Rational incorporation of selenium into temozolomide elicits superior antitumor activity associated with both apoptotic and autophagic cell death.

BACKGROUND: The DNA alkylating agent temozolomide (TMZ) is widely used in the treatment of human malignancies such as glioma and melanoma. On the basis of previous structure-activity studies, we recently synthesized a new TMZ selenium analog by rationally introducing an N-ethylselenocyanate extension to the amide functionality in TMZ structure. PRINCIPAL FINDINGS: This TMZ-Se analog showed a superior cytotoxicity to TMZ in human glioma and melanoma cells and a more potent tumor-inhibiting activity than TMZ in mouse glioma and melanoma xenograft model. TMZ-Se was also effective against a TMZ-resistant glioma cell line. To explore the mechanism underlying the superior antitumor activity of TMZ-Se, we compared the effects of TMZ and TMZ-Se on apoptosis and autophagy. Apoptosis was significantly increased in tumor cells treated with TMZ-Se in comparison to those treated with TMZ. TMZ-Se also triggered greater autophagic response, as compared with TMZ, and suppressing autophagy partly rescued cell death induced by TMZ-Se, indicating that TMZ-Se-triggered autophagy contributed to cell death. Although mRNA level of the key autophagy gene, Beclin 1, was increased, Beclin 1 protein was down-regulated in the cells treated with TMZ-Se. The decrease in Beclin 1 following TMZ-Se treatment were rescued by the calpain inhibitors and the calpain-mediated degradation of Beclin1 had no effect on autophagy but promoted apoptosis in cells treated with TMZ-Se. CONCLUSIONS: Our study indicates that incorporation of Se into TMZ can render greater potency to this chemotherapeutic drug

TMZ-Se is more apoptogenic than TMZ in tumor cells.

<p>(<b>A</b>) LN229 and T98G cells were treated with 100 µM or 200 µM of TMZ or TMZ-Se for 48 h, and apoptosis was examined by flow cytometric analysis of Annexin V and 7-AAD staining. (<b>B</b>) LN229 and T98G cells were treated with TMZ or TMZ-Se for 48h, and the levels of caspse-9, caspase-3, PARP and survivin were measured by Western blot analysis. Tubulin was used as a loading control. (<b>C</b>) LN229 and T98G cells were treated with TMZ-Se for 48h in the absence or presence of Z-VAD, and cell viability was measured by MTT assay. (<b>D</b>) 1205LU and UACC cells were treated with TMZ or TMZ-Se for 48h, and the levels of PARP and survivin were measured by Western blot. Tubulin was used as a loading control. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

TMZ-Se triggers a greater autophagic response than TMZ, and inhibition of autophagy decreases the efficacy of TMZ-Se against glioma cells.

<p>(<b>A</b>) <i>Left panels</i>: LN229 and T98G cells were treated with TMZ or TMZ-Se for 48 h, and the level of LC3 was measured by Western blot analysis. Tubulin was used as a loading control. <i>Right panels</i>: LN229 and T98G cells were treated with TMZ-Se for 48 h in the presence or absence of bafilomycinA1, and the level of LC3 was measured by Western blot analysis. Tubulin was used as a loading control. (<b>B</b>) LN229 and T98G cells were transfected with a GFP-LC3 plasmid, followed by treatment with TMZ-Se for 48h. At the end of treatment, the cells were observed under fluorescence microscope. (<b>C</b>) T98G cells treated with TMZ-Se or vehicle were harvested by trypsinization, fixed and embedded in spur resin. Ninety nm thin sections were cut and examined at 80 Kv with a JEOL 1200EX transmission electron microscope. (<b>D</b>) LN229 and T98G cells were treated with TMZ-Se for 48h in the absence or presence of 3-MA or bafilomycinA1, and cell viability was measured by MTT assay. (<b>E</b>) LN229 and T98G cells were transfected with an Atg5-targeted siRNA, and then treated with TMZ-Se for 48h. Cell viability was measured by MTT assay. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</p

Chemical structures of TMZ-Se and TMZ.

<p>Chemical structures of TMZ-Se and TMZ.</p

TMZ-Se induces calpain-mediated degradation of Beclin 1.

<p>LN229 and T98G cells were treated with 100 or 200 µM of TMZ or TMZ-Se for 48h, (<b>A</b>) the levels of Beclin 1 and Bcl-2 were measured by Western blot. Tubulin was used as a loading control; (<b>B</b>) the expression of <i>beclin1</i> mRNA was measured by qRT-PCR. (<b>C</b>) LN229 and T98G cells were treated with 100 or 200 µM of TMZ-Se for 48h in the presence or absence of 10µM of MG132, and the level of Beclin 1 were measured by Western blot. Tubulin was used as a loading control. (<b>D</b>) LN229 and T98G cells were treated with TMZ-Se for 48h in the presence or absence of 20µM ALLM (<i>left panel</i>) or 10µg/ml E64D (<i>right panel</i>), and the level of Beclin1 was measured by Western blot. Tubulin was used as a loading control. (<b>E</b>) LN229 and T98G cells were treated with TMZ-Se for 48h in the presence or absence of 20µM ALLM or 10µg/ml E64D, and cell viability was measured by MTT assay. **<i>p</i> < 0.01.</p