Targeting of EGFR, VEGFR2, and Akt by Engineered Dual Drug

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Targeting of EGFR, VEGFR2 and Akt by engineered dual drug encapsulated mesoporous silica-gold nanoclusters sensitizes tamoxifen-resistant breast cancer BN Prashanth Kumar, Nagaprasad Puvvada, Shashi Rajput, Siddik Sarkar, Madhusudan Kr. Mahto, Murali M. Yallapu, Amita Pathak, Luni Emdad, Swadesh K. Das, Rui L. Reis, Subhas C. Kundu, Paul B. Fisher, and Mahitosh Mandal Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00218 • Publication Date (Web): 22 May 2018 Downloaded from http://pubs.acs.org on May 23, 2018

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Molecular Pharmaceutics

Targeting of EGFR, VEGFR2 and Akt by engineered dual drug encapsulated mesoporous silica-gold nanoclusters sensitizes tamoxifenresistant breast cancer B. N. Prashanth Kumar,a,e Nagaprasad Puvvada,b,h Shashi Rajput,a,g Siddik Sarkar,d Madhusudan Kr. Mahto,b Murali M. Yallapu,e Amita Pathak,b Luni Emdad,d Swadesh K. Das,d Rui L Reis,f S C Kundu,f, c Paul B. Fisher,d and Mahitosh Mandal,a,* a

School of Medical Science and Technology, bDepartment of chemistry, cDepartment of

Biotechnology, Indian Institute of Technology Kharagpur, West Bengal – 721302, India. d

Department of Human and Molecular Genetics, VCU Institute of Molecular Genetics, VCU

Massey Cancer, Virginia Commonwealth University, School of Medicine; Richmond, VA23298, USA. e

Department of Pharmaceutical Sciences and Center for Cancer Research, University of

Tennessee Health Science Center, Memphis, TN, 38163, USA f

3Bs Research Group, Headquarters of the European Institute of Excellence on Tissue

Engineering and Regenerative medicine, University of Minho, Avepark -4805-017 Barco, Guimaraes, Portugal.

ACS Paragon Plus Environment

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Molecular Pharmaceutics 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

g

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Tumor Initiation and Maintenance, Sanford-Burnham Medical Research Institute, La Jolla, CA

92037, USA h

Chemical Biology, CSIR-Indian Institute of Chemical Technology, Uppal Road, Hyderabad

500007, India. Key words: Silica-gold nanoclusters, ZD6474, Epigallocatechin gallate, VEGFR2, EGFR, Akt.

ABSTRACT

Tamoxifen administration, enhanced overall disease-free survival and have diminished mortality rates in cancer patients. However, patients with breast cancer often fail to respond for tamoxifen therapy due to the development of a drug-resistant phenotype. Functional analysis and molecular studies suggest that protein mutation and dysregulation of survival signalling molecules such as epidermal growth factor receptor, vascular endothelial growth factor receptor 2 and Akt contribute to tamoxifen resistance. Various strategies, including combinatorial therapies show to chemo-sensitize tamoxifen-resistant cancers. Based on chemo-toxicity issues, researchers are actively investigating alternative therapeutic strategies. In the current study, we fabricate a mesoporous silica gold cluster nano-drug delivery system that displays exceptional tumortargeting capability, thus promoting accretion of drug indices at the tumor site. We employ dual drugs, ZD6474 and epigallocatechin gallate (EGCG) that inhibit EGFR2, VEGFR2 and Akt signalling pathways, since changes in these signalling pathways confer tamoxifen resistance in MCF 7 and T-47D cells. Mesoporous silica gold cluster nano-drug delivery of ZD6474 and EGCG sensitize tamoxifen-resistant cells to apoptosis. Western and immune-histochemical


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analyses confirmed the apoptotic inducing properties of the nanoformulation. Overall, results with these silica gold nanoclusters suggest that they may be a potent nanoformulation against chemo-resistant cancers.

1. Introduction Tamoxifen is used extensively against hormone-dependent breast cancer patients1. Since its employment for the metastatic breast cancer therapy, it also increased the prognosis of patient survival. Tamoxifen has chosen against treatment of ductal carcinoma in situ, in addition to chemoprevention of breast cancer. In a large randomized clinical trial, both relapse and mortality are reduced significantly and survival improved in women receiving tamoxifen for 10 years 2. Despite of documented therapeutic efficacy, tamoxifen administration for five years has reduced the incidence of breast cancer to 50% approximately in post-menopausal women 3. However, the success of tamoxifen treatment being is pivotally influenced by the expression of estrogen receptors (ER) in the tumors of the breast cancer. On a molecular level, tamoxifen exhibit inhibitory effects through competitive binding along with estrogen towards ER, subsequently the tamoxifen-ER complex homo-dimerizes thus disabling the functions of estrogen response mediated cell proliferation and survival 4.

The patients with ER breast cancer more prone to tamoxifen therapy. Unfortunately, the vast majority of patients undergoing tamoxifen treatment develop chemoresistant towards the drug over progress of the time and among those 50% of tumors cause the relapse of the disease due to drug resistance 5. Resistance to tamoxifen therapies is prevailing clinical issue for which notable molecular mechanisms have been reported 6. Functional screening of genes in breast cancer cells responsible for resistance demonstrates that induced expression of Akt and over-expression of


3


the PI3K/Akt axis correlate with resistance

7-8

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. Aberrant induction of epidermal growth factor

receptor (EGFR), vascular endothelial growth factor receptor 2 (VEGFR2) and its downstream signaling confers tamoxifen insensitivity in breast cancer patients 9-10. Also, atypical activation of the EGFR/VEGFR2 signaling pathway willingly encourages failure of anti-hormonal treatment in patients with breast cancer 11-12.

