Cancer Cell Apoptosis with FACS Analysis

Lecture 16 Summary

• Lecture presented by Dr. Kavitha Thirumurugan, Professor HAG, SBST at VIT Vellore Institute of Technology.
• Lecture focused on Fluorescence-activated cell sorting (FACS) analysis of apoptosis in cancer cells.

Annexin V Apoptosis Assay (MCF-7 Cells)

• Annexin V Apoptosis assay using FACS analysis on MCF-7 breast cancer cells.
• Cells treated with different concentrations of SFN, WA, or both compounds.
• Treatment was for 3 days.
• Results show significant apoptosis at higher concentrations (p<0.01, p<0.001)
• Data represents three replicates.
• X-axis displays compound concentration (µM).

Annexin V Apoptosis Assay (MDA-MB-231 Cells)

• Annexin V Apoptosis assay, using FACS on MDA-MB-231 breast cancer cells.
• Cells treated with different concentrations of SFN, WA, or both compounds.
• Treatment was for 3 days.
• Results show significant apoptosis at higher concentrations (p<0.01, p<0.001).
• Data represents three replicates.
• X-axis displays compound concentration (µM).

Flow Cytometry Analysis

• Flow cytometry used by the lecture to analyze the cell cycle in relation to apoptosis.
• Negative control group showed no distinct apoptotic peak.
• Experimental group (undergoing apoptosis) showed an apoptotic peak (Ap peak) before the G1 peak in the histogram.

Flow Cytometry as Method to Detect Apoptosis

• Flow cytometry is a high-sensitivity method for detecting apoptosis.
• It can be used to study the cell cycle simultaneously.
• General laboratories may not have the required equipment (flow cytometer).
• Cost associated with the equipment might be a factor.
• AO/EB staining may be a viable alternative.

Clonogenic Assay

• Clonogenic or Colony Forming Assay is an in vitro technique used to quantify cells capacity to grow into a colony.
• Defined by colonies of at least 50 cells.
• Assesses every cell's ability to undergo uncontrolled division.

Clonogenic Assay as method to determine Cell Reproductive Death

• Clonogenic assay is a critical method for determining cell reproductive death following treatment with ionizing radiation, or other cytotoxins.
• Only a fraction of seeded cells can form colonies.
• Colonies are developed in 1-3 weeks.
• Colonies fixed using glutaraldehyde and stained using crystal violet, then counted with a stereomicroscope.

HCT116 Colony Forming Assay Protocol

• Protocol for assessing the effects of drug treatment on HCT116 colorectal cancer cells.
• Seeding density of 10000 cells per well.
• Cells allowed to reach 30-40% confluency (before treatment)
• Treatment with specific drug concentrations in media.
• Incubation for 3 days (at 37°C, 5% CO2).
• Fixation and staining with Coomassie Blue stain after treatment.

Clonogenic Assay of HCT116 Cells (Treatment with Phloretin)

• HCT116 colorectal cancer cells are treated with phloretin (at various concentrations).
• Phloretin, a dihydrochalcone polyphenol, is found in apples, pears, strawberries.
• Phloretin inhibits CRC development by acting on glucose transporters, and activating p53.
• Over 80% of CRC are initiated by APC (adenomatous polyposis coli) mutations linked with Wnt activation.

Clonogenic assay of HCT116 cells: Image Analysis with Phloretin

• Image analysis was used to quantify the growth of HCT-116 cells after treatment with various concentrations of phloretin.
• The analysis revealed a reduction in the number of colonies as concentrations increased.
• Significant reduction in colony formation at higher concentrations (p<0.05, to p<0.0001).

Clonogenic Assay: Treatment Results and Colony Reduction with Phloretin

• Treatment with phloretin significantly reduced the number of colonies formed in HCT-116 cells (compared to control).
• This reduction was significant (p<0.0001) at the highest concentration of phloretin used.
• Demonstrates Inhibitory effect of phloretin on CRC proliferation

Flow Cytometry Results (HCT-116): Treatment with Phloretin Analysis

• Flow cytometry was employed to investigate the mechanism by which phloretin inhibits cell proliferation in HCT-116 cells.
• There was altered pattern in cell cycle distribution following treatment (increased G2/M phase accumulation).
• Accumulation in G2/M phase showed significant increase (p<0.0001)

Apoptosis Results (HCT-116): Phloretin treatment Analysis

• Assessing the percentage of early- and late-apoptotic cells in HCT-116 cells after phloretin treatment.
• Early and late apoptotic cells significantly increased with higher phloretin concentrations.
• This increase suggests phloretin treatment induces apoptosis in a concentration dependent manner.

Summary of Phloretin's Effect

• Phloretin significantly inhibits HCT-116 cell proliferation.
• Phloretin induces apoptosis in HCT-116 cells in a concentration dependent manner.

Additional experiments

• Further analysis was performed in HCT-116 and SW-480 cells to investigate phloretin-induced apoptosis, showing the significant increase of early and late apoptosis at higher concentrations.

This quiz delves into the fluorescence-activated cell sorting (FACS) analysis of apoptosis in MCF-7 and MDA-MB-231 breast cancer cells. You will explore the effects of different concentrations of SFN and WA compounds on apoptosis rates over a three-day treatment period. Test your knowledge on the methodology and findings of the Annexin V Apoptosis assay.