Novel biosynthesized gold nanoparticles as anti-cancer agents against breast cancer: Synthesis, biological evaluation, molecular modelling studies

Accepted Manuscript Novel biosynthesized gold nanoparticles as anti-cancer agents against breast cancer: Synthesis, biological evaluation, molecular modelling studies

Satish Kumar Vemuri, Rajkiran Reddy Banala, Sudip Mukherjee, Uppula Purushotham, Subbaiah GPV, A.V. Gurava Reddy, T. Malarvilli PII: DOI: Reference:

S0928-4931(18)31706-5 https://doi.org/10.1016/j.msec.2019.01.123 MSC 9381

To appear in:

Materials Science & Engineering C

Received date: Revised date: Accepted date:

14 June 2018 21 January 2019 27 January 2019

Please cite this article as: S.K. Vemuri, R.R. Banala, S. Mukherjee, et al., Novel biosynthesized gold nanoparticles as anti-cancer agents against breast cancer: Synthesis, biological evaluation, molecular modelling studies, Materials Science & Engineering C, https://doi.org/10.1016/j.msec.2019.01.123

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ACCEPTED MANUSCRIPT Novel Biosynthesized Gold Nanoparticles as Anti-Cancer Agents Against Breast Cancer: Synthesis, Biological Evaluation, Molecular Modelling Studies Satish Kumar Vemuri1,2*, Rajkiran Reddy Banala1, Sudip Mukherjee3, Uppula Purushotham4, Subbaiah GPV1, Gurava Reddy A.V1, Malarvilli T2* SMART, Sunshine Hospitals, PG Road, Secunderabad-500003, Telangana, India.

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Department of Biochemistry, Rajah Serfoji Govt College, Thanjavur, Tamil Nadu, India.

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Department of Bioengineering, Rice University, Houston, TX-77005, USA.

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Department of Chemistry, Koneru Laxmaiah education foundation, Guntur, Andha Pradesh,

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1.

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India-522502. Abstract

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Cancer therapeutics development has been a challenge due to their untoward side effects and cytotoxicity. Phytochemical anti-cancer drugs have several advantages over chemical

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chemotherapeutic drugs as they are less cytotoxic and has a greater pharmacological advantage.

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However, lack of targeting ability limits the use of phytochemicals at a great extent for a successful therapeutic strategy. Gold nanoparticles (AuNPs) have long been used to load the therapeutic cargo and provided significant advantages over conventional chemo-therapeutics. In this present study,

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we report the synthesis and testing of various biosynthesized AuNPs (b-AuNPs) using naturally

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derived phytochemicals (Curcumin: Cur, Turmeric: Tur, Quercetin: Qu and Paclitaxel: Pacli). The synthesized b-AuNPs have been well characterized by different Physicochemical techniques. Cytotoxic potential of these b-AuNPs were evaluated in different breast cancer cells either in an

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individual or in a combination forms. We have observed the maximum therapeutic activity in a combination of all four types of b-AuNPs (AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli) as compared to their pristine administration. Further, mechanistic studies of these compounds revealed that, combinations of AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli were significantly effective in inhibiting cell proliferation, apoptosis, angiogenesis, colony formation and spheroid formation predicting a synergistic effect when compared to individual treatment against different breast cancer cell lines (MCF-7 and MDA-MB 231). Interestingly the nanoconjugates alone or in combinations did not show cytotoxicity towards human embryonic

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ACCEPTED MANUSCRIPT normal kidney cell line (HEK 293), demonstrating the biocompatibility. Together the results demonstrated the potential anti-cancer properties of b-AuNPs in a combinatorial approach that could be the future of cancer nanomedicine. Keywords: Gold nanoparticles, Green synthesis, Flavonoids, Breast cancer, Anti-cancer,

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Nanomedicine.

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Introduction

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Breast cancer is the most common cancer in women and second leading cause of cancer-related death among females worldwide. Approximately 25% of breast cancers showed a higher

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prevalence in developed countries [1]. Currently, chemotherapy has been proven to be effective in clinical settings. However, toxicity associated with the chemotherapeutic drugs has limited the use

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and utility of chemotherapy. Herbal medicine is considered as an alternative therapy for cancer treatment. In addition, natural compounds have higher anti-tumor response, less toxicity and

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greater pharmacological or biological activities that provide therapeutic benefits in treating cancer [2, 3]. Paclitaxel (Taxol) is the drug of choice for chemotherapy in breast cancer due to its

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effectiveness [4-6]. Paclitaxel works as a microtubule inhibitor by attaching to microtubules and forming the framework inside living cells stabilizing them against depolymerization, resulting in

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inhibition of cell division [7, 8]. It is also capable of inducing apoptosis-mediated cell death [9]. However, the success of paclitaxel chemotherapy in cancer patients is limited by myelotoxicity

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and neurotoxicity [10,11].

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On the other hand, Turmeric (Curcuma longa L.) is popularly used in Ayurveda and dietary; which also has several biomedical applications including anti-inflammatory, anti-oxidant and anti-tumor activity [12,13]. Studies also reported that Curcumin inhibits the apoptosis, angiogenesis and metastasis by up-regulating p53, Bax, caspase-9 and down regulation of VEGF, COX2, STAT, CYCLIN-D1, and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) [14, 15, 16]. Similarly, Quercetin (Qu), is a well known dietary flavonoid and present in edible fruits, vegetables, and medicinal plants [17] shown anti tumor activity through induction of P53, caspase 3,9, Cyt C in breast cancer cell lines [18,19]. A fore mentioned study showed that combination of phytochemicals are a wise choice to use as anti-cancer agents as they are less 2

ACCEPTED MANUSCRIPT cytotoxic to normal cells [3, 20]. However, requirement of high dose, toxicity, and lack of specificity seriously hamper the use of these phytochemicals in clinical setting. Recent studies have demonstrated that coating of anticancer drugs on nanoparticles serve as excellent drug delivery systems with better efficacy [21, 22]. Recently several researchers demonstrated the applications of bioconjugated metal nanoparticles including gold nanoparticles (b-AuNPs) and silver nanoparticles (b-AgNPs) with significant therapeutic efficacy and lower

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toxicity. These metal nanoparticles conjugated with naturally available phytochemicals showed

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profound cancer theranostics ability [23, 24].

