Collection of Plant Material and Preparation of Extract
The plant material TA is scientifically well characterized belongs to clade: angiosperms, order: Caryophyllales, family: Tamaricaceae, genus: Tamarix and species: Tamarix articulata was collected in August 2019 from the deserts of Qassim province in the Kingdom of Saudi Arabia. A methanolic extract derived from the dry leaves was prepared as per the standard protocol published in our previous work . After collection, the TA parts were first air-dried in the shade to remove the moisture completely. Using sharp blades, TA were chopped into small pieces, followed by grinding in a kitchen blender to produce a fine powder. After weighing 12 g of TA powder was added to 300 mL of 100% methanol and stirred constantly with magnetic bead for 5 days at room temperature. The mixture obtained was first filtered through a cheesecloth to remove the bulk followed by filtration through a Whatman filter paper in an autoclaved glass beaker. The methanol (solvent) was completely evaporated from the plant extract mixture in a glass beaker by keeping the temperature of the hot plate at 45°C to avoid the degradation of heat labile compounds. After the complete evaporation of solvent, the fine powder of residue was left in the glass beaker are collected and stored at 4°C in stored vials for future experiments to evaluate the biological activities of the TA.
Plant metabolites from TA were extracted as decried by previously published work . Briefly, 50 mg of plant material was weighed into an Eppendorf tube and 1 ml of methanol was added, vortexed for 30 s, then centrifuged for 15 min at 5000 rpm. After centrifugation, 200 µL of the supernatant was diluted 5 folds with HPLC grade acetonitrile, and then filtered through a 0.2 µm filter. Afterwards, the metabolic filtrate was transferred into an LC vial for analysis.
LC–MS metabolomic analysis and data processing
The analysis was carried out on an LC-MS consisted of an ACQUITY UPLC I-Class System (Waters Technologies, USA) coupled to a 6500 Qtrap (AB Sciex, Canada). The chromatographic separation was performed on a Zorbax XDB C18 column (2.1×150 mm. 3.5 µm) (Agilent, USA) kept at 40°C with a flow rate of 300 µL/min. The mobile phase consisted of A (0.1% formic acid in HPLC grade water) and B (0.1% formic acid in HPLC grade acetonitrile). The linear gradient elution was as follows: 2% B (from 0 to 2), 95% B (from 2 to 24), 95% B (held for 2 min), then 4 min equilibration time. Electrospray ionization mass spectra (ESI-MS) was acquired in the positive (ES +), with an electrode voltage of 5500 V. The declustering potential was set at 90 V and the entrance potential was 10 V. Nitrogen was used as curtain gas (30 psi) and nebulizer gas on the MS. Spectra were collected with a mass range of 100–900 m/z. Data files from the LC were converted to MZxml format using MS Convert (ProteoWizard 3.0.20270). Analysis of the data was conducted using Mzmine software (version 2.53). After importing the data into MZmine, a minimum intensity cutoff of 1,000 was applied and the retention time was adjusted with a tolerance of 0.2 min. Adjusted peaks were then aligned into one mass list to facilitate identification and comparison. The KEGG Database was used to identify compounds of interest in the finalized list based on m/z with a tolerance of 30 ppm.
Culturing of Cells and Treatments
The cellular models of prostate cancer cells (PC-3, LnCaP, DU 145, C42B, and VCaP) along with benign hyperplasia normal (BPH) cells were ordered and purchased from the American Type Culture Collection (ATCC). All the purchased cell lines were cultured in their respective cell culture media [Roswell Park Memorial Institute (RPMI)-1640 (#11875101) and Minimal Essential Medium (MEM, #31095029)] in a 5% CO2 incubator. The cell culture medium procured from Thermofisher Scientific, was supplemented with L-Glutamine, and phenol red. Externally, culture media were added with 1% penicillin-streptomycin (pen-strip, #15140122), and 10% fetal bovine serum (FBS, #16000044) procured from Thermofisher Scientific. All the cell lines obtained from ATCC were regularly checked for all Mycoplasma contaminations.
