20 mg), C-DIM-8 (50 ± 5.36 mg), C-DIM-5 + doc (46 ± 3.47 mg) and C-DIM-8 + doc (45 ± 5.20 mg) compared to vehicle (100 ± 6.84 mg) ( Fig. 6A). Decreased tumor growth based on volumes was also significantly (p < 0.05) decreased in the treated compared to control mice ( Fig. 6B). A relative mean tumor volume of 150 ± 8.90 mm3 was observed in the control mice, and tumor volume decreased following treatment with doc (66.67%; 50 ± 4.77 mm3), C-DIM-5 (65.33%; 52 ± 4.80 mm3), C-DIM-8 (62.67%; 56 ± 5.80 mm3), C-DIM-5 + doc (74.67%; 38 ± 4.20 mm3), and C-DIM-8 + doc (70.67%; 44 ± 3.80 mm3) ( Fig. 6B). C-DIM-5 and C-DIM-8 nebulized formulations inhibited VEGF expression in A549 lung tumor when given alone and when combined

with doc ( Fig. 7A). This was observed as positive (dark brown) immunohistochemical BKM120 staining for VEGF on lung sections. Quantification of VEGF-positive cells was represented as percentage of the mean normalized against control ( Fig. 7B). The results showed

a decrease in VEGF staining following treatment with doc (68 ± 5.82%; Fig. 7A-II), C-DIM-5 (49 ± 5.30%; Fig. 7A-III), C-DIM-8 (54 ± 5.83%; Fig. 7A-IV), C-DIM-5 + doc (26 ± 4.25%; Fig. 7A-V) and C-DIM-8 + doc (28 ± 4.02%; Fig. 7A-VI) compared to control ( Fig. 7A-I). The decrease in VEGF expression was significant across all treatment groups relative to control and between the single and combination treatments of the same compounds (p < 0.05). However, the differences High Content Screening in VEGF expression between C-DIM-5 and C-DIM-8 and between their combinations were not significant ( Fig. 7B). Microvessel density (MVD) was determined by immunopositive staining for CD31 (Fig. 7C). Tissue sections stained dark brown for CD31 with a progressive decrease in staining observed for sections from the treatment groups compared to the control. MVD assessment of sections showed significant reduction (p < 0.05) in MVD in the groups treated with doc (182 ± 10.28 microvessels/mm2;

Fig. 7C-II and D), C-DIM-5 (164 ± 15.31 microvessels/mm2; Fig. 7 C-III and D), C-DIM-8 (158 ± 10.85 microvessels/mm2; Fig. 7 C-IV and D), C-DIM-5 + doc (106 ± 9.50 microvessels/mm2; Fig. 7 C-V and D), and C-DIM-8 + doc (118 ± 11.07 microvessels/mm2; Fig. 7C-VI and D) compared to 248 ± 25.11 microvessels/mm2 in the control ( Fig. 7C-I and D). Treatment-related however induction of apoptosis was determined by TUNEL staining which showed positive staining for DNA fragmentation as dark-brown or reddish staining (Fig. 8A). Compared to the untreated control group (Fig. 8B), there was significantly increased (p < 0.05) DNA fragmentation in mice treated with doc (38 ± 4.02%), C-DIM-5 (56 ± 6.20%) and C-DIM-8 (60 ± 5.40%), combination treatment of C-DIM-5 + doc (78 ± 8.11%) and C-DIM-8 + doc (80 ± 8.90%). Positive staining for TR3 was evident as dark-brown staining (Fig. 8C). The pattern of TR3 expression following immunostaining was similar in intensity and was evident of nuclear localization in all groups.