Regulation Of The Opioid Growth Factor Opioid Growth Factor

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Regulation Of The Opioid Growth Factor Opioid Growth Factor

Transcript Of Regulation Of The Opioid Growth Factor Opioid Growth Factor

The Pennsylvania State University The Graduate School
Department of Neural and Behavioral Sciences
REGULATION OF THE OPIOID GROWTH FACTOR – OPIOID GROWTH FACTOR RECEPTOR AXIS IN THE PROGRESSION OF TRIPLE NEGATIVE BREAST CANCER AND ITS POTENTIAL AS A THERAPEUTIC AGENT
A Thesis in Anatomy
by Beth L. Worley
© 2015 Beth L. Worley
Submitted in Partial Fulfillment of the Requirements for the Degree of
Master of Science
May 2015

The thesis of Beth L. Worley was reviewed and approved* by the following:
Patricia J. McLaughlin Professor of Neural and Behavioral Sciences Director of Graduate Program in Anatomy Thesis Advisor
Gordon L. Kauffman Stephen and Sharon Baron Professor of Surgery Professor of Humanities and Cellular and Molecular Physiology
Ian S. Zagon Distinguished Professor of Neural and Behavioral Sciences
*Signatures are on file in the Graduate School.
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ABSTRACT
Triple negative breast cancer (TNBC) is an extremely aggressive form of breast cancer characterized by tissues lacking estrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor (HER2). TNBC accounts for approximately 15% of all breast cancers diagnosed worldwide, and frequently affects younger women, women of color, and those from lower socioeconomic countries. TNBC has an increased likelihood of recurrence and a twofold greater mortality risk compared to other types of breast cancer, as well as a tendency to metastasize outside the breast. The financial burden for patients with TNBC is considerable, as costs are increased by 50% compared to other breast cancers.
The median survival rate for patients with metastatic TNBC is only 13 months. Lack of hormone receptors and frequent de novo or acquired chemotherapy resistance renders almost all therapies ineffective. Thus, there is a critical need to develop TNBC treatments that target the underlying biology of tumor growth. A novel biological pathway has been studied in a wide variety of human cancers that may provide knowledge on progression of TNBC and serve as an effective treatment.
The opioid growth factor – opioid growth factor receptor axis is a determinant of cell proliferation in both normal cells and neoplasia. OGF inhibits cell proliferation by upregulating the cyclin dependent inhibitory kinases p16 and p21, arresting cells in the transition from G0/G1 phase to S phase of the cell cycle and halting DNA synthesis.
The proposed experiments investigated the presence of the OGF-OGFr axis in MDAMB-231 TNBC cells in vitro and in vivo and assessed whether expression of peptide or receptor changes with cancer progression, and whether OGF serves as an effective treatment for TNBC.
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For cell culture studies, log phase cells were compared with confluent and post-confluent cultures to mimic early and advanced stages of cancer growth.
Semi-quantitative immunohistochemistry and western blot analysis comparing log phase and confluent cells, as well as small and large tumors, revealed that both OGF and its receptor are significantly reduced in advanced stages of squamous cell carcinoma of the head and neck, as well as ovarian, cancers. These observations were extended and confirmed in human breast cancer using MDA-MB-231 and MCF-7 cancer cell lines grown in vitro and in vivo. A mechanism of action involving OGF inhibition of growth was demonstrated utilizing BrdU incorporation that indicated a decrease in DNA synthesis in TNBC cells treated with OGF. Confluent cells were less responsive to OGF treatment than log phase cells.
The data suggest that OGFr expression is reduced in TNBC with tumor progression, and that tumor progression may lead to deficits in the OGF-OGFr axis. Nonetheless, OGF was shown to significantly decrease cell growth relative to controls, indicating its potential as an effective biological treatment for TNBC.
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TABLE OF CONTENTS
List of Figures List of Abbreviations Acknowledgements
Chapter 1. Introduction 1.1 Cancer Incidence and Disease 1.1.1 Breast Cancer Incidence and Disease 1.1.2 Risk Factors 1.1.3 Current Treatments 1.1.4 Triple Negative Breast Cancer 1.2 Endogenous Opioids and the OGF-OGFr Axis 1.3 The OGF-OGFr Axis and Cancer 1.3.1 OGF Inhibition of Breast Cancer 1.4 Conclusions
Chapter 2. Objectives
Chapter 3. Materials and Methodology 3.1 Cell Culture and Growth 3.2 Animals 3.2.1 Tumor Cell Implantation and Growth 3.2.2 Tissue Collection and Euthanasia

