Andarine Plays a Robust In-vitro Anti-carcinogenic Role on A549 Cells Through Inhibition of Proliferation and Migration, and Activation of Cell-cycle Arrest, Senescence, and Apoptosis

doi:10.21203/rs.3.rs-2776621/v1

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Andarine Plays a Robust In-vitro Anti-carcinogenic Role on A549 Cells Through Inhibition of Proliferation and Migration, and Activation of Cell-cycle Arrest, Senescence, and Apoptosis

https://doi.org/10.21203/rs.3.rs-2776621/v1

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Lung cancer is a highly aggressive malignancy with limited therapeutic options and a poor prognosis. Due to the development of resistance to chemotherapeutic drugs, novel therapeutic agents are required. Androgen receptor (AR) signaling affects various genes contributing to cancer characteristics, including cell cycle progression, proliferation, angiogenesis, and metastasis. The misregulation of AR signaling has been observed in many cancers, including lung cancer. Therefore, inhibiting AR signaling using anti-androgens, AR inhibitors, or AR-degrading molecules is a promising strategy for treating lung cancer. Selective androgen receptor modulators (SARMs) are small molecule drugs with a high affinity for the androgen receptor. Commonly used cell culture techniques (MTT assay, colony-formation assay, soft-agar assay, wound healing assay, EdU staining, Annexin-V/PI staining) were employed to investigate the potential anti-carcinogenic effect of andarine on A549 cells. The expression levels of several genes involved in the cell cycle and apoptosis processes were determined by qPCR. Our findings demonstrate that andarine inhibited growth, migration, and proliferation while inducing apoptosis in lung cancer cells. Gene expression analysis revealed that andarine significantly upregulated the expression of BAX, CDKN1A, PUMA, and GADD45A while downregulating MKI67, BIRC5, and PCNA expression. Although there is no study on the utility of SARMs as inhibitors of lung cancer, we report the first study evaluating the potential anti-carcinogenic effects of andarine, a member of the SARMs, on lung cancer. Our results suggest that andarine could be considered as a promising drug candidate to test further for lung cancer treatment.

Selective androgen receptor modulator

lung cancer

anti-cancer agent

androgen receptor signaling

Lung adenocarcinoma is the most common type of lung cancer, accounting for approximately 40% of all lung cancer cases (Travis et al. 2015). Two main types of lung cancer are non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC). NSCLC is the most common type, accounting for approximately 85% of all cases, and includes adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. On the other hand, SCLC accounts for approximately 15% of all cases and is highly aggressive, with a tendency to spread quickly to other body parts.

Current therapy options for lung adenocarcinoma depend on the stage of cancer at diagnosis. Surgery is the preferred treatment for early-stage cancer, while chemotherapy and radiation therapy are used in more advanced stages of the disease (Travis et al. 2015; Socinski et al. 2018). Targeted therapy is also an option for patients with advanced-stage lung adenocarcinoma who have specific genetic mutations. However, the overall survival rate for patients with lung adenocarcinoma remains poor, with a five-year survival rate of only 15% (Travis et al. 2015). One of the main reasons for this is the limited efficacy of current therapies and the high incidence of resistance to treatment (Socinski et al. 2018). Therefore, there is an urgent need for developing new and more effective treatments for lung adenocarcinoma.

The androgen receptor (AR) is a nuclear hormone receptor that modulates the biological processes of different tissues like the reproductive system, bone, muscle, and prostate. In its inactive form, the AR is situated in the cytoplasm; however, when androgen ligands such as testosterone are present, the AR relocates to the nucleus, where it modulates the transcription of target genes involved in cell cycle regulation, protein trafficking, signal transduction, and energy metabolism (Jin et al. 2013; Davey and Grossmann 2016). SARMs have recently gained attention as potential therapeutics for various medical conditions due to their selective targeting of AR in a tissue-specific manner, resulting in reduced side effects compared to traditional androgen therapy (Gao and Dalton 2007). SARMs selectively bind to androgen receptors in the body, leading to increased protein synthesis, nitrogen retention, and cell growth in skeletal muscle and bone tissue (Bhasin and Jasuja 2009). Unlike anabolic steroids, which can also bind to androgen receptors in other tissues such as the prostate and hair follicles, androgen receptor modulators are selective in their binding, minimizing unwanted androgenic effects (Bhasin and Jasuja 2009).

