Calcium-induced apoptosis is delayed by HER1 receptor signalling through the Akt and PLCγ pathways in bladder cancer cells.

The level of extracellular calcium has been demonstrated to regulate important physiological processes like cell growth and apoptosis. We demonstrate that in the bladder cancer cell line RT4, an increased extracellular calcium level induces apoptosis and that the HER1 receptor functions as a cell survival factor and delays apoptosis. After 12 h of calcium treatment (10 mM) apoptosis was detected in the RT4 cells. Increased activation of the HER1 receptor was detected as soon as 30 min after calcium addition, and the activation decreased again after 12 h of incubation, coinciding with the time when apoptosis was detectable. Inhibition of HER1 with Gefitinib (5 μM) or Tyrphostin (AG1478) (20 μM) augmented the calcium-induced apoptosis, and with HER1 inhibition apoptosis was detectable after 6 h. Analysis of downstream signalling molecules showed an increased activation of Akt, PLCγ and MAPK in response to calcium treatment. The activation of Akt and PLCγ was abolished by inhibition of HER1 with Gefitinib (5 μM), whereas this had no effect on the activity of MAPK. In addition, incubation with inhibitors of Akt and PLCγ significantly augmented calcium-induced apoptosis, whereas this was not seen with MAPK inhibition. Finally a significant increase in PKCδ activity was observed with calcium treatment alone and was augmented further with HER1 inhibition. In conclusion we show that calcium-induced apoptosis in bladder cancer cells is delayed by HER1 receptor activation involving the Akt and PLCγ signalling pathways.