In vitro effects and mathematical modelling of CTCE-9908 (a chemokine receptor 4 antagonist) on melanoma cell survival.

CTCE-9908, a CXC chemokine receptor 4 (CXCR4) antagonist, prevents CXCR4 phosphorylation and inhibits the interaction with chemokine ligand 12 (CXCL12) and downstream signalling pathways associated with metastasis. This study evaluated the in vitro effects of CTCE-9908 on B16 F10 melanoma cells with the use of mathematical modelling. Crystal violet staining was used to construct a mathematical model of CTCE-9908 B16 F10 (melanoma) and RAW 264.7 (non-cancerous macrophage) cell lines on cell viability to predict the half-maximal inhibitory concentration (IC50). Morphological changes were assessed using transmission electron microscopy. Flow cytometry was used to assess changes in cell cycle distribution, apoptosis via caspase-3, cell survival via extracellular signal-regulated kinase1/2 activation, CXCR4 activation and CXCL12 expression. Mathematical modelling predicted IC50 values from 0 to 100 h. At IC50, similar cytotoxicity between the two cell lines and ultrastructural morphological changes indicative of cell death were observed. At a concentration 10 times lower than IC50, CTCE-9908 induced inhibition of cell survival (p = 0.0133) in B16 F10 cells but did not affect caspase-3 or cell cycle distribution in either cell line. This study predicts CTCE-9908 IC50 values at various time points using mathematical modelling, revealing cytotoxicity in melanoma and non-cancerous cells. CTCE-9908 significantly inhibited melanoma cell survival at a concentration 10 times lower than the IC50 in B16 F10 cells but not RAW 264.7 cells. However, CTCE-9908 did not affect CXCR4 phosphorylation, apoptosis,\ or cell cycle distribution in either cell line.

An exploratory study on the effect of kynurenine metabolites on sEnd-2 endothelioma cells.

Cancer is the second leading cause of mortality worldwide. The development of anticancer therapy plays a crucial role in mitigating tumour progression and metastasis. Epithelioid hemangioendothelioma is a very rare cancer, however, with a high systemic involvement. Kynurenine metabolites which include l-kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic acid and quinolinic acid have been shown to inhibit T-cell proliferation resulting in a decrease in cell growth of natural killer cells and T cells. Furthermore, metabolites such as  l-kynurenine have been shown to inhibit proliferation of melanoma cells in vitro. Considering these metabolite properties, the present study aimed to explore the in vitro effects of  l-kynurenine, quinolinic acid and kynurenic acid on endothelioma sEnd-2 cells and on endothelial (EA. hy926 cells) (control cell line). The in vitro effect at 24, 48, and 72 h exposure to a range of 1-4 mM of the respective kynurenine metabolites on the two cell lines in terms of cell morphology, cell cycle progression and induction of apoptosis was assessed. The half inhibitory concentration (IC50), as determined using nonlinear regression, for  l-kynurenine, quinolinic acid and kynurenic acid was 9.17, 15.56, and 535.40 mM, respectively. Optical transmitted light differential interference contrast and hematoxylin and eosin staining revealed cells blocked in metaphase, formation of apoptotic bodies and compromised cell density in  l-kynurenine-treated cells. A statistically significant increase in the number of cells present in the sub-G1 phase was observed in  l-kynurenine-treated sample. To our knowledge, this was the first in vitro study conducted to investigate the mechanism of action of kynurenine metabolites on endothelioma sEnd-2 cells. It can be concluded that  l-kynurenine exerts an antiproliferative effect on the endothelioma sEnd-2 cell line by decreasing cell growth and proliferation as well as a metaphase block. These hallmarks suggest cell death via apoptosis. Further research will be conducted on  l-kynurenine to assess the effect on cell adhesion in vitro and in vivo as cell-cell adhesion has been shown to increase metastasis to distant organs therefore, the inhibition of adhesion may lead to a decrease in metastasis.

L-kynurenine and quinolinic acid inhibited markers of cell survival in B16 F10 melanoma cells in vitro.