In this context, we have demonstrated the anti-cancer property of b-AuNPs in a

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combination using AuNPs-Curcumin (AuNPs-Cur), AuNPs-Turmeric (AuNPs-Tur), AuNPsQuercetin (AuNPs-Qu) and AuNPs-Paclitaxel (AuNPs-Pacli) and compared with the pristine

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drugs or nanoconjugates. Further, combination of these four sets of b-AuNPs were significantly effective in inhibiting cell proliferation, apoptosis, angiogenesis, colony formation and spheroid

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formation against different breast cancer cell lines (MCF-7 and MDA-MB 231) predicting a

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synergistic effect when compared to individual treatment..

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Materials and Methods

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Preparation of stock solutions

A stock solution of 10−2 M was prepared by dissolving 1 gm of HAuCl4.3H2O in 253.92 mL of

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autoclaved Milli-Q water [16].We have further prepared stocks of 10 mg/ml of Cur, Tur and Qu in DMSO: Water mixture (3:1). Moreover, we prepared a stock of Paclitaxel in DMSO at a concentration of 10 mg/mL. Biosynthesis of gold nanoparticles using different phytochemicals (Curcumin, Turmeric, Quercetin and Paclitaxel) Gold nanoparticles were synthesized by the reduction of HAuCl4 (200 µL, 10−2 M) using constant amount (500 µg) of Curcumin (Cur), Turmeric (Tur), Quercetin (Qu) and Paclitaxel (Pacli) extract (100 µg/mL) [22,25]. The total volume of the reaction mixture was adjusted to 5 mL in all 3

ACCEPTED MANUSCRIPT experiments. The reaction was initially heated at 50 °C for 15 minutes that helps in the reduction process. The resulting b-AuNPs (AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli) were purified by ultracentrifugation at 15,000 rpm (25,000 g) for 40 min at 15°C (Sorvall WX ultra 100, Thermo scientific) and the intensely red colored pellet of the different sets of b-AuNPs were used for further characterization and biological experiments.

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Characterization techniques

Several physicochemical techniques were used for the characterization of the four sets of b-AuNPs

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(AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli). Absorption spectra of these b-AuNPs were measured by UV–Vis spectroscopy, JASCO dual beam spectrophotometer (model V-570) in

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a quartz cuvette from 800 to 200 nm with a resolution of 1 nm. The size, shape, and morphology of the gold nanoparticles were examined using transmission electron microscopy (TEM), Tecnai

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G2 F30 S-Twin Microscope, operated at 100 kV. Selected area electron diffraction patterns were also recorded using this instrument. Inductively, coupled plasma optical emission spectrometry

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(ICP OES) was conducted to determine the concentration of gold present in b-AuNPs (iCAP-6500 DUO, Thermo Fischer Scientific, and UK IRIS intrepid II XDL, Thermo Jarrel Ash). DLS was

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utilized to determine the zeta potential of the biosynthesized gold nanoparticles (AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli) [16]. TGA analysis of the powdered AuNPs-Cur,

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AuNPs-Tur, AuNPs-Qu, AuNPs-Pacli were recorded on TGA machine (TGA/SDTA Mettler Toledo 851e system). For TGA analysis, b-AuNPs were placed in an alumina pan and heated from

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20°C to 700°C at a ramping time of 10°C/minute [26].

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Molecular Docking

Molecular docking studies were performed using autodock vina on the curcumin synthase 1 (PDBID: 3OV2) and human pirin (PDBID: 5JCT) proteins with curcumin, quercetin and paclitaxel ligands. The protein preparation was carried out using autodock with default settings. Further, ligands were optimized by using inbuilt optimization process in the autodocking. The binding site and box size was defined with default settings by using binding site residues PHE373 in 3OV2 protein and PHE45 residue in 5JCT protein.

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ACCEPTED MANUSCRIPT In vitro anti-cancer studies Cell culture Human breast adenocarcinoma cells (MCF-7 cells and MDA-MB 231) were aseptically cultured in T-25 cell culture flasks or multi-well cell culture plates in Hi-Gluta XL™ Dulbecco’s Modified

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Eagle’s Medium, (High Glucose) supplemented with 10% fetal bovine serum, 50 units/ml

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penicillin, and 50 µg/ml streptomycin. 24 h after seeding, cell lines were treated with 10.0 μM of either of AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli (optimized dose) when treated

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alone or with the combinations Curcumin-Turmeric (CT), Curcumin-Quercetin (CQ), TurmericQuercetin (TQ), Curcumin-Paclitaxel (PC), Turmeric-Paclitaxel (PT), Quercetin- Paclitaxel (PQ), (PCT),

Curcumin-Turmeric-Quercetin

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Curcumin-Turmeric-Paclitaxel

(CTQ),

Curcumin-

Quercetin-Paclitaxel (PCQ), Curcumin-Turmeric-Quercetin-Paclitaxel (PCTQ) 5.0 μM of

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multiple agents for combination therapy. These doses were selected based on published work [27] and the dose curves were generated in our laboratory. Cell lines were processed for various tests

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at indicated post-treatment periods (36 h for cell Apoptosis; 36 h for cell cycle; 36 h for quantitative

Cell viability assay/MTT Assay

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PCR).

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MCF-7, MDA-MB231 and HEK293 cell lines were plated in 96-well plates. The cell lines were treated with vehicle control or different doses of AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-

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Pacli (10.0 μM), and with combinations of CT, CQ, TQ, PC, PT, PQ, PCT, CTQ, PCQ and PCTQ (5.0 μM) for 36 h. We have selected 5 μM of dose in combination therapy which is exactly half of

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the dose of the nanoconjugates during their pristine administration. The reason behind this selection is to avoid unwanted cytotoxicity in normal cells as in combination of b-AuNPs it is much more cytotoxic than its pristine form. After 36 h, cell viability was analyzed using MTT assay by published protocol and absorbance was recorded at 475 nm with reference to 660 nm. [3]

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ACCEPTED MANUSCRIPT Apoptosis by Annexin-V FITC assay The Annexin-V assay was used to quantify the number of apoptotic cells by flow cytometry. In apoptotic cells, phosphatidyl serine is translocated from the inner to the outer leaflet of the plasma membrane. Annexin V labeled with a fluorophore can identify apoptotic cells by binding to phosphatidylserine exposed on the outer leaflet. Briefly, MCF-7 and MDA-MB 231 cells were

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treated with AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli (10.0 μM), and with

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combinations of CT, CQ, TQ, PC, PT, PQ, PCT, CTQ, PCQ and PCTQ (5.0 μM) for 36 h. Further,

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the cells were trypsinized, washed once in PBS, and the pellets were collected after centrifugation and resuspended in binding buffer. To the resuspended solution, 5 μL fluorescein isothiocyanate

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(FITC)-labeled Annexin-V and 10 μL propidium iodide (PI) were added. The resulting mixtures were incubated in dark for 10 min at room temperature and the fluorescence of the cells was

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determined immediately with a flow cytometer [16].

