J Cancer Prev 2022; 27(2): 112-121
Published online June 30, 2022
© Korean Society of Cancer Prevention
1Department of Biology, Faculty of Science, Thaksin University, Phatthalung, 2Department of Basic Science and Mathematics, Faculty of Science, Thaksin University, Songkhla, 3Department of General Science and Liberal Arts, King Mongkut’s Institute of Technology Ladkrabang Prince of Chumphon Campus, Chumphon, Thailand, 4Department of Cogno-Mechatronics Engineering, Pusan National University, Busan, Korea
Correspondence to :
Chutima Kaewpiboon, E-mail: Chutima.firstname.lastname@example.org, https://orcid.org/0000-0003-2519-3796
Young-Hwa Chung, E-mail: email@example.com, https://orcid.org/0000-0001-5738-7400
*These authors contributed equally to this work as co-correspondence authors.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Considering that presence of cancer stem cell (CSC) subpopulation in tumor tissues confers anticancer drug resistance, we investigated whether human A549 lung cancer cells resistant to etoposide possess CSC-like phenotypes. Furthermore, it is known that these malignant tumor features are the leading cause of treatment failure in cancer. We have thus attempted to explore new therapeutic agents from natural products targeting these malignancies. We found that formoxanthone C (XanX), a 1,3,5,6-tetraoxygenated xanthone from Cratoxylum formosum ssp. pruniflorum, at a non-cytotoxic concentration reduced the expression of the signal transducer and activator of transcription 1 (STAT1) and histone deacetylase 4 (HDAC4) proteins, leading to inhibition of CSC-like phenotypes such as cell migration, invasion, and sphere-forming ability. Moreover, we found that treatment with STAT1 or HDAC4 small interfering RNAs significantly hindered these CSC-like phenotypes, indicating that STAT1 and HDAC4 play a role in the malignant tumor features. Taken together, our findings suggest that XanX may be a potential new therapeutic agent targeting malignant lung tumors.
Keywords: Formoxanthone C, Multidrug resistant-cancer, Cancer stem cell-like phenotypes, Signal transducer and activator of transcription 1, Histone deacetylase 4
Lung cancer represents the most common malignancy and is responsible for the leading cause of cancer-related mortality and death worldwide . Unfortunately, the success of chemotherapy is hindered by the development of multidrug resistance (MDR) in cancer. The increase of drug efflux out of cells mediated by P-glycoprotein (P-gp) is a main mechanism on drug resistance in cancer cells . Furthermore, cancer cells resistant to chemotherapy treatment usually demonstrate aggressive characteristics, such as accelerated metastasis to distant organs or tissues and self-renewal into multiple cell types . Recent studies have shown up-regulation of P-gp mediated by signal transducer and activator of transcription 1 (STAT1)-histone deacetylase 4 (HDAC4) in the etoposide-resistant human lung cancer cell line (A549RT-eto) .
STAT1 is one of the seven mammalian members of the STAT family and a master transcription factor for IFN-γ intracellular signaling, leading to anti-oncogenesis in part by upregulating caspases , cyclin-dependent kinase inhibitor 1A , and the IFN-regulatory Factor 1 (IRF1)/p53 pathway , while down-regulating the BCL2 family . Recent reports have also shown that constitutive overexpression of STAT1 is correlated with protection of tumor cells from chemotherapeutic drugs such as doxorubicin  or cisplatin . Furthermore, other studies demonstrated that STAT1 promotes the epithelial-mesenchymal transition (EMT) and manifestation of cancer stem cell (CSC)-like phenotypes such as faster cell migration, aggressive invasion and enhanced sphere-forming ability [11,12].
Histone deacetylases (HDACs) play roles in the maintenance and function of chromatin by regulating the acetylation state of histones . However, recent studies suggest that HDACs also regulate the acetylation state of many non-histone targets [14-16]. Notably, HDAC4 downregulation inhibits EMT and reduces the characteristics of CSC . Furthermore, STAT1-HDAC4 signaling induced by EMT  and doxorubicin resistance in human lung cancer cells overexpressing cancer upregulated gene 2 .
