| BackgroundThyroid cancer,the most common endocrine malignancy,has increased substantially worldwide.Well-differentiated thyroid carcinoma(WDTC)comprises the majority of thyroid cancers,which accounts for 90%of all thyroid cancer cases.The biologic behavior is inert and the overall prognosis is good in patients with WDTC.Notably,thyroidectomy and thyroid hormone suppression therapy with thyroid cancer remnant and metastasis ablation by radioiodine treatment are widely accepted as routine therapies for WDTC.Among them,radioactive 131 iodine treatment plays a vital role,especially for patients with metastasis.The ability of thyroid cancer cells to take up iodine enables the use of radioactive iodine(RAI)for targeted killing of RAI-avid thyroid carcinoma.However,some patients progress to dedifferentiated thyroid cancer(DeTC)during the treatment.The capacity of iodine uptake in patients with DeTC is weakened to varying degrees or even lost,that is,radioiodine resistance occurred.In addition,tumor growth speeds up and DeTC is prone to recurrence and metastasis,especially lymph node metastasis.Due to radioiodine resistance,radioiodine therapy is difficult to achieve in patients with DeTC.And,DeTC is resistant to chemotherapy and conventional external beam radiotherapy.Once metastasis occurs in DeTC,its treatment becomes tricky.Therefore,how to improve the ability of radioiodine uptake and to inhibit the invasion and metastasis for patients with DeTC is particularly important.Endogenous reverse transcriptase(RT)is highly expressed in poorly differentiated cells,undifferentiated cells,transformed cells,aberrant cells and embryonic tissue,while under-expressed or not expressed in differentiated and non-pathological tissues.This suggests that RT may be involved in cell proliferation and differentiation.Nevirapine,a non-nucleoside reverse transcriptase inhibitor,has been proved to be effective in inducing differentiation and inhibiting growth of melanoma and prostate cancer cells.But,few studies suggest its effects on thyroid cancer,especially on DeTC.In view of the above theories,this study takes dedifferentiated thyroid cancer WRO 82-1 and dFTC-133 cells as research objects,and studies the effects and mechanisms of nevirapine on cell proliferation,metastasis and radioiodide uptake.And,tumor xenografts in nude mice were constructed with WRO 82-1 cells to futher explore these effects.The purpose of this study is to explore the role and underling mechanisms of nevirapine in patients with DeTC,and to provide new ideas and basis for treatment of clinical patients.Part I The effects of nevirapine on radioiodide uptake of dedifferentiated thyroid cancer and its mechanismObjectives1.To observe the effect of nevirapine on radioiodide uptake of dedifferentiated thyroid cancer cells.2.To clarify the mechanism of nevirapine regulating radioiodide uptake of dedifferentiated thyroid cancer cells.3.To further verify the effect of nevirapine on radioiodide uptake of dedifferentiated thyroid cancer in animal experiments.Methods1.Human dedifferentiated thyroid cancer WRO 82-1 cells and human follicular thyroid cancer FTC-133 cells were cultured and passage.2.The dedifferentiated FTC-133(dFTC-133)cell line was established.The dFTC-133 cell line was established by radiation with 131I.Briefly,the FTC-133 cells were seeded in 6-well plates incubated with 15 μCi Na131I for 3 days and then cultured in activity free medium by graded dilutions for 3 months,afterwards wells with one cell clone were selected and cells were further cultured.Cells with lowest radioiodine uptake were defined as dFTC-133 and their molecules related to iodide-transport were detected.3.Cell Counting Kit-8 assay was used to assess cell proliferation of WRO 82-1 and dFTC-133.After incubation with nevirapine(100,200,350 and 500 μM)or the same volume of 0.1%DMSO(as control)for 24,48 and 72 hours,CCK8 reagent was added into each well,and the cells were incubated at 37℃ for 3 hours.Cell viabilities were measured through absorbance(optical density)by a microplate reader at a wavelength of 450 nm.4.Annexin V-FITC apoptosis assay was used to evaluate WRO 82-1 and dFTC-133 apoptosis.The cells seeded in six-well plates were treated with 100,200,350 and 500 pM nevirapine or the same volume of 0.1%DMSO(as