Tamoxifen-resistant breast carcinomas often overexpress EGFR/VEGFR2

13-15

. One study

showed the expression of EGFR and VEGFR2 can be chosen for predicting the extent of resistance towards tamoxifen drug

15

. Also, in this study it has been showed that EGFR

expression is inversely co-related to ERα in tamoxifen resistant cancers. Thus revealing that expression of EGFR is elevated in tamoxifen resistant cancer subsequently there was concurrent decrease in ERα expression. Seminal studies showed the growth of cells through estrogen independent mechanisms was regulated through autocrine signaling of VEGF/VEGFR2. This in turn activates the signaling of the downstream that promotes resistance against tamoxifen in breast cancer 16. This relationship is consistent with those reported in other studies 13-15.

Tamoxifen resistance often ensues as a result of the concomitant activation of multiple survival proteins and overlapping signaling that regulate cancer proliferation and survival. Considering this relationship, we choose two agents that target two major signaling that play vital role in survival and growth of breast cancer: (i) EGFR (ii) VEGFR2 signalling pathway and PI3K/Akt activation. Drug combinations targeting different proteins and displaying differential toxicity profiles play a predominantly noticeable role during therapy against multiple cancers

17-18

. To

overcome chemo-resistance and reduce drug side effects during single agent therapies,


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combination chemotherapy represents a promising approach. This can include the application of chemo-sensitizers with cytotoxic activity, thereby providing efficacy and reduced toxicity

17, 19

.

Previous studies showed the co-delivery of tariquidar and paclitaxel through PLGA nanoparticles overcome chemo-resistance and augmented paclitaxel pharmacological effects against chemoresistant breast cancer

19

. We now elucidate the molecular mechanisms underlying this

resistance, which provide a rationale for combining specific EGFR/VEGFR2 and Akt inhibitors. This is an attempt to overcome tamoxifen resistance. We anticipate that co-delivery of ZD6474, an inhibitor of dual tyrosine kinase i.e. EGFR/VEGFR2 along with epigallocatechin gallate (EGCG), a known Akt inactivator. This strategic approach might provide new dimension to existing tamoxifen resistant therapies.

Clinical complications associated through drug delivery include poor drug acceptance, lacking target specificity, variable PK/PD mechanisms and bio-availability. It denotes puzzling elements in the efficacious implication of its therapeutic enactments. To address these subjects, delivery of multiple drugs through nanoparticles may offer a sustainable approach to effect multiple pathways at molecular level through activation of apoptosis in cancer cells without affecting normal tissues

20-21

. Here, we propose the encapsulation of cancer therapeutic drugs ZD6474

with EGCG in silica gold nanoclusters (ZEAuSSi), can subdue the deficiencies linked with standard drug delivery methods and induced resistance. Additionally, the modulation of VEGF/VEGFR2 and Akt signaling through ZD6474 and EGCG can enhance the cancer inhibition in tamoxifen resistant breast cancer cells (MCF 7/TAM and T-47D/TAM). Further, to escalate the effectiveness of current nanoparticle with dual drugs; a targeted tumor-specific strategy is necessary. In this view, we have chosen pH specific delivery of drugs which seem to


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have maximum propitious outcomes and is broadly being tested as a tumor targeting strategy 2223

.

Noble metal-based fluorescent gold nanocluster formulation is gaining significant attention as promising biocompatible nanoprobes for clinical diagnostics including fluorescence, near infrared photoluminescence and optical chirality

24-25

. Highly, luminescent gold nanoclusters

have an ultra-small size along with monodispersible nature associated with core size below 3 nm and contain many gold atom associations (Aux)

25-26

. The ultra-small sized gold nanoclusters

exhibit unique optical luminescence since the restraining of free electrons resulting in high quantum energies. Presently, thiolated compounds and proteins are generally used to produce gold nanoclusters due to their green synthetic approach, biocompatibility and reduced toxicity in comparison with quantum dots of heavy metals

27-28

. The utilization of inherent

fluorescence/luminescence of nanocarriers in drug delivery, biomedical applications for real time monitoring and intracellular/in vivo tracking offers an added advantage in development of novel nanoformulations

25

. For this reason, mesoporous silica nanocarrier-based gold nanoclusters

might serve as promising candidates in the treatment of chemo-resistant cancers.

In this report, ZD6474 and EGCG encapsulated in mesoporous silica gold nanoclusters are employed to assess the therapeutic effects against tamoxifen resistant breast cancer cells. Also, its inherent fluorescence is utilized in tracing its intracellular localization. 2 Material and Methods 2.1 Reagents


