Reagents and chemicals
The CRYP used in the study was donated by Prof. Kwesi Mensah Boadu, Faculty of Pharmacy and Pharmaceutical Sciences, Kwame Nkrumah University of Science and Technology (KNUST), Kumasi, Ghana. It was dissolved in phosphate buffered saline (PBS) to a stock concentration of 43050 μM, filter sterilized and divided into aliquots, wrapped in aluminum foil and stored frozen at -20 °C until used. The CRYP stock solution was diluted to the desired concentration in normal growth medium when necessary. The foetal bovine serum (FBS) (cat # F2442) was purchased from Sigma-Aldrich, USA. Dulbecco’s Modified Eagles Medium (DMEM) (high glucose, L-glutamine, sodium pyruvate) (cat # 1-26F58-1) was purchased from BioConcept Ltd, Switzerland. Minimum essential medium (MEM) non-essential amino acids (NEAA) (cat # 0823) was purchased from ScienCell, USA. Opti-MEM reduced serum medium (cat #31985-070) was purchased from Gibco Life Technologies, USA while penicillin-streptomycin (cat #15140) was purchased from Gibco by Invitrogen, UK. Thiazolyl blue tetrazolium bromide powder (MTT powder) (cat # M5655-1G) was purchased from Sigma Aldrich, USA. The MTT powder was dissolved in PBS to a stock concentration of 5 mg/ml, filter sterilized and divided into working aliquots, wrapped in aluminum foil and stored at -20°C until used. Isopropyl alcohol (> 99.5% purity, cat # 67-63-0) was purchased from Dae-Jung Chemicals and Metals, Siheung-Si, Gyeongii, Korea. Cignal Finder Reporter Array Plates (cat # CCA-901L) were purchased from SA Bioscience, USA. Attractene Transfection Reagent (cat # 301005) was purchased from Qiagen, USA. The Dual Luciferase Reporter Assay System (cat# E1960) was purchased from Promega, USA.
The HEK 293 cells (cat # CRL-1573) used in the study was purchased from the American Type Culture Collection (ATCC), USA. The cells were grown in DMEM high glucose-containing L-glutamine, sodium pyruvate supplemented with 10% v/v heat-inactivated FBS, 1% v/v MEM non-essential amino acids, 100 IU/ml of penicillin and 100 μg/ml of streptomycin. The cultures were maintained at 37 °C in 5% carbon dioxide (CO2) under humidified condition.
HEK 293 cells were grown to about 60% confluence and then treated with increasing concentrations of CRYP (0-10 μM). The cytotoxic effect of CRYP was evaluated using MTT assay at 24, 48- and 72-hours post-treatment following the manufacturer’s instruction. Briefly, the cell culture media containing the CRYP was carefully removed. Fifty microliters (50 µl) of serum-free media and 50 µl of MTT solution were added into each well. Solvent control wells or blank wells containing 50 µl of the MTT reagent and 50 µl of cell culture media (no cells) were included in the assay. The plates were incubated for 3 hours at 37°C. After incubation, 150 µl of isopropyl alcohol (MTT solvent) was added into each well. The plates were wrapped in aluminum foil and shaken on Edmund Buhler orbital shaker (Edmund Buhler GmbH, Germany) for 15 minutes. The optical density (OD) of the wells was read at 590 nm using iMark Microplate Reader (Bio-Rad, USA).
