Chemicals, antibodies and kits
Growth medium (RPMI-1640), Fetal Calf Serum (FCS), Trypsin, Penicillin, Streptomycin, DMSO, RNase, Proteinase K, RIPA buffer, Acrylamide, Bisacrylamide, SDS, Ammonium persulfate (APS), N, N, N’, N’ Tetramethylethylenediamine (TEMED), 2-Mercaptoethanol, Tris base, Propidium iodide, Protease inhibitor cocktail, Sodium Deoxycholate, EGTA, Salmon sperm DNA, Bovine Serum Albumin, Quanti-Pro BCA assay kit, NaCl, EDTA, Hexadimethrine bromide and Lentiviral constructs were obtained from Sigma Aldrich Pvt. Ltd. (St. Louis, MO, United States). Six well plates were obtained from Iwaki (Japan), Triton X-100, H3K9ac, H3K14ac, H3K18ac, H3K27ac, H4K5 ac, H4K12ac, H4K16ac and H4K20 me3 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Immobilon Western Chemiluminescent HRP Substrate, Protein A Agarose beads, H3 and H4 antibodies were purchased from Millipore (Billerica, MA, USA). Anti-HDAC 1, Anti-HDAC 2, Anti-HDAC3, and Anti-HDAC8 antibodies and HDAC isoform specific fluorometric drug screening kit were procured from Biovision (Milpitas, CA, USA). SAHA and NaB were purchased from Selleck Chemicals Ltd (Houston, TX, USA).
Cell culture and growth conditions
Cell lines HL-60 and MOLT-4 were procured from European Collection of Authenticated Cell Cultures (ECACC), UK. Chemoresistant cell line Doxo-K562 was a kind gift from Dr. Ajay’s Lab in CSIR-IIIM, India. All cell lines were maintained in RPMI-1640 medium supplemented with 10% FCS and culture media was supplemented with 1% Penicillin. Cells were cultured in humified conditions at 37°C in CO2 incubator (New Brunswick, Galaxy 170R, Eppondroff) with internal atmosphere conditions of 95% humidity and 5% CO2.
Histone extraction and Western blot analysis
These experiments were performed as described previously [16] with certain modifications. HL-60, MOLT-4 and DOXO-K562 cells were seeded, incubated and harvested at different time points (6-48 h).
Measurement of HDAC isoform specificity
This assay was carried out as described previously [16] with certain modifications. SBAK-GHA and SAHA were used in experimental setup.
DNA content and cell cycle analysis
Cell Cycle analysis was performed as described previously [16] with certain modifications. HL-60, MOLT-4 and DOXO-K562 cells were seeded, incubated and harvested at different time points (6-72 h for HL-60) and (24-48 h for MOLT-4 and DOXO-K562).
Lentiviral Transduction
For stable and transient transduction protocol, procedure of Sigma-Aldrich was followed with certain modifications. 1.6 x 104 HL-60 cells were added in fresh media to corresponding number of wells for construct in a 96-well plate. Triplicate wells for each lentiviral construct and control were used. 4-6 h of incubation at 37°C in a humidified incubator in an atmosphere of 5-7% CO2 was carried. The number of cells plated was accommodated to a confluency of 70% upon transduction. Hexadimethrine bromide (final concentration 8 mg/ml) to each well was added just before lentiviral addition. Multiplicity of infection (MOI) of different isoforms of Class 1 HDACS i.e. HDAC 1, HDAC 2, HDAC 3, HDAC 8, Non target control (NTC) and Luciferase positive control were standardized and then used singly or in combination for stable and transient transduction. The cells were incubated for 48-72 h before adding Puromycin for selection (kill curve for Puromycin was drawn and 5µg/ml was used) in case of stable cell line formation, Puromycin addition continued every 3-4 alternative days for one month. Knockdown of the selected target gene by stable transduction could not be validated by immunoblotting and microscopy due to the death of cells (data not shown). However, incubation period of 48-72 h for transient transduction was carried out without Puromycin addition. Cells were harvested and knockdown of the target genes were validated by immunoblotting and microscopic based expression studies.
