YXQ-EQ Induces Apoptosis and Inhibits Signaling Pathways Important for Metastasis in Non-Small Cell Lung Carcinoma Cells

Lung cancer is the most common malignant disease and the leading cause of cancer related mortality worldwide [1]. Non-small cell lung cancer (NSCLC) comprises the majority of lung cancer cases, with most NSCLC patients having advanced stage disease at diagnosis [2]. The 5-year survival rate for NSCLC patients is only approximately 15%, with metastasis as the primary cause of death [3]. Conventional standard of care therapies for metastatic NSCLC include chemotherapy, targeted therapy and immunotherapy [4]. However, clinical efficacy of these therapies is limited by the development of intrinsic or acquired drug resistance [5]. Novel approaches are therefore needed for effective treatment of NSCLC.

External Qi therapy of traditional Chinese medicine (TCM) has long been one of the medical practices in China and managed by the Chinese health authorities [6]. The concept of External Qi (of Qigong) refers to the technology and ability of “Qi deployment” therapy and health preservation of TCM [6-11]. Clinical observations over the past 30 years and ongoing studies have documented significant beneficial effects of external Qi of Yan Xin Qigong (YXQ-EQ) on patients with cancer and other diseases [12-16]. Studies over the past three decades have demonstrated influence of YXQ-EQ on both the structural and functional properties of biomolecules [17-22]. Furthermore, YXQ-EQ has been shown to induce apoptosis in cancer cells while protect neurons from oxidative stress induced cell death via modulating signaling pathways and gene expression [6-11, 23]. These studies provide some molecular and cellular insights into the observed clinical benefits of YXQ-EQ.

We have previously shown that YXQ-EQ induces apoptosis and alters the expression of multiple genes involved in cell migration and energy metabolism in small cell lung cancer (SCLC) cells [10]. In the present study, we examined the effect of YXQ-EQ on NSCLC A549 cells. We report that YXQ-EQ exerted potent cytotoxic effect on A549 cells and inhibited EGF/EGFR and TGF-β/SMAD signaling that are important for NSCLC metastasis.

Human lung adenocarcinoma A549 cells (ATCC, Manassas, VA) were cultured in DMEM containing 10% FBS, 50 µg/ml penicillin and 100 µg/ml streptomycin. For EGF and TGF-β signaling studies, A549 cells were serum starved overnight and treated with EGF (10 ng/ml) or TGF-β1 (10 ng/ml) for 15 min and then harvested for immunoblot analyses.

Cell viability assay was performed as previously described [7, 8]. Briefly, cells were plated in 96-well plates at 2 x 10³ cells/well and incubated overnight prior to treatment with YXQ-EQ for 5 min. Cell viability was determined using an MTS [3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay with a CellTiter 96 Aqueous One Solution Cell Proliferation Assay kit (Promega, Madison, WI).

Clonogenic assay was performed as previously described [9, 11]. Briefly, A549 cells were suspended in growth medium and plated in 6-well plates at 1 x 10³ cells/well. Medium was changed every 3 - 4 days. Following two weeks after YXQ-EQ treatment, colonies were fixed with methanol, stained with crystal violet and counted.

Cell apoptosis was analyzed as previously described [9]. Briefly, cells were collected after trypsinization and incubated in Annexin V binding buffer containing FITC-Annexin V (BD Bioscience, San Jose, CA) and propidium iodide (PI) for 15 min. Stained cells were subjected to flow cytometric analysis and cell apoptosis was assessed using Flowjo.

Cell migration and invasion assay was performed in Transwell Boyden chambers and chambers coated with Matrigel as described previously [9]. Overnight serum starved A549 cells in DMEM containing 0.1% BSA were added to the inserts. The lower chambers were supplemented with 600 µl of DMEM containing 0.1% BSA with or without EGF (25 ng/ml). After incubation for 16 h, cells that did not migrate were carefully wiped off. Migrated cells were fixed with methanol, stained with crystal violet, and counted in 4 randomly selected fields then averaged.

Whole cell lysates were prepared and proteins of interest were detected by immunoblot analyses as described previously [9, 11]. Antibodies recognizing the following proteins were sourced from Cell Signaling Technology (Danvers, MA): AKT (catalog #9272), pAKT (Ser473, catalog #4060), ERK1/2 (catalog #4695), pERK1/2 (Thr202/Tyr204, catalog #4370), BCL-xL (catalog #2764), XIAP (catalog #2042), survivin (catalog #2808), PARP (catalog #9542), casapase-3 (catalog #9662), SMAD2/3 (catalog #8685), pSMAD2 (Ser465/467, catalog #3108), pSMAD3 (Ser423/425, catalog #9520). Other antibodies included EGFR (catalog #A300-388A, Bethyl Laboratories, Montgomery, TX), pEGFR (Tyr1068, catalog #44788G, Invitrogen, Grand Island, NY), and actin (catalog #A2228, Sigma-Aldrich, St. Louis, MI).

