Evaluation of (ɳ⁶-p-cymene) ruthenium diclofenac complex as anticancer chemotherapeutic agent: interaction with biomolecules, cytotoxicity assays.

Journal of Biomolecular Structure and Dynamics ISSN: 0739-1102 (Print) 1538-0254 (Online) Journal homepage: http://www.tandfonline.com/loi/tbsd20 6 Evaluation of (ɳ -p-cymene)Ruthenium diclofenac complex as Anticancer Chemotherapeutic Agent: Interaction with Biomolecules, Cytotoxicity Assays Rais Ahmad Khan, Hamad A. Al-Lohedan, Mohammad Abul Farah, Mohd. Sajid Ali, Ali Alsalme, Khalid Mashay Al-Anazi & Sartaj Tabassum To cite this article: Rais Ahmad Khan, Hamad A. Al-Lohedan, Mohammad Abul Farah, Mohd. 6 Sajid Ali, Ali Alsalme, Khalid Mashay Al-Anazi & Sartaj Tabassum (2018): Evaluation of (ɳ -pcymene)Ruthenium diclofenac complex as Anticancer Chemotherapeutic Agent: Interaction with Biomolecules, Cytotoxicity Assays, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2018.1528180 To link to this article: https://doi.org/10.1080/07391102.2018.1528180 Accepted author version posted online: 26 Sep 2018. Submit your article to this journal View Crossmark data Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=tbsd20 Evaluation of (ɳ6-p-cymene)Ruthenium diclofenac complex as Anticancer Chemotherapeutic Agent: Interaction with Biomolecules, Cytotoxicity Assays. Rais Ahmad Khan,α Hamad A. Al-Lohedan,φ Mohammad Abul Farah,§ Mohd. Sajid Ali,φ Ali Alsalme,α Khalid Mashay Al-Anazi,§ Sartaj Tabassum,φ,* φ Surfactant Research Chair, αDepartment of Chemistry, College of Sciences, King Saud ip § t University, P.O. Box 2455, Riyadh 11451, KSA. Department of Zoology, College of Sciences, King Saud University, Riyadh 11451, KSA. us cr *Corresponding Author: tsartaj62@yahoo.com, Mobile No. +966 530128012 Abstract an The designing of metal-based anticancer therapeutics agents can be optimized in a better M and rapid way if the ligands utilized have standalone properties. Therefore, even when the organometallic/coordination complex (i.e., metallodrug) gets dissociated in extreme d conditions, the ligand can endorse its biological properties. Herein, we have synthesized pt e and characterized ɳ6-p-cymene ruthenium diclofenac complex. Furthermore, the ruthenium complex interactions with HSA and ct-DNA have been studied using various ce spectroscopic studies viz., UV, fluorescence, and circular dichroism and exhibited Ac significant binding propensity. Furthermore, in-vitro cytotoxicity assays were carried out against human breast cancer “MCF-7” cell line. The ɳ6-p-cymene ruthenium diclofenac complex registered significant cytotoxicity with an IC50 value of ~25.0 µM which is comparable to the standard drugs. The ɳ6-p-cymene ruthenium diclofenac complex was able to decrease the MCF-7 cell proliferation and induced significant levels of apoptosis with relatively low toxicity. Keywords: Ruthenium diclofenac complex; Biomolecular interaction; cytotoxicity against MCF7; Apoptosis. 1. Introduction Metallo-chemotherapeutics is a well-established conventional selection for a wide range of diseases since ancient times, particularly for antibacterial, antifungal, analgesic, ip t antipyretic and anti-cancer. After the serendipitous discovery of cisplatin and its derivatives, (Rosenberg, B., et.al.1967, 1969, 1977, 1978) the field of metal-based cr chemotherapeutics got triggered, and since then the field is evolved stalwartly. However, us the acquired, intrinsic resistance and adverse side-effects have marred the remarkable an success of platinum-based drug candidates (Gasser & Metzler-Nolte, et al., 