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Objective To investigate the effects of cyclopamine (CYP) on endometrial carcinoma (HEC-1A) cell survival and on induction of cell apoptosis .Methods HEC-1A cells were treated with various doses of CYP (0, 5,10, 20 and 40 μmol/L) for 24 h respectively .Then,the inverted microscope was used to observe cell morphology .Cell proliferation and apoptosis were tested by CCK-8 assay and AO/EB bi-labelling assay.The apoptosis rate of HEC-1A was analyzed using flow cytometric analysis , and the key gene expression of Bax and Bcl-2 was detected by quantitative PCR .Results The HEC-1A cells exhibited dramatic morphological changes after treatment with CYP and in a dose-dependent manner .CYP significantly inhibited HEC-1A cell proliferation using CCK8 assays(P<0.05), and induced cell death by AO/EB bi-labelling assay.Moreover,flow cytometry analysis showed that CYP treatment resulted in HEC-1A cell apoptosis, and that a higher concentration of CYP induced severer cell apoptosis (P<0.05).Meanwhile, CYP treated HEC-1A cells exhibited up-regulated expression of Bax and down-regulated expression of Bcl-2 according to Q-PCR.Conclusion Our findings indicatee that CYP can inhibit HEC-1A cell proliferation and induce cell apoptosis .
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Objective:To study the effects of Hedgehog signal transduction pathway on the cell proliferation, apoptosis and connexin 32 (Cx32)and connexin 43 (Cx43)membranous distribution of breast cancer cells,and to explore its mechanism in the cell proliferation and metastasis of breast cancer.Methods:The breast cancer MCF-7 cells at logarithmic growth period were divided into cyclopamine groups and blank control groups. The MCF-7 in cyclopamine groups were treated with 5,10,20,30 and 40μmol·L-1 for 24,48 and 72 h;MTT assay was applied to detect the inhibitory rate of proliferation of MCF-7 cells. After the MCF-7 cells were treated with 0 (negative control group)and 25μmol·L-1 cyclopamine for 48 h,flow cytometry was employed to determine the apoptotic rate and to analyze membranous distribution of Cx32 and Cx43 in the MCF-7 cells.Results:Compared with blank control group,the inhibitory rates of proliferation in cyclopamine groups were increased (P<0.05), and the inhibitory effect of proliferation was increased with the increasing of cyclopamine doses and prolongation of treatment time.After treated with 25μmol·L-1 cyclopamine,the apoptotic rate of MCF-7 cells was higher than that in blank control group (P<0.05).The positive expression rates of Cx32 and Cx43 48 h after treatment were higher than those in negative control group (P<0.05).Conclusion:Hedgehog signal transduction pathway can inhibit the apoptosis and mediate membranous distribution of Cx32 and Cx43 in breast cancer cells.
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AIM:To investigate the effect of cyclopamine , a Hedgehog ( Hh) signaling pathway inhibitor , on the biological behavior of intrahepatic cholangiocarcinoma cell line RBE .METHODS:The proliferation of RBE cells was detected by cell counting with Typan blue staining and MTT assay , and the apoptosis was analyzed by the flow cytometry . The Transwell invasive cabin assay was used to detect the invasion ability , and Western blot was used to determine the pro-tein expression of Gli 1 and MMP-9 in the RBE cells before and after cyclopamine treatment .RESULTS:Cyclopamine in-hibited the growth of RBE cells in a time-and dose-dependent manner .After cyclopamine treatment for 24 h, 48 h and 72 h, the apoptotic rates were significantly higher than those in control group .In control group , the number of cells invading through the Matrigel of invasion chamber was 154.52 ±13.61, while in experimental group it was 62.00 ±12.17, indica-ting that the invasion ability of the cells declined significantly .Furthermore , Western blot showed that the protein levels of Glil and MMP-9 in the RBE cells were decreased after treatment with cyclopamine for 24 h and 48 h.CONCLUSION:Blockage of the Hh signaling pathway with cyclopamine suppresses the proliferation , promotes the apoptosis and inhibits the invasion ability of RBE cells .
