Fludarabine and Cladribine Induce Changes in Surface Proteins on Human B‑Lymphoid Cell Lines Involved with Apoptosis, Cell Survival, and Antitumor Immunity

Fludarabine and cladribine are purine analogues used to treat hematological malignancies. Alone or in combination with therapeutic antibodies, they are effective in treating patients with chronic lymphocytic leukemia and non-Hodgkin's lymphoma. However, the mechanisms of action of these drugs are not well understood. Plasma membrane proteins perform a variety of essential functions that can be affected by malignancy and perturbed by chemotherapy. Analysis of surface proteins may contribute to an understanding of the mechanisms of action of purine analogues and identify biomarkers for targeted therapy. The surface of human cells is rich in N-linked glycoproteins, enabling use of a hydrazide-coupling technique to enrich for glycoproteins, with iTRAQ labeling for quantitative comparison. A number of plasma membrane proteins on human leukemia and lymphoma cells were affected by treatment with a purine analogue, including decreases in CD22 (an adhesion and signaling molecule) and increases in CD205 (a “damaged cell marker”) and CD80 and CD50 (T-cell interaction molecules). Purine analogues may affect B-cell receptor (BCR) signaling and costimulatory molecules, leading to multiple signals for apoptosis and cell clearance. Fludarabine and cladribine induce differential effects, with some cell survival proteins (ECE-1 and CD100) more abundant after fludarabine treatment. Cell surface proteins induced by fludarabine and cladribine may be targets for therapeutic antibodies

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

Conditioned medium derived from RV-infected HBEC had increased concentrations of protein and IL-6; and caused a decrease in isoprenaline induced cAMP from ASMCs without an effect on ASMC viability.

<p>(A–B) HBEC (n = 4) were uninfected (Control) or exposed to: UV inactivated RV (UVi-RV) or replication competent RV (RV) at an MOI = 2 for 24 hours. The concentration of total protein and IL-6 in the supernatant was measured using a BCA assay and ELISA respectively. (C–D) ASMCs (n = 14) were treated with conditioned medium from HBEC (n = 3) that were uninfected, (Control) or exposed to: UV inactivated RV (UVi-RV) or replication competent RV (RV) at an MOI = 2 for 3 days. Isoprenaline induced cAMP was measured using a cAMP functional assay and ASMC viability was measured using a MTT assay. Data represent mean ± SEM. Statistical differences were examined for using 1-way ANOVA with Bonferroni post test comparison to control treatment *p<0.05.</p

The combination of TLR 3 and 7/8 agonists, and RNA extracted from RV-induced conditioned medium or RV stock caused PGE₂ induction and β₂ adrenoceptor desensitization on ASMCs, but not RNA extracted from HBECs.

<p>Control and RV-induced conditioned medium was generated from HBEC (n = 2) and pooled. ASMCs (n = 6) were treated with this pooled control or RV-induced conditioned medium; or untreated (BEGM), poly I:C (50 µg/mL), imiquimod (30 µg/mL) and poly I:C & imiquimod (50 µg/mL, 30 µg/mL respectively) in BEGM or in the presence of the control conditioned medium for 3 days (A, B). (Figure C-F) Control and RV-induced conditioned medium was generated from HBEC (n = 3) and pooled. Total RNA was extracted from: control- (53.91 ng/µL), RV-induced conditioned medium (300.25 ng/µL), RV stock (567.35 ng/µL) and cell lysate collected from a sub-confluent 75 cm² flask of HBEC (300 ng/µL) using a miRNeasy Mini purification kit and amount of RNA quantified using a spectrophotometer. ASMCs (n = 6) were treated with pooled control conditioned medium (control), or total extracted RNA collected from those sources in the presence of control conditioned medium for 3 days. PGE₂ was measured using an ELISA (A, C) and isoprenaline induced cAMP was measured using a cAMP functional assay (B, D-F). Data represent mean ± SEM. Statistical differences were detected using 1-way ANOVA with Bonferroni post test comparisons to respective BEGM, control conditioned medium only (Control) or Poly I:C *p<0.05, #p<0.05.</p

PGE₂ was successfully depleted from RV-induced conditioned medium from HBEC; but it still caused RV-induced β₂ adrenoceptor desensitization and further induced PGE₂ from ASMCs.

<p>HBEC (n = 3) were uninfected (Control) or exposed to: UV inactivated RV (UVi-RV) or replication competent RV (RV) at an MOI = 2 for 24 hours to generate conditioned medium. Conditioned medium was depleted of PGE₂ using affinity chromatography. The original conditioned medium, conditioned medium depleted of PGE₂ and PGE₂ eluted product were collected and the levels of PGE₂ (A) was measured using ELISA. ASMCs (n = 6) were then treated with the original conditioned medium, conditioned medium depleted of PGE₂ or PGE₂ eluted product in BEGM for 3 days. Isoprenaline induced cAMP was measured using a cAMP functional assay (B) and the level of PGE₂ released by ASMCs due to each component was measured using ELISA (C). Data represent mean ± SEM. Statistical differences were detected using a 2-way ANOVA (A&B) and 1-way ANOVA (C) with Bonferroni post test comparisons to the respective control conditioned medium *p<0.05; respective components of the original conditioned medium #p<0.05.</p