Iridoid glycosides-Kutkin, Picroside I, and Kutkoside from Picrorrhiza kurroa Benth inhibits the invasion and migration of MCF-7 breast cancer cells through the down regulation of matrix metalloproteinases

2.3.1 Preparation of extract

Methanolic extract (PE) of P. kurroa was prepared by the following method: Accurately weighed 50 gm of the powdered drug was extracted with methanol (100 mL × 4) under reflux on a water bath. The extract was filtered through a Whatman I filter paper, pooled, and concentrated under vacuum to dryness.

2.3.2 Isolation of active constituents

We isolated Kutkin (KS) (Scheme 1c), Picroside I (PS) (Scheme 1a) and Kutkoside (KS) (Scheme 1b) using column chromatography. The isolated compounds were characterized by recording melting point using DSC, UV, IR, and Mass spectra and identified by comparing with the reported data (Singh et al., 1993).

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Scheme 1

(a–c) Structures of Picroside I, Kutkoside and Kutkin.

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2.5.1 MTT assay

MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is taken up by the viable cells and reduced to formazan by the “Succinate-tetrazolium reductase” system that belongs to the mitochondrial respiratory chain functioning in metabolically active cells. Formazan formed, is a purple colored water-insoluble product that is largely impermeable to cell membranes, thus resulting in its accumulation within the healthy cells which is solubilized by adding DMSO. The optical density (OD) of purple colored solution developed was read using a conventional micro plate reader at 570 nm (maximum absorbance). The ability of cells to reduce MTT provides an indication of the mitochondrial integrity and activity, which, in turn, may be interpreted as a measure of viability and/or cell number (Tripathi and Mait, 2005). Exponentially growing cancer cells were harvested from 75 mm² flask (TPP, Switzerland) and a stock cell suspension was prepared. 5 × 10⁴ cells/mL were seeded in a 96-well tissue culture plate with 200 μL of DMEM and incubated for 24 h. The dry extract and the isolated compounds (Kutkin, Picroside I and Kutkoside) were dissolved in a minimum amount of DMSO and were prepared in DMEM without phenol red. All the samples were first sterilized using PVDF membrane filters of 0.22 μm. Paclitaxel was used as a positive control and was used at 1 μM, 5 μM concentrations. Cells were treated with different concentrations, 50 μg/mL and 100 μg/mL of PE and 5 μM, 10 μM of isolated compounds (PS, KS, and KT) in 100 μL volume prepared in media without phenol red and with a positive control and were incubated for 48 h. The cells in the control group received no drug treatment. Each treatment was performed in triplicates. After the treatment, drug containing media was removed and washed twice with 100 μL of PBS (Phosphate buffer saline). To each well of the 96-well plate, 20 μL of MTT reagent (Stock: 5 mg/mL) was added and incubated for 4 h at 37 °C. Plates were shaken for 10 min and inverted with gentle tapping on tissue paper to remove the media. To solubilize formazan crystals in the wells, 100 μL of 100% DMSO was added to each well. Plates were placed on a Rotary shaker and agitated for 15–20 min. The optical density was measured by an Enzyme Linked Immunosorbent Assay (ELISA) plate reader at 570 nm with a reference wavelength of 630 nm. O.D of each well was read and expressed as percentage cell survival (absorbance of treated wells/absorbance of control wells × 100). Results were expressed as Mean ± S.E. O.D values (proportional to cell survival) was plotted against the extract concentrations. From the results, the active extract/s was considered to be significant which gave less than 50% survival at the exposure time of 48 h. The PE was further diluted in medium to produce 5 concentrations of 5, 25, 50, 75, and 100 μg/mL. 100 μL/well of each concentration was added to the plates in triplicate and incubated for further 48 h. Its cytotoxic effect was determined by MTT assay. The inhibitory concentration IC₅₀ of the crude extract was determined as the drug concentration that decreased 50% of the O.D of the control (untreated) cells.