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OBJECTIVE: To investigate the effect of inhibitting sonic hedgehog (Shh) signaling pathway with cyclopamine pretreatment on proliferation of rat cortical neural stem cells (NSCs) after oxygen-glucose deprivation/reoxygenation (OGD/R) injury in vitro. METHODS: The suspended culture was used for the isolation and purification of NSCs in neonatal Sprague-Dawley (SD) rats. The third passage NSCs for adherent culture were deprived oxygen and glucose for 150 min and recovered oxygen and glucose for 24 h. There were three groups, including normal, model and cyclopamine pretreatment groups. NSCs were identified with immunofluorescence. CCK-8 assay was used to examine cell viability. The proliferation of NSCs was measured with BrdU assay and flow cytometry cell cycle. Western blot was used to detect the protein expressions of Ptc-1, Smo and Gli-1. RESULTS: There were high expression of nestin protein in suspended and adherent cultured cells. The cell vitalities in model and cyclopamine groups were decreased significantly compared with the normal group. Especially, there was less cell vitality in cyclopamine group (P<0.05). There was significantly increased for NSCs proliferation and upregulated for Ptc-1,Smo and Gli-1 proteins in the model group. On the contrary, compared with the model group, NSCs proliferation and the expressions of Ptc-1, Smo and Gli-1 proteins in cyclopamine group were significantly decreased (P<0.05). CONCLUSION: Cyclopamine pretreatment can inhibit NSCs proliferation after OGD/R injury. The result suggests that Shh signaling may participate in the regulation of NSCs proliferation after injury.
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Objective To explore the effect of cyclopamine (Cyc) on cerebral ischemia-reperfusion injury in rats. Methods Sixty male Sprague-Dawley (SD) rats were randomly divided into 3 groups (n=20): sham operation group, cerebral ischemia-reperfusion group (Con group) and cerebral ischemia-reperfusion+Cyc group (Cyc group). Cyc (10 mg/kg) or absolute ethyl alcohol was intraperitoneally injected in animals 3 h after cerebral ischemia for 7 d. Neurological deficit was assessed by Longa scale on day 1 and 14 after cerebral ischemia. At 24 h after cerebral ischemia, the cerebral water content, cerebral infaction area, pathological changes and cell apoptosis were evaluated by dry-wet method, 2,3,5-triphenyltetrazolium (TTC) staining, H-E staining and TUNEL method, respectively. Immunochemical method was used to examine the protein expression of NeuN and caspase-3 at 24 h after ischemia and glial fibrillary acidic protein (GFAP) expression on day 14 after ischemia. Results Neurological deficit score, cerebral water content, cerebral infaction area, TUNEL positive cell counting, protein levels of caspase-3 and GFAP in Cyc and Con groups were significantly higher than those in sham operation group (P<0.05), and the above parameters in Cyc group were also significantly higher than those in Con group (P<0.05). NeuN protein expressions in Cyc and Con groups were significantly lower than those in sham operation group (P<0.05), and NeuN protein in Cyc group was also significantly lower than that in Con group (P<0.05). Cellular and interstitial edema, neurocyte deformation, pyknosis and necrocytosis were more severe in Cyc group than in Con group. Conclusion Cyclopamine can aggravate rat cerebral ischemia-reperfusion injury.
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AIM:To investigate the effect of cyclopamine on Hedgehog (HH) signaling, phenotypic transfor-mation and matrix accumulation induced by aristolochic acid (AA) in renal tubular epithelial cell NRK-52E.METHODS:NRK-52E cells were randomly divided into control group (treated with solvent only), AA group (treated with AA at con-centrations of 1, 5, 10 mg/L) and cyclopamine group (treated with AA at concentration of 10 mg/L plus cyclopamine at concentrations of 1, 5, 10μmol/L).After cultured for 24 h, the mRNA expression of Ptch1, Smo,α-SMA, E-cadherin, ZO-1, BMP-7, type I collagen and type III collagen was quantified by real-time PCR.The protein levels of Shh and TGF-β1 were detected by ELISA .Immunofluorescence staining was used to evaluate the expression of Ptch 1, Smo,α-SMA, E-cadherin and type III collagen in the NRK-52E cells.RESULTS: AA increased the expression of TGF-β1, α-SMA and type III collagen, decreased the expression of E-cadherin and ZO-1 protein, and down-regulated the expression of Ptch1, Shh and Smo mRNA in the NRK-52E cells, indicating that AA activated HH signaling , and phenotypic transformation and matrix accumulation occurred in AA-treated NRK-52E cells.Treatment with cyclopamine inhibited HH signaling by decrea-sing Smo expression and increasing Ptch 1 expression.Moreover, cyclopamine also down-regulated the expression of TGF-β1,α-SMA, type I collagen and III collagen , and up-regulated the expression of BMP-7, ZO-1 and E-cadherin.CON-CLUSION:AA induces phenotypic transformation and matrix accumulation in renal tubular epithelial cells , which can be inhibited by cyclopamine treatment .The possible mechanism is that cyclopamine suppresses the activation of HH signaling , resulting in the reduction of epithelial-to-mesenchymal transition and matrix deposition .
