Bexarotene inhibits the viability of non-small cell lung cancer cells via slc10a2/PPARγ/PTEN/mTOR signaling pathway
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Thirty to 40 % of non-small cell lung cancer (NSCLC) patients developed higher hypertriglyceridemia in the process of treatment with bexarotene. And bioinformatics studies discovered that the expression of slc10a2 was increased in high-grade hypertriglyceridemia patients. So, we will explore the mechanism which may involve in this process.
We constructed slc10a2 overexpressed A549 cells and H1299 cells as cell models, normal A549 cells and H1299 cells as control. Then we explored the effects of slc10a2 on A549 cells and H1299 cells behaviors, including proliferation, invasion and apoptosis. The expression of apoptotic related genes and anti-cancer genes also been detected.
We found that the proliferation and migration were inhibited and the apoptosis of NSCLC cells was accelerated by bexarotene. In addition, overexpressed slc10a2 in NSCLC cells can further suppress the proliferation and migration, and promote apoptosis under the treatment of bexarotene. On the contrary, the opposite results were obtained after slc10a2 gene was silenced in NSCLC cells treated with bexarotene. Moreover, the expression of caspase 3, caspase 7, PTEN, P21, P53, LKB1, TSC2 were increased and the expression of Bcl-2, cyclin D1, c-FLIP were declined in NSCLC cells and slc10a2 overexpressed NSCLC cells with the treatment of bexarotene, and the opposite situations were seen after slc10a2 gene was silenced in NSCLC cells. The further studies revealed the increased expression of slc10a2 activated the expression of peroxisome proliferator-activated receptor γ (PPARγ), then up-regulated PTEN expression and down-regulated mTOR expression.
These results suggest that bexarotene inhibits the viability of lung cancer cells via slc10a2/PPARγ/PTEN/mTOR signaling pathway.
KeywordsNon-small cell lung cancer A549 cells H1299 cells Bexarotene slc10a2 PPARγ
One-way analysis of variance
Cell Counting Kit-8
Non-small cell lung cancer
Peroxisome proliferator-activated receptor
Peroxisome proliferator responsive element
Retinoid X receptors
The incidence of lung cancer is rapidly increasing in the world, and it has become the first leading cause of cancer death, especially in China . Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, accounting for almost 80% . In clinic trials, bexarotene showed both satisfactory safety and promising efficacy for the treatment of advanced NSCLC patients [3, 4]. However 30–40% of the patients appeared to be more sensitive to bexarotene treatment and developed higher hypertriglyceridemia. Interestingly, survival analysis in high-grade hypertriglyceridemia patients revealed significantly longer survival compared to the patients in the control, low-grade hypertriglyceridemia and middle-grade hypertriglyceridemia groups [5, 6].
Slc10a2 is a member of solute carrier family 10 of the sodium/bile acid co-transporter apical sodium-dependent bile acid transporter (ABST) , which plays a key role in the enterohepatic circulation through its reabsorption of bile acids from the ileum and indirectly conduces to cholesterol homoeostasis [12, 13]. ASBT is able to inhibit the concentration of plasma triglyceride and increase the concentration of HDL (high-density lipoprotein) cholesterol , and now it has aroused much concern as a drug target for the pharmacological treatment of hypercholesterolaemia [15, 16].
The goal of this study is to explore the role of slc10a2 in the treatment of NSCLC with bexarotene. We hypothesis that bexarotene inhibits the viability of NSCLC cells (e.g. A549 cells and H1299 cells) via increasing the expression of slc10a2. In this study, we have successfully constructed slc10a2 overexpressed A549 cells and H1299 cells, and the proliferation, apoptosis, migration behaviors were detected in slc10a2 overexpressed A549 cells and H1299 cells respectively. Moreover, we also explored the expression of apoptosis genes, anti-apoptosis genes, tumor suppressor genes in slc10a2 overexpressed A549 cells and H1299 cells. Furthermore, the possible mechanism which involved in this process was discovered.