Melanoma is an aggressive malignancy and remains a major cause of skin cancer mortality, highlighting the need for new treatment strategies. Recent findings revealed that L-kynurenine and quinolinic acid induce cytotoxicity and morphological changes in B16 F10 melanoma cells in vitro. This paper highlights the effects of L-kynurenine and quinolinic acid at previously determined half-maximal inhibitory concentrations on cell cycle progression, cell death and extracellular signal-regulated protein kinase inhibition. Melanoma, B16 F10 and murine macrophages, RAW 264.7 cells were used in this study, as both cell lines express all the enzymes associated with the kynurenine pathway. Post exposure to the compounds at half-maximal inhibitory concentrations, transmission electron microscopy was used to assess intracellular morphological changes. Flow cytometry was used to analyse cell cycle progression and quantify apoptosis via the dual staining of Annexin V and propidium iodide and cell survival via extracellular signal-regulated protein kinase. L-kynurenine and quinolinic acid at half-maximal inhibitory concentrations induced intracellular morphological changes representative of cell death. Flow cytometry revealed alterations in cell cycle distribution, increased apoptosis and significantly inhibition of cell survival. L-kynurenine and quinolinic acid are exogenous kynurenine compounds which inhibited cell survival through extracellular signal-regulated protein kinase inhibition, induced cell cycle alterations and induced apoptosis in B16 F10 melanoma cells.

Quantifying assays: inhibition of signalling pathways of cancer.

Inhibiting a signalling pathway concerns controlling the cellular processes of a cancer cell's viability, cell division and death. Assay protocols created to see if the molecular structures of the drugs being tested have the desired inhibition qualities often show great variability across experiments, and it is imperative to diminish the effects of such variability while inferences are drawn. In this paper, we propose the study of experimental data through the lenses of a mathematical model depicting the inhibition mechanism and the activation-inhibition dynamics. The method is exemplified through assay data obtained from an experimental study of the inhibition of the chemokine receptor 4 (CXCR4) and chemokine ligand 12 (CXCL12) signalling pathway of melanoma cells. The quantitative analysis is conducted as a two step process: (i) deriving theoretically from the model the cell viability as a function of time depending on several parameters; (ii) estimating the values of the parameters by using the experimental data. The cell viability is obtained as a function of concentration of the inhibitor and time, thus providing a comprehensive characterization of the potential therapeutic effect of the considered inhibitor, e.g. $IC_{50}$ can be computed for any time point.

An in vitro investigation of l-kynurenine, quinolinic acid, and kynurenic acid on B16 F10 melanoma cell cytotoxicity and morphology.

The metastatic behavior of melanoma has accentuated the need for specific therapy targets. Compounds, namely l-kynurenine ( l-kyn), quinolinic acid (Quin), and kynurenic acid (KA) previously displayed antiproliferative and cytotoxic effects in vitro against cancer cells. Despite the growing interest in these compounds there are limited studies examining the in vitro effects on melanoma. In B16 F10 melanoma cells, RAW 264.7 macrophage cells, and HaCat keratinocyte cells, postexposure to the compounds, crystal violet staining was used to determine the half-maximal inhibitory concentration (IC50 ), whereas polarization-optical transmitted light differential interference contrast and light microscopy after hematoxylin and eosin (H&E) staining was used to assess morphological changes.  l-kyn, Quin, and KA-induced cytotoxicity in all cell lines, with  l-kyn being the most cytotoxic compound.  l-kyn and KA at IC50 -induced morphological changes in B16 F10, RAW 264.7, and HaCat cell lines, whereas Quin had effects on B16 F10 and RAW 264.7 cells but did not affect HaCat cells.  l-kyn, Quin, and KA each display different levels of cytotoxicity, which were cell line specific.  l-kyn was shown to be the most potent compound against all cell lines and may offer future treatment strategies when combined with other viable treatments against melanoma.

The tryptophan-kynurenine pathway in immunomodulation and cancer metastasis.

