Molecular mechanism underlying chemosensitization of doxorubicin by selenium
Doxorubicin is an effective drug against breast cancer. However, the favorable therapeutic response to doxorubicin is often associated with severe toxicity. Our study was aimed at developing a strategy of increasing doxorubicin sensitivity so that low doses may be used without compromising efficacy. By combining doxorubicin with selenium, we observed a synergistic effect on the induction of apoptosis in the MCF-7 breast cancer cells. Selenium was capable of depressing doxoruicin-induced Akt phosphorylation, which is important in mediating the synergy between selenium and doxorubicin, as evidenced by several facts. Selenium was no longer able to sensitize cells to doxorubicin under a condition in which Akt was constitutively activated. Selenium reduced the abundance of phospho-GSK3β (inactive form) induced by doxorubicin, while chemical inhibition of GSK3β activity compromised the apoptotic response to the selenium/doxorubicin combination. Additional experiments showed that selenium increased the transactivation of FOXO proteins and the expression of Bim (a target gene of FOXO3A). The functional significance of Bim was confirmed by the observation that RNA interference of Bim markedly reduced the potency of selenium/doxorubicin to activate the intrinsic apoptotic pathway. In addition, the death receptor pathway was activated independent of the intrinsic pathway. Because FOXO proteins have been reported to modulate FasL and TRAIL expression, we next investigated how doxorubicin/selenium, either individually or in combination, modulated the expression of TRAIL and FasL signaling molecules. We also monitored caspase-8 activation in response to neutralizing/blocking antibodies against ligands/receptors in order to assess the contribution of a given pathway. We found that TRAIL signaling was not involved in the activation of caspase-8 by the combination. Although FasL was not changed by all treatments, we found that doxorubicin enhanced Fas oligomerization (i.e. activation) independent of FasL. Selenium, on the other hand, increased the expression of FADD, the latter is a key adaptor molecule responsible for recruitment of caspase-8 to the Fas oligomer. The significance of the above changes was confirmed by the detection of more caspase-8 in either the Fas immunoprecipitate or FADD immunoprecipitate obtained from cells treated with the doxorubicin/selenium combination. The role of FADD upregulation was supported by the finding that knocking down FADD by siRNA attenuated the ability of doxorubicin/selenium to activate caspase-8.