O6-Benzylguanine (BG) enhances
cisplatin [cis-diammine dichloroplatinum (II)]-induced cytotoxicity and apoptosis in
head and neck cancer cell lines by an unknown mechanism. We investigated the effect of
cisplatin with and without BG on two targets of damage:
DNA and the endoplasmic reticulum (ER). We chose three
cancer cell lines to ascertain the mechanism of BG-enhanced cytotoxicity: SQ20b head and neck and SKOV-3x
ovarian cancer cell lines, where BG enhanced
cisplatin cytotoxicity, and A549
nonsmall cell lung cancer line, where BG did not enhance
cisplatin cytotoxicity. All three lines had an increase in DNA damage when BG was added to
cisplatin treatment, as evidenced by increased platination and phosphorylated
histone H2AX formation. The increase in
cisplatin-induced DNA damage
after treatment with BG plus
cisplatin is not sufficient to increase cytotoxicity or apoptosis in A549 cells. We evaluated the effect of
cisplatin on the ER and observed increased
caspase 12 cleavage in SQ20b and SKOV-3x cells, but not in A549 cells,
after treatment with BG plus
cisplatin versus
cisplatin alone. Growth arrest and DNA damage inducible (GADD) 153, an ER stress-response gene, is up-regulated
after treatment with BG plus
cisplatin compared with
cisplatin alone in SQ20b and SKOV-3x cells, but not in A549 cells. ER stress-induced apoptosis is an integral part of the mechanism by which BG enhances
cisplatin. Inhibition of ER stress in the SQ20b cell line by
salubrinal, an inhibitor of eIF2alpha dephosphorylation, or GADD153
small interfering RNA, abrogated BG-enhancement of
cisplatin cytotoxicity and apoptosis through
caspase 3 and 12 cleavage. These data indicate GADD153 up-regulation plays an important role in BG-enhanced
cisplatin cytotoxicity and apoptosis.