REGULAR ARTICLE
Special Issue onBeyond Classical Chemistry
Photoactivated cytotoxicity induced by heterobimetallic Ru(II)-Pt(II) polypyridyl complexes in MCF-7 cells
SUSHMA SINGHa,d, MOKSHADA VARMAb, BHUPENDRA SHRAVAGEc, PRASAD KULKARNIb and AVINASH KUMBHARa,*
aDepartment of Chemistry, Savitribai Phule Pune University, Pune 411007, India
bBioprospecting group, Agharkar Research Institute, G. G. Agarkar Road, Pune 411004, India
cDevelopmental Biology Group, Agharkar Research Institute, G. G. Agarkar Road, Pune 411004, India
dDepartment of Chemistry, Indian Institute of Technology Bombay, Powai, Mumbai 400076, India E-mail: avinash.kumbhar@unipune.ac.in
MS received 8 March 2021; revised 26 April 2021; accepted 6 May 2021
Abstract. The heterobimetallic Ru(II)-Pt(II) polypyridyl complexes [Ru(bpy)2(BPIMBp)PtCl2]2?(3) and [Ru(phen)2(BPIMBp)PtCl2]2? (4) with their parent complexes [Ru(bpy)2BPIMBp]2?(1) and [Ru(phen)2- BPIMBp]2?(2) possessing bridging ligand (BPIMBp = 1,40-Bis-{(2-pyridin-2-yl)-1H-imidazol-1-yl)methyl}- 1,10-biphenyl) were used for photocytotoxicity against MCF-7 cells.The parent ruthenium complexes (complexes 1-2) exhibited a negligible increase in viscosity of DNA while heterobimetallic Ru(II)-Pt(II) system exhibited a decrease in viscosity of DNA, indicating covalent interaction (through cis-PtCl2unit). The Ru(II)-Pt(II) system co-precipitated with CT-DNA confirmed the covalent binding. In electrophoretic mobility studies, the interaction of Ru(II)-Pt(II) system with plasmid DNA led to retardation of negative supercoiled form, followed by positive supercoiled migration in the dark that indicated the ability to form covalent adducts with DNA similar to cisplatin. The complexes 1-4 showed enhanced photocleavage of plasmid DNA on blue light (*450 nm) irradiation. In a cell-based study, all the complexes exhibited photoactivated cytotoxicity in MCF-7 cancer cells when exposed to visible light of*450 nm as compared to low toxicity in absence of irradiation. Additionally, the complexes containing platinum showed induction of autophagy in GFP-LC3 expressing MCF-7 cells. Overall our study shows that the inclusion of photosensitizer Ru(II) polypyridyl complexes to cis-PtCl2moiety improves anticancer properties of complexes.
Keywords. Photosensitizer; heterobimetallic complexes; photocytotoxicity; autophagy.
1. Introduction
Photodynamic Therapy (PDT) employs two main components such as a) light and b) photosensitizing (PS) molecules.1Currently, PDT is gaining interest as a technique that involves selective destruction of cancer cells over normal cells.1 The photosensitizers (PS) on irradiation of suitable light produce cytotoxic reactive oxygen species (ROS). The mechanism of PDT is divided into two types A) Type I and B) Type II. Type I mechanism involves the generation of superoxide (O2•-) or hydroperoxyl radicals (HO2•) via photoinduced electron transfer. The Type II
mechanism involves singlet oxygen (1O2) on energy transfer from a photosensitizer. These ROS selectively destroy the intracellular organelles where it is gener- ated. Therefore, the preferential uptake of photosen- sitizers in cancer cells makes PDT exclusively targeting cancer cells.1–3 Photofrin is the first FDA approved drug for PDT.2 However, its application is limited due to skin photocytotoxicity. This led researchers to develop second-generation pyrrole- based PS such as chlorins, bacteriochlorins, phthalo- cyanines, pheophorbides, bacteriopheophorbides, and texaphyrins, etc. that generates intracellular cytotoxic ROS.2,4 Apart from tetrapyrrole derivatives such as
*For correspondence
Supplementary Information: The online version contains supplementary material available athttps://doi.org/10.1007/s12039-021- 01935-0.