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Tetraethyl orthosilicate (Sigma Aldrich Ltd, 98 %), NH3 (Merck Ltd, 25%), cetyltrimethyl ammonium bromide (Merck Ltd, 99 %), mercaptopropyl silane (Sigma Aldrich Ltd, 95 %), tetra chloroauric acid (SRL Pvt Ltd, 49 %), and Bovine serum albumin (Hi-Media, 98 %) were used in the present studies. Rest of other reagents chosen in current study were of analytical grade (not purified) unless mentioned. Drug standard solutions of ZD6474 (10 mM) and Epigallocatechin gallate (EGCG) (10 mM) were solubilized in dimethyl sulfoxide (Sigma-Aldrich), stored at 20ºC (Sigma Aldrich), and further dilutions were prepared in nutrient growth medium before use. In immunoblotting studies, the antibodies employed were: mouse monoclonal anti-Ki67, anti-βactin (Sigma-Aldrich), rabbit monoclonal p-anti-Akt, anti-Akt, p-anti-VEGFR2, anti-VEGFR2, p-anti-EGFR, anti-EGFR anti-Bcl-2, anti-p53, Secondary antibodies included HRP- goat antirabbit IgG, and HRP-goat anti-mouse (Cell signaling technology, Beverly, MA, USA). Also, for protein profiling studies antibodies were diluted and used according to manufacturer instructions. Assay Kits like TUNEL (Roche Applied Science), and other fluorescent dyes such as Propidium iodide, DAPI and MTT reagent were purchased from the Sigma Aldrich and respective diluent solutions were prepared in 1X phosphate buffered saline (PBS) (1 mg/ml). These diluted stocks were stored at 4 °C and protected from light exposure. RNase A stock solutions of 10 mg/ml were prepared and stored at -20 °C.

2.2 Synthesis of mesoporous SiO2 In current study, mesoporous silica nanoparticle synthesis was carried out using the template method at RT 29. Here, CTAB (0.21 gms) added to 5 ml of ethanol, 3.85 ml of water, and 1.4 ml ammonia solution under vigorous stirring for 15 min. The resultant mixtures were mixed with 0.39 g TEOS through drop-wise addition and held for 3 h at room temperature causing a white


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precipitate to form. The resultant products were centrifuged, collected and subsequently washed with dilute HCl to remove CTAB. The resultant product was dried out at 50 °C for 12 h in vacuum oven, which results in the development of mesoporous silica nanoparticles. 2.3 Synthesis of thiolated silica nanoparticles The functionalization of mesoporous silica nanoparticles with thiol groups was achieved by dispersing 100 mg of mesoporous silica nanoparticles in ethanol (20 ml) and subsequently 100 µl of MPTMS was added under argon gas atmosphere. The reaction mixture was allowed to stir for 30 min and left for 3 h. The resultant products were centrifuged, collected, and ethanol rinsed. They were parched out at 50 °C in a vacuum oven for 12 h 30.

2.4 Synthesis of gold nanoclusters Synthesis of gold nanoclusters were reported previously

31

. In brief, 1ml of 10 mM aqueous

gold solution was dissolved with a BSA stock of 20 mg/ml followed by addition of sodium ascorbate (1 µM) and 100 µl of NaOH (1 M) 25 µl at 37 °C through continuous stirring till 7 h, results the formation of gold nanoclusters. A similar procedure was repeated to produce the thiolconjugated mesoporous silica nanostructures (AuSSi) in the presence of thiolated silica nanoparticles (50 mg).

2.5 Synthesis of EGCG-loaded gold nanoclusters on thiolated silica nanoparticles 5.2 mg of EGCG was dissolved in an ethanol solution and 50 mg of SSiO2 nanospheres dispersed and dried out to eliminate the solvent at 37 °C. This resultant sample mixed with 1ml of gold nanoclusters then the resultant samples were conjugated with gold nanoclusters on a silica surface (EAuSSi).


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2.6 Synthesis of ZD6474-loaded gold nanoclusters on thiolated silica nanoparticles 50 mg of SSiO2 nanospheres was dispersed in a PBS solution and then 1 ml of gold nanocluster solution was added to this dispersed 4.8 mg of ZD6474 solution. The resultant sample was named ZAuSSi. Also, identical steps were followed for the synthesis of dual drug formulations (ZEAuSSi).

2.7 Characterization The mesopores of SiO2 were determined through low angle Powdered X-ray diffractometer (XRD) (Bruker AXS Diffractometer, D8) over a Cu Kα radiation (λ=0.15406 nm)

32

. Brunauer

Emmett Teller (BET) measurements were performed for validating the surface area using N2 adsorption desorption isotherms at 77 K by Qunatachrome (Autosorb-1, Model: ASI-C-9). The distribution of mesoporous size was determined by Barrett Joyner Halenda (BJH) method. High resolution transmission electron microscope (JEOL JEM-2100 model, Japan) was chosen to examine the morphology of the resultant particles with 200 kV 33. The surface morphology and elemental analysis of the samples was observed using a field-emission scanning electron microscope (FESEM, JSM 7600F, JEOL, Tokyo, Japan) equipped with energy-dispersive spectrometer (EDAX) with operating voltage of 20 kV

32

. Surface charge and hydrodynamic

radius of the various samples were measured through zeta potential and dynamic light scattering measurement using Zetasizer-4, Malvern instruments, U K 34-36.

2.8 Cell lines, culture conditions and establishing tamoxifen resistant breast cancer cells


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Human breast cancer cell lines MCF 7 and T-47D were purchased from the cell repository institute NCCS, Pune, India. The obtained cells were grown in DMEM medium with 10% FBS and 1% of penStrep solution. Further, the cells were allowed to grow in humidified chamber with 5 % CO2 for the continuation of studies. Tamoxifen-resistant cell lines (MCF 7/TAM and T-47/TAM) were developed by continuous exposure of the wild type MCF 7 and T-47D cells towards the tamoxifen with increasing concentrations as described earlier

33, 37

. The drug resistance in MCF 7and T-47D cells were

initiated from 0.1 µM (initial concentration) and continued till 30 µM of TAM concentration. The passage number of the cells during resistance development were from 18 to 43, till the completion of this study. After one year of continuous drug exposure to cells, the expression of AKT1 and VEGF mRNA were prominently raised in both MCF 7/TAM and T-47D/TAM cells in comparison to their respective wild type counterparts. The MCF 7/TAM and T-47D/TAM cells were cultured DMEM medium as described in previous section.

