Thesis (M.Sc.(Biochemistry)) -- University of Limpopo, 2019. / Retinoblastoma binding protein 6 (RBBP6) is the protein encoded by the Retinoblastoma Binding Protein 6 (RBBP6) gene that is located in chromosome 16p12.2. There is a growing list of newly discovered RBBP6 hypothetical splice variants but there are only three RBBP6 splice variants that are well documented. RBBP6 has been previously implicated in the regulation of cell cycle and apoptosis but little is known about the expression and regulation of the human RBBP6 splice variants during cell cycle progression and breast cancer development. This study was aimed at determining the expression pattern of RBBP6 alternatively spliced variants during arsenic trioxide-induced cell cycle arrest and apoptosis in breast cancer MCF-7 cells. It was also aimed at determining RBBP6 specific microRNAs and how they are regulated in MCF-7 breast cancer cells. MCF-7 cells were maintained and subjected to arsenic trioxide-induced cell cycle arrest and apoptosis. The MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) and the Muse™ Count & Viability assays were used to evaluate the effect of arsenic trioxide on the viability of MCF-7 cells. Cell cycle arrest using 11 μM arsenic trioxide and apoptosis using 32 μM arsenic trioxide were analysed using the MUSE® Cell Analyzer, light and fluorescence microscopy. Arsenic triode-induced apoptosis was analysed using the Muse™ Annexin V & Dead Cell Kit, MultiCaspase and MitoPotential assays using the Muse™ MultiCaspase Kit and Muse™ MitoPotential Kit. Arsenic trioxide-induced cell cycle arrest was analysed using the Muse™ Cell Cycle Kit. Semi-quantitative analysis of RBBP6 variants was carried out using the conventional Polymerase Chain Reaction (PCR), while the quantitative analysis was done using the Real-Time Quantitative PCR. The localization of RBBP6 isoforms was done using Immunocytochemistry (ICC). Web based Bioinformatics tools were used to identify RBBP6-specific microRNAs. The MTT results showed that arsenic trioxide decreased the viability of the MCF-7 cells in a dose-dependent manner. The Muse™ Cell Cycle analysis showed that 11 μM of arsenic trioxide induced G2/M cell cycle arrest in MCF-7 cells, while the Muse™ Annexin V & Dead Cell assay showed that 32 μM of arsenic trioxide induced the extrinsic apoptotic pathway in MCF-7 breast cancer cells. Using the conventional PCR, the MCF-7 cells were found to express the RBBP6 variant 1 transcript but lacks the expression of variant 2 and 3 transcripts, contrary to the kidney embryonic Hek 293 cells that exhibited the expression of RBBP6 variant 1, 2 and 3. Additionally, arsenic trioxide downregulated RBBP6 variant 1 in breast cancer cells during cell cycle arrest and apoptosis. The Real-Time PCR confirmed that MCF-7 cells lowly express RBBP6 variant 3. On the other hand, the ICC analysis showed that RBBP6 isoform 1 is localized and highly expressed in MCF-7 breast cancer cells. The Web based Bioinformatics tools showed that RBBP6 variant 1 specific microRNAs are down regulated in MCF-7 breast cancer cells. These results together showed that As2O3 is effective against MCF-7 cells and also regulated the expression of RBBP6 variants, especially, variant 1. This study showed that there are RBBP6 variants that are involved in breast cancer progression and there are those that may be involved in breast cancer suppression. Targeting these RBBP6 variants for therapeutic development is a promising strategy. In conjunction with RBBP6 expression, arsenic trioxide should be further explored as a breast cancer drug.