Andarine, also known as S4 or GTX-007, is a SARM developed in the late 1990s as a potential treatment for muscle wasting, osteoporosis, and benign prostatic hyperplasia (BPH) (Gao et al. 2004). Selective binding as a non-steroidal SARM to androgen receptors in many tissues, including skeletal muscle and bone tissue, results in anabolic effects such as increased muscle mass and strength, improved bone density, and reduced body fat. In addition to its potential use in altering muscle and fat mass, andarine has been investigated for its potential therapeutic applications. One area of interest is its potential use in treating BPH, a condition characterized by the enlargement of the prostate gland, which can cause urinary symptoms such as difficulty urinating and increased urgency. Andarine has been shown to inhibit the growth of prostate cells, suggesting its potential for use in the treatment of BPH (Gao et al. 2004). Another potential therapeutic application of andarine is in the treatment of muscle wasting in cancer patients. Muscle wasting is a common complication of cancer and its treatment, and can lead to a decrease in physical function, quality of life, and survival. Andarine has been shown to increase muscle mass and strength in animal models of cancer-induced muscle wasting, suggesting its potential as a treatment for this condition (Gao et al. 2005)

SARMs, including andarine, have also been investigated for their potential use in cancer. For instance, androgen deprivation therapy (ADT) is a standard treatment for advanced prostate cancer. However, ADT can have significant side effects, including muscle wasting and bone loss, contributing to morbidity and mortality. Therefore, it has been suggested that SARMs could play a critical role in suppressing the growth and proliferation of prostate cancer cells (Nyquist et al. 2021). Studies have shown that SARMs can reduce the growth of prostate cancer cells in vitro and inhibit the growth of prostate tumors in animal models (Kawanami et al. 2018; Nyquist et al. 2021). Inhibition of the growth of prostate cancer cells by SARMs was provided by inducing apoptosis and inhibiting androgen receptor signaling (Chisamore et al. 2016). In addition, other SARMs have demonstrated promising results in preclinical studies as potential treatments for breast cancer and other hormone-related cancers (Solomon et al. 2019). Several studies have investigated the role of AR in lung cancer. In NSCLC, AR has been shown to be expressed in both tumor and normal lung tissues, and its expression is associated with a worse prognosis in NSCLC patients (Recchia et al. 2009; Mikkonen et al. 2010; Chang et al. 2014). Moreover, AR expression has been detected in a subset of SCLC cell lines and tumors, and AR knockdown has been shown to inhibit proliferation and induce apoptosis in these cell lines (Nazha et al. 2022; Gockel et al. 2022). These findings suggest that AR may be a potential therapeutic target for NSCLC and SCLC.

In this study, we investigated the potential anti-cancer effects of andarine on A549 cells. To our knowledge, no previous studies have investigated the activity of andarine on lung cancer. Our research aimed to elucidate the mechanisms by which andarine exerts its anti-neoplastic activity on lung cancer cells. Our results demonstrate that andarine treatment reduced cell viability, tumorigenic potential, cell proliferation, and migration capacity of A549 cells. Additionally, we observed a significant increase in the apoptosis rate and cell cycle arrest rate at the G0/G1 phase following the administration of andarine. Furthermore, we examined the molecular mechanism underlying the anti-tumorigenic effect of andarine by comparing the gene expression profiles of andarine-treated and untreated A549 cells. Based on our findings, we propose that the negative regulation of proliferation and increased apoptosis induced by andarine treatment led to the interference of A549 cells' carcinogenic capacity.

2.1 Cell Culture and Chemicals

The human lung adenocarcinoma cell line A549 (cat. no. CRM-CCL-185, ATCC) was procured from internal resources. The cells were grown in Dulbecco's Modified Eagle's Medium (DMEM) (cat. no. D6429, Sigma-Aldrich), which was supplemented with 10% fetal bovine serum (FBS) (cat. no. A4736401, Thermo Fisher Scientific) and 1% penicillin/streptomycin (cat. no. 15140122, Thermo Fisher Scientific). The cells were incubated at 37°C in a humidified cell culture incubator containing 5% CO₂. Passaging of cells was carried out once they reached 80% confluency. An inverted microscope was employed to monitor cell proliferation routinely. Andarine ((S)-3-(4-Acetylaminophenoxy)-2-hydroxy-2-methyl-N-(4-nitro-3-trifluoromethylphenyl) propionamide, S4, GTx-007; product no. 78986, Sigma Aldrich) was dissolved in dimethyl sulfoxide (DMSO) to prepare stock solutions. These solutions were subsequently diluted in a complete culture medium to achieve the required concentrations for subsequent assays.

