Biomarkers of Breast Cancer Apoptosis Induced by Chemotherapy and TRAIL

Treatment of breast cancer is complex and challenging due to the heterogeneity of the disease. To avoid significant toxicity and adverse side-effects of chemotherapy in patients who respond poorly, biomarkers predicting therapeutic response are essential. This study has utilized a proteomic approach integrating 2D-DIGE, LC–MS/MS, and bioinformatics to analyze the proteome of breast cancer (ZR-75-1 and MDA-MB-231) and breast epithelial (MCF-10A) cell lines induced to undergo apoptosis using a combination of doxorubicin and TRAIL administered in sequence (Dox-TRAIL). Apoptosis induction was confirmed using a caspase-3 activity assay. Comparative proteomic analysis between whole cell lysates of Dox-TRAIL and control samples revealed 56 differentially expressed spots (≥2-fold change and <i>p</i> < 0.05) common to at least two cell lines. Of these, 19 proteins were identified yielding 11 unique protein identities: CFL1, EIF5A, HNRNPK, KRT8, KRT18, LMNA, MYH9, NACA, RPLP0, RPLP2, and RAD23B. A subset of the identified proteins was validated by selected reaction monitoring (SRM) and Western blotting. Pathway analysis revealed that the differentially abundant proteins were associated with cell death, cellular organization, integrin-linked kinase signaling, and actin cytoskeleton signaling pathways. The 2D-DIGE analysis has yielded candidate biomarkers of response to treatment in breast cancer cell models. Their clinical utility will depend on validation using patient breast biopsies pre- and post-treatment with anticancer drugs

Biomarkers of Breast Cancer Apoptosis Induced by Chemotherapy and TRAIL

Treatment of breast cancer is complex and challenging due to the heterogeneity of the disease. To avoid significant toxicity and adverse side-effects of chemotherapy in patients who respond poorly, biomarkers predicting therapeutic response are essential. This study has utilized a proteomic approach integrating 2D-DIGE, LC–MS/MS, and bioinformatics to analyze the proteome of breast cancer (ZR-75-1 and MDA-MB-231) and breast epithelial (MCF-10A) cell lines induced to undergo apoptosis using a combination of doxorubicin and TRAIL administered in sequence (Dox-TRAIL). Apoptosis induction was confirmed using a caspase-3 activity assay. Comparative proteomic analysis between whole cell lysates of Dox-TRAIL and control samples revealed 56 differentially expressed spots (≥2-fold change and <i>p</i> < 0.05) common to at least two cell lines. Of these, 19 proteins were identified yielding 11 unique protein identities: CFL1, EIF5A, HNRNPK, KRT8, KRT18, LMNA, MYH9, NACA, RPLP0, RPLP2, and RAD23B. A subset of the identified proteins was validated by selected reaction monitoring (SRM) and Western blotting. Pathway analysis revealed that the differentially abundant proteins were associated with cell death, cellular organization, integrin-linked kinase signaling, and actin cytoskeleton signaling pathways. The 2D-DIGE analysis has yielded candidate biomarkers of response to treatment in breast cancer cell models. Their clinical utility will depend on validation using patient breast biopsies pre- and post-treatment with anticancer drugs

iTRAQ-Based Proteomic Profiling of Breast Cancer Cell Response to Doxorubicin and TRAIL

Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC–MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer

iTRAQ-Based Proteomic Profiling of Breast Cancer Cell Response to Doxorubicin and TRAIL

Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC–MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer

iTRAQ-Based Proteomic Profiling of Breast Cancer Cell Response to Doxorubicin and TRAIL

Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC–MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer

iTRAQ-Based Proteomic Profiling of Breast Cancer Cell Response to Doxorubicin and TRAIL

Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC–MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer

iTRAQ-Based Proteomic Profiling of Breast Cancer Cell Response to Doxorubicin and TRAIL

Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC–MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer

iTRAQ-Based Proteomic Profiling of Breast Cancer Cell Response to Doxorubicin and TRAIL

