Micelle Mixtures for Coadministration of Gemcitabine and GDC-0449

Subscriber access provided by RMIT University Library

Article

Micelle Mixtures for Co-administration of Gemcitabine and GDC-0449 to treat Pancreatic Cancer Melek Karaca, Rinku Dutta, Yildiz Ozsoy, and Ram I. Mahato Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00971 • Publication Date (Web): 16 Mar 2016 Downloaded from http://pubs.acs.org on March 17, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 44

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

Molecular Pharmaceutics

Micelle Mixtures for Co-administration of Gemcitabine and GDC-0449 to treat Pancreatic Cancer 1

Melek Karaca, 1Rinku Dutta, 2Yildiz Ozsoy and 1*Ram I. Mahato

1

Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, NE 68198

2

Department of Pharmaceutical Technology, Faculty of Pharmacy, Istanbul University, Istanbul, Turkey

*Address for correspondence: Dr. Ram I. Mahato Department of Pharmaceutical Sciences University of Nebraska Medical Center 986025 Nebraska Medical Center Omaha, NE 68198-6025 Tel: (402) 559-5422; Fax: (402) 559-9543 E-mail: [email protected]

1 ACS Paragon Plus Environment


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

ABSTRACT Hedgehog (Hh) signaling plays an important role in the development and metastasis of pancreatic ductal adenocarcinoma (PDAC). Although gemcitabine (GEM) has been used as a first-line therapy for PDAC, its rapid metabolism and short plasma half-life restrict its use as a single chemotherapy. Combination therapy with more than one drugs is a promising approach for treating cancer. Herein, we report the use of methoxy poly(ethyleneglycol)-block-ploy(2-methyl-2-carboxyl-propylenecarbonate)-graftdodecanol (mPEG-b-PCC-g-DC) copolymer for conjugating GEM and encapsulating an Hh inhibitor, Vismodegib (GDC-0449) into its hydrophobic core for treating PDAC. Our objective was to determine whether the mixed micelle formulation of these two drugs could show better response in inhibiting Hh signaling pathway and restraining the proliferation and metastasis of pancreatic cancer. The in vivo stability of GEM significantly increased after conjugation, which resulted in its increased antitumor efficacy. Almost 80 % of encapsulated GDC-0449 and 19 % conjugated GEM were released in vitro at pH 5.5 in 48 h in a sustained manner. The invasion, migration and colony forming features of MIA PaCa-2 cells were significantly inhibited by micelle mixture carrying GEM and GDC-0449. Remarkable increase in PARP cleavage and Bax proved increased apoptosis by this combination formulation compared to individual micelles. This combination therapy efficiently inhibited tumor growth, increased apoptosis and reduced Hh ligands PTCH-1, Gli1, and lowered EMT-activator ZEB-1 when injected to athymic nude mice bearing subcutaneous tumor generated using MIA PaCa-2 cells compared to monotherapy as observed from immunohistochemical


Page 2 of 44

Page 3 of 44

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


analysis. In conclusion, micelle mixtures carrying GEM and GDC-0449 have the potential to treat pancreatic cancer.

Key Words: pancreatic cancer, hedgehog signaling, gemcitabine, GDC-0449, polymeric micelles, combination therapy.



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

1. INTRODUCTION Amongst the malignant cancers leading to fatal mortality, pancreatic ductal adenocarcinoma (PDAC) deserves special mention and expected to become the second among cancer-related deaths in the United States before 2030.1,2 Surgical resection is the only beneficial option for early and advanced stage PDAC that further requires adjuvant therapeutic approaches for patients developing metastasis and postsurgery local recurrence.2,3 Despite various therapeutic approaches, 5-year survival rate remains < 5 % due to the development of chemoresistance and toxicity.4 Gemcitabine (GEM), (2’,2’-difluorodeoxycytidine; dFdC) has been used as a chemotherapeutic agent for more than 15 years.5,6 Its anti-tumor action is manifested due to blocking of the chromosomal replication and inducing apoptosis through caspase signaling.7,8 However, overall clinical potential of GEM is compromised by rapid metabolism into its inactive metabolite 2’,2’-difluorodeoxyuridine (dFdU) by cytidine deaminase and fast kidney excretion thus requiring higher doses to meet therapeutic level at the tumor site that leads to undesirable side effects and toxicity.9 The modest results of the drug regimen also arise from the hydrophilic nature of this drug that prevents it from prolonged release and subsequent extravasation to cancerous tissues.10 Delivery approaches to increase the overall intracellular GEM concentration via improved pharmacokinetics and chemoresistance have been widely applied.11-14 Nanoformulations have been extensively applied to address the plasma instability, metabolic inactivation and subsequent deamination of GEM into its inactive uracil derivatives. Both conjugation and encapsulation strategies have been explored to overcome the barriers 4 ACS Paragon Plus Environment