Inflammatory cytokines and chemokines are known to promote tumor cell survival, invasion, the formation of blood and lymphatic vessels, and hence, metastasis. We previously showed that a pro-inflammatory pathway regulated by Toll-like Receptor-4 (TLR4) can be activated in human breast cancer (BC) cell lines by a clinical chemotherapeutic drug, paclitaxel (PXL). Prior data showed that PXL treatment of TLR4+ tumors in vivo increased inflammation and tumor spread. Here, we used two BC models based on MDA-MB-231 and HCC1806 cell lines. Transcript expression of 123 cytokines in vitro and in vivo was determined by qRT-PCR. We found that 18 and 26 cytokines were upregulated by nanoparticle albumin-bound paclitaxel (nab-PXL) in 231TLR4+ cells and in 1806TLR4+ cells, respectively. Upregulation of cytokines was observed in cultured cells and in tumor models in vivo. Furthermore, fourteen cytokines (11.3% of total) were induced by nab-PXL in both tumor models suggesting that these targets are upregulated by PXL regardless of genetic makeup of tumor cells. We also confirmed the expression of these cytokines on protein level by ELISA. We found that expression of CCL20 and CXCL1 proteins is consistent with transcript expression detected by qRT-PCR. Taken together, CCL20 and CXCL1 can be the potential targets for further study, and they may have the capacity to recruit myeloid-derived lymphatic endothelial cell progenitors to the tumor site to promote lymphangiogenesis in breast cancer.

archives@tulane.edu / Transpapillary drug delivery is a novel drug administration technique that integrates the non-invasive, passive aspect of transdermal drug delivery with the targeted approach of intraductal drug delivery by capitalizing on the mammary ducts to serve as an entry point, conduit and reservoir. Although these channels have been identified as a primary transport route, their contribution to overall tissue penetration has not been quantified. By combining two fluorescence techniques, we were able to quantitatively assess the various transport routes of small molecules and drug delivery vehicles following in vitro diffusion. Analysis of fluorescent images of porcine nipple cross-sections following diffusion of model hydrophilic and lipophilic fluorescent dyes indicated that both molecules penetrated the nipple via the stratum corneum and mammary ducts, however the lipophilic molecule targeted the ducts more so than the hydrophilic molecule. Encapsulating either dye within a liposome enhanced the ductal-associated fluorescence and reduced (hydrophilic dye) or did not affect (lipophilic dye) the stratum corneum-associated fluorescence. This suggests the capability of liposomes to selectively target and improve diffusion within the ductal channels. Encapsulation of the lipophilic dye within an oil-in-water nanoemulsion, however, either substantially increased penetration via both routes or only moderately improved transductal penetration, depending on the specific formulation. The in vivo distribution and efficacy of transpapillary diffusion was evaluated by first establishing an intraductal estrogen receptor positive breast cancer model. Results from in vivo imaging elucidated two growth rates, either slow or fast, which were discernable 14 days post-injection. A pilot therapeutic efficacy study using 4-hydroxytamoxifen was then performed; however due to a small sample size, the results were inconclusive. In vivo transpapillary diffusion of a small, lipophilic molecule was confirmed, as illustrated following application of a fluorescent dye. We conclude that transpapillary drug delivery is a viable in vitro administration technique for which the penetration routes can be tailored with drug carriers on a formulation-dependent basis. Furthermore, the feasibility of intraductally establishing estrogen receptor positive lesions and tracking their growth using in vivo imaging was validated. However, the use of this model to assess in vivo efficacy of transpapillary diffusion merits further evaluation. / 1 / Samantha Kurtz

Breast cancer is the leading cause of death among Latinas women. Several barriers persist when accessing health care and utilization of healthcare services such as annual mammograms, leading to a late stage diagnosis or death related to breast cancer illness. The purpose of this study was to examine disparities in breast cancer experiences within Latina communities in the United States. The Health Belief Model served as the foundation of this qualitative grounded theory study. The research questions explored; access to breast care services that encourage early breast cancer detection; breast care diagnostics such as exams, mammograms and biopsies; and views of availability to breast care exams, diagnostics and treatment options improving health outcomes. The participants were females who self-identified as Hispanic and 19 years of age or older and resided in north east part of the United States. Participants must have discovered a breast tumor, engaged in the decision-making process to seek biopsy, and had a breast cancer diagnosis. A total of 12 Latina women were recruited for 60 minutes recorded interviews Later, the interviews were transcribed.. Findings of the study showed the participants perceived the disease as serious leading to death; cultural context, insurance status may not have contributed to susceptibility to the disease. This study benefits Latina women, and other vulnerable female populations in the United States diagnosed with breast cancer. The social change implications of the study can influence program initiatives that seek to improve equitable access to care, breast care services and the quality of life. It provides insight to practice approaches regarding access to care, service utilization, and development of program initiatives.