Page Number vii viii xi
1 1 1 2 3 5 6 7 8 9
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12 12 13 13 13
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3.3 Tissue Preparation

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3.3.1 Semi-quantitative Immunohistochemistry

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3.3.2 Protein Isolation and Western Blotting

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3.4 DNA Synthesis

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3.5 Statistical Analyses

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Chapter 4. Results

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4.1 OGF-OGFr Detection in Human Breast Cancer Cells

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4.2 OGF-OGFr Detection in Tumor Tissue

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4.3 The Inhibitory Effects of OGF on Cell Growth

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Chapter 5. Discussion

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References

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LIST OF FIGURES

Figure 4.1 Figure 4.2 Figure 4.3 Figure 4.4 Figure 4.5 Figure 4.6 Figure 4.7

The Presence and Function of the OGF-OGFr Axis in Human Triple

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Negative Breast Cancer Cells

The Presence and Function of the OGF-OGFr Axis in Human Breast

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Cancer Cells

Western Blot Validation of OGFr Protein Expression in Human Breast 21 Cancer Cells

The Presence of the OGF-OGFr Axis in MDA-MB-231 TNBC Tumors 23 Grown in Nude Mice

The Inhibitory Action of OGF on DNA Synthesis

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OGF and Taxol Inhibition of Human Breast Cancer Cell Growth

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OGF Inhibition of Human Breast Cancer Cell Growth

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AI ANOVA BCS BRCA1 BRCA2 BrdU BSA °C cm d DMEM DNA ER et al. g G µg G0 G1 G2

LIST OF ABBREVIATIONS
aromatase inhibitor analysis of variance breast-conserving surgery breast cancer 1, early onset (gene) breast cancer 2, early onset (gene) bromodeoxyuridine bovine serum albumin degrees Celsius centimeter day Dulbecco’s modified Eagle’s medium deoxyribonucleic acid estrogen receptor and others gram gauge microgram G zero/resting phase of cell cycle gap 1 phase of cell cycle gap 2 phase of cell cycle
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GAPDH h [3H] H&E HER2 IACUC IHC IgG l L-15 LDN M M phase mg ml mm mM µm NGS NTX OCT OGF

glyceraldehyde 3-phosphate dehydrogenase hour tritium hematoxylin and eosin human epidermal growth factor receptor International Animal Care and Use Committee immunohistochemistry immunoglobulin G length Leibovitz’s medium low-dose naltrexone molar mitotic phase of cell cycle milligram milliliter millimeter millimolar micrometer normal goat serum naltrexone optimal cutting temperature embedding compound opioid growth factor
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OGFr p16 p21 PBS PMSF PPE PR % π RIPA S SCCHN SDS SPB SEM TNBC w

opioid growth factor receptor cyclin-dependent kinase inhibitor 2A cyclin-dependent kinase inhibitor 1 phosphate buffered saline phenylmethylsulfonyl fluoride preproenkephalin A gene progesterone receptor percent pi radioimmunoprecipitation assay buffer synthesis phase of cell cycle squamous cell carcinoma of the head and neck sodium dodecyl sulfate Sorensen’s phosphate buffer standard error of the mean triple-negative breast cancer width

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OgfTnbcBreast CancerPresenceCells