The animal protocol for this experiment has been approved by the Committee of Animal Care and Use, LSUHSC in accordance with National Institutes of Health (NIH) guidelines. All procedures were conducted under sterile conditions. Mice were allowed access to sterile food and water ad libitum. b) Maximum Tolerated Dose Groups of 3 or 4 female SCID mice were randomized to receive

TQ alone or in combination with CDDP. TQ was prepared by dissolving in cremophor: ethanol: PBS (1:1:4) and CDDP was prepared by dissolving in PBS. TQ was given at 5, 10 and 20 mg/kg subcutaneously (s.c.) on Monday, this website Wednesday and Friday for 3 weeks either alone or in combination with CDDP at 5 mg/kg i.p once a week for 3 weeks. MTD was considered the highest dose in which no mortality was observed. At the end of three weeks mice were sacrificed and liver and kidneys were harvested for histological analysis. c) Mouse xenograft experiment For the xenograft study, female SCID mice (age 5-6 weeks old) were shaved on the flank two days prior to injection with tumor cells. Xenografts were obtained by injecting NCI-H460 2 × 106 cells subcutaneously into the check details right flank of

each mouse. Tumors were allowed to grow for one week and when tumor volume reached approximately 20 mm3 mice were randomized to 6 groups with 10 mice in each group. Tumor volume was calculated using the formula V = (L × W2) × 0.5 where V= volume, L = length, W = width. Mice were randomized into following 6 treatment groups with 10 mice in each group and treated for 3 weeks. 1) Control   2) TQ alone 5 mg/kg (s.c) M, W, F   3) TQ alone 20 mg/kg (s.c) M, W, F   4) CDDP alone 2.5 mg/kg (i.p) every Monday   5) CDDP 2.5 mg/kg+TQ5 mg/kg (combination)   6) CDDP 2.5 mg/kg+TQ20 mg/kg (combination)   Tumor volume and body weight was measured M, W, F for three weeks during the course of study.

At the end of three weeks (Day 26) mice were sacrificed by CO2 asphyxiation in a pre charged chamber and tissue samples were obtained for histological analysis. Mean tumor weight was also calculated after harvesting tumors. 7) NF-κB Pregnenolone expression in lung cancer xenografts NF-κB in the xenografts was assayed by Western blot analysis on snap-frozen xenograft tumor specimens which were pooled in duplicate for a total of 5 samples per group. Briefly, aliquots of the xenograft samples were lysed in Radio-Immunoprecipitation Assay_RIPA (Buffer) containing protease and phosphatase inhibitor cocktails and EDTA (Thermo Scientific, Rockford, IL). Proteins (40 μg) were resolved by SDS-PAGE [17] and the proteins were transferred electrophoretically to PVDF membranes (0.45 μm, Millipore Corp., Billerica, MA) [18]. Western blot assays were conducted using antibodies against phospho-Ser529 NF-κB (1:200) and unphosphorylated NF-κB (1:500), followed by the appropriate secondary antibodies and enhanced chemiluminescent detection [19]. The intensities of immunostained proteins were determined using Image J software http://​rsb.​info.​nih.