Chemicals, Reagents, and Antibodies
Dimethyl sulphoxide (DMSO, #D2650), phenylmethylsulphonyl fluoride (PMSF, #52332), Propidium iodide (PI, #4170), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT, #M5655), triton X-100 (#T8787), paraformaldehyde (#158127), 4’,6-Diamidino-2-phenylindole dihydrochloride (DAPI, #D8417) and trypan blue (#T6146) were ordered and purchased from Sigma Aldrich. However, primary antibodies were ordered and bought from cell signaling technology, these being β-actin (#4970), PI3K-p110α (#4249S), Akt (#4691S), p-Akt-Ser473 (#4060), TGF-β (#3711S), SMAD2 (#5339S), SMAD3 (#9523S), TIMP1 (#8946S), MMP-2 (#40994S), E-cadherin (#14472S), Snail (#3879S), Vimentin (#5741S), and MMP-9 (#13667S). From Santacruz Biotechnology, secondary antibodies, namely anti-rabbit IgG (SC-2359) and anti-mouse IgG (SC-2005)-coupled with enzyme horseradish peroxidase (HRP) were procured.
Preparation of TA Stock Solution
The TA stock solution in DMSO (10 mg/ml) was prepared, aliquoted, and stored in autoclaved microcentrifuge tubes (1.5 ml) at -20°C. At the time of the experiment stock concentration was diluted with culture media to prepare a working concentration (10 -10000 µg/mL (10 -10000 µg/mL for exposing prostate cancer cells to varying doses of TA extract.
Cell Proliferation Assay/Cell Cytotoxicity Assay
The proliferation of prostate cancer cells was determined by the most used MTT assay . Briefly, a panel of prostate cancer and transformed cells (PC-3, LnCaP, DU 145, and BPH) were subjected to harvesting and seeded at a density of 2×103 cells in each well of a 96-well plate. The seeded cells were exposed to various doses of TA extract (10–10000 µg/mL) along with DMSO control. After incubation lasting 24 h in a 5% CO2 incubator, cells were flooded with 20 µl of 2.5 mg/mL MTT dye and incubated for 3–4 h in a 5% CO2 incubator at 37°C. After incubation, it is necessary to remove the media containing MTT dye and add DMSO to dissolve formazan crystals. These were formed when cells were incubated in MTT dye. The purple-colored solution obtained after proper mixing or gentle shaking of the 96-well plate was measured at 570 nm to record absorbance. The number of viable cells was analyzed as a percentage cell proliferation of treated cells compared to the untreated DMSO control group.
Colony Formation Assay
LnCaP cells 1×103 plated in the 6-well plates were treated with varying concentrations of TA extract (67.5, 125, 250, and 500 µg/mL) along with control DMSO and staurosporine (25 nM) . The treated cells were incubated in 5% CO2 at 37°C for 5 days. Next, the medium was discarded, and the treated cells were washed with pre-cooled PBS. Following this the cells were fixed in ice-cooled methanol for 20 min and then stained with 0.5% crystal violet solution for about 15 min. The cellular colonies stained in each well of the 6-well plate were photographed with a phase-contrast microscope, and the cells in each colony were counted.