control)for 72 hours,then harvested and stained with annexin V-fluorescein isothiocyanate and propidium iodide(NeoBioscience,China)according to the manufacturer’s instructions.Finally,the apoptotic rates were obtained by flow cytometry.5.Lentiviruses carrying shRNA targeting human PAX8 lentiviral vectors(GV248)were constructed to knock down the expression of PAX8.The lentiviruses were used to infect WRO 82-1 and dFTC-133 cells in the presence of Polybrene.Forty-eight hours later,the stable clones were selected by puromycin and the expression of PAX8 in the infected cells was verified by qRT-PCR and western blot analysis.6.The effect of nevirapine on the levels of thyroid differentiation-related genes(NIS,TSHR and TPO)and thyroid transcription factors(TTF-1,TTF-2,PAX8)mRNA were evaluated by qRT-PCR.Both cells were treated with 100 μM and 200 1μM nevirapine or the same volume of 0.1%DMSO(as control)for 72 hours.Total cell RNA was extracted using TriZol reagent and then converted to synthesize cDNA using the First Strand cDNA Synthesis Kit.The expression of iodine uptake related genes(NIS and TSHR)and thyroid transcription factor PAX8 mRNA was detected by qRT-PCR on an ABI PRISM 7500 Real-time PCR System using the SYBR Green.7.The effect of nevirapine on localization and expression of NIS protein was detected by confocal immunofluorescence(IF)and western blot.The effect of nevirapine on the protein levels of PAX8,TSHR,cAMP and pCREB(Ser133)was evaluated by western blot.Different concentrations of nevirapine(100 μM,200 μM or the same volume of 0.1%DMSO)were added to both cells for 24,48,and 72 hours.Then,the expression of NIS protein in the cytoplasm and cell membrane was detected by western blot.In order to further explore the effect of nevirapine on the localization of NIS,IF assay was used to observe the localization of NIS.To explore the mechanism of nevirapine on up-regulation of NIS expression,both cells were treated with different concentrations of nevirapine(100 μM,200 μM or the same volume of 0.1%DMSO)for 72 hours,then the expression of PAX8,TSHR,cAMP and pCREB(Ser133)protein was detected by western blot.The cAMP inhibitor SQ22536 was added in both cells to inhibit TSHR/cAMP/CREB/PAX8 pathway,the inhibition effect of the pathway was observed by western blot.The protein expression of PAX8 and NIS was further detected by western blot,in order to determine whether nevirapine up-regulated NIS protein expression by activating the TSHR/cAMP/CREB/PAX8 pathway.8.The effect of nevirapine on iodine uptake of both cells was detected by in vitro radioactive 125 iodine uptake assay.After treatment with nevirapine(100 μM and 200 μM)or 0.1%DMSO as control in 24-well plates,the cells were incubated with 2 μCi Na125I in 5 mM nonradioactive NaI for 30 minutes at 37℃.The cells were then washed with cold HBSS and lysed with 500μl formic acid for 20 minutes.The radioactivity was measured in the cell lysates by a gamma counter.The radioactivity was normalized to the number of viable cells at the beginning of the experiment and expressed as cpm per 106 cells.9.Tumor xenografts were constructed by subcutaneous injection of WRO 82-1 cells into nude mice.The male 4-week-old nude mice were fed under specific pathogen-free conditions for 1 week to accommodate the experimental conditions.The nude mice were then inoculated subcutaneously with WRO 82-1 cells(1×106/mouse).Ten days after tumor implant,nevirapine(150 mg/kg/day)was administered orally five days a week.10.The effect of nevirapine on iodine uptake of tumor xenografts was detected by in vivo radioactive 125 iodine uptake assay.Three weeks after nevirapine treatment,10 μCi Na125I were injected intraperitoneally.Animals were sacrificed 4,24 and 48 hours after Na125I injection.Iodine uptake was measured in a gamma counter normalized by weight and expressed as a radioactivity ratio of tumor to thyroid.11.The expression of NIS protein in tumor xenografts and human normal thyroid tissues was detected by immunohistochemical staining(IHC).The tumor xenografts and patient thyroid tissues were fixed in 4%formalin for 1 day.After dehydration and paraffin embedding,the samples were sliced into 4μm thick sections and mounted on glass slides.Antigen recovery was performed by pressure-cooking the slides in