Journal: ACS Omega

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and Its Semisynthetic Derivatives on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.1021/acsomega.6c00504

Figure Lengend Snippet: Effect of brazilin and derivatives on cell viability of breast cancer and nontumorigenic epithelial cells. Dose–response effects of DMSO and submaximal concentrations of brazilin and derivatives on breast cancer and nontumorigenic cell proliferation. Cell counting assay of proliferating cells treated with increasing concentrations (2.5–40 μM) of brazilin or derivatives is shown for (a-c) MDA-MB-231, (d-f) MCF7, and (g-i) MCF10A cells. Cells cultured in the presence of the DMSO vehicle were used as controls (see Supplementary Figure 1a,b additional DMSO controls). Live cells were quantified by Trypan blue exclusion at 24, 48, 72, and 96 h. The data represents the percentage of cell viability and are plotted as mean ± SE. Data represent three independent biological replicates. * p < 0.05, ** p < 0.001, *** p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317), MDA-MB-231 (CRM-HTB-26), and MCF7 (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Cell Counting, Cell Culture

Journal: ACS Omega

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and Its Semisynthetic Derivatives on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.1021/acsomega.6c00504

Figure Lengend Snippet: Four-parameter logistic dose–response curve fitting and morphological effects of brazilin and brazilin-(OAc)­3 in breast cell lines. (a–c) Dose–response curves for brazilin in MDA-MB-231 (a), MCF7 (b), and MCF10A (c) cells. (d–f) Dose–response curves for brazilin-(OAc) 3 in MDA-MB-231 (d), MCF7 (e), and MCF10A (f) cells. Curves were generated from cell viability measurements at 48 h and fitted using nonlinear regression (four-parameter logistic model; 4PL for MDA-MB-231 and MCF7, three-parameter logistic model with a fixed slope; 3PL for MCF10A) to estimate the concentration–response relationship and determine IC 50 and IC 10 values, representing the compound concentration required to reduce cell viability by 50% and 10%, respectively. (g) Representative brightfield microscopy images of MDA-MB-231, MCF7, and MCF10A cells after 96 h treatment with 20 μM brazilin or brazilin-(OAc) 3 . Images were captured using a NIKON ECLIPSE Ts2 microscope. Scale bar = 200 μm. Images illustrate representative fields corresponding to the cell counting assays used to generate growth curves. See Supplementary Figure 3 for images at 48 and 72 h. Data are derived from four independent biological replicates ( n = 4) and is plotted as mean ± SE.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317), MDA-MB-231 (CRM-HTB-26), and MCF7 (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Generated, Concentration Assay, Microscopy, Cell Counting, Derivative Assay

Journal: ACS Omega

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and Its Semisynthetic Derivatives on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.1021/acsomega.6c00504

Figure Lengend Snippet: Brazilin and its derivatives modulate apoptosis-related gene and protein expression in breast cell lines. Cells were treated with 20 μM brazilin or its derivatives for 48 h. (a) Steady state mRNA expression analyses was conducted by RT-qPCR to determine the expression of apoptosis-related genes: (a) BAX , (b) BAK , (c) BCL2 , and (d) BCL2L1 . GAPDH was used as control for normalization. Data are presented as mean ± SE from three independent biological replicates and are compared to nontreated control cells. Representative immunoblots and corresponding quantifications of apoptosis-associated proteins are shown for MDA-MB-231 (e–g), MCF7 (h–j), and MCF10A (k–m) cells, including BAX, BCL-2 and cleaved PARP. The BAX/BCL-2 ratio and cleaved PARP levels were quantified and are presented as mean ± SE from three independent biological replicates. Staurosporine (50 nM) was used as a positive control for apoptosis, and GAPDH served as a loading control. Statistical significance was determined relative to untreated cells and is indicated as * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317), MDA-MB-231 (CRM-HTB-26), and MCF7 (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Expressing, Quantitative RT-PCR, Control, Western Blot, Positive Control

Journal: ACS Omega

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and Its Semisynthetic Derivatives on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.1021/acsomega.6c00504

Figure Lengend Snippet: Antioxidant potential and pro-oxidant effects of brazilin and derivatives in breast cancer and nontumorigenic cells. (a) Radical scavenging activity of brazilin, brazilin-(OMe) 3 , and brazilin-(OAc) 3 (nontreated and 2.5–80 μM) was assessed using DPPH synthetic radicals. The percentage of scavenged radicals is plotted as mean ± SE; derivatives antioxidant potential is compared to brazilin antioxidant potential with a statistical significance of * p < 0.05, ** p < 0.01, *** p < 0.001, data from three independent replicates. (b) Cytoplasmic ROS levels were measured in MDA-MB-231, MCF7, and MCF10A cells after 48 h treatment with 20 μM of each compound by fluorescent detection of DHE oxidation. ( c – h ) Mitochondrial ROS (MitoSox) and mitochondrial membrane potential (TMRE) were evaluated in (c,f) MDA-MB-231, ( d , g ) MCF7, and ( e , h ) MCF10A cells treated for 48 h with 20 μM or 40 μM of brazilin or derivatives. FCCP was used as a positive control for mitochondrial depolarization. The percentage of fluorescence intensity of three independent biological replicates was plotted. Data show means ± SE of three independent biological replicates imaged. * p < 0.05, ** p < 0.001, *** p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317), MDA-MB-231 (CRM-HTB-26), and MCF7 (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Activity Assay, Membrane, Positive Control, Fluorescence

Journal: ACS Omega

Article Title: Cell Death Induced by Homoisoflavonoid Brazilin and Its Semisynthetic Derivatives on MDA-MB-231 and MCF7 Breast Cancer Cell Lines

doi: 10.1021/acsomega.6c00504

Figure Lengend Snippet: Validation of differentially expressed genes in breast cancer and nontumorigenic epithelial cells treated with brazilin and derivatives. Differentially expressed genes (DEG) identified by RNA-seq in MDA-MB-231 cells. (a) ATF3 , (b) H2BC21 , (c) SOX4 , (d) MTRNR2L8 , (e) MTRNR2L1 , (f) MTRNR2L2 , and (g) GCLM , were validated by RT-qPCR in MDA-MB-231, MCF7, and MCF10A cells treated with 20 μM brazilin or derivatives for 48 h. Data are expressed as mean ± SE from three independent biological replicates. * p < 0.05, ** p < 0.001, *** p < 0.001.

Article Snippet: Mammary gland-derived cell lines MCF10A (CRL-10317), MDA-MB-231 (CRM-HTB-26), and MCF7 (HTB-22) were purchased from the American Type Culture Collection (ATCC, Manassas, VA).

Techniques: Biomarker Discovery, RNA Sequencing, Quantitative RT-PCR