J. Chem. Sci. (2021) 133:89 Indian Academy of Sciences
https://doi.org/10.1007/s12039-021-01935-0Sadhana(0123456789().,-volV)FT3](0123456789().,-volV)
Photofrin, TOOKAD and 5,10,15,20-Tetrakis(3- hydroxyphenyl)chlorin (mTHPC, Temoporfin),5 some metal-based photosensitizers6, 7 especially ruthenium Ru(II)-based compounds8–10 have displayed intracel- lular ROS generation on exposure to light that makes them potential candidates for PDT. Glazer and co- workers have developed [Ru(bpy)2(6,60-dmb)]2? or [Ru(bpy)2(2,20-biq)]2? complexes where 6,60-dmb = 6,6’-dimethyl-2,20-bipyridine and 2,20-biq = 2,20- biquinoline, that form covalent adducts with DNA on exposure of visible light.11,12 These complexes pho- toeject ligand 6,60-dmb and 2,20-biq and act as pho- tocisplatin agents that are phototoxic to hypoxia/
anoxia cells.11,12 Gasser and co-workers have done extensive work on developing Ru(II) polypyridyl complexes as photosensitizers for PDT.13–15 They have developed ruthenium polypyridyl complexes that absorb 1-and 2-photon light and are significantly
photocytotoxic against 2D monolayer and 3D multi- cellular spheroid.16,17 These complexes target multi- resistant tumour in mice.16,17Recently, McFarland and co-workers have reported Ru-based PS (TLD1433) (Figure 1A) which displays Type II PDT properties that generate intracellular singlet oxygen (1O2)and remarkable photocytotoxicity against several human cancer cells.18 Additionally, TLD1433 has success- fully entered Phase II clinical trial for PDT in the treatment of bladder cancer. This finding opens a door for the researchers to look for other metal-based PS for the treatment of other cancers such as breast cancer.
Breast cancer is one of the leading causes of cancer death. Therefore, there is a need for a technique like PDT for treating such cancer urgently.19 MCF-7 cells are relatively resistant to the well-known drug cis- platin as compared to other breast cancer cells.20This is due to the reason that MCF-7 expresses a high level
(A)
(B)
Ru N
N N
N
N N
2+
[Ru(bpy)2BPIMBp]2+(1) N
N
N N
Ru N
N N
N
N N
2+
[Ru(phen)2BPIMBp]2+(2) N
N
N N
Ru N
N N N N
N
2+
[Ru(bpy)2(BPIMBp)PtCl2]2+(3) N
N
N
N Pt
Cl Cl
Ru N
N N N N
N
2+
[Ru(phen)2(BPIMBp)PtCl2]2+(4) N
N
N
N Pt
Cl Cl
Figure 1. (A) Chemical structure of TLD1433 (B) Complexes1-4used in present study31.
of the anti-apoptotic BCL-2 protein.21 Anti-apop- totic BCL-2 protein blocks apoptosis and is expressed in 40-80% of breast cancer patients.21 In that case, apoptotic deficient cells rely on autophagy for cell death. Autophagy can serve as both cell survival and cell death mechanism.22,23 Autophagy dealing with cell death mechanism involves degra- dation of cytoplasmic components.24 Cisplatin up- regulates autophagy thereby preventing cancer cell death. However, the use of chemosensitizer as autophagy modulating agents can overcome the resistance to cisplatin.25
Heterobimetallic agents could be a potential alter- native to conventional metallodrugs for PDT.26–29 As the heterobimetallic agents can be fine-tuned by careful selection of different metals, bridging ligand and co-ligands. The synergistic effect of different metal complex moieties can enhance inherent prop- erties and therapeutic potential. In addition to this, heterobimetallic complexes such as Ru(II)/Pt(II) sys- tem display remarkable mechanisms of action result- ing in a distinctive profile of activity. Several light- absorbing Ru(II)-polypyridyl chromophores coupled with DNA binding Pt(II) site have been reported with exceptional electrochemical and spectroscopic prop- erties and effective Photo Dynamic Therapeutic (PDT) activity. Sakai and co-workers have reported [Ru(bpy)2{m-bpy(CONH-(CH2)-NH2)2}PtCl2]2? where bpy = 2,2’- bipyridine, that exhibited distinctive photophysical properties and cleaved pBR322 DNA on visible light irradiation in air-saturated condi- tions.26 Brewer and co-workers have synthesized a new heterobimetallic complex [(tpy)RuCl(dpp)PtCl2]? where tpy = 2,2’:6’,2’’-terpyridine; dpp =2,3-bis(2- pyridyl)pyrazine with interesting electrochemical and photophysical properties that not only cleave DNA on visible light irradiation but also exhibit antibacterial activity.27 The same group has also