| Summary: | A Thesis submitted to the Faculty of Health Sciences (Internal Medicine), University of the Witwatersrand, in fulfillment of the requirements for the degree of Doctor of Philosophy.
Johannesburg, 2017 === South Africa has the largest ARV treatment programme in the world, wherein highly active antiretroviral treatment (HAART) has improved the health related quality of life (HRQoL) in HIV/AIDS patients. On the contrary, cancers not previously associated with HIV/AIDS (non-Aids defining cancers; NADCs) have been shown to be increasing, compared to the AIDS defining cancers (ADCs). Lung cancer, as a NADC has been documented in the HIV/AIDS population as a leading malignancy. The poor understanding of the association between ARV drugs and lung cancer places a burden on public health, both globally and in South Africa (SA). Furthermore, the deregulation of the cell-cycle is one of the hallmarks of cancer, including lung cancer. The main aim of this study was to elucidate the effects of HAART components Efavirenz (EFV) and Lopinavir/ritonavir (LPV/r) on cell-cycle related genes in an in vitro lung cancer model. To achieve this, cellular based, molecular and Bio-Informatics approaches were employed. First, the cytotoxic effects of EFV (at 4, 13, 26, 50 μM) and LPV/r (at 10, 32, 50, 80 μM), for 24h, 48h and 72h on normal lung fibroblasts (MRC-5) and lung adenocarcinoma (A549) cells, were evaluated using the Alamar Blue (AB) assay. This was then followed by cell-impedance “xCELLigence” real-time cell analysis (RTCA) assay. This was done to determine the effects of EFV (at 4, 13, 50 μM) and LPV/r (at 10, 32, 80 μM) on cell viability, cell death and proliferation. Cell-cycle analysis using propidium iodide (PI) by Fluorescence-activated cell sorting (FACS) was done to quantify DNA present at each of the cell-cycle stages of the cell-cycle in response to ARV treatment. Subsequently, an apoptosis assay using Annexin V FITC and Propidium iodide (PI) dual staining by FACS was carried out to confirm and quantify the ARVs potential apoptotic effects. Then, 4′,6-diamidino-2-phenylindole (DAPI) staining was used to assess changes in nuclear morphology exerted by the ARVs’ effects. A more in depth interrogation of the cell-cycle was performed using a focussed gene array panel of some 84 human cell-cycle related genes. First, total RNA was isolated from both treated and untreated MRC-5 and A549 cells and reverse transcribed to cDNA for use as template in the PCR array reactions. From the array gene expression results, by convention a ±2 fold up-or-down-regulation was used as the basis of target selection. Following this, a real-time quantitative PCR (RT-qPCR) validation of selected genes of interest was done to quantify and confirm the PCR array results. This was followed
by in-silico Bio-informatics analysis to map the molecular pathways regulated by the identified targets. For this purpose, STRING, Database for Annotation, Visualization and Integrated Discovery (DAVID), Reactome and Ingenuity Pathway Analysis (IPA) databases were used.
Interestingly, double-edged oncogenic properties of both EFV and LPV/r at different concentrations were identified. The proliferative effects of EFV at 4, 13μM and LPV/r at10 μM, were elucidated, while 26, 50μM of EFV, and 32μM of LPV/r had slight inhibitory effects on cell proliferation. LPV/r at concentrations of 50 and 80μM exerted cytotoxic effects on the cells, as demonstrated by the AB and xCELLigence RTCA assays. Cell-cycle analysis using PI staining, particularly showed cell-cycle arrest at 32μM LPV/r, and a shift to G2/M by 13μM EFV, plasma relevant doses, compared to the untreated cells. An increasing apoptosis percentage was observed with increasing LPV/r concentrations, that is, 80μM LPV/r raised the apoptosis percentage almost two-fold compared to 32μM. This was coupled by necrosis, observed in a time-dependant manner. DAPI staining confirmed loss of nuclear integrity post ARV exposure, suggesting that both EFV and LPV/r impose damage to the genomic DNA. To further assess the observed changes in nuclear morphology, the effects of EFV and LPV/r on the expression of an arrayed panel of human cell-cycle genes in cancer and normal lung cells was determined. Significantly differentially expressed targets were identified and further quantified and confirmed by RT-qPCR. Such targets included ATM, p53, cyclin-dependant kinase inhibitors (CDKIs), such as, p21, aurora kinase B (AURKB), Mitotic Arrest Deficient-Like 2 (MAD2L2) and the apoptosis related gene, caspase 3 (CASP3). Bio-Informatics analyses revealed close and direct protein-protein interactions (PPIs) between these targets, notably, with change in interaction between the gene products involved in DNA repair mechanisms, observed between ARV treated and untreated groups, as illustrated by STRING interactions. DAVID, Reactome and IPA analysis showed changes in expression of genes related to stress and toxicity and DNA damage response genes. In particular, ATM, p53 and its downstream targets such as GADD45A (growth arrest and DNA damage inducible alpha) gene were up-regulated by ARV treatment, while cyclin/CDK activity was down-regulated, resulting in reduced cell proliferation. Thus in summary, both EFV and LPV/r altered the expression levels of cell-cycle related genes, influencing overall cellular health, acting to either inhibit or stimulate cell proliferation. This suggests EFV’s and LPV/r’s proliferative and
inhibitory roles in the proliferation of lung cells. Moreover, future directions can include the transfection of lung cells with HIV provirus followed by treatment of the cells with the same ARVs under study. This could be substantiated by including HIV positive patient samples on and off ARV drug treatment with lung cancer, including HIV negative patients with cancer as one of the controls. === MT2018
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