2.2 Cell Viability Assay

Cell viability was determined using the CellTiter 96® Non-Radioactive Cell Proliferation Assay Kit (cat. no. G4000, Promega). A549 cells were seeded at a density of 0.1x10⁵ cells per well in 100 µL culture medium in a 96-well plate. After 24 hours, the culture medium was replaced with various concentrations (0.0001 mM to 0.4 mM) of andarine in the culture medium and incubated for another 24 hours. The negative control was the culture medium, and the positive control was the culture medium with 10% DMSO. The dye solution was added to each well, and after 4 hours, the solubilization solution/stop mix was added to each well to stop the reaction. Absorbance was measured at 570 nm wavelength. The same procedure was followed with a density of 0.05x10⁵ cells for 48 hours. The IC50 values were calculated by fitting dose-response curves using the “drc” package in the R programming language as implemented elsewhere (Yavuz and Demircan 2022).

2.3 Colony Formation Assay

The study employed the colony formation assay (CFA) to evaluate the ability of cells to form colonies following drug exposure. At the outset, 2x10³ cells were seeded in 100 µL of culture medium in a 96-well plate, and after 24 hours, the culture medium was replaced. After that, the medium was replaced every two days until the cells reached 80% confluency or higher. The cells were then fixed with 100% methanol for 20 minutes at room temperature, stained with 0.2% Crystal Violet for 15 minutes, washed twice with ddH2O to eliminate background staining, and the plate was left to dry. Digital images were captured using a bright-field microscope, and the ColonyArea plugin of ImageJ software was used to determine colony number and intensity.

2.4 Wound Healing Assay

The in vitro wound healing assay evaluated cell migration response following drug treatment. Initially, 1x10⁵ cells were seeded in a 24-well plate containing 1 mL of culture medium. After 24 hours of incubation, the culture medium was replaced with andarine-containing culture medium with a concentration of 0.078 mM, as determined by the 48-hour IC50 value. The culture plate was then incubated until a confluent cell monolayer was formed. Cell-free zones were generated across the confluent monolayer using a 200 µL pipette tip. Sequential digital images were captured at 0, 6, 24, and 48 hours following wound creation using a bright-field microscope (SOPTOP ICX41). Wound areas for each well were calculated using the “MRI_Wound_Healing_Tool” plugin in ImageJ software.

2.5 Soft Agar Assay

A soft agar colony formation assay was employed to assess the growth of A549 cells that do not require a surface for attachment. A mixture of 5% agar solution and culture medium was used to create a bottom layer in a 12-well plate. After solidification, 13x10³ cells were added to a cell suspension containing a mixture of agar solution and either culture medium (at the 24-hour IC50 value) for the control and treatment groups, respectively. The top layer was created by adding a second mixture of 5% agar solution and medium, followed by the addition of culture medium or andarine-containing culture medium. The cells were incubated for 30 days, and colony number and size were determined by capturing images of the colonies using a bright-field microscope (SOPTOP ICX41) and analyzing them with the ImageJ software using the "ParticleSizer" plugin.

2.6 Apoptosis Assay

In order to evaluate the apoptotic effects of andarine, an Alexa Fluor®488 Annexin V/Dead Cell Apoptosis kit (cat. no. V13242, Thermo Fisher Scientific) was used. A549 cells were initially seeded in 1 mL of culture medium at a density of 1x105 in a 12-well plate and incubated at 37°C with 5% CO₂ for 24 hours. The culture mediums were then replaced with either culture medium alone or andarine-containing medium (IC50: 0.22 mM). The cells were collected, washed in cold phosphate-buffered saline (PBS), and resuspended in 100 µL of 1X annexin-binding buffer. FITC Annexin V and propidium iodide (PI) working solution were added to each tube, and the cells were incubated at room temperature for 15 minutes. Following this, 400 µL of 1X annexin-binding buffer was added, and the cells were analyzed by flow cytometry (BD Accuri™ C6 Plus).

