Circadian Timing of Curcumin Efficacy and Nuclear Transport Properties of Cancer Cells
Understanding how the circadian clock controls delivery and activity of chemotherapies could optimize cancer treatments. Most anti-cancer drugs are targeted to the nucleus and their movement through the nuclear envelope could be controlled by the circadian clock. Circadian regulation of cell toxicity was tested by measuring the ability of the phytochemical curcumin to kill C6 glioma cells at different phases of the circadian cycle. Mitotic and cell death events were counted every 5 minutes for 5 days. An initial low curcumin treatment (5 µM) caused an elevated cell death rate in C6 cells at rhythmic intervals with a period of 24.4 hours. The rhythm was disrupted at 10 µM. These results revealed a sensitive phase of the circadian cycle that could be exploited in therapies based on curcumin or its analogs. Curcumin’s persistence in cells was imaged for at least 24 hours using autofluorescence. The observed higher stability of the curcumin congeners demethoxycurcumin and bisdemethoxycurcumin suggested that they too may produce sustained cancer cell toxicity.
Circadian regulation of anti-cancer agents may also occur in the nuclear transport mechanism through nuclear pore complexes (NPCs). The transport mechanism could be regulated by cell calcium ion stores. A single-molecule imaging technique provided the kinetic parameters as well as spatial locations of transported molecules at altered calcium ion store concentrations. This three-dimensional, high-speed, super-resolution imaging indicated that transport was altered. It is possible that previously described circadian calcium oscillations regulate NPC activity.
Quantitative RT-PCR was used to test whether with the circadian clock controls expression of the Crm1 gene. CRM1 protein is a nuclear exporter that may generate circadian rhythms in cargo concentrations in the cytoplasm. The phase of the circadian rhythm detected suggested that Crm1 could be controlled by the circadian clock protein BMAL1. To test whether transport of an anti-cancer agent into the nucleus might be under circadian control, the nuclear accumulation rate of doxorubicin fluorescence was measured in C6 cells. Cyclic accumulation was observed with a period near 24 hrs, suggesting that nuclear entry may be clock regulated. These results could help in determining optimal daily drug delivery times during cancer therapy.