Most cancer cells rely on aerobic glycolysis to support the mitochondrial oxidative phosphorylation system (OXPHOS). The persistent oxic-anoxic cycle exerts selection pressures which lead to constitutive activation of glycolysis even in the presence of abundant oxygen. Expression of hypoxia-inducible factor 1 (HIF1) increases following hypoxia in neoplastic cells. This leads to the induction of pyruvate dehydrogenase kinase 1 (PDK1). The latter inactivates pyruvate dehydrogenase (PDH) that converts pyruvate to acetyl-coenzyme A for delivery to the tricarboxylic acid cycle (TAC). Dichloroacetic acid (DCA) is an inhibitor of PDK that forces cells into oxidative phosphorylation thereby suppressing cancer growth. 2-Ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-3-ol-17-one (C9), along with a few other 17β-estradiol analogs, are a novel class of in silico-designed inhibitors of microtubule dynamics. These newly designed and synthesized antimitotic compounds induce G2/M arrest and apoptosis by docking to colchicine binding site between α- and β-tubulin. These compounds are 5 to 20 times more potent than their source molecule, 2-methoxyestradiol (2ME). To improve bioavailability C9 has been in silico-modified at carbon positions C2, C3 and C17 compared to 2ME. The approach to investigate the anticancer potential of the in silico-designed antimitotic C9 in combination with the glycolytic inhibitor DCA in vitro is novel. Human breast carcinoma cell line MCF-7 and non-tumorigenic breast cells MCF-12A were used as an experimental model system. The present study demonstrated that DCA (7.5 mM) in combination with C9 (130 nM) selectively inhibited half of MCF-7 cells‘ population (50.8%). Under the same treatment conditions, MCF-12A cells displayed high number of cell survival (70% cell growth). Qualitative morphological studies revealed decreased cell density in both cell lines, as well as hallmarks of apoptosis and autophagic processes including formation of apoptotic bodies, DNA fragmentation and autophagic vacuoles. Cell cycle- and apoptosis quantification analyses revealed C9+DCA treatment induced apoptosis in both cell lines and exhibited selectivity towards tumorigenic cells. Presence of autophagosome was observed and microtubule-associated protein 1 light chain 3 (II) (LC3-II) expression was elevated. Reduction of mitochondrial membrane potential depolarization in tumorigenic MCF-7 cells was demonstrated, but not in MCF-12A cells. Oxidative stress tests suggested the combination treatment C9+DCA is able to induce lysosomal rupture and/or mitochondrial damage in tumorigenic MCF-7 cells. Kinase inhibition studies revealed that transient activation of c-Jun N-terminal kinase (JNK) plays an important role in cell proliferation. However, C9+DCA stimulated prolonged JNK activation and, in turn, promoted Bcl-2 phosphorylation, thereby facilitating autophagic and apoptotic cell death. C9+DCA induced expression of a number of genes related to stress in MCF-7 treated cells including TP53BP1, MDM2 and BBC3/PUMA. Genes related to cell motility and maintenance of the cytoskeleton such as ACTG1, MAP7, TUBA1, TUBA6, TUBA8 and TUBB2A genes were down-regulated. In MCF-12A cells, treatment of C9+DCA induced expression of multidrug resistance gene ABCB1. Moreover, genes involved in reactive oxygen species metabolism FTH1, GSTA2, NOS2A, SMOX, SOD1 and SOD2 were also up-regulated. In conclusion, the novel 17β-estradiol derivative, C9, in combination with DCA is a potent antiproliferative treatment. This study addressed the mechanisms of combination treatment at the basis of molecular and cellular level, warranting further research projects to develop viable and functional combination treatment as clinically useable anticancer agents. / Thesis (PhD)--University of Pretoria, 2014. / lk2014 / Physiology / PhD / Unrestricted

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Cancer therapy traditionally seeks to achieve complete tumor eradication via induction of cancer cell death by chemotherapeutic agents or radiation. An alternative strategy is to induce cytostasis, i.e. to arrest proliferation of cancer cells, perhaps in parallel with conventional chemotherapy. Such an alternative strategy could provide prolonged survival with less severe consequences of cytotoxic agents. To be truly effective, a chemotherapeutic drug should exert its action on biochemical targets specific for neoplastic cells while leaving the normal cells unaffected. Therefore, the knowledge of tumor cell-specific biochemical and signaling pathways is a pre-requisite for development of new, prospective anticancer drugs. In this study, we concentrated on the energy metabolism which is remarkably different in tumor and healthy cells. Cancer cells generate ATP mainly through the glycolytic pathway, and depend far less on oxidative phosphorylation (the Warburg effect). The way cancer cells generate energy reflects their need for energy as well as building blocks required for fast biosynthesis. Glycolysis, in contrast to oxidative phosphorylation, enhances biosynthetic pathways thus accelerating progression of tumor cells through the cell cycle. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) occupies a central position in the glycolytic pathway thus playing a critical role in the energy metabolism of cancer cells. Along with its enzymatic activity, GAPDH is a multifunctional protein which acts as a signaling and regulatory molecule in several cellular mechanisms. Based on the fact that glycolysis plays a pivotal role in survival of cancer cells, we hypothesized that down-regulation of GAPDH protein would alter the cancer cell proliferation, and cellular sensitivity of cancer cells to chemotherapy. The goal of this study was to evaluate GAPDH as a potential molecular target for treatment of cancer. In this project, our aims were: 1) To determine the effect of GAPDH level on cell proliferation and cell cycle progression of human carcinoma cells; 2) To elucidate the molecular mechanism(s) causing proliferation arrest in GAPDH-depleted cells; 3) To identify the chemotherapeutic agents exhibiting cytotoxic effect against non-dividing, senescent cells; 4) To analyze molecular dynamics of nuclear GAPDH and its mutant variants in the context of chemotherapy-induced stress. Towards these aims, we developed an experimental model where the level of GAPDH in human carcinoma cells was modulated by RNA interference (RNAi) technology. In vitro experiments were performed in this model system to evaluate the energy status, and signaling pathways in cancer cells after GAPDH depletion. Human carcinoma isogenic cell lines with different levels of GAPDH protein were analyzed for the sensitivity to various chemotherapeutic agents. Using site-mutagenesis, we prepared mutated variants of GAPDH and estimated their enzymatic activity. We also prepared constructs where GAPDH cDNA was fused with green fluorescent protein (EGFP) cDNA, and transiently expressed them in human cancer cells, to assess GAPDH localization and biological effects. We analyzed intranuclear localization and dynamic characteristics of GAPDH and its variants in the live cells using image confocal technologies (e.g. FRAP). In our study, we demonstrated that GAPDH is a molecular target with clinical potential for senescence-based tumor suppression. Our experiments revealed that depletion of GAPDH induces energy crisis and proliferation arrest in human carcinoma cells. We elucidated the molecular mechanisms initiated by GAPDH depletion, and demonstrated that GAPDH-depleted cells acquire the accelerated senescence phenotype. Moreover, we found chemotherapeutic agents cytotoxic to the senescent cells, a finding that opens a way to combination chemotherapy with therapy-induced senescence agents. Our results on dynamic characteristics of intranuclear GAPDH and its mutant forms indicate that in the nucleus, GAPDH interacts with biomolecules yet to be identified. The results of this study suggest a novel, prospective molecular target for pharmacotherapeutic intervention in cancer management. / Pharmaceutical Sciences