2012; Barry, & Sadler, 2013; Galanski et al., 2003; Hill & Speer, 1982; Khan et al., 2014; Kelland et M al., 2007). In lieu of the problem of undesirable side effects and to improve efficacy, ruthenium pt e d complexes emerged as the potential class of anticancer agents. Among the Ru(III) compounds, NAMI-A (a metastasis agent), (imidazolium trans-[tetrachlorido(dimethyl (III)] and KP1019, (indazolium trans- ce sulfoxide)(1H-imidazole)ruthenate [tetrachloridobis(1H-indazole)ruthenate(III)] and KP1339, (sodium (indazolium trans- Ac [tetrachloridobis(1H-indazole)ruthenate(III)] exhibited potential activity against human tumor models, are subjected to phase II clinical trials.(Hartinger, et al., 2012; 2008; 2006.) In recent decades, fluxional ruthenium arene complexes have emerged as versatile scaffolds for the design of the new metallodrugs (Mehta, et.al. 2017, Yadav, et al, 2018, Yaun, et. al., 2013). In this area of half sandwich-organoruthenium, groups of scientist viz, Sadler et al. (Zhang, & Sadler et.al. 2017; Bruijnincx, & Sadler et. al., 2009), Dyson et al., (Nazarov, et. al., 2014; Murray, et.al, 2016) and Keppler et al., (Hartinger, et.al, 2006; Renfrew, et al., 2009) with their co-workers have marked a tremendous success with piano-stool type geometry. RAPTA-C [(ɳ6-p-cymene)Ru(PTA)Cl2], PTA; 1,3,5triaza-7-phosphatricyclo[3.3.1.1]decane and RM175 [Ru(ɳ6-biphenyl)(ethylene- diammine)Cl]+ are the representatives of this class and hit the clinical trials with good ip t impact. Merely having slight modifications in the structures of the two they exhibited extremely different biological properties. These lead molecular frameworks has been cr extensively modified at both the ends viz., arenes part as well as the co-ligand/s to build us up a structural-activity relationship (SAR) and decorated with different functionalities to an get target specificity of the drug candidates. Interestingly, combination therapy has also emerged in scientist are working on the M already existing drugs mainly NSAIDs, and combining with the metal centers and studying the synergistic effect of metallodrugs, which have shown significant potential. pt e d In organo-ruthenium complexes, Turel et al.(Hudej et al., 2012; Turel et al.,2010; Kljun et al., 2011) synthesized Ru(arene) complexes of antibacterial compounds viz, nalidixic ce acid, ofloxacin. Likewise, Aman et al. 2014, has developed ruthenium arene compounds Ac with oxicam moieties namely, piroxicam and meloxicam (see fig 1). Ru Cl O Ru Cl O O O O N S R O O N O H N OS(CH3)2 cr Cl Ru Cl us O ip t N O R Turel et al. Aman et al. an Cl Present work Figure 1. Structures of the representatives of this class of ɳ6-p-cymene ruthenium M complexes pt e d Thus standing on this, we have studied the ɳ6-p-cymene ruthenium derived diclofenac complex (1) as a potential metallo-chemotherapeutic agent. The binding affinity of ce ruthenium complex with DNA and HSA has also been studied using various spectroscopic techniques and calculating various binding parameters. Furthermore, this Ac ruthenium complex was studied against MCF-7 human breast cancer cell lines via cytotoxicity assays, studying the apoptotic potential and morphological changes induced by the potential metallo-drug. 2. Experimental Section 2.1. Material and methods The sodium salt of ct-DNA (D1501, Type I, fibers) and HSA essentially fatty acid-free (≥98%) were purchased from Sigma, USA. Tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl) was of analytical grade and also obtained from Sigma. Fetal bovine serum (FBS), trypsin/EDTA and