Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2021 / Lung cancer is the most diagnosed cancer with an estimated 3 million deaths expected by 2035. Bioactive phytochemicals present in plants are preferred as anticancer therapeutic agents, due to their ability to differentiate between cancerous and normal cells. One such plant, Senna italica, is traditionally used to treat diabetes, malaria, constipation, jaundice, fever and sexually transmitted diseases. Several studies have reported on its anti-proliferative potential against different types of cancers. However, there is scanty information regarding its molecular mechanism of action against different types of cancers, more especially lung cancer. This study, therefore, aims to determine the differential expression profiles of apoptotic genes in lung A549 cancer cells induced by treatment with S. italica leaf and root extracts in an attempt to understand its purported anticancer molecular mechanism of action. The leaves and roots of S. italica were dried in the dark and extracted with ethyl acetate and methanol. Screening for the presence of secondary metabolites was performed using thin layer chromatography and various standard chemical-based tests. The total phenolic and flavonoid compounds were evaluated using gallic acid and quercetin equivalence assays. The antioxidant activity of S. italica extracts was determined using DPPH free radical scavenging and ferric ion reducing power assays. The cytotoxicity of both leaf and root extracts on lung A549 cancer cells was evaluated using 3-(4,5- dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide (MTT) and further confirmed by Muse cell count and cell viability assays. The proliferation of cells, after treatment with different concentrations of the extracts, was examined using the Ki67 proliferation assay. Genotoxicity was determined to assess the potential damage caused by the extracts on the DNA using a MUSETM multicolour DNA damage kit following manufacturer’s protocol. The morphological change of cells treated with different concentrations of S. italica ethyl acetate root extract was analysed using acridine orange/ ethidium bromide (AO/EB) dual staining assay and examined under fluorescent light. The total number of cells undergoing apoptosis was also determined using the Annexin V assay. The expression of 84 key genes, involved in programmed cell death or apoptosis, was determined using the Human Apoptosis RT² Profiler PCR array kit. Senna italica methanol extract had a high content of plant materials in both leaves and roots compared to the ethyl acetate extract. A higher phenolic content was observedxii mainly in the leaf extract and a higher flavonoid content was observed in the root extract. Phytochemicals, such as phenols, tannins, flavonoids, terpenoids and steroids, which are known to exhibit anti-cancer activity against cancerous cells were abundant in the ethyl acetate leaf and root extracts as compared to the methanol leaf and root extracts. Additionally, the ethyl acetate root extract exhibited more antioxidants and radical scavenging activity in comparison to the methanol root extract. The IC50 of ethyl acetate root extract was determined to be 200µg/ml. Both methanol and ethyl acetate root extracts had little to no effect on the viability of lung A549 lung cancer cells. The results were confirmed by cell count and viability assay results. The cytotoxicity of ethyl acetate root extract was also evaluated against the normal kidney HEK-293 cells, which displayed little cytotoxic effect. The proliferation results indicated that S. italica ethyl acetate root extract has the potential to reduce the proliferation of lung A549 cancer cells. The ethyl acetate root extract was found to induce late apoptosis in A549 cells, but the genotoxicity data indicated that the DNA double strand breaks (DSBs) were repairable. The results further showed an expression of different genes that inhibit apoptosis, such as XIAP in lung A549 cells, following treatment with S. italica ethyl acetate root extract. In conclusion, the ethyl acetate root extract displayed a promising anti-cancer therapeutic potential, and thus warrants further investigation to elucidate the identity of the inherent chemical components that are responsible for the observed biological activity. / University of Limpopo and SAMRC

Lung cancer is the leading cause of cancer mortality and morbidity. Patients with advanced lung cancer have distressful symptoms like dyspnea. It has severe negative impact on the quality of life. Airway stenting has become widespread for palliation of airway stenosis in patients with metastatic airways disease. Although it provides improvement in symptoms, actual survival benefit is limited with severe potential complications. Appropriate patient selection in terms of site of tumor, type of stent placement is needed to achieve maximum benefit for patients. Here we will discuss 2 patients with advanced lung cancer who received bronchial stent for intractable dyspnea. Although there was dramatic improvement in symptoms and quality of life, both died shortly. Was there any benefit of stenting remained unanswered.