2.7 EdU Assay

The Click-iT™ EdU Cell Proliferation Kit (cat. no. C10337, Thermo Fisher Scientific) was used to measure the capacity of cells to proliferate after andarine treatment. 10,000 cells were seeded in a 96-well plate in 100 µL of medium and allowed to adhere overnight at 37°C. After 24 hours of incubation with andarine-containing medium, 10 µL of EdU was added to each well, and the cells were incubated for an additional 2 hours. The cells were then fixed with 100% methanol, washed with bovine serum albumin in phosphate-buffered saline, and permeabilized with the saponin-based reagent. EdU detection was accomplished by preparing the Click-iT® reaction cocktail according to the manufacturer's instructions and adding it to the wells. Following incubation at room temperature, the cells were stained with Hoechst dye, and images were captured using a fluorescence microscope. The number of EdU- and Hoechst-positive cells was calculated using ImageJ software.

2.8 Analysis of the Cell Cycle

PI staining was utilized to analyze the cell cycle profile of the cells that underwent treatment with andarine. The cells were counted and seeded into a 12-well plate, where 1x10⁵ cells were added into a 100 µL medium. The plate was then incubated for 24 hours, and the culture medium was replaced with either the normal medium or andarine-containing medium for another 24 hours. Following the incubation, the cells were harvested and washed in PBS. Next, the cells were fixed in 70% ethanol for 30 minutes at 4°C. After fixation, the cells were washed twice with PBS and then treated with 50 µL of a 100 µg/mL stock RNase A solution for 30 minutes at 37°C to prevent RNA staining. Finally, 200 µL PI from a 50 µg/mL stock solution was added to each well, and the cells were analyzed using flow cytometry (BD Accuri™ C6 Plus).

2.9 Senescence-associated B-galactosidase (SA β-gal) staining

To detect senescent cells following andarine treatment, SA-β-gal staining (cat. no. 9860, Cell Signaling Technology) was performed. In brief, 25x10³ cells were seeded in a 24-well plate (Thermo Fisher Scientific) with 0.5 ml of medium for the assessment of senescence. The following day, medium with or without andarine was added to form treatment and control groups. After a 24-hour incubation, the medium was replaced with fresh medium, and cells were allowed to recover for 3 days. Cells were then washed with 1X PBS and fixed in 1X fixative solution for 15 minutes at room temperature. Subsequently, cells were washed twice with 1X PBS, and 0.25 ml of B-galactosidase staining solution, prepared according to the manufacturer’s protocol, was added to each well. The plate was sealed with parafilm to prevent evaporation and incubated overnight at 37°C in a dry incubator without CO₂. Following the overnight incubation, cells were examined under a light microscope (SOPTOP ICX41). To evaluate the impact of 24-hour andarine administration on cellular senescence, cells were seeded at 50x10³ cells/well confluency in a 24-well plate with 0.5 ml of medium. The assay was performed as described above, except for the recovery step.

2.10 cDNA Synthesis and qRT-PCR

To explore the molecular link between observed cellular effects upon andarine treatment and cell division and apoptosis pathways, qRT-PCR was conducted using primers specific to cell-cycle-associated genes. In summary, 3x10⁵ cells were cultured in a 6-well plate (Thermo Fisher Scientific) using 2 mL of culture medium. After a 24-hour incubation period, the medium in each well was substituted with fresh culture medium or a medium containing andarine. After an additional 24-hour incubation period, the cells were collected, and RNA isolation was conducted using previously described methods (Sibai et al. 2019; Yavuz and Demircan 2023). The isolated RNAs were then converted to cDNA, and real-time qRT-PCR was performed using gene-specific primers presented elsewhere (Yavuz and Demircan 2022). The GAPDH housekeeping gene was used for the data normalization. Data were analyzed by implementing the 2^−ΔΔCt method. Data were illustrated on a bar chart utilizing the ‘ggplot2’ package in the R environment as described before (Demircan et al. 2019).

2.11 Statistical Analysis

The results obtained from the assays were expressed as mean ± SD. The normality of the data was assessed using the Shapiro–Wilk test. The data obtained from the cell viability assay was analyzed using the One-Way ANOVA test, followed by Tukey's test. The significance of other data was determined using the t-test. P-values less than 0.05 were considered statistically significant. The statistical analyses and graphical representations were performed in the R environment using the ‘Statix’ package.