penicillin-streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Trypan blue, phosphate buffered saline (PBS), dimethyl sulfoxide (DMSO), ethidium bromide, acridine orange, and Dulbecco’s Modified Eagle’s medium (DMEM), were obtained from Sigma-Aldrich (St Louis, MO, USA). Cell Titer ip t 96® Non-radioactive cell proliferation assay kit was obtained from Promega (Madison, WI, USA). Annexin V-FITC apoptosis detection kit was purchased from BD Biosciences cr (San Diego, USA). Culture wares and other consumables used in this study were us procured from Nunc, Denmark an 2.2. Biological Studies 2.2.1. HSA Binding Studies M HSA binding studies were carried out using UV-visible, fluorescence quenching and circular dichroism methods and the detailed experimental procedures for these studies pt e d have been described elsewhere (Alsalme, et al., 2016; Tabassum et al. 2017; Mach et al., 1995; Yusuf, et. al., 2018; Afzal, et al., 2018). ce 2.2.2. DNA Binding Studies UV-visible spectroscopy, in the range of 225 to 350 nm, was used to understand the Ac binding of ɳ6-p-cymene ruthenium diclofenac complex (1) with DNA. Increasing concentration of ct-DNA was titrated against 30 x10-6 mol dm-3 of ɳ6-p-cymene ruthenium diclofenac complex. A fixed amount of ct-DNA (0-30 x10-6 mol dm-3) was taken in the blank and baseline was corrected before each measurement. The binding mode of the ɳ6-p-cymene ruthenium diclofenac complex was seen by competitive binding assay using EtBr and DAPI dyes. The circular dichroism studies of ct-DNA in the presence of complex were carried out similarly as described in case of HSA binding. (Mach, et al., 1995; Tabassum et al., 2012; Khan et al., 2014; Yusuf, et. al., 2018; Afzal, et al., 2018) 2.2.3. Cell cultures, and in-vitro Cytotoxicity, Apoptosis Experiments The MCF-7 human breast cancer cell culture, cytotoxicity and apoptosis experiments ip t were carried out by using standard protocols as adopted by us (Farah et al., 2016) with slight modifications, for more detail see SI. cr 3. Result and discussion us 3.1. Synthesis and Characterization an To synthesize the ruthenium ɳ6-arene NSAID compound (1), the sodium salt of diclofenac reacted with (ɳ6-p-cymene) ruthenium dichloride dimer was stirred in dry M methanol and few drops of dimethyl sulfoxide. The mixture was stirred at 80 °C for 10 h, and the reaction mixture kept on slow evaporation. The yellow color precipitation in yield pt e d of 68% was obtained. Unfortunately, after several attempts, we are unable to grow suitable single crystals for X-ray study. However, the ruthenium compounds synthesized ce were characterized by several spectroscopic and analytical methods. Stability in aqueous medium and dmso is an essential requirement for drug candidates. Since dmso/water was Ac used to make a stock solution for biological studies. The ruthenium compound was found quite stable in DMSO while in H2O; the chlorine atom gets hydrolyzed and forms an aqua complex of ruthenium over a period of 1h (Fig 2). ONa+ Cl O O H N (p-cymene)RuCl2 dimer (MeOH) Cl o few drops of DMSO, 10 h, 80 C H N Ru O OS(CH3)2 Cl Cl Cl ip t Figure 2. Schematic representation of the (ɳ6-p-cymene)ruthenium complex of diclofenac us cr (1). The FT-IR spectrum of the complex 1 showed νasym (C-O) and νsym (C-O) at 1567 and an 1459 cm-1, respectively which is characteristic for the bidentate coordination of carboxylate group of the ligand with a metal center. Since the difference between [νasym M (CO) -νsym (CO)] is 108 cm-1, which is less than 150 cm-1 and thus confirms the bidentate d binding