citrate buffer(pH 6.0)twice for 8 minutes each time after deparaffinization and rehydration.After blocking endogenous peroxidase activity by 3%hydrogen peroxide,the sections were incubated with rabbit anti-NIS antibody at 4℃ overnight,followed by incubation with anti-rabbit secondary antibody at 37℃ for 50 minutes.NIS staining was detected using a DAB kit whereas cell nuclei were counterstained with hematoxylin.Results1.Low doses of nevirapine significantly inhibited cell proliferation in a dose-and time-dependent manner without inducing apoptosis in dedifferentiated thyroid cancer cells.After 24 hours serum starvation,both cells were incubated with nevirapine(100,200,350 and 500μM)for 72 hours.Nevirapine did not increase both cells apoptosis in the presence of 100 μM and 200μM nevirapine(all P>0.05),but suppressed both cells proliferation in a dose-and time-dependent manner.When the concentration of nevirapine reached 200 μM and both cells were incubated with nevirapine for 72 hours,the viability rates of WRO 82-1 and dFTC-133 cells were 74.9±3.1%and 77.4±2.8%(both P<0.05).2.Nevirapine significantly increased expression of thyroid differentiation-related genes TSHR and NIS and thyroid transcription factor PAX8 in dedifferentiated thyroid cancer cells.To assess the effect of nevirapine on thyroid differentiation specific genes in WRO 82-1 and dFTC133 cells,the expression of thyroid differentiation-related genes(TPO,TSHR and NIS)and thyroid transcription factors(TTF-1,TTF-2 and PAX8)were detected by qRT-PCR experiments.The results demonstrated that nevirapine significantly increased the levels of PAX8,TSHR and NIS mRNA,but failed to up-regulate the expressions of TTF-1,TTF-2 and TPO mRNA.After 72 hours of 200μM nevirapine treatment,the expressions of NIS mRNA were approximately 1.9-fold and 1.8-fold,the mRNA levels of TSHR were about 3.7 times and 3.0 times,and the levels of PAX8 mRNA were approximately 2.9-fold and 2.8-fold compared with that in controls in WRO 82-1 and dFTC-133 cells,all had statistical significances(all P<0.05).3.Nevirapine upregulated the expression of plasma membrane-localized NIS in dedifferentiated thyroid cancer cells.To determine the effect of nevirapine on NIS protein translocation,the changes in levels of membranous and cytoplasmic NIS proteins were conducted by immunoblotting with NIS-specific antibody.Treatment with nevirapine resulted in a marked increase of plasma membrane-localized NIS protein in WRO 82-1 cells and dFTC-133 cells in dose-and time-dependent manner.The expression of NIS protein(membrane/cytoplasm)was increased by nevirapine for 72 hours in dose-dependent manner,with an increase of 1.8-fold in WRO 82-1 cells and 1.7-fold in dFTC-133 cells with 200 μM nevirapine compared with that of control group(P<0.05).Further results showed that the expression of NIS protein(membrane/cytoplasm)was upregulated by nevirapine(200 μM)in a time-dependent manner,with an increase of 1.3-fold in WRO 82-1 cells and 1.6-fold in dFTC-133 cells with 200 μM nevirapine for 72 hours,respectively,compared with that treated for 24 hours(P<0.05).The localization and expression of NIS were then explored by immunofluorescence experiment.The results showed that nevirapine increased both cytoplasmic and cytomembrane-located NIS expression,the latter was more significant.4.Nevirapine increased radioiodide uptake in differentiated thyroid cancer cells.The effect of nevirapine on NIS-mediated radioactivity was investigated in WRO 82-1 cells and dFTC-133 cells.As expected,nevirapine-treated groups showed significantly increase in radioactive countings compared with control group in a concentration and time-dependent manner,1.4-fold and 1.7-fold in WRO 82-1 cells,1.4-fold and 2.1-fold in dFTC-133 cells with 100 and 200 μM nevirapine for 72 hours,and 1.3-fold,1.4-fold and 1.8-fold in WRO 82-1 cells,1.3-fold,1.6-fold and 2.1-fold in dFTC-133 cells with 200 μM nevirapine for 24,48 and 72 hours(all P<0.05).5.Nevirapine significantly increased the expression of PAX8 protein,and PAX8 was a key factor mediated up-regulation of NIS protein and increased radioiodide uptake in both cells.The expression of PAX8 protein by Western blotting was engaged to demonstrate its role in response to nevirapine.It was found that nevirapine-treated