HDAC INHIBITOR-TARGETED THERAPY WITH NANOMEDICINE INCREASES THE EFFICACY OF PACLITAXEL IN TRIPLE NEGATIVE BREAST CANCER LIM CHEN SIEW B.Eng (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF ENGINEERING BY RESEARCH DEPARTMENT OF CHEMICAL AND BIOMOLECULAR ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION! ! I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously _ Lim Chen Siew 13 January 2014 ACKNOWLEDGEMENTS I would like to take this opportunity to thank my supervisors, Assistant Professor David LEONG and Professor FENG Si-Shen, for their constant guidance and encouragement throughout my Masters studies I would also like to extend my thanks to the other members of the group for their kind advices and assistance: MI Yu, ZHAO Jing, Rajaletchumy Veloo KUTTY, TAN Guang Rong, Dalton TAY, Marcella GIOVANNI, CHIA Sing Ling and Magdiel Inggrid SETYAWATI And also to the lab technologists and operators whom I greatly appreciate their help and services Last but not least, I would like to thank my family and friends for their unwavering support and understanding i! TABLE OF CONTENTS ! ACKNOWLEDGEMENTS .i TABLE OF CONTENTS ii ABSTRACT .iv LIST OF TABLES v LIST OF FIGURES vi LIST OF SYMBOLS vii INTRODUCTION 1.1 Triple Negative Breast Cancer 1.2 Therapeutic Resistance 1.3 Cancer and Epigenetics 1.3.1 Histone Deactylases (HDACs) 1.4 Epigenetics-targeted therapy using inhibitors of HDACs 1.5 Combination Chemotherapy with Paclitaxel 1.6 Challenges in Conventional Chemotherapy 1.7 Nanomedicine 1.8 Problem Statement 10 MATERIALS AND METHODS 11 2.1 Materials 11 2.2 Formulation of paclitaxel- and vorinostat-loaded DSPEPEG2000/TPGS micelles 12 2.3 Characterization of micelles 12 2.3.1 Micelle shape, size and size distribution and surface charge 12 2.3.2 Drug encapsulation efficiency 12 2.3.3 Critical micelle concentration (CMC) 13 2.4 Drug release profile 13 2.5 in vitro studies 14 ii! 2.5.1 Cell cultures 14 2.5.2 Cellular uptake 14 2.5.3 MTT cell viability 15 2.5.4 Cell cycle analysis 16 2.5.5 Caspase-3 activity 16 2.5.6 Scratch wound-healing assay 17 2.6 Statistical analysis 17 RESULTS AND DISCUSSIONS 18 3.1 Characterization of micelles 18 3.1.1 Micelle shape, size and size distribution and surface charge 18 3.1.2 Drug encapsulation efficiency 20 3.1.3 Critical micelle concentration (CMC) 21 3.2 Drug release profile 23 3.3 in vitro studies 24 3.3.1 Cellular uptake 24 3.3.2 MTT cell viability 27 3.3.3 Cell cycle analysis 31 3.3.4 Capase-3 activity 33 3.3.5 Scratch wound-healing assay 34 CONCLUSIONS 36 REFERENCES 37 ! ! ! iii! ABSTRACT Triple negative breast cancer is often associated with poor prognosis and high relapse, which are linked to drug resistance to chemotherapy Drug resistance is a stumbling block in successful cancer treatment and metastatic cancers due to chemoresistance accounts for more than 90% of cancer deaths It is thus crucial to develop new strategies to overcome drug resistance and enhance efficacy especially in the early setting of TNBC when it is chemo-sensitive and controllable Epigenetic aberrations play an important role in modulating resistance and by relying on epigenetics-targeted therapy, these defects could be reversed to their normal state and prevented from passing on to future generations In this study, a novel system involving the co-encapsulation of vorinostat, a histone deactylase inhibitor, and paclitaxel in mixed micelles consisting of vitamin E TPGS and DSPE-PEG2000 was developed to achieve maximal therapeutic response by targeting different mechanisms of action Results showed that the TPGS/DSPE-PEG2000 mixed micelles exhibited enhanced cellular uptake and stability In vitro investigations also suggested that the micelle system led to improved pharmacokinetics and enhanced anticancer activity as the IC50 value decreased from 3.071 in the free drugs formulation to 0.520 μg/ml The cell cycle profile also showed a significant and sustained cell cycle arrest in the G2/M phase at 93% Inhibition of cell migration activity was observed where the wound area only recovered by 2.93 ± 0.01 % compared to 100% in untreated cells Significant caspase-3 activity involved in apoptosis was also found iv! LIST OF TABLES ! Table 1: Size distribution and zeta potential of dual drug loaded DSPEPEG2000/TPGS micelles and TPGS micelles 19 Table 2: Encapsulation efficiency and drug