Figure 6
From: Radiosensitization by a novel Bcl-2 and Bcl-XL inhibitor S44563 in small-cell lung cancer
Radiation induced the expression of anti-apoptotic proteins and sensitized SCLC to S44563. (a) H69 xenografts were given 3 Gy radiation from day 1 to day 4 (IR), twice (IR*2) when tumors recurred upon the first course of radiation and then were excised when the tumors recurred again. Protein lysates were obtained and blots were stripped with excised for Bcl-2 and Bcl-XL antibodies expression analysis. Protein/GAPDH ratio (from 0 to 1) were quantified and reported at the top of each lane. (b) H69 xenograft tumor curves after treatment with fractionated X-ray irradiation and S44563, alone or in concomitant and sequential combination. When tumors reached appropriate size, the mice were randomized into 5–10 mice per group and treated with either S44563 100 mg/kg i.p., × 5 days or X-ray irradiation 2 Gy × 4, × 1 week, or their combination as in Figure 5. A group in which mice were received S44563 only within the 5 days of the completion of radiotherapy was added. Standard errors are shown (columns, mean; bars, standard error deviation; *P<0.05, permutational t-tests two-way ANOVA test). (c) TNF and LTB gene expression induction following radiation in H16 and H69 cells using real-time PCR. H146 and H69 cells were treated with 2 Gy radiation and harvested either 2 or 24 h after the treatment (columns, mean; bars, standard error deviation; *P<0.05 as compared to GAPDH level). Each experiment has been carried out in triplicate. The pictures are representative of three experiments. (d) Accumulation of p65/RelA in cell nuclei after irradiation. Nuclear extracts from H146 cells were immunoblotted with specific antibodies to show p65/RelA. Blots were stripped and reprobed with mouse monoclonal anti-nucleoplasmin (NPM) as a control protein loading. Unirradiated cells served as a control. Data from three experiments were combined. (e) H146 cells were given one 3-Gy fraction and were then harvested 24 h later for Bcl-2 and, Bcl-XL immunoblotting. GAPDH was used as the loading control. (f) H146 cells and H196 cells were transfected with silencing RNA (siRNA) against p65/RelA (Santa Cruz, sc-29410, final concentration: 15 nmol/l ) using Amaxa Nucleofector Technology for 24 h and then were irradiated at a dose of 6 Gy. Total lysates were obtained 24 h after irradiation and blots were stripped with p65/RelA, Bcl2 and Bcl-Xl proteins. GAPDH were used as loading control