cells presented significantly increased expressions of PAX8,2.7 times in WRO 82-1 cells and 2.2 times in dFTC-133 cells with 200μM nevirapine for 72 hours compared with control group(all P<0.05).Furthermore,to verify these results,we established PAX8 knockdown cells by lentivirus.Nevirapine failed to increase the expression of NIS protein when PAX8 was knocked down(P<0.05).After the expression of PAX8 was knocked down by lentivirus,the expression of NIS protein and the iodine uptake rate of cells were significantly reduced.These results demonstrated that PAX8 was related to the up-regulation of NIS protein and the increased iodine uptake rate,and was a key factor that mediated NIS expression and iodine uptake by nevirapine.6.Nevirapine increases NIS-mediated radioiodide uptake by activation of TSHR/cAMP/CREB/PAX8 signaling pathway in dedifferentiated thyroid cancer cellsThe expressions of thyroid stimulating hormone receptor(TSHR),cyclic adenosine monophosphate(cAMP)and phosphorylation level of cAMP-response element binding protein(pCREB(Ser133))were upregulated siginificantly by nevirapine(all P<0.05).Furthermore,nevirapine-induced expressions of pCREB(Ser133),PAX8,NIS and radioiodide uptake were inhibited by SQ22536,a specific cAMP inhibitor(all P<0.05),which meant that the upregulation of NIS-mediated radioiodide uptake by nevirapine was dependent of TSHR/cAMP/CREB/PAX8 signal pathway.7.Nevirapine inhibited tumor growth in athymic mouse xenografts of WRO 82-1 cells.To further confirm the results of the above in vitro experiments,we constructed nude mouse xenograft models and conducted in vivo experiments.Three weeks after nevirapine treatment,51.3±3.1%reduction in tumor growth was recorded in nevirapine-treated mice compared with that of control mice(P<0.05).8.Nevirapine elevated NIS protein expression and iodine accumulation in nude mouse xenografts of WRO 82-1 cells.The positive rates of NIS were 39.5%±1.9%in normal thyroid tissues,30.4%±1.8%in xenografts of the nevirapine-treated group and 18.2%±0.8%in xenografts of the control group.The positive rate of NIS in nevirapine-treated group was 1.7-fold compared with that in untreated group and 0.8-fold compared with that in normal thyroid tissues,which indicated that nevirapine increased the level of NIS protein significantly(P<0.05),although the expression of NIS in nevirapine-treated xenografts was lower than that in normal thyroid tissues(P<0.05).Consistently,nevirapine-treated xenografts exhibited a significant augment in the ratio of radioactivity between tumor and thyroid,which reached the maximum at 48 hours after Na125I injection,0.93±0.04(P<0.05),while control xenografts displayed a constant low level of radioiodide uptake.Conclusions1.Nevirapine significantly inhibited proliferation of dedifferentiated thyroid cancer WRO 82-1 and dFTC-133 cells.2.Nevirapine significantly improved radioiodide uptake in both cells.The mechanism was to upregulate the expression of NIS protein and to promote the membrane localization of NIS protein by activating the TSHR/cAMP/CREB/PAX8 pathway.3.Nevirapine significantly inhibited tumor growth and significantly elevated the expression of NIS protein and radioiodide uptake in athymic mouse xenografts of WRO 82-1 cells.Part Ⅱ The effects of nevirapine on migration and invasion of dedifferentiated thyroid cancerObjectives1.To explore the effect of nevirapine on metastasis in nude mouse xenografts of WRO 82-1 cells.2.To observe the effect of nevirapine on migration and invasion of dedifferentiated thyroid cancer cells.3.To investigate the possible mechanism of nevirapine regulating migration and invasion of dedifferentiated thyroid cancer cells.Methods1.Tumor xenografts were constructed by subcutaneous injection of WRO 82-1 cells into nude mice.The male 4-week-old nude mice were fed under specific pathogen-free conditions for 1 week to accommodate the experimental conditions.The nude mice were then inoculated subcutaneously with WRO 82-1 cells(1×106/mouse).Ten days after tumor implant,nevirapine(150 mg/kg/day)was administered orally five days a week.2.The distant metastasis of differentiated thyroid cancer was detected by hematoxylin-eosin(HE)staining.The tissues were immersion-fixed in 4%paraformaldehyde for 24 hours,embedded in paraffin,and