reported [(Ph2phen)Ru(BL)PtCl2]2?where BL = 2,3-bis(2-pyr- idyl)pyrazine (dpp) or 2,3-bis(2-pyridyl)quinoxaline (dpq) in which incorporation of Ph2phen to the com- plex offered enhanced spectroscopic and photophysi- cal properties. In addition to this, Pt site binds to pUC18 DNA which results in a decrease in DNA migration similar to cisplatin and Ru(II) helps in photocleavage of pUC18 DNA via an oxygen-depen- dent mechanism.28 The heterobimetallic complex [(Ph2phen)2Ru(dpp)PtCl2]2?, displays enhanced DNA modification, antiproliferative activity towards F98 malignant glioma cells on visible light irradiation as compared to cisplatin.29 Recently, Gasser and coworkers have reported Ru(II)-Pt(IV) conjugates combining both chemotherapy and PDT which were
employed to target drug-resistant cancers.30 Till date, only a few ruthenium-platinum polypyridyl complexes and their biological activity as photosensitizing agents have been investigated so far. Therefore, in the same line, heterobimetallic Ru(II)-Pt(II) polypyridyl com- plexes have been synthesized as photosensitizers for the application of photodynamic therapy (PDT). The previously reported31 heterobimetallic ruthenium(II)- platinum(II) polypyridyl complexes of the type [Ru(bpy)2(BPIMBp)PtCl2]2? (3) and [Ru(phen)2(- BPIMBp)PtCl2]2? (4) using suitable precursors [Ru(bpy)2BPIMBp]2? (1) and [Ru(phen)2BPIMBp]2?
(2), where BPIMBp = 1,40-Bis-{(2-pyridin-2-yl)-1H- imidazol-1-yl)methyl}-1,10-biphenyl) have been uti- lized as photosensitizing agents (Figure1B) for photo induced cytotoxicity in cancer cell. The design approach involved conjugation of Ru(II) polypyridyl moiety (photosensitizer) with cis-PtCl2 (nucleotide covalent binder) by bridging ligand (Figure 2). The structural activity may contribute to their biological activity by targeting cancer cells that can lead to their application in photodynamic therapy. In present study, the complexes were used with the assumption that the two different metal units would act by different mechanism, attenuating overall activity. The interac- tion of complexes (1-4) with CT-DNA was studied using various biophysical and biochemical techniques.
DNA cleavage activity of complexes 1-4 in dark as well as DNA photocleavage activity in visible light (blue light, *450 nm) was also studied. The autop- hagy inducing ability and light-induced photocyto- toxicity of the complexes1-4against MCF-7 cells was also studied.
2. Experimental
2.1 Methods and materials
All chemicals used in this work were of analytical grade and used as received. Calf-thymus DNA (CT- DNA), Plasmid pBR322 DNA, Tris(hydrox- ymethyl)aminomethane (Tris), sodium chloride (NaCl), potassium hydrogen phosphate (K2HPO4), potassium dihydrogen phosphate (KH2PO4), agarose (superior grade type I gelling temp. 38–40 C) and ethidium bromide (EtBr) were of molecular biology grade and obtained from SRL (India). Dulbecco’s modified eagle medium (DMEM), Dulbecco’s phos- phate-buffered saline (DPBS), fetal bovine serum (FBS) and penicillin-streptomycin solution were pur- chased from Invitrogen. G418 solution was purchased from Roche. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) was purchased from Sigma-Aldrich, USA.MCF-7 and NIH3T3 cells were obtained from National Centre for Cell Science, Pune, India. MilliQ water was autoclaved and used for the preparation of the buffers.
2.2 Photophysical studies
UV-Visible spectra of complexes 1-4 in acetonitrile were recorded on Shimadzu UV-1800 Spectropho- tometer in the range 200-800 nm in 10 mm path-length cuvette. Steady-state emission experiments were car- ried out on a Jasco spectrofluorometer using quartz.
Emission quantum yields (/) were calculated by integrating the area under the luminescence curves and by using equation (1) where OD is the optical density of the compound at the excitation wavelength (450 nm) and A is the area under the emission spectral curve. The standard used for the luminescence quan- tum yield measurements was [Ru(bpy)3]Cl2.32
/Sample¼ fODStandardASampleg=fODSample
AStandardg /Standard ð1Þ
2.3 Biological investigation
2.3a DNA binding studies: All experiments involving the interaction with calf thymus DNA (CT- DNA) were carried out in phosphate buffer (pH = 7.2).
