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Objective@#To investigate the effect of the down-regulated expression of pituitary tumor-transforming gene 1 (PTTG1) on the senescence of human castration-resistant prostate cancer LNCaP-AI cells.@*METHODS@#Human castration-resistant prostate cancer LNCaP-AI cells were induced in vitro and transfected with siRNA targeting PTTG1 (the siRNA-PTTG1 group), the reagent lip3000 only (the mock group) or siRNA negative control vector (the NC group). All the cells were cultured in fetal bovine serum (FBS) or charcoal-stripped bovine serum (CSS) and counted with the cell counting chamber. The senescence characteristics of the transfected LNCaP-AI cells were examined by senescence-associated β-galactosidase (SA-β-Gal) staining, and the expressions of the senescence-related β-galactosidase-1-like proteins (Glb1), the cyclin-dependent kinase inhibitors p-21CIP1 and p-27Kip1, and the chromatin-regulating heterochromatin protein 1γ (HP1γ) were detected by Western blot.@*RESULTS@#The expression of PTTG1 in the human prostate cancer LNCaP-AI cells was significantly reduced in the siRNA-PTTG1 group compared with those in the mock and NC groups (0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05). Culture with FBS markedly increased while that with CSS decreased the number of LNCaP-AI cells transfected with siRNA, but both FBS and CSS enhanced the proliferation of the LNCaP-AI cells in the mock and NC groups. SA-β-Gal staining revealed that reducing the expression of PTTG1 induced a remarkably higher positive rate of the LNCaP-AI cells in the siRNA-PTTG1 than in the mock and NC groups (［63.5 ± 2.35］% vs ［11.3 ± 1.24］% and ［12.4 ± 1.15］%, P < 0.05). The siRNA-PTTG1 group, in comparison with the mock and NC groups, showed a significantly down-regulated expression of PTTG1 (0.21 ± 0.01 vs 0.56 ± 0.02 and 0.61 ± 0.02, P < 0.05), but up-regulated expressions of p-21CIP1 (0.32 ± 0.03 vs 0.20 ± 0.02 and 0.21 ± 0.03, P < 0.05), p-27Kip1 (0.38 ± 0.02 vs 0.20 ± 0.03 and 0.22 ± 0.01, P < 0.05), Glb1 (0.24 ± 0.01 vs 0.13 ± 0.01 and 0.15 ± 0.01, P < 0.05), and HP1γ (0.41 ± 0.01 vs 0.26 ± 0.01 and 0.27 ± 0.02, P < 0.05) in the LNCaP-AI cells.@*CONCLUSIONS@#Down-regulated expression of PTTG1 induces senescence of human castration-resistant prostate cancer LNCaP-AI cells.
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Objective@#To investigate the effects of down-regulation of PTTG1 expression on the proliferation, invasiveness and apoptosis of androgen-independent human prostate cancer LNCaP-AI cells and their sensitivity to androgen antagonists.@*METHODS@#Human prostate cancer LNCaP-AI cells were transfected with siRNA targeting the PTTG1 gene using the Lipofectamine 2000 transfection reagent. The proliferation, invasiveness and apoptosis of the cells were detected by MTT, Transwell assay and flow cytometry, respectively. The protein expressions of PTTG1, p-Akt, and p-ERK were determined by Western blot and the mRNA expression of PTTG1 measured by agarose gel electrophoresis.@*RESULTS@#The siRNA expression vector markedly down-regulated the expression of PTTG1, which effectively suppressed the proliferation of the LNCaP-AI cells, with the inhibition rates of (19.47 ± 2.12), (24.01 ± 2.13) and (48.02 ± 2.22)% at 24, 48 and 72 hours, respectively, after transfection, with statistically significant differences among the three groups (P <0.05). The number of the cells passing through the polycarbonate film was remarkably decreased at 24, 48 and 72 hours (74.67 ± 9.85, 56.44 ± 8.66 and 37.33 ± 6.14) as compared with the baseline (111.11 ± 13.47) (P <0.01), while the apoptosis rate of the cells was significantly increased at 24, 48 and 72 hours (18.32 ± 0.94), (19.94 ± 1.30) and (21.73 ± 1.88)% in comparison with the baseline (［2.17 ± 0.49］%)， (P <0.05). PTTG1 siRNA combined with androgen antagonist flumatide exhibited even more significant