 1'10-phenanthroline are typically used to shelter the complexation of metals. In 1929, the antitumor activity of organolead and organotin(IV) complexes was checked in mouse which produced results with slight contradiction. 31 Later in 1972, it was confirmed that triphenytin acetate retard the growth of tumor cells in mice, not the chlorides. 31 Subsequently, enormous number of organotin compounds were synthesized and tested in vitro and in vivo for various cancerous cells, like murine leukemia (P388 and L1210). 32 In the last two decades a series of organotin(IV) carboxylates have been synthesized with phenyl, butyl, methyl derivatives and were screened for biological studies especially antitumor studies. 33 In the present study, triorganotin (IV) complexes with aromatic carboxylate ligand were designed and synthesized. Further, their anticancer potential was explored against cancer cell lines (HeLa and MCF-7), while safety profile was investigated by testing the same compounds for normal cells (BHK-21). The pro-apoptotic mechanism of the active compound was explored through fluorescence microscopy, analysis of cell cycle, activation of caspase-9 and -3, production of reactive oxygen species, release of lactate dehydrogenase, DNA binding studies and by measuring mitochondrial membrane potential.
Reagents i.e. 3,4-dimethoxybenzoic acid, sodium bicarbonate, trimethyltin(IV) chloride, tributyltin(IV) chloride and triphenyltin(IV) chloride were procured from Aldrich (USA) and were used without additional purification. All the solvents used were purchased from E. Merck (Germany) and dried according to the standard procedure given in literature. 34 The characterization techniques used for the synthesized compounds are CHN analysis, FT-IR, NMR ( 1 H, 13 C and 119 Sn) and single crystal X-ray diffraction. Melting points of the synthesized compounds were determined using Gallenkamp (UK) electrothermal melting point apparatus.
Bruker Tensor II was used to record the FT-IR spectra, 1 H, 13 C NMR spectra were recorded on Bruker-300 MHz FT-NMR and for 119 Sn NMR, Bruker DRX-500 MHz Spectrometer was used. DMSO-d6, CDCl3 and Acetontirle-d3 were used as solvents for NMR measurements. 35 The resonant frequency of the nucleus compared to magnetic field denoted as chemical shift is shown in ppm while the J (coupling constants) values are given in Hertz. The spin multiplicities of signals for 1 H NMR are given as (s = singlet, d = doublet, t = triplet, m = multiplet). Crystal analysis was conducted using Bruker Smart Apex II single X-ray diffractometer (MoKα source).
For the synthesis of the sodium salt of the ligand, equimolar aqueous solution of sodium hydrogen carbonate (NaHCO3) was added dropwise to the methanolic suspension of HL and stirred for two hours at room temperature to get a clear solution. The solution was then rotary evaporated under reduced pressure. The white product obtained was vacuum dried. For the synthesis of organotin(IV) carboxylate complexes, equimolar suspended solution of sodium salt of ligand was taken in two neck round bottom flask and solution of triorganotin(IV) chloride (R3SnCl) was added drop wise, then refluxed for 7 h in dry toluene. The solution was cooled at room temperature, filtered and rotavap at low pressure to get the desired product. The product was then recrystallized in hexane : chloroform (3:1). Fig. 1 shows the schematic method of the desired syntheses of the sodium salts of the ligand and organotin (IV) complexes while Fig. 2 shows the numbering scheme of the compounds.
The computational calculations were accomplished by DFT approach to optimize the structures in the gas phase [36]. 36 The Gaussian 09 package was used to visualize all the theoretical results. 37 The chemical shift (δ) values were calculated from the optimized geometries by employing Gauge Independent Atomic Orbital (GIAO) with B3LYP functional and LANL2DZ basis set. 38 Natural bond orbital (NBO) analysis was performed using similar functional and basis set to check the charge density on individual atom.
Redox activity of the synthesized compounds was done on Corrtest CS Electrochemical Workstation, China. Equimolar concentrations (1 mM each) for all compounds were taken in 80% dimethyl sulfoxide solution having 0.1 M TBAP (tetrabutylammonium perchlorate) as supporting electrolyte using three electrode electrochemical cell. The glassy carbon (GC) was used as working electrode with surface area 3mm (0.03 cm 2 ), Ag/AgCl as reference electrode and Platinum wire as counter electrode. Prior to experimental work, working electrode (GC) was washed several times with aq.Al2O3 on a nylon pad with double distilled water.
