Nasopharyngeal carcinoma (NPC) is a common cancer in Southeast Asia. While external radiotherapy is the mainstay of treatment, adjuvant chemotherapy is required in advanced disease. Current chemotherapy heavily relies on cisplatin and docetaxel. The disease relapse rate is relatively high with poor survival chance for recurrent or metastatic disease. Development of novel therapeutic strategies against the disease is clearly needed. Bortezomib and suberoylanilide hydroxamic acid are respectively classified as proteasome inhibitor and histone deacetylase inhibitor. Bortezomib and SAHA induce apoptosis in various cancers including renal cell carcinoma, hepatoma and mantle cell lymphoma. However, the effect of bortezomib and SAHA on NPC cells was not mentioned. We sought to study the molecular and cellular effects of the bortezomib and SAHA on NPC cells hoping to look for drug alternatives in NPC treatment. Since SAHA reactivates EBV in NPC cells, the combined effect of bortezomib and SAHA on EBV lytic cycle was also evaluated. NPC proliferation was assessed by MTT assay. 5 EBV-positive NPC cell lines authenticated by Short Tandem Repeats (STR) profiling were used as most NPC in Chinese contains EBV. Isobologram and combination index analysis confirmed that the anti-proliferative effect on NPC mediated by the drug combination was synergistic. 30 nM bortezomib and 5μM SAHA were chosen for further studies on apoptosis because the synergism of the drugs was maximal at these concentrations. NA and C666-1 were chosen for further studies because C666-1 was the only NPC cell line that consistently harboured native NPC and the combination index was lowest in NA among the rest of the NPC cell lines. Bortezomib led to apoptosis in NPC cells. The effect was more pronounced after the addition of SAHA as evidenced by greater TUNEL positive population and earlier cleavage of poly ADP ribose polymerase (PARP). In previous cancer studies, ROS induction was commonly suggested pathways of bortezomib and SAHA’s antiproliferative effects. Staining with dichlorofluorescein diacetate (DCFH-DA) revealed enhanced reactive oxygen species (ROS) level in cells treated with both drugs. At the same time, addition of N-acetyl cysteine, a ROS scavenger, markedly reduced their effect on cytotoxicity. SAHA is known for its effect on EBV lytic cycle induction. Yet, the addition of bortezomib diminished SAHA-induced viral load, lytic protein expression and EBV infectivity. The expression of Latent Membrane Protein 1 (LMP1) was much lower in NPC treated with both drugs than in NPC treated with SAHA alone, which would reduce NF-κB activation. This, together with reduced EBNA1 expression upon treatment with both drugs, would theoretically reduce oncogenic activity. In conclusion, bortezomib and SAHA induced ROS-driven apoptosis of NPC in a synergistic manner and bortezomib inhibited SAHA-induced EBV lytic cycle. It suggests that bortezomib and SAHA are potential drug candidates for the treatment of nasopharyngeal carcinoma. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine

abstract / Pathology / Master / Master of Philosophy

Small cell lung cancer (SCLC) is characterized by prompt response to chemotherapy and radiotherapy but relapsing with drug resistance and distant metastasis, leading to poor overall prognosis. New anticancer agents and regimens are drastically needed for SCLC treatment. Arsenic trioxide (ATO), a traditional Chinese medicine used as a poison for thousands of years, has been tested in many hematological and solid cancers both in vitro and in vivo, with promising effects. In order to establish the scientific ground for future clinical application of ATO in SCLC, this study aimed to investigate the anticancer effect and mechanism of ATO in SCLC using in vitro and in vivo models, either as a single agent or in combination with standard chemotherapy. In addition, an ATO-acquired resistant cell line (H841-AR) derived from SCLC cell line H841 was used to explore potential mechanisms of ATO resistance and cross-resistance to other chemotherapeutic drugs. In the first part of this study, ATO was shown to exert cytotoxic effect in all of the chosen SCLC cell lines. Various cellular mechanisms were triggered upon ATO exposure: redox status disturbances (hydrogen peroxide (H2O2) generation, glutathione (GSH) depletion and thioredoxin 1 (Trx1) down-regulation), mitochondrial membrane depolarization (MMD), DNA damage, apoptosis and necroptosis. In concert with this, Bcl-2 was down-regulated accompanied by MMD, release of AIF and SMAC, DNA degradation, XIAP inhibition and caspases activation. Adoption of N-acetyl-L-cysteine (NAC) and buthionine sulfoximine (BSO) demonstrated GSH depletion and reactive oxygen species (ROS) generation played the pivotal role to mediate cytotoxic effect of ATO in SCLC. In the second part of this study, when combined with chemotherapeutic agents, ATO displayed synergistic and antagonistic interaction with cisplatin and etoposide respectively in SCLC cell line models. The beneficial combination of ATO and cisplatin was also substantiated by tumor xenograft models. Augmented GSH depletion and suppressed drug efflux mechanism were found to explain the synergistic effects. In the last part of this study, H841-AR was generated as an acquired multi-drug resistant (ATO, cisplatin and etoposide) cell line to investigate the potential resistance mechanisms and possible future drug combinations. Comparing H841-AR cells with parental H841 cells using cDNA microarray, a long list of genes was altered in ATO-resistant cells. At least 20 up-regulated and 45 down-regulated genes were short-listed as candidates with a cut-off at 5-fold change. Interestingly, qPCR data has shown that 5 selected up-regulated genes in H841-AR cells were also highly expressed in DMS79 cells with intrinsic ATO resistance compared to the relatively sensitive cell lines, indicating that these genes might be associated with ATO resistance in SCLC. In summary, ATO was shown to be an active anticancer agent in SCLC, either alone or in combination with cisplatin. The major mechanisms of action of ATO and its synergism with cisplatin in SCLC were elucidated. Genetic data derived from an acquired resistant (to ATO, cisplatin and etoposide) SCLC cell line may help to uncover the mechanisms of resistance to ATO, allowing possible future drug combinations. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Molecular Biology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Surgery / Master / Master of Surgery

Several diseases, including cancer, have been associated with high membrane type-1 matrix metalloproteinase (MT1-MMP) expression levels. MT1-MMP together with a non-membrane-bound, soluble MMP, MMP-2, also associated with many other biological functions, have been implicated in breast cancer progression, invasion and metastasis, and poor prognosis. Researchers who ran early clinical trials that employed broad-spectrum MMP inhibitors (MMPIs) lacked understanding of the intricate physiological and patho-physiological roles that MMPs play in tissues. In addition, structural similarities between MMPs hamper selective inhibition. Selective inhibition of MT1-MMP is of particular interest as MT1-MMP is overexpressed in target cancer cells relative to normal cells and is key in signalling for invasion. The inhibition profiles of two structurally similar synthetic pyrimidine-class MMPIs, with increased bioavailability and stability compared to hydroxamates tested in earlier clinical trials, TF 17-2 and TF 22d, were assessed. TF 17-2 and TF 22d were applied to a normal MCF-10A breast epithelial cell line and its premalignant H-ras(V12)-transfected MCF-10AneoT derivative to assess their efficacy for inhibiting MT1-MMP-mediated normal and premalignant cell migration, and indirectly, invasion. Both inhibitors form a co-ordination complex with the zinc ion of the catalytic site of MT1-MMP and MMP-2 with greater affinity for MT1-MMP. The computational molecular docking package, AutoDock Vina, was used for in silico predictions of binding affinities that could potentially substitute for in vitro kinetic assays when assessing inhibitor potential for inhibiting target MMPs. The binding of the two MMPIs was assessed using AutoDock Vina and compared to established kinetic data. The AutoDock Vina program was found to be an unreliable predictor for assessing relative efficacy of inhibition. During in vitro applications, analysis of the induction of apoptosis and metabolic effects were assessed using flow cytometry and the MTS assay, respectively. These showed no significant toxicity. Effects of inhibitors on collective and single cell migration in the normal and premalignant cell model, assessed using time lapse live cell imaging, cell morphology and labelling for vinculin and F-actin (for focal adhesions, FAs) showed that the TF 17-2 and TF 22d inhibitors reduced the collective cell migration of MCF-10A cells in scratch assays. Live-cell analysis of single cell migration, however, showed that TF 22d increased cell migration rates, and reduced the size of FAs and actin stability in MCF-10AneoT cells, resulting in a predominently rounded cell morphology in the premalignant cell line. TF 17-2, on the other hand was seen to be a relatively selective inhibitor of premalignant cell migration and resulted in MCF-10AneoT cells re-establishing larger focal - v - adhesions due to more stable F-actin networks resembling those of the non-transfected MCF-10A cell line, but reduced MCF-10AneoT cell migration most markedly. FA size and velocity of movement seemed inversely related in the normal and premalignant cells. The results of the current study suggest that, TF 17-2 seemed to have the greater therapeutic potential than TF 22d for inducing phenotype reversion, inhibition of dissemination, invasion and metastasis. Three promising selective pharmacological actions of TF 17-2 on the premalignant MCF10AneoT cell line include the suppression of proliferation, induction of increased in metabolic activity (possibly indicating cell stress) and a decrease in premalignant cell migration. A lack of cytotoxicity, however, suggests that TF 17-2 would need to be administered with an ancillary chemotherapeutic agent. This study showed that MMPIs directed against MT1-MMP, may still represent an effective strategy for inhibiting the migration of premalignant cells expressing high levels of MT1-MMP, and suggests further studies on this topic may be profitable. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2013.

