We also tested the levels of ER (Estrogen Receptor alpha), since it is known that: melatonin downregulates ER in MCF-7 cells , doxorubicin upregulates ER , and high levels of TWIST1 correlate with low levels of ER . of breast cancer, angiogenesis and clock genes. Moreover, melatonin regulates the levels JNJ0966 of TWIST1-related microRNAs, such as miR-10a, miR-10b and miR-34a. Since TWIST1 plays a pivotal role in the epithelial to mesenchymal transition, acquisition of metastatic phenotype and angiogenesis, our results suggest that inhibition of TWIST1 by melatonin might be a crucial mechanism of overcoming resistance and improving the oncostatic potential of doxorubicin in estrogen-dependent breast malignancy cells. < 0.001 vs. C; b, < 0.05 vs. C; c, < 0.01 vs. D (10 nM); d, < 0.01 vs. D (1 nM). 2.2. Effects of Melatonin and Doxorubicin on Cell Migration and Invasion in MDA-MB-231 and MCF-7 Cells We next investigated the effects of doxorubicin and melatonin around the migratory capacity of MCF-7 and MDA-MB-231 cells by using wound-healing assays. As shown in Physique 2A,B, doxorubicin treatment did not alter cell migration in MCF-7 cells, whereas melatonin significantly decreased cell migration either alone or in combination with doxorubicin. In marked contrast, neither doxorubicin nor melatonin had any effect on the migratory capacity of MDA-MB-231 cells (Physique 2C,D). When the invasive potential was tested, we found that doxorubicin enhanced the invasive potential of MCF-7 cells, whereas addition of melatonin counteracted this stimulatory effect (Physique 2G,H). In contrast, the invasive potential of MDA-MB-231 was not altered by doxorubicin, and melatonin treatment did not have JNJ0966 any significant effect (Physique 2E,F). Open in a separate window Physique 2 Effect of melatonin and doxorubicin on migration and on the invasive potential of MCF-7 and MDA-MB-231 cells. A, C Effects of melatonin (1 nM) and doxorubicin (1 M) on MCF-7 (A) or MDA-MB-231 (C) cell migration analyzed through the wound JNJ0966 healing assay. Quantification of MCF-7 (B) or MDA-MB-231 (D) cell migration was expressed as mean SEM. Representative microphotographs of initial and after 24 h are shown. (E,G) Effects of melatonin (1 nM) and doxorubicin (1 M) on MCF-7 (E) or MDA-MB-231 (G) invasive potential. Representative images from the 3D invasion assays of cell spheroids embedded into a collagen matrix at initial (= 0 h) and final time (= 24 h) for the different treatments are shown. (F,H) Graphs represent the quantification of the invasive area of MDA-MB-231 (F) or MCF-7 (H) cells at the indicated occasions. Data was expressed as mean SEM. A, C: Scale bar: 500 m; E, G: Scale bar: 100 m. 2.3. Effects of Doxorubicin and Melatonin around the Expression of Cancer-Related Genes We used the human breast PIK3C2G malignancy RT2 Profiler PCR Array to assess the expression changes in MCF-7 cells upon treatment with doxorubicin (1 M) either alone or combined with a physiological dose of melatonin (1 nM). The RT2 Profiler PCR Array allows the simultaneous analysis of 84 genes involved in various different key processes for breast JNJ0966 cancer biology, such as angiogenesis, cell adhesion, proteases, breast malignancy classification markers, signal transduction, cell cycle, transcription factors, apoptosis, DNA damage and repair. As shown in Table 1, doxorubicin alone upregulated the expression of 27 genes JNJ0966 and downregulated 17 genes. Table 1 The table summarizes the distribution of breast cancer gene categories induced or repressed in MCF-7 cells treated with doxorubicin (1 M), doxorubicin plus melatonin (1 nM) or melatonin (1 nM) for 6 h. Pathway-focused gene expression profiling was performed using the Human Breast Malignancy RT2 Profiler PCR Array. The number of up and downregulated genes in each category is usually indicated. (phosphatase and tensin homolog), (constitutive photomorphogenic 1) and (cyclin-dependent kinase inhibitor.