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OBJECTIVE: To evaluate the apoptosis effect of HDAC inhibitor trichostatin A (TSA) combination with Hedgehog (Hh) signaling inhibitor cyclopamine (CYA) on the PANC-1 cells line. METHODS: Hochest 33258 staining was used to measure apoptosis of PANC-1 cells treated with TSA and CYA respectively or cooperatively. The 24 h IC50 of PANC-1 cells line responding to TSA and CYA were detected by CCK8. The mRNA expression of the gene relative with apoptosis and Hh signaling were evaluated by SYBR GREEN based FQ-PCR (fluorescent quantization polymerase chain reaction). The proteins associated with apoptosis (Bcl-2 and activated caspase-3, activated caspase-8, activated caspase-9) and Hh signalling pathway (Gli1, Smo and Ptc-1) were measured by Western blotting (WB). Gli1 was also detected by cell immunofluorescence. RESULTS: TSA and CYA could induce apoptosis of PANC-1 cells by Hochest 33258 stain, respectively or combinatively. The 24 h IC50 of PANC-1 cells line treated with chemical were (0.51±0.07) μmol·L-1 (TSA) and (33.6±2.3) μmol·L-1 (CYA), respectively; the combination index (CI) of TSA and CYA on the PANC-1 is 0.835. Combination usage of TSA and CYA caused more significantly decreased expression of Bcl-2 mRNA and increased Bax mRNA as compared with the TSA or CYA treatment. TSA (0.5 μmol·L-1) inhibited the expression of Gli1 mRNA, TSA promoted the inhibition effect of CYA on Hh pathway, down-regulation of Gli1, Smo mRNA and up-regulation of Gli3 mRNA. Western blotting showed that combination usage of TSA and CYA induced more significant activation of activated caspase-3, activated caspase-8, activated caspase-9 and reduction of Bcl-2, Gli1, Smo and Ptc-1 expression than TSA or CYA treatment. Decreased Gli 1 also confirmed by immunofluorescence. CONCLUSION: Combination usage of TSA and CYA could induce PANC-1 cells line apoptosis and synergistically inhibit Hh pathway.
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Objective To investigate the effects of cyclopamine on the proliferation ,apoptosis and the expression of PSAmRNA in prostate cancer LNCaP cells .Methods LNCaP cells were interfered with different concentrations of cyclopamine (1 ,5 ,10 ,15μmol/L) at the different timepoints(24 ,48 ,72 h) .The poliferation inhibition was measured by the MTT assay ;the apoptotic mor-phological changes were observed by the Hoechst33258 staining method ;the apoptosis rate was examined by the flow cytometry ;the effects of PSAmRNA gene expression was detected by the FQ-RT-PCR .Results 5 ,10 ,15μmol/L cyclopamine groups had obvious inhibition effect on the LNCaP cell proliferation ,which had statistically significant difference compared with the control group (P<0 .01) .10 μmol/L group reached IC50 at 48 h ;the apoptosis rates at 24 ,48 ,72 h in the 10 ,15 μmol/L groups were 37 .21% , 57 .38% ,57 .98% and 21 .16% ,71 .31% ,72 .90% respectively ,the difference had statistically significant difference compared with the control group(P<0 .01) .The cellular apoptotic morphological changes were significantly enhanced with the increase of cyclo-pamine concentration and the extension of action time .The expression level PSAmRNA gene exhibited the obvious decreasing trend with the increase of cyclopamine concentration and was significantly decreased compared with the control group (P< 0 .01) .The PSAmRNA gene expression was extremely low in 10 ,15 μmol/L cyclopamine at different time period .Conclusion Cyclopamine significantly inhibits the proliferation of LNCaP cells ,induces apoptosis and obviously down-regulates the PSAmRNA gene expres-sion in LNCaP cells .Certain concentration of cyclopamine may be effective for treating advanced prostate cancer .