effects in inhibiting the proliferation and promoting the apoptosis of the LNCaP-AI cells than either used alone, and in a flumatide dose-dependent manner. The inhibition and apoptosis rates of the LNCaP-AI cells treated with 50 nmol/L flumatide were (27.13 ± 3.52) and (3.94 ± 0.48)%, and those treated with siRNA + 50 nmol/L flumatide were (67.51 ± 5.13) and (19.93 ± 1.72)%, respectively, both with statistically significant differences between the two groups (P <0.05). The inhibition and apoptosis rates of the cells treated with 100 nmol/L flumatide were (43.72 ± 3.90) and (5.33 ± 0.66)%, and those treated with siRNA + 100 nmol/L flumatide were (73.19 ± 4.78) and (23.43 ± 1.76)%, respectively, both with statistically significant differences between the two groups (P <0.05).@*CONCLUSIONS@#The siRNA expression vector can down-regulate the expression of PTTG1, which can inhibit the proliferation and invasiveness of LNCaP-AI cells, promote their apoptosis, and increase their sensibility to androgen antagonists. Suppressing the expression of PTTG1 may enhance the effect of androgen-deprivation therapy on advanced prostate cancer.
Subject(s)
Humans , Male , Androgen Antagonists , Pharmacology , Apoptosis , Cell Line, Tumor , Cell Proliferation , Down-Regulation , Neoplasm Invasiveness , Prostatic Neoplasms , Drug Therapy , Metabolism , Pathology , RNA, Small Interfering , Metabolism , Securin , Genetics , Metabolism , Time Factors , TransfectionABSTRACT
<p><b>Objective</b>To explore the expression of pituitary tumor transforming gene 1 (PTTG1) during the transformation of prostate cancer from androgen-dependent (ADPC) to androgen-independent (AIPC).</p><p><b>METHODS</b>We established an AIPC cell model LNCaP-AI by culturing the androgen-dependent LNCaP cell line in the hormone-deprived medium for over 3 months. The cell model was verified and the PTTG1 expression in the LNCaP cells was detected by Western blot and RT-PCR during hormone deprivation.</p><p><b>RESULTS</b>The AIPC cell model LNCaP-AI was successfully established. The PTTG1 expression was gradually increased in the LNCaP cells with the prolonged time of hormone deprivation and the expressions of matrix metalloproteinases MMP-2 and -9 were elevated at the same time.</p><p><b>CONCLUSIONS</b>The expression of PTTG1 is increased gradually in AIPC, which may be a target of gene therapy for advanced prostate cancer.</p>
Subject(s)
Humans , Male , Blotting, Western , Cell Line, Tumor , Gene Expression Regulation, Neoplastic , Matrix Metalloproteinase 2 , Metabolism , Matrix Metalloproteinase 9 , Metabolism , Neoplasms, Hormone-Dependent , Prostatic Neoplasms , Genetics , Securin , GeneticsABSTRACT
Objective: To explore the role of pituitary tumor-transforming gene (PTTG) in invasion of breast cancer cells. Methods: The expression level of PTTG protein in selected breast cancer cells with different invasion abilities was detected by Western blotting. The expression level of PTTG protein in MDA231 cells with high invasion ability after transfection with siRNA targeting PTTG (named as siPTTG/MDA231 cells) was detected by Western blotting. The invasion ability of siPTTG/MDA231 cells was measured by Transwell cell invasion assay. The expression levels of phospho-Akt (p-Akt) and phospho-AMP-related protein kinase (AMPK)-related protein kinase 5 (p-ARK5) in siPTTG/MDA231 cells induced by epithelial growth factor (EGF) were examined by Western blotting. Results: The expression level of PTTG protein in MDA231 cells with high invasion ability was higher than those in MCF7 and T47-D cells with low invasion abilities (both P 0.05). Conclusion: The expression level of PTTG protein in breast cancer cells with high invasion ability is high. The invasion ability of MDA231 cells after inhibition of PTTG expression is decreased, and this effect may be related to the regulation of expression levels of p-Akt and p-ARK5 proteins.
