Sulforaphane Inhibits Cancer Stem Cells In Lung Cancer Through Suppressing β-catenin Signaling And Upregulating MiR-214Expression

BackgroundSulforaphane (SF) is a natural compound converted from glucoraphanin, a major glucosinolated in broccoli/broccoli sprouts. It is traditionally applied as a drug that can prevent cancer because of its function of inhibiting phase1metabolism enzymes that convert procarcinogens to carcinogens and inducing phase2metabolism enzymes that promote excretion of carcinogens. Subsequent studies revealed that SF could modulate diverse cellular activities, such as inhibiting the growth of cancer cells, inducing apoptosis and cell cycle arrest and suppressing angiogenesis and metastasis by different signaling pathway.Hypothesis of cancer stem cells holds that tumors contain a small number of tumor-forming and self-renewing cancer stem cells (CSCs) within a population of nontumor-forming cancer cells. CSCs were identified in many human cancers including lung cancer. Cancer stem cells hypothesis also suggests that CSCs are resistant to chemotherapy and may contribute to tumor metastasis and tumor recurrence after treatment. Therefore, removal of CSCs is very crucial for effective cancer therapy. Several pathways including Hedgehog, Notch and, of particular interest, Wnt/β-catenin, have been identified to be critical to the self-renewal behavior of both normal stem cells and CSCs. It has been reported that Wnt/p-catenin signaling promotes self-renewal of many kinds of CSCs through regulating expression of the pluripotency gene c-Myc and Nonog.MicroRNAs (miRNA),21-23nucleotide non-protein coding RNAs, act as powerful regulators of gene expression at the post-transcriptional level by targeting specific mRNA degradation or by suppression or activation of translation. Accumulating evidences have demonstrated that miRNAs influence a wide range of biological processes including development, lifespan, metabolism, and tumorigenesis. Transcription of miRNA gene is regulated at multiple levels in response to differentiation and mitogenic signaling. Some miRNAs are characteristic of specific differentiated cell types, whereas others are specifically expressed in stem and progenitor cells during early development. Recent evidence has shown that miRNAs can function as tumor suppressors or oncogenes and were involved in the differentiation and quiescence of CSCs in various types of tumors.PurposeThe aim of this study was to evaluate efficacy of sulforaphane to inhibit lung CSCs and further explore the underlying mechanisms.Method1. MTT cell proliferation assayCells were cultured and measured for cell growth by using MTT assay. Cells were cultured for3days in medium with different concentrations of sulforaphane. Then MTT was added in this medium for2hours before treatment was finished. The plates was added with DMSO and swinged for10minites in37℃.At last the plate was measured in490nm wavelength. 2. Apoptosis assaySulforaphane induced apoptosis in H460cells, measured by FACS and Annexin V-FITC/PI analyses. Annexin, a kind of calcium dependent phospholipid binding protein, is widely locates in eukaryotic cell cytoplasm and involves in intracellular signal transduction. But only Annexin V is reported to modulate activity of PKC. Annexin V can selectively bind to phosphatidyl serine. Phosphatidyl serine is mainly distributed in the inside of the cell membrane, and cytoplasm adjacent side. In cells with early apoptosis, phosphatidyl serine in different cell types is valgus to the cell surface, which is the lateral cell membrane. Exposure of phosphatidyl serine to the cell surface can lead to coagulation and inflammatory reaction. Annexin V binding to phosphatidyl serine in cell surface can blocked activity of phosphatidyl serine in procoagulation and inflammatory reaction. With the green the fluorescent probe FITC labeled Annexin V, Annexin V-FITC can be used by flow cytometry and fluorescence microscope simply and directly for detecting valgus of phosphatidyl serine,the important characteristics of apoptosis. Propidium iodide can be stained necrotic cells or late apoptotic cells which loss cell membrane integrity, shows red fluorescence. Since cell membrane integrity has been lost in the necrotic cells,,Annexin V-FITC can enter into the cytoplasm, and located on the inside of the cell membrane of the phosphatidyl serine.So the necrotic cells showed green fluorescence. H460cells were treated with different concentrations of sulforaphane (0,5,10μmol/L) for72hours, and cells were digested into single cells.Then Annexin V-FITC Kit was used according to the manufacture procedure.3. Tumorsphere cultureStem/progenitor cells are enriched in tumorsphere of many cancers,based on the unique ability of stem/progenitor cells to grow and form spheres in serum-free medium.H460cells were cultured in tumorsphere forming medium with different concentration of SF (1.0,2.0and5.0μmol/L).Cells were cultured in a density of1000-1500cells/ml.7days after treatment, the number of tumor sphere was counted and diameter of tumor sphere was measured in microscope. In the second passage, cells from primary tumorsphere were digested and cultured in a density of800-1000cells/ml without sulforaphane.4. Side population assaySide population(SP) cells are some special cells which are observed during separating haemopoiesis stem cell/progenitor cell from blood with Hoechst dying and flow cytometry. SP cells exist in many of adult tissues, embryo and many tumor cell lines, and