Hexane fraction of Adenophora triphylla var. japonica root extract induces apoptosis of human lung cancer cells by inactivating Src/STAT3 pathway

Abstract The aim of this study was to investigate the anticancer effect of Adenophora triphylla var. japonica (AT) root extract on human non-small cell lung cancer (NSCLC) cells and the mechanism involved in such effect. Among three fractions of AT root extract, hexane fraction (HAT) significantly decreased the proliferation of NSCLC cells. Besides, HAT treatment dose-dependently inhibited colony formation of NSCLC cells. These effects were associated with apoptosis induction evidenced by increased chromatin condensation, accumulation of sub-G1 DNA content and annexin V-positive cells, and enhanced expression of apoptotic proteins, including cleaved-caspases and cleaved-poly (ADP-ribose) polymerase (PARP). Notably, phosphorylation levels of signal transducer and activator of transcription 3 (STAT3) and Src were decreased by HAT. Transfection with STAT3 or Src for constitutive activation reversed the anti-proliferative effect of HAT on H1299 cells. Taken together, our findings suggest that HAT-induced apoptosis in NSCLC cells is mediated by inhibition of Src/STAT3 pathway. Graphical Abstract


Introduction
Despite a declining trend in the incidence of lung cancer, lung cancer is still the second most commonly diagnosed cancer and the leading cause of cancer-related mortality worldwide (Sung et al. 2021). Approximately 85% of lung cancer patients have nonsmall cell lung cancer (NSCLC), the most common histological subtype of lung cancer (Molina et al. 2008). Platinum-based combination therapy has been the standard therapy for advanced NSCLC (Hanna et al. 2017). In addition, various targeted therapies and immunotherapy have improved clinical outcomes of NSCLC patients. However, platinum-induced severe side effects, the occurrence of drug resistance, and high cost are major obstacles for effective application of these drugs (Herbst et al. 2018). Therefore, it is urgent to develop more effective and less toxic therapeutics to treat NSCLC.
The root of Adenophora triphylla var. japonica (AT) has been traditionally used in Oriental medicine for managing symptoms of inflammatory airway diseases (Park 2019). Recent pharmacological studies have reported that AT extracts possess antiobesity and anti-fungal activities (Lee et al. 2013;Kim and Kim, 2021). Notably, AT extract exhibits anti-cancer effects by inducing apoptosis and G1 arrest of several cancer cells (Lee et al., 2000). Our previous study has also demonstrated that AT extract can suppress the migration of lung cancer cells by regulating tumour-associated macrophages (Park 2019). However, the molecular mechanism involved in the anticancer effect of AT remains unclear. Thus, the objective of the current study was to investigate the anticancer potential of AT extract in various NSCLC cell lines and postulate the molecular target of AT extract.

Effects of various fractions of at extract on viability of NSCLC cells
We investigated which fraction of AT extract had the strongest cytotoxicity to human NSCLC cells. Four kinds of NSCLC cells were treated with various fractions of AT extract, including hexane fraction (HAT), ethyl acetate fraction (EAT), and butanol fraction (BAT). Among the three fractions, HAT exhibited the highest cytotoxicity to all four NSCLC cell lines (Figures S1A-S1D, Supplementary material). Therefore, we conducted the following experiments using HAT.

HAT inhibits proliferation and colony formation of NSCLC cells
To evaluate the effect of HAT on the proliferation of NSCLC cells, we conducted trypan blue exclusion assay. Our results clearly showed that HAT treatment attenuated proliferation of all four NSCLC cell lines ( Figures S2A-S2D). In addition, HAT treatment dosedependently decreased the number of colonies of H1299 and H460 cells ( Figure S3A-S3D).

HAT induces apoptosis of NSCLC cells
To determine whether the anti-proliferative and anti-colony formation effects of HAT were associated with apoptosis induction, we next conducted DAPI staining. As shown in Figure S4A, the proportion of condensed and fragmented nuclei, a morphological characteristic of apoptotic nuclei, was markedly increased after HAT treatment ( Figure  S4A). For validation of our results, apoptosis was detected by flow cytometry. HAT treatment increased percentages of annexin V-positive cells and sub-G1 phase cells, respectively ( Figures S4B and S4C). Consistently, expression levels of cleaved caspase-3 and cleaved PARP known to be general markers of apoptosis were up-regulated by HAT treatment ( Figure S4D). Interestingly, the cleavage of caspase-8 was not increased while that of caspase-9 was significantly increased by HAT in a time-dependent manner ( Figure S4D). It is well-recognized that caspase-8 is cleaved by the extrinsic apoptotic pathway which needs the binding of ligands to death receptors, while caspase-9 is activated by the intrinsic apoptotic pathway which involves the collapse of mitochondrial function (Wong 2011). These findings suggest that HAT can induce apoptosis of NSCLC cells via the intrinsic mitochondrial pathway.