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Clonogenicity assay

Approximately 100 ± 5 cells of MCF-7 and MDA-MB 231 cell lines were plated per well in a 6-

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well plate. Both the cell lines were treated with 10μM AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli and with combinations of and with combinations of CT, CQ, TQ, PC, PT, PQ, PCT,

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CTQ, PCQ and PCTQ (i.e. 5.0 μM of each) for 24 h. After 24 h the culture medium was replenished and subsequently changed once every 3 days without treatment. After 10 days the medium was

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removed and cells were fixed in methanol, stained with 0.1% crystal violet and colonies were counted (>50 normal appearing cells considered one colony). All experimental groups were taken

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in triplicates [28].

Cell cycle analysis

MCF-7 and MDA-MB 231 cell lines were cultured in 60 mm dishes and treated with optimized dose(s) of single agent AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli (10.0 μM), and with combinations of CT, CQ, TQ, PC, PT, PQ, PCT, CTQ, PCQ and PCTQ (5.0 μM). Cells were harvested 36 h after treatment and prepared for cell cycle examination probing with propidium iodide (PI) for 15-20 minutes in the dark at room temperature. Cell cycle phase distribution was

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ACCEPTED MANUSCRIPT estimated using a Becton-Dickinson flow cytometer (BD Biosciences, Heidelberg, Germany) per the manufacturer’s instructions. Result was showed as percent cells in G0/G1, S, and G2/M [28]. Spheroid migration of breast cancer cell lines MCF-7 and MDA-MB 231 cell lines were cultured for 24h. Both cell lines (3 × 105) were mixed

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in complete medium, and cultured onto 1.2% agarose-coated 96-well plates. Then, the plates were

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kept on a shaker at 400 rpm for 48 h. Each spheroid was moved to individual wells of 12 well plates, and cultured for 24 h with or without treatments AuNPs-Cur, AuNPs-Tur, AuNPs- Qu and

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AuNPs-Pacli (10.0μM), and with combinations of CT, CQ, TQ, PC, PT, PQ, PCT, CTQ, PCQ and PCTQ (5.0μM) breast cancer cell lines. Spheroids were photographed at 10X magnifications using

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a Motic digital camera. The observed results were stated as a percentage of the migration of treated

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and untreated cell lines [29]. Quantitative RT-PCR

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CRC cell lines were treated with DMSO or the optimized concentration of Curcumin and its

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analogues for 24-36 h. Cell line or tissue total RNA were extracted with RNASure mini isolation kit Nucleo-pore, Genetix, cDNA solution with Emerald Amp® GT PCR Master Mix (Takara) instructions and cDNA were prepared using Thermo fisher cDNA synthesis kit with primers3. The

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analysis of gene expression was performed using gene specific primers (Table 1). The qRT-PCR

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steps were: 1) Denaturation at 95 °C for 3 min, 2) 30 cycles at 95 °C for 1 min, 3) 57 °C for 30 s (depending on primer sets), 4) 72 °C for 1 min, and 5) Extension at 72 °C for 7 min. Melting curve examination verified a single product. Relative expression quantities were evaluated and

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normalized by comparing to action.

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ACCEPTED MANUSCRIPT Table 1: List of primers employed for assessing the gene expression of the proposed genes (Hypoxia inducible factor (HIF1α), vascular endothelial growth factor (VEGF), STAT-3, Cyclin kinase inhibitor (Cyclin D1), Caspase-9 and GAPDH) using real-time PCR. Immunocytochemistry

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MCF-7 and MDA-MB231 cell lines were plated in 12-well chamber slides and then treated as

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previously described. Cell lines were kept in 4% formalin (for fixation) for 30 min and subsequently cell lines were permeabilized with Triton-X100. Cell lines were blocked with 3%

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Bovine serum albumin (BSA) in Phosphate buffered saline (PBS) for 30 min. Then the cell lines were incubated with primary antibody for Caspase-9 (1:1000 dilutions) for 4 h at RT. The cell

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lines were washed with 1XPBS for three times for every 5 min and followed by Alexa Fluor®conjugated secondary antibodies was added. Subsequently, DAPI were used for nuclear staining

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and slides then mounted. The slides were examined by fluorescence microscope [16].

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Results:

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Physicochemical characterization analysis

Figure 1 A: Biosynthesis and UV-Visible spectroscopy studies of b-AuNPs. The ruby red color supports the formation of gold nanoparticles upon reduction, B: Characterization

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studies of AuNPs by Transmission Electron Microscope (TEM) and Dynamic light scattering

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(DLS) studies.

The figure No: 1 confirms the formation of gold nanoparticles for all the four different sets

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(AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli) as all the four sets showed ruby red color due to the reduction of choloroauric acid in the presence of natural phytochemicals. The TEM images of the AuNPs-Cur, AuNPs-Tur, and AuNPs-Qu, AuNPs-Pacli showed monodispersed spherical nanoparticles Figure 1-A showed the absorption spectra of all the four sets of AuNPs at λmax ~530-540 nm, which is attributed to the surface plasmon excitation of small spherical sized AuNPs. The TEM images of the AuNPs-Cur, AuNPs-Tur, and AuNPs-Qu, AuNPs-Pacli showed monodispersed spherical nanoparticles. TEM analysis was carried out to determine the size of all 8

ACCEPTED MANUSCRIPT the four different sets of b-AuNPs. The average size of AuNPs-Cur, AuNPs-Tur, and AuNPs-Qu, AuNPs-Pacli was found to be 5-25 nm, 3-20 nm, 15-60 nm and 15-45 nm; respectively, which is attributed to the surface plasmon excitation of small spherical sized AuNPs [30]. DLS studies were carried out to calculate the hydrodynamic diameter and zeta potential. DLS results showed the size of the nanoparticles were for AuNPs-Cur, AuNPs-Tur, and AuNPs-Qu, AuNPs-Pacli is 74.5 nm, 53 nm, 105 nm and 87.6 nm; respectively (Figure 1B). It is important to mention that the size of

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the b-AuNPs obtained from DLS is more than the size obtained from TEM.