Breast cancer is a molecularly heterogeneous disease, and predicting response to chemotherapy remains a major clinical challenge. To minimize adverse side-effects or cumulative toxicity in patients unlikely to benefit from treatment, biomarkers indicating treatment efficacy are critically needed. iTRAQ labeling coupled with multidimensional LC–MS/MS of the enriched mitochondria and endoplasmic reticulum fraction, key organelles regulating apoptosis, has led to the discovery of several differentially abundant proteins in breast cancer cells treated with the chemotherapeutic agent doxorubicin followed by the death receptor ligand, TRAIL, among 571 and 801 unique proteins identified in ZR-75-1 and MDA-MB-231 breast cancer cell lines, respectively. The differentially abundant proteins represent diverse biological processes associated with cellular assembly and organization, molecular transport, oxidative stress, cell motility, cell death, and cancer. Despite many differences in molecular phenotype between the two breast cancer cell lines, a comparison of their subproteomes following drug treatment revealed three proteins displaying common regulation: PPIB, AHNAK, and SLC1A5. Changes in these proteins, detected by iTRAQ, were confirmed by immunofluorescence, visualized by confocal microscopy. These novel potential biomarkers may have clinical utility for assessing response to cancer treatment and may provide insight into new therapeutic targets for breast cancer

IGFBP-3 decreases vascular leakiness in the OIR model at P17.

<p><b>A–P</b> Retinal whole mounts following intravascular perfusion of HRP in OIR control plasmid injected eyes (A–D), contralateral uninjected eyes (<b>E–H</b>), OIR IGFBP-3 plasmid injected eyes (<b>I–H</b>) and control animals (<b>M–P</b>) in the peripheral and mid-peripheral regions. In the contralateral uninjected eye, HRP had leaked from within the vessel lumen and was evident in the tissue parenchyma as a brown background staining. The outlines of the vessels were not sharp with diffuse HRP reaction product (brown color) in the parenchyma. The control plasmid injected eyes had more leaking. In contrast, both the IGFBP-3-injected eyes and P17 control eyes showed high levels of contrast between the vessel lumen and the tissue parenchyma, illustrating that the HRP reaction product was well retained within the vessel lumen and indicating an intact BRB since HRP has a similar molecular weight to albumin. <b>Q</b> The HRP average intensity was determined within the vessel lumen and in the immediate adjacent parenchyma where luminal values acted as the denominator. The superficial and deep vascular plexii were captured and analyzed using LMS 510 software to provide a relative quantitative index of HRP retention, where an index of 1, is assumed for age-matched controls. During the hypoxic phase of OIR, in the neovasculature of the contralateral uninjected eyes had a HRP leakage index of 0.875±0.006 in the superficial plexus and 0.890±0.014 in the deep plexus (P<0.05, Kruskal-Wallis test). The HRP leakage index in control plasmid injected retinas were 0.847±0.016 in superficial plexus and 0.833+0.033 in deep plexus (P<0.05, Kruskal-Wallis test). In contrast, IGFBP-3 plasmid injected eyes had a HRP leakage index of 1.023±0.025 in the superficial plexus and 1.070±0.051 in the deep layer compared with an index of 1 for the age-matched control eyes (not statistically significant) indicative of the enhanced barrier function of the neovascularization of the OIR model after IGFBP-3 injection. The barrier properties of the vessels in IGFBP-3 injected eyes was found to be significantly (P<0.05) higher than contralateral uninjected eyes or plasmid-injected eyes, similar to that observed in healthy P17 control eyes in both superficial and deep vascular plexuses. Calibration in B is applied through A to P.</p

IGFBP-3 stimulates NO release in intact arteries by activating scavenger receptor-B1.

<p><b>A</b>. Intraluminal IGFBP-3 increased NO generation, determined by DAF-FM fluorescence, in rat PCAs pressurized at 70 mmHg: i) bright field image of pressure-mounted artery; ii) DAF-FM fluorescence image of the arterial segment with vehicle intraluminally applied; iii) image of an artery treated with IGFBP-3, iv) image of an artery treated with L-NAME and IGFBP-3; and v) image of an artery treated with SRB1-Ab and IGFBP-3. Shown in the left was color scale for DAF-FM fluorescence. <b>B</b>. Quantification of the effect of IGFBP-3 in the presence or the absence of different blockers on myogenic tone and NO generation. DAF-FM fluorescence, expressed as arbitrary fluorescence units (AFU) was significantly increased (*P<0.02, n = 5) by IGFBP-3 compared to vehicle-control. Effects of IGFBP-3 were inhibited by 300 µM L-NAME (γ P<0.03, n = 4) or 1∶100 SRB1-Ab (Ψ P<0.02, n = 4).</p