Page 4 of 44

Page 5 of 44

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


for attenuated delivery of GEM. Particularly, polymer-drug conjugates resulted better efficacy with the use of modified polyethylene glycol (PEG) to increase its circulation half-life, enhanced permeability and retention (EPR) effect and thus augmenting extravasation to tumor tissues.15 Squalene (SQ) conjugated GEM encapsulated in liposomes has shown promising anticancer efficacy in leukemia cancer model.16 For elevated intracellular drug fate, Bildstein et al demonstrated that SQdFdC application lead to achieve better pharmacokinetic profiles and increased tumor accumulation in resistant cancer cells. However, these formulations were taken up by the cells of reticuloendothelial system (RES), leading to high accumulation in the liver and spleen.17 Consequent increased toxicity demands new delivery approaches. In our previous study, we conjugated GEM to methoxypoly(ethylene)-block-poly(2-methyl-2-carboxylpropylene carbonate) (mPEG-b-PCC) copolymer, which self-assembled into micelles and resulted in its improved plasma stability, enhanced internalization and apoptosis.18 Cancer stem cells (CSCs), stromal depletion and excess production of the extracellular matrix (ECM) proteins lead to the pathology of PDAC and its metastasis.19 Correlation between PDAC and hedgehog (Hh) signaling has been well established in various studies, where dysregulation of this pathway has resulted in significant disturbances in the cancer microenvironment.20 Hh signaling is responsible for cellular differentiation during embryonic development in non-pathological conditions and when activated leads to various cancers including PDAC.21 Hh antagonists, such as cyclopamine, vismodegib (GDC-0449) and erismodegib block Hh signaling pathway by binding to the Smoothened (SMO) thus disturbing the downstream intracellular cascade, leading to the inactivation of the transmembrane protein PTCH-1 and Gli transcription factors.22 5 ACS Paragon Plus Environment


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

When Hh is repressed alone, tumor progression continued thus inferring that only Hh inhibition is unable to ameliorate the metastasis.23,24 GDC-0449 at a very high dose (100 mg/kg) suppressed Hh signaling but increased the angiogenesis subsequently.25 In fact, combination with GEM/GDC-0449/nab-paclitaxel was superior than GEM/nabpaclitaxel.26 Bahra et al. have demonstrated the in vivo efficacy of the combination therapy of cyclopamine and GEM administrated intraperitoneally causing synergistic effect in PDAC tumor growth reduction.21 Feldmann et al. efficiently used the combination of these two drugs in orthotopic xenograft model to illustrate the reduction in primary tumor size and thus disseminating metastasis.20 Co-administration of GEM and IPI-926 (a semisynthetic derivative of cyclopamine) resulted in enhanced GEM efficacy extending the median survival of mice from 11 days to 25 days and this combination treatment significantly decreased liver metastasis.27 This study proved tumor perfusion was augmented in the presence of the stromal depletion agent, the Hh inhibitor that indirectly increased the intracellular concentration of GEM metabolite. However, the SMO inhibitor monotherapy did not affect the overall cellular proliferation significantly and particularly in metastatic PDAC, GDC-0449/GEM combination in pilot clinical trial did not improve the median overall survival or progression free survival compared to GEM alone.28 Thus, to have an effective treatment regimen, a balanced Hh inhibition with a chemotherapy can be a pivotal approach. We hypothesize a rationale design of formulation strategy for sustained release of combination drugs could serve as an inevitable alternative for effective treatment. Additionally, relationship between epithelial-to-mesenchymal transition (EMT) and Hh signaling has been established in various reports.29-32 EMT is a developmental process 6 ACS Paragon Plus Environment

