Effects of Prodigiosin on Breast Cancer Cell Viability
Effects of Prodigiosin on Breast Cancer Cell Viability.
ABSTRACT
The Luteinizing hormone-releasing Hormone (LHRH); also known as a gonadotropin-releasing Hormone can be utilized for targeted therapy with cytotoxic analogs, in which prodigiosin is linked to the [D-Lys6] LHRH.
Our studies demonstrate receptor-mediated actions of the cytotoxic LHRH analog in LHRH receptor-positive breast cancer cells. Chemotherapy today is the only systematic therapy for patients with triple-negative breast cancer cells. About 50% to 64% of human breast cancer-expressed receptors for LHRH might be used as a target.
This thesis focused on a new conjugate in which prodigiosin was used as a cytostatic compound and an analog or Luteinizing hormone-releasing Hormone (LHRH) as a targeting moiety was synthesized. The molecules of the peptide were modified to allow its connection to the prodigiosin via a spacer.
The objectives of our study were to synthesize a bioconjugate of LHRH analog [D-Lys]-LHRH and prodigiosin targets and inhibits breast cancer cell growth in vitro and in vivo.
Prodigiosin was synthesized by bacteria (Serratia marcescens (subsp. marcescen)). High-performance liquid chromatography (HPLC) analysis on the purity was determined to be 92.8% and the ultraviolet (UV) reading was 535nm.
Adhesion force measurements were also carried out to determine the adhesion force between the conjugate and the breast cancer cells.
This had the highest measurement as compared to the bare, prodigiosin coated and LHRH-coated tips. The adhesion force of the conjugates was measured (80±4nN) which gave the highest peak. The conjugates were prepared linking prodigiosin through its 14-0-hemiglutarate to [D-Lys6] LHRH.
The results showed that the use of LHRH peptide as a targeting moiety in the anticancer drug delivery substantially enhanced the efficacy of prodigiosin which leads to amplified apoptosis induction in the tumor and minimized the side effects of the anticancer drug on healthy organs.
In conclusion, prodigiosin conjugate with LHRH exhibited an antiproliferative effect for which further testing will be implemented.
TABLE OF CONTENTS
DECLARATION……………………………………………………………………………………………………………..II
ABSTRACT………………………………………………………………………………………………………………….III
ACKNOWLEDGMENT…………………………………………………………………………………………………IV
DEDICATION………………………………………………………………………………………………………………..V
LIST OF FIGURES……………………………………………………………………………………………………..VIII
LIST OF TABLES…………………………………………………………………………………………………………..X
CHAPTER ONE………………………………………………………………………………………………………………1
1.0 Introduction………………………………………………………………………………………………………………..1
1.1 Scope of Work………………………………………………………………………………………………………..4
CHAPTER TWO……………………………………………………………………………………………………………..5
2.0 Literature Review…………………………………………………………………………………………………..5
2.1 Introduction…………………………………………………………………………………………………………..5
2.2 Prodigiosin Production by Serratia marcescens………………………………………………………….5
2.2.1 Prodigiosin Structure………………………………………………………………………………………..6
2.2.2 Effects of Growth Conditions on Yield of Prodigiosin………………………………………….7
2.3 Luteinizing Hormone-Releasing Hormone (LHRH)……………………………………………………7
2.3.1 Chemistry of Drug-Peptide Conjugation……………………………………………………………..8
2.3.2 Drug-LHRH (GnRH) Analog Peptide Conjugates………………………………………………10
2.4 Conjugation of Prodigiosin-LHRH………………………………………………………………………….10
2.5 Cell Culture………………………………………………………………………………………………………….10
2.6 Breast Cancer Cell Culture Methods……………………………………………………………………….11
2.7 LC-50: Rate of Cell Growth and Measurement of Cell Death…………………………………….12
2.8 Trypan – Blue Assay……………………………………………………………………………………………..13
2.9 Clonogenic Assay…………………………………………………………………………………………………13
2.10 UV-Vis Spectrometry…………………………………………………………………………………………….15
2.11 Atomic Force Microscope (AFM)…………………………………………………………………………..15
CHAPTER THREE………………………………………………………………………………………………………..20
3.0 Prodigiosin Extraction from Serratia marcescens……………………………………………………..20