Phosphate buffer of 0.2 M (pH = 7.2) was prepared using 2 M K2HPO4and 2 M KH2PO4 solution. A solution of CT-DNA in the buffer gave a ratio of 1.8-1.85:1 at 260 nm and 280 nm indicated that the DNA is sufficiently free from proteins. The concentration of DNA was determined spectrophotometrically by assuming R260 =
6600 dm3mol-1cm-1. All the complexes are partially soluble in water/phosphate buffer. The test samples were prepared as a stock solution in 100% DMSO and were stored at -20C. Further dilutions were prepared on the day of the experiment as required.
2.3b Absorption titration: Absorption titration was performed using metal complex concentration (10 lM) and increasing the calf thymus DNA concentration (0-90 lM) in phosphate buffer on Shimadzu UV-1800 Spectrophotometer. The data was fitted to equation (2).
½DNA
½eaef¼ ½DNA
½ebefþ 1
Kb½ebef ð2Þ
To estimate the intrinsic binding constant Kb, where [DNA] is the concentration of DNA in base pairs,eais the extinction coefficient observed for the MLCT absorption at the given DNA concentration, ef is the extinction coefficient of the complex free in solution andebis the extinction coefficient of the complex fully bound. A plot of [DNA]/[ea-ef] vs. [DNA] gave a slope of 1/[eb-ef] and Y-intercept equal to 1/Kb[eb-ef] respectively. The intrinsic binding constant Kb is cal- culated by the ratio of the slope to the intercept.
2.3c Steady-state emission titration: This experiment was performed by maintaining the concentration of metal complex concentration fixed to which increasing concentration of the stock DNA solution was added. The concentration of the metal complex was 10 lM with an increase in the concentration of DNA 0-90 lM. After the addition of DNA to the metal complex, the solution was excited to their MLCT band between 430 to 450 nm and the emission was measured at wavelength 500 to 750 nm.
The excitation and emission slit width were 5 nm each.
Figure 2. The design approach and structure-activity relationship of complexes3and4for biological activity.
2.3d Viscosity measurement: This experiment was performed using a capillary (micro) viscometer at 25 C maintained in Julabo 31A visco bath. The flow time of solutions in phosphate buffer (pH = 7.2) was recorded in triplicate for each sample and average flow time was calculated. Data was presented as (g/go)1/3 versus [complex]/[DNA], where g is the viscosity of DNA in the presence of complex and go is the viscosity of DNA.
2.3e Covalent binding assay: The covalent binding assay was carried out in a buffer containing phosphate buffer (pH 7.2) using the reported procedure.33 DNA (500lM) was incubated in presence of the complexes 1-4 (50lM) at 37C for 3 h. NaCl and ethanol were added to quench the reaction and precipitate the DNA after different time intervals. The solutions were centrifuged and the supernatant was assayed spectrophotometrically on Shimadzu UV-1800 spectrophotometer to determine the levels of free and bound complexes.
2.3f Gel electrophoretic mobility study: The electrophoretic mobility studies of pBR322 DNA were carried out by 1% agarose gel electrophoresis as described earlier.34, 35 The stock solutions of complexes 1-4 were prepared in DMSO which was further diluted in water for the experiment. Plasmid pBR322 DNA (100 ng) was treated with different concentration of complexes1-4and the mixtures were incubated for 1 h at 37 C. The concentrations of the complexes and DNA were adjusted to the final sample volume of 10lL using deionized water. To investigate the interaction of complexes 1-4 with plasmid DNA presence of visible light (430-480 nm). The experiments were carried out by incubating a mixture of plasmid DNA and complexes 1-4 for 30 min at 37 C, followed by irradiation of blue visible light for 30 min at 37 C. The reactions after incubation were quenched by the addition of 2 ll gel loading dye (0.25% bromophenol blue, 0.25% xylene cyanol, 40% glycerol and 2 mM EDTA). The samples were subjected to electrophoresis for 1.5 h at 60 V on 1% agarose gel in TBE (Tris-Boric acid-EDTA) buffer (pH 8.3). The gel was stained with 0.5lg/mL ethidium bromide and visualized by UV light and photographed for analysis using AlphaImager 2200.
2.4 Cell-based studies
2.4a (Photo) cytotoxicity study: The photocytoto- xicity of complexes 1-4 was studied using MTT
(3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazo- lium bromide) reagent by MTT assay according to literature procedure.29 The cytotoxicity of complexes was studied in both dark and following photolysis to evaluate the photodynamic activity of the complexes.