No description available.

Small Cell Lung Cancer (SCLC) is a highly aggressive neuroendocrine tumor with a 5% survival rate over 5 years. Though SCLC comprises 13% of all cases of lung cancer the median survival time of 14.5 months has seen little improvement over the last four decades. Standard treatment relies on DNA damaging agents such as Cisplatin/Etoposide (EP) which induce a high response rate of 60-70%. Despite this initial response, nearly all patients will relapse rendering first-line therapies ineffective. Furthermore, SCLC has been shown to develop chemotherapy cross-resistance in which resistance to first-line chemotherapies will confer resistance to additional DNA damaging agents thereby reducing treatment efficacy and duration of response. Cross-Resistance constitutes a major clinical issue whose underlying mechanisms remain a mystery. The modest improvements in SCLC patient outcomes over the decades may be partially explained by the existing systems of study. Current methodologies of SCLC study rely on cell lines, patient samples, and Genetically Engineered Mouse Models which have little functional correlation to clinical outcomes. While few sources have proposed Patient Derived Xenograft (PDX) systems as an improved alternative, significant data remains sparse. Without a robust model system which accurately recapitulates patient outcomes, molecular pathways driving resistance cannot be uncovered. Here we present the generation of 34 SCLC PDX models which maintain both genomic and functional fidelity. Furthermore, treatment of a 30-model subset with first-line chemotherapy EP and a novel chemotherapy Olaparib/Temozolomide (OT) allowed for functional and molecular comparison between groups. Our findings demonstrate incomplete independent resistance mechanisms between EP and OT treatment with a small overlap of 31 genes involved in glycolysis and xenobiotic metabolism.

Lung cancer affects many individuals each year and accounts for many deaths around the globe. Lung cancer screening is a preventative health measure that has the ability to detect lung cancer earlier. The purpose of this project was to focus on the education of nursing staff within a community health system, with subsequent implementation of an electronic health record clinical decision support system, to create a direct referral pathway to lung cancer screening, delivered through patient education. The concept of prevention was the framework for this project design, which was further organized around the plan-do-study -act model, while taking into consideration the health belief model and theory of interpersonal relations. Using systemized dashboard reports within the electronic health record software, specific variables were targeted for data collection and analyzed for the purpose of this project. Final data demonstrated an increase of triple the programmatic volume of the previous year, directly following the implementation of the above initiative. Further comparative statistics bespeak to the significant needs of the community regarding tobacco dependence and lung cancer screening. High-risk individuals who are current or former smokers will benefit from this initiative by receiving education about lung cancer screening and tobacco dependence treatment while within the care of the community based health system. A nursing-driven pathway to preventative care could also serve other cancer screening programs effectively, as well as be applied to a variety of chronic disease comorbidities to make a significant positive social change.

Improvements in the medical field have given many cancer patients and survivors better odds of long-term survival. As more patients become survivors, the demand for psychological treatment becomes greater. The most prevalent concern of survivors is getting help with a psychosocial condition known as fear of recurrence (FOR). Prior to this study, few researchers had explored how having a more aggressive cancer influences the development of FOR. The purpose of this quantitative study was to determine whether cancer stage and type (a measurement of severity) are predictive of FOR development in the high-risk cancer groups lung and bronchus and female breast. The theoretical framework guiding this research was based on Mishel's theory of uncertainty in illness, which states that uncertainties about illness recurrence can cause survivors to experience breakdown in their lives (whether psychological and/or physical). The fear of cancer recurrence inventory (FCRI) survey was administered to 97 lung and bronchus and female breast cancer survivors; the survivors were asked to rate their level of discomfort about the possibility of a cancer recurrence. Data were analyzed using multiple linear regression. The results indicated that cancer type and severity both impacted the development and severity of FOR in lung and bronchus and female breast cancer survivors. Furthermore, regardless of the cancer type, stage of cancer, age of the survivor, or years in remission, survivors reported clinical levels of FOR in all areas of concern. Practitioners can use the current findings to work towards developing better intervention and treatment programs that promote quality survivorship and reduce the risk and rate of FOR in high risk cancer populations.