2.5.2 Propidium iodide staining

PI binds to DNA by intercalating between the bases with little or no sequence preference. Once the dye is bound to nucleic acids, its fluorescence is enhanced 20–30-fold. Propidium iodide is suitable for fluorescence microscopy, confocal laser scanning microscopy, flow cytometry, and fluorometry. PI is membrane impermeant and is generally excluded from viable cells. PI is commonly used for identifying dead cells in a population and as a counterstain in multicolor fluorescent techniques. (According to the manufacturer’s protocol) Cells (5 × 10⁵ cells/mL) were cultured at 37 °C for 3 days in medium that contained 10% FBS. After 24 h media was aspirated. Cells were treated with different concentrations, 50 μg/mL and 100 μg/mL of the crude extract and 5 μM and 10 μM of the isolated compounds in 100 μL volumes were prepared in media without phenol red and were incubated for 48 h. The cells in the control group received no drug treatment. At the end of the treatment, the drug containing media was removed. Cells were thoroughly washed 2–3 times with 3 mL of PBS to remove any trace amount of drug and fresh 10% DMEM was added. The cells were harvested, washed with PBS, fixed with 75% ethanol at 4 °C for 2 h, and treated with 0.25 mg/mL RNAse (Sigma) at 37 °C for 1 h. After washing, the cells were stained with 500 μg/mL PI at room temperature for 10 min. Digital images were taken under inverted fluorescence microscope, Olympus 1X51 with Prog Res Software from Jenoptix.

2.6.1 Wound scratch migration assay

Cell migration is a complex phenomenon that requires the coordination of numerous cellular processes. The commonly used scratch wound healing assay of tissue-culture cell monolayers is by mechanically scratching the cell substrate with a sterile plastic pipette tip, a characteristically sized wound is created. Wound width is the measure of the distance covered by the cells at different concentration after a particular time point study. When wounded or scratched, the cell monolayer’s respond to the disruption of cell–cell contacts and to growth factors at the wound margin by healing the wound through a combination of proliferation and migration. These processes reflect the behavior of individual cells as well as the properties of the cell sheet as a surrogate tissue. They have been used with multiple cell types and, as the monolayer heals the wound in a characteristic manner, used to study cell polarization, matrix remodeling, cell migration, and numerous other processes.

A cell migration assay was performed using a 24-well plate as previously described (Kim et al., 2003). 1 × 10⁴ cells/mL were seeded in 24-well plates, prepared in DMEM, and incubated for 24 h. Cells were treated with different concentrations, 50 μg/mL and 100 μg/mL of the crude extract and 5 μM and 10 μM of the isolated compounds in 100 μL volume prepared in media without phenol red and with a positive control and were incubated for 48 h. The cells in the control group received no drug treatment. At the end of the treatment, a physical wound was given to all the plates in the presence of drug containing media with a sterile plastic micro tip in a linear fashion throughout the center of the plate. This media was removed and the cells were thoroughly washed 2–3 times with 2 mL of PBS to remove any trace amount of drug and the dislodged cells. One of the plates was immediately fixed in methanol. The rest of the plates were incubated with freshly prepared 10% DMEM for 24 h. After incubation, media was aspirated out; cells were washed twice with 2 mL of PBS, fixed with 75% ethanol at 4 °C for 2 h. After washing, the cells were stained with 500 μg/mL of dye at room temperature for 10 min. Digital images were obtained with an inverted fluorescence microscope.

2.6.2 Gelatin zymography assay

Zymography is an electrophoretic method based on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) for measuring activity of proteolytic enzymes. The gel is impregnated with a protein substrate depending on the activity of the enzyme to be studied (e.g. type III gelatin) that is commonly chosen for studying gelatinases. Gelatin is degraded by the proteases resolved during the incubation period (conditioned media). Samples for Zymography are prepared in the standard SDS–PAGE treatment buffer but without boiling, and without a reducing agent (β-mercaptoethanol). Following electrophoresis, the SDS is removed from the gel (or Zymogram) by incubation in 2.5% Triton X-100. Staining the gel with Coomassie (Sigma) and destaining with 40% methanol, 10% acetic acid allows the bands of proteolytic activity to be detected as clear bands of lyses against a blue background. Within a certain range, the band intensity can be related linearly to the amount of protease loaded (Hauzenberger et al., 1999).

