Chemical structures of TMZ-Se and TMZ.

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

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 induces calpain-mediated degradation of Beclin 1.

<p>(<b>A</b>) LN229 and T98G cells were transfected with Beclin1siRNA followed by TMZ-Se treatment, and the levels of LC3, Beclin 1 and PARP were examined by Western blot. Tubulin was used as a loading control. (<b>B</b>) LN229 and T98G cells were transfected with a Beclin 1-targeted siRNA, followed by treatment with TMZ-Se for 48h. Cell viability was measured by MTT assay. *<i>p</i> < 0.05; **<i>p</i> < 0.01.</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

Effects of TMZ-Se and TMZ on phosphorylation of H2AX, and on activity of Akt-mTOR-S6K pathway.

<p>(<b>A</b>) T98G, LN229, 1205LU and UACC903 cells were treated with 100 µM or 200 µM of TMZ or TMZ-Se for 48h, and the level of phospho-H2AX was measured by Western blot. Tubulin was used as a loading control. (<b>B</b>) LN229 and T98G cells were treated with TMZ or TMZ-Se for 48 h, and the levels of p-Akt, Akt, p-mTOR, mTOR, p-S6K, and S6K were measured by Western blot analysis. Tubulin was used as a loading control.</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

Differential Gene Expression in Tamoxifen-Resistant Breast Cancer Cells Revealed by a New Analytical Model of RNA-Seq Data

<div><p>Resistance to tamoxifen (Tam), a widely used antagonist of the estrogen receptor (ER), is a common obstacle to successful breast cancer treatment. While adjuvant therapy with Tam has been shown to significantly decrease the rate of disease recurrence and mortality, recurrent disease occurs in one third of patients treated with Tam within 5 years of therapy. A better understanding of gene expression alterations associated with Tam resistance will facilitate circumventing this problem. Using a next generation sequencing approach and a new bioinformatics model, we compared the transcriptomes of Tam-sensitive and Tam-resistant breast cancer cells for identification of genes involved in the development of Tam resistance. We identified differential expression of 1215 mRNA and 513 small RNA transcripts clustered into ERα functions, cell cycle regulation, transcription/translation, and mitochondrial dysfunction. The extent of alterations found at multiple levels of gene regulation highlights the ability of the Tam-resistant cells to modulate global gene expression. Alterations of small nucleolar RNA, oxidative phosphorylation, and proliferation processes in Tam-resistant cells present areas for diagnostic and therapeutic tool development for combating resistance to this anti-estrogen agent.</p> </div

Most differentially-expressed genes revealed by both significance tests.

<p>Most differentially-expressed genes revealed by both significance tests.</p

NGS identification and comparison of differentially-expressed genes in TamR cells by the Fisher's exact test.

<p>(<b>A</b>) Total RNA from human breast cancer cell lines MCF-7 (TamS) and MTR-3 (TamR) were collected and subjected to the next generation sequencing process. (<b>B</b>) Gene expression followed a Poisson distribution with significantly differentially-expressed genes two standard deviations from the mean in the traditional method. The new method used the FET significance test. The change of the normalized smRNA exon reads (<b>C</b>) and intron reads (<b>D</b>), and exon reads for mRNA genes (<b>E</b>) from TamS to TamR cells is plotted against the mean expression between these two types of cells for the new method. Purple dots represent significantly expressed genes as determined by FET; gray dots represent genes with similar expression. The red horizontal line at zero provides visualization for the signs of differential expression.</p