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Objective This article aims to study the impact of cyclopamine,a Hedgehog signaling pathway inhibitor,on the proliferation and apoptosis of QBC939 cholangiocarcinoma cells.Methods The proliferation of QBC939 cells was detected with the MTT assay,and the apoptotic rate was analyzed with the flow cytometry assay.RT-PCR and Western blow were used to detect the expressions of tumor-related genes and proteins in QBC939 cells before and after cyclopamine treatment.Results Our results show that cyclopamine inhibited the growth of QBC939 cells in time and dose dependent manners.After a 5,10,or 20 μmol/L cyclopamine treatment for 48 hours,QBC939 cells showed increased apoptotic rates significantly higher than those in the control group (P＜0.01).Furthermore,cyclopamine down regulated the mRNA and protein levels of PTCH1,GLI1,and EGFR in QBC939 cells.Conclusion Therefore,blockage of the Hedgehog signaling pathway with cyclopamine could suppress the proliferation and promote the apoptosis of QBC939 cholangiocarcinoma cells.
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Objective To investigate the regulatory effects of hedgehog pathway on intestinal epithelial barrier function under hypoxia.Methods The IEC-6 cells of rats were divided into 3 groups:the normoxia group (21％ oxygen concentration),the hypoxia group (2％ oxygen concentration) and the hypoxia + cyclopamine group (cells pretreated by 5 mmol/L of cyclopamine,and then exposed in an atmosphere with 2％ oxygen concentration).The mRNA expressions of IHH,PTCH and GLI-1 were detected,and the transepithelial electrical resistance (TER) was determined.The protein expressions of tight junction proteins (ZO-1,Occludin,Claudin-1) and IHH were assayed by using the Western blot.All data were analyzed using the one-way analysis of variance or LSD-t test.Results The relative mRNA expressions of IHH,PTCH and GLI-1 were 0.056 ± 0.009,0.459 ± 0.087,0.142 ± 0.023 in the normoxia group,and 0.303 ± 0.052,0.678 ± 0.073,0.483 ± 0.061 in the hypoxia group,with significant difference between the 2 groups (t =-14.05,-11.85,-6.52,P ＜ 0.05).The relative protein expressions of IHH in the normoxia group and the hypoxia group were 0.39 ±0.06 and 0.91 ±0.15,with a significant difference between the 2 groups (t =-8.08,P ＜ 0.05).The TERs of the normoxia group,the hypoxia group and the hypoxia + cyclopamine group were (134 ± 5) Ohm/cm3,(100 ± 6) Ohm/cm3 and (118 ± 5) Ohm/cm3,with significant difference between the 3 groups (F =1.04,P ＜ 0.05).Compared with the normoxia group,the TER of the hypoxia group was decreased by 27.7％ (t =7.84,P ＜ 0.05) ; compared with the hypoxia group,the TER of the hypoxia + cyclopamine group were increased by 16.4％,but it was still significantly lower than the normoxia group (t =4.23,P ＜ 0.05).The expressions of ZO-1,Occludin and Claudin-1 were 1.18 ± 0.24,0.80 ±0.13 and 0.90 ±0.09 in the normoxia group,and 0.58 ±0.08,0.32 ±0.05 and 0.50 ±0.09 in the hypoxia group,and 0.92 ± 0.21,0.43 ± 0.10 and 0.82 ± 0.11 in the hypoxia + cyclopamine group,with significant difference between the 3 groups (F =4.95,2.88,10.09,P ＜0.05).The expressions of ZO-1,Occludin and Claudin-1 in hypoxia group were decreased by 48.7％,40.0％ and 55.6％ when compared with the normoxia group (t =12.86,9.35,18.90,P ＜0.05).The expressions of ZO-1,Occludin and Claudin-1 in the hypoxia + cyclopamine group were increased by 59.9％,35.2％ and 65.1％ when compared with the hypoxia group (t =5.63,2.92,6.66,P ＜ 0.05).Conclusion Hedgehog signal pathway could be activated under hypoxia,and then the expressions of tight junction proteins are decreased,which finally induces the injury of intestinal epithelial barrier function.