Cignal Finder 45-Pathway Reporter Array and Reverse Transfection
We used the Cignal Finder 45 Pathway Reporter Array Plate (Qiagen and SA Biosciences, USA) to simultaneously assess the effects of CRYP on 45 different signalling pathways. These 45 signalling pathways cover research areas such as cancer, immunology, development and toxicology. The Cignal Finder 45-Pathway Reporter Array has 45 pathway reporters dried and coated down in duplicate wells of the 96-well plate with the remaining 6-wells containing positive and negative controls. Each reporter consists of an inducible transcription factor-responsive construct and a constitutively expressing Renilla luciferase construct. The inducible transcription factor-responsive construct encodes the firefly luciferase reporter gene and monitors both the increase and decrease in the activity of the coupled transcription factor in a said signalling pathway while the Renilla construct encodes the Renilla luciferase reporter gene which serves as an internal control to which firefly activity is normalized. To determine the effects of CRYP on the 45 signalling pathways, the reporter constructs were reverse transfected into HEK 293 cells following the manufacturer’s instruction. Briefly 50 µl of Opti-MEM® was added into each well of the Cignal Finder Array plate to resuspend the reporter constructs. The plate was incubated at room temperature for 5 minutes. The Attractene transfection reagent was diluted in Opti-MEM after which 50 µl of diluted Attractene was added into each well containing 50 µl of the diluted nucleic acids. Cells were suspended to a density of 8 × 105 cells/ml in Opti-MEM® containing 10% of FBS and 0.1mM NEAA and 50 µl of the cell suspension was added to each well of the Cignal Finder Array plate containing the reporter constructs and the transfection reagent. The cells were incubated at 37°C in a 5% CO2 incubator for 18 hours. Following reverse transfection, the cells were treated with assay medium (Opti-MEM® supplemented with 0.5% FBS, 0.1mM NEAA, 100 U/ml Penicillin and 100 µg/ml Streptomycin) containing 5 µM CRYP for another 18 hours. A control experiment in which the cells were reverse transfected but not treated with cryptolepine was done in parallel with the treated experiment. The cells were lysed passively and dual luciferase expression was determined using the dual luciferase reporter assay system (Promega, USA, cat no E1960) following the manufacturer’s protocol. After preparing the cell lysates, 20 μl of the aliquot was employed for luminescence measurement using Berthold Orion luminometer (Berthold Detection Systems, Germany).
Reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR)
HEK 293 cells were cultured and treated with increasing concentrations of CRYP (0-5 µM) for 24 hours. Total RNA was then extracted using the Gene JET RNA purification kit (Thermo Scientific, Germany) following the manufacturer’s instruction. The quantity and the purity of the total RNA was verified by spectroscopy (Nano Drop 1000, Thermo Scientific). The purity was later confirmed by 1% agarose gel electrophoresis using ethidium bromide as a stain. The total RNA was converted to cDNA as we previously described (19). The cDNA was stored frozen at -80 °C until used in the qPCR. IRF1, PR, HIF1-α and STAT3 target genes were amplified using the Maxima Probe/Rox qPCR master mix (Thermo Scientific, Germany). The primers and probes used were designed and synthesized by Biomers, Germany (Table 1). The probes of the target genes and the endogenous control Beta-actin (B-actin) were labelled with different fluorescent reporter dyes at the 5′ end and quencher dyes at the 3′ end and this allowed the target genes to be amplified in the same tube in a duplex qPCR reaction. After optimizing the primer and probe PCR conditions, a duplex qPCR was performed in a 20 μl reaction volume that contained 0.3 μM forward and reverse primers of the target genes, 0.2 μM of the target probes, 0.2 μM forward and reverse primers of the B-actin, 0.2 μM of the B-actin probe and 2.0 μl of 1:5 dilution of the cDNA samples. The qPCR cycling conditions were as we previously described(19). The qPCR reaction products were analyzed using the Applied Biosystems StepOne Plus Manager Software. The relative quantification of the target genes was calculated using the Relative Standard Curve method.
All experiments were conducted three (3) times at different times in duplicate or triplicate wells where applicable. Data were entered into an excel spread sheet and then analysed using excel and the appropriate data management software. One-way Analysis of variance (ANOVA) and student t-tests were used to analyse the level of significance between the treated and untreated groups where applicable. P-value ≤ 0.05 was considered as statistically significant.
Table 1: Primers and probe sequences
Sequences of primers & probes
Forward primer: 5’-TCACCCACACTG