Page 6 of 44

Page 7 of 44

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


where the polar, non-motile phenotype of epithelial cells changes to non-polar, motile mesenchymal cells thus accelerating tumor progression and metastasis.33 Tumor sensitivity to cytotoxic agents can be increased via reversal of EMT by inhibiting Hh signaling which has been demonstrated in a study where blocking of Hh signaling abrogated resistance of non-small lung cancer cells to erlotinib and cisplatin.22 Suppression of EMT was effective in increasing the sensitivity to GEM treatment.34 Several clinical studies showed the association between increased epithelial features and improved survival in different tumor types.35,36 Islam et al. demonstrated the benefits of targeting Hh signaling pathway that resulted in reversed EMT phenotype and decreased tumorigenicity of bladder cancer.37 In our previous studies, we have demonstrated the application of micelle formulation to increase the bioavailability of the hydrophobic drug, GDC-0449.38 Herein, we report, the use of micelle mixtures carrying GEM (mPEG-b-PCC-g-GEM-g-DC) micelles and GDC0449 (mPEG-b-PCC-g-DC). When these micelle mixtures were injected subcutaneously pancreatic tumor bearing mice the synergistic effect of these two drugs showed comparable inhibition of tumor growth. Combination therapy with micelle mixtures can potentially elude multiple resistance mechanisms that limit the activity of individual anticancer drugs. 2. MATERIALS AND METHODS 2.1.

Materials and Reagents

Gemcitabine and Vismodegib (GDC-0449) were purchased from Ark Pharma (Libertyville,

IL)

and

LC

Laboratories

(Woburn,

MA),

respectively.

2,2-

bis(hydroxymethyl) propionic acid, methoxy polyethylene glycol (mPEG; Mn= 5000, 7 ACS Paragon Plus Environment


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

Page 8 of 44

polydispersity index (PDI)=1.03), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide HCL (EDC), 1-hydroxybenzotriazole (HOBt), 1,8-diazabicycloundec-7-ene (DBU), benzyl bromide and all other reagents were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. 2.2.

Synthesis and Characterization of Copolymers

Monomer 2-methyl-2-benzyloxycarbonyl-propylene carbonate (MBC) was synthesized in two steps as white crystals according to our previous report.18 Ring opening polymerization was performed with mPEG and MBC for the synthesis of mPEG-MBC in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) catalyst at the room temperature for 3 h under nitrogen atmosphere. mPEG-MBC was in tetrahydrofuran (THF): methanol (1:1, v/v)) hydrogenated in presence of 10 wt % palladium on charcoal (Pd/C) to obtain the copolymer containing carboxyl pendant groups (mPEG-b-PCC). Finally, GEM and/or dodecanol (DC) were conjugated by carbodiimide coupling reaction at the room temperature for 48 h to obtain mPEG-b-PCC-g-DC and mPEG-b-PCC-gGEM-g-DC using EDC/HOBt as reported previously.18 These final products were precipitated in isopropyl alcohol followed by diethyl ether. mPEG-b-PCC-g-GEM-g-DC was dissolved in acetone and dialyzed against water (M.W. Cut off 2000 Da) followed by lyophilization overnight (Fig. 1). Copolymers were characterized by

1

H NMR,

recorded with a Bruker (500 MHz, T=25 °C) using deuterated dimethyl sulfoxide (DMSO-d6, Cambridge Isotope Laboratories, Inc., MA) Critical micelle concentration (CMC) of both polymers (mPEG-b-PCC-g-GEM-g-DC and mPEG-b-PCC-g-DC) was measured according to pyrene probe method (λexcitation = 338 and 333 nm and λemission = 390 nm) by spectrofluorometer. Plots of intensity ratio of two


Page 9 of 44

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


excitation wavelengths (I338/I333) versus logarithm of polymer concentration were used to calculate the critical micelle concentration (CMC) values of the micelles (Fig. S3). 2.3.

Preparation of Micelles

GEM conjugated micelles and GDC-0449 loaded micelles were prepared by thin film hydration as described previously.18,38 Briefly, 20 mg of copolymers were dissolved in 0.4 mL of chloroform. Organic solvent was evaporated under pressure followed by storing overnight at vacuum. Rehydration of the film with phosphate buffered saline (PBS; pH 7.4), followed by vortexing, sonication, centrifugation at 5,000 g (5 min) and filtration (0.22 µ) were performed for preparing micelles. Micelle mixtures were prepared by mixing the individual micelle formulations (containing required equivalent amount of drugs; GEM micelles and GDC-0449 micelles) at the ratio of 50:50 (Fig. 1). 2.4.

