Potassium and Chloride Ion Channels in Cancer: A Novel Paradigm for Cancer Therapeutics.

Cancer is a collection of diseases caused by specific changes at the genomic level that support cell proliferation indefinitely. Traditionally, ion channels are known to control a variety of cellular processes including electrical signal generation and transmission, secretion, and contraction by controlling ionic gradients. However, recent studies had brought to light important facts on ion channels in cancer biology.In this review we discuss the mechanism linking potassium or chloride ion channel activity to biochemical pathways controlling proliferation in cancer cells and the potential advantages of targeting ion channels as an anticancer therapeutic option.

Potassium channels activity unveils cancer vulnerability.

"No cell could exist without ion channels" (Clay Armstrong; 1999). Since the discovery in the early 1950s, that ions move across biological membranes, the idea that changes of ionic gradients can generate biological signals has fascinated scientists in any fields. Soon later (1960s) it was found that ionic flows were controlled by a class of specific and selective proteins called ion channels. Thus, it became clear that the concerted activities of these proteins can initiate, arrest, and finely tune a variety of biochemical cascades which offered the opportunity to better understand both biology and pathology. Cancer is a disease that is notoriously difficult to treat due its heterogeneous nature which makes it the deadliest disease in the developed world. Recently, emerging evidence has established that potassium channels are critical modulators of several hallmarks of cancer including tumor growth, metastasis, and metabolism. Nevertheless, the role of potassium ion channels in cancer biology and the therapeutic potential offered by targeting these proteins has not been explored thoroughly. This chapter is addressed to both cancer biologists and ion channels scientists and it aims to shine a light on the established and potential roles of potassium ion channels in cancer biology and on the therapeutic benefit of targeting potassium channels with activator molecules.

A novel role for involvement of N-methyl-D-aspartate (NMDA) glutamate receptors in sperm acrosome reaction.

Ionotropic glutamate receptors are expressed in mouse and human spermatozoa. However, the possible role of these receptors has not been reported in the sperm acrosome reaction. This study was conducted to demonstrate the function of N-methyl-D-aspartate (NMDA) glutamate receptors in the acrosome reaction of mouse spermatozoa. Epididymal spermatozoa from adult mice were release in a culture medium. The sperm suspension was then divided into six groups: (1) spermatozoa at 0 min, (2) spermatozoa at 60 min (control), (3) spermatozoa treated with NMDA glutamate receptor agonist (L-glutamate, LG), (4) spermatozoa treated with α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainite glutamate receptor agonist (kainic acid), (5) spermatozoa treated with NMDA glutamate receptor antagonist (MK-801)+LG and (6) spermatozoa treated with ethylene glycol tetraacetic acid (EGTA, as a calcium chelator)+ LG. The sperm samples were examined for the acrosome reaction and intracellular calcium concentration. After 60 min, LG but not kainic acid significantly increased both the acrosome reaction and intracellular calcium levels in the spermatozoa compared with the control group. Co-administration of MK-801 or EGTA+LG could significantly reverse the effect of LG in the acrosome reaction and the level of intracellular calcium as compared to the LG group. The possibility that LG induced the acrosome reaction and elevated inter-cellular calcium concentration in mouse spermatozoa and that MK-801 could reverse the effects of LG, may suggest the involvement of NMDA glutamate receptors, at least in the initiation of the acrosome reaction in vitro.

Iodine-131 radiolabeling of poly ethylene glycol-coated gold nanorods for in vivo imaging.

Gold nanorods (GNRs) can be used in various biomedical applications; however, very little is known about their in vivo tissue distribution by radiolabeling. Here, we have developed a rapid and simple method with high yield and without disturbing their optical properties for radiolabeling of gold rods with iodine-131 in order to track in vivo tissue uptake of GNRs after systemic administration by biodistribution analysis and γ-imaging. Following intravenous injection into rat, PEGylated GNRs have much longer blood circulation times.

MEKK1 Regulates Chemokine Expression in Mammary Fibroblasts: Implications for the Breast Tumor Microenvironment.

