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Table of Contents
ORIGINAL ARTICLE
Year : 2023  |  Volume : 66  |  Issue : 4  |  Page : 189-199

Calycosin inhibits gemcitabine-resistant lung cancer cells proliferation through modulation of the LDOC1/GNL3L/NFκB


1 Center of Stem Cell and Precision Medicine, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation; College of Medicine, Tzu Chi University, Hualien, Taiwan
2 Department of Post-Baccalaureate Medicine, College of Medicine, National Chung Hsing University, Taichung, Taiwan
3 GeneReach Biotechnology Corp, Taichung, Taiwan
4 Clinical Laboratory, Chung Shan Medical University Hospital; School of Medical Laboratory and Biotechnology, Chung Shan Medical University, Taichung, Taiwan
5 College of Medicine, Tzu Chi University; Department of Hematology and Oncology, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan
6 Integration Center of Traditional Chinese and Modern Medicine, Hualien Tzu Chi Hospital; Department of Chinese Medicine, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation; School of Post-Baccalaure Chinese Medicine, College of Medicine, Tzu Chi University, Hualien, Taiwan
7 Department of Biological Science and Technology; Ph.D. Program for Biotechnology Industry, China Medical University, Taichung, Taiwan
8 Department of Nursing, MeiHo University, Pingtung, Taiwan
9 Graduate Institute of Chinese Medical Science, China Medical University, Taichung, Taiwan
10 Division of Colorectal Surgery, Department of Surgery, Taichung Veterans General Hospital, Taichung; Faculty of Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan
11 Cardiovascular and Mitochondrial Related Disease Research Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien; Graduate Institute of Biomedical Sciences, China Medical University, Taichung; Department of Biological Science and Technology, Asia University, Taichung; Center of General Education, Buddhist Tzu Chi Medical Foundation, Tzu Chi University of Science and Technology, Hualien; Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan

Date of Submission19-Jan-2023
Date of Decision24-Apr-2023
Date of Acceptance02-Jun-2023
Date of Web Publication22-Aug-2023

Correspondence Address:
Prof. Chih-Yang Huang
Cardiovascular and Mitochondrial Related Disease Research Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien
Taiwan
Dr. Ming-Cheng Chen
Division of Colorectal Surgery, Department of Surgery, Taichung Veterans General Hospital, Taichung
Taiwan
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cjop.CJOP-D-23-00009

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  Abstract 


Lung cancer is the most common malignant cancer worldwide. Combination therapies are urgently needed to increase patient survival. Calycosin is a phytoestrogen isoflavone that has been reported previously to inhibit tumor cell growth, although its effects on lung cancer remain unclear. The aim of this study was to investigate the effects of calycosin on cell proliferation and apoptosis of gemcitabine-resistant lung cancer cells. Using calycosin to treat human lung cancer cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0 GEMR) and examine the effects on the cells. Cultured human lung cancer cells (CL1-0) and gemcitabine-resistant lung cancer cells (CL1-0 GEMR) were treated with increasing concentrations of calycosin. Cell viability and apoptosis were studied by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide, flow cytometry, and TUNEL assays. Western blots were used to measure the expression levels of proliferation-related proteins and cancer stem cell proteins in CL1-0 GEMR cells. The results showed that calycosin treatment inhibited cell proliferation, decreased cell migration ability, and suppressed cancer stem cell properties in CL1-0 GEMR cells. Interestingly, in CL1-0 GEMR cells, calycosin treatment not only increased LDOC1 but also decreased GNL3L/NFκB protein levels and mRNA levels, in concentration-dependent manners. We speculate that calycosin inhibited cell proliferation of the gemcitabine-resistant cell line through regulating the LDOC1/GNL3L/NFκB pathway.