2.9 Hemocompatibility study The native drugs (EGCG and ZD6474), and AuSSi, EAuSSi, ZAuSSi, and ZEAuSSi nanoparticles, were dispersed in 1X PBS. Hemo-compatibilities of the aforementioned nanoparticles were performed

38-39

. The extent of hemolysis was measured in terms of percent

hemolysis with respect to controls.

2.10 Cell proliferation studies The effect on cell proliferation of the single drug- and dual drug-loaded nanocarriers was evaluated through MTT studies. MCF 7/TAM and T-47D/TAM cells (3.5 × 103/well) were


10

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seeded in a 96 well tissue culture plate and allowed to adhered and grow for overnight. Then AuSSi, EAuSSi, ZAuSSi and ZEAuSSi nanocarriers at varying concentrations were added to wells and incubated for 48 h. Then nanoparticles containing medium was changed with 100 µl MTT (1 mg/ml) reagent and kept for 3-4 h. The medium with MTT was replaced with 100 µl DMSO and absorbance reading was measured at λmax 570 nm 40-41.

2.11 Cell-cycle analysis The percent apoptosis during nanocarriers treated cells were evaluated by cell cycle analysis through flow cytometric studies. Briefly, cells (2 × 105) after 48 h nanoparticle treatment were rinsed two times with 1X PBS to eliminate unbound nanocarriers. Further, cells were harvested and incubated with PI-RNase mixture (PI: 40 µg/ml, RNase A: 100 µg/ml RNase A) at 37°C till 30 min 42. The cell phase distributions were explored through flow cytometer FACSCalibur and CellQuest Pro software (BD Biosciences, Franklin Lakes, NJ).

2.12 Exploring uptake mechanisms of nanoparticles through endocytic inhibitors The molecular interactions of cytoplasmic membrane during nanoparticle uptake was examined in presence of endocytic inhibitors through flow cytometric studies. The MCF 7/TAM and T-47D/TAM cell lines (2 × 105 cells/ml) were treated with inhibitor of clathrin vesicles formation (chlorpromazine-10 µg/ml) or a caveolae inhibitor (genistein-200 µM) at 37 °C till 1 h 43

. Subsequently, these treated cells were subjected for nanoparticle treatment for 4 h. Further,

after the nanoparticle treatment cells were rinsed with 1X PBS thrice, harvested and were analyzed using flow cytometer (BD Biosciences).


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2.13 Measurement of secreted VEGF levels in serum The measurement of VEGF was evaluated through enzyme linked immunosorbent assay (ELISA) method. Tamoxifen resistant breast cancer cells MCF 7/TAM and T-47D/TAM at a seeding density of (4.5 × 105/well) in 6 well plates were seeded and grown for overnight. The following day medium was changed with fresh medium (devoid of serum). The cells were subjected to AuSSi, EAuSSi, ZAuSSi, or ZEAuSSi nanocarrier treatment and their respective medium was collected after 24 h 44. Subsequently, the extracellular VEGF levels were quantified through VEGF ELISA kit (DVE00, R&D Systems, Minneapolis, MN, USA) as per manufacturer’s protocol. The absorbance readings of the samples were measured at 570 nm through Microplate reader (model 550 Bio-Rad, Hercules, CA, USA).

2.14 Immunoblot studies In this study, cells were subjected to drug nanoformulations exposure for 24 h, harvested and cell pellets were collected and suspended in NP-40 lysis buffer. Further, these cell lysates were processed for protein profiling studies

45

to elucidate the molecular effects of nanoparticles on

various cellular signaling and regulatory proteins.

2.15 In vivo investigations Tumor responses to AuSSi, EAuSSi, ZAuSSi, and ZEAuSSi nanocarriers were studied using nude mice models. These in vivo studies were performed after the protocol approvals from the institutional animal care and use committee (VCU School of medicine, Richmond, VA, USA). These mice were accommodated and adapted to pathogen free environment at institutional animal facility till one week before the implantation of MCF 7/TAM cells. The breast cancer


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cells (MCF 7/TAM) cells were trypsinized at their logarithmic growth phase and at a density of 5 × 106 cells were implanted subcutaneously (s.c.) in athymic female BALB/c nude mice

44

.

Tumors were allowed to grow for 14-days and all of the mice were then weighed. Tumor sizes were measured as described previously 46. Subsequently, the mice were divided into 4 different groups with 3 mice in each of the group. The group with vehicle treatment (PBS) i.e., control group-1 was injected intratumorally. The group 2 mice were treated with EAuSSi nanoparticles (2.5 mg/kg/day -body weight) twice in a week intravenously (i.v.) for 5 weeks. Group 3 mice received ZAuSSi nanoparticles (60 mg/kg/day -body weight) twice a week (i.v.) for 5 weeks. Group 4 animals were treated with ZEAuSSi nanoparticles (360 mg/kg/day -body weight) 2 times a week (i.v.) till 5 weeks. These drug doses were selected from previous studies. Tumors volumes and body weights of the all mice groups were measured. The treatment was continued till 5 weeks and after that mice were euthanized and tumor were collected for immunohistochemical studies and and FE-SEM investigations.

2.16 Immunohistochemical studies Immunohistochemical studies (IHC) of tumor tissues were probed with for apoptotic and regulatory proteins with their respective antibodies such as p-EGFR, EGFR, p-VEGFR2, VEGFR2, p-Akt, Akt, Bcl-2 and Bax. Immunohistochemical analysis were conducted as shown earlier 47. The microscopic images were imaged at 10X magnification.