Breast tumors contain both transformed epithelial cells and non-transformed stroma cells producing secreted factors that can promote metastasis. Previously, we demonstrated that the kinase MEKK1 regulates cell migration and gene expression, and that transgene-induced breast tumor metastasis is markedly inhibited in MEKK1-deficient mice. In this report, we examined the role of MEKK1 in stroma cell gene expression and the consequent effect on breast tumor cell function. Using a heterotypic cell system to quantify the effect of stroma cells on breast tumor cell function, we discovered that MEKK1-/- fibroblasts are significantly less effective at inducing tumor cell invasion than MEKK1+/+ fibroblasts. Expression array analysis revealed that both baseline and tumor cell-induced expression of the chemokines CCL3, CCL4, and CCL5 were markedly reduced in MEKK1-/- mammary fibroblasts. By focusing on the role of MEKK1 in CCL5 regulation, we discovered that MEKK1 kinase activity promotes CCL5 expression, and inactive mutant MEKK1 strongly inhibits CCL5 transcription. CCL5 and the other MEKK1-dependent chemokines are ligands for the GPCR CCR5, and we show that the CCR5 antagonist Maraviroc strongly inhibits fibroblast-induced tumor cell migration. Finally, we report that fibroblast growth factor 5 (FGF-5) is secreted by MDA-MB 231 cells, that FGF-5 activates MEKK1 effectors ERK1/2 and NFκB in fibroblasts, and that chemical inhibition of NFκB inhibits CCL5 expression. Our results suggest that MEKK1 contributes to the formation of a breast tumor microenvironment that supports metastasis by promoting expression of stroma cell chemokine genes in response to tumor cell-induced paracrine signaling.

Compensatory expression of NRF2-dependent antioxidant genes is required to overcome the lethal effects of Kv11.1 activation in breast cancer cells and PDOs.

Potassium channels are important regulators of cellular homeostasis and targeting these proteins pharmacologically is unveiling important mechanisms in cancer cell biology. Here we demonstrate that pharmacological stimulation of the Kv11.1 potassium channel activity results in mitochondrial reactive oxygen species (ROS) production and fragmentation in breast cancer cell lines and patient-derived organoids independent of breast cancer subtype. mRNA expression profiling revealed that Kv11.1 activity significantly altered expression of genes controlling the production of ROS and endoplasmic-reticulum (ER) stress. Characterization of the transcriptional signature of breast cancer cells treated with Kv11.1 potassium channel activators strikingly revealed an adaptive response to the potentially lethal augmentation of ROS by increasing Nrf2-dependent transcription of antioxidant genes. Nrf2 in this context was shown to promote survival in breast cancer, whereas knockdown of Nrf2 lead to Kv11.1-induced cell death. In conclusion, we found that the Kv11.1 channel activity promotes oxidative stress in breast cancer cells and that suppression of the Nrf2-mediated anti-oxidant survival mechanism strongly sensitized breast cancer cells to a lethal effect of pharmacological activation of Kv11.1.

Molecular Activation of the Kv11.1 Channel Reprograms EMT in Colon Cancer by Inhibiting TGFß Signaling via Activation of Calcineurin.

Control of ionic gradients is critical to maintain cellular homeostasis in both physiological and pathological conditions, but the role of ion channels in cancer cells has not been studied thoroughly. In this work we demonstrated that activity of the Kv11.1 potassium channel plays a vital role in controlling the migration of colon cancer cells by reversing the epithelial-to-mesenchymal transition (EMT) into the mesenchymal-to-epithelial transition (MET). We discovered that pharmacological stimulation of the Kv11.1 channel with the activator molecule NS1643 produces a strong inhibition of colon cancer cell motility. In agreement with the reversal of EMT, NS1643 treatment leads to a depletion of mesenchymal markers such as SNAIL1, SLUG, TWIST, ZEB, N-cadherin, and c-Myc, while the epithelial marker E-cadherin was strongly upregulated. Investigating the mechanism linking Kv11.1 activity to reversal of EMT into MET revealed that stimulation of Kv11.1 produced a strong and fast inhibition of the TGFß signaling. Application of NS1643 resulted in de-phosphorylation of the TGFß downstream effectors R-SMADs by activation of the serine/threonine phosphatase PP2B (calcineurin). Consistent with the role of TGFß in controlling cancer stemness, NS1643 also produced a strong inhibition of NANOG, SOX2, and OCT4 while arresting the cell cycle in G0/G1. Our data demonstrate that activation of the Kv11.1 channel reprograms EMT into MET by inhibiting TGFß signaling, which results in inhibition of motility in colon cancer cells.

