Diosgenin induces cell cycle arrest and apoptosis in human lung cancer cells (A549). Treatment with Diosgenin caused G0/G1 cell cycle arrest and increased the sub-G0 hypodiploid population in a dose-dependent manner. It also reduced mitochondrial membrane potential and increased caspase 3 expression, indicating induction of the intrinsic apoptosis pathway. Flow cytometry with Annexin V/PI staining confirmed a dose-dependent increase in apoptosis with Diosgenin treatment. The study suggests that Diosgenin has potential as an anti-cancer agent for lung cancer by blocking cell cycle progression and triggering mitochondrial apoptosis.

1. Diosgenin induces G0/G1 phase cell cycle
arrest and apoptosis in human non small
cell lung cancer cell line A549
Supriya Nath
Abstract
Diosgenin, a purified steroidal sapogenin from the plants under genus Dioscorea (e.g. Yam) and seeds of Trigonella
foenum graecum (Domestic Fenugreek) inhibits cell proliferation and induces apoptosis in various cell line models such as
laryngocarcinoma and melanoma. Though any of the proapoptotic roles of Diosgenin has not been reported yet in case of human
lung carcinoma. That s why we have investigated to screen Diosgenin as a pro apoptotic molecule in case of human lung cancer
cell line A549. Hemolytic assay and MTT assay was performed to determine the less toxic and better effective dose. Cell cycle
distribution prominently shows a G0/G1 phase cell cycle arrest in A549 cell line in a dose dependent manner. The flowcytometric
analysis using AnnexinV-FITC confirms the induction of apoptosis in a dose dependent manner in addition to that Mitochondrial
membrane potential was also reduced accross the mitochondrial membrane which was evaluated using Rhodamine 123. Finally
the western blot analysis of Caspase 9 more surely directs us towards the intrinsic pathway for apoptosis. Though more study is
required to confirm the whole mechanism of the apoptosis triggered by Diosgenin in A549 cells. From here we can conclude that
Diosgenin can trigger the apoptosis of A549 cells by blocking the cell in G0/G1 phase and then by triggering the mitochondrial
pathway for apoptosis.
1. Introduction
Arrest of the cell cycle often leads to various modes of cell death; apoptosis is one of them having least or no effect
on neighbouring tissues than necrosis. Loss of apoptosis is one of the first achievements of a presumptive tumour
to survive [1]. That s why targeting apoptosis of a transformed cell, is a novel way to treat various cancers [2].
Lung cancer is one of the leading fatal cancers. According to the report of WHO, there are 1.8 million new cases
of lung cancer in 2012 worldwide [3]. Among all types of lung carcinomas, Non- small cell lung carcinoma has a
poor prognosis and difficult to detect at its early stages [4]. Treating this type of carcinomas with herbal compounds
may have lesser side effects. Diosgenin which is a steroidal sapogenin isolated from ancient Chinese medicine and
vegetable Yam and seeds of domestic Fenugreek, inhibits migration of human breast cancer MDA-MB-231 cells by
suppressing Vav2 activity [5]. Diosgenin also induces G2/M cell cycle arrest and apoptosis in human hepatocellular
carcinoma cells [6]. The purified, steroidal sapogenin also induces ROS- dependent autophagy and cytotoxicity via
mTOR signalling pathway in chronic myeloid leukemia cells [7]. Not only anti tumour or anticancer effect, Diosgenin
also have in vivo protective effects against Doxorubicin-induced cardiotoxicity [8]. Recent study shows that Diosgenin
inhibits hTERT gene expression of NSCLC cell-line A549 which is a cause of its cell cycle arrest [9]. Our present
study is aimed to explore the induction of apoptosis of A549 cells using Diosgenin by the intrinsic pathway. Intrinsic
pathway of apoptosis starts from mitochondrial outer membrane permeabilisation [10] [11]. Thereafter leaking of
cytochrome-c from leaky mitochondria and binding with Apaf-1 molecule initiates the recruitment of the initiator
caspase-9 [12]. Activation of caspase-9 effects by activating other downstream executioner caspases, like caspase-3
[13]. Activated caspases-3 induces the apoptotic- chromatin condensation and DNA denaturation, thus apoptosis is
induced. However the mechanism of initiation of the mitochondrial outer membrane permeabilisation in response to
Diosgenin treatment yet to be explored in this case.