DNA interaction with foremost effective compound C2 were performed following a published strategy. 39,40 DNA stock solution was prepared by dissolving 5 mg herring sperm-DNA (hs-DNA) in 10 ml of distilled water which was then estimated for purity by taking ratio of absorption at 260 and 280 nm. Ratio was found in between 1.6 and 1.9 which indicated that DNA is pretty pure to carry out assay. Different concentrations of compound from 0 to 400 µM were first allowed to react with a fixed DNA concentration in order to obtain a reasonable concentration of compound that can be tested with varying DNA concentrations to evaluate DNA binding. End concentration of test compound C2 in each well of 96-well UV microplate was kept 200 µM with varying (from 0 µM to 392 µM) end concentration of hs-DNA. After an incubation of 30 min in dark at room temperature, the UV absorption spectra were recorded using a FLUOstar Omega microplate reader (BMG Labtech, Germany).
Cytotoxic capability of compounds was determined in MCF-7 and HeLa cells by MTT cell viability assay as described earlier [41, 42], while BHK-21 cells were used to study effect of these compounds on non-cancerous cells. Briefly, 1 × 10 4 cells/well in a sterile 96-well culture microtiter plate were seeded and incubated in a 5% CO2 incubator at 37 ˚C for 24 hours.
Compounds were initially tested at 100 µM end concentration and then their dilutions were made to calculate IC50 values while 1% DMSO was used as control. After 24 hours, cells were treated with 0.2 mg/mL MTT reagent for 4 hours. 10% acidified SDS (sodium dodecyl sulfate) solubilizing solution in propanol (1:1) was added to solubilize formazan crystals. After 30 minutes on gyratory shaker, plate was placed in microplate reader to measure optical density. IC50 values were calculated from three independent experiments as previously reported. 43
Release of an endogenous enzyme lactate dehydrogenase into extracellular fluid i.e culture medium upon treatment with the compound C2 at concentrations of 3.25 µM and 6.5 µM was assessed by Pierce LDH Cytotoxicity Assay Kit (Thermo Scientific). Briefly, 1 x 10 4 cells were seeded in a clear, flat bottom, sterile, polystyrene 96-well culture plate, kept overnight in incubator and treated with the compound. After 24 hours, plate was centrifuged at 1500 rpm for 3 minutes and supernatant was transferred to a new sterile 96-well plate. After addition of substrate mix, plate was placed in dark for 30 minutes at room temperature and absorbance was measured at 490 nm and 680 nm. Cytotoxicity was measured as described earlier. 44
Morphological analysis of cells was performed using fluorescence imaging technique. Briefly, 2 × 10 5 cells/well were incubated overnight and treated with compound (C2) at 3.25 µM and 6.5 µM concentrations. After 24 hours, cells were washed with sterile PBS (phosphate buffered saline) and fixed with 4% formalin and 0.1% Triton X-100. Then 10 µL of propidium iodide or 4′,6-diamidino-2-phenylindole (DAPI) dye was added and kept in dark for 10 minutes.
Images were captured as previously reported. 40
The ROS production in HeLa cells after treatment with compound (C2) was visualized by fluorescence microscope. Cells (2 × 10 5 ) were treated with 3.25 µM and 6.5 µM end concentration of the compound. After 24 hours, cells were fixed, treated with 2',7'dichlorodihydrofluorescein diacetate (H2DCF-DA) dye, placed in dark for 10 minutes and observed under fluorescence microscope as previously reported. 45
Effect of potent compound on distribution of cell cycle was analyzed by flow cytometer.