The 105cells/mL were seeded in 96-well plate. MCF-7 cells were grown in DMEM medium supplemented with 10% FBS and 1% antibiotic (penicillin and streptomycin) at 37 C in a 5% CO2 humidified atmosphere for 24 h before treatment. The 10 mM stock solutions of the complexes were prepared in DMSO solvent. Then, the cells were treated with complexes at different concentration from 20-80 lM (20, 40, 60 and 80 lM) dissolved in plain DMEM.
After 24 h incubation, the treatment media was removed from wells. The control cells were treated with plain DMEM without FBS and complexes. For cytotoxicity assay, the plates were incubated in the dark for 1 h. For photocytotoxicity, the cells were incubated in the dark for 15 min and then were incubated for 30 min using lab-built light LED array (430-480 nm, 0.5 mW/cm2, 1 J/cm2). A final 15 min dark incubation was performed after photoirradiation.
The solutions were then aspirated and the cells were washed with plain DMEM. After incubation for 24 h, MTT was added to each well and incubated for 3 h.
The purple formazan crystals formed were dissolved by the addition of 180lL of DMSO for 5 min. Finally, the absorbance of the solution was measured using a multimode reader (Biotek Synergy) at 570 nm. The IC50 values of control and photo-irradiated cells were obtained.
Figure 3. Effect of an increasing amount of EtBr and complexes 1-4 on the relative viscosity at DNA at 25C, [DNA] = 100lM.
2.4b Cytotoxicity assay of complexes 1-4 against NIH3T3 cells: Cell viability assay of complexes 1-4 was performed in non-cancerous NIH3T3 cells using MTT reagent. The 15 mM stock solutions of the complexes were prepared in DMSO solvent. Cells were treated with complexes at a concentration of 20, 40, 60, 80, 100 and 150 lM prepared in DMEM without FBS. DMSO in media without FBS (1.5%) was used as a control. MTT assay was carried out after 24 h incubation. A MTT solution (20lL, 5 mg/mL) was prepared in PBS pH 7.4 then added to each well and incubated for 3h. The purple formazan crystals formed were dissolved by the addition of 180lL of DMSO for 5 min. Finally, the absorbance of the solution was measured using a multimode reader (Biotek Synergy) at 570 nm. IC50was determined by using ED50V10 excel add-on software.
2.4c Effect of complexes 1-4on autophagy in GFP- LC3 expressing MCF-7 cell line: MCF-7 cells were cultured in DMEM containing 10% FBS and penicillin (100 U/mL) and streptomycin (100 lg/mL) in a
humidified 5% CO2 incubator at 37 C. For determining the effect of complex 1-4 on autophagy, MCF-7 cells were stably transfected with pAutophagSENSE (Clonetech, USA) by the calcium phosphate transfection method. This construct drives the expression of GFP-LC3 under the CMV promoter.
After additional 24 h incubation, the cells were transferred to the medium containing G418 at 300 lg/mL. After 4 weeks of G418 selection, stable GFP- LC3 expressing MCF-7 cells were obtained. The MCF-7 cells were treated for 4 h with 20lM complex 1-4 and cisplatin. Cell images were captured using a Confocal fluorescence microscope (Leica, TCS, SP8, Germany). The p-value was determined by one-way Anova using GraphPad Prism 6.
3. Results and Discussion
The synthesis of complexes is already reported;31 however, the1H, 196Pt-NMR and ESI-MS spectra are given in supporting formation (Figure S12-S20, Sup- plementary Information). The result and discussion on photophysical studies i.e. molar absorption coefficient vs. wavelength, fluorescence spectra, the quantum yield of complexes 1-4 in acetonitrile is given in supporting information (Figure S1 and Table S1, Supplementary Information).
The interaction of complexes 1-4 with CT-DNA using absorption titration and steady-state fluorescence titration is discussed in the Supplementary Informa- tion. (Figure S2 and S3, Supplementary Information) The hyprochromism and intrinsic binding constantsKb of complexes on binding with DNA are tabulated in Table S2, Supplementary Information.