2. Objectives
These are the following objectives of our study :
1. Whether Diosgenin is able to kill A549 lung Carcinoma.
2. Whether this killing dose is non toxic to normal cell.
3. Whether Diosgenin causes any type of cell cycle arrest.
4. Whether Diosgenin cytotoxicity is due to its Necrotic activity or Apoptotic activity.
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2. 3. Experimental Design
Figure 1: Work Plan
4. Materials
4.1. Cell Culture
Non small cell lung carcinomas A549 were purchased from National Centre for Cell Science (NCCS, Pune, India).
Lung carcinoma cell line A549 cells were cultured in DMEM medium (purchased from Gibco), containing 100mg/mL
streptomycin (purchased from Invitrogenn),10% FBS (Gibco), 100U/mL Penicillin and maintained at 37◦C with
5% CO2 at a humidified atmosphere. Blood cells for haemolytic assay were obtained from Goat. Peripheral Blood
Mononuclear cells (PBMC s) were isolated from peripheral human blood.
4.2. Reagents
Purified Diosgenin (Molecular formula C27H42O3, Molecular Weight 414.62). MTT 3-(4, 5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide salt, Propidium Iodide all are purchased from Sigma-Aldrich, U.S.A. AnnexinV-FITC
Apoptosis Detection Kit (eBioscience, U.S.A). Rhodamine 123 and H2DCFDA were purchased from (Life Technologies,
Invitrogen). Antibodies for Caspase 9 & 3 were brought from Santa Cruz, U.S.A and Annexin V was purchased from
eBiosciences, U.S.A.
5. Methods
5.1. Hemolytic Assay
Fresh human blood was centrifuged at 4000Xg for 10 min and the cell pellet was washed thrice and re-suspended in
10mM PBS at pH 7.4 to obtain a final concentration of 1.6×109 erythrocytes/ml. Equal volumes of erythrocytes were
incubated with varying concentrations of Diosgenin and kept on rocker at 37◦C for 1hr. Samples were then subjected
to centrifugation at 3500Xg for 10 min at 4◦C. RBC lysis was measured by taking absorbance at an OD of 540 nm.
Complete haemolysis (100%) was determined using 1% Triton X 100 as a positive control and 1X PBS as a negative
control. Hemolytic activity of the Diosgenin was calculated in percentage using the following Equation
H = 100×(Op − Ob)/(Om − Ob) (1)
Here, Op is the optical density of given peptide concentration, Ob is the optical density of PBS and Om is the optical
density of Triton X 100.
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3. 5.2. Cell Viability Assay
The cytotoxicity of Diosgenin was tested on A549 cells by the MTT-assay. Briefly, the cells were seeded in a 96-well
micro titre plate (1×105 cells/well in 100 µL of DMEM medium) and then incubated with different concentration
of Diosgenin. After 24 hr of exposure to the phytochemicals, 50 µl of MTT (5 mg/5 mL) was added to each well,
and the cells were incubated in the dark at 37◦C for an additional 4 hrs. Thereafter, the medium was removed, the
formazan crystals dissolved in 200 µL of dimethyl sulfoxide, and the absorbance measured at 570 nm.
5.3. Cell Cycle Phase Distribution Analysis
For the determination of cell cycle phase distribution of nuclear DNA, A549 cells (1×106 cells) were harvested. Then,
cells were fixed with 100% ice chilled methanol for 2 minutes and kept in 4◦C. After that RNase was added at a
concentration of () and was kept in 37◦C fo 45 minutes and after that nuclear DNA was labelled with Propidium
Iodide (PI, 125µg/mL). Cell cycle phase distribution of nuclear DNA was determined using Tali Image based
Cytometer (Invitrogen).
5.4. Measurement of Mitochondrial Outer Membrane Potential
For the measurement of Mitochondrail Membrane Potential ( ψm /MMP ), In Vitro culture of A549 cells was done in
a 12 well cell culture plate. The cells were treated for 24 hrs with doses of Diosgenin, trypsinised, harvested and
washed twice with PBS (pH - 7.4). Cells were treated in dark with Rhodamine 123 (10µM) for 30 minutes and again
washed thrice with PBS (10mM, pH - 7.4). The cell pellet was resuspended in PBS and the fluorescence intensity was
measured using Tali Image Based Cytometer (Invitrogen).