mode of the carboxylate moiety. The FT-IR spectrum exhibited absorption band pt e at 1157, 976, 908 cm-1 exhibiting a significant shift in ν(S-O) corresponding to free DMSO (1005 cm-1). Thus ascertains coordination of DMSO through sulfur to the metal ce center. The band at 438 cm-1 is attributed to the ν(Ru-O) (see Fig. S1, in ESI). Ac The 1H NMR of the complex 1 in DMSO-d6 resulted in significant shifts when compared to the ligand. Complexation with ligand is confirmed by the disappearance of the carboxylic O-H signal from 10.27 ppm and appearance of the p-cymene protons additional signals. An upfield shift of the signals of 0.7-0.5 ppm of the ligand was observed in the ligand upon complexation. The band associated with the DMSO coordinated with the metal center and in solution due to the dissociation of the labile chlorido is also quite evident and which leads to the formation of a most stable product which is ascertained by the ESI-MS of the complex (see Fig. S2-S6 in SI). The UV-Vis spectrum of the ligand exhibited the band at around ~275 nm (n-π* transition) and upon complexation, the complex 1 displayed the band complex at ~260 nm with a significant shift of around 15 cm-1 and the new band appears at 323 nm ip t (LMCT band) which confirms the coordination. The emission spectrum of the ruthenium complex was also taken in solution and found exhibit signals at ~360 nm (see Fig S8, S9 cr in SI). us 3.2. Biological Studies an 3.2.1. HSA Binding Studies The difference UV-visible spectra of HSA with various concentrations of the complex 1 d M are shown in Fig 3. pt e 0.8 Ac ce Absorbance 1.2 Native HSA HSA + complex (1:1) HSA + complex (1:2) 0.4 0.0 240 260 280 300 Wavelength (nm) 320 Figure 3. Difference UV – visible spectra of HSA-complex binding. [HSA]=3 μM HSA gives a peak at 280 nm which can be used to see the changes during its binding with the ligand (Mach et al., 1995). The increased intensity of the protein-Ru-Diclofenac system in comparison to the pure HSA is an indication of the complex formation between HSA and Ru-diclofenac; moreover, a noticeable red shift of the absorption maximum is due to the involvement of electrostatic interaction. ip t The fluorescence intensity of HSA decreases gradually (Ali et al. 2017) with the increase in the concentration of Ru-Diclofenac complex (Fig. 4). Since there is a significant cr redshift of about 11 nm in the maximum fluorescence emission at a wavelength, it is M 2000 Fluorescence intensity (a) an 3000 2400 1600 d 1200 800 400 320 pt e Fluorescence intensity us proposed that the involvement of electrostatic interactions (Mandeville et al., 2009). 340 360 380 (b) 2500 2000 1500 1000 500 400 320 Wavelength (nm) 340 ce Wavelength (nm) 2000 (c) Fluorescence intensity Ac 1800 1600 1400 1200 1000 800 600 400 320 360 340 360 Wavelength (nm) 380 400 380 400 Figure 4. Effect of complex 1 on the fluorescence emission spectra (λex = 295 nm) of HSA at (a) 25 °C, (b) 35 °C and (c) 45 °C. [HSA] = 3 µM, [complex] = 0, 2.5, 5, 7.5, 10, µM. Various fluorescence and binding parameters have been calculated using Figs. 5 & 6 and ip t Eqs. (S1-S3) (For equations, see SI) and their values are summarized in Table 1 an 1.3 us 25 °C 35 °C 45 °C 1.4 1.2 M F0/F cr (Lakowicz, et al., 1999; Anand et al., 2010). 1.0 0.5 pt e 0.0 (A) d 1.1 1.0 6 ( ) ce 10 [Q] M -0.6 log (F0-F)/F Ac -0.4 1.5 2.0 -1 25 °C 35 °C 45 °C -0.8 -1.0 -1.2 -1.4 (B) -6.3 -6.2 -6.1 -6.0 -5.9 -5.8 -5.7 log [Q] Figure 5.