3.1 Introduction………………………………………………………………………………………………………….20
3.2 Materials……………………………………………………………………………………………………………..20
3.3 Experiments………………………………………………………………………………………………………..21
3.3.1 Media Preparation for Serratia marcescens inoculation………………………………………21
3.3.2 Extraction and purification of Prodigiosin…………………………………………………………21
3.3.3 Estimation of Prodigiosin………………………………………………………………………………..22
3.4 Results and Discussion………………………………………………………………………………………….23
3.5 Summary / Conclusion………………………………………………………………………………………….23
CHAPTER FOUR………………………………………………………………………………………………………….28
4.0 Conjugation of Prodigiosin and LHRH……………………………………………………………………28
4.1 Introduction………………………………………………………………………………………………………….28
4.2 Materials……………………………………………………………………………………………………………..28
4.3 Prodigiosin – LHRH Conjugation…………………………………………………………………………..29
4.4 Breast Cancer Cell Culture…………………………………………………………………………………….29
4.5 Fluid /Drugs Concentration……………………………………………………………………………………30
4.6 Effect of Prodigiosin on LC 50 and Clonogenic Assay………………………………………………30
4.7 Detection of prodigiosin –LHRH improvement – LC50/clonogenic……………………………31
4.8 Cell Adhesion force measurement and characterization of Conjugated Prodigiosin-LHRH
to Breast Cancer Cell and Normal Cells…………………………………………………………………………….31
4.9 Results and Discussion………………………………………………………………………………………….33
4.10 Conclusion and Summary………………………………………………………………………………………35
CHAPTER FIVE……………………………………………………………………………………………………………42
5.0 Summary and Conclusion………………………………………………………………………………………42
5.1 Recommendations for Future Work…………………………………………………………………………43
REFERENCES………………………………………………………………………………………………………………44
INTRODUCTION
Cancer is a broad term for over 200 different diseases. The increasing incidence of cancer [1] has stimulated research on the development of novel therapies that would be effective in the treatment and reduction in the rate of re-occurrence.
Currently, cancer is the second leading cause of death after cardiovascular diseases [2, 3]. Among the 200 different classes of diseases, breast cancer is the second most common cause of cancer death in women [3]. By 2030, cancer will become the leading cause of death as suggested by current trends [2, 3].
The changes that occur in genes cause one cell or a few cells to grow and multiply uncontrollably leading to cancer. Where cancer starts it is a primary tumor and sometimes when it spreads to other parts of the body it is called a secondary tumor or metastasis.
The conventional treatment of cancer can affect the body systems, such as blood circulation, lymphatic and immune systems, and the hormone system [1]. The conventional treatments include surgery, radiation, chemotherapy, hormone therapy, immune therapy, and targeted therapy (drugs that specifically interfere with cancer cell growth) [4].
Drug delivery refers to the approaches, formulations, technologies, and systems for transporting a pharmaceutical compound in the body as needed to safely achieve its desired therapeutic effect. The use of localized drug delivery has so many advantages as compared to conventional treatments.
This treatment can avoid harm to normal cells, dramatically reduces side effects, high efficacy, and controlled drug release. Cancer is a group of diseases characterized by the uncontrolled growth and spread of abnormal cells.
Cancer is believed to be caused by external factors such as tobacco smoking, infectious organisms, an unhealthy diet, and internal factors, such as inherited genetic mutations, hormones, and immune conditions. Research shows that cancer has resulted in more deaths than death from AIDS, tuberculosis, and malaria combined [5].
The Global burden of cancer continues to increase largely because of the aging and growth of the world population. This is due to the increasing adaptation of cancer-causing behaviors, particularly smoking in economically developing and developed countries [5].
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CSN Team.