3.1 Viscosity measurement
Viscosity measurement is critical to determine differ- ent types of DNA binding modes as it is sensitive to Figure 4. Plot of complexes1-4binding to CT-DNA as a
function of time; where r = bound complex to nucleotide concentration
Figure 5. Agarose (1%) gel electrophoresis of plasmid DNA in dark, incubation time = 1 h, at 37C. TBE buffer, pH = 8.3. Form I – supercoiled DNA, Form II – nicked circular plasmid DNA, Form III-nicked linear DNA. Complex1: Lane 1 – DNA control, lane 2 – DNA?1(0.2lM), lane 3 – DNA?1(0.5lM), lane 4 – DNA?1(2lM), lane 5 – DNA?1(6 lM), lane 6 –DNA?1(8lM), lane 7 – DNA?1(10lM), lane 8 – DNA?1(15lM), lane 9 – DNA?1(20lM), lane 10 – DNA?1(25lM).
length changes of DNA helix.36 The intercalation of organic drugs or metal complexes into DNA causes a distinctive increase in the viscosity of DNA due to the unwinding of DNA strands and hence, an increase in overall DNA molecular length. While a molecule that binds in the DNA grooves cause less or no change in viscosity of DNA solution. In contrast, covalent bin- ders such as cisplatin, are known to kink DNA through covalent binding which leads to shortening of the length of DNA helix and cause a decrease in relative viscosity of DNA. The effect of EtBr (ethidium bro- mide, a known DNA intercalator) and complexes 1-4 on the viscosity of DNA was studied in order to assess the binding mode of complexes with DNA. The addition of EtBr to DNA increases the relative vis- cosity of DNA dramatically. However, increasing concentration of complex 1-2 exhibited a negligible increase in viscosity of DNA, suggesting complexes
are binding to the surface of DNA and hence exhibiting groove binding mode which is consistent with results of absorption titration, and steady-state emission titration. While complexes3and4showed a significant decrease in the viscosity (Figure 3), sug- gesting an interaction of complexes 3-4 via covalent interaction through a cis-PtCl2 unit of complexes resulting in kinking or bending of DNA, similar to other covalent binding molecules especially cisplatin analogues.36, 37
3.2 Covalent binding assay
The degree of covalent binding of complexes to DNA was assayed spectrophotometrically using the method described by Barton and co-workers.33 Solutions of calf-thymus DNA were incubated at 10:1 nucleotides:
Figure 7. Agarose (1%) gel electrophoresis of plasmid DNA in dark; incubation time = 1 h, at 37C. TBE buffer, pH = 8.3. Form I – supercoiled DNA, Form II – nicked circular plasmid DNA. Complex3: Lane 1 – DNA control, lane 2 – DNA
?3(0.2lM), lane 3 – DNA?3(0.5lM), lane 4 – DNA?3(2lM), lane 5 – DNA?3(6lM), lane 6 –DNA?3(8lM), lane 7 – DNA ?3 (10lM), lane 8 – DNA?3(15lM).
Figure 6. Agarose (1%) gel electrophoresis of plasmid DNA on visible light irradiation (30 min, 430-480 nm, 0.5 mW/
cm2, 1 J/cm2); incubation time = 30 min, at 37C. TBE buffer, pH = 8.3. Form I – supercoiled DNA, Form II – nicked circular plasmid DNA, Form III-nicked linear DNA. Complex1: Lane 1 – DNA control, lane 2 – DNA?1(0.2lM), lane 3 – DNA?1(0.5lM), lane 4 – DNA?1(2lM), lane 5 – DNA?1(6lM), lane 6 –DNA?1(8lM), lane 7 – DNA?1 (10lM), lane 8 – DNA?1(15lM), lane 9 – DNA ?1 (20lM), lane 10 – DNA control.
Figure 8. Agarose (1%) gel electrophoresis of plasmid DNA on visible light irradiation (30 min, 430-480 nm, 0.5 mW/
cm2, 1 J/cm2); incubation time = 30 min, at 37C. TBE buffer, pH = 8.3. Form I – supercoiled DNA, Form II – nicked circular plasmid DNA, Form III – nicked linear DNA. Complex3: Lane 1 – DNA control, lane 2 – DNA?3(0.2lM), lane 3 – DNA?3(0.5lM), lane 4 – DNA?3(2lM), lane 5 – DNA?3(6lM), lane 6 –DNA?3(8lM), lane 7 – DNA?3 (10lM), lane 8 – DNA?3(15lM), lane 9 – DNA ?3 (20lM), lane 10 – DNA control.