(A) Stern-Volmer plots of HSA interaction with complex 1 at various temperatures. [HSA] = 3 µM, λex=295 nm. (B) Plot of log (F0 - F)/F as a function of log [complex]. [HSA] = 3 µM, λex=295 nm. Decreasing trend of the Stern-Volmer constant demonstrates the involvement of the static ip t type of quenching mechanism. Binding parameters were computed using Eq 3 and Fig. 5 cr (B) the data are displayed in Table 1. There was approximately 1:1 binding between HSA and complex 1. The thermodynamic parameters (change in enthalpy (∆H), entropy (∆S) us and free energy change (∆G)) were calculated adopting the widely used Van't Hoff an equation Usman et al. 2017, for which the plot is given in Fig. 6. M 20 d 10 pt e ln Kb 15 ce 5 Ac 0 0.0032 ( ) 0.0033 -1 1/T K Figure 6. Van't Hoff plot of HSA interaction with the complex 1. [HSA] = 3 µM, λex=295 nm. The values of thermodynamic parameters, calculated by Eqs. S4 and S5 (for equations, see SI) using Fig. 6 are given in Table 1, and it is clear from the negative values of ∆G that the binding of complex 1 with HSA is a spontaneous process. The interaction is also a highly exothermic process with a substantial ordering of the system as revealed by the Ac ce pt e d M an us cr ip t negative values of both ∆H and ∆S. Table 1 Stern-Volmer quenching constants, binding parameters and thermodynamic parameters for the interaction of HSA with complex 1 at various temperatures. λex= 295nm Binding parameters Thermodynamic Parameters Ksv (M-1) Kq (M-1s-1) R2 n K (M-1) R2 ∆G (KJ mol-1) ∆H (KJ mol-1) ∆S (J mol-1 K-1) 288 1.97 x 105 3.45 x 1013 0.9924 1.1 13.6 x 105 0.9996 -36.87 -187.27 -504.69 298 1.25 x 105 2.19 x 1013 0.9247 1.1 12.0 x 105 0.9529 -31.82 308 1.20 x 105 2.10 x 1013 0.9921 0.9 1.1 x 105 0.9921 -26.78 us cr ip t Stern-Volmer quenching constants an The far-UV CD is a useful method to determine the secondary structure of proteins (Ali M et al. 2016). The far-UV CD spectra of HSA in absence and presence of complex 1 are pt e 60 d given in Fig.7. Native HSA HSA+Complex (1:1) 0 ce CD (mdeg) 30 Ac T (K) -30 -60 -90 190 200 210 220 230 240 250 Wavelength (nm) Figure 7. Far-UV CD spectra of HSA in the presence of complex 1 at 25 °C and pH 7.4. [HSA] = 3 µM. It is exhibited from the figure that ellipticity of HSA is decreasing in the presence of the complex. Hence, it can be deduced that HSA partially unfolds in the presence of complex 1 (Feng et al., 1998). ip t 3.2.2. DNA Binding Studies UV-visible spectroscopy is an important technique to obtain the information about cr ligand-biomolecules interactions. Difference UV-visible spectra of ct-DNA and complex us 1 interaction are given in Fig.8 addition of ct-DNA to the 1 cause’s slight hypochromism Ac ce pt e d M an in the spectra. 0.5 0.50 0.48 0.46 0.3 0.44 0.42 0.2 0.40 260 270 280 0.1 300 350 400 450 Wavelength (nm) -20 an -40 -60 A0/(A-A0) 500 cr 250 us 0.0 ip t Absorbance 0.4 M -80 -100 d -120 pt e -140 10.0 15.0 20.0 -1 4 10 /[ct-DNA] (M ) ce 5.0 Figure 8. (a) UV–visible absorption spectra of ct-DNA (30 µM) in the presence of Ac increasing concentrations of the complex 1 (0-30.0 µM) at 25 °C. (b) Figure showing Benesi Hildebrand plot. The changes in the absorbance at 278 nm were utilized to calculate the apparent association constant, Kapp, of 1 and ct-DNA interaction. 1 A obs  A 0  1 Ac  A0  1 K app ( A c  A 0 )[ ct  DNA ] (4) A graph of 1/(Aobs – A0) versus 1/[ct-DNA] yielded a Benesi Hildebrand (B-H) plot with a slope equal to 1/Kapp (AC – A0) and an intercept equal to 1/(AC – A0). From the plot, the values of Kapp was found to be 1.85 x 104 M-1. From the collective information obtained from the DAPI and EtBr displacement assays, Fig 9. It is proposed that the complex 1 bound at the interfacial region of minor groove ip t and intercalation site. cr (a) us 3000 an 2000 1000 400 450 500 550 600 Wavelength (nm) 650 pt e Ac 100 (b) ce Fluorescence Intensity d 0 M Fluorescence intensity 4000 50 0 550 600 650 Wavelength (nm) 700 40 (c) 0 -20 ip 250 300 Wavelength (nm) 350 us 200 t DNA DNA+Complex -40 cr CD (mdeg) 20 Figure 9.