2.17 statistical analysis


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The statistical studies was conducted in this study using graph pad prism 5 software. Data shown as the mean ± S.D. The statistical significance was established through one way analysis of variance (ANOVA). ***P ZAuSSi > EAuSSi > ZD6474 > EGCG > AuSSi = control untreated animals (Figure 8 A and B). Moreover, ZAuSSi, EAuSSi, and ZEAuSSi displayed profound tumor regression in nanocarrier treatments in


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comparison to free drug groups. The results from the current study were consistent with our in vitro assays.

3.17 Tumor retention and immunohistochemical analysis To validate tumor localization of ZEAuSSi, we performed EDX (Energy dispersive X ray spectroscopy) mapping and spectra. This affirms nanocarrier’s presence at the target site (Figure 8 C, D and E). EDX is one of the accepted methods in detecting elemental presence in in vivo samples. EDX mapping results revealed the presence in silica (red), gold (yellow), sulphur (green), carbon (pink), fluorine (violet), nitrogen (blue), chlorine (Navy blue) and oxygen (orange) elements in tumors and their corresponding morphologies (Figure 8 E). This was determined through FE-SEM. The results of western blotting analysis were further validated through immuno-histochemical observations. These immuno-histochemical results affirmed through apoptotic and survival proteins profiles such as Ki-67 in tumors treated with nanoformulation (Figure 9). These results annotate apoptotic potential of the nanoformulation. These results correlated directly with the in vitro experiments. Further, the immuno-histochemical studies reveal the targeted effects of the ZAuSSi, EAuSSi and ZEAuSSi by inhibiting EGFR, VEGFR and Akt activation. Results show the phosphorylation levels of EGFR, VEGFR and Akt are drastically reduced when compared to the untreated tumor and AuSSi controls (Figure 10). 4. Discussion Chemotherapy remains the principal treatment modality employed in combating cancer. With prolonged exposure, chemotherapy can result in diminished efficacy and therapy-resistance. Drug resistance frequently develops due to mutations in originally sensitive cells exposed to


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chemotherapeutic drugs during the course of the treatment. Additionally, survival signaling activation while death signaling at downstream level inactivation also promote chemoresistance. Substantial efforts have been expended in attempting to cure chemo-resistant cancer through development of newer more effective drugs. These drugs often precisely inhibit signaling proteins with aberrant expression in cancer cells. Therapeutic drugs and their combinations have been used to treat patients with chemo-resistant cancers, but they frequently provoke undesirable symptoms and side-effects including drug toxicity and other hematological complications. Unfortunately, these effects can frequently outweigh the therapeutic benefits of the therapy. To address these issues, nanoparticle-mediated drug delivery systems emerge as a viable approach.

Epigallocatechin gallate is profoundly available catechin among the constituents of the green tea with numerous functional bioactivities such as antioxidant, chemo-preventive and anticarcinogenesis activities

52-53

. It has been shown to inhibit various signaling such as protein

kinases, inactivation of transcriptional factors 54, suppress the growth factor mediated signaling, and also promote cell cycle arrest resulting in apoptosis

55-57

. Additionally, EGCG exerts its

apoptotic activity in breast cancer by specifically suppressing Akt activation that aids in cell survival 58. Various nanoformulations of EGCG have been designed with different architectures that encapsulate EGCG and tested in various cancer types. Recently, Radhakrishnan and Tsai et al. prepared different nanoformulations with EGCG encapsulation and evaluated activity against prostate cancer

59-60

. Many EGCG nanoformulations are documented for breast cancer therapy

are documented so far

61

. We believe that this therapeutic formulation of EGCG is first of its

kind to be employed in breast cancer therapy.


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ZD6474 (vandetanib) is a heteroaromatic-substituted anilinoquinazoline that potently inhibits tyrosine kinase activity of VEGFR2. Accordingly, it blocks VEGF promoted the cell proliferation, migration ability of endothelial cells. Also, it hinders the EGFR’s tyrosine kinase activity that regulates proliferation of cancer cells, migration and angiogenesis in various cancers 62-63

. By exploiting the pharmacological behavior of the above two drugs, we design a

nanoformulation (nanoclusters) that can sequentially release the two drugs. They target the aberrantly expressed molecules and eventually affect their downstream signaling in tamoxifenresistant cells. So far one report is documented the generation of nanoformulations with the ZD6474 chemotherapeutic drug from our group, previously 64.

We synthesize nanoclustered silica gold particles as vehicle that can entrap two drugs spatially separated in a single nanocarrier. This composite structure permits sequential release of the two drugs. EGCG is entrapped in the core and ZD6474 is localized peripherally through gold-amine interactions in the nanocluster. XRD results confirm the mesoporous nature of these silica nanocarriers. From nanoparticle characterization studies, we demonstrate the pH responsive behavior of ZEAuSSi nanoclustered silica gold particles are resistant to dissociation in neutral to alkaline normal tissue environments. In contrast, the acidic tumor environment promotes the dissociation of the nanoclusters, which suggest the surface release of the peripheral drug (ZD6474). The semi-dissociated nanoclusters are strongly attracted due to –SH groups present on the silica. This enhances cellular internalization through both caveolae and clathrin-mediated endocytosis 65. Thus, ZEAuSSi may be a potential nanocarrier to achieve localized drug delivery through sustained release in the in vivo tumor environment.


