Action of econazole on Ca2+ levels and cytotoxicity in OC2 human oral cancer cells

Abstract

Background/purpose: Econazole is an antifungal drug. Antifungal activity of econazole against non-dermatophyte molds was reported. Econazole inhibited Ca²⁺ channels and stimulated cytotoxicity in lymphoma and leukemia cells. Ca²⁺ cations are crucial second envoy that triggers various processes. This research was aimed to investigate action of econazole on Ca²⁺ levels and cytotoxicity in OC2 human oral cancer cells.

Materials and methods: Cytosolic Ca²⁺ levels ([Ca²⁺]_i) were detected employing fura-2 as a probe in a RF-5301PC spectrofluorophotometer (Shimadzu). Cytotoxicity was determined using 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] (WST-1) to detect fluorescence changes.

Results: Econazole at 10-50 μmol/L provoked [Ca²⁺]_i raises. Forty % of 50 μml//L econazole-induced signal was diminished when external Ca²⁺ was eliminated. The Ca²⁺ influx provoked by econazole was suppressed by different degrees by store-induced Ca²⁺ influx suppressors SKF96365 and nifedipine; GF109203X (a protein C [PKC] inhibitor); an extracellular signaling pathway (ERK) 1/2 blocker PD98059, and phospholipase A2 suppressor aristolochic acid, but was enhanced by phorbol 12-myristate 13 acetate (PMA; a PKC activator) by 18%. Without external Ca²⁺, econazole-caused [Ca²⁺]_i raises were abolished by thapsigargin. In contrast, econazole partially suppressed the [Ca²⁺]_i raises caused by thapsigargin. U73122 fell short to change econazole-caused [Ca²⁺]_i responses. Econazole (10-70 μmol/L) elicited cytotoxicity in a dose-dependent fashion. Blockade of 50 μmol/L econazole-induced [Ca²⁺] rises with BAPTA/AM enhanced econazole-induced cytotoxicity by 72%.

Conclusion: Econazole evoked [Ca²⁺]_i raises and provoked cytotoxicity in a concentration-dependent manner in OC2 human oral cancer cells. In Ca²⁺-containing solution, BAPTA/AM enhanced 50 μmol/L econozole-induced cytotoxicity.

Keywords: Cellular calcium; Cytotoxicity; Econazole; Fura-2; OC2 human oral cancer cells.

Figure 1

Effect of econazole on [Ca²⁺]_i (A) The solution contained Ca²⁺. (B) Assays were performed in Ca²⁺-deprived solution. (C) Response-concentration association of econazole-provoked [Ca²⁺]_i elevations. Y axis (control) represented percentage of the accumulated part beneath responses (25–250 s) of [Ca²⁺]_i elevations evoked by 50 μmol/L econazole in Ca²⁺-containing solution. Findings were reported as the mean ± standard error of three separate experiments. ∗P value < 0.05: significant differences in contrast to open loops. Ca²⁺: calcium ions; [Ca²⁺]_i: cytosolic free Ca²⁺ level; sec: second.

Figure 2

Effect of Mn²⁺ on econazole-induced Ca²⁺ incursion. One min prior to assays, MnCl₂ (50 μmol/L) was administered to a medium containing Ca²⁺. The y axis represents arbitrary fluorescence collected at excitation wavelength of 360 nm. Trace a: no econazole present. Trace b: in the presence of econazole (20 μmol/L). Data were shown as the mean ± standard error of three independent assays. Sec: second; Mn²⁺: manganese; MnCl₂: manganese (II) chloride.

Figure 3

In chemical-pretreated sets, chemicals were added 60 s before addition of 20 μmol/L econazole. Results were presented as percentage of first column which was integrated district beneath the responses (25–200 s) of econazole-evoked [Ca²⁺]_i rises (control). Data were reported as the mean of standard error of three separate assays. ∗P value < 0.05 in contrast to first column. [Ca²⁺]_i: cytosolic free Ca²⁺ level; sec: second.

Figure 4

(A) (B) Thapsigargin (1 μmol/L) and econazole (20 μmol/L) were administered to cells as illustrated. These analyses were conducted in Ca²⁺-deprived solution. Findings were reported as the mean ± standard error of three independent assays. Ca²⁺: calcium ions.

Figure 5

Action of U73122 on econazole-caused Ca²⁺ discharge. The following assays were conducted in Ca²⁺-deprived solution. (A) ATP (10 μmol/L) was administrated to cells as shown. (B) U73122 (2 μmol/L) and ATP (10 μmol/L) fell to change resting [Ca²⁺]_i and econazole (20 μmol/L)-provoked [Ca²⁺]_i raises. (C) In the existence of 2 μmol/L U73343 (incubated for 1 min), 10 μmol/L ATP provoked a considerable Ca²⁺ response. Findings were indicated as the mean ± standard error of three separate examinations. ATP: adenosine triphosphate; Ca²⁺: calcium ions; [Ca²⁺]_i: cytosolic free Ca²⁺ level; U73122: 1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; U73343: 1-[6-[[(17b)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-2,5-pyrrolidinedione. Min: minute.

Figure 6

(A) After overnight incubation of cells with econazol (0–70 μmol/L), the viability was investigated. Results were represented as percentage of control which meant increases in cell count in the presence of econazole. Cell counts of control had 99,222 ± 180 cells/well prior to assays and 14,888 ± 145 cells/well after overnight incubation. (B) To investigate whether [Ca²⁺]_i raises altered cytotoxicity, 5 μmol/L BAPTA/AM was administered to cells 1 h prior to econazole (50 μmol/L) treatment overnight in the presence of Ca²⁺. Results were represented as percentage of control which meant increases in cell count after overnight incubation without econazole or BAPTA/AM. Cytotoxicity assay was performed. Data were shown as the mean ± standard error of three independent experiments. ∗P value < 0.05 in contrast to first bar. ^#P value < 0.01 in contrast to third bar. BAPTA/AM: 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester); Ca²⁺: calcium ions; [Ca²⁺]_i: cytosolic free Ca²⁺ level; H: hour.