(a) Fluorescence titration of CT-DNA and DAPI complex with the complex 1. (b) Fluorescence titration of EtBr and ct-DNA with complex 1. (c) CD spectra of DNA in an absence and presence of the complex 1. The concentration of [DNA] =30 µM and M [complex] =10 µM. d 3.2.3. Cytotoxicity Assay pt e The percent viability of cells exposed to different concentrations of complex 1 and ruthenium dimer (R) (5.0-50.0 µM). The IC50 values estimated at 24 h post-treatment in ce MCF-7 for complex 1 is about 25.0 µM and for R is ~400 µM. A significant (p < 0.05) decrease in the cell viability was observed which was also concentration dependent. At Ac the highest concentration of 50.0 µM cell proliferation was inhibited by 77% and 59% with complex 1 and R (ruthenium salt) treatment, respectively. While at lowest concentration of 5.0 µM 79% and 88% percent cell growth was registered for complex 1 and R treatment, respectively. These data suggest that complex 1 induced higher cytotoxicity in MCF-7 cells as compared to standard drugs (cisplatin IC50= 28±0.6 μM, Morais et el, 2012). Ruthenium complexes reported earlier exhibited IC50 values against MCF7 cell line, [Ru(bpy)2 p-CPIP]2+, IC50 = 64.4 μM; [Ru(bpy)2 p-NPIP]2+, IC50 = 86.5 μM; [Ru(phen)2 p-NPIP]2+, IC50 = 38.9 μM (Perdisatta et al 2018) in comparision to these complex 1 exhibited significantly good activity. 3.2.4. Morphological changes analysis To evaluate any morphological changes in MCF-7 cells induced by complex 1, cells were ip t treated with two concentrations below IC50 value for complex 1 (5.0 and 10.0 µM) for 24h. Fig. 10 shows inverted microscopic images of morphological alterations observed in cr MCF-7 cells. The untreated control cells (Fig. 10 A) reached about 95-100% confluence us contained a typical shape and was found attached to the surface. Conversely, in the an treated groups the cells lost their normal epithelial cell morphology, becoming elongated, and some of them found swelled condition. A significant decrease in cell population was ce pt e d M observed in complex 1 treated MCF-7 cells (Fig. 10 B-C). Figure 10. Phase contrast inverted microscopic observation of MCF-7 cells for Ac morphological alterations induced by complex 1 (B, C). (A) Control (B) 5.0 µM (C) 10.0 µM. Magnification: 100X 3.2.5. Detection of Apoptosis by flow cytometry The percentages of early and late apoptotic and necrotic cells were measured using flow cytometry to quantify the levels of detectable phosphatidylserine on the outer membrane of apoptotic cells (Evens et al., 2004). Representative results in the form of dot plots were pt e d M an us cr ip t presented in fig. 11. Figure 11. Flow cytometric analysis of MCF-7 cells exposed to different concentrations ce of complex 1 (B, C) for 24 h. Representative dot plots showing the percentage of viable cells, early apoptosis, late apoptosis and necrotic cells (A) Control, (B) 5.0 µM (C) 10.0 Ac µM (D) Bar diagrams showing the percentage of apoptosis observed by flow cytometric analysis of MCF-7 cells. All data are expressed as mean ± SE. * Significant (p < 0.05) compared with controls. The treatment of MCF-7 with complex 1 revealed that a significant decrease (p < 0.05) in the population of viable cells and increase in the percentage of apoptotic cells was observed. While in the untreated control, the only small percentage of apoptotic cells was observed (Fig. 11 A). As seen in figure 11, MCF-7 cells when exposed to 1, the percentage of early apoptosis cells increased to 7.2% in 5.0 µM to 14.2% 10.0 µM concentrations. While, the percentage of late apoptosis cells reached 9.5% and 7.8% in 5.0 µM and 10.0 µM, respectively (Figure 11 B-C). 