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This is because DLS provides the hydrodynamic diameter of the nanoparticles that includes any nanoparticles surface coating by phytochemicals or assembly of water molecules around the

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nanoparticles. However, TEM provides the accurate size of the metallic part of the nanoparticles. We also measured the zeta potential for the all four sets of b-AuNPs. Zeta potential gives the information about surface charge or surface potential of the nanoparticles. High positive or

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negative zeta potential data indicates the colloidal stability of nanoparticles. Herein, we analyzed

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the zeta potential value for AuNPs-Cur, AuNPs-Tur, and AuNPs-Qu, AuNPs-Pacli. Result shows highly negative zeta potential [AuNPs-Cur: -16.2±0.2 mV, AuNPs-Tur: -30.8±0.8

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mV, and AuNPs-Qu: -23.2±0.8 mV and AuNPs-Pacli: -19.9±0.4 mV] for all the four sets that supports the colloidal stability of the nanoparticles (Figure S2). In order to investigate the

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concentration of the gold (Au) in the different sets, ICP-OES analysis was performed. The result demonstrated that the amount of Au present in the following samples (AuNPs-Cur, AuNPs-Tur,

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AuNPs-Qu and AuNPs-Pacli) was 0.075, 0.084, 0.108 and 0.072 mg/mL; respectively. TGA analysis was further carried out in order to determine the organic content in the surface of the

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nanoparticles and results were presented in a tabular for below (Table 2, Figure S1). Table 2: Determination of organic content in b-AuNPs sample by TGA and ICP-OES analysis Molecular docking Figure 2: In-silico studies: Molecular docking and binding site interaction studies.

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ACCEPTED MANUSCRIPT The representation of the binding site interactions along with residue name and distances (in Å) of Curcumin, Quercetin and paclitaxel ligands in the 3OV2 and 5JCT proteins. Residues within the 5Å distance from ligand are represented in bonds and Curcumin/Quercetin ligands represented in CPK model. (Figure 2).

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Cytotoxicity studies

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MTT assay

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Figure 3 (A-C): MTT assay for assessing the cytotoxicity of individual and combinatorial b-

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AuNPs treatments in cancer cell lines (MCF-7 and MDA-MB 231) and normal (HEK 293). We have carried out in vitro studies for assessment of anticancer efficiency of AuNPs-Cur, AuNPsTur, AuNPs-Qu and AuNPs-Pacli alone and and with combinations (Curcumin-Turmeric (CT),

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Curcumin-Quercetin (CQ), Turmeric-Quercetin (TQ), Curcumin-Paclitaxel (PC), TurmericPaclitaxel (PT), Quercetin- Paclitaxel (PQ), Curcumin-Turmeric-Paclitaxel (PCT), Curcumin(CTQ),

Curcumin-Quercetin-Paclitaxel

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Turmeric-Quercetin

(PCQ),

Curcumin-Turmeric-

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Quercetin-Paclitaxel (PCTQ)) were performed by MTT assay in cancer cells (MCF-7, MDA-MB 231) and normal cells (HEK 293) after 36h of exposure (Figure-3C).

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Combination of AuNPs-PCTQ (5.0 μM) demonstrated excellent synergistic cytotoxic effect when compared to individual treatment in various breast cancer cell lines (MCF-7 and MDA-MB 231)

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(3A-B). We have selected 5 μM of dose in combination therapy which is exactly half of the dose of the nanoconjugates during their pristine administration. The reason behind this selection is to

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avoid unwanted cytotoxicity in normal cells as in combination of b-AuNPs it is much more cytotoxic than its pristine form. However, normal cells did not show any cytotoxicity at the similar conditions. It is important to mention that we have also performed the in vitro cytotoxicity using 10 μM of combination of b-AuNPs in normal HEK 293 cells resulting in normal cell killing. Hence, we avoided the higher dose (10 μM) for combination therapy.

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ACCEPTED MANUSCRIPT Gene expression studies Quantitative Reverse transcriptase-PCR studies Figure 4: Gene expression studies of varied genes (Hypoxia inducible factor (HIF1α), vascular endothelial growth factor (VEGF), STAT-3, Cyclin kinase inhibitor (Cyclin D1),

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Caspase-9 and GAPDH) in MCF-7 and MDA-MB 231 cell lines after treatment with different

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b-AuNPs in combination or pristine mode of treatment.

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The expression of Caspase 9, HIF, VEGF, Cyclin D, and STAT genes was studied in both MCF7 and MDA-MB 231 cell lines with AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs- Pacli

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treatment alone and with combinations CT, CQ, TQ, PC, PT, PQ, PCT, CTQ, PCQ, PCTQ in MCF-7 and MDA-MB 231 cell lines. We observed that the combinational treatments significantly

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down regulated HIF-1α, VEGF, CYCLIN D1 and STAT-3 genes and up regulated the Caspase-9 expression in comparison to the individual treatments in both MCF-7 and MDAMB 231 cell lines.

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The individual and combinational treatments gave significant results in case of MCF-7 cell line

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when compared to the MDA MB 231 cell line.

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Apoptosis by ANEXIN-5 and PI binding assay.

Figure 5: Apoptotic assay- Percentage of cells undergoing apoptosis in different breast

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cancer cell lines (MCF-7 and MDA-MB 231) after treatment with different b-AuNPs in combination or pristine mode of treatment (Annexin V – PI assay).

