Role of Cytochrome C in Apoptosis During Breast Cancer Treatment

Xavier University of Louisiana XULA Digital Commons Festival of Scholars 2021 Role of Cytochrome C in Apoptosis During Breast Cancer Treatment Teresa Beamon Department of Chemistry

Xavier University of Louisiana XULA Digital Commons

Festival of Scholars

2021

Role of Cytochrome C in Apoptosis During Breast Cancer

Treatment

Teresa Beamon

Department of Chemistry, Xavier University of Louisiana

Royce Hooks

Department of Chemistry, Xavier University of Louisiana

Degrick Cheatham

Department of Chemistry, Xavier University of Louisiana

Navneet Goyal

Department of Chemistry, Xavier University of Louisiana

Tulasi Ponnapakkam

Department of Chemistry, Xavier University of Louisiana

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Recommended Citation

Beamon, Teresa; Hooks, Royce; Cheatham, Degrick; Goyal, Navneet; Ponnapakkam, Tulasi; and Foroozesh, Maryam, "Role of Cytochrome C in Apoptosis During Breast Cancer Treatment" (2021) Festival of

Scholars 15

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Authors

Teresa Beamon, Royce Hooks, Degrick Cheatham, Navneet Goyal, Tulasi Ponnapakkam, and Maryam Foroozesh

This book is available at XULA Digital Commons: https://digitalcommons.xula.edu/xula_fos/15

Role of Cytochrome C in Apoptosis During

Breast Cancer Treatment

Teresa Beamon, Royce Hooks, Degrick Cheatham , Navneet Goyal, Tulasi Ponnapakkam, & Maryam Foroozesh

Department of Chemistry, Xavier University of Louisiana, 1 Drexel Dr., New Orleans, LA 70125

Research reported in this presentation was supported by the National Institute of General Medical Sciences of the National Institutes of Health under award numbers TL4GM118968 and 5RL5GM118966, NIMHD-RCMI grant number 5G12MD007595, and the Louisiana Cancer Research Consortium at Xavier University of Louisiana The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health

Introduction

Materials & Methods

Results

Acknowledgements

• Globally, breast cancer is the most frequently diagnosed cancer in women, with

an estimated 2.2 million new cases diagnosed in 2020 (1)

• There were approximately 685,000 deaths reported in 2020 from breast cancer

worldwide (1)

• The 5-year prevalence of breast cancer in all ages is reported to be 7.7 million (1)

• About 1 in 8 U.S women (about 12%) will develop invasive breast cancer over the

course of her lifetime (1)

• Chemo and radiation therapies are thought to primarily exert anti-tumor effects

through the activation of apoptosis, or programmed cell death pathways (2)

• Analog 315 is a novel ceramide which was developed in our laboratory for the

potential treatment of breast cancer

• The development of novel agents that can induce programmed cell death or aid

in overcoming resistance are expected to improve patients’ outcomes and prolong their survival

• Therefore, this study focuses on determining the pathways involved in the

treatment of breast cancer cells with ceramide analog 315

Conclusion

• Cytochrome C is known for its function as a key participant in supporting the ATP

synthesis pathway, which takes place in the mitochondria (3)

• There are two distinct apoptotic pathways, intrinsic (mitochondria-mediated), and

extrinsic (death receptor-mediated) (4)

• After the ejection of cytochrome C from the mitochondria into the cytosol, a chain

reaction causes high enzyme activity, leading to cell death Because of this, higher rate of cell death is expected to be observed in the cell cultures that are treated with the ceramide analog used in this experiment (5)

• This experiment was expected to show the involvement of the intrinsic pathway in the

ceramide-mediated apoptosis However, the very preliminary data obtained does not support this pathway

• Additional studies will be performed to further understand the mechanisms of action

of this ceramide drug

y = 0.4166x + 0.2234 R² = 0.8957

0 0.5 1 1.5 2 2.5

OD 450

Figure 1: Cytochrome C Standard Curve

Abstract

Ceramide Analog:

• The drug used for this assay was ceramide analog 315,

(S,E)-3-hydroxy-2-(2-hydroxybenzylidene)amino-N-tetradecyl propanamide, synthesized in our laboratory

• Analog 315 was dissolved in DMSO (dimethyl sulfoxide) and used to prepare varying

concentrations with the addition of cell culture medium

Cell Culture:

• Human breast cancer MCF-7-TNR cells were donated by Tulane University and grown

in DMEM (Dulbecco modified Eagle’s medium) enhanced with 1%

penicillin-streptomycin and 10% FBS (fetal bovine serum)

Cytochrome C Apoptosis Assay

• TNR cells were plated at 400,000 in a 6-well plate allowing adhesion overnight

• The next day cells were washed with warm PBS

• The control cells were treated with 3 mL of a 10mL

media and 40µL DMSO mix

• The treated cells were treated with 3 mL of a 10mL

media and 40µL analog 315 mix (40µM)

• The plate was incubated for 48 hours

• After being harvested, cold PBS was added to each well

• Cells were scraped, removed, and pelleted

• Extraction buffer was added to the pellet, and the lysate was used for this assay

• Using Cytochrome C ELISA Kit, both treated and control lysate samples were analyzed

• Standard curve was prepared by diluting the standard provided by the vendor to 2.5

ng/mL, 1.25 ng/mL, 0.625 ng/mL, 0.312 ng/mL, 0.156 ng/mL, and 0.078 ng/mL

• The rest of the procedure was followed using Cytochrome C ELISA Kit

• The unknown values were calculated using the standard curve

0 1 2 3 4 5 6 7 8 9 10

Control Concntration Treated Concentration

Figures 2: Cytochrome C Levels in TNR Cell line

Drug Design

CONTROL

TREATED

Breast cancer is the leading cause of death in women Various current cancer

treatments include surgery, chemotherapy, immunotherapy, hormone therapy, and

radiation therapy Because of prolonged exposure, resistance to chemotherapy often

arises Chemotherapeutic drugs and radiation therapy have been shown to increase

intracellular ceramide levels Ceramide, a bioactive sphingolipid, is a powerful

tumor suppressor molecule that is thought to induce apoptosis and inhibit

proliferation

As part of our ongoing efforts toward the synthesis of a potent anti-cancer drug,

ceramide analog 315,

(S,E)-3-hydroxy-2-(2-hydroxybenzylidene)amino-N-tetradecyl propanamide has been synthesized Ceramide analog 315 has been

shown to induce apoptosis in vitro, and decrease tumor volume and size in in vivo

studies In the present study, an attempt is made to corelate the cytochrome C levels

with cell death during the treatment of chemo-resistant breast cancer cells with

analog 315

Fig 2 displays the average concentration of cytochrome C found in control cells and treated cells

1 Globocan Cancer Today https://wwwuiccorg/news/globocan-2020-new-global-cancer-data 2020.

2 Alfarouk KO, Stock CM, Taylor S, Walsh M, Muddathir AK, Verduzco D, et al Resistance to cancer

chemotherapy: failure in drug response from ADME to P-gp Cancer cell international

3 Cytochrome c ELISA Kit Catalog No KH01051

4 Jan, Rehmat, and Gul-E-Saba Chaudhry “Understanding Apoptosis and Apoptotic Pathways Targeted

Cancer Therapeutics.” Advanced pharmaceutical bulletin vol 9,2 (2019): 205-218

doi:10.15171/apb.2019.024

5

https://www.thermofisher.com/us/en/home/life-science/antibodies/antibodies-learning-center/antibodies-resource-library/cell-signaling-pathways/cellular-apoptosis-pathway.html

References

Fig 1 displays the observed optical density of each dilution plotted against the cytochrome c

standard curve The standard accurately reflects cytochrome C content in samples.

*P <0.05

[Treated]= 7.46 ng/mL +/-SE

[Control]= 8.80 ng/mL +/-SE

*