Objectives: The present investigation aimed at examining if post-cancer treatment with a potentized homeopathic drug Condurango 30C which is generally used to treat oesophageal malignancy could also show an ameliorating effect through apoptosis induction on lung malignancy induced by benzo[a]pyrene (BaP) in white rats (without any supplementation Group 2 olive-oil fed: animals received normal food and water supplemented with olive oil (solvent of BaP) Group 3 placebo fed: normal animals received a drug vehicle (30% ethyl alcohol as a placebo) orally once daily for 1 2 and 3 months respectively after malignancy development in BaP-fed rats Group 4 only drug-treated: normal animals received Condurango 30C orally once daily for 1 2 and 3 months respectively after four months of malignancy development Group 5 Carcinogen (BaP)-treated: animals received BaP orally 2 days (Tuesday and Friday) Vax2 a week for 1 month and then a normal diet and water Group 6 BaP+placebo-treated: animals received a placebo orally once daily for 1 2 and 3 months respectively after development of lung malignancy Group 7 BaP+Condurango 30C-treated: animals received Condurango 30C orally once daily for 1 2 and 3 months respectively after development of lung malignancy. in BaP-fed rats Group 4 only drug-treated: normal animals received Condurango 30C orally once daily for 1 2 and 3 months respectively after four months of malignancy development Group 5 SB 415286 Carcinogen (BaP)-treated: animals received BaP orally 2 days (Tuesday and Friday) a week for 1 month and then a normal diet and water Group 6 BaP+placebo-treated: animals received a placebo orally once daily for 1 2 and 3 months respectively after development of lung malignancy Group 7 BaP+Condurango 30C-treated: animals received Condurango 30C orally once daily for 1 2 and 3 months respectively after development of lung malignancy. The experimental data were collected after 1 (5th ) 2 (6th ) and 3 (7th ) months to investigate the possible efficacy of Condurango 30C. At the ends of the experimental periods the animals were sacrificed humanely by cervical dislocation. 2.3 Preparations and administrations of BaP and Condurango 30C BaP (dissolved in olive oil) at a dose of 50 mg/kg body weight was fed to each rat through gavage [4]. Condurango 30C was supplied by Boiron Laboratory Lyon France. One ml of Condurango 30C was diluted with 20 ml of double- distilled water to make the stock answer and each rat was SB 415286 fed 0.06 ml orally from the stock at a time with the aid of a fine pipette [13]. 2.4 Scanning electron microscopy (SEM) and histology of lung Lung samples were fixed with 2.5% glutaraldehyde dehydrated with graded acetone (50%-100%) and observed by using an S530-Hitachi SEM instrument (Department of University Science Instrumentation Centre Burdwan University) after gold coating [14]. Formalin-fixed lung sections (5μm) from each group were stained with hematoxylin- eosin double staining [15] and were evaluated by using a light microscope. 2.5 Lung cell perfusion annexinV-FITC/PI DNA fragmentation and caspase-3 activation assays The lung tissues were minced within 2% Roswell Park Memorial Institute-1640 media (Himedia India) and the lung epithelial cells were flushed gently using a hypodermic syringe. The media-containing cells were spun down at 1 SB 415286 SB 415286 0 G and the supernatant-containing lung epithelial cells were used for further study [14]. The rate of apoptosis of the perfused cells (3 x 107 cells/ well) was assessed by using AnnexinV- fluorescein isothiocyanate/ propidium iodide (FITC/PI) through circulation cytometry (FACS Callibur BD Bioscience USA) [16]. DNA was extracted by using the standard phenol-chloroform method was separated in 2% agarose gel and was visualized under an UV transilluminator. Perfused lung cells were incubated with caspase-3 main and FITC-tagged secondary antibodies (Santa Cruz Biotechnology USA). Caspase-3 (Cas-3) activity was analyzed by using circulation cytometry (Callibur BD Bioscience USA). 2.6 Preparation of lung and liver tissue homogenates and semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) Lung and liver tissues were homogenized and homogenates were collected after centrifugation [16]. Total RNA was extracted from each lung by using trizol (Himedia India) and expressions of different apoptotic genes were analyzed by using semi-quantitative RT-PCR [17]. The primer sequences are offered in (Table ?(Table1)1) The band intensities were analyzed densitometrically by using Image J software (Germany). Table. 1 Primer names and sequences 2.7 Localization of protein distribution by SB 415286 immunohistochemistry and analysis of protein expression by using a Western blot An immunohistochemical study was performed [18] with caspase-3 and epidermal growth factor receptor (EGFR) main antibodies and HRP-conjugated secondary antibodies (Santa Cruz Biotechnology USA). Haematoxylin was used to counterstain for observation under a light microscope (Leica Germany). The expressions of EGFR and PARP1 were analyzed by using Western blots [16]. The band intensities were analyzed densitometrically by using Image J software (Germany). 2.8 Statistical analysis Data were analyzed and the signi?cance of the differences between the mean values was determined by using the one-way analysis of variance (ANOVA) with Fisher’s least significant difference (LSD) post-hoc.