Novel therapeutic agents from natural products against anti-cancer drug resistance becomes the major challenge in cancer therapies. Natural products from plants have been a rich source of chemotherapeutic agents . One of them is formoxanthone C (XanX), a xanthone from the green fruit of
This study was initiated to investigate whether A549RT-eto cancer cells harbor other CSC-like phenotypes in addition to drug resistance and whether the STAT1-HDAC4 signaling pathway is involved in the manifestation of such additional phenotypes. Furthermore, we explored whether formoxanthone C (XanX) could effectively inhibit the malignant phenotypes through suppression of STAT1-HDAC4 in A549RT-eto cells.
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Human lung cancer A549 cells (ATCC, Manassas, VA, USA) and A549RT-eto cells, which were developed and generously provided by the Laboratory of Biochemistry, Chulabhorn Research Institute, Thailand, as described elsewhere . The cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA), supplemented with 10% FBS, 1% penicillin, and streptomycin (Gibco), at 37°C in a humidified atmosphere of 5% carbon dioxide in air.
The cells were plated into 24 well plates at (1 × 105 cells per well) for overnight to achieve 80% to 90% confluency before XanX treatment. Then, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (CellTiter 96 Non-Radioactive Cell Proliferation assay; Promega Corporation, Madison, WI, USA) was used to measure cell survival, as previously described . Dye solutions containing tetrazolium were added to the cells in the 24-well plate and incubated for 2 hours. The absorbance of the formazan produced by living cells was measured at a wavelength of 570 nm (Victor3; PerkinElmer, Waltham, MA, USA). The relative percentage of cell survival was calculated by the mean optical density of treated cells (ODT) and the mean optical density of control cells (ODC) with the following formula: Cell survival rate = (ODT/ODC).
For immunoblotting, antibodies against STAT1 (#9172) and phospho-STAT1 (#9171) were acquired from Cell Signaling Biotechnology (Beverly, MA, USA). Anti-P-gp (Calbiochem, San Diego, CA, USA) and anti-actin (C4), -Klf4 (sc-166229), -HDAC4 (sc-5245) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Anti-E-cadherin (ab15148), -N-cadherin (ab18203), -Vimentin (ab137321), -Bmi1 (ab126783), -Sox2 (ab97959), -Nanog (ab80892) and -Oct4 (ab109183) were obtained from Abcam (Cambridge, MA, USA).
The cells were harvested and lysed with lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl [pH 7.5] containing 0.1 mM Na2VO3, 1 mM NaF, and protease inhibitors [Sigma Aldrich, St. Louis, MO, USA]). For immunoblotting, proteins from whole-cell lysates were resolved by 10% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and then transferred to nitrocellulose membranes. After that the membranes were incubated with PBS containing 0.05% Tween 20 and 5% skimmed milk for 1.5 hours at room temperature. After washing, the membranes incubated separately with primary antibodies (1:1,000 or 1:2,000 dilution) for overnight at 4°C. After washing, the membranes were incubated with the secondary antibodies (1:2,000 dilution), horseradish peroxidase–conjugated rabbit anti-goat or mouse Immunoglobulin G, at room temperature. Immunodetection was performed using chemiluminescence reagent and exposed for an enhanced chemiluminescence assay using LAS 4000 mini (GE Healthcare, Durham, NC, USA). Densitometric analysis of protein bands from all experiments were performed using ImageJ software (https://imagej.nih.gov/ij/) for image analysis . The visualization of β-actin was used to ensure equal sample loading in each lane. All experiments were repeated at least 3 times.
The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with cold ethanol for 15 minutes, blocked with 10% bovine serum albumin for 30 minutes, and treated with primary antibodies (1:100 dilution) for 30 minutes at room temperature. After incubation, the cells were washed three times with PBS, incubated with Alexa Fluor 418-conjugated goat anti-mouse or donkey anti-rabbit antibody (1:500 dilution; Molecular Probes, Eugene, OR, USA) in PBS for 30 minutes. at room temperature, and washed three times with PBS. For nuclear staining, the cells were incubated with 4′, 6-diamidino-2-phenylindole (DAPI) for 5-10 minutes in the dark and washed three times with PBS. The stained cells were mounted using PBS containing 10% glycerol and photographed using a fluorescence microscope (Axio Observer D1; Zeiss, Oberkochen, Germany).