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From cellular uptake studies, we have performed time dependent studies by choosing ZEAuSSi, results indicated accumulation inside tumor cells following an extended incubation time (180 min). Results of flow cytometric studies confirmed the ZEAuSSi cellular-uptake in the acidic tumor milieu, which peaks at 3 h after treatment. The morphological results confirmed the blue to red transition with the subsequent release of drugs which was similar to our previous studies 66

. Overall our results support the cellular uptake of ZEAuSSi, thus causing the release of

ZD6474 and EGCG at the tumor acidic microenvironment of the cancer cells. These agents inhibited phosphorylation of EGFR, VEGFR2 and Akt and downstream promoted apoptosis (Figure 11). We believe that ZEAuSSi may circulate in vascular system for extended time and accumulate at tumor site by passive targeting due to leaky tumor vasculature, as documented in our previous study 33. These events will aids in maintenance of optimal therapeutic indices of the drug at the tumor sites for longer times. On the other side, leaky tumor vasculature facilitates the penetration of ZEAuSSi towards the core of the tumor tissue and subsequently, due to the tumor acidic microenvironment evokes the disintegration of the nanocarrier resulting in the release of drugs. The induced expression of pro-survival and anti-apoptotic proteins triggers cancer cell proliferation and survival. This process is the underpinning of tumorigenesis and cancer progression. Excessive DNA damage can induce apoptosis, where the expression of Bcl2 family of proteins regulates the intrinsic pathway of apoptosis. At molecular level, DNA damage activates p53 related signaling this in turn leads to the pro-apoptotic expression counters the functionality of anti-apoptotic Bcl2 family such as Bcl2 69, Bax 72

70

, Bak

67-68

71

, this

and Bim

. The inhibition of Bcl2 promotes caspase activation resulting in programmed cell death 73-74. In

addition, the Bax/Bcl2 ratio is crucial to determine the cellular fate to survive or undergo


27


apoptosis

75-76

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. Our Western blot and immunohistochemical results indicated that there was

inhibition of Bcl2 expression and induction of pro-apoptotic proteins such as Bax, Bim, Bak and p53 after ZEAuSSi treatment. This supports the role of ZD6474 and EGCG in inducing programmed cell death.

Activation of growth regulatory signaling contributes to the proliferation and survival of cancer cells. EGFR signaling is one of the growth regulatory pathway that promotes cell survival by activating Akt signaling. Ligand-receptor based growth signaling like epidermal growth factor (EGF) binds to EGF receptors (EGFR) that promotes oncogenesis and cell survival

77-78

.

Similarly, of the growth regulating signaling includes vascular endothelial growth factor (VEGF) binds to VEGF receptor 2 (VEGFR2) that stimulates the vasculature in the tumors 79. In cancer cells, the expression of EGFR induce VEGF signaling. In addition, the upregulation of VEGF signaling and its activation is not dependent on the signaling of the EGFR. Both EGFR and VEGFR2 signaling contributes to tamoxifen chemoresistance. Besides their mitogenic roles, EGFR and VEGFR2 activate of PI3K/Akt pathway in the downstream as a pro-survival mechanisms. Molecularly, phosphorylation of EGFR/VEGFR2 stimulate PI3K protein that induce Akt phosphorylation

80

. The activated Akt promotes cell survival and proliferation, thus

contributing to tumor growth. Therefore, targeting the VEGF and VEGFR2 signaling pathways is a recognized strategy for treating solid tumors. Our western blotting and immunohistochemical analyses showed treatments with ZEAuSSi have reduced EGFR and VEGFR2 phosphorylation. They in turn reduced Akt phosphorylation that led to the programmed cell death through p53 dependent mechanisms in tamoxifen resistant cells. The in vitro results observed through cell proliferation, cell phase distribution and microscopic studies are


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overlapping with our in vivo outcomes. From in vivo studies nanoclustered treatments in mice bearing with MCF 7/TAM tumor xenografts, treatments including AuSSi exhibited negligible tumor growth reduction, while treatments with EAuSSi, ZAuSSi, and ZEAuSSi prominently inhibited Ki-67 (Proliferation marker), subsequently promoted apoptosis validated through TUNEL studies and tumor growth inhibition. Altogether, these results suggest that the delivery of ZD6474 and EGCG through nanoclusters targets cell survival in tumors via concomitant induction of apoptosis.

Conclusions This report explains a novel therapeutic strategy that includes concomitant delivery of dual drugs (ZD6474 and EGCG) in tamoxifen resistant breast cancer through unique silica gold nanoclusters. The novel method of synthesizing silica gold clusters through the Gel-Sol method using CTAB is described. The synthesis route of nanoclusters encompassing one or two drugs concurrently with ideal characteristic features including pH responsiveness, enhanced drug pay loading abilities and sustained drug release. Using this therapeutic approach, we simultaneously target the activated forms of EGFR, VEGFR2 and Akt, which promote the progression and survival of tamoxifen resistant breast cancer cells. Our experimental results clearly show the inhibition of VEGFR2, EGFR and Akt by the combined action of ZD6474 (dual tyrosine kinase inhibitor) and Epigallocatechin gallate.

This strategy using dual drug delivery with gold

nanoclusters sensitizes the cells to apoptosis. Our investigations document the tumor targeting abilities of ZEAuSSi in cells of tamoxifen resistant breast cancer. This treatment modality might be a promising therapeutic approach in chemo-resistant cancer of the breast. The combinatorial


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chemotherapy for targeting multiple molecular pathways through our nanoclusters might serve as a global future therapeutic strategy for chemoresistant cancers.

