FIGURE CAPTIONS
Figure 1.Antiproliferative effects of CIGB-552, CDDP and Paclitaxel in NCI-H460 cells. A)Proliferation inhibition induced by CIGB-552 and CDDP (A) or Paclitaxel(B) alone and combined at selected concentrations in µM.C) Color surface interaction map, based on CI values fromin vitro drug combination assay for CIGB-552 with CDDP and Paclitaxel under concomitant and pre-treatment settings. * p<0.05, ** p<0.01 for the combination in comparison to individual treatments (One way ANOVA and Dunnet post test).
Figure 2. Cell cycle profile of NCI-H460 cells treated with CIGB-552 (37.5µM), CDDP (5 µM) and the combination of both drugs for 12 (A) and 24 hours (B). The DNA content was assessed by Propidium Iodide (PI) staining and flow cytometry. The percentage of cells in each cell cycle phase is represented in the corresponding histograms.
Figure 3. Apoptosis induction in NCI-H460 cells mediated byCIGB-552 (37.5 µM) and CDDP (5 µM) alone and combined. A) Western Blott of apoptosis-related proteins in response to 12 and 24 hours of exposure to the products. Staurosporin (STS) 1 µM, was used as positive control. B) Cell distribution on the Annexin V/Propidium Iodide (AV/PI) double staining assessed by flow cytometry after 48 of exposure to the drugs. STS (1 µM) was also included as positive control for the experiment. C) Percentage of apoptotic cells (AV + AV/PI) from the double staining assay in response to each treatment. * p<0.05, for the combination in comparison to individual treatments (One way ANOVA and Dunnet post test).
Figure 4. Superoxide anion ( O2•−) accumulation assays in response to CIGB-552 (37.5 µM) and CDDP (5 µM) alone and in combination. A)Fluorescence microscopy images from Hydroethidine (HE) staining in NCI-H460 (A) and MRC-5 cells (B ) to detect O2•− accumulation in response to 1 hour exposure to the products. C)HE fluorescence intensity as fold of control in NCI-H460 cells in response to the treatments, quantified from microscopy images with ImageJ 1.41 software. ** p<0.01 for the combination in comparison to individual treatments (One way ANOVA and Dunnet post test). D) HE positive cells in percent, after 12 hours of exposure to the products in NCI-H460 cell line, assessed by flow cytometry. * p<0.05 for the combination in comparison to individual treatments (Unpaired T test). H2O2(2.5 mM) was included as a positive control for all experiments.
Figure 5. In vivo antitumor activity of CIGB-552 (1 mg/kg s.c.) and CDDP (0.4 mg/kg i.p.) in a TC-1 lung cancer model in C57/BL6 mice. A) Mean tumor volume of treated and control mice from 10 to 24 days post-implantation of TC-1 cells. B) Tumor volume of animals from the four experimental groups at day 21 post-implant. Control animals only received saline solution (PBS 1X s.c.) * p<0.05 for the combination in comparison to individual treatments (Unpaired T test).C) Mean body weight of treated and control mice measured once a week during the three weeks of the experiment. D) Kaplan-Meier survival curves of control and treated animals from implantation to 60 days post-implant. Log-rank analysis showed statistical differences between treated and control animals (p<0.01).
Figure 6. Hypothetical mechanism proposed to explain the synergic effects between CIGB-552 and CDDP in lung cancer cells. Both drugs target oxidative stress and apoptosis in tumor cells but acting through different pathways. CDDP intercalates in the DNA inducing DNA damage and cell death by apoptosis and at the same time, it causes a progressive reactive oxygen species (ROS) accumulation and oxidative stress as a result of its enzymatic biotransformation. On the other hand, CIGB-552 stabilizes and accumulates COMMD1 leading to the downregulation of the NF-kB signaling and consequently it inhibits the transcription of antiapoptotic and pro-survival genes. This effect enhances the CDDP-induced apoptosis and decreases chemoresistance. In addition, the COMMD1-mediated inhibition of SOD1 enzyme induced by CIGB-552 also triggers oxidative stress. The sustained and synergic oxidative stress activation by both products eventually leads to apoptotic cell death as well. CDDP: Cisplatin, CTR1: High affinity copper uptake protein 1; CIGB-552: anticancer cell penetrating peptide; COMMD1: Copper Metabolism Murr 1 Domain Containing Protein 1; SOD1: Superoxide Dismutase 1; p50/p65: NF-kB subunits. Black arrows indicate individual effects and red arrows indicate synergic effects. Red crosses indicate inhibition.