| BackgroundOsteosarcoma is a most common high-grade malignant bone tumor occurringfrequently in children and adolescents. At the moment anti-osteosarcoma drugs havecytotoxicity and severe side effects which are harmful to our health. Nearly a half ofyoung adult survivors of childhood cancer have at least one major adverse outcome oftheir health status as a result of their cancer therapy. Early toxicity consists ofhematological toxicity, acute liver (MTX) and renal toxicity (CDP and IFO). Longersurvival has led to the increase in late chemotherapy toxicity. The main late toxicities are:cardiac, second tumor, sterility, chronic renal failure and neurologic toxicities (mainlyototoxicity due to CDP). It is urgent clinically to develop safe and efficient agents withhypotoxicity.Pterostilbene(trans-3,5-dimethoxy-4’-hydroxystilbene, PTE), a natural compound occurring from our daily diet appears to be a safe and efficient agent. Pterostilbene,dimethylated analog of resveratrol from blueberries, is known to have diversepharmacologic activities, including anticancer, anti-inflammation, antioxidant,anti-proliferative and analgesic activities PTE has been shown to have potent antitumoractivity with low toxicity in various cancer types, including hepatoma, breast cancer,prostate cancer, pancreatic cancer and chronic myelogenous leukemia, among others. Inmost circumstances, PTE was as potent or significantly more potent than resveratrol,indicating that PTE might show better biological activity due to its increasedbioavailability, as the substitution of a hydroxy group with a methoxy group increases itslipophilicity. However, the effects of PTE in human osteosarcoma have not been clarified.In addition, various molecules and signaling pathways are involved in the anti-tumoreffects of PTE, including adenosine monophosphate-activated protein kinase (AMPK),cytosolic Ca2+overload, ROS, autophagy, Wnt signaling, and lysosomal membranepermeabilization, among others. The molecular mechanisms underlying the effects of PTEremain largely unknown. The JAK/STAT pathway is pivotal for the transduction of amultitude of signals that are critical for development and homeostasis in mammals. TheJAK family of proteins consists of JAK1, JAK2, JAK3and tyrosine kinase2, all of whichshare similar structures and functions. JAK activation plays an important role in cellproliferation, differentiation, migration and apoptosis. Constitutively activated JAKsphosphorylate critical cellular substrates, such as the STAT family, which includesSTAT3, that are associated with oncogenic signaling pathways. Constitutive activation ofSTAT3plays a critical role in cell growth and survival in human solid tumor malignanciesand in the up-regulation of the anti-apoptotic genes encoding Mcl-1and Bcl-xL proteins inhuman cancer cells. PTE was also shown to inhibit STAT3phosphorylation, a marker ofaccelerated tumorigenesis, and decrease pancreatic tumor growth in vivo. Importantly,activated JAK2/STAT3signaling has been extensively validated as a new molecular targetfor the treatment of human solid tumors. The mechanisms of PTE in human osteosarcomahave not been clarified Part I Antitumor activity of PTE against humanosteosarcoma cellsObjectiveTo investigate the effects of PTE on proliferation and apoptosis in humanosteosarcoma cells; to investigate the effects of PTE on migration and adhesion in humanosteosarcoma cells; to investigate the effects of PTE on cell cycle.Methods1. Cell culture and treatmentThe human osteosarcoma cell line, SOSP-9607(9607), were grown in Dulbecco’smodified Eagle’s medium, supplemented with10%fetal bovine serum, L-glutamine (2mM), penicillin (100units/ml), streptomycin (100units/ml), and HEPES (25mM). Thecells were maintained in the presence of5%CO2at37°C.The PTE stock solution was prepared in DMSO and diluted with culture mediumimmediately prior to the experiment. The control group was treated with DMSO (0.01%).First, the cells were treated with PTE (1,2, and4μM).2. Analysis of cell viabilityAfter the cells were treated and washed with PBS,100μL of0.5mg/mL MTTsolution in phenol red-free DMEM was added to the cells, and the samples were incubatedfor4h at37°C. Finally,100μL ofN, N-dimethylformamide was added to each well, andthe samples were incubated for15min at37°C with shaking. The wells were measured at490nm using a microplatereader and cell viability was expressed as an optical density(OD) value. In addition, cell morphology was observed under an inverted/phase contrastmicroscope, and pictures were taken with an Olympus BX61camera. 