Post-cancer Treatment with Condurango 30C Shows Amelioration of Benzo[a]pyrene-induced Lung Cancer in Rats Through the Molecular Pathway of Caspa- se-3-mediated Apoptosis Induction

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  • ABSTRACT

    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 cancer, could also show an ameliorating effect through apoptosis induction on lung cancer induced by benzo[a]pyrene (BaP) in white rats (Rattus norvegicus).

    Methods:

    Lung cancer was induced after four months by chronic feeding of BaP to rats through gavage at a dose of 50 mg/kg body weight for one month. After four months, the lung-cancer-bearing rats were treated with Condurango 30C for the next one (5th), two (5th-6th) and three (5th-7th) months, respectively, and were sacrificed at the corresponding time- points. The ameliorating effect, if any, after Condurango 30C treatment for the various periods was evaluated by using protocols such as histology, scanning electron microscopy (SEM), annexinV-FITC/PI assay, flow cytometry of the apoptosis marker, DNA fragmentation, reverse transcriptase-polymerase chain reaction (RT-PCR), immunohistochemistry, and western blot analyses of lung tissue samples.

    Results:

    Striking recovery of lung tissue to a near normal status was noticed after post-cancerous drug treatment, as evidenced by SEM and histology, especially after one and two months of drug treatment. Data from the annexinV-FITC/PI and DNA fragmentation assays revealed that Condurango 30C could induce apoptosis in cancer cells after post-cancer treatment. A critical analysis of signalling cascade, evidenced through a RT-PCR study, demonstrated up-regulation and down-regulation of different pro- and anti-apoptotic genes, respectively, related to a caspase-3-mediated apoptotic pathway, which was especially discernible after one-month and two- month drug treatments. Correspondingly, Western blot and immunohistochemistry studies confirmed the ameliorative potential of Condurango 30C by its ability to down-regulate the elevated epidermal growth factor receptor (EGFR) expression, a hallmark of lung cancer.

    Conclusion:

    The overall result validated a positive effect of Condurango 30C in ameliorating lung cancer through caspase-3-mediated apoptosis induction and EGFR down-regulation.


  • KEYWORD

    apoptosis , benzo[a]pyrene (BaP) , caspase-3 , Condurango 30C , homeopathy , lung cancer

