Synuclein gamma expression enhances radiation resistance of breast cancer cells

Abstract

Resistance to therapy is a major obstacle for the effective treatment of cancer. Expression of synuclein-gamma (SNCG) has been associated with poor prognosis and resistance to therapy. While reports on SNCG overexpression contributing to chemoresistance exist, limited information is available on the relationship between SNCG and radioresistance of cancer cells. Here we investigated the role of SNCG in radiation resistance in breast cancer cells. siRNA mediated knockdown of SNCG (siSNCG) markedly reduced SNCG protein level compared to scrambled siRNA (siScr) treatment. Furthermore, siSNCG treatment sensitized Estrogen Receptor-positive breast cancer cells (MCF7 and T47D) to ionizing radiation at 4 to 12 Gy as evidenced by the significant increase of apoptotic or senescent cells and reduction in clonogenic cell survival in siSNCG treated cells compared to siScr treated cells. On the other hand, we established an in vitro model of SNCG ectopic expression by using a triple-negative breast cancer cell line (SUM159PT) to further investigate the radioprotective effect of SNCG. We showed that ectopic expression of SNCG significantly decreased apoptosis of SUM159PT cells and enhanced clonogenic cell survival after radiation treatment. At the molecular level, after irradiation, the p53 pathway was less activated when SNCG was present. Conversely, p21^Waf1/Cip1 expression was upregulated in SNCG-expressing cells. When p21 was down-regulated by siRNA, radiosensitivity of SNCG-expressing SUM159PT cells was dramatically increased. This suggested a possible connection between p21 and SNCG in radioresistance in these cells. In conclusion, our data provide for the first time experimental evidence for the role of SNCG in the radioresistance of breast cancer cells.

Keywords: biomarker; breast cancer; radiation; resistance; synuclein gamma.

Figure 1. Expression of SNCG in breast cancer cell lines

(A) Expression was analyzed by qRT-PCR using total RNA from different breast cancer cell lines. Levels of SNCG expression were normalized to those of RPLP0 internal control. Graph shows fold enrichment over normal immortalized breast epithelial hTERT-HME1 cells. Data represent the mean (± standard deviation, SD) of three independent experiments. (B) Representative immunoblot analysis (n=3) was performed on whole cell lysate for SNCG expression using anti β-actin antibody as a loading control.

Figure 2. Inhibition of SNCG expression increases T47D cell radiosensitivity

(A) siSNCG-treated T47D cells showed reduced RNA (left panel) and protein (middle and right panels) expression compared to siScramble (siScr)-treated T47D cells. Relative expression of SNCG mRNA (left panel) was assessed by qRT-PCR analysis performed in triplicate (normalized against RPLP0). Representative immunoblot analysis (middle panel) was performed on whole cell lysate for SNCG expression using anti β-actin antibody as a loading control. Bar graph (right panel) shows quantitative analysis of scanning densitometric values of SNCG protein as ratio to β-actin protein. Data represent mean values ± standard error of the mean of two (RNA) and three (protein) independent experiments. (B) siSNCG-treated T47D cells showed increased apoptosis after radiation treatment (8 and 12 Gy) compared to siScramble (siScr)-treated T47D cells. Flow cytometry analysis was carried out to detect apoptotic and necrotic cells. Histogram shows percentage of apoptotic cells 72 hours after irradiation (left panel). Data represent mean values ± standard error of the mean of four independent experiments. Representative experiment of flow cytometry analysis (n=4) shows the percentage of Annexin-V and propidium iodide staining of T47D cells irradiated or not at a dose of 8 or 12 Gy (right panel). (C) Radiation sensitivity was determined from the number of viable cells at different times after irradiation at 4, 8, and 12 Gy using the resazurin-based cell viability assay. The upper panel shows representative growth curves of siScr- or siSNCG-treated cells. Curves from three independent experiments were used as basis for calculation of doubling time in hours (hr) (lower panel) ^** = P value < 0.01; ns = not significant.