The cells were plated into 24 well plates at (1 × 105 cells per well) for overnight to achieve 60% to 70% confluency before small interfering RNA (siRNA) transfection as previously described , Human STAT1-siRNAs (100 nM ; Bioneer, Daejeon, Korea), human HDAC4-siRNAs (100 nM; Bioneer), or negative control siRNAs (100 nM; Bioneer) were mixed with Lipofectamine 2000 (Invitrogen). The cells were incubated with the transfection mixture for 24 hours and then rinsed with RPMI-1640 medium containing 10% FBS. The cells were incubated for 24 hours before harvest.
As described previously , the cells were cultured in tumor sphere medium consisting of serum-free DMEM/F12 medium (Gibco), 20 ng/mL human recombinant epidermal growth factor (EGF) (Invitrogen), 10 ng/mL basic fibroblast growth factor (Invitrogen), 5 g/mL insulin (Sigma Aldrich, St. Louis, MO, USA) and 0.4% bovine serum albumin (Sigma Aldrich), then cells at density of 1,000 cells/well suspended in sphere medium with or without XanX were plated in 24-well ultra-low attachment plates. Two, four or six day later, tumor sphere formation was visualized and photographed under a microscope. The size and the number of spheroids were analyzed under a light microscope. Spheroids more than 50 µm in size served as a criterion for sphere formation.
The cells (1 × 105 cells/well) were plated in growth medium in 24-well plates and incubated for 24 hours. After confirming the formation of a complete monolayer almost 100%, the cells were wounded by scratching lines with a standard 200-µL plastic tip as described previously . The wound was generated and then the cells were washed with PBS. The medium with or without XanX was added the cells and incubated for 24 hours. Migration and cell movement throughout the wound area were visualized and photographed under a phase-contrast microscope at 0 and 24 hours. The cell wound closure rate was calculated using the following equation: Wound closure = [1-(wound area at Tt/wound area at T0)] × 100, where Tt is the time passed since wounding and T0 is the time the wound was created. The experiments were performed in triplicate.
The cell invasive capacity was measured using Transwell Filter (8-µm pore size; Corning, Corning, NY, USA) with Matrigel (BD Biosciences, San Jose, CA, USA). The cells treated with XanX or untreated at density of 5 × 104 cells/well were transferred to each upper chamber of Transwell in 200 µL of serum-free medium. As mentioned elsewhere , 500 µL of complete medium was added to each bottom chamber of Tranwell with the same concentration of XanX. After incubation for 24 hours, the cells in the upper chambers were removed, and the invading cells in the membrane were stained with hematoxylin-eosin. The stained cells were photographed and counted under a light microscope in at least six randomly selected fields.