Bexarotene was obtained from Aladdin (Shanghai, China). Cell Counting Kit-8 was ordered from Dojindo (Japan). Cell culture plates and Transwell plates were ordered from Corning (NY, USA). Crystal violet was obtained from Beyotime (Haimen, China). Annexin V/fluorescein isothiocyanate (FITC) apoptosis detection kit was obtained from Beyotime biotech company (China). Gentamicin, Fetal bovine serum, glutamine, and RPMI 1640 medium were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The primary antibodies including slc10a2, PPARγ, mTOR and PTEN (Abcam, Cambridge, UK), GAPDH (Thermo, Walteham, Washington, USA). The pcDNA3 and pcDNA3-slc10a2 plasmid, slc10a2-shRNA, GW9662 were obtained from Shanghai Funeng Biological Technology, Co., LTD.
The human NSCLC cell lines A549 cells (CRM-CCL-185™) and H1299 cells (CRL-5803™) was obtained from American Type Culture Collection (Rockville, MD). Cells were maintained in RPMI 1640 media plus 10% fetal bovine serum, 1% glutamine and 0.05 mg/ml gentamycin sulfate at 37 °C and 5% CO2.
The construction of slc10a2 overexpressed A549 cells and H1299 cells
Before transfection, A549 cells and H1299 cells were seeded into 6-well plate at a density of 2 × 106 cells/well, after cells grow above 80% areas, A549 cells were transfected with 8 mg pcDNA3-slc10a2 plasmid using Lipofect2000 transfection Reagent according to manufacturer’s instructions, pcDNA3 treatment as control. The transfection medium was replaced by regular growth medium after 5 h transfection, and at each time point, the cells were used to observation using inverted fluorescence microscope.
A549 cells, H1299 cells and slc10a2 overexpressed A549 cells, H1299 cells were seeded in a 96-well plate, 5 × 103 cells per well. After 12 h culture, the cultured medium was replaced by conditional medium, which was added with bexarotene, bexarotene in combination with slc10a2-shRNA, bexarotene in combination with GW9662 respectively. At indicated time point, ten microliter CCK-8 was added each well and continually incubated for 4 h. Then the optical density (OD) value (450 nm) was determined by an enzyme-linked immunosorbent assay plate reader (Bioreader).
Transwell migration assay
A549 cells and slc10a2 overexpressed A549 cells were starved for 12 h, then resuspended in serum-free medium, and adjusted to 1 × 106 cells/ml. One hundred microliters of A549cells or slc10a2 overexpressed A549 cells were placed in the upper chamber of Transwell plates. Serum-free RPMI 1640 medium with bexarotene, bexarotene in combination with slc10a2-shRNA, was added to the lower chamber respectively, RPMI 1640 medium as control. Prior to the addition of cells suspension, preheated serum free RPMI 1640 medium (300 μl) was added to the upper chamber. After 24 h incubation, the invaded cells were collected from lower chambers, then stained with crystal violet.
The apoptosis of A549 cells, H1299 cells and slc10a2 overexpressed A549 cells, H1299 cells was analyzed using the Apoptosis Detection Kit according to the manufacturer’s instructions. Cells were seeded in 6-well plate (1 × 105 cells/well) with different medium, including RPMI 1640 medium plus bexarotene, RPMI 1640 medium plus bexarotene in combination with slc10a2-shRNA for 2 days. At indicated times, cells were digested then resuspended in 300 μL binding buffer solution which containing 5 ul Annexin V-FITC and 5 ul PI solution, then incubated in the dark for 20 min at room temperature. Finally flow cytometry (FACScan; BD Biosciences) was used to analyzed the apoptotic rate of each kind of cells.
