To our knowledge, the consequences of elevated Pi on behavior of breast cancer cells have been poorly resolved. cell cycle progression, without apoptosis event. We found that Pi causes cells to accumulate in G1 phase inside a time-dependent manner. Accordingly, G1 build up was associated with a decrease of cyclin A and cyclin E and an increase of cell cycle inhibitors p21 and p27 protein levels, respectively. Moreover, the Pi-induced antiproliferative effect was dynamically accompanied by profound changes in ERK1/2 and STAT3 protein and phosphorylation levels in response to Pi. Altogether, our data represent the first evidence of Pi acting as a novel signaling molecule in MDA-MB-231 breast cancer cells, capable of eliciting a strong antiproliferative action and suggest that targeting Pi levels at local sites might represent the rationale for developing novel strategies for therapeutic intervention in triple-negative breast malignancy. for 5?min, and pellets were washed once with Nocodazole ice-cold PBS and centrifuged for a further 5?min. Pellets were resuspended in 0.5?mL of DNA staining solution (50?g/mL of propidium iodide [PI] and 100?g of RNase A in PBS), and incubated at 37C for 1?h in the dark. Samples were transferred to 5-mL Falcon tubes and stored on ice until assayed. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) interfaced with a Hewlett-Packard computer (mod. 310) for data analysis performed with the ModiFIT Cell Cycle Analysis software. For the evaluation of intracellular DNA contents, at least 20,000 events for each point were analyzed, and regions were set up to acquire quantitative data of cells that fell into the normal G1, S, and G2 regions and with fragmented DNA (sub-G1 or apoptotic events).12,14 Preparation of cell lysates Cell extracts were prepared as follows. Briefly, three to five volumes of RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 10?g/mL aprotinin, leupeptin, and 1?mM phenylmethylsulfonyl fluoride were added to recovered cells. After incubation on ice for 1?h, samples were centrifuged at 18,000 in an Eppendorf microcentrifuge for 15?min at 4C and the supernatant (SDS total extract) was recovered. Some aliquots were taken for protein quantification according to Bradford method (Bradford, 1976); others were diluted in 4Laemmli buffer, boiled, and stored as samples for immunoblotting analysis.16 Immunodetection of proteins Typically, we employed 20C40?g of total extracts for immunoblotting. Proteins from cell preparations were separated by SDS-PAGE and transferred onto nitrocellulose linens (Schleicher & Schuell, Dassel, Germany) by a Mini Trans-Blot apparatus BioRad (Hercules, CA). II Goat anti-rabbit or anti-mouse antibodies, conjugated with horseradish peroxidase (BioRad), were used as a detection system (ECL) according to the manufacturer’s instructions (Amersham Biosciences, Amersham, United Kingdom).17 Statistical analysis Experiments were performed three times with replicate samples, except where otherwise indicated. Data are plotted as meanSD (standard deviation). The means were compared using analysis of variance (ANOVA) plus Bonferroni’s values of less than 0.05 were considered significant. National Institutes of Health Image J 1.42Q (NIH, Bethesda, MD) software was used for densitometric analysis. Results Pi inhibits proliferation of human MDA-MB-231 breast malignancy cells The triple-negative human breast cancer cell line MDA-MB-231 is usually a well-established and widely used model system of highly aggressive breast malignancy cells.18,19 To evaluate the consequences of elevated Pi on behavior of breast cancer cells, first we looked at the impact of Pi on proliferation of MDA-MB-231 cells. To this purpose, first we performed doseCresponse experiments. Throughout our experiments, we used a spectrum of final concentration of Pi in agreement with most of the published studies on Pi-triggered effects.9C13 MDA-MB-231 cells were incubated with increasing (2.5, 5, and 10?mM) concentrations of Pi for 72?h, and then cell proliferation was determined by conventional MTT assay and by direct cell number counting. Physique 1A shows that Pi causes a statistically significant reduction of cell viability ( em p /em 0.05) in a dose-dependent manner of 12%, 35%, and 40% at 2.5, 5, and 10?mM concentrations, respectively. Open in a separate windows FIG. 1. Effects of inorganic phosphate (Pi) around the proliferation of MDA-MB-231 breast malignancy cells. (A) DoseCresponse. MDA-MB-231 cells were cultured in medium supplemented with 2.5, 5, and 10?mM Pi or not (control) for 72?h. (B) Time-course. MDA-MB-231 cells were cultured in medium supplemented with 5?mM Pi or not (control) for 24, 48, 72?h. Then, cell viability was measured by MTT assay. (C) MDA-MB-231 cells were plated at 5105/10?cm plate, cultured in medium supplemented with 5?mM Pi or not (control) for 24, 48, 72?h and cell number recorded. Data represent the average of three impartial experiments. The means and SD are shown. * em p /em 0.05 vs. control untreated cells. Next, we performed time-course experiments. MDA-MB-231 cells were exposed to 5?mM Pi (submaximal dose) for up to 72?h, after which cell proliferation was determined by conventional MTT assay and by direct cell number counting. Figure 1B, shows that Pi caused a statistically significant reduction of cell.