INTRODUCTION: The activation of the kynurenine pathway in cancer progression and metastasis through immunomodulatory pathways has drawn attention to the potential for kynurenine pathway inhibition. The activation of the kynurenine pathway, which results in the production of kynurenine metabolites through the degradation of tryptophan, promotes the development of intrinsically malignant properties in cancer cells while facilitating tumour immune escape. In addition, kynurenine metabolites act as biologically active substances to promote cancer development and metastasis. METHODS: A literature review was conducted to investigate the role of the tryptophan-kynurenine pathway in immunomodulation and cancer metastasis. RESULTS: Evidence suggests that several enzymes and metabolites implicated in the kynurenine pathway are overexpressed in various cancers. As such, the tryptophan pathway represents a promising target for cancer treatment. However, downstream signalling pathways, including aryl hydrocarbon receptor activation, have previously induced diverse biological effects in various malignancies, which resulted in either the promotion or the inhibition of metastasis. CONCLUSION: As a result, a thorough investigation of the kynurenine pathway and its regulatory mechanisms is necessary in order to properly comprehend the effects of kynurenine pathway activation involved in cancer development and metastasis.

Chemokines as possible therapeutic targets in metastatic melanoma.

BACKGROUND: Cutaneous melanoma is a relentless form of cancer which continues to rise in incidence. Currently, cutaneous melanoma is the leading cause of skin cancer-related mortality, which can mainly be attributed to its metastatic potential. The activation of chemokine axes is a major contributor to melanoma metastasis through its involvement in promoting tumour cell migration, proliferation, survival, and adhesion. This review will focus on the role of chemokines in melanoma and possible therapeutic strategies to alter chemokine activation and subsequently inhibit the activation of signalling cascades that may promote metastasis. METHODS: A literature review was conducted to evaluate chemokines as possible therapeutic targets in metastatic melanoma. RESULTS: The crosstalk between signalling pathways and immune responses in the melanoma microenvironment resembles a complex and dynamic system. Therefore, the involvement of governing chemokine axes in the promotion of cutaneous and metastatic melanoma demands a proper understanding of the tumour microenvironment in order to identify possible targets and develop appropriate treatments against melanoma. CONCLUSION: Even though chemokine axes are regarded as promising therapeutic targets, it has become increasingly evident that chemokines can play a critical role in both tumour inhibition and promotion. The inhibition of chemokine axes to inhibit signalling cascades in target cells that regulate metastasis should, therefore, be carefully approached.

The involvement of a chemokine receptor antagonist CTCE-9908 and kynurenine metabolites in cancer development.

Cancer is the second leading cause of mortality worldwide. Skin cancer is the most common cancer in South Africa with nearly 20,000 reported cases every year and 700 deaths. If diagnosed early, the 5-year survival rate is about 90%, however, when diagnosed late, the 5-year survival rate decreases to about 20%. Melanoma is a type of skin cancer with an estimated 5-year survival rate of approximately 90%. Neuroblastoma is a paediatric cancer with a low survival rate. Sixty percent of patients with metastatic disease do not survive 5 years after diagnosis. Despite recent advances in targeted therapies, there is a crucial need to identify reliable prognostic biomarkers which will be able to contribute to the development of more precision-based chemotherapeutic strategies to prevent tumour migration and metastasis. The compound, CTCE-9908 inhibits the binding of CXC chemokine ligand 12 (CXCL12) to the CXC chemokine receptor 4 (CXCR4) receptor leading to reduced metastasis. Kynurenine metabolites are derived tryptophan, which is an essential amino acid. Kynurenine metabolites inhibit T-cell proliferation resulting in cell growth arrest. For this reason, chemokines receptors represent potential targets for the treatment of cancer growth and metastasis. In this review paper, the role of the CXCL12/CXCR4 signalling pathway in the development of cancer is highlighted together with the current available treatments involving the CTCE-9908 compound in combination with microtubule inhibitors like paclitaxel and docetaxel.

Pellagra in South Africa from 1897 to 2019: a scoping review.