RT quantitative-PCR analysis
The total RNA of A549 cells and H1299 cells were isolated by using RNeasy kit according to the manufacturer’s protocol (Qiagen, Valencia, CA). Briefly, total RNA (1 μg) was used as a template to prepare cDNA (Invitrogen), and was amplified by Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). A master mix was prepared for each PCR reaction, which included Platinum SYBR Green qPCR SuperMix-UDG, forward primer, reverse primer, and 10 ng of template cDNA. PCR was performed with the following thermocycling conditions: An initial 5 min at 95 °C, followed by 40 cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s.  The forward and backward primer sequences for Bcl-2 was 5’-CCGATCAGTGGAGCTGAAGAA-3′(sense) and 5’-GCCACAGGATGTTCTCGTCA-3′(antisense), cyclinD1:5’-CAAGGCCTGAACCTGAGGAG-3′(sense) and 5’-CTTGGGGTCCATGTTCTGCT-3′(antisense), c-FLIP:5’-GAGTGCCGGCTATTGGACTT-3′(sense) and 5’-GCGCTTCTCTCCTACACCTC-3′(antisense), Caspase-3:5’-GCGGTTGTAGAAGTTAATAAAGGT-3′(sense) and 5’-TACCAGACCGAGATGTCATTCC-3′(antisense), Caspase-7:5’-CGTGGGAACGGCAGGAAGT-3′(sense) and 5’-CGGGTGGTCTTGATGGATCG-3′(antisense), PTEN:5’-CAGGATACGCGCTCGGC-3′(sense) and 5’-TCAGGAGAAGCCGAGGAAGA-3′(antisense), P21:5’-AGTCAGTTCCTTGTGGAGCC-3′(sense) and 5’-CATTAGCGCATCACAGTCGC-3′(antisense), P53:5’-GTGCTCAAGACTGGCGCTAAA-3′(sense) and 5’-CAGTCTGGCCAATCCAGGGAAG-3′(antisense), LKB1:5’-GACCTGCTGAAAGGGATGCT-3′(sense) and 5’-GACCTGCTGAAAGGGATGCT-3′(antisense), TSC2:5’-TCTGAACATGTGGTCCGCAG-3′(sense) and 5’-TCTGAACATGTGGTCCGCAG-3′(antisense).
A549 cells and H1299 cells were treated with 1 mM, 5 mM, 10 mM bexarotene for 24 h, then the cells were harvested, then total protein from tissue or cell was extracted using radioimmunoprecipitation lysis buffer containing 1 mM phenylmethanesulfonylfluoride and the protein concentration was determined using the Bradford method (Beyotime Institute of Biotechnology, Nantong, China) according to the manufacturer’s instructions. Proteins (20 μg) were separated by 10% SDS-PAGE and transferred onto a nitrocellulose membrane. After blocking with 5% non-fat milk for 1 h at 4 °C, then membrane was incubated with the primary antibody at 4 °C overnight. Membranes were washed 3 times with 0.25% PBST and then incubated with the peroxidase-conjugated secondary antibody for 2 h at room temperature. After washed 3 times, the specific protein bands were detected using the enhanced chemiluminescence reagents .
All data were expressed as mean ± S.D. Differences between the groups were analyzed using one-way analysis of variance (ANOVA) using SPSS 13.0 (SPSS Inc, Chicago, IL, USA). p-values less than 0.05 were considered statistically significant.
The construction of slc10a2 overexpressed NSCLC cells
Slc10a2 plays an important role in the proliferation of NSCLC cells with the treatment of bexarotene
Slc10a2 plays an important role in the invasion of NSCLC cells with the treatment of bexarotene
Slc10a2 plays an important role in the apoptosis of NSCLC cells with the treatment of bexarotene
Slc10a2 plays an important role in tumor suppressor with the treatment of bexarotene
Slc10a2 via PPARγ plays an important role in the proliferation of NSCLC cells with the treatment of bexarotene
Slc10a2 via PPARγ plays an important role in tumor suppressor with the treatment of bexaroten
Bexarotene inhibits the viability of NSCLC cells via slc10a2/PPARγ/PTEN/mTOR signaling pathway
Three types of PPARs have been identified: alpha, gamma, and delta, they are a group of nuclear receptor proteins which function as transcription factors regulating the expression of genes . Among the three phenotypes, PPARγ has been attracting tremendous attention, the previous studies revealed that PPARγ plays essential roles in the regulation of cellular differentiation, development, and metabolism (carbohydrate, lipid, protein), and tumorigenesis [20, 21].
A mass of studies have demonstrated that PPARγ agonists via inhibiting the expression of cyclinD1, cyclinB, cyclinE, CDK4 and CDK2 and increasing the expression of CDKN1A to prevent cell cycle from G1 to S phase to prohibit tumor cells proliferation [22, 23, 24, 25, 26]. In this study we found that bexarotene worked as a PPARγ agonists, which was capable of enhancing the expression of PPARγ, then the expression of cyclinD1 was suppressed and the proliferation of A549 cells was prohibited, these results were consistent with the previous studies.