3.1 Andarine effectively reduced A549 viability

The viability of A549 cells was significantly reduced by andarine treatment at 24 and 48 hours, as determined by MTT assay. Various concentrations of andarine (0, 0.0001, 0.001, 0.01, 0.05, 0.1, 0.2, 0.4 mM) were used in the experiments. Andarine showed significant inhibitory effects on cell growth at doses between 0.2–0.4 mM for 24-hour (p < 0.001) and between 0.05–0.4 mM for 48-hour periods (p < 0.05 for 0.05 mM, p < 0.01 for 0.1 mM and p < 0.001 for the rest) (Fig. 1a-b). Specifically, after 24-hour treatment, 0.2 mM andarine led to a 1.42-fold decrease in cell viability. For 48-hour treatment, cell viability decreased in a dose-dependent manner by 1.23-fold, 1.26-fold, and 3.57-fold with 0.05, 0.1, and 0.2 mM andarine treatments, respectively. Cells were not viable when treated with 0.4 mM andarine. The IC50 values were calculated as 0.23 mM and 0.15 mM after 24- and 48-hour drug exposure, respectively. These results indicate that andarine restricts the growth of LUAD cells, even at low doses, as demonstrated by the MTT assay.

3.2 Andarine remarkably inhibited colony formation and spheroid forming ability of A549 cells

In order to further understand the effect of andarine on tumorigenicity, colony numbers were counted, and colony intensity was measured using the "ColonyArea" plugin. Treatment with 0.1 mM or higher doses of andarine resulted in significant inhibition of colony formation ability. A 2.6 times fold decrease in the number of formed colonies after treatment with 0.1 mM of andarine was detected compared to the approximately 1188 ± 42 colonies detected in control wells (Fig. 1c) (p < 0.001). Almost no colonies were observed after treatment with 0.2 or 0.4 mM of andarine.

The study further evaluated the effect of andarine treatment on the tumorigenicity of A549 cells by performing a soft agar assay. Results showed that 0.22 mM andarine significantly reduced the average spheroid number and feret diameter at the examined time points (Fig. 1d). Control cells formed 89 spheroids with a feret diameter of 25.5 µm, while in the andarine treatment group, the number of spheroids was 46. Feret diameter was 11 µm (p < 0.05) on the 6th day. On the 19th day, control cells formed 215 spheroids with a feret diameter of 39 µm, while andarine-treated cells formed 105 spheroids with a feret diameter of 15 µm (p < 0.05 for spheroid number, p < 0.01 for spheroid size). At the end of the assay, control cells formed 326 spheroids which were 51.52 µm in feret diameter, while andarine treatment cells formed 221 spheroids with 51.52 µm feret diameter (p < 0.05 for spheroid number, p < 0.001 for spheroid size). The results indicate that 0.22 mM andarine treatment significantly reduced A549 tumorigenicity.

3.3 Andarine significantly impaired with migration capacity of A549 cells

The migration capacity of A549 cells was measured using the wound-healing assay, which showed that andarine had a significant anti-migratory effect (Fig. 2a). The control cells closed 7% of the wound area in 6 hours, while the andarine-treated cells closed only 1% of the wound area (p < 0.01). During the following 18-hour incubation, the andarine-treated cells demonstrated a 1.5-fold decrease in migration capacity compared to the control group (p < 0.001) (Fig. 2a). These results suggest that andarine has a potent anti-migratory effect on A549 cells.

3.4 Andarine stimulated apoptosis while inducing G0/G1 cell cycle arrest and reduction in proliferation rate

As a next step, we aimed to investigate the effect of andarine on apoptotic activity in A549 cells. Following treatment with andarine (0.22 mM) for 24 hours, apoptotic activity was analyzed using flow cytometry. The results revealed that andarine significantly increased apoptotic activity in A549 cells, evident by a 6.8-fold increase in apoptotic cell numbers (p < 0.001) (Fig. 2b). In addition to the increased apoptotic rate, we considered that the growth-limiting effect of andarine may also be attributed to reduced cell proliferation and cell cycle arrest. Analysis of cell cycle distribution using flow cytometry and PI staining showed that andarine induced G0/G1 arrest (p < 0.001) in A549 cells, with a significant increase in the proportion of G0/G1 cells (1.35-fold) (Fig. 2d). Additionally, an EdU incorporation assay was employed to examine the cell proliferation rate whose results demonstrated that andarine significantly reduced cell proliferation (p < 0.001), with a 2.81-fold decrease in the ratio of EdU-positive cells after 24 hours of andarine treatment (Fig. 2c).