have the abilities of self-renewal and multiple differentiating, which are similar to stem cell. Many researches have indicated that SP cells represented a new type of stem cells. In this study,lung cancer cells were detached from the dishes with Trypsin-EDTA(invitrogen) and suspended at1×x103cels/mL in Hank’s balanced salt solution(HBSS) supplemented with2%fetal calf serum and10mmol/L Hepes. These cells were then incubated at37℃for90minutes with201～g/mL Hoechst33342(Sigma),either alone or in the presence of50pmol/L verapamil(Sigma), which is an inhibitor of verapamil-sensitive ABC transporter. After90minutes incubation, the cells were centrifuged immediately for5minutes at300g,4℃and resuspended in ice-cold HBSS. The cells were kept on the ice to inhibit eflux of Hoechst dye. Then we added1pg/ml Propidium iodide (BD Pharmingen)to discriminate dead cells. Cell dual-wavelength analysis and purification were performed on a dual-laser FACS Vantage Ⅱ (BD).The Hoechst33342was excited at355nm UV light and collect blue fluorescence with a450,20band-pass (BP) filter and red fluorescence with a675nm edge filter long-pass (EFLP). A610nm dichroic mirror short-pass (DMSP) was used to separate the emission wavelengths. PI positive dead cells were excluded from the analysis. 5. Animal experiment:Luciferase reporter plasmid with Nanog promoter was Constructed. H460cells were transfected with Nanog promoter-luciferase reporter plasmid and purified by G418. Animal studies were conducted in strict accordance with the principles and procedures approved by the Committee on the Ethics of Animal Experiments of Southern Medical University.5-week-old female Nude mice (BALB/C nu/nu) were fed autoclaved water and laboratory rodent chow. A volume of100μ1of PBS mixed with Matrigel (BD Biosciences) containing2×106H460cells was transplanted into the flanks of mice by subcutaneous injection. The second day after the cell injection, activity of luciferase in these transplanted tumor was measured and the negative animals were excluded. Positive animals were randomly separated into two groups:one group was i.p. injected with control (0.9%NaCl solution) and the other group was injected with50mg/kg SF (dissolved in0.9%NaCl solution) daily for2weeks. Activity of luciferase was detected by living animal Imaging equipment every3days using luciferin (Sigma), according to the manufacturer’s protocol.6. Western blotting method was used to measure the change of β-catenin after SF treated. H460cells with stable over-expression of activated β-catenin protein were constructed by transfection of S33Y plasmid in H460cells. The change of the pluripotency maintaining factors (Nanog, Oct4, Sox2and c-Myc) in H460cells treated with SF was also detected by immunofluorescence.7. MiRNA level was measured by QPCR and the effect of SF on miRNAs and targeted genes by western blotting; RNA22and gene promoter sequence software were used to analyze the relationship of relative miRNAs and genes.8. Dual Luciferase genes reportor assay was applied to assess the relationship between miRNA and presumed target genes.Results 1. SF inhibited the growth of H460and H446cell efficiently (1C50was11.2and9.8μmol/L, respectively), and induced cell apoptosis(P=0.003).2. SF inhibited primary tumor sphere and second tumor sphere dose-dependently (p<0.05). Notably, SF suppressed the formation of tumor sphere at a very low concentration that was-10-fold lower than the IC50of SF. SF also dose-dependently eliminated SP cells (P<0.05) and a very low concentration of SF markably suppressed the SP cells, which was similar to what happened in tumor sphere formation assay.3. SF dose-dependently suppressed the expression of the pluripotenc maintaining factors(Nanog, Oct4, Sox2and c-Myc) in vitro.4. SF efficiently inhibited self-renewal of cancer stem cells in lung cancer in animal transplant model (F=242.06, P=0.000).5. Expression of β-catenin was suppressed dose-dependently and time-dependently after H460cells were treated with SF. H460-S33Y cell line which stably expressed activated β-catenin was attained.Nanog and c-Myc were suppressed in H460-S33Y cells treated with sulforaphane, suggesting that other mechanism existed.6. The transcript of miR-214increased at least8-fold than control after H460cells were treated with SF for3days. Inhibition of miR-214led to up-regulation of c-Myc and Sox2.Combined treatment of sulforaphane and miR-214inhibitor in H460abolished the effect of the miR-214inhibitor on c-Myc and Sox2.7. RNA22analysis showed that miR-214might directly combine to the CDS of c-Myc mRNA. Sox2gene promoter analysis showed that there were five putative c-Myc response elements contained in Sox2gene promoter.8. The result of dual-luciferase reporter assay showed that miR-214directly binded to the CDS of c-Myc and decreased the activity of luciferase.Conlusion 1. SF efficiently suppressed lung cancer cells and specially preferred to suppress cancer stem cells at low concentration in vitro and in vivo.2. SF suppressed self-renewal of cancer stem cells in lung cancer through suppressing β-catenin-dependent and (3-catenin-independent pathwany.3. We firstly demonstrated that SF suppressed expression of c-Myc and Sox2through up-regulating miR-214.4. We firstly demonstrated that miR-214suppressed expression of c-Myc by directly combining to the CDS of c-Myc mRNA.5. We suggested that a relationship of interacted regulation might exist between c-Myc and Sox2. c-Myc could bind to the Sox2promoter and regulate Sox2expression.