Malaria accounts for 198 million cases worldwide; with a high mortality rate. 584000 deaths were reported in 2013. Malaria is a re-emerging disease globally due to drug resistance, parasite recrudescence and non-availability of a vaccine. Chloroquine, quinine and antifolates served as frontline antimalarial drugs for decades. Development of resistance to chloroquine and antifolates, and the decreased efficacy of mefloquine, and even quinine, in malaria-endemic regions, has led to artemisinin derivatives evolving as frontline drugs. Artemisinin is a potent antimalarial compound and clears around 104 parasites per cycle. Despite being a potent antimalarial, artemisinin derivatives suffer from poor pharmacokinetic properties and short half lives. This has led to the development of artemisinin-based combination therapies (ACTs) using a partner drug with a longer half-life. However, resistance to ACTs has been reported in the last few years, perhaps due to lack of adherence to prescribed regimens or suboptimal treatment and the use of counterfeit drugs. Therefore there is an urgent need to develop an alternative ACT which overcomes these limitations. This thesis entitled “Adjunct therapy with curcumin for the treatment of malaria: studies in a murine model” describes the antimalarial activity of curcumin and artemisinin and the adjunct role of curcumin in the prevention of parasite recrudescence and cerebral malaria. The thesis is divided into three chapters: The first chapter entitled “Introduction: Malaria and anti-malarial drugs” consists of a brief introduction of malaria, the parasite life cycle and currently known antimalarial drugs. During the course of infection, the Plasmodium undergoes sporogony in the mosquito, and merogony and schizogony in the human host. All these life cycle stages are briefly described with depictions. A major part of this chapter is dedicated to describe antimalarial compounds under the following headings 1. Quinoline derivatives 2. 4-aminoquinolines 3. Antifolates 4. Artemisinin derivatives 5. Antibiotics and 6. Curcumin. The second chapter is aimed at examining the ability of curcumin-arteether (a synthetic derivative of artemisinin) combination therapy in preventing parasite recrudescence in a murine model through immunomodulation employing various immunological, molecular biological, and biochemical techniques. The use of suboptimal doses of antimalarial drugs leads to recrudescence or relapse of malaria (reappearance of the parasite in blood after antimalarial regimen). In the present study we have addressed this issue by the use of curcumin as an adjunct molecule with α,β arteether (a synthetic derivative of artemisinin). We have studied recrudescence in a Swiss mice model. A suboptimal dose was standardized by the use of different doses of α,β arteether (AE) ranging from 250µg to 1500 µg. We found 750 µg to be a suboptimal dose and studied the adjunct nature of curcumin when animals were treated with AE suboptimal dose or AE+curcumin (AC) combination treatment and monitored the survival of animals. Our results clearly demonstrate that ~95% of animals treated with the suboptimal AE dose died of recrudescent malaria but there was almost 100% survival of AC-treated animals; these animals were under observation for at least 3 months. We have studied the effect of curcumin in a recrudescence model at the molecular level. Curcumin by itself has antimalarial activity, but only in combination with α,β arteether prevented recrudescence. Our results indicate that curcumin has immunomodulatory activity. Serum cytokine analysis and spleen mRNA analysis for proinflammatory and anti-inflammatory mediators indicate that AC treatment effectively reduced both mRNA and serum cytokine levels of IFNγ, TNFα, IL-12 and effectively increased both mRNA and serum levels IL-10 and antibodies of the IgG subclass. Using TLR2 and IL-10 knockout animals, we have conclusively demonstrated that TLR2 is involved in the production of IL-10, and IL-10 is required for the AC-mediated protection of animals during the recrudescence period. We conclude that curcumin is able to prevent parasite recrudescence essentially by switching the Th1 response to a Th2 response. The third chapter deals with the study the effect of areether-curcumin (AC) combination therapy in the prevention of Experimental Cerebral Malaria. Although malaria mortality rates have decreased by an impressive 47% between 2000 and 2013, it is still a major affliction of mankind (WHO 2014). Plasmodium falciparum infection causes human cerebral malaria (HCM). The mortality rate in HCM is unacceptably high (15–20%), despite the availability of artemisinin-based therapy. HCM is characterized by a rapid progression from headache, general malaise, and prostration to hemiparesis, ataxia, unrousable coma, and death. Paediatric HCM deaths are mostly due to respiratory arrest. Alternatively, death