3.2.6. Apoptotic morphological changes in MCF-7 cells ip t MCF-7 cells were exposed to complex 1 for 24 h as mentioned above and stained with acridine orange/ ethidium bromide (AO/EB) dye to test if the increase in cell death was Ac ce pt e d M an us cr due to apoptosis (Fig. 12). Figure 12. Apoptotic morphological changes in MCF-7 cells observed under fluorescence microscopy. (A) Control (B) 5.0 µM 1 (C) 10.0 µM 1. Magnification: 200X. (F) Quantification of normal, apoptotic and necrotic cells recorded in more than 300 cells. All data are expressed as mean ± SE for three independent experiments. * Significant (p < 0.05) compared with control. Note: White arrows: Viable cells (living cells); Yellow arrows: Apoptotic cells; Red arrows: Necrotic cells AO/EB staining facilitates typical apoptotic nuclear morphology such as nuclear shrinkage, DNA condensation, and fragmentation. Around 93.7% of viable cells were prominently evident in untreated MCF-7 cells. Control cells showed uniformly ip t distributed green fluorescence (AO stain) with no morphological changes and no red cr fluorescence (Fig 12A). The percentage of viable cells, however, decreased significantly (p < 0.05) in both the treatments. As shown in Fig 12 B-C, and D. Quantification of us apoptotic and necrotic cells in total 300 cells in each group were performed which an revealed that complex 1 treatment induced highest percentage of apoptotic cells (26.2%). The MTT assays, AO/EB staining, and flow cytometry analysis showed similar M concentration and time-dependent effects on MCF-7 cells. There was a difference in the d percentage of viable cells because MTT is based on mitochondrial function assays that pt e detected cell death earlier than others, while apoptosis is indicating assays (AO/EB and flow cytometry) detected cell death later in the process, which is in correlation with other ce reports (Curčić et. al., 2012; Oh et. al., 2004). The AO/EB method improves the detection Ac of apoptosis and can distinguish between late apoptotic and dead cells. (Liu et al., 2015) 4. Conclusion A new methodology was adopted for the search of the potential new drug with known bioactive compounds. In our studies, we have designed and synthesized a new potential metallodrug with use of sodium diclofenac (an anti-inflammatory drug) as bioactive ligand and coordinated it with the metal core, i.e., ruthenium(p-cymene) with known anticancer properties. The binding propensity of the ruthenium complex 1 with model protein (HSA) and ct-DNA was investigated. The ruthenium complex 1 binding results exhibited significant binding propensity via interfacial binding mode. Furthermore, the ruthenium complexes were studied for cytotoxicity against MCF-7 (breast cancer cell lines) and the IC50 values are compared with the standard drug cisplatin and earlier ip t reported ruthenium complexes and ruthenium complex 1 exhibited significantly good activity. From these experimental data, we infer that the ruthenium complex 1 possess cr potential to act as anti-cancer agent. 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