load of dual drug loaded DSPEPEG2000/TPGS micelles and TPGS micelles 20 Table 3: IC50 values of different formulations in MDA-MB-231 after a 24hour incubation 29 v! LIST OF FIGURES Figure 1: Schematic illustration of the formulation of paclitaxel- and vorinostat-loaded DSPE-PEG2000/TPGS mixed micelles 11 Figure 2: Transmission electron microscopy image of paclitaxel- and vorinostat-loaded DSPE-PEG2000/TPGS mixed micelles 19 Figure 3: (A) Excitation spectra of pyrene in DSPE-PEG2000/TPGS micelles and (B) plot of fluorescence intensity ratio I336/I330 from the excitation spectra versus DSPE-PEG2000/TPGS concentration 22 Figure 4: Drug release profile of (A) paclitaxel and (B) vorinostat in dual drug-loaded TPGS and DSPE-PEG2000/TPGS micelles over 169 hours * p < 0.05 24 Figure 5: Quantitative cellular uptake efficiency of C6, C6-loaded TPGS micelles and C6-loaded DSPE-PEG2000/TPGS micelles at varying concentrations after a (A) 0.5-hour and (B) 2-hour incubation * p < 0.05 compared with C6 25 Figure 6: Cellular uptake and intracellular localization of (A) negative control, (B) C6 and (C) C6-loaded DSPE-PEG2000/TPGS micelles in MDA-MB-231 cells after a 2-hour incubation 27 Figure 7: (A) Effect of concentration of the blank micelles on the viability of MDA-MB-231 cancer cells and (B) effect of concentration of the indicated formulations on the viability of MDA-MB-231 cancer cells after incubation for 24 hours * p < 0.05 compared with control 30 Figure 8: (A) Cell cycle distribution by flow cytometry where MDA-MB231 cancer cells were treated with the indicated formulations for 24 hours and (B) the effect of the different formulations on MDAMB-231 cancer cells arrested in the various cell cycle phases 32 Figure 9: Fold-increase in caspase-3 activity of the indicated formulations compared with control in MDA-MB-231 cells after treatment for 24 hours * p < 0.05 compared with control 33 Figure 10: (A) Cell migration in a scratch wound-healing assay where MDAMB-231 cancer cells were treated with the indicated formulations for 24 hours and (B) the effect of the different formulations on wound closure in percentage * p < 0.05 compared with control 35 vi! LIST OF SYMBOLS ABCB1 ATP-binding cassette sub family B member ACN Acetonitrile ATCC American type culture collection ATP Adenosine triphosphate C6 Coumarin CDKN2A Cyclin-dependent kinase inhibitor 2A CLSM Confocal laser scanning microscope CMC Critical micelle concentration CpG Cytosine-phosphate-guanine CSC Cancer stem cell CTCF Corrected total cell fluorescence DAPI 4',6-diamidino-2-phenylindole DLS Dynamic light scattering TPGS D-α-tocopheryl polyethylene glycol 1000 succinate DCM Dichloromethane DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DSPE-PEG2000 1,2-distearoyl-sn-glycero-3-phosphoethanolamine polyethylene glycol 2000 EDTA Ethylenediaminetetraacetic acid EPR Enhanced permeability and retention ER Estrogen FBS Fetal bovine serum FDA Food and drug administration FETEM Field emission transmission electron microscope vii! HAT Histone acetyltransferase HER2 Human epidermal growth factor HPLC High performance liquid chromatography HSP-90 Heat shock protein 90 IC50 Inhibitory concentration 50 HDAC Histone deactylase HDACi Histone deactylase inhibitor Ixxx Intensity at xxx nm MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide MWCO Molecular weight cut-off NaOH Sodium hydroxide P Paclitaxel P+S Paclitaxel and vorinostat Pmic Paclitaxel-loaded DSPE-PEG2000/TPGS micelles Pmic + Smic Paclitaxel-loaded DSPE-PEG2000/TPGS micelles and vorinostat-loaded DSPE-PEG2000/TPGS micelles (P+S)mic Paclitaxel- and vorinostat-loaded DSPE-PEG2000/TPGS micelles P-gp P-glycoprotein PBS Phosphate buffered saline pCR Pathological complete response PEG Poly(ethylene) glycol PFA Paraformaldehyde PI Propidium iodide PR Progesterone RES Reticuloendothelial system RNase A Ribonuclease A viii! 3.3.3 Cell cycle analysis Cell cycle analysis of the various formulations was studied using propidium iodide flow cytometry and analyzed by the ModFit LT software to gain a better understanding of the biochemical pathways that result in cell death Figure 8(A) illustrates the cell cycle distribution after MDA-MB-231 cancer cells were treated with 0.25 μg/ml of the indicated formulations for 24 hours The relative proportion of the cell cycle phases associated with each formulation