Indigenous plants have long been used by African populations in their cultural lives and health care. Sutherlandia frutescens (SF) is a popular traditional medicinal plant found in various parts of southern Africa and used for treatment or management of different diseases, including cancer and HIV/AIDS. In this study, the biochemical effects of various dilutions (1/50, 1/150, 1/200, and 1/300) of SF 70% ethanol (SFE) and deionised water (SFW) extracts in cancerous H9 and normal T cells were examined. Untreated, 70% ethanol-treated and camptothecin (CPT, 20jiiM) treated cells were used as reference samples for comparison. Cytotoxicity, apoptotic enzymes activity, oxidant scavenging and antioxidant promoting abilities, cellular morphology and cytokine signalling effects were assessed using the methylthiazol tetrazolium (MTT) assay, adenosine triphosphate (ATP) assay, caspase-3/-7 activity assay, thiobarbituric acid reactant substance (TBARS) and glutathione (GSH) assays, fluorescence microscopy and an ELISAbased cytokine analyses assay respectively. Sutherlandia frutescens ethanol and water extract dilutions (1/50 and 1/200) were shown to be cytotoxic to H9 T cells in a dose- and time-dependent manner with the SFE extract having an average IC50 of 1/40 after 24 hours while SFW extract reached a similar IC50 only after 48 hours. In normal T cells, the SFE extract induced proliferation after 24 hours but this was reverse after 48 hours. The SFW extract dilutions did not significantly change cell viability after 24 hours but significantly increased cell viability after 48 hours. Both SFE and SFW extracts dilutions induced a dose- and time-dependent inhibition of caspase-3/-7 activity in both H9 and normal T cells. Both types of extracts were also shown to efficiently remove lipid peroxides from supernatants of treated cell lines, with SFW extract having a more lasting effect. In the GSH assay, the SFE and SFW extract dilutions reduced GSH levels in H9 T cells, with the SFW extract dilutions being more effective. In normal T cells, the higher dilutions (1/150 and 1/300) of SFW extract increased GSH levels significantly while lower dilutions (1/50) of both SFE and SFW extracts significantly inhibited GSH levels. Lower dilutions (1/50) of SFE and SFW extracts induced chromatin condensation in both H9 and normal T cells after 48 hours incubation. Using treated peripheral blood mononuclear cells (PBMCs) supernatants, SFE and SFW extract dilutions were shown to reduce the levels of pro-inflammatory cytokines IL 1 p and TNF-a in a dose-dependent manner. These results further confirmed the anticancer abilities of SF and showed that higher concentrations of this medicinal plant can be toxic to normal T cells in vitro while lower concentrations can stimulate the immune cells. Therefore further studies should be conducted with regards to the effects of SF on the immune system in both in vitro and in vivo systems. / Thesis (M.Med.Sci.)-University of KwaZulu-Natal, 2008.