Scratch Motility (Wound Healing) Assay
Briefly, prostate cancer cells (LnCaP) were harvested from the culture flask followed by seeding of 5.5×105 cells in each well of the 6-well plate . After overnight incubation in a 5% CO2 incubator at 37°C, a complete matt of the monolayer was formed of the cells. In the middle of each well, a scratch was made with a sterile 200 ul microtip to create a wound. The 6-well plate with serum-free media was employed to wash out detached cells while creating a wound. After clearing detached cells, wound-created wells were exposed to varying concentrations of TA extract, i.e., 67.5, 125, 250, and 500 µg/mL in the presence of untreated control and positive control camptothecin 2 µM for 24 h. Next, the cells were analyzed under a microscope, and then photographs were taken of areas where the wound was created at 0 h. The percentage of wound closure was measured by using the formula as follows:
% age of wound closure = [1-(area of wounding after 24 h/area of the wound at 0 h) x 100%]
Transwell Boyden Chamber Invasion Assay
Briefly, 1.25 × 106 LnCaP cells were harvested, counted, and added uniformly to the culture medium without serum in upper insert of the Transwell Boyden chamber plate and subsequently subjected to varying doses of TA extract (67.5, 125, 250, and 500 µg/mL). This occurs in the presence of control (untreated) and camptothecin 2 µM as a positive control. The lower chamber was filled with 10% serum-containing media as the chemoattractant. Re-suspended cells exposed to varying concentrations of TA extract tried to move towards the lower chamber containing 10% serum media. After 24 h, the porous Matrigel-coated polycarbonate insert membrane was removed and the attached cell inside the insert was completely wiped off with a swab of cotton. These cells adhered to the bottom outside of the upper insert were fixed in pre-cooled methanol for 15 min. This was followed by staining the cells with 0.25% crystal violet solution in PBS. Then this stained porous biological membrane was put under a microscope to examine cells and subsequently photographed. The cells which can cross the membrane were photographed and counted under a phase-contrast microscope.
Prostate (LnCaP) cancer 5×105 cells were harvested and plated in a 6-well plate as performed previously . Cells were exposed to varying concentrations of TA extract (67.5, 125, 250, and 500 µg/mL) along with control DMSO and camptothecin (2 µM) as a positive control for 24 h. The conditional medium obtained from each treated well along with untreated control and positive control were collected in separate autoclaved 2 ml microcentrifuge tubes. Each sample was subjected to protein estimation by the standard Bradford method. The equal protein concentration of all samples was mixed with loading dye. The samples were loaded in the wells of SDS-PAGE gel containing 0.1% gelatin and the gel could resolve by setting the voltage initially at 80 V and after samples entering the resolving gel set the voltage at 100 V for 3 h at 4°C. The resolved gelatin gel was washed with a 2% Triton X-100 buffer to remove SDS. The gel with incubation buffer was incubated to renature the matrix metalloproteinases for 24 h at 37°C. Following this, the gel with Coomassie brilliant blue solution was stained for 1 to 2 h, followed by distaining of gel with a mild destaining solution to ensure clear bands appeared. The gelatinase activity was determined by analyzing clear transparent bands against the dark blue background after Coomassie brilliant blue staining.
Briefly, 50 × 105 cells (LnCaP) were plated in each well of the 6-well plate and they were allowed to adhere overnight to the bottom surface of the well in a 5% CO2 incubator at 37°C. The attached LnCaP cells were exposed to varying doses of TA extract (67.5, 125, 250, and 500 µg/mL) along with DMSO vehicle . After 24 h, the treated cells were gently washed with pre-cooled PBS, scraped, and harvested the cells in a sterile microcentrifuge tube. 500 µl of lysis buffer which contained PMSF and protease cocktail inhibitor were added, and cells repeatedly agitated intermittently for 10 seconds and kept on ice for 2–3 min. This was done 5 times to ensure complete lysis. The cell lysate solution was centrifuged at a speed of 12000 g for 15 min at 4°C to collect the supernatant. Using the Bradford method, the collected supernatant was subjected to the process of estimating protein concentration. 15–20 µg protein from each TA exposed cell sample was calculated for loading in SDS-PAGE gel and resolving the gel. This followed the protocol mentioned in the above reference. Then SDS-PAGE gel was transferred onto the PVDF membrane followed by incubation of this membrane in a blocking solution containing 5% fat-free milk. This served to block nonspecific sites. The PVDF membrane was incubated with primary antibody solution for 3–4 h at room temperature or overnight at 4°C. Subsequently, the PVDF membrane was washed with TBST 6 times followed by incubation with secondary antibody labeled with enzyme horseradish peroxidase for 1 h 30 min at room temperature. After incubation was completed the PVDF membrane was washed with TBST 6 times and each washing lasted 5 min. After washing, cleaning, and drying the PVDF membrane, immunoreactive substance enhanced the process of chemiluminescence to detect the bands on the PVDF membrane. The chemiluminescent signal on the PVDF membrane with the x-ray films was captured after proper exposure under dark conditions or with inbuilt chemiluminescent gel doc.