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  • [Table. 1] Primer names and sequences
    Primer names and sequences
  • [Fig. 1] Scanning electron microscopy of rat lungs of different groups [(A) and (B)]. (A) [200X] (i) and (B) [500X] (viii) - untreated control group with regular tissue rearrangement, (A) [200X] (ii)-(iv) and (B) [500X] (ix)-(xi) - BaP-induced cancerous groups at 5th, 6th and 7th month intervals, respectively, where alveolar spaces were narrowing, (A) [200X] (v)-(vii) and (B) [500X] (xii)-(xiv) ? Condurango-30C-treated groups at 5th, 6th and 7th month intervals, respectively. (C) Histological sections of rat lungs (40X): (i) normal lung section, (ii)-(iv) BaP-induced lung sections at 5th, 6th and 7th months and (v)-(vii) Condurango-30C-treated lung sections at 5th, 6th and 7th month intervals, respectively.
    Scanning electron microscopy of rat lungs of different groups [(A) and (B)]. (A) [200X] (i) and (B) [500X] (viii) - untreated control group with regular tissue rearrangement, (A) [200X] (ii)-(iv) and (B) [500X] (ix)-(xi) - BaP-induced cancerous groups at 5th, 6th and 7th month intervals, respectively, where alveolar spaces were narrowing, (A) [200X] (v)-(vii) and (B) [500X] (xii)-(xiv) ? Condurango-30C-treated groups at 5th, 6th and 7th month intervals, respectively. (C) Histological sections of rat lungs (40X): (i) normal lung section, (ii)-(iv) BaP-induced lung sections at 5th, 6th and 7th months and (v)-(vii) Condurango-30C-treated lung sections at 5th, 6th and 7th month intervals, respectively.
  • [Fig. 2] (A-1) Flow cytometric analysis of perfused lung cells by using annexin V-FITC/PI. (i) normal, (ii)-(iv) BaP-induced lung cancer at 5th, 6th and 7th month intervals (Cancer1, Cancer2, Cancer3) and (v)-(vii) BaP+Condurango 30C treated groups at 5th, 6th and 7th month intervals (30_1, 30_2, 30_3), respectively. Live cells are denoted as `Live’, early apoptosis is denoted by `Early’, late apoptosis is signified by `Late’ and PI positive cells (dead cells) is denoted by Q1. FL1H- filter of annexin V -FITC and FL2H- filter of PI. Table (2A-2) and graphical representation (2A-3) below give the apoptosis percentages. (B) DNA fragmentation of rat lung DNA of different groups. (i) Fragmented DNA (L3) occurred after Condurango-30C treatment at 5th month interval, whereas cancerous DNA sample (L2) became slightly smeared. (ii) Representation of the increase in the DNA fragmentation (L5) after Condurango-30C treatment at the 6th month interval, whereas the cancerous DNA (L4) showed increased smearing. (iii) A slight decrease in the DNA fragmentation (L7) after Condurango-30C treatment with increased smearing of cancerous DNA (L6) at the 7th month interval. L1- is normal DNA.
    (A-1) Flow cytometric analysis of perfused lung cells by using annexin V-FITC/PI. (i) normal, (ii)-(iv) BaP-induced lung cancer at 5th, 6th and 7th month intervals (Cancer1, Cancer2, Cancer3) and (v)-(vii) BaP+Condurango 30C treated groups at 5th, 6th and 7th month intervals (30_1, 30_2, 30_3), respectively. Live cells are denoted as `Live’, early apoptosis is denoted by `Early’, late apoptosis is signified by `Late’ and PI positive cells (dead cells) is denoted by Q1. FL1H- filter of annexin V -FITC and FL2H- filter of PI. Table (2A-2) and graphical representation (2A-3) below give the apoptosis percentages. (B) DNA fragmentation of rat lung DNA of different groups. (i) Fragmented DNA (L3) occurred after Condurango-30C treatment at 5th month interval, whereas cancerous DNA sample (L2) became slightly smeared. (ii) Representation of the increase in the DNA fragmentation (L5) after Condurango-30C treatment at the 6th month interval, whereas the cancerous DNA (L4) showed increased smearing. (iii) A slight decrease in the DNA fragmentation (L7) after Condurango-30C treatment with increased smearing of cancerous DNA (L6) at the 7th month interval. L1- is normal DNA.
  • [Fig. 3] Evaluation of caspase-3 activation by flow cytometric analysis: (A) (i) normal, (ii)-(iv) BaP-induced lung cancers at 5th, 6th and 7th month intervals, and (v)-(vii) BaP+Condurango-30C-treated groups at 5th, 6th and 7th month intervals, respectively. M1 denotes the percentage of cells showing caspase-3 activation. (B) Standard deviations are represented graphically.
    Evaluation of caspase-3 activation by flow cytometric analysis: (A) (i) normal, (ii)-(iv) BaP-induced lung cancers at 5th, 6th and 7th month intervals, and (v)-(vii) BaP+Condurango-30C-treated groups at 5th, 6th and 7th month intervals, respectively. M1 denotes the percentage of cells showing caspase-3 activation. (B) Standard deviations are represented graphically.