is shown in Figure 8(B) The results suggested that the blank micelles did not cause cell cycle arrest as no significant changes in the cell cycle distribution was observed when compared to the negative control Formulations that include paclitaxel such as P, Pmic, P + S, Pmic + Smic and (P + S)mic induced G2/M arrest with an accumulation of cells in the G2/M phase of more than 90% This suggested that paclitaxel is very effective in inducing G2/M arrest when used as a free drug, in combination therapy or in nanocarrier system However, the results also showed that encapsulating paclitaxel in micelles or combination chemotherapy with paclitaxel did not show any significant increase in the percentage of cells arrested in the G2/M phase compared to the paclitaxel free drug This implied that although nanocarrier system or combination chemotherapy resulted in lower drug doses required, they had no effect on the cell cycle distribution It was also observed that vorinostat-loaded micelles, Smic resulted in G1/S phase arrest, which could be mediated by the retinoblastoma pathway that targets cyclin-dependent kinase inhibitor p21 encoded by CDKN2A that is upregulated in TNBC In combination chemotherapy, results showed that the presence of vorinostat did not alter the cell arrest profile of paclitaxel since Pmic + Smic and (P + S)mic still showed a sustained G2/M arrest profile 31 A Blank micelles Control P P+S S Pmic (P + S)mic Smic Pmic + Smic 100% B 90% 80% Percentage(%) 70% 60% 50% Dip G2 40% Dip S 30% Dip G1 20% 10% 0% Control Blank micelles P P mic S S mic P + S P mic + (P + S) S mic mic Formulations Figure 8: (A) Cell cycle distribution by flow cytometry where MDA-MB-231 cancer cells were treated with the indicated formulations for 24 hours and (B) the effect of the different formulations on MDA-MB-231 cancer cells arrested in the various cell cycle phases 32 3.3.4 Capase-3 activity Caspase-3 is a protein in the cysteine-aspartic acid protease (caspase) family where its activation plays a role in the execution-phase of apoptosis that causes the degradation of intracellular elements It can occur via both the extrinsic death ligand and the intrinsic mitochondrial pathways In this fluorescence-based assay, free fluorophore i.e AFC is released and detected when the substrate DEVD-AFC is cleaved by the presence of activated caspase-3 (Porter & Jänicke, 1999) The fluorescence of AFC from the indicated formulations were then compared with that of the control where there was an absence of activated caspase-3 to give the fold increase in caspase-3 activity as shown in Figure It was observed that (P+S)mic exhibited the greatest fold-increase in caspase-3 activity i.e 1.55-fold which implied that the combined micelle formulations led to the most significant apoptotic response In general, micelle formulations yielded a greater foldincrease in caspase-3 activity than the free drug formulations i.e 1.35-fold increase in Pmic compared to 1.27-fold increase in P and 1.40-fold increase in Smic compared to 1.28-fold increase in S This showed that by encapsulating drugs in micelles, higher apoptotic response due to caspase-3 activity could be achieved Blank micelles with a 1.03-fold increase were observed to exhibit Caspase-3 Activity (-fold increase) minimal apoptotic response 1.5 * * * P S P+S * * P mic S mic * * 0.5 Control Blank Micelles P mic+S (P+S)mic mic Formulations Figure 9: Fold-increase in caspase-3 activity of the indicated formulations compared with control in MDA-MB-231 cells after treatment for 24 hours * p < 0.05 compared with control ! 33 3.3.5 Scratch wound-healing assay A scratch wound-healing assay was performed to investigate the effect of various drug formulations at an equivalent concentration of 0.01 μg/ml on the inhibition of cell migration after 24 hours A scratch wound was created on the cell monolayer and in the absence of induced treatment, cells will move to close the gap Figure 10(A) shows the images acquired at the start of the experiment as well as after 24 hours to compare the changes in the area of the wound gap White dotted lines were drawn to illustrate the boundary of the gap for area calculation The changes in the area at hours and 24 hours were analyzed using ImageJ and used to calculate the percentage of wound closure by taking the quotient of the difference in wound gap