L’identification de la notion de driver oncogene et le développement de molécules capables de cibler leur activité a entrainé d’importants changements dans le traitement des cancers CBNPC.L’EGFR est un récepteur transmembranaire à activité TK (Tyrosine Kinase) permettant latransmission de signaux extracellulaires jusqu’au sein de la cellule grâce à l’activation de voies de signalisations. Parmis ces voies de signalisation on retrouve les voies de prolifération et de survie cellulaire. Des mutations activatrices dans le domaine TK confèrent à ce dernier un rôle de driver oncogene. Dans ce domaine l’EGFR a joué un rôle avant gardiste. Ainsi de nombreuses molécules ciblant l’activité de l’EGFR muté (EGFR-TKI, gefitinib) ont été développées. L’utilisation de ces molécules en clinique a représenté une vraie révolution dans la prise en charge des patients. Cependant ces derniers développent inéluctablement des mécanismes de résistance. L’analyse transcriptomique de modèles ayant acquis une résistance aux EGFR-TKI a mis en évidence une dérégulation de l’expression des transcrits. Par ailleurs des résultats del’équipe ont montré que l’expression des protéines SR (facteurs d’épissage impliqués dans la régulation de nombreux transcrits) était dérégulée dans les CBNPC. Sur la base de ces résultats nous avons émis l’hypothèse que l’épissage alternatif des transcrits médié par les protéines SR pourrait jouer un rôle dans la résistance acquise par les tumeurs pulmonaires en réponse aux EGFR-TKI.Au sein du laboratoire des clones résistants ont été générés après exposition chroniqueau gefitinib de modèles cellulaires d’adénocarcinomes pulmonaires exprimant une mutation activatrice de l’EGFR.Nous avons tout d’abord mis en évidence une accumulation de l’expression de SRSF2 dans les clones résistants comparativement à la lignée sensible. Dans deux clones résistants au gefitinib, issus de la lignée d’adénocarcinome pulmonaire sensible PC9 exprimant un EGFR mutant Del19, nous montrons que la neutralisation de la protéine SRSF2 sensibilise les clones à l’apoptose induite par le gefitinib. Une analyse RNA-seq nous a permis d’identifier Atg16L1 comme un acteur potentiel de la resensibilisation à l’apoptose médiée par SRSF2. La neutralisation de SRSF2 entraine une modulation de l’épissage de l’exon8 de la protéine Atg16L1 en réponse à un traitement au gefitinib. Et la neutralisation de l’expression des transcrits ARN d’ATG16L1 comportant l’exon8 sensibilise les clones résistant à l’apoptose induite par le gefitinib. Ce switch d’épissage entraine une modulation de l’activité autophagique des cellules en réponse au gefitinib. Nous montrons qu’une expression majoritaire des transcrits comportant l’exon 8 favorise une inhibition de l’autophagie en réponse au gefitinib. De plus les modèlesrésistants pour lesquels on observait une resensibilisation à l’apoptose suite à une neutralisation des transcrits contenant l’exon 8, conservent leur phénotype de résistance lorsque dans ces mêmes conditions l’activité autophagique est inhibée. L’ensemble de ces travaux met en avant l’existence d’un switch d’épissage de la protéine Atg16L1 au niveau de son exon 8 contribuant à une inactivation de l’autophagie corrélée avec un phénotype de résistance à l’apoptose en réponse à un traitement par EGFR-TKI. Enfin SRSF2 participerait à la modulation de cet épissage en réponse à un traitement au gefitinib. / Identifying what is a driver oncogene and developping small molecules that are able to targetits activity led to drastic changes in NSCLC treatments. EGFR is a transmembrane receptorwith Tyrosine Kinase (TK) activity allowing signal transmission from the environment towardsthe inner of the cell by signaling pathways activation. Among those signaling pathways arefound survival and proliferation pathways. Activating mutations make EGFR a driver onco-gene, which was the first protein to be identified as such. Hence numerous chemical compoundtargeting mutated EGFR (EGFR-TKI, gefitinib) have been developped. Their use in clinicsrepresent a huge improvement for patients care. However resistance mechanisms ultimatelyoccur. Transcriptomic analyses of acquired resistant models to EGFR-TKI have shown thattheir RNA transcripts expression is abnormal. Moreover results from the team have demons-trated that SR proteins (splicing factor) expression is deregulated in NSCLC. Based on thoseresults we hypothesized that SRSF2 mediated alternative splicing of mRNA could be involvedin resistance mechanims acquired by lung carcinoma in response to EGFR-TKIThe lab developped resistant clones by chronic exposure to gefitinib of EGFR mutated lungadenocarcinoma cellular models.We first observed the accumulation of the expression of SRSF2 protein in resistant clonescompared to the sensitive cell line. Secondly, sensitivity to gefitinib induced apoptosis of twoclones was resored when neutralising SRSF2. A RNA-seq analysis led us to identify Atg16L1 aspotentially being involved in the SRSF2-mediated sensitization to gefitinib-induced apoptosis.SRSF2 neutralisation modulates Atg16L1 splicing in response to gefitinib. Neutralisation ofExon 8 containing transcripts of Atg16L1 sensitizes resistant clones to gefitinib induced apop-tosis. This alternative splicing switch modulates autophagic activity of the cells in reponseto gefitinib. We have shown that exon8 containing transcripts favor autophagy inhibition inreponse to gefitinib. This work emphasize the role of Atg16L1 alternative splicing switch ofexon8 in autophagy inhibition and its correlation with a resistant phenotype in response toEGFR-TKI. SRSF2 may participate in the modulation of this alternative splicing switch inreponse to gefitinib.