Acknowledgments Prashanth Kumar B.N. and Shashi Rajput are the research fellowship recipients from the Council of scientific and Industrial Research (CSIR), India. Nagaprasad Puvvada is the recipient of DSTInspire faculty program (GAP0631). Paul B. Fisher holds the Thelma Newmeyer Corman Chair in Cancer Research at the VCU Massey Cancer Center, Virginia Commonwealth University, School of Medicine, Richmond, VA USA. S. C. Kundu holds ERA Chair Professor of European Commission Programme (FoReCaST- grant number 668983) at 3Bs Research Group University of Minho, Portugal. Conflict of Interest Disclosure The authors declare no competing financial interest. Funding sources This work was supported by Council of Scientific and Industrial Research (CSIR), Department of Biotechnology (DBT) and Department of Science and Technology (DST), India.

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Figure legends: Figure 1: Characterization of mesoporous silica gold nanoclusters:(A) Low angle XRD pattern of meso-SiO2 and the corresponding TEM image as an inset; (B) FTIR pattern of SiO2, SSiO2, AuSSi, EAuSSi, ZAuSSi, ZEAuSSi; (C) UV-Visible absorption spectra of EAuSSi, ZAuSSi, ZEAuSSi; and (D) Comparison of fluorescence of Au clu, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi.

Figure 2: BET surface isotherms of SiO2 spheres (A) and (B) BJH pore size distribution

Figure 3: Characterization of mesoporous silica gold nanoclusters: (A) TEM images of Au clusters and ZEAuSSi, (B) pH dependent surface potential of AuSSi, EAuSSi, ZAuSSi and ZEAuSSi , and (C) EDX mapping of ZEAuSSi through FE-SEM.

Figure 4: Size and Drug release profiles of mesoporous silica gold nanoclusters: (A) Hydrodynamic radius at various pH of AuSSi, EAuSSi, ZAuSSi and ZEAuSSi; and (B) Drug


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release from samples at pH 7 and 5.2 for ZAuSSi, EAuSSi, ZEAuSSi (Where Er and Zr corresponds to EGCG and ZD6474 release from ZEAuSSi sample).

Figure 5: Synthesis, biocompatibility and cellular uptake studies of ZEAuSSi by flow cytometry and epi-fluorescence microscopic observations: (A) Schematic representation of ZD6474 and EGCG adsorption in silica gold nanoclusters (ZEAuSSi); (B) Blood compatibility of ZD6474, EGCG, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi analysed by hemocompatibility assay; (C) The cells were treated with ZEAuSSi and incubated for varying time intervals (60, 120, 180 min). Quantitative assessment of uptake of nanocarriers by flow cytometry in MCF 7/TAM and T-47D/TAM cell lines; (D) Analysis of uptake pathways of nanocarriers using endocytic inhibitors (i.e.; clathrin and calveoli) by flow cytometer； and (E) Qualitative analysis of cellular localization by epifluorescence microscope. (scale bars: 10 µm at 20 ×) Mean ± SD .

Figure 6: Overexpression protein profile, cytotoxicity assessment and SubG1 phase distribution analysis in nanocluster treated samples: (A) Western blot analysis of overexpressed VEGFR2, EGFR and Akt levels of the MCF 7/TAM and T-47D/TAM cell lines with respect to MCF 7 and T-47D (left) and densitometric analysis (right); (B) Proliferation assays of AuSSi, EAuSSi, ZAuSSi and ZEAuSSi on MCF 7/TAM and T-47D/TAM cells were measured using MTT assays; and (C) Apoptotic activity of control, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi on MCF 7/TAM and T-47D/TAM were determined by flow cytometric phase distribution study. Value of percent apoptosis (subG1 phase) is provided in the inset of each


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figure. Data presented as means ± S.D. P < 0.05. Each individual experiment was repeated three times.

Figure 7: Morphological analysis, VEGF quantification and phosphorylation profiles of AuSSi, ZAuSSi, EAuSSi and ZEAuSSi on breast cancer cells: (A) SEM analysis; (B) Extracellular VEGF levels in different treatment groups by ELISA assay; and (C) Suppression of protein phosphorylation of EGFR, VEGFR2 and Akt with the downstream regulation of p53mediated apoptotic proteins as evaluated by Western blot analysis.

Figure 8: Regression of MCF 7/TAM tumors in mice treated with ZD6474, EGCG, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi: Tumor mass and volumes in the various silica gold nanoformulated groups diminished significantly at 21 days after treatment (P < 0.05). (A) Tumor mass; (B) Tumor volume; (C) FE-SEM image of the tissue sample of ZEAuSSi treated tumor; (D) EDX mapping; and (E) spectra of the tissue section of ZEAuSSi treated tumor.

Figure 9: Immunohistochemistry of control, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi in MCF 7/TAM tumors of mice: Paraffin-embedded sections of tumors in mice were processed, and Immunohistochemical (IHC) analysis was carried out for Bcl2, Bax, Ki-67 and TUNEL. Compared with tumors from the control mice, treatment with ZAuSSi and EAuSSi decreased the number of Ki-67-positive cells and increased the number of TUNEL-positive cells. The changes are significantly prominent in tumors treated with ZEAuSSi. IHC results are representative of three independent experiments. Bar represents 10 µm.


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Figure 10: Immunohistochemistry of control, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi in MCF 7/TAM tumors of mice: Paraffin-embedded sections of tumors in mice were processed, and Immunohistochemical (IHC) analysis was carried out for p-EGFR, EGFR, p-VEGFR, VEGFR, p-Akt, and Akt. The changes are significantly prominent in tumors treated with ZEAuSSi, when compared to EAuSSi and ZAuSSi treated tumors. Immunohistochemical results are representative of three independent experiments. Bar represents 10 µm.

Figure 11: Sequential release of ZD6474 and EGCG from mesoporous silica gold nanoclusters in cells.