Keywords: Calycosin, gemcitabine resistant, lung cancer, NFκB signaling, proliferation


How to cite this article:
Li CC, Lu CY, Hsu CH, Hsieh DJ, Wang TF, Ho TJ, Kuo WW, Day CH, Liao SC, Chen MC, Huang CY. Calycosin inhibits gemcitabine-resistant lung cancer cells proliferation through modulation of the LDOC1/GNL3L/NFκB. Chin J Physiol 2023;66:189-99

How to cite this URL:
Li CC, Lu CY, Hsu CH, Hsieh DJ, Wang TF, Ho TJ, Kuo WW, Day CH, Liao SC, Chen MC, Huang CY. Calycosin inhibits gemcitabine-resistant lung cancer cells proliferation through modulation of the LDOC1/GNL3L/NFκB. Chin J Physiol [serial online] 2023 [cited 2023 Sep 26];66:189-99. Available from: https://www.cjphysiology.org/text.asp?2023/66/4/189/384126




  Introduction Top


Lung cancer is one of the most prevalent human cancers and also the leading cause of cancer death worldwide.[1] It can be classified into two categories based on the histological and clinical characteristics: small cell lung cancer and non-small cell lung cancer (NSCLC); the latter accounts for approximately 85% of lung cancers.[2] Although there are many advanced diagnostic and treatment modalities for lung cancer, the 5-year overall survival rate remains <15% when patients progress to the late stage.[3]

For lung cancer treatment options, including surgery, radiation therapy, chemotherapy, and targeted therapy. Therapeutic modalities recommendations depend on several factors, including the type and stage of cancer.[4],[5] If the patients are with epidermal growth factor receptor (EGFR) or anaplastic lymphoma kinase (ALK) mutation, they might be suitable for tyrosine kinase inhibitors (TKIs) treatment. More trials have suggested that for advanced NSCLC patients with EGFR or ALK mutant tumors, initial targeted therapy with a TKI instead of chemotherapy may be the best choice of treatment.[5],[6] In addition, chemotherapy is the most common and effective therapy for NSCLC patients who cannot undergo surgery and gemcitabine (2´deoxi-2´,2´-difluorocytidinemonohydrochloride [the beta isomere]) is probably the most valuable agent for treating early and advanced stages of NSCLC.[7] Gemcitabine is a novel deoxycytidine analog with structural similarities to cytosine arabinoside[7] and is used as a first-line chemotherapeutic agent for NSCLC,[8] breast cancer,[9] bladder cancer,[10] and ovarian cancer.[11] Patients usually have a good initial response to gemcitabine-based chemotherapy but develop resistance over time. Gemcitabine resistance can be either intrinsic or acquired and result from molecular and cellular changes including nucleotide metabolism enzymes, inactivation of apoptosis pathways, high expression of drug efflux pumps, and activation of cancer stem cells or the epithelial-to-mesenchymal transition pathway.[12] Currently, the exact molecular mechanism of resistance to gemcitabine is not fully understood. To improve the survival and the prognosis of cancer patients, a better understanding of the mechanism responsible for gemcitabine resistance, and the discovery of new therapeutic strategies, are needed.

Calycosin, a representative bioactive compound, is the major isoflavonoid in Huangqi Radix Astragali Mongolici (synonyms: Astragalus membranaceus (Fisch.) Bunge and Astragalus membranaceus (Fisch.) unge var. Mongholicus), a traditional Chinese herbal medicine.[13] It has been shown to possess therapeutic activities against breast cancer,[14] osteosarcoma[15] and lung cancer.[16],[17] Some studies indicated that calycosin might be a potent antitumor agent.[16],[18],[19] For example, previous studies have shown that calycosin can induce tumor cell apoptosis, and inhibit cell proliferation, invasion, and angiogenesis, by affecting the expression of estrogen receptor β and some tumor-related genes such as AKT, insulin-like growth factor 1 receptor, mitogen-activated protein kinase, Bcl-2, caspase-3, and RAS dexamethasone-induced 1.[14] However, the role of calycosin in lung cancer cells resistant to gemcitabine remains unclear. Thus, we conducted the current study to determine whether calycosin affected the growth and migration of human lung cancer gemcitabine-resistant cells. Specifically, we investigated the effects of calycosin on proliferation, apoptosis, and migration in parental and gemcitabine-resistant human lung cancer cells and explored the underlying mechanisms involved in the biological actions of calycosin.


  Materials and Methods Top


Cell culture

The CL1-0 cell line was a poorly differentiated lung adenocarcinoma. The gemcitabine-resistant cell line (CL1-0 GEMR) was generated by seeding CL1-0 cells in 10-cm plates, then treating them with 1 μM gemcitabine for 12 days, replacing the medium every 72 h with fresh medium containing the drug. Following the same procedure, the cells were challenged for 6 months with 1–10 μM gemcitabine to continue enhancing drug resistance. Human fetal lung fibroblast (HFL) 1 cells were purchased from the American Type Culture Collection (Rockville, MD, USA). HFL1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS); cells between the 16th and 20th passages were used for experiments. CL1-0 and CL1-0 GEMR cells were maintained in RPMI 1640 with 10% FBS plus antibiotics at 37°C in a humidified atmosphere of 95% air/5% CO2.