Proteins are large biomolecules that have great therapeutic potential in treating many human diseases. However, chemical/enzymatic degradation, denaturation, and poor penetration into cells are some of the challenges for clinical use of intracellular proteins. Previously, our group has developed cationic lipid-coated magnesium phosphate nanoparticle (LP MgP NP-CAT) formulations to enhance the intracellular delivery of the negatively charged protein catalase. The goal of the current research is to develop a formulation to deliver cytochrome c (CytC), a positively charged protein into lung cancer cells A549. Specifically, this thesis research prepares and tests liposome-coated magnesium phosphate nanoparticle for delivery of cytochrome c (CytC LP/MgP). CytC LP/MgP was designed, prepared and characterized, showing that it had an average diameter around 150 nm and ζ-potential around +30 mV. The morphology of CytC LP/MgP was validated by transmission electron microscopy. CytC LP/MgP successfully led to the attachment of CytC to A549 cells, as supported by fluorescence imaging. Intracellular delivery of CytC alleviated the cytotoxicity of cationic lipids in A549 cells, as suggested by the MTS assay on cell viability, which could facilitate the clinical use of cationic lipids in drug delivery systems.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18155号 / 医博第3875号 / 新制||医||1002(附属図書館) / 31013 / 京都大学大学院医学研究科医学専攻 / (主査)教授 上本 伸二, 教授 平岡 眞寛, 教授 安達 泰治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

リポジトリの登録にあたっては、Peer reviewされた最終版のみ可能であり、その際には下記の出版社のウェブサイトのアドレスを記載することが求められる。当該論文は2014年6月の出版であり、12ヶ月を経過していることから、公開には差し支えはない。http://clincancerres.aacrjournals.org/content/20/12/3087.full / 京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19617号 / 医博第4124号 / 新制||医||1015(附属図書館) / 32653 / 京都大学大学院医学研究科医学専攻 / (主査)教授 小川 誠司, 教授 野田 亮, 教授 伊達 洋至 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM