Clement Penny

Overview of the role of nanotechnological innovations in the detection and treatment of solid tumors.

Nanotechnology, although still in its infantile stages, has the potential to revolutionize the diagnosis, treatment, and monitoring of disease progression and success of therapy for numerous diseases and conditions, not least of which is cancer. As it is a leading cause of mortality worldwide, early cancer detection, as well as safe and efficacious therapeutic intervention, will be indispensable in improving the prognosis related to cancers and overall survival rate, as well as health-related quality of life of patients diagnosed with cancer. The development of a relatively new field of nanomedicine, which combines various domains and technologies including nanotechnology, medicine, biology, pharmacology, mathematics, physics, and chemistry, has yielded different approaches to addressing these challenges. Of particular relevance in cancer, nanosystems have shown appreciable success in the realm of diagnosis and treatment. Characteristics attributable to these systems on account of the nanoscale size range allow for individualization of therapy, passive targeting, the attachment of targeting moieties for more specific targeting, minimally invasive procedures, and real-time imaging and monitoring of in vivo processes. Furthermore, incorporation into nanosystems may have the potential to reintroduce into clinical practice drugs that are no longer used because of various shortfalls, as well as aid in the registration of new, potent drugs with suboptimal pharmacokinetic profiles. Research into the development of nanosystems for cancer diagnosis and therapy is thus a rapidly emerging and viable field of study.

In Vitro Inhibition of Angiogenesis by Antibodies Directed against the 37kDa/67kDa Laminin Receptor

The 37kDa/67kDa laminin receptor (LRP/LR) is a central receptor mediating interactions between tumour cells and the basement membrane and is thereby a key player in adhesion and invasion, essential processes in metastatic cancer. To affect continued tumour growth, tumours induce angiogenesis for the constant delivery of nutrients and oxygen. This study aims to determine the blocking effect of the anti-LRP/LR specific antibody, W3 on the angiogenic potential of HUVE (human umbilical vein endothelial) cells. Flow cytometric analysis revealed that 97% of HUVE cells display cell surface LRP/LR. An angiogenesis assay was conducted employing HUVE cells seeded on the basement membrane reconstituent Matrigel™ supplemented with the pro-angiogenic factor vascular endothelial growth factor (VEGF). Post 18h incubation at 37°C tubular structures, namely tube lengths were assessed. Treatment of established tubular structures with 100 µg/ml anti-LRP/LR specific antibody completely blocked angiogenesis. Our findings suggest a central role of the 37kDa/67kDa LRP/LR in tube formation and recommends anti-LRP/LR specific antibodies as potential therapeutic tools for treatment of tumour angiogenesis.

Anti-LRP/LR specific antibodies and shRNAs impede amyloid beta shedding in Alzheimer's disease

Alzheimers disease (AD) is the most prevalent form of dementia. The amyloid beta (Aβ) peptide is the predominant candidate aetiological agent and is generated through the sequential proteolytic cleavage of the Amyloid Precursor Protein (APP) by beta (β) and gamma (γ) secretases. Since the cellular prion protein (PrPc) has been shown to regulate Aβ shedding, we investigated whether the cellular receptor for PrPc, namely the 37 kDa/67 kDa Laminin Receptor (LRP/LR) played a role in Aβ shedding. Here we show that LRP/LR co-localises with the AD relevant proteins APP, β- and γ-secretase, respectively. Antibody blockage and shRNA knock-down of LRP/LR reduces Aβ shedding, due to impediment of β-secretase activity, rather than alteration of APP, β- and γ-secretase levels. These findings indicate that LRP/LR contributes to Aβ shedding and recommend anti-LRP/LR specific antibodies and shRNAs as novel therapeutic tools for AD treatment.

Recent advances in the design of drug-loaded polymeric implants for the treatment of solid tumors

Introduction: The effective treatment of solid tumors continues to be a great challenge to clinicians, despite the development of novel drugs. In order to improve the clinical efficacy of existing chemotherapeutic agents, researchers have considered the possibility of site-specific solid tumor treatment. The greatest advantage of localized delivery is the significantly fewer side effects experienced by patients. Recently, in situ forming implants have attracted considerable interest. These polymeric systems are injected as solutions into tumor sites and the injected solution forms an implant as a result of local environmental stimuli and hence removes the need for surgical implantation. Areas covered: This review summarizes the attempts that have been made to date in the development of polymeric implants for the treatment of solid tumors. Both in situ forming implants and preformed implants, fabricated using natural and synthetic polymers, are described. In addition, the peri- or intra-tumoral delivery of chemotherapeutic agents based on implants inserted surgically into the affected region is also discussed along with a short coverage of implants having an undesirable initial burst release effect. Expert opinion: Although these implants have been shown to improve the treatment of various solid tumors, the ideal implant that is able to deliver high doses of chemotherapeutics to the tumor site, over prolonged periods with relatively few side effects on normal tissue, is yet to be formulated.