A549 cells were treated with TGF-β1 (10 ng/ml) for 48 h in growth medium to induce EMT. Morphological changes of cells were observed under an inverted microscope.

Data are presented as mean ± SD. Statistical significance was determined using two-tailed Student’s t-test. P < 0.05 was considered significant.

To examine any cytotoxic effect of YXQ-EQ, A549 cells were treated with YXQ-EQ for 5 min and cell viability was determined using an MTS assay 24 h later. YXQ-EQ treatment resulted in pronounced reduction (by approximately 93%) in cell viability (Fig. 1A). Furthermore, YXQ-EQ treated A549 cells failed to form colonies in contrast to the many colonies that developed from control untreated A549 cells (Fig. 1B). These findings demonstrated a potent cytotoxic effect of YXQ-EQ on A549 cells.

To better understand the cytotoxic effect of YXQ-EQ on A549 cells, we next examined if YXQ-EQ influenced the PI3K/AKT and MAPK/ERK1/2 pathways that are known to be critical for A549 cell survival [24]. Immunoblot analyses revealed significant suppression of phosphorylation of AKT (Ser473) and ERK1/2 in YXQ-EQ treated A549 cells (Fig. 2A). Furthermore, the expression of anti-apoptotic proteins BCL-xL, survivin and XIAP was reduced in YXQ-EQ treated A549 cells (Fig. 2B). We next investigated if YXQ-EQ exerted its cytotoxic effect by inducing apoptosis in A549 cells. Apoptosis was measured using Annexin V-FITC/PI staining and flow cytometric analysis 20 h after YXQ-EQ treatment. Apoptotic cells consist of Annexin V positive/PI negative early apoptotic cells and Annexin V positive/PI positive late apoptotic cells. A small fraction (approximately 5%) of apoptotic cells were detected in control untreated A549 cells (Fig. 2C and D). However, approximately 65% of the YXQ-EQ treated A549 cells underwent apoptosis (Fig. 2C and D). Apoptosis in YXQ-EQ treated A549 cells was further confirmed by the cleavage of pro-caspase-3 and PARP as revealed by immunoblot analyses (Fig. 2E).

EGFR is known to be overactive in lung cancer cells due to overexpression or gain-of-function mutations [25-27]. The binding of EGFR with its ligands activates several signaling cascades important for lung cancer cells, principally the RAS/RAF/MEK/ERK pathway and the PI3K/AKT pathway [28, 29]. To investigate if YXQ-EQ influenced EGFR signaling in A549 cells, we examined the effect of YXQ-EQ on EGF/EGFR mediated AKT and ERK1/2 activation. As expected, treatment of A549 cells with EGF only increased phosphorylation of EGFR and downstream effectors AKT (Ser473) and ERK1/2 (Fig. 3A). Furthermore, EGF treatment alone promoted A549 cell migration and invasion (Fig. 3B-E). Treatment of A549 cells with YXQ-EQ for 5 min prior to EGF incubation abrogated EGF stimulated phosphorylation of EGFR (Tyr1068), AKT (Ser473) and ERK1/2 (Fig. 3A). YXQ-EQ treatment prior to EGF incubation not only completely inhibited EGF induced migration (Fig. 3B and C) and invasion (Fig. 3D and E) but also significantly reduced migration and invasion below the basal levels in A549 cells.

Tumor metastasis is a major challenge in the management of NSCLC. EMT of cancer cells is associated with increased motility and a landmark of carcinoma progression during the metastatic phases. TGF-β is known to induce EMT in cancer cells [30, 31]. TGF-β transduces signals via binding to the heteromeric TGF-β receptor (TGF-βR) composed of type I and type II serine/threonine kinases, which in turn phosphorylate and activate downstream effectors SMAD2 and SMAD3 [31, 32]. Compared to untreated cells, SMAD2/3 expression and phosphorylation were markedly reduced in YXQ-EQ treated A549 cells (Fig. 4A), indicating that YXQ-EQ might negatively regulate TGF-β/TGF-βR signaling. To further address this notion, we examined the effect of YXQ-EQ on TGF-β1 induced SMAD2/3 phosphorylation in A549 cells. As expected, TGF-β1 treatment increased phosphorylation of SMAD2 and SMAD3 in these cells (Fig. 4B). TGF-βl induced SMAD2/3 phosphorylation was abrogated by 5 min of YXQ-EQ pretreatment followed by the same TGF-β1 incubation (Fig. 4B). As reported previously [33], A549 cells underwent EMT after TGF-β1 treatment for 48 h (Fig. 4C and D). To examine the effect of YXQ-EQ on TGF-β1 induced EMT, A549 cells were treated with YXQ-EQ for 5 min prior to incubation with TGF-β1. It was observed that A549 cells had lost their intact morphology and died 48 h post YXQ-EQ treatment, regardless of treatment with TGF-β1 (Fig. 4E and F).