area and the wound gap area at 0-hour which was illustrated in Figure 10(B) Blank micelles, S and Smic did not cause inhibition of cell migration with a 100% wound closure It may be because they did not cause a significant effect on the growth factors at the concentration of 0.01 μg/ml Comparing the two drugs individually, it was found that P had a much more significant effect on the blockage of cell migration than S which was probably due to the fact that P is also an inhibitor of angiogenesis that is involved in down-regulating vascular endothelial growth factor (VEGF) which is expressed in MDA-MB-231 When combined together especially in a micelle system, results showed that the (P+S)mic exhibited the most significant inhibition of cell migration compared to other formulations with a closure of the wound by 2.93 ± 0.01 % compared with 100% in the untreated cells This implied that the targeting of different mechanisms of action greatly enhanced the therapeutic potential and was thus more successful in preventing cell growth and metastasis The wound closure was also more significant than that of Pmic + Smic (i.e 15.97 ± 0.03 %) This implied that co-encapsulation of drugs within a single nanocarrier was more effective (i.e 5.45-fold) in inhibiting growth factor production (P+S)mic was also more successful in blocking cell invasion than single therapy system in both free drugs and micelle formulations i.e 34.12-fold more effective than S and Smic as well as 15.71-fold and 8.97-fold more efficient than P and Pmic respectively 34 A 120% B Wound Closure (%) 100% 80% 60% * *! 40% *! 20% *! *! 0% Formulations Figure 10: (A) Cell migration in a scratch wound-healing assay where MDAMB-231 cancer cells were treated with the indicated formulations for 24 hours and (B) the effect of the different formulations on wound closure in percentage * p < 0.05 compared with control 35 CONCLUSIONS Both paclitaxel and vorinostat drugs were successfully loaded in DSPEPEG2000/TPGS mixed micelles for combination chemotherapy to achieve therapeutic effects in triple negative breast cancer cells DSPE- PEG2000/TPGS micelles were found to have a lower CMC value of 0.0218 ± 0.0006 mg/ml and a more negative surface charge of -22.5 ± 2.9 mV than TPGS micelles, which contributed to its stability and enhanced cellular uptake efficiency Drug encapsulation efficiencies of paclitaxel and vorinostat were also significantly higher in DSPE-PEG2000/TPGS micelles at 97.6 ± 0.3% and 99.5 ± 2.7% respectively In vitro investigation was thus focused on comparing DSPE-PEG2000/TPGS micelles with other free drugs combinations It was observed that the dual drug micelle system led to an improved pharmacokinetics than dual drug conventional chemotherapy when the IC50 value decreases from 3.071 to 0.520 μg/ml and overall, combinatory chemotherapy exhibited better anti-cancer effects than single drug treatment Cell cycle analysis also showed that although vorinostat-loaded micelles induced G1/S arrest probably due to the retinoblastoma pathway, the cell cycle profile of paclitaxel was not altered and the dual drug mixed micelles still resulted in a significant and sustained cell arrest in the G2/M phase of 93% Furthermore, the dual drug mixed micelles showed the most enhanced caspase-3 activity with a 1.55-fold increase compared to the control as well as an inhibition of cell migration with a wound closure by 2.93 ± 0.01% compared with 100% with no treatment in the scratch wound-healing experiment 36 REFERENCES Abdullah, L N., & Chow, E K.-H (2013) Mechanisms of chemoresistance in cancer stem cells Clinical and Translational Medicine, 2(1), doi:10.1186/2001-1326-2-3 Alexis, F., Pridgen, E M., Langer, R., & Farokhzad, O C (2010) Nanoparticle Technologies for Cancer Therapy In M Schäfer-Korting (Ed.), Drug Delivery (Vol 197, pp 55 – 86) Berlin, Heidelberg: Springer Berlin Heidelberg doi:10.1007/978-3-642-00477-3 Angelucci, A., Mari, M., Millimaggi, D., Giusti, I., Carta, G., Bologna, M., & Dolo, V (2010) Suberoylanilide hydroxamic acid partly reverses resistance to paclitaxel in human ovarian cancer cell lines Gynecologic Oncology, 119(3), 557–63 doi:10.1016/j.ygyno.2010.07.036 Arnedos, M., Bihan, C., Delaloge, S., & Andre, F (2012) Triple-negative breast cancer: are we making headway at least? 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