The cells undergoing apoptosis was quantified by the Annexin-V binding assay. The Annexin-V (Apoptotic cells) phosphatidyl serine (Dead cells) showed that AuNPs-Cur, AuNPs-Tur, AuNPsQu and AuNPs-Pacli (10.0μM) treated cells have the ability to induce apoptosis (39%) in MCF-7 cell line, though the degree of the impact varied with the treatment and the cell line (Figure 5A.B). The total number of cells undergoing apoptosis in MCF-7 was more compared to MDA-MB 231 cells. The anti cancer activity of the proposed combination at reduced dose levels of AuNPs-CTQ 11

ACCEPTED MANUSCRIPT and AuNPs-PCTQ (5.0μM) treated cells showed higher apoptosis (46%) compared to individual treatments in MCF-7 cells and MDA-MB 231 cells (5A-B).

Colony formation assay

Figure 6: Effects of individual and combinations of b-AuNPs on colony formation of MCF-7

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and MDA-MB 231 cell lines.

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The figure 6 (A-B) shows the ability of immortal cells (MCF7 and MDA-MB 231) to form a colony in the presence of AuNPs-Cur, AuNPs- Tur, AuNPs-Qu, AuNPs-Pacli alone and in combinations

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[AuNPs-CTQ and AuNPs-PCTQ (5μM)] when the cells were stained using crystal violet stain. The anchorage-dependent colony formation assay showed a significant reduction in colony

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formation in MCF7 cells (7A-B) when treated with AuNPs-Cur, AuNPs- Tur, AuNPs-Qu, AuNPsPacli individually and in combinations AuNPs-CTQ and AuNPs-PCTQ. Similar treatments in

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MDA-MB 231 cells (7A-B) did not have that significant effect in controlling the cells from colony formation. The combination of AuNPs-CTQ and AuNPs-PCTQ certainly has anti-cologenic

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efficiency in both the cell lines. The results obtained were statistically significant (**P<0.01).

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Cell Cycle Analysis:

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Figure 7 (A-B): Effect of individual and combinatorial AuNPs (Curcumin, Turmeric,

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Quercetin and Paclitaxel) treatment on cell cycle in MCF-7 and MDA-MB 231 cell lines. MCF-7 and MDA-MB 231 cells were treated with biosynthesized nanoparticles individually (AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli) at 10.0 μM/mL and in the combination of AuNPs-CTQ and AuNPs-PCTQ at 5.0 μM/mL for 36 h. Untreated cells were kept as control. The results obtained were statistically significant (**P<0.01). (Figure 7A-B) The cell cycle analysis demonstrates alteration in the cell cycle phases in both MCF-7 and MDA-MB 231 cell lines on treatment with individual or combinations i.e. AuNPs-Cur, AuNPs-Tur, AuNPs-Qu, AuNPs-Pacli, AuNPs-CTQ and AuNPs-PCTQ.

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Table 3: Cell cycle analysis of MCF-7 and MDA-MB 231 cell lines treated with AuNPs-Cur, AuNPs- Tur, AuNPs-Qu, AuNPs-Pacli alone and in combinations [AuNPs-CTQ and AuNPs-PCTQ (5μM)], 36 hr). The percent cells in each phase of the cell cycle were analyzed using a Becton-Dickinson flow cytometer. The table 3 represents percentage of cells with fractional DNA content (apoptotic cells) and cells in G1 , S, and

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G2/M phases of the cell cycle.

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Percentage of cells in different cell cycle phases observed after 36 h of treatment with AuNPs

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individually and in combinations in both cell lines (Table 3). Findings from cell cycle analysis demonstrate significant percentage of MCF-7 cells got arrested in G0/G1 phase on treatment with AuNPs treatment i.e. both individual and combinations. Whereas the similar treatment pattern in

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MDA-MB 231 cell line showed significant percentage of cells were arrested in both G0/G1and S phases, indicating the effectiveness of AuNPs in inducing cell arrest in various phases of cell cycle

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both individual and combinations.

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Spheroid Formation assay

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Figure 8 (A-B): Spheroid migration Assay-Effect of AuNPs-Cur, AuNPs-Tur, AuNPs-Qu, AuNPs-Pacli, AuNPs-CTQ and AuNPs-PCTQ on spheroid forming and migration ability of

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MCF-7 and MDA-MB 231 cell lines.

The above figure describes the effect of individual and combinatorial AuNPs (Curcumin,

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Turmeric, Quercetin and Paclitaxel) on the formation of spheroid and migration ability of immortal cells. The spheroid model human breast cancer cell lines were analyzed in the homotypic tumor

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cell line but underwent a spontaneous transformation towards invasive morphology around 24 h in 3D cultures was observed in MCF-7 and MDA-MB 231 cells. The onset of morphological transformation into the stellate, invasive phenotype was dependent on cell density. Cell death could be more in AuNPs- Tur, AuNPs-Qu and AuNPs-Pacli (10.0μM) in MCF-7 cells compare to MDAMB 231 cells combination of drugs significantly death compare to individual treatments of both the cell lines. (Figure 8 A and B) The concentration of gold in MCF-7 cells was further determined by ICP-OES analysis. The results showed enhanced uptake of gold particles in MCF-7 cells in the

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ACCEPTED MANUSCRIPT combinatorial treatment compared to the pristine nanoparticles treatment. The results were given in a tabular form below. Table 4: Cellular uptake of b-AuNPs in MCF-7 cells by ICP-OES analysis. The concentration of gold was represented as pg/cell value. All the cells were treated with same amount of b-AuNPs for

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24 hours for uptake analysis.

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Discussion

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In the present study, we have demonstrated the synthesis of gold nanoparticles by using previously published green synthesis method by Balakrishnan et al [16]. AuNPs-Cur, AuNPs-Tur, AuNPs-

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Qu and AuNPs-Pacli were employed for studying the anticancer efficiency in multidrug-resistant cancer cell lines. These synthesized nanoparticles were characterized by several physicochemical

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techniques. All the four different b-AuNPs showed nearly monodispersed spherical nanoparticles. DLS studies further showed that the size of the nanoparticles is less than 100 nm, which is ideal

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for the biomedical applications. Zeta potential data further support the colloidal stability and mono dispersity of the nanoparticles. The structure of these plant based phytochemicals is well known

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and these contain poly phenolic or alcoholic groups that facilitate nanoparticle synthesis due to the ambient redox reactions. Moreover, these phenolic or alcoholic compounds play a crucial role in

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the stabilization of gold nanoparticles upon dative binding between –OH and Au. The presence of surface coating by these phytochemicals provides extra stability against air oxidation or

