Kevin Dzobo

The Role of Tumor Microenvironment in Chemoresistance: To Survive, Keep Your Enemies Closer

Chemoresistance is a leading cause of morbidity and mortality in cancer and it continues to be a challenge in cancer treatment. Chemoresistance is influenced by genetic and epigenetic alterations which affect drug uptake, metabolism and export of drugs at the cellular levels. While most research has focused on tumor cell autonomous mechanisms of chemoresistance, the tumor microenvironment has emerged as a key player in the development of chemoresistance and in malignant progression, thereby influencing the development of novel therapies in clinical oncology. It is not surprising that the study of the tumor microenvironment is now considered to be as important as the study of tumor cells. Recent advances in technological and analytical methods, especially ‘omics’ technologies, has made it possible to identify specific targets in tumor cells and within the tumor microenvironment to eradicate cancer. Tumors need constant support from previously ‘unsupportive’ microenvironments. Novel therapeutic strategies that inhibit such microenvironmental support to tumor cells would reduce chemoresistance and tumor relapse. Such strategies can target stromal cells, proteins released by stromal cells and non-cellular components such as the extracellular matrix (ECM) within the tumor microenvironment. Novel in vitro tumor biology models that recapitulate the in vivo tumor microenvironment such as multicellular tumor spheroids, biomimetic scaffolds and tumor organoids are being developed and are increasing our understanding of cancer cell-microenvironment interactions. This review offers an analysis of recent developments on the role of the tumor microenvironment in the development of chemoresistance and the strategies to overcome microenvironment-mediated chemoresistance. We propose a systematic analysis of the relationship between tumor cells and their respective tumor microenvironments and our data show that, to survive, cancer cells interact closely with tumor microenvironment components such as mesenchymal stem cells and the extracellular matrix.

Pharmacogenomics Implications of Using Herbal Medicinal Plants on African Populations in Health Transition

The most accessible points of call for most African populations with respect to primary health care are traditional health systems that include spiritual, religious, and herbal medicine. This review focusses only on the use of herbal medicines. Most African people accept herbal medicines as generally safe with no serious adverse effects. However, the overlap between conventional medicine and herbal medicine is a reality among countries in health systems transition. Patients often simultaneously seek treatment from both conventional and traditional health systems for the same condition. Commonly encountered conditions/diseases include malaria, HIV/AIDS, hypertension, tuberculosis, and bleeding disorders. It is therefore imperative to understand the modes of interaction between different drugs from conventional and traditional health care systems when used in treatment combinations. Both conventional and traditional drug entities are metabolized by the same enzyme systems in the human body, resulting in both pharmacokinetics and pharmacodynamics interactions, whose properties remain unknown/unquantified. Thus, it is important that profiles of interaction between different herbal and conventional medicines be evaluated. This review evaluates herbal and conventional drugs in a few African countries and their potential interaction at the pharmacogenomics level.

Inhibition of CYP2B6 by Medicinal Plant Extracts: Implication for Use of Efavirenz and Nevirapine-Based Highly Active Anti-Retroviral Therapy (HAART) in Resource-Limited Settings

Highly active antiretroviral therapy (HAART) has greatly improved health parameters of HIV infected individuals. However, there are several challenges associated with the chronic nature of HAART administration. For populations in health transition, dual use of medicinal plant extracts and conventional medicine poses a significant challenge. There is need to evaluate interactions between commonly used medicinal plant extracts and antiretroviral drugs used against HIV/AIDS. Efavirenz (EFV) and nevirapine (NVP) are the major components of HAART both metabolized by CYP2B6, an enzyme that can potentially be inhibited or induced by compounds found in medicinal plant extracts. The purpose of this study was to evaluate the effects of extracts of selected commonly used medicinal plants on CYP2B6 enzyme activity. Recombinant human CYP2B6 was used to evaluate inhibition, allowing the assessment of herb-drug interactions (HDI) of medicinal plants Hyptis suaveolens, Myrothamnus flabellifolius, Launaea taraxacifolia, Boerhavia diffusa and Newbouldia laevis. The potential of these medicinal extracts to cause HDI was ranked accordingly for reversible inhibition and also classified as potential time-dependent inhibitor (TDI) candidates. The most potent inhibitor for CYP2B6 was Hyptis suaveolens extract (IC50 = 19.09 ± 1.16 µg/mL), followed by Myrothamnus flabellifolius extract (IC50 = 23.66 ± 4.86 µg/mL), Launaea taraxacifolia extract (IC50 = 33.87 ± 1.54 µg/mL), and Boerhavia diffusa extract (IC50 = 34.93 ± 1.06 µg/mL). Newbouldia laevis extract, however, exhibited weak inhibitory effects (IC50 = 100 ± 8.71 µg/mL) on CYP2B6. Launaea taraxacifolia exhibited a TDI (3.17) effect on CYP2B6 and showed a high concentration of known CYP450 inhibitory phenolic compounds, chlorogenic acid and caffeic acid. The implication for these observations is that drugs that are metabolized by CYP2B6 when co-administered with these herbal medicines and when adequate amounts of the extracts reach the liver, there is a high likelihood of standard doses affecting drug plasma concentrations which could lead to toxicity.