Despite the existence of breast cancer screening that could promote early diagnosis and survival of breast cancer, high mortality rates of breast cancer persist among Filipino women. The purpose of the qualitative study was to describe the perceptions, beliefs, and behaviors of Filipino women working as Overseas Filipino Workers (OFWs) in Saudi Arabia. Face-to-face interviews were conducted with 20 Filipino women between the ages of 40 to 60 years who were recruited voluntarily using purposeful sampling technique. Guided by the structures of health belief model (HBM), the study used an inductive coding technique to elicit common themes from the raw data. The study established that the participants' screening behaviors were influenced by family history of breast cancer, the financial and emotional burden of the disease and its treatment, the benefit of early detection, mobility to participate, culture and language barriers, and the social media. The study also demonstrated that the desire to participate in breast cancer screening is influenced by the participants' perception of susceptibility and perception of severity to breast cancer. The findings of the study could create a positive social change as it may inform the practice of public health providers, influence the drafting of informed policies for comprehensive breast health care, and improve access to preventive health services for Filipino women OFWs. Furthermore, the study could empower Filipino women in their personal health decision making, especially when working in other countries where good health is the working capital and a precondition for survival.

Breast cancer is a serious illness that often has fatal consequences. Adherence to the recommendations for breast cancer surveillance is poorly practiced among African American women. The purpose of this phenomenological study was to seek individual professed barriers to breast cancer screening among African American women to better understand why breast cancer continues to be one of the principal basis of mortality among African American women. The theoretical framework for this study was the behavioral model of health services use. Purposeful selection was used to invite 14 African American women to participate in the in-depth interview process. Interview data were transcribed and then coded for recurring themes and meaning. The findings of this study demonstrate that these women's perceived barriers to breast cancer screening were lack of information, a belief that genetics dictates who gets breast cancer, embarrassment, a norm of people not going for health checkups, the procedure of breast cancer screening, and fear. Participants noted that the improved method of mammography may promote utilization within the population. Breast cancer disparities among African American women may decline if healthcare providers promote awareness of the availability and accessibility of breast cancer prevention resources and if African American women understand the barriers to breast cancer prevention and change their own screening practices.

Cancer-related fatigue (CRF) is a persistent and debilitating problem for many breast cancer survivors. Although many CRF measurement tools are available, no consensus exists on the most appropriate tool to use for breast cancer survivors. The purpose of this project was to identify the best method of assessing CRF in breast cancer survivors. The practice-focused question inquired about the most appropriate way to assess fatigue in breast cancer survivors. The central concepts of the project were CRF and cancer survivorship. This project was informed by the theory of health as expanding consciousness and Mishel's theory of uncertainty in illness. The sources of evidence included multi-database searches and literature from professional organizations. Results were tracked using preferred reporting items for systematic reviews and metasystems and a literature review matrix. The search identified 14 sources, which were assessed for quality using the grading of recommendations, assessment, development, and evaluation process. The results of this systematic review did not support the use of any particular assessment tool; however, 2 clinical practice guidelines recommended screening using a numerical severity scale followed by detailed assessment of clinically significant fatigue using available assessment tools. Screening can be implemented into the survivorship clinic, allowing nurses to identify potentially clinically significant fatigue so that further workup is done and interventions are implemented. Identifying, assessing, and intervening for clinically significant fatigue can improve the quality of life for breast cancer survivors, contributing to positive social change.