Colony formation assay

The colony formation assay was performed with CL1-0 and CL1-0 GEMR cells. Briefly, 0.25% trypsin was used to harvest cells. Thereafter, a single cell suspension was dispersed in 10% FBS. The cells were seeded into 6- or 24-well plates at a density of 500 cells/well and cultured in a 5% CO2 atmosphere at 37°C for 2–4 weeks. When cell clones appeared, they were fixed with 4% paraformaldehyde for 10–45 min, stained with Giemsa or 0.5% crystal violet for 10–40 min, and counted under a light microscope. Colonies number were counted using ImageJ software (version 1.47; National Institutes of Health, Bethesda, MD, USA). Triplicate wells were measured for each treatment group.[20]

Cell viability assay

Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, St. Louis, MO, USA). Cells were seeded in 24-well plates and treated with 0, 50, 100, 150, 200, or 250 μM calycosin. After 24 h, 0.5 mg/mL MTT solution were added to each well for 2 h. The purple MTT formazan product was dissolved in 300 μL of dimethyl sulfoxide, then transferred to a 96-well plate. Finally, the absorbance at 540 nm was detected using a microplate reader; cell viability is presented as a percentage of the control.[21],[22]

Annexin V apoptosis assay

For the flow cytometry Annexin V apoptosis assay, CL1-0 and CL1-0 GEMR cells were treated with 0, 50, 100, 150, 200 and 250 μM of calycosin for 24 h. The cells were collected and stained using an Annexin V-fluorescein isothiocyanate apoptosis detection kit (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol.[23] Flow cytometry was performed at the FACS Core Facility, China Medical University, Taiwan, on a FACS Canto™ system (BD Biosciences). Cells were gated to obtain single cells in each quadrant of the fluorescein isothiocyanate versus propidium iodide plot. For convenience, only 10,000 of the 50,000 cells visualized were counted.

In situ terminal deoxynucleotidyl transferase dUTP nick-end labeling assay

TUNEL staining for cells was based on fluorescein-dUTP labelling of DNA strand breaks. Cells were analyzed by fluorescence microscopy as described by the manufacturer (Roche Molecular Biochemical, Mannheim, Germany). TUNEL-positive cells were considered apoptotic.

Migration assay

For the cell migration assay, CL1-0 or CL1-0 GEMR cells (5 × 104) treated with 0, 50, 100, 150, or 200 μM calycosin were suspended in 200 μL of OPTI-MEM medium and seeded into the upper Transwell chamber (8.0-μm pore size filter; Corning, Canton, NY, USA). Six hundred microliters of OPTI-MEM containing 10 μL/mL fibronectin (Sigma-Aldrich, Shanghai, China), a chemoattractant, were then added to a 24-well plate. After incubating 6 h at 37°C, cells that migrated into the lower chamber membrane were fixed with methanol and subjected to Giemsa staining. Cells in the lower chamber membrane were counted microscopically (six fields per filter).[24]

Western blot analyses

Protein concentrations of the extracted cell lysates were determined and 40 μg of protein from each sample were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, then transferred to a polyvinylidene difluoride membrane (Merck Millipore, Darmstadt, Germany). The membrane was then blocked with 5% skim milk in Tris-buffered saline (TBS) and incubated overnight with the primary antibody in 5% milk in TBS containing 0.1% Tween-20 (TBST).[25],[26],[27],[28] The primary antibodies were: anti-c-myc (Santa Cruz Biotechnology, Santa Cruz, CA, USA; sc-40), anti-cyclin D1 (Santa Cruz Biotechnology; sc-246), anti-cyclooxygenase 2 (Cox 2) (Cell Signaling Technology, Danvers, MA, USA; #4842), anti-Oct4 (Cell Signaling Technology; #2750), anti-Nanog (Cell Signaling Technology; #4903), anti-Sox 2 (Cell Signaling Technology; #4900), anti-phospho (p) NFκB (Cell Signaling Technology; #3033L), anti-NFκB (Abcam, Cambridge, MA, USA; ab32536), anti-pIκBα (Cell Signaling Technology; #2859), anti-IκBα (Santa Cruz, USA; sc-1643), anti-GNL3L (Abcam; ab94862), anti-LDOC1 (Abcam; ab86126), and anti-β actin (Santa Cruz Biotechnoogy; sc-47778) at 4°C overnight. After washing with TBST, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody and developed using a chemiluminescent horseradish peroxidase substrate (Merck Millipore).

