Background
Lung cancer is the leading cause of cancer death worldwide and its incidence is still on the increase [
1]. About 89% of lung cancer patients are non-small cell lung cancer (NSCLC) [
2] and 20–30% of these patients were diagnosed at a locally advanced stage [
3]. In most NSCLC patients, the oncogenic AKT, ERK and STAT3 signaling pathways are constitutively activated. It has been reported that 50–70% overexpression of phosphorylated AKT [
4], 70% expression of activated ERK [
5] and over 50% high levels of activated STAT3 [
6] were observed in NSCLC. Aberrant activation of these three signaling pathways results in uncontrolled proliferation, apoptosis resistance and other oncogenic cascades in lung cancer cells [
4,
6‐
8]. Therefore, there has been increasing research interest in identifying novel therapeutics to target these oncogenic signaling pathways for effectively treating NSCLC patients [
4,
9,
10].
PI3K-AKT-mTOR and RAS-RAF-MEK-ERK are two main downstream signaling pathways driven by
epidermal growth factor receptor (
EGFR) mutations or abnormal fusion of
echinoderm microtubule-
associated protein-
like 4 and
anaplastic lymphoma kinase (
EML4-
ALK) genes [
4]. STAT3 (signal transducer and activator of transcription 3), an important point of convergence for various signaling pathways [
6] is also required for the oncogenic effects of NSCLC-associated
EGFR mutations [
11]. The relatively limited subset of NSCLC carrying the above genetic mutations can be effectively treated by the tyrosine kinase inhibitors (TKIs)-mediated targeted therapy [
4,
12,
13]. However, most NSCLC patients do not harbor these genomic events and the 5-year survival rate remains dismal [
13]. More novel targeted agents to suppress these oncogenic pathways are imperatively needed.
Taiwanofungus camphoratus (syn.
Antrodia camphorata) is a widely used medicinal polypore fungi, which belongs to the Polyporaceae, Basidiomycotine family, and grows in a unique host, the endemic perennial tree
Cinnamomun kanehirai (Bull camphor tree) in Taiwan [
14,
15]. It is a well-known folk medicine and has been used as a local remedy to treat abdominal pain, diarrhea, drug intoxication, hypertension, and skin itching as well as to improve immune system and liver function [
15,
16]. On the other hand, many studies have demonstrated its anticancer effects in the aspects of anti-proliferation, apoptosis induction and anti-invasion [
14,
16‐
18]. Instead of the commonly used ethanol, we use
n-hexane to extract the
T. camphoratus and further separate the extract into eight fractions (HS1–HS8) by silica gel chromatography. We have isolated the most potent active fraction (HS7) according to its anti-proliferative activities against a panel of human cancer cell lines, including, lung (CL1-0), prostate cancer (PC3) and hepatocellular carcinoma (HepG2, Hep3B and Huh7) cells (see Additional file
1). Our previous study had demonstrated the effects of HS7 on the apoptosis induction and Wnt/β-catenin signaling inhibition in human colon cancer cells [
14]. In the present study, we explore its effects on the aforementioned AKT-mTOR, ERK and STAT3 signaling pathways in a human NSCLC cell line CL1-0, which harbors wild-type EGFR and is resistant to EGFR TKIs [
19]. The results show that HS7 vigorously suppresses the signaling pathways described above and arrests the cell growth, accompanied with induction of cyclin-dependent kinase (CDK) inhibitors such as p15, p21 and p27. Our findings suggest the potential of HS7 as an alternative medicine for the treatment of NSCLC.
Methods
The information of experimental design, statistics, and resources used in this study are attached in Minimum standards of reporting checklist (Additional file
2).
Cell culture
The CL1-0 human lung adenocarcinoma cell line was kindly provided by Dr. Shine-Gwo Shiah (NHRI, Miaoli, Taiwan) and the MRC-5 normal fetal human lung fibroblasts were purchased from Bioresource Collection and Research Center (Hsinchu, Taiwan). CL1-0 cells were maintained in RPMI1640 (Gibco, CA, USA) and MRC-5 cells were in MEM (Gibco, CA, USA) medium supplemented with 10% fetal bovine serum and 1× penicillin–streptomycin–glutamine (Gibco, CA, USA). Cells were cultured at 37 °C in a water-jacketed 5% CO2 incubator.