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Objective To explore the expression of pituitary tumor transforming gene (PTTG) and c-myc gene in patients with non-Hodgkin lymphoma (NHL),its relationship with NHL and its clinical significance.Methods PTTG mRNA and c-myc mRNA levels in bone marrow mononuclear cell (BMMNC) isolated from 38 NHL patients and 10 chronic lymphadenitis patients were quantified by reverse transcriptionpolymerase chain reaction (RT-PCR).Results mRNA expression of PTTG and c-myc gene in BMMNC was significantly higher in NHL patients than those in normal controls (PTTG:0.567 7±0.270 7 vs 0.071 2± 0.020 1,t =4.706,P ＜ 0.05; c-myc:0.352 6±0.185 4 vs 0.107 3±0.043 5,t =3.303,P =0.002).The expression of PTTG and c-myc gene in peripheral T cell lymphoma and diffuse large B cell lymphoma showed no significant difference (PTTG:0.556 8±0.211 3 vs 0.602 8±0.244 6,t =0.640,P =0.527; c-myc:0.350 1± 0.177 6 vs 0.361 0±0.190 2,t =0.302,P =0.765).Both expression of PTTG and c-myc were positively related to NHL clinical stage and IPI.Expression of PTTG mRNA was positively related to the expression of c-myc mRNA.Conclusion There was overexpression of PTTG and c-myc in NHL,which indicates that PTTG might be involved in tumorigenesis of NHL through direct or indirect activation of c-myc oncogene.
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ObjectiveTo investigate the expression and relationship of pituitary tumor transforming gene (PTTG) and p53 protein in large intestinal adenoma,large intestinal carcinoma and normal large intestinal mucosa tissues.MethodsImmunohistochemistry was employed to determine the expression of PTTG and p53 protein in 50 cases with large intestinal adenoma tissues,42 cases with large intestinal carcinoma tissues and normal large intestinal mucosa tissues.The relationship of the expression of PTTG and p53 protein with the clinicopathological characteristics was analyzed.ResultsThere was no positive expression of p53 protein in normal large intestinal mucosa tissues,while the positive rate of PTTG expression was 7.14％(3/42).The positive rates of PTTG and p53 protein expression were 82.00％(41/50) and 90.00％(45/50) in large intestinal adenoma tissues,88.10％ (37/42) and 95.24％ (40/42) in large intestinal carcinoma tissues.The positive rates of PTTG and p53 protein over expression were 45.24％(19/42) and 69.05％(29/42) in large intestinal carcinoma tissues.The positive rates of PTTG and p53 protein expression in large intestinal carcinoma tissues were higher than those in large intestinal adenoma tissues and normal large intestinal mucosa tissues,the positive rates of PTTG and p53 protein expression in large intestinal adenoma tissues were higher than those in normal large intestinal mucosa tissues,and there were significant differences(P ＜ 0.05).The expression of PTTG was not correlated with p53 protein in large intestinal carcinoma tissues(P＞ 0.05 ),while the positive relationship was found between the expression of PTTG and p53 protein in large intestinal adenoma tissues (P ＜ 0.05 ).The over expression of PTTG was significantly correlated with lymph node metastasis (P ＜ 0.01 ),but the over expression of p53 protein was not correlated with lymph node metastasis(P ＞ 0.05) in large intestinal carcinoma tissues.Conclusions The expression of PTTG is significantly correlated with p53 protein in large intestinal adenoma tissues,and their co-expression may be used as markers for carcinogenesis of large intestinal adenoma tissues.The over expression of PTTG and p53protein is found in large intestinal carcinoma,and the over expression of PTTG is correlated with lymph node metastasis.The over expression of PTTG may be used as a marker for lymph node metastasis of large intestinal carcinoma.
