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

Effects of TMZ-Se and TMZ on tumor growth in mouse glioma and melanoma xenograft models.

<p>(<b>A and B</b>) The human glioma cells LN229 (1×10⁵ cells in 15 µl of DMEM medium) were injected into the brains of 6-week-old male BALB/c nude mice at 4 mm depth under anesthesia with chloralic hydras (4%, 2ml/kg, ip). Three days after tumor cell implantation, mice were randomly divided into three groups (15 mice/group). Treatments were begun on day 4. TMZ-SE (15 mg/kg), TMZ (15 mg/kg) or vehicle (10% DMSO in saline) was given p.o. daily for 2 weeks. (<b>A</b>) At day 7 and day 21 after tumor cell implantation, the mice were euthanized, and the brains were fixed in 10% buffered formalin, embedded in paraffin, and then stained with hematoxillin-eosin (H&E). The images shown are the representative of 5 mice from each group; (<b>B</b>) The kaplan-Meier survival curves, n = 10; (<b>C</b>) Nude mice (swiss^nu/nu) were inoculated s.c. with UACC903 human melanoma cells (1×10⁶ cells/100 µl/mouse). When the tumors reached 50∼100 mm³ in volume, TMZ or TMZ-Se (15 mg/kg) was administered i.p. on days 1, 3, 5, 7 and 9. Tumor sizes and body weight of the animals were measured every other day. The differences between treatments were analyzed using a two-sample <i>t</i>-test. The survival curves of the tumor - bearing mice subjected to different treatments were estimated using Kaplan-Meier method and compared by log-rank statistic analysis.</p

Chemical structures of TMZ-Se and TMZ.

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