Preparation of ethanol and n-hexane extracts of T. camphoratus and the active fraction HS7
As shown in our previous study [
14], the fruiting body-like
T. camphoratus (Voucher Number TC-2004-09-001) was cultivated and provided by Well Shine Biotechnology Development Co. (Taipei, Taiwan). Briefly, air-dried ground powder of cultivated
T. camphoratus was extracted exhaustively with
n-hexane or ethanol. The
n-hexane extract was then further separated to eight fractions (HS1–HS8) by silica gel chromatography, and the seventh fraction (HS7) exerted the most potent effect on the growth inhibition of a screening panel of cancer cell lines (CL1-0, PC3, HepG2, Hep3B and Huh7). After lyophilization, the stock solutions of ethanol and
n-hexane extracts and HS7 dissolved in dimethyl sulfoxide (DMSO) at concentration of 100 mg/mL were made. They were diluted in phosphate-buffered saline (PBS) prior to use. The final concentrations of DMSO were all below 0.2%.
Reagents
The sulforhodamine B (SRB) dye for cell viability assay, propidium iodide for cell cycle analysis and the EGFR-TKI gefitinib (Iressa) were from Sigma-Aldrich (St. Louis, MO, USA). The MEK-ERK pathway inhibitor U0126 and PI3K-AKT pathway inhibitor LY294002 were from Cell Signaling Technology (Danvers, MA, USA). The JAK-STAT3 pathway inhibitor AG490 was from Calbiochem (La Jolla, CA, USA).
Cell viability assay by sulforhodamine B (SRB) staining
According to the method described by Vichai and Kirtikara [
20], SRB dye-binding assay was used to determine the viability of cancer cells. CL1-0 and MRC-5 cells were seeded in a 96-well plate at a density of 2 × 10
3 cells/well in 10% FBS-RPMI or MEM medium. After 24 h of incubation, cells were treated with agents as indicated or PBS only for another 72 h. Cells were then harvested and fixed by 10% trichloroacetic acid (TCA). After fixing, cells were washed by distilled water and stained viable cells by 0.4% (w/v) SRB dye dissolved in 1% acetic acid. After staining for 30 min, the unbound dye was then washed away by 1% acetic acid and the plate was air-dried. The cell-bound SRB dye was then dissolved in 200 μL of 10 mM Tris base and the absorbance was read on a microplate reader (Molecular Devices, CA, USA) at a wavelength of 562 nm. The absorbance was directly proportional to the cell number over a wide range.
Cell cycle analysis
Propidium iodide (PI) staining and flow cytometry were used to determine the cell cycle distribution. One day after being seeded in a six-well plate (105 cells/2 mL/well), CL1-0 cells were treated with different doses of HS7 for 72 h. At harvest, cells were washed with PBS, incubated with 0.25% Trypsin EDTA at 37 °C for 5–10 min and then suspended in medium at a concentration of 1 × 106 cells/tube. After being washed with PBS and centrifuged at 1200 rpm at 4 °C for 5 min, cells were resuspended in 500 μL PBS and fixed with 70% ethanol followed by gentle vortexing. Cells were allowed to stand overnight at − 20 °C. Fixed cells were spun down and washed with PBS. The cells were suspended in 500 μL PI (2 μg/mL)/Triton X-100 (0.1% v/v) staining solution with RNase A (200 μg/mL) at room temperature for 20 min and then analyzed by a flow cytometer (FACSCalibur™, BD Bioscience, CA, USA). Approximately 10,000 counts were made for each sample. The percentages of cell-cycle distribution were calculated by CellQuest software (BD Bioscience, CA, USA).
