Evaluation of the mechanism of enhancement of cisplatin cytotoxicity by O(6)-benzylguanine.

摘要

Cisplatin has been widely used as a chemotherapeutic agent for nearly four decades; however, its usefulness in treating head and neck, gynecologic, and lung cancers has been limited by acquired resistance. Our lab has previously determined that a nontoxic pharmacologic agent, O6-benzylguanine (BG), modulates cisplatin-induced apoptosis and cytotoxicity in head and neck cancer cell lines. In this thesis, the mechanism by which BG enhances cisplatin-induced cytotoxicity was evaluated, as was the tissue specificity of this enhancement. Initially, I tested the role of BG in affecting transport of cisplatin through three copper transport proteins. Expression of copper influx protein hCtr1 and the copper efflux proteins ATP7A and ATP7B was unrelated to the ability of BG to enhance cisplatin cytotoxicity, thereby effectively ruling out transport as a mechanism for BG. I showed that BG not only enhances cisplatin in head and neck cell lines but also in ovarian and other gynecologic cell lines, but not in non-small cell lung cancer cell lines. Three of these cell lines, head and neck cancer (SQ20b), ovarian cancer (SKOV-3x), and non-small cell lung cancer (A549), were chosen for further evaluation, with the SQ20b and SKOV-3x cell lines used as positive controls for enhancement of cytotoxicity by BG, and the A549 cell line used as an example of a cell type that is negative for enhancement by BG. In all three cell lines, BG enhanced the level of DNA platination following cisplatin treatment, and the rate of repair does not appear to be affected by addition of BG, as measured by the slopes of the platinum curves over time. Additionally, in all three cell lines, there is an increase in DNA double-strand breaks (DSB) as measured by histone H2AX phosphorylation (gammaH2AX) formation over time. The negative modulator 9-methyl-O6-benzylguanine (9-methyl-BG) also enhances DNA platination in the SQ20b cell line following cisplatin treatment; however, it does not significantly increase gammaH2AX formation. This indicates that the increase in DNA damage following BG plus cisplatin treatment is not sufficient to account for the enhancement in cytotoxicity observed in the SQ20b and SKOV-3x cell lines, as similar increases in DNA damage occur in the A549 cell line.;Because we showed that the increase in cisplatin cytotoxicity following treatment with BG was independent of DNA damage, we decided to examine another target of cisplatin: the endoplasmic reticulum (ER). Cisplatin has been shown to induce apoptosis in a number of cell types through initiation of the endoplasmic reticulum stress pathway. Interestingly, microarray results from our laboratory indicated that a number of ER stress transcripts were upregulated following treatment of SQ20b cells with BG plus cisplatin as compared to treatment with cisplatin alone, but not in cells treated with 9-methyl-BG plus cisplatin. Based on these results, I evaluated the ER stress pathway as a potential target for BG to enhance cisplatin-induced cytotoxicity. BG alone significantly increases the expression the ER stress transcripts GADD34 and GADD153 in cell lines positive for enhancement by BG. This expression is further enhanced when cisplatin is added to the treatment regimen. In the A549 non-small cell lung cancer cell line, ER stress transcripts are not significantly upregulated by either cisplatin or BG plus cisplatin treatment. Additionally, ER stress-induced apoptosis, as measured by cleavage of the ER stress specific caspase, caspase 12, is significantly increased in SQ20b and SKOV-3x cell lines treated with BG plus cisplatin, but not in the A549 cell line. Inhibition of the ER stress pathway in SQ20b cells by both treatment with the eIF2alpha dephosphorylation inhibitor salubrinal and siRNA targeted against GADD153 significantly decreased enhancement in both apoptosis and cytotoxicity caused by BG plus cisplatin treatment versus cisplatin alone. This indicates that BG is acting at least partially to increase cisplatin-induced cytotoxicity by augmenting ER stress-induced apoptosis. These results confirm that cisplatin can induce apoptosis through the ER stress pathway, shown for the first time in head and neck and ovarian cancer cell lines. Additionally, the results indicate that modulation ER stressors, including cisplatin, with agents such as BG may be a viable method by which to circumvent resistance associated with chemotherapy treatment.