Role Of Mitochondrial Ca2+ Uniporter In Radiation-Induced Cell Damage

Radiotherapy for head and neck cancer induces significant acute- and long-term damage in other tissues within the treatment area, such as salivary gland. TRPM2 is activated by irradiation (IR) and Ca2+ entry via this channel contributes to irreversible loss of salivary gland function (Liu et al. 2013, Nat. Commun., http://dx.doi.org/10.1038/ncomms2526). Here we have examined the possible mechanism(s) involved in long-term disruption of salivary gland cells. IR of HSG cells caused an increase in mitochondrial reactive oxygen species (mtROS) which remained elevated up to 24 hours post-IR. The elevation in mtROS was attenuated by removing extracellular Ca2+ during IR or by blocking the mitochondrial permeability transition pore (MPTP) with cyclosporin A (CsA) or suppression of mitochondrial Ca2+ uniporter (MCU) with siMCU. Consistent with this, the mitochondrial [Ca2+] was significantly increased by IR and this increase was attenuated in cells treated with siMCU or by removing external Ca2+ during IR. Additionally, mitochondrial membrane potential (Ψm) was depolarized in irradiated HSG cells during first 2h and gradually returned to normal. IR-induced decrease in Ψm was blocked by treating cells with CsA, siMCU, or Gd3+. Thus, IR-induced increase in plasma membrane Ca2+ permeability, primarily via TRPM2, leads to elevation in mtCa2+ and mtROS as well as a decrease in Ψm. Furthermore, IR also induced a time-dependent activation of Caspase 3 and a decrease in cell viability. Treatment of cells with siMCU or Caspase 3 inhibitor, zVAD, conferred significant protection of cell viability up to 96 hours post-IR. Together, our data indicate that mitochondria are likely a major target for IR with mitochondrial disruption and caspase activation leading to irreversible changes in cell function.