Reverse transcription and quantitative polymerase chain reaction

The total RNA was extracted using the GeneJET RNA Purification Kit (Thermo Scientific, Vilnius, Lithuania) followed the manufacturer's protocol. Quantitative reverse-transcription polymerse chain reaction employed the GScript First-Strand Synthesis Kit (GeneDirex, Cat. MB305-0050) and GNL3 L, LDOC1, NFκB specific primers, the analysis was performed on a Quantstudio 5 real-time polymerase chain reaction (PCR) System (Applied Biosystems, Foster, CA, USA). β-actin was used as an endogenous control, to determined the relative expression levles of target genes using the 2−ΔΔCt method. The primers for quantitative PCR analyses were shown in [Table 1].
Table 1: Primers for quantitative polymerase chain reaction analysis

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Statistical analysis

Statistical analysis of all results was performed using paired two-way analysis of variance with Kruskal–Wallis test. All results reflect the mean ± standard error of the mean of data obtained from at least three independent experiments. Statistical significance was defined as P < 0.05.


  Results Top


Calycosin inhibits proliferation of CL1-0 and CL1-0 GEMR cells

To evaluate the anti-proliferative effects of calycosin, HFL-1, CL1-0, and CL1-0 GEMR cells were treated with increasing concentrations of calycosin (0, 50, 100, 150, 200, and 250 μM) for 24 h and cell viability was determined by the MTT assay [Figure 1]. Compared with the negative control HFL1 cells that exhibited no toxicity, calycosin significantly inhibited the proliferation of CL1-0 and CL1-0 GEMR cells in a concentration-dependent manner. Notably, both 200 and 250 μM calycosin induced more death in CL1-0 GEMR cells than CL1-0 cells.
Figure 1: Effects of calycosin on the proliferation of HFL1, CL1-0, and CL1-0 GEMR cells. Cells were treated in triplicate with, or without, calycosin at the indicated concentrations for 24 h and the viability of cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide assays. Data are representative of three independent experiments. *P < 0.05 versus control (0 μM).

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Calycosin induces apoptosis in CL1-0 and CL1-0 GEMR cells

Flow cytometric analysis was performed to determine whether calycosin induced apoptosis in CL1-0 and CL1-0 GEMR cells. As shown in [Figure 2]a and [Figure 2]c, 8.9% of untreated CL1-0 cells underwent late apoptosis. However, after treatment with 50, 100, 150, 200, and 250 μM calycosin for 24 h, the apoptosis percentages were 8.7, 7.6, 10, 12.7, and 18.5%, respectively. Untreated CL1-0 GEMR cells showed a 3.8% late apoptosis rate. However, after treatment with 50, 100, 150, 200, and 250 μM calycosin for 24 h, the apoptosis percentages were 5.2, 4, 9.3, 34.6, and 54.8%, respectively [Figure 2]b and [Figure 2]c. We also determined the extent of early plus late apoptosis in CL1-0 and CL1-0 GEMR cells treated with calycosin [Figure 2]d.
Figure 2: Effects of calycosin on cell apoptosis of CL1-0 and CL1-0 GEMR cells. CL1-0 (a) and CL1-0 GEMR (b) cells were treated with calycosin (50, 100, 150, 200, and 250 μM) and incubated for 24 h. Apoptotic cells were detected using Annexin V and PI staining. Apoptotic rate of late stage (c) and early with late stage (d) obtained from three independent experiments and presented as the mean ± standard deviation. P < 0.05 versus control (0 μM).