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aggregation (Fig 1 A,B and Table 1& Fig S1A, S2). Molecular docking studies were carried out to analyze the protein-ligand interactions. In

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this study, we selected Curcumin synthase 1 from Curcuma longa (IPDBID: 3OV2) and Human pirin (PDBID: 5JCT) proteins which are targets for the breast cancer along with Curcumin, Quercetin and paclitaxel ligands. The docking results reveal strong binding energy of ligands with the 3OV2 and 5JCT proteins. In general, all ligands considered in this study showed similar binding strength within the difference of ~0.4 kcal/mol in 3OV2 protein. However, the Curcumin showed strong binding energy (-8.90 kcal/mol) with 3OV2 protein and with 5JCT protein it showed -8.70 kcal/mol binding energy. On the other hand, Quercetin also showed similar binding strength as Curcumin ligand with 3OV2 (-8.80 kcal/mol) and 5JCT (-8.30 kcal/mol) proteins. The

14

ACCEPTED MANUSCRIPT paclitaxel ligand has strong binding strength than other ligands with 3OV2 protein, however, with 5JCT protein this ligand showed weaker interaction than Curcumin and Quercetin. The preceding analysis on binding energies clearly shows that the Curcumin ligand binds strongly with both 3OV2 and 5JCT proteins. (Fig 2 supporting information Table 2-S-2). To understand the mode of ligand binding at atomistic level we analyzed protein-ligand

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interactions in detail. In 3OV2 protein-ligand binding pocket, Curcumin ligand formed hydrogen

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bonds with MET59 and ALA210 residues within 5Å distance from ligand. Along with these

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interactions, Curcumin also stabilized with aromatic π-π (PHE265) and CH-π interactions (PHE267). Furthermore, we can also observe CH-O type of interactions in Curcumin with PHE267 and PHE265 residues. In 5JCT protein binding site also Curcumin ligand stabilized though π-π

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interaction with PHE53 residue, CH-π interaction with PHE45 and ALN45 residue within 4Å distance from the ligand molecule. In addition, Curcumin showed strong hydrogen bonding

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interactions formed with PRO54, TRP117, HIS58 and ASP43 residues in the binding site. All these hydrogen bonding interactions are formed within 4Å distance from ligand. Further, Quercetin

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ligand also showed strong hydrogen bonding and other non-covalent interactions such as π-π, CH-

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π, NH-π and CH-O interactions. Finally, Curcumin have showed more strong interactions with binding site residues in the 3OV2 and 5JCT proteins. In conclusion, this analysis reveals that various non-covalent interactions and strong hydrogen bonding interactions of Curcumin results

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strong activity than paclitaxel and Quercetin towards breast cancer. (Fig 2).

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The efficacy of AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli on cell viability was determined by various in vitro assays. The IC50 values of AuNPs-Cur, Tur, Qu and Pacli in both

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MCF-7 and MDA-MB 231 cell lines were found as 10.0 μM/mL, respectively, and IC50 values of the combination of AuNPs-Cur, Tur, Qu and Pacli was found as 5.0μM/mL, for MCF-7 and MDAMB 231 cell lines respectively [31,32]. In the present study we were successful in inducing cytotoxicity in breast cancer cells on treatment with AuNPs-Cur, AuNPs-Tur, AuNPs-Qu and AuNPs-Pacli at the concentration of 10μM, however the treatment with AuNPs-Pacli have no significant cytotoxicity towards MDA-MB 231 cell line i.e due to the absence of surface markers and resistance towards the Paclitaxel [33]. When comparison was made between the individual AuNPs (Cur, Tur, Qu and Pacli) and combination of AuNPs (CT, CQ, TQ, PC, PT, PQ, PCT, CTQ, PCQ and PCTQ ) in both cell lines (MCF-7 and MDA-MB 231), it was found that 15

ACCEPTED MANUSCRIPT combination treatments were exerting better apoptotic and cytotoxicity in both MCF-7 (46%) and MDA-MB 231 (39%) cell lines. Similarly, when we treated the normal cell line (HEK-293) with these individuals and combinations, we could not see any cytotoxicity being induced. Hence, we conclude that the AuNPs (Cur, Tur, Qu and Pacli) have protective property, but specifically targets the cancer cells. Our findings are in concurrence with the results published by Zamzami et al [34].

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(Fig 3 A-C).

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Apoptosis is a process of programmed cell death [35]. illustrates the characteristic features

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of apoptotic cells, i.e. cell shrinkage, nuclear fragmentation, pyknosis and extensive plasma membrane blebbing leading to the formation of apoptotic bodies which will be subsequently

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phagocytosed by macrophages [36]. Bcl-2 is considered as an anti-apoptotic protein, as it controls the apoptosis process by governing the mitochondrial outer membrane permeabilization (MOMP). The Bcl-2 family proteins activate caspase-9 and caspase-3, triggering the apoptosis cascade [37].

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The role of cysteine aspartic acid protease (caspases) family for the initiation of apoptotic pathways is well reported [38, 39]. It was also mentioned that amongst all caspases known; the caspase-3

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was mostly implicated in several apoptotic pathways.

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Angiogenesis is a physiological process for the formation of new blood vessels from preexisting vessels and is considered to be a vital process that regulates endothelial cell growth,

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migration and survival of cells [40].VEGF is the major regulator of angiogenesis in breast cancers. NF-kB controls the transcription of several angiogenic factors including VEGF [41]. In the present

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report the treatments with AuNPs (Cur, Tur, Qu and Pacli) individually and combination of AuNPs (CT, CQ, TQ, PC, PT, PQ, PCT, CTQ, PCQ and PCTQ ) have shown potential in down regulating

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the expression of multiple angiogenic factors and preventing the development of hypoxia driven aggressive tumors. As such these compounds may be used as effective anti-angiogenic agents for clinical use in breast cancer treatment in comparison to the commercial drugs targeting the VEGF [42] (Fig 4). These trials have assessed therapeutic property of b-AuNPs which could specifically target VEGF or its receptors. The challenge in inhibiting angiogenesis relates to the presence of nonVEGF pro-angiogenic pathways that can contribute to resistance. Curcumin has been shown ability to inhibit multiple pathways including HIF-1a and STAT-3 [43,44]. One of the objectives of this 16