Briefly, HeLa cells (2 × 10 5 cells/mL) were seeded, given an overnight incubation and treated for 24 hours by compound (C2) at 3.25 µM and 6.5 µM concentrations. Pellet was obtained after harvesting the cells and washed three times with PBS. Cells were allowed to fix in 70% ethanol and kept at -20 o C for 24 hours. Cell pellet obtained after centrifugation was resuspended in propidium iodide solution, kept in dark for 30 minutes and analysed as earlier mentioned. 46
Activation of apoptosis inducer by active compound (C2) was assessed using fluorometric assay kit by Abcam. 10 × 10 5 cells were incubated for overnight and then treated with compound (C2) at concentrations of 3.25 µM and 6.5 µM. After an 18 hours treatment, cells were harvested and lysed. After determination of protein content, lysate was treated with substrates and fluorescence was measured as previously reported. 43
Apoptotic agents which target mitochondria cause a decrease in mitochondrial membrane potential. 1 x 10 5 cells were seeded and treated with compound at its 3.25 µM and 6.5 µM concentrations. After 24 hours, cells were harvested, treated with 0.25 µM JC-1 (5′, 5′, 6′, 6′tetrachloro-1′, 1′, 3′, 3′-iodide) dye, shifted to a black-well 96-wells plate, kept in dark for 20 minutes and emission of fluorescence was measured at 590 nm for j-aggregates and 520 nm for j-monomers. Results were calculated as previously reported. 47
The synthesized carboxylate compounds (C1-C3) were characterized through CHN analysis, FT-IR, multinuclear ( 1 H, 13 C, 119 Sn) NMR and single X-ray crystallographic techniques. The melting point indicated the purity of the compounds. The structures of the compounds (C1-C3) along with numbering scheme are shown in Fig. 2. FT-IR spectra of the synthesized compounds were recorded in the range of 4000-400 cm -1 . In the IR spectra of compounds C1-C3, the disappearance of OH group in the range of 3400-3300 cm -1 confirm the formation of complexes. The sharp band detected at 1727 cm -1 (C1), 1740 cm -1 (C2) and 1736 cm -1 (C3) is assigned to carbonyl group (C=O). For the binding mode of (COO) to metal was calculated from the difference of asymmetric and symmetric stretching frequencies of carboxylate moiety.
The magnitude of ∆ν falls in the range 230-250 cm -1 showing monodentate nature of the binding. 48,49 The absorption bands in the range 424-450 cm -1 for compounds 1-3 attribute to stretching frequency of (Sn-O) linkage further confirmed the formation of the compounds.
The NMR ( 1 H, 13
The crystal data and refinement parameters of C1 is shown in Table S1, while selected bond lengths and bond angles are mentioned in Table 1. C1 has monoclinic crystal system having space group P21/c and adopted distorted trigonal-bipyramidal geometry defined by C3SnO2, where, C3 are the carbon atoms of tributyl groups of organotin moiety and O2 are the oxygen atoms of carboxylate ligand. It is worth mentioning that complex 1 exists as two independent molecules 1a (having Sn1 atoms) and 1b (having Sn2 atoms) in one unit cell (Fig. 3). However, in the trigonal-bipyramidal geometry, distortion around metal center is quantified by τ value viz 0.777 (1a) and 0.743 (1b) [τ = (β-α)/60 and β > α, are the largest angles around coordination center and τ =1.0, for an ideal trigonal-bipyramidal and τ = 0.0 for an ideal square pyramidal geometry] which shows greater degree of distortion in 1b than that of 1a. 52,53 This can be due Another significant feature of 1a and 1b is their superamolecular packing supported by various secondary bond forces. In both independent co-existing molecules, the carboxylate moiety is also acting as bridging ligand between two Sn(IV) centers and generating zig-zag pattern of a polymeric 1D chain. While substituted phenyl groups of adjacent carboxylate ligands are located at alternative positions of 1D chain and are perpendicular to each other. However, in Moreover, polymeric chains of 1a are also connected with 1b by non-covalent interactions like C---H{C30---H8B= 2.889 Å, C21---H30A= 2.867 Å}, π---H{C18---H43A= 2.855 Å}, H---H{H22B---H29B= 2.378 Å} and O---H{O7---H21C= 2.668 Å, O3---H43A= 2.582 Å} to further cement the superamolecular architecture (Fig. 6).