In the present study, we show that YXQ-EQ treatment exerted profound cytotoxic effect on lung cancer A549 cells and inhibited EGF/EGFR and TGF-β1/SMAD signaling pathways that play an important role in lung cancer metastasis. These findings are in agreement with previously reported potent cytotoxicity of YXQ-EQ toward cancer cells and inhibition of multiple signaling pathways by YXQ-EQ in cancer cells [7-11]. These studies together provide some important molecular and cellular insights into the clinically observed anti-cancer activities of YXQ-EQ.

EGFR is expressed in 60% of NSCLC and mutated in approximately 10 - 35% of NSCLC [25-27]. The EGF/EGFR signaling is involved in a wide variety of cellular events, including proliferation, migration, and invasion [34, 35]. Growing evidence show that activation of AKT and ERK1/2 is critical for survival and proliferation driven by EGFR signals, either activated by EGFR ligands or activating EGFR mutations, in NSCLC [28, 29]. EGFR tyrosine kinase inhibitors (TKIs) are effective in treatment of lung cancer with activating EGFR mutations and currently the standard treatment option for advanced NSCLC; however, often resistance to TKIs develops within months after the start of treatment [34, 36]. TKIs alone have limited effect on NSCLC with wild type EGFR [35, 37] and dual inhibition of EGFR and AKT is required to induce apoptosis and inhibit growth of EGFR wild type NSCLC cells [38, 39]. A549 cells express wild type EGFR that is important for A549 cell growth [40]. Concomitant inhibition of AKT and ERK1/2 synergistically inhibits A549 cell growth and induces apoptosis in vitro and in vivo [24, 41, 42]. In agreement with these findings, YXQ-EQ induced apoptosis was associated with dual inhibition of AKT and ERK1/2 activation in A549 cells.

Lung cancer has a high potential for metastasis and this poses a challenge to lung cancer therapy. As reported previously [43, 44], EGFR activation with EGF enhanced A549 cell migration and invasion. AKT activation is required in EGF mediated lung cancer cell migration and invasion [43, 44]. Furthermore, ERK1/2 is also involved in A549 cell migration and invasion [45, 46]. These findings suggest that inhibition of AKT and ERK1/2 activation may be at least partly involved in the mechanism of inhibition of EGF mediated A549 cell migration and invasion by YXQ-EQ.

TGF-β is a pleiotropic cytokine known to drive cancer progression by multiple mechanisms including promotion of angiogenesis and metastasis, and suppression of anti-tumor immunity [47, 48]. TGF-β induced EMT increases the potential for tumor metastasis by enhancing motility and invasion of cancer cells [30, 49]. TGF-β induces EMT in cancer cells including A549 cells by activating SMAD2/3, AKT, and ERK1/2 [33, 50-52]. Inhibition of TGF-β/SMAD signaling by YXQ-EQ reported here suggests that YXQ-EQ may also exert anti-tumor effects by inhibiting the pro-tumor activities of TGF-β.

It remains to be investigated how YXQ-EQ inhibits EGF/EGFR and TGF-β/SMAD signaling. This may involve structural alterations of the receptors imposed by YXQ-EQ, since YXQ-EQ has been shown to influence the molecular structure of proteins and enzyme activity in previous studies [20-22]. The suppression of activation of AKT and ERK1/2 by YXQ-EQ may result from direct inhibition of the kinases themselves and/or indirectly from inhibition of upstream receptors or mediators. For example, YXQ-EQ may inhibit AKT activation via suppressing PI3K enzyme activity, shown in a previous study [22]. While more studies are needed to elucidate the mechanisms underlying YXQ-EQ’s cytotoxic effect and inhibitory effect on EGF/EGFR and TGF-β/SMAD signaling in A549 cells, findings presented here suggest that YXQ-EQ may have anti-NSCLC activity via inhibiting multiple signaling pathways important for NSCLC cell survival and metastasis.

This work was supported in part by Yan Xin Foundation.

The authors declare to have no competing interests.