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Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Cancer cells require large amounts of iron to maintain their proliferation. Iron metabolism is considered a hallmark of cancer, making iron a valid target ...

Northumbria University

The anticancer activity of a series of novel synthesized, hydroxypyridone-based metal chelators (analogues of L-mimosine) was evaluated in an in vitro model of melanoma consisting of malignant melanoma (A375), non-melanoma epidermoid carcinoma (A431) and immortalized non-malignant keratinocyte (HaCaT) cells. More specifically, we have demonstrated that the L-enantiomer of a methylated analogue of L-mimosine (compound 22) can exert a potent anticancer effect in A375 cells when compared to either A431 or HaCaT cells. Moreover, we have demonstrated that this analogue has the ability to i) promote increased generation of reactive oxygen species (ROS), ii) activate both intrinsic and extrinsic apoptosis and iii) induce perturbations in cell cycle growth arrest. Our data highlights the potential of compound 22 to act as a promising therapeutic agent against an in vitro model of human malignant melanoma.

Anticancer activity of a novel methylated analogue of L-mimosine against an in vitro model of human malignant melanoma.

Cancer cells accumulate iron to supplement their aberrant growth and metabolism. Depleting cells of iron by iron chelators has been shown to be selectively cytotoxic to cancer cells  and . Iron chelators are effective at combating a range of cancers including those which are difficult to treat such as androgen insensitive prostate cancer and cancer stem cells. This review will evaluate the impact of iron chelation on cancer cell survival and the underlying mechanisms of action. A plethora of studies have shown iron chelators can reverse some of the major hallmarks and enabling characteristics of cancer. Iron chelators inhibit signalling pathways that drive proliferation, migration and metastasis as well as return tumour suppressive signalling. In addition to this, iron chelators stimulate apoptotic and ER stress signalling pathways inducing cell death even in cells lacking a functional p53 gene. Iron chelators can sensitise cancer cells to PARP inhibitors through mimicking BRCAness; a feature of cancers trademark genomic instability. Iron chelators target cancer cell metabolism, attenuating oxidative phosphorylation and glycolysis. Moreover, iron chelators may reverse the major characteristics of oncogenic transformation. Iron chelation therefore represent a promising selective mode of cancer therapy.

Reversing oncogenic transformation with iron chelation.

Melanoma is an aggressive and highly metastatic type of skin cancer where the design of new therapies is of utmost importance for the clinical management of the disease. Thus, we have aimed to investigate the mode of action by which a novel methylated analogue of L-Mimosine (e.g., L-SK-4) exerts its therapeutic potency in an in vitro model of malignant melanoma. Cytotoxicity was assessed by the Alamar Blue assay, oxidative stress by commercially available kits, ROS generation, caspase 3/7 activation and mitochondrial membrane depolarisation by flow cytometry, expression of apoptosis-related proteins by western immunoblotting and profiling of lipid biosynthesis by a metabolomic approach. Overall, higher levels of ROS, sphingolipids and apoptosis were induced by L-SK-4 suggesting that the compound's therapeutic potency is mediated through elevated ROS levels which promote the upregulation of sphingolipid (ceramide) biosynthesis thus leading to the activation of both extrinsic and intrinsic apoptosis, in an experimental model of malignant melanoma.

A novel methylated analogue of L-Mimosine exerts its therapeutic potency through ROS production and ceramide-induced apoptosis in malignant melanoma.

Iron is an essential micronutrient due to its involvement in many cellular processes including DNA replication and OXPHOS. Tumors overexpress iron metabolism linked proteins which allow for iron accumulation driving high levels of proliferation. Our group has designed novel iron chelator SK4 which targets cancer's "iron addiction." SK4 comprises of two key moieties: an iron chelation moiety responsible for cytotoxicity and an amino acid moiety which allows entry through amino acid transporter LAT1. We selected LAT1 as a route of entry as it is commonly overexpressed in malignant tumors. SK4 has previously demonstrated promising results in an  model for melanoma. We hypothesized SK4 would be effective against a range of tumor types. We have screened a panel of tumor-derived cell lines from different origins including breast, prostate, ovarian and cervical cancer for SK4 sensitivity and we have found a range of differential sensitivities varying from 111.3 to >500 μM. We validated the iron chelation moiety as responsible for inducing cytotoxicity through control compounds; each lacking a key moiety. Following the screen, we conducted a series of assays to elucidate the mechanism of action behind SK4 cytotoxicity. SK4 was shown to induce apoptosis in triple negative breast cancer cell line MDA MB 231 but not ovarian cancer cell line SKOV3 suggesting SK4 may induce different modes of cell death in each cell line. As MDA MB 231 cells harbor a mutation in p53, we conclude SK4 is capable of inducing apoptosis in a p53-independent manner. SK4 upregulated NDRG1 expression in MDA MB 231 and SKOV3 cells. Interestingly, knockdown of NDRG1 antagonized SK4 in MDA MB 231 cells but not SKOV3 cells suggesting SK4's mechanism of action may be mediated through NDRG1 in MDA MB 231 cells. In conclusion, we have shown tagging iron chelators with an amino acid moiety to allow entry through the LAT1 transporter represents a double pronged approach to cancer therapy, targeting "iron addiction" and amino acid metabolism dysregulation.