may be due to parasite-mediated injury to a sensitive location; a small lesion due to parasite in brain stem can cause sudden respiratory arrest. In HCM, cytoadherence of pRBCs in brain microvasculature has been implicated as a major contributing factor for CM pathology. The failure of a large number of adjunct therapies in HCM demands the development of new intervention strategies. An effective adjunct therapy is urgently needed. Experimental Cerebral Malaria (ECM) in mice manifests many of the neurological features of HCM. In this study, we have demonstrated the efficacy of curcumin and PLGA nanocurcumin in the treatment of Experimental Cerebral Malaria (ECM), using the Plasmodium berghei ANKA-infected mouse model (C57BL/6). Curcumin/PLGA nanocurcumin alone can prevent the onset of ECM. We have shown that curcumin/PLGA nanocurcumin can prevent CD8+ T cell, CXCR3+ CD8 T cell and parasite-infected RBC (pRBC) sequestration in the brain. These are also the essential parameters underlying HCM. We have also demonstrated that curcumin effectively inhibits T cell proliferation in spleen. We have explained the anti-inflammatory effects of curcumin by showing the inhibition of NF-B in both brain and spleen, which is a plausible explanation. But, curcumin/PLGA nanocurcumin treated animals died later due to build up of parasitemia in blood and subsequent anemia. Moreover, a combination therapy with arteether and curcumin given even after the onset of neurological symptoms can completely cure and protect the animals against mortality. We have tested AC-combination after the onset of symptoms to mimic patient conditions in HCM, since the murine regimens reported were not successful in the treatment of HCM. Our results clearly demonstrate that AC treatment even after the onset of symptoms ensures 100% survival. Since the bioavailability of curcumin is reported to be poor, we have also tested the efficacy of PLGA nanocurcumin and find that it is superior to native curcumin in terms of therapeutic effects. It is concluded that curcumin would be an ideal adjunct drug to be used with the artemisinin derivatives to treat malaria, including cerebral malaria.

Breast cancer is the most common malignancy diagnosed among women and is the first cause of neoplastic death in women globally. In the last decade our understanding of breast cancer biology has increased and led to the development of a number of targeted therapies, one of which is targeting the cell apoptosis pathway. One of the new targeting pathways under investigation, which was found to be involved in both cell apoptosis and cell proliferation processes, is the sphingolipid signalling pathway. The sphingolipid pathway represents a group of intracellular and extracellular bioactive lipid molecules, including ceramide, ceramide- 1-phosphate, sphingosine, and sphingosine-1-phosphate (S1P). In my research, I focused on the role S1P plays in breast cancer and its potential application as a therapeutic agent. I examined the effects of S1P on the apoptosis, proliferation, and cytotoxicity of different types of breast cancer cell lines in vitro. In addition, I evaluated the effect of both low and high doses of S1P when co-administrated with anticancer drugs commonly used in breast cancer treatment in vitro and in vivo. Moreover, I studied the S1P cellular distribution following exogenous administration. My results demonstrate that S1P can selectively induce apoptosis in breast cancer cells without harming normal breast cells and that S1P is more effective against aggressive breast cancer cells. Another major finding of my study is that S1P can increase the efficacy of chemotherapies against human breast cancer cells. Although S1P cannot directly substitute the current chemotherapies, S1P may function as a good candidate for combination therapy. Furthermore, my work showed that the pro-apoptotic and anti-proliferative effect of S1P is correlated with its intracellular action and that chronic exposure of exogenous S1P in vivo is not toxic to the major organs. Certainly, S1P inclusion in breast cancer treatment modalities may decrease the morbidity and mortality of breast cancer patients and improve clinical outcomes. Further investigations are required to understand the mechanism by which S1P induces apoptosis and inhibits cell proliferation.