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To confirm apoptotic cell death, we performed the TUNEL assay in CL1-0 and CL1-0 GEMR cells treated with calycosin. The death [Figure 3]a and [Figure 3]b, green fluorescence] of both CL1-0 and CL1-0 GEMR cells was increased following treatment with increasing concentrations of calycosin for 24 h [Figure 3]c. We also found that calycosin induced more cell death in CL1-0 GEMR cells than CL1-0 cells at the same calycosin concentrations.
Figure 3: Calycosin treatment reduced CL1-0 and CL1-0 GEMR cells apoptosis. Representative images of TUNEL staining of CL1-0 (a) and CL1-0 GEMR (b) cells treated with indicated concentrations of calycosin. DAPI stain was used to label the nucleus. (c) Quantitative analysis of the number of TUNEL-positive cells in each dosage of calycosin treatment. P < 0.05 versus control (0 μM).

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Calycosin inhibits colony formation of CL1-0 GEMR cells

The colonyformation assay was used to examine whether calycosin affected cancer stem cell properties of CL1-0 GEMR cells. The results [Figure 4]a showed a concentration-dependent decrease in the number of colonies and a smaller colony size in CL1-0 GEMR cells treated with calycosin compared to the untreated control cells. Quantitative results from this assay [Figure 4]b demonstrated that calycosin significantly and concentration-dependently inhibited the CL1-0 GEMR reproductive potential compared with the control group, consistent with the results from the MTT assay [Figure 1].
Figure 4: Calycosin inhibited colony formation in CL1-0 GEMR cells. (a) CL1-0 and CL1-0 GEMR cells were incubated with different concentration of calycosin for 24 h and assayed for colony formation. (b) Quantitative analysis of the colony formation number of cells in each concentration of calycosin treatment. P < 0.05 versus CL1-0 GEMR control (0 μM).

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Calycosin reduces the migration abilities of CL1-0 and CL1-0 GEMR cells

Calycosin significantly inhibited the migration abilities of CL1-0 and CL1-0 GEMR cells in a concentration-dependent manner [Figure 5]. Consistent with our other results, calycosin had a stronger effect on the migration ability of CL1-0 GEMR compared to CL1-0 cells.
Figure 5: Dose-dependent inhibitory effects of calycosin on the migration of CL1-0 and CL1-0 GEMR cells detected by transwell assay. (a) Cells were cultured in triplicate with the indicated concentration of calycosin for 24 h. The migrated cells were stained with Giemsa staining and counted under a light microscope. Quantitative analysis of migrated cells of CL1-0 (b) and CL1-0 GEMR (c) cells were expressed as the mean ± standard deviation. P < 0.05 versus control (0 μM).

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Calycosin treatment alters cell proliferation and cancer stem cell property proteins in CL1-0 GEMR cells

Expression of the cell proliferation markers, c-myc, cyclin D1, and Cox2, was decreased in a concentration-dependent manner [Figure 6]a, [Figure 6]b, [Figure 6]c, [Figure 6]d; this was consistent with the MTT results [Figure 1]. Because the quantitative results from the clonogenic assay [Figure 4]b demonstrated that calycosin significantly inhibited CL1-0 GEMR cancer stem cell properties, we performed western blots to detect the protein levels of several cancer stem cell markers. We found that Oct4, Nanog, and Sox2 were all decreased as the concentration of calycosin increased from 50 to 200 μM [Figure 6]a and [Figure 6]e, [Figure 6]f, [Figure 6]g.
Figure 6: An analysis of c-myc, cyclin D1, Cox2, Oct4, Nanog and Sox2 of calycosin-treated CL1-0 GEMR cells. (a) Western blotting of c-myc, cyclin D1, Cox2, Oct4, Nanog and Sox2, extracted from calycosin-treated CL1-0 GEMR cells; β-actin: internal control. (b-g) Statistical significance was defined as *P < 0.05 versus control (0 μM).

