Pro-apoptotic, anti-metastatic, and anti-telomerase activity of Tinospora cordifolia and its active polysaccharide arabinogalactan during Benzo(a)pyrene-induced lung carcinogenesis

Abstract

Background: The present study aims to unravel the pro-apoptotic, anti-metastatic, and anti-telomerase activity of aqueous extract of Tinospora cordifolia stem (Aq.Tc) and its active component arabinogalactan (AG) during Benzo(a)pyrene [B(a)P]-induced lung tumorigenesis in mice.

Materials and methods: Lung tumors were induced in male BALB/c mice using B(a)P as a carcinogen. Animals were administered twice with 50 mg/kg b.wt (i.p.) dosage of B(a)P at the 2^nd and 4^th week of the study. Mice were orally treated with Aq.Tc and AG on alternate days at a dose of 200 mg/kg b.wt and 7.5 mg/kg b.wt, respectively, for continuous 22 weeks.

Results: Oral administration of animals with Aq.Tc and AG suppressed the development of lung carcinogenesis by modulating the mRNA and protein expressions of different apoptotic genes; bcl-2, bax, caspase 3, and caspase 9. The pro-apoptotic proficiency of Aq.Tc and AG was further confirmed by DNA agarose gel electrophoresis showing fragmentation in B(a)P + Aq.Tc group and smear formation in B(a)P + AG group. In contrast to the control group, an increase in tumor invasion factors such as matrix metalloproteinases-2 (MMP-2) and MMP-9 was also observed in B(a)P treated animals. Nevertheless, Aq.Tc and AG treatment effectively mitigated the B(a)P-induced upregulation of MMP-2 and MMP-9. The activity of the telomerase enzyme was also observed to be upregulated in B(a)P treated animals which consecutively found to get normalized with the parallel administration of Aq.Tc and AG.

Conclusion: Aq.Tc and AG successfully mitigated the altered expression of apoptosis, metastasis, and telomerase activity-associated genes during pulmonary carcinogenesis.

Keywords: Apoptosis; Tinospora cordifolia; lung cancer; metastasis; telomerase.

Figure 1

Gross morphological and histopathological changes in pulmonary tissue at 22^nd week of the treatment regime (a) Control; (b and c) B(a)P group; RL: Right lung; LL: Left lung. Arrow indicates presence of tumors. sa: single squamous epithelium av: alveolar sacs sb: small bronchiole ad: alveolar destruction, ep: extensive proliferation of alveolar epithelium and presence of hyperchromatic nuclei in the alveolar wall; i: Inflammatory cellular infiltrations N; thinning of alveolar walls)

Figure 2

mRNA expression of Apoptotic genes in various treatment groups. (a) RT-PCR gel showing mRNA expression. (b) caspase 9 graph, (c) bcl-2 graph, (d) bax graph, (e) Caspase 3 graph. Values expressed as: Mean ± SD (n= 4). Data analyzed using one-way ANOVA (post hoc test). a1: P ≤ 0.001; a2: P ≤ 0.01 significant to control group, b1: P ≤ 0.001; b2: P ≤ 0.01 significant compared to Aq.Tc group, c1: P ≤ 0.001; c2: P ≤ 0.01 significant to AG group, d1: P ≤ 0.001; d2: P ≤ 0.01 significant to B(a)P group. RT-PCR: Reverse transcription-polymerase chain reaction, SD: Standard deviation, AG: Arabinogalactan

Figure 3

Quantitative mRNA expression of MMP-2 and MMP-9 genes in various treatment groups. (a) RT-PCR gel depicting mRNA expression. (b) Fold change of mRNA expression of MMP-2 (c) MMP-9. Values are expressed as: Mean ± SD (n= 4). Data is analysed by one-way ANOVA (post hoc test). a1: P ≤ 0.001; a2: P ≤ 0.01significant compared to the control group, b1: P ≤ 0.001 significant compared to Aq.Tc group, c1: P ≤ 0.001 significant compared to AG group, d1: P ≤ 0.001 significant compared to B(a)P group. MMP-2: Matrix metalloproteinases-2, MMP-9: Matrix metalloproteinases-9, RT-PCR: Reverse transcription-polymerase chain reaction, SD: Standard deviation, AG: Arabinogalactan

Figure 4

Enzymatic activity of MMP-9 and MMP-2 during B(a)P induced lung tumorigenesis. (a) Representative activity of MMP-2 and MMP-9. (b) Gelatinolytic activity of MMP-9 and (c) MMP-2. Data is analysed using one-way ANOVA followed by post hoc test. a1: P ≤ 0.001; a2: P ≤ 0.01significant compared to the control group, b1: P ≤ 0.001; b2: P ≤ 0.01 significant compared to Aq.Tc group, c1: P ≤ 0.001; c2: P ≤ 0.01 significant compared to AG group, d1: P ≤ 0.001; d2: P ≤ 0.01 significant compared to B(a)P gr. MMP-2: Matrix metalloproteinases-2, MMP-9: Matrix metalloproteinases-9, AG: Arabinogalactan

Figure 5

DNA fragmentation assay of lung tissue from different treatment groups Lane I – DNA ladder, Lane II – Control, Lane III – Aq.Tc, Lane IV – AG, Lane V – B(a)P, Lane VI – B(a)P + Aq.Tc, Lane VII – B(a)P + AG

Figure 6

Effect of Aq.Tc and AG on telomerase enzyme activity during B(a)P induced pulmonary tumorigenesis Values are expressed as: Mean ± SD (n= 5) and analysed using one-way ANOVA followed by LSD post hoc test. a1: P ≤ 0.001 significant as compared to the control group, b1: P ≤ 0.001; b2: P ≤ 0.01 significant as compared to Aq.Tc group, c1: P ≤ 0.001; c2: P ≤ 0.01 significant as compared to AG group, d1: P ≤ 0.001 significant as compared to B(a)P group. AG: Arabinogalactan, SD: Standard deviation, LSD: Least significant difference