Several wild-type poxviruses have emerged as potential oncolytic viruses (OVs), including orf virus (OrfV), and vaccinia virus (VV). Oncolytic VVs have been modified to include attenuating mutations that enhance their tumour selective nature, but these mutations also reduce overall viral fitness in cancer cells. Previous studies have shown that a VV (Western Reserve) with its E3L gene replaced with the E3L homologue from, OrfV (designated VV-E3LOrfV), maintained its ability to infect cells in vitro, but was attenuated compared to its parental VV in vivo. Our goal was to determine the safety and oncolytic potential VV-E3LOrfV, compared to wild type VV and other attenuated recombinants. VV-E3LOrfV, was unable to replicate to the same titers and was sensitive to IFN compared to its parental virus and other attenuated VVs in normal human fibroblast cells. The virus was also less pathogenic when administered in vivo. Viral replication, spread and cell killing, as measures of oncolytic potential in vitro, along with in vivo efficacy, were also observed.. The Parapoxvirus, OrfV has been shown to have a unique immune-stimulation profile, inducing a number of pro-inflammatory cytokines, as well as potently recruiting and activating a number of immune cells. Despite this unique profile, OrfV is limited in its ability to replicate and spread in human cancer cells. Various strategies were employed to enhance the oncolytic efficacy of wild-type OrfV. A transient transfection/infection screen was created to determine if any of the VV host-range genes (C7L, K1L, E3L or K3L) would augment OrfV oncolysis. Combination therapy, including the use of microtubule targeting agents, Viral Sensitizer (VSe) compounds and the addition of soluble VV B18R gene product were employed to see if they also enhance OrfV efficacy. Unfortunately, none of the strategies mentioned were able to enhance OrfV.

A.baumannii é um importante patógeno em infecções nosocomiais principalmente por sua capacidade de se tornar resistente aos antimicrobianos. Surtos de A.baumannii resistente aos carbapenêmicos (ABRC) têm sido descritos em todo mundo. Devido à emergência de resistência aos antimicrobianos e ausência de novas opções de tratamento, as polimixinas reemergiram como opção de terapia contra infecções causadas por A.baumannii. O uso de polimixina é associado a maior mortalidade e menor eficácia comparada a outros antimicrobianos. Alguns estudos in vitro têm avaliado a combinação de polimixina com outros antimicrobianos a fim de aumentar a eficácia dos tratamentos. O objetivo deste estudo foi avaliar o sinergismo entre a polimixina B com outros antimicrobianos em isolados de ABRC, pelo método de Curvas Tempo-Morte bacteriana (Time- Kill Curves). Os isolados foram provenientes de banco de amostras e foram avaliadas as combinações de polimixina B com carbapenêmicos (imipenem e meropenem), tigeciclina, rifampicina, amicacina e ceftazidima. As combinações foram testadas nos tempo 0, 30’, 1,4,12 e 24 h. Sinergismo entre polimixina B foi demonstrado contra todos antimicrobianos para ambos isolados, exceto para ceftazidima e imipenem no isolado 1. Nosso estudo mostrou que tigeciclina, amicacina e rifampicina são agentes mais ativos combinados com polimixina B, sendo assim estes agentes podem apresentar efeito benéfico em combinação com a polimixina no tratamento de ABRC. / A.baumannii is an important pathogen in nosocomial infections primarily for its ability to become resistant to antimicrobials. Outbreaks carbapenem- resistant A.baumannii (CRAB) has been described worldwide. Due to the emergence of antimicrobial resistance and the absence of new treatment options, the polymyxins reemerged as an option therapy against infections caused by A.baumannii. The use of polymyxin is associated with higher mortality and lower effectiveness compared to other antimicrobials. In vitro studies have evaluated the combination of polymyxin with other antimicrobial agents to enhance the effectiveness of the treatments. This study was to evaluate the synergy between polymyxin B with other antimicrobials in isolates from ABRC, by Time-Kill Curves. The isolates were from stool samples and were evaluated combinations of polymyxin B with carbapenems (imipenem and meropenem), tigecycline, rifampin, amikacin and ceftazidime. The combinations were tested at time 0, 30 ', 1,4,12 and 24 h. Synergism between polymyxin B was demonstrated against all antimicrobials for both isolates, except for ceftazidime and imipenem in isolated 2. Our study showed that tigecycline, amikacin and rifampicin more active agents are combined with polymyxin B, and thus these agents may have a beneficial effect in combination with a polymyxin in treating CRAB.