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Calycosin inhibits the proliferation of CL1-0 GEMR cells via the LDOC1/GNL3L/NFκB pathway

Western blot assays were conducted to examine the molecular mechanism by which calycosin inhibited the proliferation of CL1-0 GEMR cells. After a 24 h and exposure to increasing concentrations of calycosin (0, 50, 100, 150, and 200 μM), the expression levels of pNFκB in CL1-0 GEMR cells were significantly decreased in a concentration-dependent manner [Figure 7]a and [Figure 7]b. The pIκBα expression level was increased when calycosin treatment reached 100 μM. This was consistent with the trend of pNFκB, but it decreased at higher calycocin concentrations (150 and 200 μM), which might be the result of cell death. Interestingly, GNL3L, a protein upstream of NFκB, was also decreased as the concentration of calycosin increased [Figure 7]a and [Figure 7]d. This supports the potential mechanism of calycosin-induced apoptosis. The tumor suppressor protein, LDOC1, which is up-stream of GNL3L, was increased as the concentration of calycosin increased [Figure 7]a and [Figure 7]e. Finally, we examined the mRNA levels of GNL3L, LDOC1, and NFκB in CL1-0 GEMR cells treated with increasing concentrations of calycosin. We found that the mRNA level of GNL3L was decreased along with an increased mRNA level of LDOC1 [Figure 8]. We speculate that the inhibition of cell proliferation by calycosin might be through regulating the LDOC1/GNL3L/NFκB axis.
Figure 7: An analysis of pNF-κB, pIκBα, GNL3L, and LDOC1 of calycosin-treated CL1-0 GEMR cells. (a) Western blotting of pNF-κB, pIκBα, GNL3L and LDOC1, extracted from calycosin-treated CL1-0 GEMR cells. β-actin: Internal control. (b-e) Statistical significance was defined as *P < 0.05 versus control (0 μM).

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Figure 8: The mRNA levels of GNL3L, LDOC1, and NFκB in CL1-0 GEMR cells treated with calycosin. Cells were treated for 24 h with the indicated concentrations of calycosin. Levels of GNL3L, LDOC1, and NFκB were determined using the quantitative reverse transcription-polymerase chain reaction. β-actin: Internal control. Data are expressed as the mean ± standard deviation. *P < 0.05 versus control (0 μM); #P < 0.05 versus control (0 μM).

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  Discussion Top


In the present study, we detected the in vitro biological effects of calycosin on human CL1-0 and CL1-0 GEMR cells for the first time. The results showed that calycosin markedly inhibited cell proliferation, induced apoptosis, reduced colony formation, and suppressed cell migration in both cell lines in concentration-dependent manners.

The incidence and mortality rates of lung cancer are ranked first among all cancer types. Current treatments for lung cancer do not necessarily improve patient symptoms, quality of life, or survival. Furthermore, drug resistance usually develops during the course of chemotherapy. Thus, there remains a need for more effective and less toxic therapies that induce apoptosis in tumor cells.

Gemcitabine resistance can be either intrinsic or acquired. Gemcitabine is transported into cells by sodium-dependent concentrative nucleoside transporter and sodium-independent equilibrative nucleotide transporter (hENT) mechanisms.[29],[30] Many mechanisms of gemcitabine resistance have been identified. One mechanism responsible for gemcitabine resistance is the dysregulation of proteins participating in gemcitabine metabolism pathways, including a deficiency of hENT1, down-regulation of deoxycytidine kinase, and up-regulation of ribonucleoside-diphosphate reductase large subunit 1/2.[31],[32] Another gemcitabine resistance mechanism is high expression of drug efflux pumps, such as ABC transporter family proteins.[33] Gemcitabine resistance is also associated with multiple genetic and epigenetic abnormalities. Changes in one or a few genes remain crucial for maintaining drug resistance, cell survival, and a malignant phenotype. There is evidence from in vitro and in vivo models to indicate that NFκB,[34] AKT,[33],[35] MAPK,[36] and hypoxia inducible factor 1α[37] pathways are involved in gemcitabine resistance. Nevertheless, gemcitabine has clinical potential to treat a broad spectrum of human cancers, though resistance is the major impediment to achieving satisfactory clinical outcomes.

There are many evidences suggested that cancer stem cells play a critical role in tumor progression and drug resistance cancer cells,[11],[12],[38] and the previous studies have demonstrated that gemcitabine resistance cancer cells have more potent in forming colonies and higher percentage of cancer stem cell marker.[11],[12],[39] Furthermore, the previous study reported that the activation of epithelial-to-mesenchymal transition pathway might contribute to gemcitabine resistance.[12] However, our results displayed that CL1-0 cells showed higher (Oct4) or similar (Nanog, and Sox2) stem cell markers expression compared with the CL1-0 GEMR cells, and there are more migrating CL1-0 cells than migrating CL1-0 GEMR cells. Thus, we suggested the stem cell markers and migration in our gemcitabine-resistant lung cancer cells have not significantly change compared with the CL1-0 GEMR cells, but treatment with calycosin could decrease the expression of stem cell markers, colony numbers and migration in gemcitabine-resistant lung cancer cells, suggesting calycosin has the potential effect of decreasing stem cell markers, colony numbers and migration in CL1-0 GEMR cells. Our future work need to evaluate why stem cell markers and migration in our gemcitabine-resistant lung cancer cells have not significantly change compared with the CL1-0 GEMR cells.