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Figure 1: Characterization of mesoporous silica gold nanoclusters:(A) Low angle XRD pattern of meso-SiO2 and the corresponding TEM image as an inset; (B) FTIR pattern of SiO2, SSiO2, ASSi, EAuSSi, ZAuSSi, ZEAuSSi; (C) UV-Visible absorption spectra of EAuSSi, ZAuSSi, ZEAuSSi; and (D) Comparison of fluorescence of Au clu, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi. 82x65mm (300 x 300 DPI)


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Figure 2: BET surface isotherms of SiO2 spheres (A) and (B) BJH pore size distribution 254x126mm (300 x 300 DPI)



Figure 3: Characterization of mesoporous silica gold nanoclusters: (A) TEM images of Au clusters and ZEAuSSi, (B) pH dependent surface potential of AuSSi, EAuSSi, ZAuSSi and ZEAuSSi , and (C) EDX mapping of ZEAuSSi through FE-SEM. 161x123mm (300 x 300 DPI)


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Figure 4: Size and Drug release profiles of mesoporous silica gold nanoclusters: (A) Hydrodynamic radius at various pH of AuSSi, EAuSSi, ZAuSSi and ZEAuSSi; and (B) Drug release from samples at pH 7 and 5.2 for ZAuSSi, EAuSSi, ZEAuSSi (Where Er and Zr corresponds to EGCG and ZD6474 release from ZEAuSSi sample). 73x30mm (300 x 300 DPI)



Figure 5: Synthesis, biocompatibility and cellular uptake studies of ZEAuSSi by flow cytometry and epifluorescence microscopic observations: (A) Schematic representation of ZD6474 and EGCG adsorption in silica gold nanoclusters (ZEAuSSi); (B) Blood compatibility of ZD6474, EGCG, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi analysed by hemocompatibility assay; (C) The cells were treated with ZEAuSSi and incubated for varying time intervals (60, 120, 180 min). Quantitative assessment of uptake of nanocarriers by flow cytometry in MCF 7/TAM and T-47D/TAM cell lines; (D) Analysis of uptake pathways of nanocarriers using endocytic inhibitors (i.e.; clathrin and calveoli) by flow cytometer； and (E) Qualitative analysis of cellular localization by epifluorescence microscope. (scale bars: 10 µm at 20 ×) Mean ± SD . 178x110mm (300 x 300 DPI)


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Figure 6: Overexpression protein profile, cytotoxicity assessment and SubG1 phase distribution analysis in nanocluster treated samples: (A) Western blot analysis of overexpressed VEGFR2, EGFR and Akt levels of the MCF 7/TAM and T-47D/TAM cell lines with respect to MCF 7 and T-47D (left) and densitometric analysis (right); (B) Proliferation assays of AuSSi, EAuSSi, ZAuSSi and ZEAuSSi on MCF 7/TAM and T-47D/TAM cells were measured using MTT assays; and (C) Apoptotic activity of control, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi on MCF 7/TAM and T-47D/TAM were determined by flow cytometric phase distribution study. Value of percent apoptosis (subG1 phase) is provided in the inset of each figure. Data presented as means ± S.D. P < 0.05. Each individual experiment was repeated three times. 95x100mm (300 x 300 DPI)



Figure 7: Morphological analysis, VEGF quantification and phosphorylation profiles of AuSSi, ZAuSSi, EAuSSi and ZEAuSSi on breast cancer cells: (A) SEM analysis; (B) Extracellular VEGF levels in different treatment groups by ELISA assay; and (C) Suppression of protein phosphorylation of EGFR, VEGFR2 and Akt with the downstream regulation of p53-mediated apoptotic proteins as evaluated by Western blot analysis. 173x118mm (300 x 300 DPI)


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Figure 8: Regression of MCF7/TAM tumors in mice treated with ZD6474, EGCG, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi: Tumor mass and volumes in the various silica gold nanoformulated groups diminished significantly at 21 days after treatment (P < 0.05). (A) Tumor mass; (B) Tumor volume; (C) FE-SEM image of the tissue sample of ZEAuSSi treated tumor; (D) EDX mapping; and (E) spectra of the tissue section of ZEAuSSi treated tumor. 189x104mm (300 x 300 DPI)



Figure 9: Immunohistochemistry of control, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi in MCF 7/TAM tumors of mice: Paraffin-embedded sections of tumors in mice were processed, and Immunohistochemical (IHC) analysis was carried out for Bcl2, Bax, Ki-67 and TUNEL. Compared with tumors from the control mice, treatment with ZAuSSi and EAuSSi decreased the number of Ki-67-positive cells and increased the number of TUNEL-positive cells. The changes are significantly prominent in tumors treated with ZEAuSSi. IHC results are representative of three independent experiments. Bar represents 10 µm. 210x163mm (300 x 300 DPI)


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Figure 10: Immunohistochemistry of control, AuSSi, EAuSSi, ZAuSSi and ZEAuSSi in MCF 7/TAM tumors of mice: Paraffin-embedded sections of tumors in mice were processed, and Immunohistochemical (IHC) analysis was carried out for p-EGFR, EGFR, p-VEGFR, VEGFR, p-Akt, and Akt. The changes are significantly prominent in tumors treated with ZEAuSSi, when compared to EAuSSi and ZAuSSi treated tumors. Immunohistochemical results are representative of three independent experiments. Bar represents 10 µm. 202x231mm (300 x 300 DPI)



Figure 11: Sequential release of ZD6474 and EGCG from mesoporous silica gold nanoclusters in cells. 157x117mm (300 x 300 DPI)


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