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Objective To study the effect of down-regulation of PTTG on the proliferation and migration of cutaneous squamous cell carcinoma cell line SCL-1 and its related mechanism. Methods SCL-1 cells were transfected with control siRNA or PTTG-targeting siRNA (PTTG-siRNA), or remained untransfected. After additional culture, the proliferation of SCL-1 cells as observed with cell counting kit-8 (CCK-8), and cell migration with Boyden chamber. Real-time PCR and Western blot were performed to detect the expression of matrix metalloproteinase 2 (MMP-2), MMP-9 and PTTG. Results The proliferation of SCL-1 cells transfected with PTTG-siRNA was markedly deccelarated in comparision with that of untransfected cells and those transfected with control siRNA (both P< 0.05). Real-time PCR and Western blot disclosed a significant decrease in the mRNA and protein expression of MMP-2, MMP-9 and PTTG in PTTG-siRNA-transfected SCL-1 cells compared with the other two groups of cells. As real-time PCR showed, the expressions of MMP-2, MMP-9 and PTTG in PTTG-siRNA-transfected SCL-1 cells were 0.8%, 23.2% and 21.3% of those in untransfected cells, respectively. Further more, the number of SCL-1 cells migrating through microporous membrane in the Boyden chamber was significantly smaller in PTTG-siRNA-transfected group than in untransfected group and control siRNA-trans-fected group (51.38 ± 4.71 vs 131.33 ± 6.12 and 127.72 ± 5.20, both P< 0.05). Conclusion The down-regulation of PTTG may deccelarate the proliferation and migration of SCL-1 cells and inhibit the expression of MMP-2 and MMP-9 in SCL-1 cells.
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ssion of prostate cancer. Over-expression of PTTG and high Gleason grade are independent adverse predictors of pro-gression-free survival for patients with local or locally advanced prostate cancer.
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Objective To study the relationship between fragile histidine traid (FHIT), pituitary tumor transforming gene-1 (PTTG-1) in thyroid tumor tissue. Methods The expression of FHIT and PTTG-1 were detected by immunohistocbemistry in 96 eases (56 carcinoma,40 adenoma). Results Compared with thyroid adenoma, the expression of FHIT decreased (P <0.01) ,PTTG-1 increased in thyroid carcinoma(P <0.01). The expression of FHIT is different in thyroid carcinoma in eancerometastasis to non-cancerometastasis (P < 0. 01), prognosis index (≥65) and prognosis index(< 65) (P < 0.01 and P < 0.05) ; There also was statistically significant differences between the expression of PTTG in thyroid carcinoma (P <0.05 and P <0.01). Conclusion FHIT and FTTG-1 may be an important reference significance in the differentiation of benign and malignant thyroid tumor tissue, and may serve as useful prognostic markers.
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Objective To investigate the effect of tamoxifen on pituitary tumor transforming gene ex-pression in C6 cell and tumor growth of glioma in vivo. Methods Animal models were established on 32 SD rats with C6 cells of glioma. The rats bearing with C6 glioma were divided into 4 groups randomly, which were treated without tamoxifen or with different doses of tamoxifen(0. 02 mg · kg-1 · d-1, 0. 2 mg · kg-1 · d-1, 2mg · kg-1 · d-1) once a day for 20 days. The dimension of tumors were measured, the tumor inhibition rates were caculated, and living state of the rats were observed. The expression of PTTG mRNA was detected by RTPCR. Results All kinds of doses of tamoxifen could inhibit the tumors growth in rats with C6 glioma, and the tumor volume were reduced by 47.6%, 35.5% and 21.2% in the high-, middle-and low-dose groups respec-tively, there were significantly differences among the 4 groups ( P < 0. 05 ). Low-, middle- and high- dose of tamoxifen all could inhibit the expression of PTTG mRNA, and there were significantly differences among the 4 groups in PTTG mRNA expression ( P < 0. 05 ). Conclusion Tamoxifen can inhibit PTTG expression and the growth of glioma cell line C6 in vivo significantly in dose-dependent manner, which provides a theory basis for clicinal use of tamoxifen in patients with gliomas.