Western blotting
A total of 4 × 105 CL1-0 cells/10 cm dish were incubated for 24 h after seeding and then treated as indicated in figures for 72 h. On the day of harvest, the whole-cell lysates were prepared with radioimmunoprecipitation (RIPA) lysis buffer containing 1× tyrosine phosphatase inhibitor cocktail (FC0020-0001, BIONOVAS, Toronto, Canada), 1× protease inhibitor cocktail-full range (FC0070-0001, BIONOVAS, Toronto, Canada), and 1× serine/threonine phosphatase inhibitor cocktail (FC0030-0001, BIONOVAS, Toronto, Canada). Samples of protein extract were size fractionated electrophoretically by polyacrylamide SDS-PAGE gel and transferred onto a PVDF membrane using the BioRad Mini Protean electrotransfer system (CA, USA). The membranes blots were incubated with 5% milk in PBST for 1 h to block nonspecific binding and then were incubated with primary antibodies overnight at 4 °C. The membranes were detected with an appropriate peroxidase-conjugated secondary antibody incubated at room temperature for 1 h. Intensive PBS washing was performed after each time of incubation. The immune complexes were visualized using an enhanced chemiluminescence detection system (ECL, Perkin Elmer, Waltham, MA, USA) according to the manufacturer’s instructions. Primary antibodies against ERK (sc-94), p-ERK (sc-7383), STAT3 (sc-8019), p-STAT3 (sc-7993), p-p70S6K (sc-7984-R), p15 (sc-613), tubulin (sc-5286) and GAPDH (sc-47724) were purchased from Santa Cruz Biotechnology (San Diego, CA, USA). Primary antibodies for p-AKT (#4051), p-mTOR (#2971), mTOR (#2972) and p-pRB (#9308) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies for p21 (#05-345) and HIF-1α (07-628) were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Primary antibody for p27 (#610241) was purchased from BD Transduction Laboratories (San Jose, CA, USA).
Photograph of the cells
At harvest, cells were examined under a Nikon inverted microscope. The photomicrographs were captured by a CCD camera (Nikon, Mito, Japan) adapted to the microscope.
Statistical analysis
Cell viability data are expressed as mean ± SE. Differences between the cell viabilities of control and treated groups were evaluated by one-way ANOVA followed by Dunnett’s t test. Probability value of p < 0.05 was considered statistically significant. Single asterisk (*) indicate p < 0.05; double asterisks (**) indicate p < 0.01; triple asterisks (***) indicate p < 0.001.
Discussion
The oncogenic EGFR signaling in NSCLC engages the activation of downstream effectors such as AKT-mTOR, ERK and STAT3 to promote cell proliferation, cell survival, and tumor growth [
4,
9,
31,
32]. Blockade of EGFR activation by TKIs targeted therapy has significantly changed the treatment paradigm in NSCLC [
4]. However, only a small proportion of NSCLC patients can respond to clinically used TKIs [
4,
31]. Most of the NSCLC patients do not carry the genetic alterations for the effectiveness of EGFR-TKIs treatment [
13]. Tumor samples derived from NSCLC patients can show robust activation of AKT, ERK, and STAT3 while EGFR is not activated [
31]. Alternative novel targeted therapeutics other than the EGFR-TKIs is imperatively needed. Nowadays, lots of ongoing efforts have been conducted in identifying potential therapeutics targeting the above-mentioned effectors (ERK, AKT-mTOR and STAT3), which are frequently dysregulated in NSCLC.
The anticancer activities of
T. camphoratus (syn.
A. camphorata) have been studied in numerous types of cancer cells including NSCLC [
14,
16‐
18,
33,
34]. Its potential for the treatment of NSCLC has been shown in a preclinical evaluation in which the significant tumor suppression and apoptosis induction were observed [
33]. To further develop the application of
T. camphoratus for NSCLC treatment, we isolate a more potent active fraction (HS7) from its
n-hexane extract. For the demanding of novel targeted therapeutics mentioned above, we investigate and demonstrate the substantial inhibition of ERK, AKT-mTOR and STAT3 signaling pathways by HS7 in EGFR-TKI resistant CL1-0 human NSCLC cells (EGFR wild-type).
