Korean J Physiol Pharmacol.  2014 Dec;18(6):509-516. 10.4196/kjpp.2014.18.6.509.

Distinct Cellular Calcium Metabolism in Radiation-sensitive RKO Human Colorectal Cancer Cells

• Kim YT
• Jo SS
• Park YJ
• Lee MZ
• Suh CK

• KMID: 2285554
• DOI: http://doi.org/10.4196/kjpp.2014.18.6.509

Abstract

Radiation therapy for variety of human solid tumors utilizes mechanism of cell death after DNA damage caused by radiation. In response to DNA damage, cytochrome c was released from mitochondria by activation of pro-apoptotic Bcl-2 family proteins, and then elicits massive Ca2+ release from the ER that lead to cell death. It was also suggested that irradiation may cause the deregulation of Ca2+ homeostasis and trigger programmed cell death and regulate death specific enzymes. Thus, in this study, we investigated how cellular Ca2+ metabolism in RKO cells, in comparison to radiation-resistant A549 cells, was altered by gamma (gamma)-irradiation. In irradiated RKO cells, Ca2+ influx via activation of NCX reverse mode was enhanced and a decline of [Ca2+]i via forward mode was accelerated. The amount of Ca2+ released from the ER in RKO cells by the activation of IP3 receptor was also enhanced by irradiation. An increase in [Ca2+]i via SOCI was enhanced in irradiated RKO cells, while that in A549 cells was depressed. These results suggest that gamma-irradiation elicits enhancement of cellular Ca2+ metabolism in radiation-sensitive RKO cells yielding programmed cell death.

Keyword

A549 cells; Inositol-1,4,5-triphosphate receptors; Na+-Ca2+ exchanger; RKO cells; Store-operated Ca2+ influx

MeSH Terms

Calcium*
Cell Death
Colorectal Neoplasms*
Cytochromes c
DNA Damage
Homeostasis
Humans
Inositol 1,4,5-Trisphosphate Receptors
Metabolism*
Mitochondria
Calcium
Cytochromes c
Inositol 1,4,5-Trisphosphate Receptors

Figure

• 

  Fig. 1 Effects of γ-irradiation on NCX in RKO and A549 cells. The activity of NCX in cells was measured in the reverse mode of NCX induced by superfusing 0 mM Na+/2.5 mM Ca2+ solution containing 1 µM thapsigargin (Thapsi), 5 mM caffeine (CAF), and 250 µM La3+ and in the forward mode of NCX by 140 mM Na+/0 mM Ca2+ solution. (A) In RKO control cells, R340/380 increased with the reverse mode of NCX and decreased with the forward mode. (B) In γ-ray irradiated RKO cells, the second peak was observed. The slope of R340/380 changes by the forward mode of NCX was increased in γ-rays irradiated cells. (C) In A549 control cells, R340/380 changed as in (A). (D) In γ-ray irradiated A549 cells, the slope of R340/380 changes by the forward mode of NCX was increased, confirming the activity of NCX was increased by γ-irradiation (p<0.0001). Tracings in (A) to (D) represent the average values of R340/380.

• 

  Fig. 2 Effects of γ-irradiation on SOCI in RKO and A549 cells. (A) When RKO control cells were superfused with Tyrode's solution including 2 mM Ca2+ after depleting intracellular Ca2+ store, R340/380 was increased by Ca2+ influx via SOCI. (B) Ca2+ influx via SOCI was enhanced in γ-ray irradiated RKO cells, compared to control cells (***p<0.0001). (C) In A549 control cells, R340/380 was increased by Ca2+ influx via SOCI, as in (A). (D) Ca2+ influx via SOCI was decreased in γ-ray irradiated A549 cells, compared to (C) (*p<0.05). (E) In RKO cells, areas under SOCI response, which approximate the amount of Ca2+ influxed via SOCI, were increased by γ-ray irradiation (***p<0.0001). In A549 cells, areas under SOCI response were decreased by γ-irradiation (*p<0.05). The increment of areas under SOCI response in RKO cells after γ-irradiation was significantly different from that in A549 cells, which was decreased by γ-irradiation (###p<0.0001). Tracings in (A) to (D) represent the average values of R340/380.

• 

  Fig. 3 Effects of γ-irradiation on ATP-induced [Ca2+]i changes in RKO and A549 cells. (A) Application of 100 µM ATP induced transient changes in R340/380 (ATP-induced Ca2+ responses) in RKO control cells. (B) In γ-ray irradiated RKO cells, transient changes in R340/380 were enhanced, compared to control cells (1.18±0.07 vs. 0.89±0.06; p<0.0001). (C) In A549 control cells, transient changes in R340/380 were elicited by application of 100 µM ATP. (D) In γ-ray irradiated A549 cells, transient changes in R340/380 were not significantly different from those in control cells. (E) In more than 60% of A549 cells measured, multiple transient changes in R340/380 were observed with a single application of ATP. (F) The frequency of multiple transient changes was increased in γ-ray irradiated A549 cells (See Table 5). Tracings in (A) to (F) represent the average values of R340/380.

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