The garlic compound ajoene targets protein folding in the endoplasmic reticulum of cancer cells

Ajoene is a natural allylsulfur compound found in crushed garlic that arrests growth and induces apoptosis in cancer cells. To gain mechanistic insights into the cytotoxicity of ajoene in cancer cells, two fluorescently labelled ajoene analogs with dansyl‐ (DP) and fluorescein‐ (FOX) tags were synthesized. The tagged ajoenes were found to retain their activity at inhibiting proliferation and inducing apoptosis in MDA‐MB‐231 human breast‐cancer and WHCO1 human esophageal‐cancer cells. Both tagged ajoenes localized to the endoplasmic reticulum (ER) in MDA‐MB‐231 cells as observed by live cell confocal laser scanning microscopy (CLSM) and confirmed by generating an MDA‐MB‐231 cell line expressing yellow fluorescent protein (YFP) in the ER. DP appears to S‐thiolate multiple protein targets in MDA‐MB‐231 cells as observed by immunoblotting under non‐reducing conditions only; and a competition assay demonstrated that DP and Z‐ajoene in fact share the same target. Ajoene S‐thiolation interfered with protein folding and led to an accumulation of misfolded protein aggregates and activated the unfolded protein response (UPR). Consistent with this mechanism, increased levels of GRP78 and total ubiquitinated proteins were observed; and an ER‐folded protein, type‐1 collagen, was tracked to the proteasome following ajoene treatment. The intracellular protein aggregates were observed by CLSM and transmission electron microscopy (TEM). This is the first time that ajoene has been shown to target protein folding in the ER of cancer cells.

Wnt/β-Catenin and MEK-ERK Signaling are Required for Fibroblast-Derived Extracellular Matrix-Mediated Endoderm Differentiation of Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the potential to differentiate into all cells of the three germ layers, thus making them an attractive source of cells for use in regenerative medicine. The greatest challenge lies in regulating the differentiation of hESCs into specific cell lineages by both intrinsic and extrinsic factors. In this study we determined the effect of a fibroblast-derived extracellular matrix (fd-ECM) on hESCs differentiation. We demonstrate that growth of hESCs on fd-ECM results in hESCs losing their stemness and proliferation potential. As the stem cells differentiate they attain gene expression profiles similar to the primitive streak of the in vivo embryo. The activation of both the MEK-ERK and Wnt/β-catenin signaling pathways is required for the fd-ECM-mediated differentiation of hESCs towards the endoderm and involves integrins α1, α2, α3 and β1. This study illustrates the importance of the cellular microenvironment in directing stem cell fate and that the nature and composition of the extracellular matrix is a crucial determining factor.

Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro

Mesenchymal stromal/stem cells (MSCs) represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell–matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs) in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM) did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4), SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

Wharton’s Jelly-Derived Mesenchymal Stromal Cells and Fibroblast-Derived Extracellular Matrix Synergistically Activate Apoptosis in a p21-Dependent Mechanism in WHCO1 and MDA MB 231 Cancer Cells In Vitro

The tumour microenvironment plays a crucial role in tumour progression and comprises tumour stroma which is made up of different cell types and the extracellular matrix (ECM). Mesenchymal stromal cells (MSCs) are part of the tumour stroma and may have conflicting effects on tumour growth. In this study we investigated the effect of Whartons Jelly-derived MSCs (WJ-MSCs) and a fibroblast-derived ECM (fd-ECM) on esophageal (WHCO1) and breast (MDA MB 231) cancer cells in vitro. Both WJ-MSCs and the fd-ECM, alone or in combination, downregulate PCNA, cyclin D1, Bcl-2, Bcl-xL, and MMPs and upregulate p53 and p21. p21 induction resulted in G2 phase cell cycle arrest and induced apoptosis in vitro. Our data suggest that p21 induction is via p53-dependent and p53-independent mechanisms in WHCO1 and MDA MB 231 cells, respectively. Vascular endothelial growth factor, Akt, and Nodal pathways were downregulated in cancer cells cocultured with WJ-MSCs. We also demonstrate that WJ-MSCs effects on cancer cells appear to be short-lived whilst the fd-ECM effect is long-lived. This study shows the influence of tumour microenvironment on cancer cell behaviour and provides alternative therapeutic targets for potential regulation of tumour cells.