  • [Fig. 4] RT-PCR analysis of different pro- and anti-apoptotic genes: (A) L1-normal, L2-cancer, L3-Condurango-30C treatment (after 5th month); (B) L1-normal, L4-cancer, L5- Condurango-30C treatment (after 6th month) and the histograms of relative band intensities of RT-PCR bands; (C) L1-normal, L6-cancer, L7-Condurango-30C treatment (after 7th month) and histograms of relative band intensities of RT-PCR bands. The results shown in histograms are the averages ± SDs; n = 6. Signi?cance * P < 0.05 normal vs. cancer (5th (A), 6th (B) & 7th (C) month intervals) and normal vs. Condurango-30C treatment (5th, 6th & 7th month intervals), † P < 0.05 cancer (5th, 6th & 7th month intervals) vs Condurango-30C treatment (5th, 6th & 7th month intervals).
    RT-PCR analysis of different pro- and anti-apoptotic genes: (A) L1-normal, L2-cancer, L3-Condurango-30C treatment (after 5th month); (B) L1-normal, L4-cancer, L5- Condurango-30C treatment (after 6th month) and the histograms of relative band intensities of RT-PCR bands; (C) L1-normal, L6-cancer, L7-Condurango-30C treatment (after 7th month) and histograms of relative band intensities of RT-PCR bands. The results shown in histograms are the averages ± SDs; n = 6. Signi?cance * P < 0.05 normal vs. cancer (5th (A), 6th (B) & 7th (C) month intervals) and normal vs. Condurango-30C treatment (5th, 6th & 7th month intervals), † P < 0.05 cancer (5th, 6th & 7th month intervals) vs Condurango-30C treatment (5th, 6th & 7th month intervals).
  • [Fig. 5] Immunohistochemical studies of (A) caspase-3 and (B) EGFR: (A) (i) normal lung section, (ii)-(iv) BaP-induced lung sections at 5th, 6th and 7th month intervals, respectively, where caspase-3 expression was not found, (v)-(vii) Condurango-30C- treated lung sections at 5th, 6th and 7th month intervals, respectively, where the caspase-3 localization was found, especially at 5th and 6th month intervals, indicated by black spots with arrows; (B) (i) normal lung section, (ii)-(iv) BaP induced lung sections at 5th, 6th and 7th month intervals, respectively, where EGFR over-expression was clearly visible (denoted by black spots with arrows), and (v)-(vii) Condurango-30C-treated lung sections at 5th, 6th and 7th month intervals, respectively, where the downregulation of EGFR was found, especially at 5th and 6th month time-points.
    Immunohistochemical studies of (A) caspase-3 and (B) EGFR: (A) (i) normal lung section, (ii)-(iv) BaP-induced lung sections at 5th, 6th and 7th month intervals, respectively, where caspase-3 expression was not found, (v)-(vii) Condurango-30C- treated lung sections at 5th, 6th and 7th month intervals, respectively, where the caspase-3 localization was found, especially at 5th and 6th month intervals, indicated by black spots with arrows; (B) (i) normal lung section, (ii)-(iv) BaP induced lung sections at 5th, 6th and 7th month intervals, respectively, where EGFR over-expression was clearly visible (denoted by black spots with arrows), and (v)-(vii) Condurango-30C-treated lung sections at 5th, 6th and 7th month intervals, respectively, where the downregulation of EGFR was found, especially at 5th and 6th month time-points.
  • [Fig. 6] Western blot analyses of PARP1 and EGFR. (A) L1-normal, L2-cancer, and L3-Condurango-30C-treated (after 5th month). (B) L4-cancer and L5-Condurango-30C-treated (after 6th month). (C) L6-cancer and L7-Condurango-30C-treated (after 7th month). The relative band intensities shown in the histograms are averages ± SDs; n = 6. Signi?cance *P < 0.05 normal vs. cancer (5th (A), 6th (B) & 7th (C) month intervals) and normal vs. Condurango-30C treatment (5th, 6th & 7th month intervals); † P < 0.05 cancer (5th, 6th & 7th month intervals) vs. Condurango-30C treatment (5th, 6th & 7th month intervals).
    Western blot analyses of PARP1 and EGFR. (A) L1-normal, L2-cancer, and L3-Condurango-30C-treated (after 5th month). (B) L4-cancer and L5-Condurango-30C-treated (after 6th month). (C) L6-cancer and L7-Condurango-30C-treated (after 7th month). The relative band intensities shown in the histograms are averages ± SDs; n = 6. Signi?cance *P < 0.05 normal vs. cancer (5th (A), 6th (B) & 7th (C) month intervals) and normal vs. Condurango-30C treatment (5th, 6th & 7th month intervals); † P < 0.05 cancer (5th, 6th & 7th month intervals) vs. Condurango-30C treatment (5th, 6th & 7th month intervals).