Several types of malignant tumors, such as glioblastoma multiforme and melanoma, are notoriously resistant to conventional chemotherapy. Even for TMZ, the frontline drug for treatment of glioblastoma multiforme and metastatic melanoma, its therapeutic outcomes are often disappointing. In the current study, we show that TMZ-Se, a new TMZ analog developed and synthesized by our group, is superior to TMZ in inhibiting viability of glioma and melanoma cells (), and in suppressing growth of tumor xenograts in animal models (). Noteworthily, TMZ-Se also showed efficacy against TMZ-resistant tumor cells (). The cytotoxic effect of methylating agents is believed to result from formation of O6
-MeG, which causes DNA DSBs that act as a trigger of apoptotic cell death 
. Also, a number of studies have shown that selenium has a potent anti-cancer activity that correlates with its apoptosis - inducing effects 
. These observations have recently led to development of several organoselenium small molecule compounds, both in our laboratories and elsewhere, which showed promising anti-tuomr properties 
. The introduction of selenium into TMZ structure was particularly challenging because the changes in structure should not compromise its ability to cross the blood brain barrier (BBB). Therefore, we designed TMZ-Se by extending the amide functionality in TMZ so as not to disturb the ring involved in degradation to active metabolites and responsible for 100% bioavailability and apparently the BBB crossing ability of TMZ 
. The -SeCN group was used as carrier of selenium to generate a second active moiety into TMZ structure since it is known to be efficiently metabolized to a selenol (-SeH) intermediate that is responsible for the redox cycling and anticancer properties of selenium. Our results suggest that TMZ-Se acts by retaining the properties of both TMZ and Se. The efficient reduction in brain tumor xenografts clearly indicates that TMZ-Se crosses the BBB when administered orally. This suggests that TMZ-Se metabolizes similar to TMZ, leading to active intermediates by cleaving the tetrazene ring,_which apparently is responsible for its crossing through the BBB. In addition, the enhanced cytotoxicity of TMZ-Se as compared to TMZ indicates the expected usual functioning of -SeCN group, which would cleave to generate a free selenol (-SeH) thus adding the redox cycling properties of Se and enhancing selective cytotoxicity to tumor cells.
We demonstrated that rational introduction of selenium into TMZ greatly increased the inducing effect on DNA DSB () and on apoptosis (). TMZ-induced apoptosis is a late response that needs at least two cell cycles after treatment 
. For instance, apoptosis in gliomas is only visible 4–6 days following TMZ treatment 
, and TMZ - treated melanoma cells start to undergo apoptosis 72 h later 
. It appears that TMZ-Se not only possesses stronger apoptosis - inducing activity but also triggers cell death more rapidly than TMZ, as at 48 h post-treatment, more marked apoptosis were observed in tumor cells treated with TMZ-Se than those treated with TMZ ().
Autophagy is a catabolic process by which damaged organelles and proteins are sequestered into autophagosome and subsequently degraded through fusion with lysosomes. It was found in both glioma cells and tissues that TMZ treatment caused activation of autophagy 
, but the precise roles of autophagy in determining the efficacy of this drug remains uncertain. We found that our new compound, TMZ-Se, induced stronger autophagic response than TMZ in glioma cells (). There has long been debate over whether autophagy is cell-killing or cell-protective in cancers. Autophagy was originally recognized as a cell-protective mechanism under various stressful conditions, but later was also found to play a death-promoting role, causing autophagic cell death 
. Here, we demonstrated that inhibition of autophagy decreased the efficacy of TMZ-Se against glioma (), indicating that autophagy plays a pro-death role and contributes to the antitumor effects of TMZ-Se. Interestingly, we found that Beclin 1, a critical regulator of autophagy, was decreased in cells treated with TMZ-Se (), and inhibition of calpain, a Ca2+
-dependent protease involved in control of cell proliferation and apoptosis, blocked the degradation of Beclin1 induced by TMZ-Se (). Calpain-triggered degradation of Beclin 1 has been reported by Yoo BH et al
. who demonstrated that calpain activity is required for Ras-dependent down-regulation of Beclin-1 
. Additionally, we found that the decrease in Beclin 1 protein was not accompanied by a reduction in LC3 but resulted in an increase in PARP (), suggesting that TMZ-Se triggers a non-canonical, Beclin 1-independent autophagy, and the calpain-mediated degradation of Beclin 1 promotes the apoptotic response to this agent. It was previously reported that Beclin 1 participates in inhibition of apoptosis, and that silencing of Beclin 1 expression augments the mitochondrial permeabilization and apoptosis induced by Fas stimulation or doxorubicin treatment in tumor cells 
. The previous studies also demonstrated that the interaction of Beclin 1 with either Bcl-2 or Bcl-XL is essential for the anti-apoptotic effects of Beclin 1 
. We have reported that inhibition of Beclin 1 increased the caspase activities in glioma cells treated with TRAIL via reducing survivin level 
. Here, we observed a decrease in Bcl-2 expression in glioma cells subjected to TMZ-Se treatment, indicating that the decrease of Beclin 1 leads to the dissociation of Beclin1 with Bcl-2, thereby promoting the degradation of Bcl-2 and causing apoptosis. All of these results suggest that there might be new mechanisms of action involved in the antitumor effects of TMZ-se.
TMZ-Se also showed a stronger antitumor activity in both the glioma and melanoma mouse models (), suggesting that TMZ selenium derivatives may hold promise for further developing as novel chemotherapeutic agents. We are planning further pre-clinical studies including pharmacokinetic comparisons to determine whether or not TMZ-Se warrants clinical trials in the hope of developing it to a useful and effective anticancer agent.
Taken together, by introducing selenium into TMZ, we have developed a novel TMZ analog that demonstrates superior anti-tumor activity against glioma and melanoma both in vitro and in vivo. This also suggests that by modifying the amide function, we were able to retain the BBB-penetrating capability of Se-TMZ while enhancing its tumor-inhibitory property. We believe that appropriately incorporating selenium into certain conventional anticancer drugs to improve their effectiveness might represent a new strategy of drug development that is worth further investigation.