Characterization of Formulations

Mean particle size and size distribution of the micelles were determined by dynamic light scattering using a Zeta SizerTM (Malvern 3800-ZLS, Boston, MA). 2.4.1. Quantification of Gemcitabine Loading in Polymer-Drug Conjugate The degree of GEM conjugation to the copolymer was determined by alkaline hydrolysis in the presence of 1 N NaOH. The amount of GEM in the conjugated polymer was determined by HPLC-UV analytical method using Inertsil ODS 3 column (250 x 4.60 mm, 5µ) and methanol : sodium acetate buffer (20 mM, pH 5.5) mobile phase = 07:93 v/v (20 µL injection volume; 268 nm). Drug payload calculated using the following equation. Drug Payload (w/w %) = (Weight of Drug Loaded/Total weight of formulation) x 100 2.4.2. Quantification of GDC-0449 Encapsulation Efficiency and Drug Loading



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

Page 10 of 44

Micelles loaded with GDC-0449 with a 5% theoretical loading were dissolved in dichloromethane (DCM) and bath sonicated for 30 min at 37° C followed by withdrawal of the lower layer of the dual phase that includes dissolved GDC-0449 in DCM. After evaporating DCM, acetonitrile was added and vortexed for 5 min. Encapsulated drug amount was determined by HPLC using Phenomenex Aqua C18 column (250 x 4.60 mm, 5µ) with acetonitrile : water mobile phase (60:40, v/v; 1.0ml / min) at 230 nm (UV). Drug loading and encapsulation efficiency were calculated using the following equations: Encapsulation efficiency (%) = (Weight of drug encapsulated / Initial weight of drug taken) x 100 Drug loading (%) = (Weight of drug encapsulated/ total weight of formulation) x 100 2.4.3. In vitro Release of Gemcitabine and GDC-0449 from Combination Micelles In vitro release of GEM from the micelles and micelle mixtures were determined in PBS at pH 5.5 and pH 7.4 separately in dialysis bags (M.W. cutoff 2000 Da). Samples (1mL) were withdrawn at a regular interval and replaced with equivalent volume of fresh media. GEM amount in the samples was determined by HPLC-UV method. All parameters kept the same for GDC-0449 release from the micelles except the addition of 2% Tween 80 to maintain sink conditions. GDC-0449 concentration was determined by HPLC-UV as described in the earlier section. 2.5.

In Vitro Cell Culture Studies

2.5.1. Cytotoxicity Assay DMEM (containing 10% FBS and 1% antibiotic-antimycotic (streptomycin, penicillin and amphotericin B)) were used to culture MIA PaCa-2 cells in 5% CO2 at 37° C in a 95%


Page 11 of 44

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


humidified atmosphere. MIA PaCa-2 cells were seeded on a 96-well plate in 100 µL media (5 x 103 cells / well) and incubated for 24 h. The cells treated with different concentrations of GEM conjugated micelles, GDC-0449 encapsulated micelles and micelle mixture were incubated for 72 h at 37° C. MTT assay (absorbance at 570 nm and cell debris corrected by subtracting at 630 nm) was performed to assess cell viability. Cell viability was calculated using the formulation below: Cell viability (%) = (Absorbance of test sample) / (Absorbance of control) x 100 2.5.2. Western Blotting MIA PaCa-2 cells (2 x 105 cells/well) were seeded in 6-well plates containing 2 ml media. After 24 h, GEM conjugated micelles, GDC-0449 loaded micelles and micelle mixture carrying equivalent amount of GEM and GDC-0449 were added and incubated for 72 h. Untreated cells were taken as the control. After 72 h, cells were incubated with RIPA buffer and protease inhibitor cocktail (Sigma, St. Louis, MO) followed by the determination of total protein concentration by BCA protein assay kit (Thermo Scientific, Rockford, IL). 4-15% mini PROTEAN polyacrylamide gel was used for separation of 40 µg total proteins from cells treated with three different formulations and the control group followed by transferring to Polyvinylidenefluoride (PVDF) (Life Technologies Carlsbad, CA) membranes by iBlot gel transfer system. Odyssey Blocking Buffer was used to block membranes for 1 h at room temperature. Membranes were incubated overnight with rabbit polyclonal to Gli-1 (SC-20687), Bax (SC-6236), total PARP (CS9532S) (Cell Signaling Tech., Danvers, MA), goat polyclonal to β-actin (SC-1616) (1:1000), Patched-1 (SC-6147) (1:1000) (Santa Cruz Biotech., Dallas, TX). Following washing with TBST buffer, the membranes were incubated with their corresponding