The activation of PPARγ can induce tumor cell apoptosis via several different pathways. PPARγ agonists was able to up-regulate pro-apoptotic protein BAX and BAD expression, and then induced glioma cells apoptosis through releasing cytochrome C and activating the activation of caspase . Li et al. reported the activation of PPARγ was associated with a decrease of the expression of Bcl-2 and increase of the expression of P53 in human melanoma cell line A375 cells . Similarly, we found that the expression of anti-apoptotic proteins Bcl-2, cyclin D1 and c-FLIP was reduced whereas the expression of apoptotic proteins caspase-3, caspase-7 and tumor suppressor gene PTEN, P21, P53, LKB1, TSC2 were accelerated in A549 cells with the treatment of bexarotene, which was associated with the activation of PPARγ through enhancing the expression of slc10a2, resulting in promoting the apoptosis of A549 cells.
Moreover, the activation of PPARγ can reduce the invasion ability of tumor cells. Thiazolidinedione (TZD) is a synthetic agonist of PPARγ, which contains troglitazone, pioglitazone and rosiglitazone . Willson et al. discovered that after adrenocortical cancer cell lines H295R cells co-cultured with pioglitazone and rosiglitazone, the expression of MMP-2 which play an important role in cell migration was reduced, and the migration of H295R cells was significantly declined . Also Galli et al. found that TZD can effectively inhibit tumor cell invasion, after pancreatic cancer cell treated with TZD for 24 h, the activity and transcriptional level of MMP-2 were declined . And in this study, the migration ability of A549 cells was significantly shortened after treated with bexarotene, whereas the migration of A549 cells was distinctly accelerated after with the treatment of bexarotene in combination with GW9662.
PI3K/Akt/mTOR signaling pathway exists in almost all mammals, it regulates cell growth mainly through controlling the protein synthesis. PTEN as a negative regulator of this pathway via suppressing the expression of PI3K and Akt. Patel et al. revealed that PPARγ can combined with peroxisome proliferator responsive element 1 (PPRE1) and PPRE2, the upstream gene of PTEN, and increased the expression of PTEN then induced phosphorylation of Akt decreased, cells differentiation and apoptosis . And it has been preclinically showed that deficiency of TSC2 or PTEN expression induces impaired PI3K/Akt/mTOR activation, suggesting that mTOR overexpression with the loss of PTEN plays a key role in the development and progression of pancreatic neuroendocrine tumors . In this study, we discovered that bexarotene accelerated the expression of PPARγ through enhancing the expression of slc10a2, also the expression of PPARγ was promoted, while the expression of mTOR was declined, thus the viability of A549 cells was suppressed. Finally this study has some limitations, for example, PPAR agonist bexarotene induces PPARγ and slc10a2 in a dose dependent manner, but the effects of bexarotene on PPARα, PPARβ/δ expression we don’t explore. We have clarified limitations in the discussion section, and we will explore the effects of bexarotene on PPARα, PPARβ/δ expression in our future study.
In this study we found that bexarotene can suppress the proliferation, migration, and promote the apoptosis of NSCLC cells. Moreover, we demonstrated that this effects owned to the increased expression of PPARγ via enhancing the expression of slc10a2, then up-regulated the expression of PTEN and down-regulated the expression of mTOR, thus increased the expression of apoptotic genes and anti-cancer genes, and reduced the expression of anti-apoptotic genes to suppress the proliferation of NSCLC cells and promote the apoptosis of NSCLC cells .
This work was supported by Major Key Project of Shanghai Chest Hospital Science & Technology foundation (2014YZDC10600). The funding source has no role in the study design, analysis, or interpretation of the data, writing of the manuscript, or the decision to submit the manuscript for publication.
Availability of data and materials
The data and materials used in this current study are available from the corresponding author on reasonable request.
Prof. SL designed this study; Prof. YST gave final approval of the version to be published; XHA wrote and revised this paper; FM made substantial contributions to acquisition of data, analysis and interpretation of data, SPS and JJW performed experiments and collected data. All authors have read and approved the manuscript.
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