OBJECTIVE: Pellagra is a nutritional deficiency disease associated with niacin (vitamin B3) deficiency. The history of pellagra is well documented for Europe and the USA, but less is known about the prevalence in sub-Saharan African countries. This study documents the history of pellagra in South Africa, as diagnosed based on dermatological symptoms. DESIGN: Scoping review of information from scientific databases, library archives, other archives and record services and from Statistics South Africa. SETTING: South Africa, 1897-2019. PARTICIPANTS: South African. RESULTS: Pellagra was first officially recorded in South Africa in 1906, but there are earlier indications of the disease. The prevalence of pellagra peaked after it was all but eradicated in the USA and Europe. Pellagra was never as prevalent in South Africa as in Europe, the USA and Egypt, where special hospitals for pellagrins were established. However, studies on urinary excretion of metabolites conducted in 1960s and 1970s suggested a high prevalence of subclinical (sub-pellagra) niacin deficiency, especially in previously disadvantaged Black populations. As in Europe and the USA, pellagra was associated with poverty and an overdependence on maize as staple food. Malnutrition was the main cause of the disease, but alcohol abuse might have been a contributing factor. In South Africa, reports of pellagra had declined by the late 1980s/early 1990s and hardly any cases were reported by the year 2000. CONCLUSIONS: Although pellagra, diagnosed based on dermatological symptoms, appears to be largely eradicated in South Africa, it does not rule out the potential for subclinical niacin deficiency.

Comparison between plasma neopterin and the urine neopterin:creatinine ratio as inflammatory biomarkers.

BACKGROUND: Neopterin, a product of cell-mediated immunity, is a non-specific biomarker of inflammation. Plasma/serum is generally the body fluid of choice for neopterin assessment, but urine is often used as it does away with venepuncture. Analysis of urine neopterin is based on collection of a single urine sample and expressed as µmol neopterin/mol creatinine. OBJECTIVES: To examine published correlations between plasma neopterin levels and urine neopterin:creatinine ratios and to determine whether they are in diagnostic agreement. METHODS: Literature search was performed by databases and by hand. Databases included Academic Search Complete; Africa-Wide Information; AHFS Consumer Medication Information; eBook Collection (EBSCOhost); Family & Society Studies Worldwide; MasterFILE Premier; MEDLINE; TOC Premier. RESULTS: Positive correlations of varying statistical significance generally exist between plasma neopterin and urine neopterin: creatinine ratios. With a decline in renal clearance, plasma neopterin over-estimates inflammatory activity. With immune-complex renal disease, urine neopterin:creatinine ratios over-estimate systemic inflammation. The two biomarkers can differ in diagnostic validity. CONCLUSION: Correlations between plasma neopterin and urine neopterin:creatinine ratios suggest both as suitable biomarkers. However, since correlations reflect equality of means and not individual values, significant correlations, do not necessarily imply diagnostic agreement. Therefore, plasma and urine cannot summarily be assumed interchangeable for diagnostic/prognostic purposes.

Tryptophan depletion in context of the inflammatory and general nutritional status of a low-income South African HIV-infected population.

BACKGROUND: The essential amino acid tryptophan cannot be synthesised in the body and must be acquired through dietary intake. Oxidation of tryptophan, due to immune induction of the enzyme indoleamine 2,3-dioxygenase (IDO), is considered to be the main cause of tryptophan depletion in HIV infection and AIDS. We examined plasma tryptophan levels in a low-income sub-Saharan HIV-infected population and compared it to that of developed countries. Tryptophan levels were further examined in context of the general nutritional and inflammatory status. METHODS: This cross-sectional study included 105 HIV-positive patients recruited from the Kalafong Hospital in Pretoria, South Africa, and 60 HIV-negative controls. RESULTS: Patient tryptophan levels were in general markedly lower than those reported for developed countries. In contrast to reports from developed countries that showed tryptophan levels on average to be 18.8 % lower than their control values, tryptophan levels in our study were 44.1 % lower than our controls (24.4 ± 4.1 vs. 43.6 ± 11.9 µmol/l; p < 0.001). Tryptophan levels correlated with both CD4 counts (r = 0.341; p = 0.004) and with pro-inflammatory activity as indicated by neopterin levels (r = -0.399; p = 0.0001). Nutritional indicators such as albumin and haemoglobin correlated positively with tryptophan and negatively with the pro-inflammatory indicators neopterin, interleukin 6 and C-reactive protein. The most probable causes of the lower tryptophan levels seen in our population are food insecurity and higher levels of inflammatory activity. CONCLUSIONS: We contend that inflammation-induced tryptophan depletion forms part of a much wider effect of pro-inflammatory activity on the nutritional profile of HIV-infected patients.