In order to evaluate the reactivity and Lewis acid character of Sn center, Natural bond orbital (NBO) analysis were perform through density functional theory (DFT) showing electronic charge density on each atom as shown in Fig. 7. There is a positive charge on Sn(IV) atom (C1, C2 and C3) and negative charge on oxygen atoms attached to Sn(IV) atom attributed to the shifting of electron density from metal to ligand. In compound C2, Sn(IV) has greater positive charge than in C1 which means less amount of electronic density has been shifted to the coordinated oxygen. As a result, weaker Sn-O bond compared to C1 i.e. due to butyl bulky nature in C2 and phenyl in C3. Moreover, in compound C2 fewer negative charges on directly attached carbon atoms (C19, C32, C45) of butyl groups than those of methyl groups (C19, C23, C27) in compound C1 demonstrates greater electron donating power of former than the later ones as shown in Table 2. C3). Due to the presence of DNA, certain changes occurred in the electrochemical response and respective current-potential parameters indicating the compound-DNA presence in the system. These changes, along with a shift toward more positive potential, denote the electrostatic mode of interaction between the compounds and DNA. 54,55 Furthermore, for confirmation of DNA interaction with compounds, cyclic voltammogram was recorded at different scan rates, to find the diffusion coefficient (Do) whose significance is the formation of large of ions in solution. Anodic peak current vs square root of scan rate (v 1/2 ) plot was drawn for all the compounds before and after the addition of ss-DNA. The decrease in slope after the addition of ss-DNA indicates the drug-DNA adduct formation with slower rate in diffusion process and can be seen in diffusion coefficient Table 3.
In support of above argument, binding constant (K) also known as stability constant was calculated by using the following equation. 56 1
where A is an empirical constant. Expressively, the large K value compared to the values reported for different compounds 54,57 suggests the potential ability of these compounds to interact with DNA as shown in Table 3. The number of DNA base pairs {binding sites (s)} involve in the interaction with these compounds were also calculated using the following equation:
where Cf and Cb are the concentrations of free and DNA-compound bound species respectively.
Also, Cb/Cf can be represented by the equation Cb/Cf = (Io-I)/I as reported in the literature. 58 The values of binding site size show that all the three compounds integrate more than one base pair of the DNA resulting in strong interactions for these compounds.
Table 3 The drug-DNA interaction electrochemical parameters of compounds on glassy carbon vs. Ag/AgCl in a DMSO solution at a 50 mVs -1 scan rate at 25 o C.
In view of measuring electron transfer, differential pulse voltammetry was performed for all the compounds having peak currents in the order of C1 > C2 > C3 as shown in Fig. 10. The electron transfer (ET) process in all the compounds, is in close agreement with its simple and planar structures. However, there is lethargic ET process in case C3 due to bulky nature of compound that cased hindrance to the ET process. From the full width at half maximum (FWHM), the experimental W1/2 values (≈155 mV) of all the compounds propose the one electron transfer process. These values are slightly larger than the theoretical values i.e. 90 mV for one electron process, which may be due to n uncompensated resistance. 59
Apoptosis also known as programmed cell death, is activated when intracellular signals are received by cell regarding any unusual condition like DNA damage, protein damage, deprivation of growth factor and cytokine. 61 Keeping in mind the anticancer activity of Schiff base derivatives, previously reported by our group, 43 cell viability studies were carried out with newly synthesized complexes. MTT is well established in-vitro assay for determination of cytotoxicity of compounds using cell lines where viable cells are estimated on basis of conversion of water-soluble dye into water insoluble formazan crystals due to metabolic activity of reductases in living cells. Formed formazan crystals are dissolved by solubilizing solution and optical density is measured. Greater the color intensity greater will be the viability of cells and vice versa. [62][63][64] This assay is usually utilized for initial screening and determination of IC50 values of active compounds. MTT assay was employed on two cancer cell lines (human cervical cancer (HeLa) and human breast carcinoma (MCF-7) and one non-cancer BHK-21 cells using cisplatin as the positive control and results are given in Table 4. Excellent growth inhibition of cells was obtained for C2 complex carrying n-butyl ligand as linker with an IC50 values having lower micro-molar range for both MCF-7 (0.19 ± 0.05 µM) and HeLa cell lines For this reason, compound C2 was selected for further studies.
Three tin complexes (C1-C3) were examined for their anticancer activity. All the compounds showed good cytotoxic activity for MCF-7 and HeLa cells with IC50 values reported in Table 4. For the examination of safety profile of the tested compounds, cytotoxicity was also evaluated against BHK-21 cells and IC50 values were provided in Table 4. Cytotoxic activity was found higher in cancer cells as compared to normal cells.