Data are expressed as the mean ± standard error of the mean (SEM). Statistical analysis involved calculating the unpaired t-test using GraphPad InStat (GraphPad Software, San Diego, CA, USA). Values significantly different from the control are shown at *
Since we observed that human A549RT-eto exhibit upregulation of STAT1 and HDAC4 and the elevated levels of P-gp encoded by
CSCs play a major role in cancer initiation, progression, metastasis, and drug resistance  CSC-like features include sphere formation and elevated expression of stemness-related factors such as Sox2, Oct4, Klf4, Bmi1 and Nanog. We thus exploered whether A549RT-eto cells could exhibit CSC-like phenotypes. Because the sphere formation assay is considered a useful method to evaluate self-renewal of CSCs in vitro , we compared the sphere-forming ability of A549RT-eto cells with that of the parental cells. A549RT-eto cells exhibited a larger size and the greater number of spheroids compared to those of their parental cells (Fig. 1E) (***
A549RT-eto cells were shown to exhibit enhanced P-gp protein levels, which lead to MDR through upregulation of the STAT1-HDAC4 signaling . Thus, we screened natural compounds derived from medicinal plants in Thailand to reverse MDR via suppression of the STAT1-HDAC4 signaling. The compound isolated from
Conventional chemotherapy usually shows inefficiency in eradicating CSC because CSC harbor some mechanisms for anti-cancer drug resistance . Therefore, as it is a challenge to search for anticancer drugs to overcome these cancer cells bearing CSC-like phenotypes, we tested whether XanX can block CSC-like phenotypes in A549RT-eto cells. First, we performed wound-healing assays to evaluate the effects of XanX on the migration of A549RT-eto cells. For this purpose, the cells were incubated at a non-cytotoxic dose (10 μg/mL) of XanX for 24 hours. The results showed significantly decreased cell migration rates in A549RT-eto cells (Fig. 3A) (***
Since XanX treatment inhibited STAT1 and HDAC4 expression and activity as well as malignant tumor features (Fig. 2 and 3), a question arose as to whether STAT1-HDAC4 signaling is involved to silence expression of these malignant tumor features. Thus, siRNA was introduced to silence STAT1 and HDAC4. The treatment with STAT1 and HDAC4 siRNA inhibited migration of A549RT-eto cells (Fig. 4A) (***
In our study, the levels of STAT1 and HDAC4 proteins were enhanced in A549RT-eto cells compared to those in the A549 parental cells . Some studies have shown that activation of STAT1 and HDAC4 is associated with chemoresistance [4,19,30]. Thus, the suppression of STAT1 and HDAC inhibited manifestation of CSC-like phenotypes, including cell migration, invasion, and sphere formation in A549RT-eto cells. Our study further demonstrated that the decrease in STAT1 and HDAC4 levels by XanX treatment diminished the expression of mesenchymal protein markers such as N-cadherin and vimentin but enhanced the expression of the epithelial protein marker E-cadherin. The decrease in STAT1 and HDAC4 levels by XanX treatment also led to the reduced expression of stem cell transcription factors (Oct4, Bmi1, Nanog, Sox2 and Klf4) in A549RT-eto cells. The stemness-related transcription factors were found to be overexpressed in several cancers, including breast cancer, prostate cancer, and oral squamous cell carcinoma, and their expression levels are associated with malignant transformation, tumorigenicity, and tumor metastasis . On the basis of these studies, we propose that the decrease in STAT1 and HDAC4 levels can be potential resolution of anticancer agents in treating other cancers in addition to A549 lung cancer with MDR. Therefore, STAT1-HDAC4 signaling becomes one of the most suitable candidates for therapeutic targets in the management of cancer . This observation necessitates the exploration and development of anticancer agents targeting STAT1-HDAC4 signaling targeting ability.
Many approaches exploit the differences in cell surface markers to identify compounds that selectively target cancer cells. These targets include the ABC transporter superfamily, Wnt/β-catenin, TGF-β, Hedgehog, EGFR and Notch [32-34]. Recently, several compounds such as vitamins A and D, genistein, epigallocatechin gallate, sulforaphane, piperine, theanine, choline and curcumin have been shown to target malignant tumor features, particularly in combination with conventional chemotherapy drugs. They have been shown to significantly suppress self-renewal, induce differentiation, and inhibit tumor growth and metastasis . Herein, we present novel finding that XanX treatment decreases the levels of P-gp through inhibition of STAT1-HDAC4 signaling. Downregulation of STAT1-HDAC4 signaling by XanX may be a key to solve some major obstacles in malignant cancer therapy, namely cancer relapse and chemotherapy resistance.
This study was supported by IPST for research funds for DPST graduate with first replacement (No.03/2557). The study was also supported by the Korea Institute for Advancement of Technology (KIAT) grant funded by Korea Government (MOTIE) (P0008763, The Competency Development Program for Industry Specialist; and N0002310, Construction Project of Supporting Center for Commercializing Customized Nano-mold-based Technologies).
No potential conflicts of interest were disclosed.