Diosgenin induces G0/G1 Phase Cell Cycle Arrest & induces

Apoptosis in A549 Human Non Small Cell Lung Carcinoma Cell

Line

Project Submitted bySupriya Nath

Reg. No.- 2014003158 of 2014-16Department of Zoology

Under Supervision of Dr. Shamee Bhattacharjee& Dr. Debaprasad Mandal

Assistant Professors, Department of Zoology

• Diosgenin: A purified, Steroidal Sapogenin extracted from Seeds of Fenugreek and the roots of Yam…

•Previously published journals told us that it can inhibit cancer cell growth and migration on different Cancer cell lines. It was also reported that it can reduce the cardio toxicity when used in combination with Doxorubicin. Though there is no report about the pro-apoptotic Roles of

Diosgenin on Lung Carcinoma, that’s why we choose Non small Cell lung Carcinoma, A549…

Objective of the Work:These are the following objectives of our study :

To investigate the anti proliferative property of Diosgenin on A549 lung carcinoma cells.

To check the toxicity of Diosgenin on normal cells.

To study the effect of Diosgenin on cell cycle phase distribution on A549 cancer cell.

To evaluate the pro-apoptotic activity of Diosgenin on A549 cells.

Study of anti-cancer activity of

Diosgenin on A549 Cells

Study of the apoptosisA. Annexin-V assayB. Estimation of mitochondrial outer membrane potential.C. Studying pro-apoptotic proteins- Caspase expression.

Study of the anti-proliferative role of

DiosgeninA. Cell cycle phase distribution anlysis

Dose Selection:1. Hemolytic Assay2. MTT assay

Work Plan:

• Choosing the less toxic Doses: Hemolytic Assay

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Percentage cell viability at various concentrations of Diosgenin after 24 hour treatment, measured by MTT assay.

MTT assay : Determination of the IC50 value of Diosgenin on A549 Cells

•CELL CYCLE PHASE DISTRIBUTION ANALYSIS:

Result: The chosen doses of Diosgenin, 20µM & 40µM, induces cell cycle arrest on G0/G1 checkpoint upon 24h treatment.

Column Representation of the cell cycle data:

• Detection of Degree of Apoptosis and Necrosis using Flowcytometry by Annexin V- FITC & Propidium Iodide Staining after 24 hrs. treatment:

A. Control

B. 20 μM C. 40 μM

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.

Control 20 µM 40 µMAnnexin/PE( -/-)

85.83 73.81 59.19

Annexin/PE(+/-)

5.12 10.60 22.64

Annexin/PE(-/+)

1.98 1.97 1.70

Annexin/PE(+/+)

7.07 13.50 16.47

• Mitochondrial Outer Membrane Potential change Detection(Δψmit) Using Rhodamine 123

control

20 μM

40 μM

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.

• Western Blot Analysis to Detect the activity of Caspase 3

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.

•Conclusion: From the results obtained, it can be concluded that Diosgenin arrests cell cycle at the G0/G1 check point and there by triggers apoptosis of the A549 cells in vitro.

•Future Research: To get the complete picture and the total signaling pathway of the Diosgenin- treatment, in depth research is needed.

Acknowledgement: I am heartily grateful to my Teachers Dr. Shamee Bhattacharjee & Dr. Debaprasad Mandal, Dr. Samir Saha, Assistant Professors, Dr. Chiranjib Paul, Associate Professor(HOD) and Prof. Narayan Ghorai & Prof. Silanjan Bhattachharyya for their encouragement and caring attitude. I am also specially thankful to my Laboratory Seniors – Ashikur Rahaman(JRF) and Samarjit Jana (SRF), Kartick Patra (SRF), Arnab Sarkar(JRF), Subrata Karmakar(JRF), Priyanka Pal(JRF), Munia Parvin( JRF), Sonali Bhattachharya( JRF). I am also grateful to my parents for their moral support.

Thank you..