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Objective To explore the relationship between invasive growth and expression of ER、PTTG、bFGF in human prolactinomas.Methods Fourty-six prolactinomas specimens were collected and divided into invasive group and non-invasive group.Expressions of ER、PTTG、bFGF were examined by immunocytochemical method.Total RNA was extracted from specimens and ER-mRNA and PTTG-mRNA were detected by RT-PCR.Results IA of protein expression of ER、bFGF、PTTG in invasive prolactinomas was seperately 5385.1?1348.6、9874.2?2143.7、7938.5?1436.5;The optical density ratio of ER-mRNA and PTTG-mRNA was 0.71 and 0.83,difference between invasive group and non-vasive group was significant.Conclusions ER、PTTG、bFGF in human prolactinomas closely correlated with invasiveness of prolactinomas,and may play important function in prolactinomas.
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Objective To investigate the inhibition effect of pituitary tumor transforming gene(PTTG) antisense oligodeoxynucleotide(ASODN) on C6 glioblastoma in rats.Methods The C6 glioma cells were injected into the right caudate nucleus.PTTG-ASODN of 8 or 16 ?g/ml was injected into the tumor-affected area with stereotactic technique immediately,at 1st and 2nd week after inoculation of C6 cells.Three weeks after C6 cell inoculation,all rats were killed and the tumors were excised,then tumor volume was calculated and pathologically analysed,and immunohistochemical statining for GFAP, PCNA ang PTTG was performed.Results PTTG-ASODN could suppress the proliferation of C6 glioblastoma in a dose-and time-dependent manner.The inhibition effect was better when large-dose PTTG-ASODN was repeatedly used for glioblastoma as early as possible.Conclusion PTTG-ASODN can suppress the proliferation of glioblastoma,which may become a new strategy of gene therapy for glioblastoma.
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Objective:To identify the androgen-responsive genes in prostate and screen the molecular targets for further studying human prostate cancer. Methods:The potential androgen-responsive gene pituitary tumor transforming gene 1 (PTTG1) was selected which had been previously screened by cDNA microarray in rat prostate and its mRNA level was detected by Northern blot in the castrated rat prostate with and without replacement of Mibolerone. Immunohistochemistry was performed to determine the expression and location of PTTG1 in human prostate tissues. Then human androgen-dependent prostate cancer cells LNCaP were used as a model to study the regulation of PTTG1 by Mibolerone. Results: PTTG1 mRNA was hardly detectable in the prostate of 7-day castrated rats, while it was up-regulated dramatically in the prostate of 7-day castrated rats treated with Mibolerone for 2 days. It was showed that high expression of PTTG1 was localized to the epithelial cells of human prostate cancer but not to the stromal cells with Immunohistochemistry. Northern blot analysis indicated that LNCaP cells treated with 0.1 nmol/L Mibolerone for 2 days led to the high PTTG1 mRNA expression. The basic expression of PTTG1 in human androgen-independent prostate cancer cell lines PC3 or DU145 was even higher than that in the human androgen-dependent prostate cancer cells LNCaP treated with Mibolerone. Conclusion: Androgen can up-regulate the PTTG1 expression in castrated rat prostate and human prostate cancer cell LNCaP. It suggests that PTTG1 is potential to play an important role in human prostate cancer progression.
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Objective To detect the expression of pituitary tumor transforming gene (PTTG) in estrogen-induced rat prolactinoma and various types of human pituitary adenomas to explore the role of PTTG in pituitary tumor oncogenesis. Methods Rat model of estrogen-induced prolactinoma was set up, and PTTG mRNA level in rat prolactinoma was measured by RT-PCR. The expression levels of PTTG and bFGF mRNA in 32 cases of human pituitary adenomas were measured by RT-PCR as well. Results The expression level of PTTG mRNA in rat prolactinoma was obviously higher than that in control pituitary tissue(P