γ-Glutamylcysteine synthetase (γ-GCS) as a target for overcoming chemo- and radio-resistance of human hepatocellular carcinoma cells

Aims: This study uncovered that the genetically endowed intracellular glutathione contents (iGSH) regulated by the catalytic subunit of.-glutamylcysteine synthetase heavy chain (gamma-GCSh) as a prime target for overcoming both the inherited and stimuli-activated chemo-and radio-resistance of hepatocellular carcinoma (HCC) cells. Main methods: Reactive oxygen species (ROS) production and mitochondrial membrane potential (Delta psi m) were determined by the probe-based flow cytometry. The TUNEL assay was used as an index of radio-sensitivity and the MTT assay was used as an index of chemo-sensitivity against various anti-cancer agents. iGSH and gamma-GCSh activity were measured by HPLC methods. gamma-GCSh-overexpressing GCS30 cell line was established by tetracycline-controlled Tet-OFF gene expression system in SK-Hep-1 cells. Key findings: The relative radio-sensitivities of a panel of five HCC cells were found to be correlated negatively with both the contents of iGSH and their corresponding gamma-GCSh activities with an order of abundance being Hep G2 > Hep 3B > J5 > Mahlavu > SK-Hep-1, respectively. Similarly, the cytotoxicity response patterns of these HCC cells against arsenic trioxide (ATO), a ROS-producing anti-cancer drug, were exactly identical to the order of ranking instigated by the radiotherapy (RT) treatment. Next, gamma-GCSh-overexpressing GCS30 cells were found to possess excellent ability to profoundly mitigate both the drop of Delta psi m and apoptotic TUNEL-positive cell population engendered by ATO, cisplatin, doxorubicin, and RT treatments.