Chromatin Immunoprecipitation (ChIP) assay
Approximately 10x106 HL-60 and MOLT-4 cells were seeded in 100 mm dishes and cultured with different concentrations of SAHA and SBAK-GHA for 48 h. Briefly, cells were cross linked with 1% formaldehyde for 10 minutes at room temperature and cross linking reaction was stopped by adding glycine (125 mM). After centrifugation at 1000 rpm for 5 minutes, the cells were incubated in RIPA buffer in presence of protease inhibitors and sonicated to shear DNA to lengths between 200 and 800 base pairs. After sonication, centrifugation was done at 14,000 g for 20 minutes at 4°C. The supernatant was 10-fold diluted with ChIP dilution buffer (16.7 mM Tris-HCl pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100). A 10% aliquot of the total chromatin preparation was set aside and designated as the input fraction. Protein A agarose beads were blocked with salmon sperm DNA and 1 mg/ml BSA for 4 h at 4°C under constant agitation. The samples were pre-cleared by incubating with 50 μl of protein A agarose beads. Following centrifugation (1,000 rpm, 1 min, 4°C), sample supernatants were added with prescribed concentration of antibodies overnight at 4°C under constant agitation followed by addition of pre-cleared beads for 6 hours. After this step immuno-precipitates were collected. Washing was done and complexes were eluted with 200-300 μl elution buffer (0.1 M NaHCO3, 1% SDS and 10 mM DTT). After addition of 0.2 M NaCl, all samples, including input, were incubated at 65°C for 4 h to revert cross-linking. Following treatment with 100 μg/ml Proteinase-K, DNA was extracted by phenol-chloroform-isoamyl alcohol method. The immunoprecipitated DNA was quantified by Nano drop and results were analyzed by qPCR method.
Quantitative real-time PCR
Immunoprecipitated DNA was analyzed for cyclins by Real-Time PCR as per Khanday et al., (2013) with some modifications. Primers were designed using the online Primer-BLAST tool (http://www.ncbi.nlm.nih.gov/tools/primerblast/) and cross-checked for primer self-dimerization and potential hairpin formation using Oligonucleotide Properties Calculator. Details of primers are given in (Table S1).
The quantified DNA was analyzed using Applied Biosystems Step One plus TM Real-Time PCR System under the conditions of 1 cycle of 95oC for 10 min, 40 cycles of 95oC for 15s, primer-specific annealing temperature for 1 min and followed by a melt curve of 95oC for 10s, 60oC for 1 min and 95oC for 15s. The PCR mixture contained 10 µL of SYBR Green master mix (Applied Biosystems, CA, USA), 500 nM of each primer and 1.5 mM of MgCl2 and 1 µl (5-20 ng) of DNA template for 20µl reaction.
The comparative Ct method (ΔΔCT) (Livak et al., 2001) was used for quantification of DNA immunoprecipitated with different specific Abs and IgG. Measurement of ΔCt was performed in triplicates.
In vivo anticancer activity
SBAK-GHA was evaluated for its in vivo anti-cancer activity against P388 lymphocytic leukemia and L1210 lymphoid leukemia murine models. CD2F1 mice (18-23 g) from an in-house colony were used for this study. The animals were housed under standard husbandry conditions as per guide for the care and use of laboratory animals and fed with standard pellet diet and autoclaved water was given ad libitum. Approval of the Institutional Animal Ethics Committee, Indian Institute of Integrative Medicine, Jammu was sought for the study and number of animals used in all the experiments. P388 and L1210 were obtained from the peritoneal cavity of DBA/2 mice harboring 6-10 days old ascitic tumor. 2.5×106 cells were injected in the peritoneal cavity of each mouse on day 0. Intraperitoneally injected animals were randomized on the next day and assigned to different groups containing six animals in each group. Test group first was treated with SBAK-GHA at a dose of 25mg/kg i.p. test group second was treated with SAHA at a dose of 50mg/kg i.p. test group third was treated with 5-FU at a dose of 20mg/kg i.p. which served as positive control. Tumor bearing control group was administered normal saline (0.2 ml, i.p.). Treatment lasted for nine consecutive days and Median Survival Time was taken as the Parameter of Effect. Median Survival Time can be calculated by the following formula.
Median Survival Time= ∑S + 6S5 – 19N ÷ S5 – NT
Where, S5 = No. of survivors on Day 5, ∑S= Sum of daily survivors from day 6 to day 18, NT = no. of no takes (survivors beyond day 18)
Acceptable Control Median Survival Time: 9-13 days
Minimum criteria of activity: T/C < 86% Toxicity
T/C ³ 130% Moderate Activity
T/C ³ 175% Significant Activity
Statistical evaluation
The results of experiments were expressed as the Mean ± S.D. Statistical evaluation was performed using students’ unpaired t-test ***p ≤ 0.001, **p ≤ 0.01, *p ≤ 0.05 for each analysis.