The PI3K/AKT/NFκB pathway is an important survival pathway that is frequently altered in cancer. Activation of this pathway contributes to initiation and maintenance of tumors, and to resistance to many cancer treatments. Previous studies indicated that the PI3K/AKT signaling pathway has a role in the functional mechanism of the effects of calycosin.[11],[27],[33],[34] Chen et al. reported that calycosin enhanced apoptosis in ER-positive breast cancer cells via ERß-induced inhibition of the insulin-like growth factor 1 receptor, as well as regulation of the PI3K/AKT and MAPK pathways.[14] Zhao et al. published similar findings on the mechanism of the antitumor role of calycosin on colorectal cancer cells.[40] Moreover, Zhu et al. indicated that calycosin suppresses colorectal cancer progression by targeting ERβ, upregulating PTEN, and inhibiting PI3K/Akt signal pathway.[33]

Here, we found that the upstream of NFκB signaling genes, GNL3L and LDOC1, were altered with increasing concentrations of calycosin treatment, which is consistent with a previous study.[41] GNL3L is a nucleolar GTP-binding protein that promotes mammalian cell proliferation and is overexpressed in many tumor types.[42],[43] The observed downregulation of GNL3L by LDOC1 may significantly influence cell proliferation and apoptosis in mammalian cells. Recent reports suggest that LDOC1 is a novel regulator of NFκB activity,[44] and GNL3L acts as a mediator of the NFκB pathway,[45] although the mechanisms remain unknown. The present study provides evidence that GNL3L promotes NFκB dependent transcriptional activity and is suppressed by upregulation of LDOC1 following calycosin treatment.

Our results show that calycosin inhibits the proliferation and migration abilities of CL1-0 GEMR cells, and that these effects might be through the LDOC1/GNL3L/NFκB pathway. These findings improve our understanding of the anti-tumor activity and potential mechanisms of calycosin in gemcitabine-resistant lung cancer cells, and offer valuable support for the clinical application of calycosin in treating gemcitabine-resistant lung cancer.

The present study has some limitations. First, the current study was examined in vitro systems, future work is required to evaluate the in vivo effects. Second, since the present study was designed based on clinical scenario, we examined the calycosin inhibits proliferation, induces apoptosis, inhibits colony formation, reduces the migration abilities, and alters cell proliferation and cancer stem cell property proteins of CL1-0 GEMR death. Therefore, we need to examine the detailed mechanism as to how calycosin can improve the effect of gemcitabine-resistant lung cancer treatment. Third, there is a need to determine whether calycosin may be a novel therapeutic agent that can be used individually, or as part of combined therapy, in the treatment of gemcitabine-resistant lung cancer. Fourth, the current study was only examined on gemcitabine-resistant lung cancer CL1-0 cells in vitro systems, future work is required to evaluate the effects of other types of lung cancer.


  Conclusion Top


In conclusion, we found calycosin inhibits the proliferation and migration abilities of CL1-0 GEMR cells, and that these effects might be through the LDOC1/GNL3L/NFκB pathway. Notably, calycosin had a little toxic effect on nontransformed cells, which indicated that calycosin can selectively kill malignant cells but not normal cells. Thus, our data suggest that calycosin-regulated LDOC1/GNL3L/NFκB pathway could be a novel therapeutic approach for gemcitabine-resistant lung cancer patients.

Acknowledgments

Experiments and data analysis were performed, in part, through the use of the Cardiovascular and Mitochondrial Related Disease Research Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien, Taiwan.

Financial support and sponsorship

This study was supported by Hualien Tzu Chi Hospital (Buddhist Tzu Chi Medical Foundation) (IMAR-109-01-04-06; IMAR-111-01-09), and China Medical University and Asia University (CMU105-ASIA-01).

Conflicts of interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.



 
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