5.5. AnnexinV-FITC Staining Assay
Apoptosis assays was performed by Annexin V Apoptosis Kit according to the manufacturer s instruction (eBioscience,
U.S.A). Briefly, PI and Annexin V were added directly to A549 cells and incubated for 15 min at 37◦C. Cells were then
analyzed on FACSVerse (BD Biosciences). To exclude the overlapping of emission spectra, electronic compensation
of the instrument was done. After acquiring 10,000 events, data was plotted using FACSuite Software (Becton
Dickinson).
5.6. Determination of Protein Concentration
Protein in the cell lysate was measured by using Folin-Ciocalteu reagent. 5 µL of sample lysate was added to 95
µL ddH2O to make a final volume of 100 µL. The protein measurement is done by using a Blank consisting of 100
µL of ddH2O. Briefly 1mL mixture of 1 % Sodium Potassium Tartarate, 1% Copper Sulphate and 2 % Na2CO3
in 0.1 NaOH (1:50) was added and kept aside for 15 minutes and after that 100 µL of diluted Folin-Ciocalteu
reagent (1:1) was added and kept in dark for 15 minutes. After 15 minutes the Absorbance was measured at 660
nm in a spectrophotometer and protein was estimated by plotting the OD in a standard curve prepared by varying
concentration of Bovine Serum Albumin (BSA).
5.7. Western Blot Analysis
5.7.1 Denaturing and loading of protein
After measurement of the protein content in the cell lysates equal amount of sample is loaded into a 10% SDS-
Polyacrylamide gel by mixing it with loading dye and heated in a water bath at 95◦C. A known molecular weight
marker was also loaded along with the samples.
5.7.2 Protein transfer to Polyvinylidine Fluoride membrane(PVDF)
The gel containing the protein was taken out and placed above the PVDF membrane and sandwiched between
blotting papers in a transfer apparatus. The membrane and the blotting paper was soaked in transfer buffer before
use. The transfer was done by 25 Volt, 500 mA, for 45 minutes.
5.7.3 Development and Detection of proteins
After transfer the membrane was incubated in 5% BSA for 90 minutes followed by primary and secondary antibody
and streptavidin-AP conjugate and blots were then developed by using 5-Bromo-4-chloro-3-indolyl phosphate and
Nitro Blue Tetrazolium Chloride solution (BCIP-NBT).
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4. 6. Results
6.1. Selection of sub-lethal concentration of Diosgenin.
Hemolytic data in Figure 2 suggests that Diosgenin is toxic above 40µM Hence, we have selected two concentrations
(20 µM and 40 µM) for Diosgenin having less hemolytic activity and greater toxicity against Human Non Small cell
lung cancer cell line, A549.
Figure 2: Effect of treatment with Diosgenin on A549 cell viability. Percentage hemolytic activity of Diosgenin (A) and (C) on human red
blood cells. B.) Percentage cell viability at various concentration of Diosgenin (B) measured by MTT assay.
Figure 3: Cell Cycle analysis of treated and untreated A549 cells by Image based Cytometer using Propidium Iodide (PI) as DNA-Binding
Fluorochrome. Histogram display of DNA content (X-axis, PI-Fluorescence) versus counts (y-axis) has been shown. (A) represents
control group, (B) and (C) represents treated group
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5. 6.2. Diosgenin induces Cell Cycle Arrest
In order to analyse the effect of Diosgenin on A549 cells, cell cycle phase distribution analysis was performed using
Tali Image Based Cytometer (Invitrogen). Treatment with Diosgenin resulted in significant increase in the sub-G0
region (hypoploidy population).Figure 3 represents that as compared to the control group Diosgenin treatement
shows an increase in hypoploidy region dose dependently as well as the S phase as well as the G2/M phase cell
distribution is reduced in case of treatment dose dependently.
6.3. Mitochondrial disruption and reduction of Outer Membrane Potential
After staining with Rhodamine 123 staining, the dye cationic gets sequestered inside the mitochondria. As shown
in the figure-4, the 40µM dose of Diosgenin causes the shift of the median fluorescence intensity(MFI) leftward,
more then that of the 20µM dose with respect to control, in 24 hours treatment. This shift indicates mitochondrial
membrane permeabilisation by Diosgenin treatment in a dose dependent fashion.
Figure 4: Effect on integrity of mitochondrial membrane. Cells were treated with Diosgenin for 24 hour, then incubated with Rhodamine 123
and analyzed by Image based cytometer. The median fluorescence intensity values represented graphically shows disruption of the
mitochondrial membrane potential.