3.4 Andarine triggered senescence in A549 cells

Then, we interrogated the possible effect of andarine on cell aging measuring SA β-gal (Fig. 3a). Following 24 hours of andarine application, there was a significant increase in the mean density of SA β-gal in the andarine-administered group compared to the control group in A549 cells, with a fold change of 1.6 and a p-value of less than 0.01 (Fig. 3a). In a subsequent study, a 72-hour recovery period was implemented after the 24-hour andarine application, and the mean senescence density was measured. The results showed a much greater difference in senescence density between the control and andarine-administered groups, with a fold difference of 10.2 and a p-value less than 0.001 (Fig. 3a). The mean senescence density was 1.83% in the control group and 18.68% in the andarine-administered group.

3.5 Andarine modulated gene expression profile

In order to gain more insight into the molecular changes induced by andarine, we conducted a gene expression analysis of cell cycle-related genes in cells treated with andarine for 24 hours. We observed that andarine treatment caused significant upregulation of 8 genes (BAX, PUMA, TP53, GADD45A, CDKN1A, CDKN1B, CCNE1, and CDK4) and downregulation of 4 genes (AKT, BIRC5, MKI67, and PCNA) (Fig. 3b). Among these genes, GADD45A and BAX (fold change: 28.18 and 22.18, respectively) were the most upregulated, while MKI67 and BIRC5 (fold change: -23.10 and − 16.33) were the most downregulated (Fig. 3b). These results suggest that andarine treatment activated genes that promote apoptosis and cell cycle arrest while suppressing genes associated with proliferation and survival, which could explain the observed anti-carcinogenic effect of andarine.

Lung cancer is the most common cause of cancer-related deaths worldwide. According to the Global Cancer Observatory, in 2020, lung cancer accounted for 11.4% of all cancer cases worldwide, and adenocarcinoma was the most common subtype, representing 38% of all lung cancer cases (Sung et al. 2021). Lung adenocarcinoma can be further classified into several subtypes based on their histological and molecular features, and its diagnosis typically involves imaging studies and tissue biopsy (Morgensztern et al. 2010; Travis et al. 2015). Treatment options for this complex disease depend on several factors, such as the stage and type of cancer, the patient's health status, and the genetic profile of the tumor. In recent years, significant advances have been made in the development of new therapies for lung cancer, including targeted therapies and immunotherapies, particularly after molecular characterization of cancer (Herbst et al. 2018; Ettinger et al. 2021).

The current therapies for lung cancer can be broadly categorized into three main types: surgery, radiation therapy, and systemic therapy. Surgery involves the removal of the tumor and surrounding tissue, and it is typically recommended for early-stage lung cancer. Radiation therapy uses high-energy radiation to kill cancer cells and is often used as a primary treatment for patients who cannot undergo surgery. Systemic therapy includes chemotherapy, targeted therapy, and immunotherapy, and it is designed to treat cancer cells that have spread to other parts of the body (Herbst et al. 2018; Xia et al. 2019; Duan et al. 2020). Especially, drugs that target epidermal growth factor receptor (EGFR) mutations or anaplastic lymphoma kinase (ALK) rearrangements have shown significant benefits in patients with lung adenocarcinoma (Bethune et al. 2010; Roskoski 2017).

Although significant progress has been made in lung cancer treatment, there are still several limitations associated with the current therapies. These limitations include the development of treatment resistance, toxicity, and the lack of effective treatment options for specific subtypes of lung cancer (Huang and Fu 2015; Lim and Ma 2019). Resistance to chemotherapy, targeted therapy, and immunotherapy can develop through various mechanisms, such as the activation of alternative signaling pathways or the loss of target expression (Lim and Ma 2019; Liu et al. 2020). This can result in disease progression and the need for alternative treatments. Therefore, research efforts focusing on discovering more effective compounds or repurposing existing drugs in lung cancer treatment are required.

Selective estrogen receptor modulators (SERMs) and selective androgen receptor modulators (SARMs) are a class of drugs that have shown potential as novel anticancer agents (Patel and Bihani 2018). SERMs, such as tamoxifen, are currently used to treat estrogen receptor (ER)-positive breast cancer (Bartlett et al. 2019). Tamoxifen has also shown promise in ER-positive lung cancer, with a recent study demonstrating a survival benefit in patients with advanced lung cancer treated with tamoxifen (Hsu et al. 2021). Similarly, SARMs have been shown to have anticancer effects in preclinical studies (Narayanan et al. 2014). For example, the SARM enobosarm has been shown to inhibit the growth of prostate cancer cells in vitro and in vivo (Chisamore et al. 2016). These findings suggest that SERMs and SARMs have potential as novel anticancer agents and further research is needed to explore their efficacy in various cancer types. We therefore aimed at unveiling the potential anticancer activity of an under-studied SARM, andarine, on lung cancer. Our results provide strong evidence supporting andarine's anti-growth activity in A549 cells (Fig. 1), consistent with previous reports on the role of AR in lung cancer.