The kynurenine pathway activities in a sub-Saharan HIV/AIDS population.

BACKGROUND: Tryptophan is an essential amino acid for the synthesis of proteins and important metabolites such as serotonin, melatonin, tryptamine and niacin. After protein synthesis, more than 90 % of tryptophan catabolism occurs along the kynurenine pathway. The inflammation-inducible enzyme indoleamine 2,3 dioxygenase (IDO) is responsible for the first rate-limiting step in the kynurenine pathway, i.e., oxidation of tryptophan to kynurenine. Excessive IDO activity in conditions such as HIV/AIDS may lead to tryptophan depletion and accumulation of metabolites downstream from kynurenine. Little is known about the kynurenine pathway of HIV/AIDS patients in sub-Saharan regions. This study, in a low income sub-Saharan HIV/AIDS population, examined the effects of activities in the kynurenine pathway on plasma levels of tryptophan, kynurenine and the neurotoxin quinolinic acid, and on de novo synthesis of nicotinamide. METHODS: Plasma samples were obtained from a cohort of 105 HIV patients and 60 controls. Kynurenine pathway metabolites were analysed using gas chromatography - mass spectrometry. ELISA and flow cytometry were used to assess plasma inflammatory markers. RESULTS: IDO activity, depletion of tryptophan, as well as accumulation of kynurenine and the neurotoxin quinolinic acid, were not only significantly greater in the patients than in the controls, but also markedly greater than in HIV/AIDS patients from developed countries. Tryptophan levels were 12.3 % higher, kynurenine levels 16.2 % lower, quinolinic acid levels 43.2 % lower and nicotinamide levels 27,2 % lower in patients on antiretroviral treatment than in antiretroviral-naïve patients. Patients' kynurenine pathway metabolites correlated with the levels of inflammatory markers, including that of the major IDO-inducer, interferon-gamma. Indications are that the rate of de novo synthesis of nicotinamide in the kynurenine pathway correlates with increases in quinolinic acid levels up to a point where saturation of the enzyme quinolinate phosphoribosyl transferase occurs. CONCLUSIONS: Higher levels of inflammatory activity in this low income sub-Saharan HIV/AIDS population than in patients from developed countries lead to greater tryptophan depletion and greater accumulation of metabolites downstream from tryptophan with quinolinic acid levels often reaching levels associated with the development of HIV/AIDS-associated neurocognitive dysfunction. De novo synthesis of nicotinamide from quinolinic acid contributes to the maintenance of nicotinamide, and by implication NAD levels, in HIV/AIDS patients from low income populations. Antiretroviral treatment partially corrects disturbances in the kynurenine pathway.

A non-specific biomarker of disease activity in HIV/AIDS patients from resource-limited environments.

BACKGROUND: A general non-specific marker of disease activity that could alert the clinician and prompt further investigation would be of value in patients with HIV/AIDS, especially in resource limited environments. OBJECTIVE: To investigate the potential of neopterin as non-specific biomarker in patients with advanced HIV/AIDS. METHODS: Cross-sectional study in 105 HIV positive patients (75 on highly active antiretroviral treatment (HAART). Neopterin was assessed by enzyme linked immune-absorbent assay and cytokines by flow cytometry. RESULTS: Neopterin levels were significantly higher (p<0.001) for the total patient than for the control group. Significant correlations between neopterin and plasma indicators of inflammation showed neopterin to be a good indicator of active inflammatory status and of the effect of HAART on the immune system. Neopterin was superior to C-reactive protein and to individual cytokines as indicator of immune deficiency. Increased neopterin levels were associated with a decline in albumin, haemoglobin and the albumin/globulin ratio, and with increases in red cell distribution width. CONCLUSIONS: Plasma neopterin is a good non-specific biomarker of disease activity in HIV/AIDS patients. It is a good indicator of inflammatory activity, perpetuation of inflammation-associated co-morbidities, degree of immune deficiency and has predictive value for underlying disease, and for monitoring the HAART response.

Antidepressants may lead to a decrease in niacin and NAD in patients with poor dietary intake.