Breast cancer survivors (BCS) account for the largest group of cancer survivors living in the United States and they often experience lingering physical symptoms that may affect quality of life, with fatigue and pain the most commonly reported. This genetic research study was conducted within a parent R01 study, with the purpose of exploring associations between genetic variants and fatigue and pain symptoms and the Mindfulness-Based Stress Reduction for Breast Cancer (MBSR(BC)) program. The aims of this study were to: 1) identify specific genotypes involved in fatigue and pain symptoms, and 2) explore whether single nucleotide polymorphism (SNP) rs1800795 in gene IL6, SNP rs16944 in gene IL1B, and SNP rs4680 in gene COMT, moderate the effects of the MBSR(BC) intervention on fatigue and/or pain symptoms. As part of a larger R01 trial, one-hundred-fifty-eight participants were randomized to either a six-week MBSR(BC) intervention or Usual Care (UC). Data were collected at baseline, six-week, and 12-weeks on subjective measures of pain, fatigue, along with demographic and clinical history information. In addition, DNA was collected for genotyping among the 158 participants using the PCR analysis method. For Aim 1, one-way linear trend analysis of variances (ANOVAs) were implemented to explore associations between the SNPs in genes with subjective symptom measures of pain and fatigue. For Aim 2, comparison of mean scores along with linear mixed model (LMM) analyses were used to explore if the patient’s SNPs moderated the effects of the MBSR(BC) intervention on fatigue and pain symptoms. Results found the mean age of the total sample was 58.4 years and 89% were White, non-Hispanic. Although participants were randomized 1:1 to either the MBSR(BC) or UC groups, chi square analyses found that there was a significant difference for time since treatment, with the UC group being closer to treatment end (< 1 year) than the MBSR(BC) group (p < .05). No other statistically significant differences between groups for baseline demographic or clinical characteristics were found. For Aim 1, one-way linear trend ANOVAs among fatigue and pain scores and the three SNPs (COMT rs4680, IL1B rs16944, IL6 1800795) included as part of this study, fatigue and/or pain, resulted in no statistically significant associations (p > .05). Linear Mixed Model (LMM) analyses, implemented to assess the between-group interactions between pain and/or fatigue symptom, time, and SNP, resulted in no statistically significant findings for SNP rs4680 in COMT and SNP rs16944 in IL1B, however significant findings were found for the interaction between assignment (MBSR(BC) versus UC) and genotype for SNP rs1800795 in IL6. Second, a comparison of means suggests that participants in the MBSR(BC) group who had CG genotype for SNP rs1800795 in IL6 benefited more from the intervention than those with CC or GG genotypes for fatigue severity, fatigue interference, pain severity, and pain interference, with small to large effect sizes ranging from d = 0.38 to d = 0.72. Although this genetic study was exploratory in nature, the results suggests that the effects of the MBSR(BC) program may be moderated by SNPs in genes that are involved in cytokine production, which means that BCS with specific genotypes experience a greater improvement in symptoms than those with other genotypes. The results of this study also suggest that further research is needed, with larger sample sizes, to assess the genetic moderation of symptoms experienced by BCS.

No description available.

Human Papillomavirus (HPV) is a small, double stranded DNA tumour virus. Infection with HPV normally results in formation of warts. Certain types of HPV, such as type -16 and -18, are shown to have a causal role in the development of uterine cervical cancer, and are so called high risk type HPV. Recently, a role of HPV in breast cancer has been suggested, although a causal role for HPVs in human breast cancer is yet to be demonstrated. The first part of this study investigates the association of HPV with human breast cancer. The results demonstrate that 48% of breast cancers that occurred in Australian women are HPV positive and they are mainly variants of HPV-18. Further analysis shows that HPV positive breast cancer patients are significantly younger than HPV negative patients, suggesting infection with HPV increases the risk of breast cancer development. This is coincidental with increased risk of HPV infection in sexually active young women and provides evidence that HPV has a role in breast cancer development. The second part of this project investigates the mechanisms by which high risk type HPV oncogenic protein E6, transforms primary human foreskin keratinocytes (natural host cells of HPV). HPV E6 is always expressed in HPV positive cervical carcinoma and results in the degradation of the cellular tumour suppressor protein p53. It is generally believed that HPV E6 contributes to HPV transformation by degradation of p53 protein which leads to cellular immortalisation ? an early step in tumorigenic transformation. Subsequent studies, however, indicate that HPV E6 possesses other functions (such as induction of telomerase activity) which may also be involved in cellular immortalisation. The results of my investigations demonstrate: 1) that degradation of p53 protein is required but is insufficient to immortalise primary cells; 2) that HPV E6 induced telomerase activity is coincidental with an increase in cell culture passage number; 3) that multiple functions of high risk type HPV E6 protein are required for cellular immortalisation. This finding suggests HPV infection is associated with early onset of breast cancer and that multiple functions of high risk type HPV E6 protein are involved in cellular immortalisation. Further study in both of these areas should provide alternative diagnostic markers, leading to prevention and treatment strategies for HPV positive breast cancer and other cancers.