3. Analysis of cell cycleAfter treatment with PTE for24h, cells were collected by trypsinization, fixed in70%ethanol, washed in PBS, resuspended in1mL of PBS co ntaining0.02mg/mL RNaseand0.02mg/mL PI, and incubated in the dark for30min at room temperature. The cellcycle distribution was analyzed using a FACScan flow cytometer equipped with theFACStation data management system, running the Cell Quest software. The results areexpressed in a plot of fluorescence intensity vs. cell number.4. Analysis of cell mitochondrial transme mbrane potentialMMP was estimated by flow cytometry after staining with JC-1fluorescent dye.When the cell is in a normal state, MMP is high, and JC-1predominantly appears as redfluorescence. When the cell is in an apoptotic or necrotic state, the MMP is reduced, andJC-1appears as a monomer that shows green fluorescence. A change in florescence fromred to green indicates a decrease in MMP. The cells were plated in6-well plates andtreated with PTE for24h. Then, the cells were washed with PBS and incubated with JC-1working solution for20min at37°C in the dark. The cells were washed with PBS andresuspended in500μl PBS. The stained cells were analyzed by the same flow cytometerand software used for the cell cycle analysis. The results are expressed as the proportion ofcells with low MMP.5. Analysis of cell apoptosisThe apoptosis of osteosarcoma cells was detected using the fluoresceinisothiocyanate FITC-Annexin V/PI staining Kit. After being treated with PTE for24h, thecells were harvested, washed in ice-cold PBS, incubated for15min withfluorescein-conjugated Annexin V and PI, and analyzed using the same flow cytometerand software used for the cell cycle analysis. PI-negative and Annexin V-positive cellswere considered early apoptotic (lower right quadrant), while cells that were both PI-andAnnexin V-negative were considered normal (lower left quadrant).6. Analysis of wound-healingThe cells were grown to confluence, and a linear wound was created in the confluentmonolayer using a200μl micropipette tip. The cells were then washed with PBS to eliminate detached cells. After being treated with PTE for24h, wound edge movementwas monitored with a microscope. The results are expressed as the distance between thecells on each side of the scratch.7. Analysis of cell adhesionAfter being treated with PTE for24h, the cells were centrifuged and resuspended inbasal medium with10%fetal bovine serum. The treated cells were placed on a96-wellplate and incubated for30min at37°C. After the cells were allowed to adhere for30min,they were gently washed3times with PBS. The adherent cells were stained with MTT andobserved under an inverted/phase contrast microscope, and pictures were taken with anOlympus BX61camera (Japan). Finally,100μL of N, N-dimethylformamide was addedto each well, and the samples were incubated for15min at37°C with shaking. The wellswere measured at490nm using a spectrophotometer (SpectraMax190, Molecular Device,USA), and the OD value in the control group was set as100%.8. Statistical AnalysesAll experiments were performed in duplicate and repeated at least three times. Thedata are expressed as the means±the standard deviation (SD). The treatment groups werecompared by one-way variance (ANOVA) with SPSS12.0. Differences were consideredstatistically significant at P <0.05.Results1. Effects of PTE on viability and apoptosis in osteosarcoma cellsOsteosarcoma cells were treated