High resolution imaging study of interactions between the 37 kDa/67 kDa Laminin receptor and APP, beta-secretase and gamma-secretase in Alzheimer’s Disease

Alzheimers disease (AD) is the most prevalent form of dementia affecting the elderly. Neurodegeneration is caused by the amyloid beta (Aβ) peptide which is generated from the sequential proteolytic cleavage of the Amyloid Precursor Protein (APP) by the β– and γ- secretases. Previous reports revealed that the 37 kDa/67 kDa laminin receptor (LRP/LR) is involved in APP processing, however, the exact mechanism by which this occurs remains largely unclear. This study sought to assess whether LRP/LR interacted with APP, β- or γ-secretase. Detailed confocal microscopy revealed that LRP/LR showed a strong co-localisation with APP, β- and γ-secretase, respectively, at various sub-cellular locations. Superresolution Structured Illumination Microscopy (SR-SIM) showed that interactions were unlikely between LRP/LR and APP and β-secretase, respectively, while there was strong co-localisation between LRP/LR and γ-secretase at this 80 nm resolution. FRET was further employed to assess the possibility of protein-protein interactions and only an interaction between LRP/LR and γ-secretase was found. FLAG co-immunoprecipitation confirmed these findings as LRP/LR co-immunoprecipitated with γ-secretase, but failed to do so with APP. These findings indicate that LRP/LR exerts its influence on Aβ shedding via a direct interaction with the γ-secretase and possibly an indirect interaction with the β-secretase.

Anti-LRP/LR specific antibody IgG1-iS18 and knock-down of LRP/LR by shRNAs rescue cells from Aβ42 induced cytotoxicity

Alzheimers disease (AD) is characterized by neurofibrillary tangles, senile plaques and neuronal loss. Amyloid beta (Aβ) is proposed to elicit neuronal loss through cell surface receptors. As Aβ shares common binding partners with the 37 kDa/67 kDa laminin receptor (LRP/LR), we investigated whether these proteins interact and the pathological significance of this association. An LRP/LR-Αβ42 interaction was assessed by immunofluorescence microscopy and pull down assays. The cell biological effects were investigated by 3-(4,5-Dimethylthaizol-2-yl)-2,5-diphenyltetrazolium bromide and Bromodeoxyuridine assays. LRP/LR and Αβ42 co-localised on the cell surface and formed immobilized complexes suggesting an interaction. Antibody blockade by IgG1-iS18 and shRNA mediated down regulation of LRP/LR significantly enhanced cell viability and proliferation in cells co-treated with Αβ42 when compared to cells incubated with Αβ42 only. Results suggest that LRP/LR is implicated in Αβ42 mediated cytotoxicity and that anti-LRP/LR specific antibodies and shRNAs may serve as potential therapeutic tools for AD.

A Novel Salted-out and Subsequently Crosslinked Poly(Lactic-co-Glycolic Acid) Polymeric Scaffold Applied to Monolithic Drug Delivery

This study involved a statistical approach to develop a mechanistic understanding of the salting-out of poly(lactic-co-glycolic acid) (PLGA) and to evaluate the capacity to modulate the physicochemical and physicomechanical properties of PLGA by incorporating electrolytes that produce stochastic fluctuations. The correlation between the three types of salts used and the extent of PLGA chain transitions were established by structural-thermal analysis. Drug-loaded monolithic matrices are prepared by direct compressing salted-out PLGA and a model drug (melatonin). PLGA scaffolds possess fiber diameters and volumes ranging between 0.1—15 μm and 0.0075—14,000 μm3 , respectively. Texture profile analysis reveal a significant increase in the energy absorbed and matrix resilience with increased NaCl2 and AlCl3 concentrations. In vitro drug release studies were performed in phosphate buffered saline (pH 7.4; 37°C); the release media was sampled at pre-determined intervals and analyzed by UV spectroscopy. Ideal zero-order drug release profiles were observed with 20% melatonin over a 30-day period. Monolithic matrices prepared by crosslinking melatonin with PLGA reveal a superior capability to control drug release. The salting-out and subsequent crosslinking of PLGA significantly modified the physicochemical and physicomechanical properties of native PLGA and demonstrated the ability to achieve controlled drug release.