Novel iron chelator SK4 demonstrates cytotoxicity in a range of tumour derived cell lines.

LncRNAs are involved in regulatory processes in the human genome, including gene expression. The rs35705950 SNP, previously associated with IPF, overlaps with the recently annotated lncRNA AC061979.1, a 1712 nucleotide transcript located within the MUC5B promoter at chromosome 11p15.5. To document the expression pattern of the transcript, we processed 3.9 TBases of publicly available RNA-SEQ data across 27 independent studies involving lung airway epithelial cells. Epithelial lung cells showed expression of this putative pancRNA. The findings were independently validated in cell lines and primary cells. The rs35705950 is found within a conserved region (from fish to primates) within the expressed sequence indicating functional importance. These results implicate the rs35705950-containing AC061979.1 pancRNA as a novel component of the MUC5B expression control minicircuitry.

The Idiopathic Pulmonary Fibrosis-Associated Single Nucleotide Polymorphism RS35705950 Is Transcribed in a MUC5B Promoter Associated Long Non-Coding RNA (AC061979.1).

Anti-cancer therapy by iron chelation has been shown to inhibit many cellular processes including DNA replication, mitochondrial metabolism and oncogenic signalling pathways (e.g., EGFR). Iron chelator SK4 represents a double pronged approach towards treating cancer. SK4 enters through LAT1, a commonly overexpressed amino acid transporter in tumours, thus targeting iron addiction and LAT1 overexpression. The aim of this study was to characterise the mode of action of SK4 through proteomics, metabolomics, lipidomics and seahorse real-time analysis in ovarian cell line SKOV3 and triple negative breast cancer cell line MDA MB 231. Pathway enrichment of proteomics data showed an overrepresentation of metabolism related pathways. Metabolic change after SK4 exposure have been confirmed in investigations of changes in basal and maximal mitochondrial respiration using seahorse real-time analysis of mitochondrial metabolism. Metabolomics also showed an increase in AMP and glucose-1-phosphate. Interestingly, our lipidomics data show a decrease in phospholipid synthesis in the SKOV3 cells which is in contrast with previous data which showed an upregulation of ceramide driven apoptosis. In summary, our data highlight impairment of energy metabolism as a mechanism of action underlying SK4 apoptosis, but also suggest a potential role of ceramide induction in the phenotypic outcome of the cell model.

Novel Iron Chelator SK4 Drives Cytotoxicity through Inhibiting Mitochondrial Metabolism in Ovarian and Triple Negative Breast Cancer Cell Lines.

Canonical NF-κB signalling by p65 (RelA) confers chemo-resistance and poor survival in chronic lymphocytic leukaemia (CLL). The role of non-canonical NF-κB signalling (leading to RelB and p52 subunit activation) in CLL is less understood, but given its importance in other B-cell tumour types, we theorised that RelB and p52 may also contribute to the pathology of CLL.

Expression and Activity of the NF-κB Subunits in Chronic Lymphocytic Leukaemia: A Role for RelB and Non-Canonical Signalling.

 Ruthenium(II) complexes have emerged recently as candidates for anti-cancer therapy, where activity is related to lipohilicity, cellular localization, and specific interactions with biomolecules.  In this work, two novel complexes were synthesized and are reported based on the [Ru(phen)(dipyrido[3,2-:2',3'-]quinoxaline] framework.  Compared to the parent complex, annealing of cyclopenteno and cyclohexeno rings to the extended ligand substantially increased cytotoxicity towards a number of cancer cell lines, and induced apoptosis. The complexes localize in the nuclei of cancer cells and co-locate with DAPI on DNA. DNA binding studies show that both complexes bind strongly to DNA and one complex intercalates DNA like the parent, whilst the other appears to have multiple modes of interaction.  It is likely that the increased lipophilicity of the novel complexes is a key factor for increasing their cytotoxicity, rather than their DNA binding mode.

Stephany Veuger

Department of Applied Sciences,
Faculty of Health and Life Sciences,
Department of Applied Sciences, Faculty of Health and Life Sciences

Northumbria University

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Stephany Veuger

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Applied Sciences,Faculty of Health and Life Sciences,Department of Applied Sciences, Faculty of Health and Life Sciences

Northumbria University

Quantifiable and Inexpensive Cell-Free Fluorescent Method to Confirm the Ability of Novel Compounds to Chelate Iron

Department of Applied Sciences,
Faculty of Health and Life Sciences,
Department of Applied Sciences, Faculty of Health and Life Sciences