with PTE for12,24, and36h with1,2and4μM ofPTE, and cell growth was inhibited in a dose-and time-dependent manner. The IC50(50%inhibitory concentration) of PTE at24h was approximately1.81μM. As observed underthe microscope, PTE treatment resulted in a decrease in the rate of cellular attachmentcompared to the control group.After treatment with1,2and4μM of PTE for24h, the apoptotic index increased by16.75±3.91%,21.55±3.26%and35.87±4.23%, respectively (P<0.01, compared with the control group). The induction of apoptosis was found to be dose-dependent. These resultsindicate that PTE induces apoptosis in osteosarcoma cells.2. Effects of PTE on osteosarcoma cell migration and adhesionAfter incubation with PTE (1,2and4μM) for24h, the distance between thescratches significantly increased to123.07±9.05%,161.53±8.28%, and223.08±11.30%, respectively (P<0.01, compared with the control group), and the cell adhesionratio decreased significantly to62.29±9.43%,34.38±6.70%, and13.64±3.35%,respectively (P<0.01, compared with the control group). These results indicate that PTEreduces the adhesive and migratory abilities of osteosarcoma cells.3. Effects of PTE on mitochondrial me mbrane potential in osteosarcoma cellsAfter treatment with PTE (1,2and4μM) for24h, the proportion of cells with lowMMP increased significantly to8.19±2.36%,16.17±2.68%,29.45±3.10%, respectively(P<0.01, compared with the control group). These results indicate that PTE enhanced theMMP of osteosarcoma cells.4. Effects of PTE on the cell cycle in osteosarcoma cellsTo further investigate the effect of PTE on cell growth, we analyzed the effect of PTEon the cell cycle distribution of osteosarcoma cells. Compared with untreated control cells,PTE (1,2and4μM) induced an accumulation of cells in the G0–G1phase fractions. TheG0–G1phase fraction increased from33.27%in control cells to55.22%in PTE (4μM)induced cells.ConclusionPTE treatment resulted in a dose-and time-dependent inhibition of osteosarcoma cellviability. Additionally, PTE exhibited strong antitumor activity, as evidenced byreductions in tumor cell adhesion, migration and mitochondrial membrane potential. Part Ⅱ Pterostilbene exerts antitumor activity byinhibiting the JAK2/STAT3signaling pathwayObjectiveTo investigate the role of the JAK2/STAT3signaling pathway on activity of PTEagainst human osteosarcoma cells; to investigate effects of PTE on the mitochondrialapoptotic pathway and cell cycle-related proteins in osteosarcoma cells.Methods1. Cell culture and treatmentSee part I. Then, the cells were treated withPTE (4μM) in the absence or presence ofAG490(a known JAK2/STAT3inhibitor,20μM). After the treatments, the cells wereharvested for further analysis.2. Analysis of intracellular ROS generation and GSH levelsAfter being treated with PTE for24h, the cells were trypsinized and subsequentlyincubated with DCFH-DA (20μM) in PBS at37℃for2h. After incubation, the DCFHfluorescence of the cells in each well was measured using an FLX800microplatefluorescence reader at530nm as the emission wavelength and485nm as the excitationwavelength. A cell-free condition was used to determine the background, and thefluorescence intensity in the control group was defined as100%.Briefly, cells were plated at a density of1×106in100-mm culture dishes, allowed toattach overnight, and treated with PTE on the second day. The cells were collected byscraping and washed with PBS. The resulting lysates were used to determine the GSHlevels using the previously mentioned kit, according to the manufacturer’s instructions. Todetermine the GSSG levels, GSH was masked by2-vinylpyridine for1h before the assay.The samples were read at405nm at5min intervals for30min. The GSH and GSSG were evaluated by comparison with standards and normalized with protein content. The resultswere expressed as total GSH (%of control) or GSH/GSSG ratio, using the reduced formGSH or an oxidized form of GSH (GSSG) as the standard.3. Western BlottingCell samples were lysed in sample buffer, sonicated, boiled, run