Multiomic analysis identifies CPT1A as a potential therapeutic target in platinum-refractory, high-grade serous ovarian cancer.

Resistance to platinum compounds is a major determinant of patient survival in high-grade serous ovarian cancer (HGSOC). To understand mechanisms of platinum resistance and identify potential therapeutic targets in resistant HGSOC, we generated a data resource composed of dynamic (±carboplatin) protein, post-translational modification, and RNA sequencing (RNA-seq) profiles from intra-patient cell line pairs derived from 3 HGSOC patients before and after acquiring platinum resistance. These profiles reveal extensive responses to carboplatin that differ between sensitive and resistant cells. Higher fatty acid oxidation (FAO) pathway expression is associated with platinum resistance, and both pharmacologic inhibition and CRISPR knockout of carnitine palmitoyltransferase 1A (CPT1A), which represents a rate limiting step of FAO, sensitize HGSOC cells to platinum. The results are further validated in patient-derived xenograft models, indicating that CPT1A is a candidate therapeutic target to overcome platinum resistance. All multiomic data can be queried via an intuitive gene-query user interface (https://sites.google.com/view/ptrc-cell-line).

Quantitative evaluation of human sperm viability using MTT assay: A laboratory study.

BACKGROUND: 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay which evaluates cellular mitochondrial activity is widely used for the assessment of cell proliferation and viability. OBJECTIVE: This study was performed to assess human sperm viability using MTT assay. MATERIALS AND METHODS: In this laboratory study, human-ejaculated semen samples (n = 56 from different donors) were used. The sperm viability was determined using quantitative MTT assay and the sperm motility was assessed according to World Health Organization guidelines. Sperm viability and the correlation between sperm viability and motility were analyzed. RESULTS: Data revealed a marked positive correlation between MTT reduction rate and the percentage of viable spermatozoa. The Pearson's correlation coefficients also showed a significant correlation between sperm viability and motility. CONCLUSION: MTT assay which is based on mitochondrial functionality is a reliable method for evaluating human sperm viability and could be used as a diagnostic test for predicting sperm fertilization ability in clinical settings.

Effect of curcumin on kidney histopathological changes, lipid peroxidation and total antioxidant capacity of serum in sodium arsenite-treated mice.

Sodium arsenite is an environmental pollutant with the ability to generate free radicals and curcumin acts as a potent antioxidant. This study investigates the effect of curcumin on kidney histopathology, lipid peroxidation and antioxidant capacity of serum in the mice treated with sodium arsenite. Adult male mice were divided into four groups: control, sodium arsenite, curcumin and curcumin+sodium arsenite. The treatments were delivered for 5 weeks. After the treatment period, blood samples were collected and the concentrations of malondialdehyde (MDA) and total antioxidant capacity of serum were determined. Left kidney was dissected, weighed and used for histopathological and histomorphometrical studies. Sodium arsenite-treated mice showed a significant decrease in the diameter of glomerulus and proximal tubule, glomerular area, total antioxidant capacity of serum as well as a significant increase in serum concentration of MDA compared to the control group. However, no significant difference was found in kidney weight, area and diameter of Bowman's capsule as well as the diameter of distal tubule in mice treated with sodium arsenite compared to the control. In curcumin+sodium arsenite group, curcumin significantly reversed the adverse effects of sodium arsenite on the diameter of glomerulus and proximal tubule, glomerular area, total antioxidant capacity of serum and serum concentration of MDA compared to the sodium arsenite group. The application of curcumin alone significantly increased the total antioxidant capacity of serum compared to the control. Curcumin compensated the adverse effects of sodium arsenite on kidney tissue, lipid peroxidation and total antioxidant capacity of serum.

Curcumin Inhibits The Adverse Effects of Sodium Arsenite in Mouse Epididymal Sperm.