6.4. Diosgenin induces Caspase 3
We analyzed the expression of caspase - 3 in A549 cells in vitro using the same concentration of Diosgenin as above.
It was observed that treatment with Diosgenin was able to substantially increase the active caspase - 3 and decrease
the expression of procaspase - 3.
Figure 5: Western blot detection of Caspase - 3 in treated and untreated A549 cells. Equal loading of protein in the lanes was confirmed by
GAPDH. Each test was performed 3 times and images presented were typical of 3 independent experiments.
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6. Figure 6: Annexin V assay in A549 cells. In a double label system, Unfixed A549 cells were labeled with PI and Annexin V and analyzed
on a Flowcytometer. Dual parameter dot plot of FITC-fluorescence (x-axis) versus PI-fluorescence (y-axis) has been shown in
logarithmic fluorescence intensity. Quadrants: lower left, live cells; lower right, apoptotic cells; upper right, necrotic cells. The
upper and lower panels demonstrated apoptosis induction in treated and untreated A549 cells. (A) represents control group, (B) and
(C) group represents Diosgenin treated group.
6.5. Diosgenin induces Apoptosis
In order to confirm the increase in apoptotic induction by Diosgenin, we performed Annexin V/PI assay in control as
well as treated cells by staining the A549 cells with FITC - tagged Annexin V and PI and measuring the fluorescence
intensity usin Tali Image based Cytometer. Figure 6 represents a significant increase in the percentage of annexin
positive cells from 12.19 % in the A549 control group to 23.11 % in the 20 µM group to 39.11 % in the 40 µM group
respectively.
7. Discussion
The synthetic chemotherapeutic drugs have various effects and side effects. Concentrating on the side effects first;
synthetic drugs are often shows cytotoxicity to body s own cells or simply gets deposited in the organ of the tumour
tissue ultimately leading to various type of toxicities [14] [15]. In this scenario, instead of chemically synthesized
drugs, treatment with phytochemicals in its purified or extract form assures comparatively less toxicity [16] [17].
Phytochemicals which gets readily transformed into metabolites inside the body are more potent in this class in
order to be used in treating cancers. That s why choosing Diosgenin, though its apoptotic role on lung cancer
cell lines has been poorly or less understood till date. We choose 20 µM and 40 µM doses of Diosgenin as better
effective and lesser cytotoxic dose by performing haemolytic assay and also determined the IC50 value of Diosgenin
on the non small cell lung carcinoma A549 cells. From the results obtained by the cell cycle phase distribution
analysis of the fixed cells by Propidium iodide staining, using a image based Cytometer, we can conclude that
Diosgenin shows significant arrest in the G0/G1 phase transition and an increase of the hypoploid cell populations
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7. in a dose dependent manner. This event leads to two possible fate of those hypo-diploid cells, either repair or death
[18]. Annexin V- Propidium Iodide staining assay and a Differential interference contrast (DIC) microscopy were
performed to get a clear picture of the mode of cell death. Results obtained by the flowcytometric analysis showed
an increase in the percentage of both early apoptotic (Annexin V +/ PI -) and late apoptotic/necrotic (Annexin
V+/PI -) cell population with a concurrent decrease in viable cells (Annexin V -/PI -). With addition to this the
DIC micrograph shows the significant change of cell shape and chromatin condensation. In order to investigate
whether the mitochondrial pathway for apoptosis is involved, we evaluated mitochondrial membrane potential by
the retention of Rhodamine 123, the fluorescent cationic dye that is readily sequestered by active mitochondria,
depending on their transmembrane potential. In the current study, treatment with 20 µM and 40 µM doses of
Diosgenin shows significant change in the fluorescence intensity in all three sets of the experiment. Finally the
western blot analysis of the Caspase-3 shows the increasing expression of the activated Caspase-3 and a decrease
in Procaspase-3 expression in a dose dependent manner. Activation of Caspase-3 may lead to the activation of the
nucleases like DFF-45, Gelsolin, etc. which results in DNA fragmentation, chromatin condensation, membrane
blebbing and ultimately apoptosis [19]. In conclusion, our in vitro data shows that potential anticancer role of
Diosgenin against A549 cells. The anticancer activity is mediated by a decrease in proliferation and increase in
apoptosis. Moreover, Diosgenin triggers the mitochondrial pathway of apoptosis in a dose dependent way.
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