Figure 3. Inhibition of SNCG expression increases MCF7 cell radiosensitivity

(A) siSNCG-treated MCF7 cells showed reduced RNA (left panel) and protein (middle and right panels) expression compared to siScramble (siScr)-treated cells. Relative expression of SNCG mRNA (left panel) was assessed by qRT-PCR analysis performed in triplicate (normalized against RPLP0). Representative immunoblot analysis (middle panel) was performed on whole cell lysate for SNCG expression using anti β-actin antibody as a loading control. Bar graph (right panel) shows quantitative analysis of scanning densitometric values of SNCG protein as ratio to β-actin protein. Data represent mean values ± standard error of the mean of two (RNA) and three (protein) independent experiments. (B) siSNCG-treated MCF7 cells showed decreased clonogenic potential after radiation treatments (4 and 8 Gy) compared to siScramble (siScr)-treated cells. Clonogenic cell survival assay was performed and curves show the percentage of survival after irradiation (left panel). Data represent mean values ± standard error of the mean of three independent experiments, each done in triplicate. Representative pictures (n=3) of 2-week-old colonies after fixation and crystal violet staining (right panel). (C) siSNCG-treated MCF7 cells showed increased cellular senescence after radiation treatments (4 and 8 Gy) compared to siScramble (siScr)-treated cells. Cellular senescence was evaluated by the detection of SA-β-galactosidase activity and curves show the percentage of SA-β-gal positive cells as mean values ± standard error of the mean of three independent experiments (left panel). Representative images of SA-β-gal positive cells (blue) are shown (right panel) (scale bar = 100 μm). ^*** = P value < 0.005 ; ^** = P value < 0.01 ; ns = not significant.

Figure 4. Ectopic expression of SNCG in SUM159PT cells increases their resistance to ionizing radiation

(A) SUM159PT cells were transfected either with pCMV6-SNCG-GFP or pCMV6-A-GFP vector as a control (CTL) and GFP-positive cells were sorted by FACS. Representative photographs of SUM-CTL-GFP (left panels) and SUM-SNCG-GFP (right panels) cells expressing GFP (green) and stained with DAPI nuclear stain (blue); scale bar = 10 μm. (B) SNCG expression was analyzed by qRT-PCR using total RNA from SUM-CTL-GFP and SUM-SNCG-GFP (left panel). Levels of SNCG expression were normalized to those of RPLP0 internal control. Graph shows fold enrichment over SUM-CTL-GFP cells. Data represent mean values of two independent experiments performed in triplicate. Representative immunoblot analysis (n=3) was performed on whole cell lysate for SNCG-GFP fusion protein expression using anti β-actin antibodies as a loading control (right panel). (C) SUM-SNCG-GFP cells showed significant decreased apoptosis after radiation treatment (12 Gy) compared to SUM-CTL-GFP cells. Flow cytometry was done to measure apoptotic and necrotic cells. Histogram shows the percentage of apoptotic cells 72 hours after irradiation (left panel). Data represent mean values ± standard error of the mean of four independent experiments. Representative experiment of flow cytometry analysis (n=4) shows the percentage of Annexin-V and propidium iodide staining of SUM-CTL-GFP and SUM-SNCG-GFP cells irradiated or not at a dose of 12 Gy (right panel). (D) SUM-SNCG-GFP cells showed significant increased clonogenic potential after irradiation at 4 Gy compared to SUM-CTL-GFP cells. Clonogenic cell survival assay was performed; curves and bar graph show the percentage of survival after irradiation (left panel). Representative pictures of 2-week-old colonies after fixation and crystal violet staining (right panel). Data represent mean values ± standard error of the mean of three independent experiments. ^** = P value < 0.01; ^* = P value < 0.05 ; ns = not significant.

Figure 5. SNCG expression attenuates p53 pathway activation and increases p21^Waf1/Cip1 expression in SUM159PT cells

(A) Representatives pictures (n=3) of immunoblotting of phospho-p53 (Ser15), total-p53, p21, and β-actin on whole cell lysates of SUM-CTL-GFP and SUM-SNCG-GFP cells irradiated or not at a dose of 4, 8, or 12 Gy. Cells were lysed 24 hours after irradiation. (B) Bar graph shows quantitative analysis of scanning densitometric values of p21 protein as ratio to β-actin protein (left panel). Bar graph shows qRT-PCR analysis of p21 RNA (normalized against RPLP0) as fold enrichment over SUM-CTL-GFP cells (right panel). Data represent mean values ± standard error of the mean of three independent experiments. (C) sip21-treated cells showed reduced protein expression compared to siScramble (siScr)-treated cells. Representative immunoblot analysis was performed on whole cell lysate for p21 expression using anti β-actin antibody as a loading control (left panel). Bar graph shows quantitative analysis of scanning densitometric values of p21 protein as ratio to β-actin protein (right panel). Data represent mean values ± standard error of the mean of three independent experiments. (D) sip21-treated cells showed increased apoptosis after radiation treatment (8 and 12 Gy) compared to siScramble (siScr)-treated cells. Flow cytometry analysis was done to detect apoptotic and necrotic cells. Bar graph shows the percentage of apoptotic cells 96 hours after irradiation. Data represent mean values ± standard error of the mean of three independent experiments. ^*** = P value < 0.005, ^** = P value < 0.01, ^* = P value < 0.05.