BACKGROUND: The aim of this study was to investigate the effects of curcumin on epididy- mal sperm parameters in adult male Navel Medical Research Institute (NMRI) mice ex- posed to sodium arsenite. MATERIALS AND METHODS: In this experimental study, we divided the animals into four groups: control, sodium arsenite (5 mg/kg), curcumin (100 mg/kg) and curcumin+sodium arsenite. Exposures were performed by intraperitoneal injections for a 5-week period. After the exposure period, we recorded the animals' body and left testes weights. The left caudal epididymis was used to count the sperm number and analyze motility, viability, morphological abnormalities, acrosome reaction, DNA integrity, and histone-protamine replacement in the spermatozoa. One-way analysis of variance (ANOVA) followed by the Tukey's test was used to assess the statistical significance of the data with SPSS 16.0. P<0.05 was considered significant. RESULTS: Mice exposed to sodium arsenite showed a significant decrease in the num- ber, motility, viability, normal sperm morphology and acrosome integrity of spermato- zoa compared to the control group. In the curcumin+sodium arsenite group, curcumin significantly reversed these adverse effects to the point where they approximated the control. In addition, the application of curcumin alone had no significant difference in these parameters compared to the control and curcumin+sodium arsenite groups. However, we observed no significant differences in the body and the testis weight as well as the DNA integrity and histone-protamine replacement in the spermatozoa of the four groups. CONCLUSION: Curcumin compensated for the toxic effects of sodium arsenite on a number of sperm parameters in adult mice.

Effect of vitamin E on sperm parameters and DNA integrity in sodium arsenite-treated rats.

BACKGROUND: Arsenic as an environmental toxicant is able to exert malformations in male reproductive system by inducing oxidative stress. Vitamin E (Vit.E) is known as antioxidant vitamin. OBJECTIVE: The aim of this study was to investigate the harmful effects of sodium arsenite on sperm parameters and the antioxidant effects of Vit.E on sperm anomalies in sodium arsenite treated rats. MATERIALS AND METHODS: Adult male rats were divided into 4 groups: control, sodium arsenite (8 mg/kg/day), Vit.E (100 mg/kg/day) and sodium arsenite+Vit.E. Oral treatments were performed till 8 weeks. Body and left testis weight were recorded and then left caudal epididymis was cut in Ham's F10. Released spermatozoa were used to analyze number, motility, viability and abnormalities of the sperm. Sperm chromatin quality was assessed by nuclear staining using acridine orange and aniline blue. RESULTS: Body and testis weight showed no significant change in 4 groups (p>0.05). A significant decrease in the number, motility, viability and normal sperm morphology was found in sodium arsenite-treated rats compared to the control (p<0.001). Sodium arsenite had no effect on sperm DNA integrity and histon-protamine replacement (p>0.05). In sodium arsenite+Vit.E group, Vit.E could significantly compensate the harmful effects of sodium arsenite on sperm number, motility, viability and morphology compared to sodium arsenite group. In addition, sperm viability and motility was significantly increased in rats treated with Vit.E alone compared to the control and sodium arsenite+Vit.E group. CONCLUSION: Vitamin E could compensate the adverse effects of sodium arsenite on sperm parameters in adult rats.

Effect of vitamin E on sperm number and testis histopathology of sodium arsenite-treated rats.

The aims of this study were to investigate the adverse effects of sodium arsenite on the reproductive system of male rats as well as to examine whether vitamin E is able to ameliorate these effects. Adult rats were divided into four groups: 1/ control, 2/ sodium arsenite (8 mg/kg/day), 3/ vitamin E (100 mg/kg/day), and 4/ sodium arsenite +vitamin E group. Treatments were administered orally by gavage for eight weeks. After treatment, body and left testis weights were recorded and the testis was used for the histological analysis. Left cauda epididymis was used to count sperm number. Body and testis weight did not differ among the groups (p>0.05). A significant decrease (p<0.001) in sperm number and mean diameter of seminiferous tubules as well as a significant increase (p<0.001) in the mean diameter of seminiferous tubules' lumen were found in sodium arsenite group compared to those of controls. Sodium arsenite did not affect the morphology and diameter of spermatogonial nucleus (p>0.05). In the sodium arsenite + vitamin E group, vitamin E ameliorated (p<0.001) the adverse effects of sodium arsenite on sperm number as well as the diameters of tubule and lumen. In addition, the treatment of rats with vitamin E alone significantly (p<0.001) increased the diameter of seminiferous tubules and significantly (p<0.001) decreased seminiferous tubules' lumen compared to the control group. Vitamin E appeared to ameliorate the adverse effects of sodium arsenite on epididymal sperm number and some morphometrical parameters of the adult rat testis.