<p>The role of platelet-derived growth factor (PDGF) in malignancies involves both autocrine and paracrine stimulation of cells within the tumor. The interstitial fluid pressure (IFP) is one of the forces that govern the transvascular flow of fluids. In both experimental and clinical cancers, the IFP is elevated and is thought to act as a barrier for delivery of drugs. Increasing evidence points to PDGF as a positive regulator of the interstitial fluid pressure in loose connective tissue. In this thesis, the effect of PDGF receptor inhibition on the tumor IFP, transvascular transport and efficacy of anti-cancer drugs is investigated.</p><p>All studies were performed using tumor models that display extensive tumor stroma and PDGF receptor expression restricted to stroma cells. Blocking of PDGF receptor signaling significantly reduced the tumor IFP in various tumor models. In parallel, pre-treatment with PDGF antagonists increased the tumor content of cytotoxic agents without affecting the uptake in other organs. Moreover, combination treatment with PDGF receptor inhibitors and chemotherapeutic agents dramatically enhanced the anti-tumor effects of the cytotoxic drugs, whereas treatment with only PDGF receptor inhibitors did not affect the growth of the tumors. Beneficial effects on the tumor reponse to radioimmunotherapy were also produced after concomitant administration of PDGF antagonists. Importantly, anti-angiogenic effects, changes in cell composition and increased tumor cell sensitivity to cytotoxic agents were ruled out as the cause for the synergistic effects. </p><p>Studies with different temporal scheduling of PDGF receptor inhibitors demonstrated a perfect correlation between a reduced IFP, an increased transvascular transport and an enhanced therapeutic effect of cytotoxic drugs, strongly suggesting that the phenomena are causally linked.</p><p>The studies presented herein illustrate for the first time the potential of cells in the stroma compartment as a target for efforts to treat cancer. In conclusion, a novel, possibly general, strategy to enhance the effects of conventional anti-cancer drugs has been identified.</p>

The role of platelet-derived growth factor (PDGF) in malignancies involves both autocrine and paracrine stimulation of cells within the tumor. The interstitial fluid pressure (IFP) is one of the forces that govern the transvascular flow of fluids. In both experimental and clinical cancers, the IFP is elevated and is thought to act as a barrier for delivery of drugs. Increasing evidence points to PDGF as a positive regulator of the interstitial fluid pressure in loose connective tissue. In this thesis, the effect of PDGF receptor inhibition on the tumor IFP, transvascular transport and efficacy of anti-cancer drugs is investigated. All studies were performed using tumor models that display extensive tumor stroma and PDGF receptor expression restricted to stroma cells. Blocking of PDGF receptor signaling significantly reduced the tumor IFP in various tumor models. In parallel, pre-treatment with PDGF antagonists increased the tumor content of cytotoxic agents without affecting the uptake in other organs. Moreover, combination treatment with PDGF receptor inhibitors and chemotherapeutic agents dramatically enhanced the anti-tumor effects of the cytotoxic drugs, whereas treatment with only PDGF receptor inhibitors did not affect the growth of the tumors. Beneficial effects on the tumor reponse to radioimmunotherapy were also produced after concomitant administration of PDGF antagonists. Importantly, anti-angiogenic effects, changes in cell composition and increased tumor cell sensitivity to cytotoxic agents were ruled out as the cause for the synergistic effects. Studies with different temporal scheduling of PDGF receptor inhibitors demonstrated a perfect correlation between a reduced IFP, an increased transvascular transport and an enhanced therapeutic effect of cytotoxic drugs, strongly suggesting that the phenomena are causally linked. The studies presented herein illustrate for the first time the potential of cells in the stroma compartment as a target for efforts to treat cancer. In conclusion, a novel, possibly general, strategy to enhance the effects of conventional anti-cancer drugs has been identified.