Cell Proliferation (brd U) Assay
Antiproliferative activity of TA extract against a panel of prostate cancer cell lines was evaluated by using colorimetric method Brd U cell proliferation ELISA kit (Abcam #Ab126556). Briefly, 2 × 103 cells were plated in each well of 96-well plate overnight. After adhered properly to the bottom surface of well, cells were exposed to varying doses (10–10000 µg/mL) of TA extract for 24 h at 37 ˚C in 5% CO2 incubator in triplicates. After the exposure of cells with TA extract, as per the manufacturer’s instruction, cells were incubated with Brd U for 2 h at 37 ˚C to incorporate Brd U in DNA. The cells were fixed, permeabilized and desaturated, followed by gentle washing and aspiration. Cells were incubated with anti-Brd U monoclonal antibody for 1 h, at room temperature. After proper incubation and removal of unbound fraction of anti-Brd U from wells by gently washing and aspiration, wells were incubated with TMB solution containing enzyme-labeled goat antimouse antibody. After the formation of color by enzymatic reaction indicates the cell proliferation, the stop solution was added, and absorbance were recorded immediately by using multiplate reader.
Cell Counting (trypan Blue) Assay
Briefly 5 × 104 prostate cancer cells were plated in each well of 24-well plate. After properly adhered to the bottom surface, cells were exposed to dose-dependent (10–10000 µg/mL) treatment with TA extract for 24 h at 37 ˚C in 5% CO2 incubator. After the completion of time point the treated cells were harvested and stained with trypan blue dye. The dead cells were stained with trypan blue; however, live cells were easily counted by trypan blue exclusion test.
Phase-contrast microscopy was performed to detect the change in cellular morphology of prostate cancer cells after dose-dependent treatment with TA extract for 24 h. Briefly, 0.5 × 106 were seeded per well of 6-well plate and exposed to different doses of TA extract for 24 h. After the completion of treatment cells were analyzed by phase-contrast microscope inbuilt with camera to detect any change in cellular morphology of cells.
Detection Of Apoptosis By Tunel Assay
Detection of apoptotic population of prostate cancer cells after exposed to varying doses of TA extract for 24 h were detected by TUNEL (Terminal Transferase dUTP Nick End Labeling) assay apoptosis detection kit (Roche). After seeding the LnCaP cells at a density of 5 × 105 per well on the sterile coverslips placed within the wells of 6-well plate were incubated overnight in 5% CO2 at 37 ˚C. After the completion of exposure time (24 h) to various doses of TA extract (67.5, 125, 250, and 500 µg/mL), cells grown on the coverslips were processed for fixation (4% paraformaldehyde), permeabilization (4% Triton X-100) at room temperature for 10 min. Next, cells grown on the coverslips were incubated with a mixture of terminal deoxynucleotidyl transferase (TdT) and biotin-dUTP for 1 h, followed by 30 min incubation with avidin-FITC solution in dark. Subsequently, DAPI solution was added to stain the nucleus. After the completion of TUNEL staining, coverslips containing cells were put on to the clean grease-free glass slide and seal the edges with nail-polish to avoid any dry ness of the slide. The glass slides were analyzed by confocal microscope (Zeiss LSM 710 META, Germany) for the detection of TUNEL positive cells.
All the experiments in the current study were performed at least three or more times. Statistical analysis of all the independent experiments was conducted by using GraphPad Prism software. The results obtained were expressed as ± SEM. All the experiments were analyzed using one-way ANOVA. Statistical significance was expressed as p-value, wherein the p-value of less than or equal to 0.05 was significant.