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

Page 12 of 44

anti-rabbit and anti-goat IR dye conjugated secondary antibodies (1:10000) (LI-COR Biosciences, Lincoln, NE) (60 min) and visualized using LI-COR imaging system. βactin (SC-1616) protein expression level were used for normalizing protein expression levels. 2.5.3. Invasion, Migration and Colony Forming Assays Matrigel invasion assay was performed using Transwell membrane filter inserts (8 µm pore size). Invaded cells were visualized under a microscope by staining with 0.2 % crystal violet.39 Migration assay was carried out to evaluate the contribution of GDC-0449 on the migratory ability of MIA PaCa-2 cells. Cell monolayer was scraped using a micropipette tip and 48 to 72 hours after treatment with GEM, GDC-0449 and mixed micelle formulations, the residual gap length was calculated from photo micrographs.40 Clonogenic assay was carried out by treating 250 cells with micellar formulations for 10 days. Visible colonies (≥50) were counted following Crystal Violet (0.2% in 2% ethanol) staining and the % colonies was calculated compared with the control as described earlier.41 2.6. In vivo Study Animal experiments were carried out in accordance to the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Nebraska Medical Center (UNMC, Omaha, NE). Flank tumors were established in 8-10 week old male athymic nude mice by subcutaneous injection of 3 x 106 MIA PaCa-2 cells suspended in a total 200 µL of 1:1 serum-free media and Matrigel (BD Biosciences, CA). When the tumor volume reached 200-300 mm3 animals were randomly divided into


Page 13 of 44

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


four groups (n= 6): blank micelles, GEM conjugated micelles, GDC-0449 loaded micelles and combination micelles (GEM micelles: GDC-0449 micelles, 50: 50). Formulations were administered intratumorally thrice a week for 2 weeks at an equivalent dose of 40 mg/kg GEM and 10 mg/kg GDC-0449. Tumor size was measured at regular intervals using a vernier caliper. Body weight of the animals was recorded thrice a week. At the end of the study, tumor tissues were excised, weighed and snapped frozen for further analysis. 2.7. Histochemical and Immunofluorescence Assays Excised tumors embedded in OCT compound (Sakura Finetek, Torrance, CA), frozen and stored in -80 °C for further experiments. To stain Gli-1 and active caspase-3, sections (4 µm) were fixed in pre-cold acetone for 30 min and washed with PBS for 3 times. Permeabilization was done by washing the slides 2 times for 5 min in TBS + 0.025% Triton X-100. After blocking in 10% goat serum with 1% BSA in TBS for 2 h at the room temperature, sections were incubated with Gli-1 antibody (SC-20687) (1:50) (Santa Cruz Biotech., Dallas, TX) and active caspase-3 (SC-1225) (1:50) overnight at 4 °C. Next day, slides were washed with TBS + 0.025% Triton X-100 and further incubated with anti-rabbit IR dye conjugated secondary antibody. To evaluate tissue morphology, sections (4 µm) were stained with hematoxylin and eosin (H&E) and analyzed blindly. For cell proliferation, marker Ki-67 and EMT-activator ZEB-1, sections were probed with rabbit polyclonal Ki-67 antibody (1:50) (ab-15580) and rabbit polyclonal ZEB-1 antibody (1:150) (sc-25388), respectively. Sections were incubated for 45 min at the room temperature with anti-rabbit horse raddish peroxidase



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

Page 14 of 44

(HRP) conjugated secondary antibody diluted to 1:500 in 2% BSA/1X PBS solution. All stained slides were visualized under microscope (Leica, Germany). 2.8.

Statistics

Data values were expressed as the mean ± standard deviation (S.D.) Student’s t test was performed for statistical evaluation. Value of p

Micelle Mixtures for Coadministration of Gemcitabine and GDC-0449

Recommend Documents