CBP501-Calmodulin Binding Contributes to Sensitizing Tumor Cells to Cisplatin and Bleomycin

Abstract

CBP501 is an anticancer drug currently in randomized phase II clinical trials for patients with non-small cell lung cancer and malignant pleural mesothelioma. CBP501 was originally described as a unique G 2 checkpoint-directed agent that binds to 14-3-3, inhibiting the actions of Chk1, Chk2, mitogen-activated protein kinase-activated protein kinase 2, and C-Tak1. However, unlike a G 2 checkpoint inhibitor, CBP501 clearly enhances the accumulation of tumor cells at G 2 -M phase that is induced by cisplatin or bleomycin at low doses and short exposure. By contrast, CBP501 does not similarly affect the accumulation of tumor cells at G 2 -M that is induced by radiation, doxorubicin, or 5-fluorouracil treatment. Our recent findings point to an additional mechanism of action for CBP501. The enhanced accumulation of tumor cells at G 2 -M upon combined treatment with cisplatin and CBP501 results from an increase in intracellular platinum concentrations, which leads to increased binding of platinum to DNA. The observed CBP501-enhanced platinum accumulation is negated in the presence of excess Ca 2þ . Some calmodulin inhibitors behave similarly to, although less potently than, CBP501. Furthermore, analysis by surface plasmon resonance reveals a direct, high-affinity molecular interaction between CBP501 and CaM (K d ¼ 4.62 Â 10 À8 mol/L) that is reversed by Ca 2þ , whereas the K d for the complex between CBP501 and 14-3-3 is approximately 10-fold weaker and is Ca 2þ independent. We conclude that CaM inhibition contributes to CBP501 0 s activity in sensitizing cancer cells to cisplatin or bleomycin. This article presents an additional mechanism of action which might explain the clinical activity of the CBP501-cisplatin combination.

Activation of Nrf2 Pathways Correlates with Resistance of NSCLC Cell Lines to CBP501In Vitro

“…The results between the different methods correlate well (8,9, and data not shown) and differential sensitivity to CBP501 varies consistently for different cell lines (9).…”

“…As shown in our previous reports, CBP501 has at least two MOA as an anticancer drug candidate (8,9). One of these, G 2 checkpoint abrogation, occurs for treatments of longer duration and at higher doses (8).…”

“…Such direct effects might be difficult to detect by conventional microarray analysis, which can sometimes be hindered by technical limitations such as variable sensitivity in the detection of individual probes. Although our previous reports indicated that the CBP501-CDDP combination effect on short-term exposure correlated well with CDDP uptake in at least seven cell lines (9), platinum accumulation in NSCLC cell lines was not demonstrated. Therefore, there was a possibility that CBP501 affected not only enhancement of CDDP uptake but also inhibition of the Nrf2-dependent cytoprotective effect against CDDP.…”

“…One of these, G 2 checkpoint abrogation, occurs for treatments of longer duration and at higher doses (8). A second anticancer activity of CBP501, the enhancement of CDDP uptake through the binding to calmodulin, occurs for treatments of shorter duration and at lower doses (9). In this report, we focus on the latter effect and characterized the CBP501 sensitivity of many different NSCLC cell lines by treating cells for shorter duration with lower doses of CBP501.…”

“…This entails direct interaction of CBP501 with calmodulin, which leads to increased uptake of CDDP into cancer cells upon much shorter exposure to and at lower doses of CBP501 than the conditions required for G 2 checkpoint abrogation (9). CDDP uptake is known to depend on multiple transporters and channels (10).…”