Ochratoxin A (OTA), a nephrotoxic mycotoxin, is a contaminant of several agricultural food products consumed by animals and humans. Apart from renal toxicity, in particular renal tumours, OTA may also result in teratogenicity, neurotoxicity and immunotoxicity. Sutherlandia frutescens, an indigenous medicinal plant, has shown significant potential in strengthening the immune system and in cancer treatment, with minimal side effects. The objective of this study was to determine the effects of OTA in male Wistar rats and ascertain if these effects may be reduced by S. frutescens. Rats were treated by intraperitoneal injection (i.p) with either a control (EtOH:dH20;30:70), S. frutescens (1.0mg/kg body weight), OTA (0.5mg/kg body weight) or a combination of OTA and S. frutescens for a period of 1 or 7 days (n=4). Genotoxicity and metabolic activity in peripheral blood mononuclear cells (PBMCs) were quantified using single cell gel electrophoresis (SCGE) and the methylthiazol tetrazolium (MTT) assay, respectively. Lymphocyte apoptosis and mitochondrial depolarisation were measured by flow cytometry. Fluorescence microscopy was utilised to determine renal tissue apoptosis (Hoechst staining) and OTA localisation using immunohistochemistry (IRC). SDS-PAGE and Western blot were utilised to determine protein expression in kidney tissue and serum. Ochratoxin A significantly reduced PBMC viability (14%) after 7 days, compared with Day 1 (p<0.001). Lymphocyte mitochondrial depolarisation was 56.5% and 66.2% in the OTA-only and combination groups, respectively after 7 days (p<0.001). Ochratoxin A produced an increase in DNA damage compared to the control (p<0.01). The renal tissue displayed typical signs of apoptosis such as chromatin condensation. Ochratoxin A was immunolocalised within the glomerulus. The protein analysis showed a decreased expression in the kidney mitochondrial protein fraction. Ochratoxin A preferentially bound to serum albumin and a 120kDa protein in the OTA-only and co-treatment groups after the 1-and 7-day regimes. Protein band intensities significantly decreased after the 7-day co-treatment (p<0.01). The data highlights that OTA toxicity is mediated by mitochondrial dysfunction. Furthermore, OTA disruptions in immune function may play a role in renal damage. / Thesis (M.Med.Sc.)-University of KwaZulu-Natal, Westville, 2009.

No description available.

Doctor of Health Science / It is well documented that for prostate cancer patients undergoing radiation therapy there is a correlation between target volume displacement and changes in bladder and rectal volumes. However, these studies have used a methodology that has captured only a subset of all treatment positions. This research used daily Computer Tomography (CT) imaging to comprehensively assess organ volumes, organ motion and their effect on dose, something that has never been performed previously, thus adding considerably to the understanding of the topic. Daily CT images were obtained using a Siemens Primus Linear Accelerator equipped with an in-room Somatom CT unit in the accelerator suite, marketed as ‘Primatom’, to accurately position the patient prior to treatment delivery. The internal structures of interest were contoured on the planning workstation by the investigator. The daily volume and location of the organs were derived from the computer to assess and analyse internal organ motion. The planned dose distribution was then imported onto the treatment CT datasets and used to compare the planned dose to i) the actual isocentre, where the isocentre was actually placed for that fraction, ii) the uncorrected isocentre, by un-doing any on-line corrections performed by the treatment staff prior to treatment delivery, and iii) the future isocentre, by placing the isocentre relative to internal organ motion on a daily basis. The results of this study did not confirm a statistically significant decrease in rectum volumes over time (hypothesis 1), however large fluctuations in bladder volume were confirmed (hypothesis 2). Internal organ motion for the rectum and bladder was demonstrated to be related to organ filling. Ideal planning volumes for these organs have been reported to minimise systematic and random uncertainty in the treatment volumes. An observed decrease in prostate volume over time, a systematic uncertainty in the location of the prostate at the time of the planning CT scan and a significant relationship between prostate centre of volume and rectum and bladder volumes has resulted in a recommendation that patients should be re-scanned during treatment to ensure appropriate clinical target volume coverage. A significant relationship between rectal and bladder volumes and the dose delivered to these organs was found (hypothesis 3). The dose delivered to the planning target volume was not related to the rectal or bladder volumes, although it was related to the motion of these organs. Despite these results only minimal effects on the dose delivered to any of the three isocentres occurred, indicating that the planned dose was accurately delivered using the methodology presented here (hypothesis 4). However the results do indicate that the patient preparation instructions need to be improved if margins are to be reduced in the future. It is unrealistic to assume that Image Guided Radiation Therapy will ever become routine practice due to infrastructure costs and time limitations. This research will inform radiation therapy centres of the variables associated with prostate cancer treatment on a daily basis, something that has never before been realistically achievable. As a result centres will be able to devise protocols to improve treatment outcomes.