Numerous studies have investigated the role and function of AR in different human cancer cell lines. In a study using female C57BL/6 mice, transplanted tumor tissues exhibited higher expression of AR and other hormone receptors related to proliferation (Dou et al. 2017). Treatment with testosterone, an AR agonist, stimulated growth up to three times in five different lung cancer cell lines examined that had positive AR expression, according to a pioneering study on AR roles in lung cancer (Maasberg et al. 1989). The same study found that the growth stimulatory effects of testosterone were antagonized by AR antagonists, flutamide, and cyproterone acetate, which indicates that testosterone has the ability to promote lung cancer cell growth (Maasberg et al. 1989). Another AR agonist, DHT, was found to significantly increase cell growth in the H1355 lung adenocarcinoma cell line (Lin et al. 2004). As a result of studies conducted over the years, ARs can promote cell growth by binding to their agonist and inhibiting ARs in tumors may be a viable approach to stop lung cancer cell proliferation and progression, as observed in our data. Furthermore, in hepatocellular carcinoma cells, overexpression of AR has been shown to promote both anchorage-dependent and anchorage-independent growth (Zhang et al. 2018). Inhibition of AR signaling with anti-androgens like hydroxyflutamide, bicalutamide, or enzalutamide has been shown to reduce the neoplastic transformation of urothelial cells, as demonstrated by colony formation and soft agar assays (Li et al. 2017; Kawahara et al. 2017). Similarly, the present study has been demonstrated that andarine treatment at IC50 dose resulted in almost no colony formation in lung cancer cells (Fig. 1c). In addition, the soft-agar assay showed a marked reduction in colony formation at both early and late time points after andarine treatment (Fig. 1d), highlighting its potent ability to disrupt both anchorage-independent and anchorage-dependent growth of lung cancer cells.

The decrease in migration capacity observed in this study following andarine treatment supports previous reports (Fig. 2a). Preclinical studies have shown that androgen induces the migration and invasiveness of AR-positive triple-negative breast cancer (TNBC) cell lines, while AR antagonists such as bicalutamide and enzalutamide inhibit the growth of TNBC cell lines in vitro (Barton et al. 2015; Giovannelli et al. 2019). A prior study that assessed the impact of SARM on breast cancer revealed that the tested SARM possesses anti-migratory effects. Specifically, GTx-027 was observed to inhibit the migration of MDA-MB-231 cells expressing androgen receptors as early as 24 hours after treatment initiation. GTx-027 was also shown to reduce the number of cells that had migrated, most likely by inhibiting the activity of MMP13 (Narayanan et al. 2014).

An additional study using DHT observed its ability to induce cell growth in A549 cell lines through the upregulation of cyclin D1 (CCND1). This study further suggests that the activation of the mammalian target of the rapamycin (mTOR)/CCND1 pathway could be the mechanism for AR and epidermal growth factor receptor (EGFR) cross-talk, leading to lung cancer development (Recchia et al. 2009). The activation of the AR is known to regulate the G1-S transition, and the deprivation of androgen leads to cell cycle arrest at the G1 phase in prostate cancer cells (Balk and Knudsen 2008). In ovarian cancer cells, AR activation with DHT results in an increased fraction of cells in the S-phase, and treatment with anti-androgens reverses the proliferative impact (Sheach et al. 2009). Treatment with another member of SARMs, S42, significantly suppressed the proliferation of LNCaP, 22Rv1, and PC-3 prostate cancer cells, as demonstrated by BrdU assay (Kawanami et al. 2018). Other structurally similar SARMs, such as GTx-027 and GTx-024, have been shown to reduce the proliferation of AR-expressing breast cancer cells in vitro and in vivo through the activation of anti-proliferative and tumor suppressor genes and inhibition of genes involved in proliferation (Narayanan et al. 2014). In this study, andarine treatment was found to have a growth-suppressive effect on lung cancer cells mediated by the induction of apoptosis and cell cycle, and restriction of proliferation (Fig. 2b-d). Andarine significantly reduced cell proliferation and induced G0/G1 cell-cycle arrest. The observed increase in apoptosis may be attributed to the upregulation of pro-apoptotic genes BAX and PUMA, coupled with the downregulation of survival factors AKT and BIRC5 after treatment with andarine. The increased expression of cell cycle-dependent kinase inhibitors CDKN1A, CDKN1B, and GADD45A provides further evidence for cell cycle arrest at the G0/G1 phase. Additionally, the upregulation of TP53 and GADD45A may contribute to the decreased proliferation rate (Fig. 3b). These findings suggest that andarine's anti-carcinogenic activity is mediated by the modulation of gene expression associated with cancer cell proliferation and death.