ACCEPTED MANUSCRIPT study was to define the mechanism of action of the AuNPs-Cur, AuNPs-Tur, AuNPs-Qu analogs or combination of AuNPs (CT, CQ, TQ, PC, PT, PQ, PCT, CTQ, PCQ and PCTQ) that resulted in the down regulation of a number of cellular signaling molecules including HIF-1a and STAT3. NF-kB regulates HIF-1a and STAT-3 expression at the transcriptional level [45]. Our findings suggest that AuNPs-Cur, AuNPs-Tur, AuNPs-Qu alone and in combination

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with CT, CQ, TQ, PC, PT, PQ, PCT, CTQ, PCQ and PCTQ inhibited the cyclin D1 gene

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expression by blocking cell cycle progression that leads to cell death. The advantages of using

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these natural compounds in the conjugated form of with gold nanoparticles could act as an alternative for anti-breast cancer treatment in near future. (Fig 4).

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In the present study the apoptotic and dead cells in MCF-7 and MDA-MB 231 cell lines were analyzed by flow cytometry, the apoptotic cells bind to Annexin V-FITC (Green), whereas

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the propidium iodide taken up to dead cells (Red). It was observed that the combination treatments exert better apoptotic efficiency in both MCF-7 and MDA-MB 231 cell lines in comparison to

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individual treatments. The present study reports about the observed alterations in nuclear

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morphology in MCF-7 and MDA-MB- 231 cell lines. (Fig 5 A-B). In our study, the combinational treatment enhanced Caspase 9 gene expression in both cell

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lines, in comparison to individual b-AuNPs (Cur, Tur, Qu and Pacli) treatments. The cells treated with b-AuNPs showed condensed chromatin and fragmented nuclei, which clearly depicted the

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induction of apoptosis in these cells, which was not, observed in the untreated cells (negative control). Among the above treatments given only AuNPs-Pacli was not able to enhance the

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expression of caspase-9 levels significantly in MDA-MB 231 cells, as significantly as it induced in MCF-7 cell line (Supporting Information S1). The present study further demonstrates the anti-cologenic properties exerted by the bAuNPs in the immortal cell lines. The immortal cell lines in (MCF-7 and MDA-MB 231) have the ability to form colonies naturally in vivo and in vitro conditions when presented with amicable environment. We observed disaggregation of colonies on treatment with individual b-AuNPs, but significant disaggregation was noticed in combination treatment with AuNPs-Pacli in both cell

17

ACCEPTED MANUSCRIPT lines. The disaggregation was more significant in MCF-7 cell line in comparison to MDA-MB231 cell line. (Fig 6A-B) The observations from our cell cycle analysis clearly indicate the mechanism of action of these b-AuNPs and also recommend the combinational treatments. These AuNPs tends to executive differently in cell types. In case of ER positive cell line (MCF-7) most of the cells got

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arrested in the G1 phase and less percentage of cells in the sub-G1, S and G2/M phases when

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treated with individual b-AuNPs, but in case of combination treatments the high percentage of

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cells were found in sub-G1 phase in comparison to that in S and G2/M phases, indicating the ability of AuNPs in inducing apoptosis [27]. Secondly, these b-AuNPs did not execute similar mechanism

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of action in a triple negative cell line (MDA-MB 231) where the cells got arrested in both G1 and S phases when treated with individual b-AuNPs along with the combinations therapy. Moreover, the percentage of apoptotic cells increased in the combination treatments in comparison to the

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individual treatments (Fig 7A-B) [38].

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Spheroid model was created in in vitro for assessing their response towards the varied drug treatments [46]. The spheroids (MCF-7 and MDA-MB 231 cells) were found to be sensitive

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towards the individual b-AuNPs and also to the combination of CTQ and PCTQ. The spheroids showed less sensitivity towards paclitaxel AuNPs treatment when compared to the others.

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However, the treatment of b-AuNPs combinations showed enhanced disaggregation of spheroids in both cell lines. Nonetheless, its efficiency was found to be less in MDA-MB 231 cell line

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compared to MCF-7 cell line. These AuNPs exert pleiotropic mechanism of action with low

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toxicity, hence they can be considered for anti-cancer treatment. (Fig 8 A-B). The in vitro studies done by Zheng et al [27] demonstrated the synergistic effect of Curcumin and Quercetin in human gastric carcinoma cells (i.e. MCG 803 cells) and their mechanisms of action in inducing the apoptosis in carcinoma cells by inhibiting the phosphorylations of AKT and ERK via mitochondrial pathway. It is reported that Quercetin enhances the bioavailability and uptake of Curcumin into human carcinoma cells. The present study, demonstrated the usage of b-AuNPs of curcumin, turmeric and quercetin in combination with paclitaxel that did not exert any cytotoxicity in the normal cell line (i.e. HEK 293), indicating its biocompatibility. The present study also reports similar efficiencies of combinational treatments 18

ACCEPTED MANUSCRIPT in inducing apoptosis in both MCF-7 and MDA-MB 231 cells when compared to the individual treatments. Niedzwiecki, A. et.al studied the anticancer efficacy of Polyphenols and their combinations in various immortal cell lines (i.e. Prostate cancer cells, Breast cancer cells (MCF-7 and MDAMB 231, human B-cell chronic leukemia, HP V-positive head and neck squamous cell carcinoma

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and lung A549 cells). Their study demonstrated the combinations of epigallocatechin gallate

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(EGCG) with Quercetin, EGCG with resveratrol, EGCG with Curcumin, EGCG with Curcumin

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and resveratrol towards cytotoxicity, anti-cologenic survival, apoptotic and anti-tumor effects. These above mentioned combinations showed less toxicity in normal cell lines that also provided

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the scope for less concentrated dosages [47]. Similarly, our biosynthesized AuNPs has helped us in reducing the over usages of anti-cancer drugs in higher concentrations and also curb its bioavailability and side effects in cancer patients. This study is first of its kind, which reports the

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synergistic efficiency of mixture of biosynthesized AuNPs (Cur-Tur-Qu-Pacli) in inducing

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apoptosis in immortal cell lines.