metal complex ratio at 37 ˚C. A covalently bound complex co-precipitates with DNA while a non-co- valently bound complex remains in the supernatant solution. Aliquots were removed and the reaction was quenched by precipitating DNA using NaCl and ethanol. The solution was finally centrifuged. The amount of the bound and free metal complexes was assayed spectrophotometrically and a plot of the extent of covalent coordination of complexes to DNA as a function of time is shown in Figure 4. The extent of bound complexes observed from r = 0.01-0.02 for the complexes 3-4 which are consistent with the results obtained for the other reported complexes binding covalently to DNA.36
3.3 Gel electrophoresis mobility assay
The interaction of complexes with DNA was studied by an electrophoretic mobility assay performed with
plasmid pBR322 DNA. As it is well known that, the supercoiled DNA (Form I) migrates quickly through the gel due to its compact nature, and while nicked circular (Form II) DNA migrates slowly. The com- plexes 1-4 were incubated at 37 C in the dark at different concentration with pBR322 DNA. On increasing the concentration of complex 1 (Figure 5) and2(Figure S5, supplementary information) to DNA, the extent of negative supercoils decreased as indi- cated by slow mobility at concentration 25 lM (Fig- ure 5). In contrast, complex3 and 4 on incubation at 37˚C in dark exhibited altered mobility. Initially, an increase in concentration up to 6 lM of complex 3 (Figure 7) and 4 (Figure S7, Supplementary Informa- tion) to plasmid DNA led to a decrease in supercoiled DNA. However, on a further increase of complex 3 and4led to positive supercoilsviacis-PtCl2moiety in Ru(II)-Pt(II) polypyridyl complex. This resulted in increased mobility similar to cisplatin (Figure S4, Supplementary Information) and cisplatin analogues.37, 38
The effect of complex 1-4 on DNA cleavage using blue light irradiation (430-480 nm) was also studied. On irradiation of visible light, complex 1 (Figure 6, Lane 5) and 2 (Figure S6, Lane 3, Sup- plementary Information) exhibited enhanced mobility retardation at low concentration (6 lM). Similarly, for complex 3 (Figure 8) and 4 (Figure S8, Sup- plementary Information), a remarkable DNA cleav- age was observed. It was due to the synergistic effect of photoactivated ruthenium polypyridyl Figure 9. Viability assay of MCF-7 cells after treatment with complexes (A)3 and (B)4 in dark and on irradiation of visible light (430-480 nm, 0.5 mW/cm2, 1 J/cm2). MTT was performed after 24 h. Results are mean values of three biological replicates.
Table 1. IC50 value of complexes1-4 in MCF-7 cells in dark and on visible light irradiation.
Complex IC50(lM) dark IC50 (lM) visible lighta
1 298.7 46.7
2 56.4 \20
3 [300 \20
4 [300 \20
a(430-480 nm), 1 J/cm2for 30 min, MTT was performed after 24 h.
Figure 10. Effect of 20lM complexes (A)cisplatin(B)1(C)2(D)3 and (E)4on autophagy in GFP-LC3 expressing MCF-7 cell line. Scale, 25 lm.
moiety and cis-PtCl2 moiety of complexes 3-4 that resulted in complete cleavage of supercoiled DNA at low concentration (6 lM).
3.4 (Photo) cytotoxicity study
The photocytotoxicity of the complexes1-4(0-80lM) was evaluated in MCF-7 cells by MTT assay. The complexes 1-4 were treated for 15 min in dark fol- lowed by photo exposure to visible light of 450-480 nm (0.5 mW/cm2, 1 J/cm2) for 30 min by LED array.
The result showed that complexes 1, 3 and 4are less cytotoxicity in the dark while complex 2 show mod- erate cytotoxicity (Figure 9 and Figure S10, Supple- mentary Information). However, the complexes 1-4 are significantly cytotoxic to MCF-7 cell line (Figure9 and Figure S10, Supplementary Information) on exposure to blue light. The IC50 values are given in Table 1. The resultant cytotoxicity of complexes 1-4 indicated that all the complexes are cytotoxic against MCF-7 cells on irradiation of light. The result sug- gested the advantage of conjugation of Ru(II) poly- pyridyl moiety as photosensitizer to cis-PtCl2 moiety that could enhance the cytotoxicity against cisplatin- resistant cancer cells. The cytotoxic effect of com- plexes is generally governed by factors such as cell permeability, specificity to certain organelle and mechanism of action of complexes. We assume that there are multiple factors involved in the cytotoxic effect of complexes 1-4under investigation.
The cytotoxic effect of complexes1-4in normal cell line i.e. NIH3T3 (mouse embryonic fibroblast cells) was also performed. The complexes 1-4are found to be less cytotoxic against NIH3T3 cells (Figure S11, Supplementary Information). IC50 value of complexes against NIH3T3 normal cell is tabulated in Table S3, Supplementary Information.