through an8-12%Bis/Tris gel using5×MES buffer, transferred to Immobilon NC membrane and blocked in5%nonfat milk in TBST. The membranes were probed with p-JAK2, JAK2, p-STAT3,STAT3, Mcl-1, Cyclin D1, p21, p27(1:500), Bcl-xL, Bax, Bak and GAPDH antibodies(1:1000) overnight at4oC in blocking buffer. The membranes were then washed in TBSTand probed with the appropriate secondary antibodies (1:5000) in blocking buffer at roomtemperature for90min, followed by washing. Fluorescence was detected using a BioRadimaging system. The signals were quantified using the Image Lab Software.4. Extract of cytosolic Cytochrome cAfter being treated, the cells were harvested by centrifugation at1,000rpm for5min.The pellets were washed twice with ice-cold PBS, suspended with5-fold volume ice-coldcell extract buffer, and incubated for40min at4oC. Then, the cells were centrifuged at1,200rpm for10min at4oC and the final supernatant was used as cytosolic fraction ofCytochrome c. Then,5×loading buffer was added to the above obtained supernatant andthe mixture was boiled at100oC for7min. Thus, the protein solution was used foridentification of cytosolic Cytochrome c by Western blotting. The Cytochrome c proteinwas detected by using anti-Cytochrome c antibody in the ratio of1:500.5. Statistical AnalysesSee part IResults1. Effects of PTE on the expression of cell cycle-regulated proteins in osteosarcomacells To further characterize the observed G0–G1phase arrest, we assayed the expressionof Cyclin D1, p21, and p27by Western blotting. Consistent with cell cycle arrest, theexpression level of Cyclin D1decreased, while the expression levels of p21and p27increased. This finding suggests that G0–G1phase arrest by PTE is, at least in part, due toprofound alterations in the expression of regulatory cell cycle-related factors.2. Effects of PTE on ROS generation and GSH levels in osteosarcoma cellsTreatment with PTE (1,2and4μM) for24h induced a dose-dependent increase inROS generation in osteosarcoma cells (Fig.4A), with increases of255.32±12.97%,411.59±16.24%, and503.61±17.05%, respectively (P<0.01, compared to the controlgroup).After treatment with PTE for24h, we observed a dose-dependent decrease (80.51±3.06%,66.43±3.82%,52.30±3.49%, respectively) in intracellular GSH levels inosteosarcoma cells,(P<0.01, compared with the control group). PTE induced adose-dependent decrease in the ratio of GSH/GSSG in osteosarcoma cells. These resultssupport the notion that PTE treatment affects cellular redox status.3. Effects of PTE on the JAK2/STAT3signaling pathway and mitochondrialapoptotic pathway-related proteins in osteosarcoma cellsThe phosphorylated forms ofJAK2and STAT3were assessed by Western blotanalysis in osteosarcoma cells treated with PTE for24h. We found that thephosphorylation of these factors was decreased by PTE in osteosarcoma cells. Consistentwith the down-regulation of p-JAK2and p-STAT3, the expression of Mcl-1and Bcl-xLwas reduced by PTE in a dose-dependent manner. In addition, mitochondrial apoptoticpathway-related proteins (Bax, Bak, cytosolic Cytochrome c and cleaved Caspase3) wereup-regulated by PTE treatment, suggesting that this apoptotic pathway was activated.4. Effects of the combination of PTE and AG490on cell viability and theJAK2/STAT3signaling in osteosarcoma cellsCombined treatment with PTE (4μM) andAG490(a known JAK2/STAT3inhibitor,20μM) further inhibited the viability of osteosarcoma cells (P<0.01, compared with thePTE or AG490group). Thus, treatment with PTE and AG490further inhibits the phosphorylation of JAK2and STAT3.ConclusionsIn conclusion, these studies provide mechanistic evidence that PTE treatment inhibitsosteosarcoma cell growth via down-regulation of the JAK2/STAT3signaling pathway.PTE seems to regulate multiple molecular targets to produce its anti-osteosarcoma effect,including the activation of the mitochondrial apoptotic pathway and the regulation of cellcycle related proteins. |
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