In 6-well plates, 3 × 10⁴ cells/mL prepared in 10% DMEM were seeded and incubated for 24 h. Cells were treated with different concentrations, 50 μg/mL and 100 μg/mL of the crude extract and 5 μM and 10 μM of the isolated compounds in 100 μL volume were prepared in media without phenol red and with a positive control and were incubated for 48 h. The cells in the control group received no drug treatment. At the end of the treatment, the drug containing media was removed and the cells were thoroughly washed twice with 2 mL of PBS to remove any trace amount of drug. To the culture plate 2 mL of plain DMEM was added (without supplemented with 10% FBS) and incubated for 18–20 h. After incubation, the serum free media called as the conditioned media was centrifuged at 1400 rpm for 10 min to remove any cell debris. The cells in the culture were trypsinized, except that no extra fresh media was added to stop the trypsin activity instead the media collected from the culture plate itself was added. The conditioned media was concentrated by lyophilization and protein content estimated by Folin Lowry method, and stored at −80 °C until further use. The conditioned media was reconstituted using autoclaved water and to assess the gelatinase activity, samples (110 μL for 24 h and 150 μL for 48 h) were run on 10% (w/v) SDS–PAGE with certain modifications containing 2% (w/v) gelatin. Proteinase K (Sigma) served as a positive control. The gel apparatus was set up on a vertical mini gel assembly and the bottom was sealed with 0.8% agarose (HiMedia). Finally, the MMP gel was stained for 30 min with 0.25% (w/v) Coomassie blue in 10% acetic acid (v/v) and 50% methanol (v/v) and destained in 10% acetic acid (v/v) and 50% methanol (v/v). Gel documentation software was used and the images were taken.

2.6.3 Semiquantitative RT-PCR (mRNA analysis)

To check the mRNA expression of these proteases, cancer cells (2 × 10⁶ cells/mL) were treated with PE and PS, KS and KT for 18 h. The total RNA was isolated using TRIzol RNA Extraction Kit in accordance with the manufacturer’s instructions. Complementary DNA was synthesized from 1 μg of total RNA in a 20 μL reverse transcription reaction mixture. For semiquantitative PCR, aliquots of cDNA were amplified in a 25 μL PCR mixture as described previously (Zhang et al., 2004). The PCR products were size-fractionated and DNA bands were visualized by staining the gel with ethidium bromide. β-actin primers were used as normalization control. PCR was performed in the presence of 0.5 U Taq DNA polymerase (Takara, Japan) using primer sets that were specific to the following: human MMP-2 (5′-GTG CTG AAG GAC ACA CTA AAG AAG A-3′, 3′-GGA TGT TGA AAC TCT TCC TAC CGT T-5′); MMP-9 (5′-CAC TGT CCA CCC CTC AGA GC-3′, 3′-GGA ATA GCG GCT GTT CAC CG-5′); MMP-1 (5′-CCT TCT ACC CGG AAG TTG AG-3′, 5′-TCC GTG TAG CAC ATT CTG TC-3); MMP-13 (5′-GTG GTG TGG GAA GTA TCA TCA-3′, 5′-GCA TCT GGA GTA ACC GTA TTG-3′); β-actin (5′-TGT GAT GGT GGG AAT GGG TCA G-3′, 5′-TTT GAT GTC ACG CAC GAT TTC C-3′). The PCR products were separated on a 2% agarose gel containing ethidium bromide (0.5 μg/mL), visualized, and photographed using a gel documentation system.