3.5 Effect of complexes1-4on autophagy in GFP-LC3 expressing MCF-7 cell line
The role of autophagy in cancer cells is important to target drug resistance and cancer cells growth. For example, the cisplatin resistance leads to the protection of cancer cell death. Hence, the strategy for autophagy modulation to mitigate cisplatin resistance utilizing chemosensitizer was studied.25The effect of complexes 1-4on autophagy in MCF-7 cells was also monitored in the present study. Microtubule-associated protein 1A/
1B-light chain 3 (LC3) is considered as a biomarker to monitor autophagy.39 Therefore, quantification of LC3 positive puncta is an important measure of induction of autophagy.40 A robust induction of autophagy was detected as seen by the formation of GFP-LC3 puncta in MCF-7 cells upon treatment of complexes 1-4.
Confocal images displayed that complex 1-4 are responsible for the induction of autophagy as the number of puncta formed is higher than control cis- platin (Figure 10). Fluorescent enhancement of GFP- LC3 puncta were measured to ascertain the upregula- tion of autophagy. Approx. *2 and 2.5-fold fluores- cence enhancement on the treatment of complexes 1 and 2 respectively to GFP-LC3 MCF-7 cells were observed. Similarly, *3 and 2-fold fluorescence enhancement on the treatment of complexes 3 and 4, respectively to GFP-LC3 MCF-7 cells was observed as compared to cisplatin (Figure11). The result indicated that Ru(II)-Pt(II) complexes 3-4 induced autophagy.
However, taking the account of photocytotoxicity of complexes 3 and4, it can be suggested that the ruthe- nium polypyridyl complexes as photosensitizer could be able to overcome cisplatin resistance through autophagy induction in MCF-7 cells to a certain extent.
Although, these results need to be verified further with a detailed investigation.
4. Conclusions
A heterobinuclear Ru(II)-Pt(II) system of the type [Ru(N-N)2(BPIMBp)PtCl2]2? where N-N is 2,20- bipyridine (bpy) (3), 1,10-phenanthroline (phen) (4) along with parent complexes[Ru(N-N)2(BPIMBp)]2?
Figure 11. Fold enhancement of fluorescence intensity of GFP-LC3 puncta on the treatment of complexes 1-4 and cisplatin in GFP-LC3 expressing MCF-7 cells. The quan- titative data are mean ± SD; n =10 parallel assessments.
Asterisk (* and **) represents p\0.05 and is considered a significant result when compared to cisplatin while ns represents not significant.
(1-2) were used as photosensitizers and phototoxic agents for MCF-7 cells. The complex 3-4 exhibited covalent binding to DNA through cis-PtCl2 moiety revealed by covalent binding assay, viscosity mea- surement and DNA cleavage assay. The photocyto- toxicity assay revealed that heterobimetallic complexes are cytotoxic against MCF-7 cells upon exposure to visible light. The treatment of complexes 1-4 in GFP-LC3 expressing MCF-7 cells revealed induction of autophagy. The overall study demon- strates the potential of conjugation of ruthenium polypyridyl complexes (1and2) with cis-PtCl2moiety forming heterobimetallic Ru(II)-Pt(II) polypyridyl complexes (3 and 4) for the photodynamic therapy against breast cancer. Our studies underline an attempt to design the complexes to target cancer cells over normal cells and also to overcome drug resistance in cancer cells. However, detailed and carefully planned experiments are needed to decipher the mechanism to explain the photocytotoxicity of these complexes and efforts are underway to design new bimetallic com- plexes for this purpose.
Acknowledgements
SS and AK acknowledge the Central Instrumentation Facility (CIF), SPPU for Instruments facility. AK acknowledges University Grant Commission under Centre for Advance Studies CAS-IV (F.540/11/CAS-IV/2016 (SAP-I)). PK thanks SERB, India for funding (CRG/2018/001882). The authors would like to thank Dr. Yogesh Karpe, ARI for providing pAutophageSENSE vector. BS acknowledges support from Agharkar Research Institute (grant number ZOO-17). MV acknowledges the Council of Scientific and Industrial Research (CSIR) for SRF fellowship. SS acknowledges Jayant Kulkarni, JV Technologies, Pune, India for manufacturing blue LED array and also thank Institute Postdoctoral Fellow- ship (IPDF), IIT Bombay for fellowship.
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