Our study demonstrating increased levels of SA-β-gal activity after andarine treatment (Fig. 3a) confirmed previous research findings, which provide a link between androgen signaling blocking and accelerated senescence. Cellular senescence is a complex and diverse response to various stresses that plays a role in tumor suppression, tissue repair, aging, and cancer therapy. Several characteristics are linked to senescence, which include alterations in cell morphology such as enlarged granularity and flattened shape, enhanced levels of SA β-gal, increased expression of cell cycle inhibitors, senescence-related epigenetic modifications, and presentation of the senescence-associated secretory phenotype. A striking finding was a significant increase after andarine treatment in the expression level of GADD45A, a gene associated with cell cycle arrest (reviewed in (Zaidi and Liebermann 2022)). Likewise, the upregulation of CDKN1A and CDKN1B gene expression in response to andarine treatment provides further evidence to support the increased rate of senescence observed. Moreover, we also monitored an elevated rate of G1 arrest for A549 cells upon andarine administration. Since senescent cells undergo cell cycle arrest primarily at the G1 phase and senescence can help prevent cancer cell growth (Kallenbach et al. 2022), we can postulate that andarine exerts its senescence accelerating impact by increasing the expression level of cyclin-dependent kinase inhibitory genes and GADD45A which then resulted in cell cycle arrest at the G1 phase. Our findings are in line with previous studies documenting that modulation of androgen receptor signaling in prostate cancer can induce cellular senescence (Hessenkemper et al. 2014; Carpenter et al. 2021).

The effect of andarine on lung cancer has not been investigated previously. This study is the first to examine the antitumor activity of andarine on A549 cells in vitro. The results suggest that andarine has potent antineoplastic activity, evidenced by inhibited cell growth, tumorigenicity, cell migration, and proliferation while promoting apoptosis and senescence. The antitumor effect of andarine was due to the upregulation of apoptosis, downregulation of proliferation rate, and cell cycle arrest at the G0/G1 phase. The gene expression profiling of A549 cells supported these observations. These results indicate that andarine is a promising and potential anti-carcinogenic agent that warrants further testing in lung cancer animal models. Thus, it can be concluded that andarine exhibits a significant anti-carcinogenic activity in lung cancer cells, and further studies, such as high-throughput sequencing, are needed to elucidate the molecular implications of andarine treatment on A549 cells.

Funding

This study was supported by the BAGEP Award of the Science Academy.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Author contributions

Turan Demircan conceptualized and designed the study. Turan Demircan, Mervenur Yavuz and Aydn Bölük performed material preparation, data collection, and analysis. Turan Demircan wrote the first draft of the manuscript and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Data availability

Data sharing not applicable to this article as no datasets were generated or analysed during the current study.

Ethics approval

This study requires no ethics approval.

Consent to participate

Not applicable.

Consent to publish

Not applicable.

Acknowledgements: None.

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Andarine Plays a Robust In-vitro Anti-carcinogenic Role on A549 Cells Through Inhibition of Proliferation and Migration, and Activation of Cell-cycle Arrest, Senescence, and Apoptosis

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Cell Culture and Chemicals

2.2 Cell Viability Assay

2.3 Colony Formation Assay

2.4 Wound Healing Assay

2.5 Soft Agar Assay

2.6 Apoptosis Assay

2.7 EdU Assay

2.8 Analysis of the Cell Cycle

2.9 Senescence-associated B-galactosidase (SA β-gal) staining

2.10 cDNA Synthesis and qRT-PCR

3. Results

3.1 Andarine effectively reduced A549 viability

3.2 Andarine remarkably inhibited colony formation and spheroid forming ability of A549 cells

3.3 Andarine significantly impaired with migration capacity of A549 cells

3.4 Andarine stimulated apoptosis while inducing G0/G1 cell cycle arrest and reduction in proliferation rate

3.4 Andarine triggered senescence in A549 cells

3.5 Andarine modulated gene expression profile

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

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