Swertia chirayita suppresses the growth of non-small cell lung cancer A549 cells and concomitantly induces apoptosis via downregulation of JAK1/STAT3 pathway

Abstract

Lung carcinoma is the leading cause of cancer-related mortalities worldwide, and present therapeutical interventions are not successful enough to treat this disease in many cases. Recent years have witnessed a surge in exploring natural compounds for their antiproliferative efficacy to expedite the characterization of novel anticancer chemotherapeutics. Swertia chirayita is a valued medicinal herb and possess intrinsic pharmaceutical potential. However, elucidation of its anticancer effects at molecular levels remains unclear and needs to be investigated. We assessed the anticancer and apoptotic efficacy of S. chirayita ethanolic extract (Sw-EtOH) on non-small cell lung cancer (NSCLC) A549 cells during this exploratory study. The results elucidated that S. chirayita extract induced toxic effects within lung cancer cells by ~1 fold during cytotoxicity and LDH release assay at a 400 μg/ml concentration. Sw-EtOH extract elevates the level of ROS, resulting in the disruption of Δψm and release of cytosolic cytochrome c by 3.15 fold. Activation of caspases-3, -8 & -9 also escalated by ~1 fold, which further catalyze the augmentation of PARP cleavage (~3 folds), resulting in a four-fold increase in Sw-EtOH induced apoptosis. The gene expression analysis further demonstrated that Sw-EtOH extracts inhibited JAK1/STAT3 signaling pathway by down-regulating the levels of JAK1 and STAT3 to nearly half a fold. Treatment of Sw-EtOH modulates the expression level of various STAT3 associated proteins, including Bcl-XL, Bcl-2, Mcl-1, Bax, p53, Fas, Fas-L, cyclinD1, c-myc, IL-6, p21 and p27 in NSCLC cells. Thus, our study provided a strong impetus that Sw-EtOH holds the translational potential of being further evaluated as efficient cancer therapeutics and a preventive agent for the management of NSCLC.

Keywords: A549 cells; Antiproliferative; Apoptosis; JAK1/STAT3 pathway; Non-small cell lung cancer; Swertia chirayita.

Fig. 1

Illustrative representation of Swertia chirayita mediated anti-proliferative effects on human non-small lung cancer A549 cells. Sw-EtOH was found to exert its anti-cancerous effects on A549 cells by impelling the extrinsic apoptotic pathway mediated by enhanced expression of caspase-8 enrouted to activation of Fas receptors by Fas-L. Sw-EtOH also indicated towards activation of intrinsic apoptotic pathway via augmentation of intracellular ROS levels concomitantly followed by increased levels of caspase dependent cleaved PARP protein. Furthermore, Sw-EtOH was also successful in modulating the JAK-STAT signaling pathway which eventually culminated in altered expression of signals required for cell survival and proliferation (as indicated by upwards and downwards arrow).

Fig. 2

Sw-EtOH induced cytotoxicity within human lung cancer A549 cells. (A) Percent (%) cell viability of A549 cells after Sw-EtOH treatment at concentrations of 100–400 μg/ml after 24 h, (B) assessment of membrane integrity as evaluated through LDH release assay at aforesaid dosage of Sw-EtOH and (C) Phase-contrast images of Sw-EtOH treated A549 cells for 24 h respectively (pink, blue and orange arrows indicate cell, shrinkage and withering of cell organelles). Data communicated constitutes the mean ± SEM of individual experiments performed thrice in triplicates. The level of significance among different Sw-EtOH treated groups was determined using one-way Anova and Dunnett post-hoc in comparison with untreated control where *represents p < 0.05, **p < 0.01 and ***p < 0.001; Scale bar = 100 μm.

Fig. 3

(A)Sw-EtOH mediated induction of apoptosis within treated human lung cancer A549 cells. Increased levels of nuclear condensation and apoptosis are depicted within photomicrographs of vehicle control and Sw-EtOH treated (100–400 µg/ml) A549 cells stained with DAPI, P.I. and their merged fluorescence respectively indicated by red, green and white arrows. (B) Quantification of apoptosis percent (%) within A549 cells pre-treated Sw-EtOH for 24 h during Annexin V-FITC/PI. Early and late apoptotic cells were found to be stained in the lower right (L.R.) and the upper right (U.R.) quadrants respectively and (C) total percent apoptosis depicted as bar graph as evident during in Annexin V-FITC/PI assay. Data communicated constitutes the mean ± SEM of individual experiments performed thrice in triplicates. The level of significance among different Sw-EtOH treated groups was determined using one-way Anova and Dunnett post-hoc in comparison with untreated control where *represents p < 0.05, **p < 0.01 and ***p < 0.001; (Scale bar = 100 µm).

Fig. 4

Effects of Sw-EtOH on (A) caspase-3, -8 and -9 activities (B) cleaved PARP levels and (C) cytosolic cytochrome c. (D) Fluorescent micrographs showing disrupted mitochondrial membrane potential in JC-1-stained A549 cells after treatment with Sw-EtOH (100–400 µg/ml). (E) photomicrographs qualitatively elucidating augmented ROS levels within Sw-EtOH pre-treated A549 cells with different mentioned concentrations of Sw-EtOH for 6 h and subsequently stained with DCFH-DA (F) Quantification of 8-OHdG, a DNA damage marker, in A549 cells. Data communicated constitutes the mean ± SEM of individual experiments performed thrice in triplicates. The level of significance among different Sw-EtOH treated groups was determined using one-way Anova and Dunnett post-hoc in comparison with untreated control where *represents p < 0.05, **p < 0.01 and ***p < 0.001. (Scale bar = 100 µm).

Fig. 5

Sw-EtOH mediated effect on mRNA expression of (A) JAK1 (B) STAT3 (C) Bcl-xL, (D) Bcl2 (E) Mcl1 and (F) IL- 6 genes. A549 cells were treated with 0–400 µg/ml for 24 h. Fold change in expression of all target genes was normalized to GAPDH mRNA which served as internal control. Data communicated constitutes the mean ± SEM of individual experiments performed thrice in triplicates. The level of significance among different Sw-EtOH treated groups was determined using one-way Anova and Dunnett post-hoc in comparison with untreated control where *represents p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 6

Sw-EtOH induced alteration within the genes involved in cell cycle progression such as (A) cyclin D1, (B) c-Myc, (C) p21 (D) p27 and pro-apoptotic genes (E) Fas, (F) Fas-L, (G) Bax and (H) p53. Data communicated constitutes the mean ± SEM of individual experiments performed thrice in triplicates. The level of significance among different Sw-EtOH treated groups was determined using one-way Anova and Dunnett post-hoc in comparison with untreated control where *represents p < 0.05, **p < 0.01 and ***p < 0.001.