Patient and tumour characteristics as prognostic markers for oesophageal cancer: a retrospective analysis of a cohort of patients at Groote Schuur Hospital

OBJECTIVES In addition to the high incidence of squamous carcinoma of the oesophagus among South African men, the neoplasm is also characterized by an exceptionally latent course and poor prognosis. The aim of this study was to review a cohort of patients with carcinoma of the oesophagus presenting to the Groote Schuur Hospital, Cape Town and evaluate patient and tumour characteristics for their role as prognostic markers for survival. METHODS Information on patients was extracted from a database established and maintained over a 30-year period. Information for the analysis included patient demographics, clinical symptoms at presentation, tumour characteristics and treatment decisions. Statistical analyses were performed using GraphPad Prism 5 applying chi-square and Kaplan-Meier tests. RESULTS Data were available on 1868 patients. The majority of patients were Black African men and the predominant histology was squamous cell carcinoma. There were significant differences (P < 0.05) in the survival of patients with respect to race (P < 0.001), performance status (P < 0.001), weight loss (P = 0.001) and prior tuberculosis diagnosis (P = 0.007). Tumour characteristics that were significantly associated with survival were histological type, tumour size and site. Gender, prior cancer, smoking status and tumour-related pain did not show significant association with survival in patients with oesophageal cancer. Only 19.8% of the patients were candidates for potentially curative treatment. CONCLUSIONS This analysis shows that there are prominent patient and tumour characteristics that are significantly associated with survival with respect to oesophageal carcinoma. The inclusion of these factors in the initial assessment of patients may assist with appropriate treatment decisions.

Feedback regulation of the α2(1) collagen gene via the Mek–Erk signaling pathway

The extracellular matrix (ECM) provides the microenvironment that is pivotal for cell growth, motility, attachment, and differentiation. Advances in cell culture techniques have led to the development of cell‐derived ECM model systems that are more reflective of the in vivo architecture of the ECM in tissue. In this study, a fibroblast‐derived ECM (fd‐ECM) was used to study the feedback regulation of type I collagen synthesis in fibroblasts. Fibroblasts plated on a preformed fd‐ECM showed a significant decrease in the production of type I collagen and pro‐α2(1) collagen mRNA compared to cells grown in the absence of a matrix. Function‐blocking antibodies showed that this downregulation of type I collagen gene expression is mediated via α2β1 integrin. The use of several kinase inhibitors and a dominant negative ras construct (N17Ras) showed that the matrix‐mediated downregulation of COL1A2 occurs via Ras‐dependent activation of the MEK/ERK signaling pathway. Deletion analysis of the COL1A2 promoter implicated the region between −375 and −107 as containing a potential matrix responsive element. The use of Sp1 siRNA demonstrated that Sp1 is an important mediator of this feedback inhibition. This study provides some new insights into the feedback regulation of COL1A2 gene expression.

Absence of feedback regulation in the synthesis of COL1A1.

AIM Recent studies have emphasized the importance of the extracellular microenvironment in modulating cell growth, motility, and signalling. In this study we have evaluated the ability of a fibroblast derived-extracellular matrix (fd-ECM) to regulate type I collagen synthesis and degradation in fibroblasts. MAIN METHODS Fibroblasts were plated on plastic (control) or on fd-ECM and type I collagen synthesis and degradation was evaluated. MTT, western blotting, real time PCR, zymographic analysis and inhibitor assays were utilised to investigate the molecular mechanism of type I collagen regulation by the fd-ECM. KEY FINDINGS Fibroblasts plated on fd-ECM showed significant downregulation in the production of type I collagen and COL1A2 messenger ribonucleic acid (mRNA) whilst COL1A1 mRNA remained unchanged. Cells grown on fd-ECM exhibited increased matrix metalloproteases (MMPs) and their corresponding mRNAs. The use of transforming growth factor β (TGF-β) and MMP inhibitors showed that the excess COL1A1 polypeptide chains were degraded by the combined action of MMP-1, MMP-2, MMP-9 and cathepsins. SIGNIFICANCE These results show the crucial role played by proteases in regulating extracellular matrix protein levels in the feedback regulation of connective tissue gene expression.