cut into 4 μm slices.After deparaffinization and rehydration,the sections were stained with HE staining to observe the morphology by microscope.3.Human dedifferentiated thyroid cancer WRO 82-1 cells were cultured and passage.4.The effects of nevirapine on migration and invasion of dedifferentiated thyroid cancer were detected by transwell migration/invasion assay.WRO 82-1 cells were treated with 200 μM nevirapine for 72 hours.Transwell migration and invasion assay were used to observe the changes of WRO 82-1 cell migration and invasion function.5.The mRNA and protein levels of VEGFA,MMP2 and MMP9 were detected by qRT-PCR.WRO 82-1 cells were treated with 100 and 200 μM nevirapine or the same volume of 0.1%DMSO(as control)for 72 hours.Total cell RNA was extracted using TriZol reagent and then converted to synthesize cDNA using the First Strand cDNA Synthesis Kit.The expression of mRNAs related to migration and invasion(VEGFA,MMP2 and MMP9)was detected by qRT-PCR on an ABI PRISM 7500 Real-time PCR System using the SYBR Green.Different concentrations of nevirapine(100μM 200μM or the same volume of 0.1%DMSO)were added to WRO 82-1 cells for 72 hours.Then,the expression of VEGFA,MMP2 and MMP9 proteins was detected by western blot.6.The level of IL-6 mRNA was detected by qRT-RCR experiment,and the phosphorylation of JAK2 protein and STAT3 protein was detected by western blot.To explore the possible mechanism of nevirapine on inhibition of migration and invasion,WRO 82-1 cells were treated with different concentrations of nevirapine(100 μM,200 μM or the same volume of 0.1%DMSO)for 72 hours,then the level of IL-6 mRNA was detected by qRT-RCR and the phosphorylation of JAK2 and STAT3 protein was detected by western blot.Results1.Nevirapine significantly reduced the metastasis in liver significantly in nude mice xenografts.Three weeks after treatment,we were surprised to see that the tumor metastasized to liver was reduced significantly compared with that of control,and the larger metastases are accompanied by necrosis in liver of the control group.Subsequently,in order to further confirm whether the metastases are derived from dedifferentiated thyroid cancer,HE staining,to observe cell morphology,was done.The result presented the spindle cells accompanied by a large number of pathological mitotic figures and atypical giant cells,which are the pathological features of dedifferentiated thyroid cancer.From the above we can see,dedifferentiated thyroid cancer metastasized to the liver and nevirapine inhibited the metastasis.2.Nevirapine significantly inhibited the migration and invasion of WRO 82-1 cells.Transwell chamber migration/invasion experiments showed that function of migration and invasion was significantly inhibited after 72 hours of 200 μM nevirapine treatment.The migration rate was reduced to 34.38±2.35%,and the invasion rate was reduced to 31.47±2.76%(all P<0.05).qRT-RCR and western blot experiments both showed that nevirapine significantly inhibited the levels of VEGFA,MMP2 and MMP9 mRNA and protein(all P<0.05).3.The possible mechanism that nevirapine inhibited the migration and invasion of thyroid cancer might be by inhibiting IL-6/JAK2/STAT3 signaling pathway.The effects of nevirapine on mRNA expression of IL-6 were assessed by qRT-PCR and the results showed that nevirapine significantly decreased the expression of IL-6 mRNA compared with untreated cells(P<0.05).The effects of nevirapine on phosphorylation level of JAK2 and STAT3 proteins were detected by western blot and the results suggested that nevirapine up-regulated the phosphorylation levels of JAK2(Y1007+Y1008)and STAT3(Tyr705)in WRO 82-1 cells compared with untreated cells(all P<0.05).Nevirapine with 200μM reduced phosphorylation expression of JAK2(Y1007+Y1008)and STAT3(Tyr705)by 56.0%and 46.1%,respectively(all P<0.05).These results suggested that inhibition of invasion and metastasis of dedifferentiated thyroid cancer cells by nevirapine may be related to the IL-6/JAK2/STAT3 pathway.Conclusions1.Nevirapine reduced the metastasis in liver significantly in nude mice.2.Nevirapine significantly inhibited the migration and invasion of WRO 82-1 cells,which may be achieved by inhibiting IL-6/JAK2/STAT3 signaling pathway. |
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