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PURPOSE: The Hedgehog (Hh) signaling pathway is important in embryonic development including cell differentiation and proliferation. Recently, activation of this pathway has been implicated in several forms of solid cancers. We investigated sonic hedgehog (Shh) protein expression and its relation to differentiation and clinicopathologic characteristics in thyroid cancer cell lines and tissues. METHODS: FTC-236, FTC-238, and XTC-1. We made tissue microarray slides using 80 thyroid surgical specimen: 40 benign and 40 malignant lesions. Immunohistochemical staining was performed using anti-Shh antibody. mRNA expression of NIS, thyroglobulin, and CD97 were evaluated by RT-PCR. Cyclopamine was used as a Shh signal inhibitor. RESULTS: Shh expression was more prominent in TPC-1, FTC-133, and XTC-1 cell lines than the others. Cyclopamine downregulated CD97 and upregulated thyroglobulin mRNA expression, but did not induce mRNA expression of NIS. Thyroid tissues showed varied expression of Shh in both benign and malignant diseases. Shh expression was detected in 38 of 50 (76%) normal, in 18 of 25 (72%) non-neoplastic benign, in nine of 15 (60%) benign tumors, and in 31 of 40 (77%) malignant tumors. Shh over-expression was significantly less frequent in papillary thyroid carcinomas than in normal or benign thyroid tissues. In addition, Shh protein expression did not relate to clinicopathologic characteristics in papillary thyroid carcinomas. CONCLUSION: Thyroid tissues and cell lines vary in expression of Shh. Cyclopamine can induce redifferentiation in thyroid cancer cell lines. Shh protein expression, however, is unrelated to clinicopathologic characteristics in papillary thyroid carcinomas.
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Female , Pregnancy , Cell Differentiation , Cell Line , Embryonic Development , Hedgehog Proteins , Hedgehogs , RNA, Messenger , Thyroglobulin , Thyroid Gland , Thyroid NeoplasmsABSTRACT
Objective To explore the expression and significance of hedgehog signal molecules (Ptch, Smo and Gli1 ) in pancreatic cancer. Methods Two hundred SD rats were randomly divided into DMBA group ( group A, n = 90), cyclopamine intervening group ( group B, n = 90) and control group ( group C, n = 20).For group A and B, DMBA was directly implanted into the parenchyma of the pancreas to establish the model of pancreatic cancer. The rats in group B were treated with 6.25 ml/kg cyclopamine and DMSO solution intraperitoneally daily. All rats were sacrificed four months later to observe the pancreatic tissue pathologic changes, and immunohistochemistry SP was used to detect the expression of Ptch, Smo, Gli1 protein in pancreatic cancer and normal pancreatic tissue. Results The prevalence rate of pancreatic cancer in group A was 57.5％ (46/80), the maximum size of the tumor was 0.5 ～ ＞2 cm; the prevalence rate of pancreatic cancer in group B was 17.1％ ( 14/82), the maximum size of the tumor was 0.5 ～ 2.0 cm, and the difference between the two group was statistically significant (P ＜0.05). The positive expression rate of Ptch, Smo and Gli1 protein was 82.6％, 73.9％ and 65.2％ in DMBA group, and was 50.0％, 42.9％ and 28.6％ in cyclopamine group, and the difference between the two group was statistically significant ( P ＜ 0.05 ). Ptch,Smo and Gli1 protein was expressed in normal pancreatic tissue. Conclusions Direct implantation of DMBA in the parenchyma of rat pancreas can induce pancreatic cancer with a high incidence in a short time.Hedgehog signal protein expression is significantly increased, cyclopamine can inhibit the occurrence and progression of pancreatic cancer by inhibiting Hedgehog messenger expression.
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The effects of Sonic hedgehog (Shh) signaling pathway activation on S-type neuroblastoma (NB) cell lines and its role in NB tumorigenesis were investigated. Immunohistochemistry was used to detect the expression of Shh pathway components-- Patchedl (PTCH1) and Glil in 40 human primary NB samples. Western blotting and RT-PCR were used to examine the protein expression and mRNA levels of PTCH1 and Glil in three kinds of S-type NB cell lines (SK-N-AS, SK-N-SH and SHEPI), re-spectively. Exogenous Shh was administrated to activate Shh signaling pathway while cyclopamine was used as a selective antagonist of Shh pathway. S-type NB cell lines were treated with different concen-trations of Shh or/and cyclopamine for different durations. Cell viability was measured by using MTT method. Apoptosis rate and cell cycle were assayed by flow cytometry. The xenograft experiments were used to evaluate the role of Shh pathway in tumor growth in immunodeficient mice. High-level expres-sion of PTCH1 and Gill was detected in both NB samples and S-type NB cell lines. Cyclopamine de-creased the survival rate of the three cell lines while Shh increased it, and the inhibition effects of cyclopaminc could be partially reversed by shh pre-treatment. Cyclopamine induced the cell apoptosis and the cell cycle arrest in G0/G1 phase, while Shh induced the reverse effects and could partially pre-vent effects of cyclopamine. Cyclopamine could also inhibit the growth of NB in vivo. Our studies re-vealed that activation of the Shh pathway is important for survival and proliferation of S-type NB cells in vivo and in vitro through affecting cell apoptosis and cell cycle, suggesting a new therapeutic ap-proach to NB.