The term niacin is the generic name for the two compounds nicotinic acid and nicotinamide, the major dietary precursors for two important coenzymes, nicotinamide adenine dinucleotide (NAD) and its phosphorylated form, NADP. Niacin is important for the maintenance of cellular integrity and energy production and is involved in more than 500 intracellular reactions. Deficiencies of niacin may contribute to neuropsychiatric and neurodegenerative disorders. Patients who develop nutritional deficiencies as a result of poor dietary intake, especially inadequate intake of proteins and vitamins, could potentially suffer from niacin deficiency and NAD depletion. However, de novo synthesis of niacin and NAD in the kynurenine pathway of tryptophan metabolism may compensate for impaired dietary intake. The rate of synthesis of NAD and niacin from tryptophan oxidation depends on the induction of the enzyme indoleamine 2,3-dioxygenase (IDO) by pro-inflammatory cytokines such as interferon-gamma. Niacin synthesis is not limited by a decrease in tryptophan and excessive IDO activity may therefore lead to a decline in tryptophan levels. Antidepressants have an anti-inflammatory effect, including reduction of interferon-gamma and therefore inhibition of IDO, the rate-limiting enzyme of the kynurenine pathway. In theory, this could account for increased serotonin as more tryptophan becomes available for serotonin synthesis. However, the downside may be that less NAD and niacin are synthesised downstream, which could exacerbate common psychiatric problems. It is our hypothesis that patients with poor dietary intake, who are treated with antidepressants, are at risk of developing niacin/NAD deficiency with possible development of associated neuropsychiatric symptoms. We therefore propose that niacin supplementation be considered in patients with inadequate diets who are treated with antidepressants. We believe that if this does not happen, a subclinical niacin deficiency may result, which would be difficult to detect as it would cause the same symptoms of the original illness (e.g. depression). Niacin deficiency should be considered and ruled out in all patients with treatment-resistant depression, who have a poor response to antidepressants. This is potentially a cost-effective and easy intervention, which could be examined in a randomized controlled trial.

Autonomic correlates at rest and during evoked attention in children with attention-deficit/hyperactivity disorder and effects of methylphenidate.

OBJECTIVE: The aim of this study was to assess autonomic nervous system functioning in children with attention-deficit/hyperactivity disorder (ADHD) and to examine the effects of methylphenidate and focussed attention. METHOD: Children with ADHD (n = 19) were tested while they were stimulant free and during a period in which they were on stimulants. On both occasions, autonomic nervous system functioning was tested at baseline and during focussed attention. Autonomic nervous system functioning of control subjects was also tested at baseline and during focussed attention. Autonomic nervous system activity was determined by means of heart rate variability (HRV) and skin conductivity analyses. Attention was evoked by means of the BioGraph Infiniti biofeedback apparatus. HRV was determined by time domain, frequency domain and Poincaré analysis of RR interval data. Skin conductivity was determined by the BioGraph Infiniti biofeedback apparatus. RESULTS: The main findings of this study were (a) that stimulant-free children with ADHD showed a sympathetic underarousal and parasympathetic overarousal of the sympathovagal balance relative to control subjects; (b) methylphenidate shifted the autonomic balance of children with ADHD towards normal levels; however, a normal autonomic balance was not reached, and (c) stimulant-free children with ADHD exhibited a shift in the sympathovagal balance towards the sympathetic nervous system from baseline to focussed attention; however, methylphenidate appeared to abolish this shift. CONCLUSIONS: Stimulant-free children with ADHD have a parasympathetic dominance of the autonomic balance, relative to control subjects. Methylphenidate attempts to restore the normal autonomic balance in children with ADHD, but inhibits the normal autonomic nervous system response to a cognitive challenge. CLINICAL APPLICATIONS: These results indicate that methylphenidate may have a suppressive effect on the normal stress response. Although this may be of benefit to those who interact with children who suffer from ADHD, the implications for the physiological and psychological well-being of the children themselves are debatable. Further research is needed. LIMITATIONS OF THE STUDY: Only 19 children with ADHD and 18 control subjects were tested. Further studies should include prior testing in order to exclude children with possible co-existing learning disabilities. Cognitive function and emotional responses of children with ADHD were not tested.