Selected compound C2 was further studied and evaluated for its ability to interfere in cell cycle regulation in cancer cells. As shown in Fig. 13, compound has shown different DNA content at different stages of cell cycle progression as compared to untreated cells. Compound caused an arrest in G2/M phase that's why lower DNA content in G0/G1 phase has been found, showing that compound C2 have caused cell cycle arrest at G2/M phase which is in compliance with a previous study. 65 Negative Control C2 (IC50) C2 (2×IC50) Fig. 13 Cell cycle analysis by flow cytometer. Distribution of cells in G0/G1, S and G2/M phases in untreated cells, compound at IC50 and 2x IC50 concentaraion.
Organotin and Schiff base complexes are important in cancer therapy due to their apoptosis inducing characteristics. 60 Propidium iodide PI is a fluorescent molecule that attach with the DNA. PI binds with DNA in the dead cells because the plasma membrane of dead cells becomes permeable for foreign molecules. 66 Similarly, DAPI staining is used to check out apoptosis in cells. 67,68 PI and DAPI staining were performed and images were captured (Fig. 14) which were compared and analyzed. DNA fragmentation, nuclear condensation and cell shrinkage were detected while untreated cells showed no change in morphology. Cytotoxic potential of compound C2 was confirmed by morphological observations using propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) as staining dyes.
Organotin compounds usually possess the ability to produce reactive oxygen species in cancer cells. 60 Exogenous and endogenous stimuli can generate ROS in cells. Endogenous ROS development, particularly superoxide anion, are generated mainly during the activity of the mitochondrial electron transport chain. 69 When antioxidant detoxification processes do not maintain low accepted ROS rates, excess cellular ROS levels may be deleterious and trigger oxidative stress. 6 Large amount of cellular ROS levels can damage proteins, nucleic acids, lipids, membranes, and organelles such as mitochondria and directly associated with both carcinogenic and anticarcinogenic mechanisms. 70,71 The ability of compound C2 to induce reactive oxygen species was observed in HeLa cells using 2`,7`-dichlorofluorescin diacetate (H2DCF-DA) which produced a fluorescent probe dichlorofluorescein (DCF) when came in contact to reactive oxygen species. Fluorescence produced was detected by green filter of fluorescence microscope at wavelength of 530 nm after exciting at 488 nm. Compound C2 has ability to produce ROS in cancer cells to show its anticancer property as shown in Fig. 14. and hence ability to induce oxidative stress related apoptosis.
Cysteine proteases are enzymes present in the cell and have numerous functions. These caspases are mainly involved in apoptosis. After re ceiving apoptotic signals, some precursors of caspases are generated to activate initiator caspases which in turn activate executioner caspases. 72 This process increases intracellular Ca +2 ion concentration and successively regulate the activation of DNA binding transcription factor, and as a result ROS production.
Afterwards, the release of cyt-c from mitochondrial membrane causes activation of initiator
Mitochondria are cell power house which mainly produce energy for cells. Some of the antiproliferative agents are able to directly target mitochondria and cause a decrease in mitochondrial membrane potential which leads to mitochondrial dysfunction and consequently apoptosis through intrinsic pathway. 19 In cancer cells, mutations in mitochondrial genes cause alterations in bioenergetics and biosynthesis that's why cancer cells adopt glycolysis to meet their needs and high lactate production is seen in these cells. 19,74 Cancer cells need higher energy which is produced by mitochondria. Dysfunctioning of mitochondria in cancer cells is gaining popularity and molecules targeting mitochondria are now being discovered. 75 In this experiment, cells treated with 1% DMSO were used as negative control and cell treated with carbonyl cyanide m-chlorophenylhydrazone (CCCP) at its 50 µM final concentration as positive control. As in Fig. 16, compound (C2) has shown a dose dependent decrease in red/green ratio in both HeLa and MCF-7 cells indicating that C2 also targets mitochondria to achieve apoptosis in cancer cells which resembles to a previous study.
Three new triorganotin(IV) compounds with aromatic carboxylate ligands were synthesized which were evaluated for their antiproliferative properties using two cancerous (Hela and MCF-7) and a non-cancerous (BHK-21) cell line. All compounds showed antitumoral property while most potent compound C2 was evaluated for its pro-apoptotic mechanism. Compound C2 has induced apoptosis at micromolar concentration in cancer cells which was visualized by fluorescence microscopy using PI and DAPI staining, noticed through high ROS production using H2DCF-DA analyzed by cell cycle arrest through flow cytometry, detected by caspase-