The interplay between these pathways is complex in CL1-0 cells. Our results show that treatments with synthetic inhibitors of these pathways such as LY294002, U0126 and AG490 induce feedback or compensatory activation of parallel circuits in CL1-0 cells. This phenomenon is similar to that reported in other previous studies of synthetic inhibitors. The feedback activation of AKT plays an important role in the unsatisfactory clinical results of mTOR inhibitor in cancer treatment [
35,
36]. Combining with AKT inhibitor to enhance the therapeutic effects of mTOR inhibitor for NSCLC treatment was thus suggested [
35]. Our results show the simultaneous inhibition of p-AKT and mTOR signaling cascade (p-mTOR, p-p70S6K and HIF-1α) by HS7, implying its potential for this therapeutic strategy. On the other hand, targeting components of ERK signaling (RAS-RAF-MEK-ERK) also has been proposed for NSCLC treatment [
37]. However, activation of alternative signaling pathways almost always occurs after inhibition of ERK [
37]. Like that reported in the study by Hayashi et al. [
38], inhibition of p-ERK by U0126 accompanied with increased p-AKT is also observed in our result. Combination with inhibitor of another parallel signaling tract such as AKT pathway has been studied in animal model [
39] and clinical trial [
37] for NSCLC treatment. Regarding the simultaneous suppression of both ERK and AKT-mTOR pathways by HS7, it might have the advantage over the individual use of synthetic ERK signaling inhibitors for NSCLC therapy.
Inhibition of STAT3 signaling may be effective for treatment of NSCLC irrespective of the EGFR mutation status [
9]. In addition, activation of STAT3 also has been shown to participate in the resistance of NSCLC cells to erlotinib (EGFR-TKI) [
9,
40] and radiation [
9,
41]. An old FDA-approved anthelmintic drug niclosamide was recently found to overcome acquired erlotinib resistance and reverse radioresistance through suppression of STAT3 in NSCLC xenografts [
40,
41]. In this perspective, the significant inhibition of STAT3 pathway by HS7 might also have the potential to reduce the resistance of NSCLC cells to EGFR-TKIs or radiotherapy. Further future investigation is warranted.
The success of targeted therapy can be limited by the ultimately developed resistance of cancer cells through mutation of the target kinase, signaling redundancy, feedback activation of pathway components, compensatory activation of parallel circuits and so forth [
32]. One strategy to improve the efficacy is combination therapy [
5,
22]. Therefore, simultaneously co-targeting the signaling pathways of such as AKT and ERK [
5,
22,
39], mTOR and ERK [
42], STAT3 and mTOR [
43] have been proposed to improve the success of NSCLC targeted therapy. According to this strategy, the aforementioned multi-targeting activity of HS7 is worth of further development for its integrative use in NSCLC targeted therapy. The point of convergence targeted by HS7 to exert its multi-targeting effects is remained to be elucidated. It is also possible that the multi-components in HS7 are responsible for its multi-targeting activity. Compounds including polysaccharides, ergostan-type triterpenoids, a sesquiterpene, and phenyl and biphenyl derivatives had been isolated from
T. camphoratus (syn.
A. camphorata) [
44,
45]. It is worthy to identify and quantify the active components in the HS7 fraction for the future development.
Like that observed in gefitinib (iressa)-treated cells [
28,
29], inhibition of proliferative signaling cascades by HS7 is also accompanied with induction of p15, p21 and p27, and decrease of pRb phosphorylation. It is reported that suppression of proliferative signaling like ERK and AKT-mTOR could inhibit the degradation of p21 and p27, respectively [
46,
47], and up-regulate the p15 mRNA level [
28]. HS7 might also modulate the proteolysis or transcription of these CDK inhibitors by the similar ways, resulting in inhibition of pRb phosphorylation. On the other hand, in addition to constrain the cell-cycle progression, HS7 also induced apoptosis at dose of 25 μg/mL, and massive apoptosis induction (46% sub-G1 fraction) could be observed at higher dose up to 50 μg/mL (see Additional file
3). The profound inhibition of these targeted signaling effectors by HS7 did have apoptotic killing effect on CL1-0 cells.
Authors’ contributions
ICL, GML, CJY and KJB conceived the study and wrote the manuscript. CFY, CHL and JLY performed the experiments and data analysis. JMC and HLL reviewed literature and interpreted the results. SEC and JWP revised the manuscript. KJB and CJY equally contributed to the paper. All authors read and approved the final manuscript.