Anticancer activity of HAT is mediated by inactivation of src/STAT3 pathway
We next clarified the molecular mechanism underlying the anticancer activity of HAT in NSCLC cells. It is known that Src and signal transducer and activator of transcription 3 (STAT3) are critically involved in tumour growth, drug resistance, and metastasis, finally contributing to poor prognosis of cancer (Johnson et al. 2018;Martellucci et al. 2020). STAT3 is a transcription factor activated by various upstream kinases, including Janus family tyrosine kinases (JAKs) and non-receptor tyrosine kinase Src. It regulates a variety of target genes involved in cancer progression (Johnson et al. 2018). As shown in Figure S5A, phosphorylation levels of STAT3 and Src were commonly downregulated by HAT treatment in H1299 and A549 cells ( Figure S5A), indicating that HAT could inhibit the Src/STAT3 pathway in NSCLC cells.
To determine whether the inactivation of STAT3 or Src was essential for the anticancer potential of HAT, we transfected H1299 cells with STAT3 or Src for their constitutive activation (STAT3 CA or Src CA). Phosphorylation levels of STAT3 and Src were significantly increased by transfection with STAT3 CA and Src CA, respectively ( Figure  S5B). Notably, Src CA-or STAT3 CA-transfected cells were much more resistant to HAT treatment compared with empty vector-transfected cells ( Figure S5C). Overall, HATinduced cell death was reversed more strongly by Src CA transfection than by STAT3 CA transfection. Our observations clearly suggested that inactivation of Src/STAT3 axis mediated the anticancer effect of HAT in NSCLC cells.

Gas chromatography/mass spectrometry (GC/MS) analysis of HAT
To identify phytochemical constituents of HAT, we conducted GC/MS analysis. b-sitosterol and lupeol are general compounds found in AT extract (Kim, Cho, et al. 2014;Kim, Zhao, et al. 2014). The chromatogram of HAT showed peaks at retention time of 14 min (peak 1) and 14.8 min (peak 2), corresponding to those of b-sitosterol and lupeol, respectively ( Figure S6 and Table S1, Supplementary material). MS data showed that molecular weights of peak 1 and b-sitosterol were the same at m/z 414.6 [M þ H] þ . Molecular weights of peak 2 and lupeol were also the same at m/z T426.6 [M þ H] þ ( Figure S6 and Table S1). Taken together, these results clearly suggested that that b-sitosterol and lupeol were present in HAT.
It is known that b-sitosterol can exert anti-cancer effects by inducing apoptosis and G1 arrest in cancer cells (Sayeed and Ameen 2015;Wang et al. 2017). Zhang et al. (2020) have reported that b-sitosterol-loaded solid lipid nanoparticles can ameliorate arthritis by down-regulating the expression of STAT3. However, no study has reported that the activity of Src is regulated by b-sitosterol. Lupeol, a triterpenoid contained in AT, possesses anti-inflammatory, anti-viral, anti-hyperglycaemic, and anti-cancer effects (Siddique and Saleem 2011). It exhibits strong anticancer effects in various cancer cells, including NSCLC cells, by suppressing cell proliferation and inducing apoptosis (Siddique and Saleem 2011). Notably, lupeol can trigger apoptotic cell death in NSCLC cells and hepatocellular carcinoma cells by blocking STAT3 activity (Min et al. 2019;Siveen et al. 2014). Dephosphorylation of Src following lupeol treatment in several cancer cells has also been reported (Siveen et al. 2014;Kangsamaksin et al. 2017). These studies collectively suggest that lupeol present in HAT might mediate the anticancer activity of HAT. More studies are warranted to determine the specific compound(s) that mediates the anticancer potential of HAT in NSCLC cells.

Experimental
See the supplementary material.

Conclusions
Our results showed that HAT treatment dose-dependently inhibited the proliferation and colony formation of NSCLC cells by inducing apoptosis. The phosphorylation levels of STAT3 and Src were decreased by HAT. Transfection with STAT3 or Src for constitutive activation reversed the anti-proliferative effect of HAT, suggesting the involvement of STAT3 and Src in the anticancer effects of HAT.

Disclosure statement
The authors report there are no competing interests to declare.

Funding
No funding was received.