SUPPLEMENTARY MATERIAL
Anti-metastatic Effect of Deoxyelephantopin from Elephantopus
scaber in A549 Lung Cancer Cells in vitro
Farha A. K.a, Dhanya S.R.b, Mangalam S Nairb and Remani Pa*
aDivision
of Cancer Research, Regional Cancer Centre, Thiruvananthapuram, Kerala,
India., bCSIR-National Institute for Interdisciplinary Science and Technology,
Thiruvananthapuram, Kerala, India.
Corresponding author: Email: [email protected]
Abstract
In this study, we focused on the in vitro anti-metastatic effects of Deoxyelephantopin
(DOE), a sesquiterpene lactone from Elephantopus scaber on lung cancer A549 cells.
DOE significantly decreased the metastatic potential of A549 cells as demonstrated by
transwell invasion and migration assay. DOE inhibited the expression of MMP-2, MMP9, uPA and uPAR at transcript level. TIMP-2 mRNA levels was up-regulated in A549
tumor cells without any change in TIMP-1 expression after DOE treatment. DOE
inhibited the protein levels of p-ERK1/2 and p-Akt in A549 cells but it activated p-JNK,
p-p38 protein expression. NF-kB and IKBα expression were down-regulated in DOE
treated cells. All these results demonstrated that DOE has shown anti-metastatic activity
against A549 tumor cells.
Key
words:
Deoxyelephantopin;
Elephantopus scaber; TIMP
anti-migratory;
anti-invasive;
MMP;
uPA;
Experimental
Chemicals and reagents
Dulbecco’s Modified Eagles Medium (DMEM), 1X antibiotic-antimycotic solution, Ready
Mix Taq PCR reaction mix, ethidium bromide and crystal violet were obtained from
Sigma-Aldrich (USA). Fetal bovine serum (FBS) was purchased from Pan Biotech
(Germany). Antibodies ERK1/2, p- ERK1/2, JNK, p-JNK, p38, p-p8, Akt, p-Akt and βactin were procured from Cell Signaling Technology (USA).
Isolation of Deoxyelephantopin from E. scaber
E. scaber was collected from Trivandrum district in Kerala, India. A voucher specimen
(TBGT-25419) was deposited in the herbarium of Jawaharlal Nehru Tropical Botanical
Garden and Research Institute (JNTBGRI), Palode, Trivandrum. E. scaber as whole
plant was extracted using chloroform at room temperature and solvent was completely
removed under vacuum. DOE (Figure 1) was isolated via fractionation of E. scaber
chloroform extract as previously described (Geetha et al. 2011). A stock solution of
DOE was prepared in DMSO (1mg/ml), and stored at -200C. Before use, the solution
was freshly prepared by diluting with DMEM to the desired concentration. In order to
evaluate the effects of DOE on the metastatic activity of A549 cells, it was necessary to
choose a non-toxic effective dose. We have previously reported that DOE exhibited
antitumor effects on A549 cells with an IC50 value of 12.28 µg/ml DOE at 48 h of
incubation (Kabeer et al., 2013). For all experiments, the concentration used was 12.28
µg/ml DOE. IC50 value at 48 h incubation was chosen for subsequent experiments since
the viability of cells remained above 70% when the cells were treated at this dose for 24
h. The maximum incubation time for most of the anti-metastatic assays was only up to
24 h. Final concentration of DMSO used in treated group was 0.6%. The above
concentration of DMSO did not affect the viability of A549 cells.
Cell culture and maintenance
The lung cancer cell line A549 was obtained from National Centre for Cell Sciences
(NCCS), Pune, India. Cells were grown in DMEM supplemented with 10% FBS and 1X
antibiotic-antimycotic solution and maintained at 37 0C in a humidified atmosphere
containing 5% CO2.
In vitro wound closure assay
A549 cells were seeded in 6-well plate and incubated to attain 80% confluence. An
injury line was made with a pipette tip on cell monolayer and washed with PBS to
remove floating cells. The cells were then treated with DOE and incubated for 48 h. The
scratched area was photographed in different time intervals (0, 24 and 48 h) in 3
randomly selected fields (100X magnifications) in 3 independent experiments.
