The effect of iron on the expression levels of calcium related gene in cisplatin resistant epithelial ovarian cancer cells

Abstract

Aim: Anticancer drugs (chemotherapeutics) used in cancer treatment (chemotherapy) lead to drug resistance. This study was conducted to investigate the possible effect of iron on calcium homeostasis in epithelial ovarian cancer cells (MDAH-2774) and cisplatin-resistant cells of the same cell line (MDAH-2774/DDP).

Methods: To develop MDAH-2774/DDP cells, MDAH-2774 (MDAH) cells were treated with cisplatin in dose increases of 5 μM between 0 μM and 70 μM. The effect of iron on the viability of MDAH and MDAH/DDP cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test at the end of 24 h incubation.

Results: At increasing iron concentrations in MDAH and MDAH/DDP cells, the mRNA gene of fifteen genes [inositol 1,4,5-triphosphate receptor (IP₃R)1/2/3, ryanodine receptor (RYR)1/2, sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA)1/2/3, Na⁺/Ca²⁺ exchange (NCX)1/2/3, and plasma membrane Ca²⁺ ATPase (PMCA)1/2/3/4] associated with Ca²⁺ differences in expression were determined by quantitative reverse transcription-polymerase chain reaction. Changes in IP₃R2, RYR1, SERCA2, NCX3, PMCA1, and PMCA3 gene expressions were observed in iron treatment of MDAH/DDP cells, while changes were detected in iron treatment of MDAH cells in IP₃R1/2/3, RYR1/2, SERCA1/2/3, NCX2/3, and PMCA1 expressions.

Conclusions: This changes in the expression of calcium channels, pumps, and exchange proteins in the epithelial ovarian cancer cell line and in cisplatin-resistant epithelial ovarian cancer cells suggest that iron may have an important role in regulating calcium homeostasis. Due to differences in the expression of genes that play of an important role in the regulation of calcium homeostasis in the effect of iron, drug resistance can be prevented by introducing a new perspective on the use of inhibitors and activators of these genes and thus cytostatic treatment strategies.

Keywords: Cisplatin; Na+/Ca2+ exchange; calcium; drug resistance; inositol 1,4,5-triphosphate receptor; iron; ryanodine receptor; sarco/endoplasmic reticulum Ca2+ ATPase.

Figure 1.

The viability of MDAH and MDAH/DDP cells after treatment with iron for 24 h was determined by performing the MTT test as described in the Materials and Methods section. Values were results from three independent experiments. Student’s “t-test” was done

Figure 2.

Gene expression of the IP3R1/2/3, RYR1 and RYR2 Ca²⁺ channels in the ER in MDAH and MDAH/DDP cells treated with increasing iron concentrations. The levels of change in mRNA gene expression rates for each sample were determined with respect to actin by RT-PCR method. A: IP3R1; B: IP3R2; C: IP3R3; D: RYR1; E: RYR2

Figure 3.

Gene expression of SERCA1/2 and SERCA3 Ca²⁺ pumps in the ER at increasing iron concentrations in MDAH and MDAH/DDP cells. The levels of change in mRNA expression rates for each sample were determined with respect to actin by RT-PCR method. A: SERCA1; B: SERCA2; C: SERCA3

Figure 4.

Gene expressions of NCX1/2 and NCX3 Ca²⁺ exchange carriers found in plasma and nuclear membrane at increasing iron concentrations in MDAH and MDAH/DDP cells. The levels of change in mRNA expression rates for each sample were determined with respect to β-actin by RT-PCR method. A: NCX1; B: NCX2; C: NCX3

Figure 5.

Gene expression of PMCA1/2/3 and PMCA4 channels in the plasma membrane at increasing iron concentrations in MDAH and MDAH/DDP cells. The levels of change in mRNA gene expression rates for each sample were determined with respect to P-actin by RT-PCR method. A: PMCA1; B: PMCA2; C: PMCA3; D: PMCA4