(A) MDA-MB-231 cells were cultured in medium supplemented with 5?mM Pi or not (control) for 24, 48, and 72?h. Pi acting as a novel signaling Rabbit Polyclonal to Heparin Cofactor II molecule in MDA-MB-231 breast cancer cells, capable of eliciting a strong antiproliferative action and suggest that targeting Pi levels at local sites might represent the rationale for developing novel strategies for therapeutic intervention in triple-negative breast malignancy. for 5?min, and pellets were washed once with ice-cold PBS and centrifuged for a further 5?min. Pellets were resuspended in 0.5?mL of DNA staining solution (50?g/mL of propidium iodide [PI] and 100?g of RNase A in PBS), and incubated at 37C for 1?h in the dark. Samples were transferred to 5-mL Falcon tubes and stored Nocodazole on ice until assayed. Flow cytometric analysis was performed using a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA) interfaced with a Hewlett-Packard computer (mod. 310) for data analysis performed with the ModiFIT Cell Cycle Analysis software. For the evaluation of intracellular DNA contents, at least 20,000 events for each point were analyzed, and regions were set up to acquire quantitative data of cells that fell into the normal G1, S, and G2 regions and with fragmented DNA (sub-G1 or apoptotic events).12,14 Preparation of cell lysates Cell extracts were prepared as follows. Briefly, three to five volumes of RIPA buffer (PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) containing 10?g/mL aprotinin, leupeptin, and 1?mM phenylmethylsulfonyl fluoride were added to recovered cells. After incubation on ice for 1?h, samples were centrifuged at 18,000 in an Eppendorf microcentrifuge for 15?min at 4C and the supernatant (SDS total extract) was recovered. Some aliquots were taken for Nocodazole protein quantification according to Bradford method (Bradford, 1976); others were diluted in 4Laemmli buffer, boiled, and stored as samples for immunoblotting analysis.16 Immunodetection of proteins Typically, we employed 20C40?g of total extracts for immunoblotting. Proteins from cell preparations were separated by SDS-PAGE and transferred onto nitrocellulose linens (Schleicher & Schuell, Dassel, Germany) by a Mini Trans-Blot apparatus BioRad (Hercules, CA). II Goat anti-rabbit or anti-mouse antibodies, conjugated with horseradish peroxidase (BioRad), were used as a detection system (ECL) according to the manufacturer’s instructions (Amersham Biosciences, Amersham, United Kingdom).17 Statistical analysis Experiments were performed three times with replicate samples, except where otherwise indicated. Data are plotted as meanSD (standard deviation). The means were compared using analysis of variance (ANOVA) plus Bonferroni’s values of less than 0.05 were considered significant. National Institutes of Health Image J 1.42Q (NIH, Bethesda, MD) software was used for densitometric analysis. Results Pi inhibits proliferation of human MDA-MB-231 breast malignancy cells The triple-negative human breast cancer cell line MDA-MB-231 is usually a well-established and widely used model system of highly aggressive breast malignancy cells.18,19 To evaluate the consequences of elevated Pi on behavior of breast cancer cells, first we looked at the impact of Pi on proliferation of MDA-MB-231 cells. To this purpose, first we performed doseCresponse experiments. Throughout our experiments, we used a spectrum of final concentration of Pi in agreement with most of the published studies on Pi-triggered effects.9C13 MDA-MB-231 cells were incubated with increasing (2.5, 5, and 10?mM) concentrations of Pi for 72?h, and then cell proliferation was determined by conventional MTT assay and by direct cell number counting. Figure 1A shows that Pi causes a statistically significant reduction of cell viability ( em p /em 0.05) in a dose-dependent manner of 12%, 35%, and 40% at 2.5, 5, and 10?mM concentrations, respectively. Open in a separate windows FIG. 1. Effects of inorganic phosphate (Pi) around the proliferation of MDA-MB-231 breast malignancy cells. (A) DoseCresponse. MDA-MB-231 cells were cultured in medium supplemented with 2.5, 5, and 10?mM Pi or not (control) for 72?h. (B) Time-course. MDA-MB-231 cells were cultured in medium supplemented with 5?mM Pi or not (control) for 24, 48, 72?h. Then, cell viability was measured by MTT assay. (C) MDA-MB-231 cells were plated at 5105/10?cm plate,.