Lipid accumulation and alkaline phosphatase activity in human preadipocytes isolated from different body fat depots

Abstract Background: Alkaline phosphatase (ALP) controls intracellular lipid accumulation in human preadipocytes, but it is not known whether ALP is expressed in all body fat depots, or whether it has a similar role at all sites. Design: Cross-sectional. Setting and subjects: Subjects undergoing breast reduction and abdominal fat biopsies operations at Charlotte Maxeke Johannesburg Academic Hospital. Outcome measures: This study compared intracellular lipid accumulation and ALP activity in the presence and absence of ALP inhibitors in preadipocytes that were obtained from different adipose depots. Abdominal and mammary gland preadipocytes were isolated from women and induced to differentiate in culture. ALP activity and intracellular lipid levels were measured at baseline and after 12 days of differentiation in the presence and absence of the ALP inhibitors, histidine and levamisole. Results: ALP activity was detected in nondifferentiated abdominal (134 ± 7.5 mU/mg protein) and mammary gland (136 ± 9.6 mU/mg protein) preadipocytes. Its activity had increased significantly (p-value < 0.0005 for both) by day 12 of differentiation (388 ± 55 for abdominal and 278 ± 28 mU/mg protein for mammary). Preadipocytes treated with histidine had lower fat accumulation (p-value < 0.0005) and ALP activity (p-value < 0.005) than nontreated cells on day 12, while those treated with levamisole had lower fat accumulation (p-value < 0.005), but elevated ALP activity (p-value < 0.05), compared to nontreated cells. Lipid accumulation (p-value < 0.005) and ALP activity (p-value < 0.05) were higher in abdominal than mammary gland preadipocytes by day 12. Conclusion: ALP is involved in the control of intracellular lipid accumulation in human preadipocytes that are isolated from both adipose depots. The ability of levamisole to inhibit this process while activating ALP, suggests that this molecule acts via an ALP-independent pathway, while histidine attenuates both lipid deposition and ALP activity.

Aberrant Apoptotic Response of Colorectal Cancer Cells to Novel Nucleoside Analogues

Despite the increased understanding of colorectal cancer and the introduction of targeted drug therapy, the metastatic phase of the disease remains refractory to treatment. Since the deregulation of normal apoptosis contributes to the pathogenesis of colorectal cancer, novel nucleoside analogues were synthesized here and evaluated for their ability to induce apoptosis and cause cell death in two colorectal adeno-carcinoma cell lines, Caco-2 and HT-29. Three novel nucleoside analogues assessed here showed cytotoxic activity, as measured by the MTT assay against both cell lines: the IC50 values ranged between 3 and 37 μM, with Caco-2 cells being more sensitive than HT-29 cells. Compared to camptothecin, the positive control, the nucleoside analogues were significantly less toxic to normal unstimulated leukocytes (p>0.05). Moreover, the nucleosides were able to induce apoptosis as measured by an increase in caspase 8 and caspase 3 activity above that of the control. This was additionally supported by data derived from Annexin V-FITC assays. Despite marginal changes to the mitochondrial membrane potential, all three nucleosides caused a significant increase in cytosolic cytochrome c (p>0.05), with a corresponding decrease in mitochondrial cytochrome c. Morphological analysis of both cell lines showed the rapid appearance of vacuoles following exposure to two of the nucleosides, while a third caused cellular detachment, delayed cytoplasmic vacuolisation and nuclear abnormalities. Preliminary investigations, using the autophagic indicator monodansylcadaverine and chloroquine as positive control, showed that two of the nucleosides induced the formation of autophagic vacuoles. In summary, the novel nucleoside analogues showed selective cytotoxicity towards both cancer cell lines and are effective initiators of an unusual apoptotic response, demonstrating their potential to serve as structural scaffolds for more potent analogues.

CD133/EpCAM Cancer Stem Cell Markers of Tumour Stage in Colorectal Cancer Cells

In solid tumours, a discreet population of tumour associated cancer stem cells (CSCs) are proposed to drive and sustain tumour development and be responsible for tumour relapse. Colorectal cancer stem cells express cellspecific surface markers, including amongst others, CD133, EpCAM, CD44, CD166, and CD94f. In the present study, we aimed to characterisecellpopulations in the human colon adenocarcinoma cell lines, SW1116, HT29 and DLD1, expressing both CSC markers CD133 and EpCAM. These cell lines represent early, mid and late stages of colorectal tumours, respectively. Up to 107 SW1116, HT29 and DLD1 cells, co-stained with anti-CD133 and anti-EpCAM, were evaluated using flow cytometry. We report here progressive increasing proportions of cells coexpressing the CD133/EpCAM epitopes in the respective cell lines. In the SW1116 cell line, 2.42 ± 0.20 percent of cells were CD133+EpCAM+, in the HT29 cell line, 5.13 ± 0.17 percent of cells were CD133+EpCAM+, and in the DLD1 cell line, 10.30 ± 0.2 percent of cells were CD133+EpCAM+. These data suggest the frequency of CD133/ EpCAM marker expression may be associated with tumour stage and aggression.