Migration assay
A549 cells (5x104) were seeded in transwell chamber (BD Biosciences, USA) with or
without DOE. DMEM containing 10% FBS was placed in the bottom chamber as
chemoattractant. The plates were incubated at 370C for 24 h. Migration was assessed
after removing non-migrating cells from the upper chamber using a cotton swab. Cells
on the bottom of the filter were stained with 0.5% crystal violet for 10 min and counted
(three fields of each triplicate filter) using an inverted microscope (Olympus 1X51,
Japan).
In vitro invasion assay
Matrigel invasion assays were performed using 24-well BD BiocoatTMMatrigel Invasion
Chamber (BD Biosciences, USA). The matrigel coated filters were rehydrated by adding
DMEM and kept in a humidified incubator at 370C and 5% CO2 for 2 h. The medium was
removed after rehydration. Cells (2.5x104/0.5ml) treated with or without indicated
concentration of DOE were placed in the upper part of the transwell chamber and
incubated for 24 h at 370C. DMEM containing 10% FBS was placed in the lower
chamber as chemoattractant. The non–invading cells on the upper chamber were
removed by scrubbing with cotton swabs. The cells that invaded to the lower surface of
the membrane were fixed with methanol and stained with 0.5% crystal violet in 25%
methanol for 10 min. The number of invaded cells was counted under light microscope
in 3 randomly selected fields (400X magnification).
RNA extraction and Reverse Transcription-PCR
Total RNA was extracted from A549 cells using TRI Reagent (Sigma-Aldrich, USA)
according to the manufacture’s protocol. The RNA was checked qualitatively by
electrophoresis in 2% agarose gel and quantitatively by spectrophotometry. For RTPCR, cDNA was synthesized from 1µg of total RNA using High capacity cDNA reverse
transcription kit (Applied Biosystem, USA) according to the manufacturer’s protocol. The
cDNA was amplified by PCR with the following primers (Table.S1). PCR products were
analysed by agarose gel electrophoresis stained with ethidium bromide.
Western Blotting
A549 cells (106) were treated with DOE for 48 h and total cell lysates were prepared.
Protein samples (50 μg) were separated on 8% SDS-PAGE and transferred onto a
PVDF membrane (Millipore, India). The membranes were blocked with 5% BSA in PBS
containing 0.1% Tween 20 for 1 h and then incubated with primary antibodies (ERK1/2,
p-ERK1/2, JNK, p-JNK, p38, p-p38, Akt, p-Akt and β-actin) overnight at 40C. After
washing with TBST, the membranes were incubated with HRP-conjugated secondary
antibody for 2 h and specific proteins were detected by enhanced chemiluminescence
detection kit (Thermo scientific, USA).
Statistical analysis
Data were expressed as mean ± standard deviation. Student's t-test was used to
determine the level of significance at P <0.05.
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Table S1: List of primers used in RT-PCR
Figure S1: Effect of DOE on tumor cell migration and invasion property (A) In vitro
wound closure assay. Cells were maintained in 6-well plate overnight. Wound was
introduced by scraping with a pipette tip. The plates were maintained with or without
DOE for 48 h. The photographs were taken in different time intervals (B) Transwell
migration and invasion assay of A549 cells treated with DOE. Cells were seeded in
transwell chamber and incubated with or without DOE for 24 h. Migrated and invaded
cells were detected by crystal violet staining (400X magnification). Data are mean ± SD
values from three independent experiments. *, P < 0.05 and **, P < 0.01 versus
untreated control.
Figure S2: Effect of DOE on the migration and invasion-associated genes and protein
expression levels in A549 cells. (A) The levels of MMP-2, MMP-9, uPA, uPAR, TIMP-1,
TIMP-2 and GAPDH. Total RNA was extracted from DOE treated cells and RNA
samples were reverse-transcribed for RT PCR. (B) The protein expression of ERK, pERK, JNK, p-JNK, p38, p-p38, Akt, p-Akt and β-actin. Cells were treated with DOE for
48 h, and the proteins levels were assessed by western blotting. Data represents mean
± SD of three experiments.
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Anti-metastatic Effect of Deoxyelephantopin from Elephantopus