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Conclusion

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Four distinct types of biosynthesized AuNPs (b-AuNPs) was prepared using nature derived phytochemicals (Cur, Tur, Qu and Pacli). All the b-AuNPs were thoroughly characterized by

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several analytical techniques. Anti-cancer potential of these b-AuNPs was determined in various breast cancer cells (MCF-7 and MDA-MB 231). Combinations of AuNPs‐ Cur, AuNPs‐ Tur,

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AuNPs‐ Qu and AuNPs‐ Pacli were found to be therapeutically most effective in inhibiting breast cancer cell proliferation, apoptosis, angiogenesis, colony formation and spheroid formation predicting a synergistic effect when compared to individual treatment of these phytochemicals or the nanoparticles. Together the results demonstrated the potential anti-cancer activities of bAuNPs in a combinatorial approach that could be the future of cancer nanomedicine due to low toxicity and high therapeutic activity. Acknowledgments: We want to thank Mr. Sourav Das and Dr. Chitta Ranjan Patra (CSIR-IICT) and Mr Kranthi Kiran, Dr. Saurabh Kumar Srivastava, Dr. Kiran Kumar Bokara from CSIR-

19

ACCEPTED MANUSCRIPT CCMB for their guidance during the experimentation. Dr. Sudip Mukherjee acknowledges Dr. Omid Veiseh (postdoc mentor) and Rice University for providing continuous support and research facility. Conflict of interest: There is no conflict of interest and we did not any receive any grant or funds

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for carrying out the work.

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ACCEPTED MANUSCRIPT Table 1: List of primers employed for assessing the gene expression of the proposed genes (Hypoxia inducible factor (HIF1α), vascular endothelial growth factor (VEGF), Signal transducer and activator of transcription 3 (Stat-3), Cyclin kinase inhibitor (Cyclin D1), Caspase-9 and GAPDH) using real-time PCR. Sequence direction 5’-3’

Product size

HIF-1α-sense

CAG AGC AGG AAA AGG AGT CA

231 bp

-antisense

AGT AGC TGC ATG ATC GTC TG

Caspsase-9 sense

CTCAGACCAGAGATTCGCAAAC

antisense

GCATTTCCCCTCAAACTCTCAA

GAPDH sense

GGCTCTCCAGAACATCATCCCTGC

antisense

GGGTGTCGCTGTTGAAGTCAGAGG

VEGF-sense

GCT ACT GCC ATC CAA TCG AG

-antisense

CTC TCC TAT GTG CTG GCC TT

CyclinD1-sense

TTC AAA TGT GTG CAG AAG GA

-antisense

GGG ATG GTC TCC TTC ATC TT

STAT-3 -sense

GCT TCC TGC AAG AGT CGA AT

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Gene

118 bp

250 bp

208 bp

221 bp

87 bp

ATT GGC TTC TCA AGA TAC CTG

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-antisense

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Table 2: Determination of organic content in b-AuNPs sample by TGA and ICP-OES analysis Organic content (μg for 1 mg bAuNPs)

% attachment

AuNPs-Curcumin

30

5.8

AuNPs-Paclitaxel

12.5

2.5

AuNPs-Quercetin

78

15.6

AuNPs-Turmeric

64.5

13.1

Concentration of phytochemicals (μg/mL of b-AuNPs)

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Sample name

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0.6

1.56 1.29

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PT

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M

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0.25

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Table 3: Cell cycle analysis of MCF-7 and MDA-MB 231 cell lines treated with AuNPs-Cur, AuNPs- Tur, AuNPs-Qu, AuNPs-Pacli alone and in combinations [AuNPs-CTQ and AuNPsPCTQ (5μM)], 36 hr). The percent cells in each phase of the cell cycle were analyzed using a Becton-Dickinson flow cytometer. The table 3 represents percentage of cells with fractional DNA

Sub-G0

G0-G1

S

Sub-G0

G0-G1

S

G2-M

0.0

15.4

8.6

1.5

63.7

34.4

0.4

3.0

59.1

35.4

2.4

M 0.0

82

8.6

9.7

75.9

AuNPs-Curcumin

3.1

74.8

13.0

9.1

AuNPs-Turmeric

4.8

68.8

06.9

9.3

AuNPs-Quercetin

3.4

72.6

10.6

5.3

9.3

55.1

33.6

2.1

AuNPs-Paclitaxel

14.4

75.5

09.0

1.1

13.4

58.1

28.1

0.3

AuNPs-CTQ

14.6

64.8

10.9

9.7

10.7

63.1

23.2

2.8

0.21 20.9

1.252.6

12.6

14.0

46.5

39.2

1.4

AN

AuNPs-PCTQ

US

Control

CR

groups

G2-

IP

Experimental

MDA-MB 231

T

MCF-7

AC

CE

PT

ED

M

content (apoptotic cells) and cells in G1 , S, and G2/M phases of the cell cycle.

27

ACCEPTED MANUSCRIPT

Table 4: Cellular uptake of b-AuNPs in MCF-7 cells by ICP-OES analysis. The concentration of gold was represented as pg/cell value. All the cells were treated with same amount of b-AuNPs for

Gold concentration (pg/cell)

AuNPs-Cur

79.1

AuNPs-Qu

79.2

AuNPs-Tur

88.1

AN

US

CR

Samples

IP

T

24 hours for uptake analysis.

95.8

AuNPs-Cur-Qu-Tur

113

144.1

AC

CE

PT

ED

AuNPs-Cur-Qu-Tur-Pacli

M

AuNPs-Pacli

28

ACCEPTED MANUSCRIPT Highlights:

1. Biosynthesis of gold nanoparticles (b-AuNPs) by green synthesis method and their

characterization. 2. Demonstration of anti-cancer properties of b-AuNPs (AuNPs-Cur, AuNPs-Tur, AuNPs-

T

Qu and AuNPs-Pacli) individually and in combinations.

IP

3. Molecular docking studies demonstrating the protein binding of varied phytochemicalsindicating the target oriented therapy

CR

4. Results demonstrate that b-AuNPs were able to execute better anti-cancer properties than the known anti-cancer or chemotherapy drug (Paclitaxol).

US

5. The b-AuNPs showed least Cytotoxicity in normal cell line (HEK 293), hence these b-

AC

CE

PT

ED

M

AN

AuNPs could be an alternative for the existing pristine drugs.

29