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Objective To investigate the in vitro effects of KAAD-cyclopamine, a specific inhibitor of hedgehog signaling pathway, on the growth and apoptosis of human squamous cell carcinoma cell line A431. Methods A431 cells were cultured and treated with KAAD-cyclopamine(0.5, 1, 2, 5 μmol/L).Then, MTT assay was used to detect the proliferation of A431 cells, and light microscopy to observe cell morphology at different time points with a 24-hour interval. Flow cytometry was used to assess cell cycle,and annexin-V/propidium iodide double staining to evaluate the apoptosis in these cells after 48 hours of treatment with KAAD-cyclopamine. Results KAAD-cyclopamine of 0.5, 1, 2 and 5 μmol/L inhibited the proliferation of A431 cells by (7.0±2.3)%, (20.6±2.8)%, (48.3±3.4)% and (61.6±3.3)%, respectively (F = 49.92, P＜0.01 ). Furthermore, in the presence of KAAD-cyclopamine of 5 μmol/L, on day 1, 2, 3, 4,and 5 the proliferation of A431 cells was suppressed by (18.5±2.6)%, (56.1±3.7)%, (65.4±2.8)%,(71.2±1.9)% and (75.9±3.0)%, respectively, the difference was significant among these time points(F =16.32, P＜0.01 ). Statistical analysis showed that KAAD-cyclopamine downregulated the growth of A431 cells in a dose-and time-dependent manner (r = 0.91, 0.86, P＜0.01 and 0.05, respectively). Light microscopy revealed typical morphological changes of cell damage in A431 cells. KAAD-cyclopamine in creased the percentage of cells in G1 phase from (51.8±2.9)% to(76.2±1.8)% (F = 26.34, P＜0.01 ), the proportion of hypoploid cells from (1.7±0.3 )% to (8.7±0.2)% (F = 6.32, P＜0.05 ), which suggested that KAAD-cyclopamine could arrest A431 cells in G1 phase of the cell cycle. The apoptosis ratio in KAAD-cyclopamine-treated cells was significantly higher than that in the untreated control [ (46.2±2.8)% vs (18.5±3.1 )%, F = 32.01, P＜0.01 ]. Tomatidine treatment did not affect the proliferation or apoptosis of A431 cells (both P＞0.05).Conclusion KAAD-cyclopamine can markedly suppress the proliferation and induce apoptosis of A431 cells.
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Objective To investigate the expressions of Hedgehog sisnaling pathway genes in hepatocellular carcinoma tissues(HCC),and the effect of specific Hedgehog pathway inhibitor(KAADcyclopamine)on the growth of HCC cells and the expressions of Hedgehog genes. Methods The expression of Hedgehog signaling pathway components(Ihh,Ptch,Smo and Gli)was investigated in 14 HCC tissue slices,4 HCC cell lines and a normal hepatic cell line by using immunochemistry.The expression of Ihh,Ptch,Smo and Gli proteins was investigated in 9 HCC tissue specimens and 6 normal hepatic tissue specimens by using Western blotting.The expression of Ihh、Ptch、Smo、Gli and Hip genes was investigated by RT-PCR.Results The positive ratio of Gli,Ptch,Ihh and Smo were 42.9%,71.4%,71.4% and 85.7% in 14 HCC tissue slices,respectively.The expressions of Gli protein and Gii gene were up regulated while the expression of Hip gene was down regulated in HCC specimens compared with normal hepatic tissue specimens.Hedgehog signaling pathways in HCC cell lines HepG2,Bel-7402 and QGY-7701 were activated;KAAD-cyclopamine,a specific inhibitor of the Hedgehog signaling pathway,down regulated cell growth and the expressions of Ptch and Gli genes in the 3 HCC cell lines(Ptch gene:tHepG2=3.78,tBel-7402=9.03,tQGY-7701=5.63;Gli gene:tHepG2=9.61,tBel-7402=4.15,tQGY-7701=20.30,P＜0.05 in each group).The expression of Hip gene was up regulated in QGY-7701 after treated with KAAD-cyclopamine(t=4.70,P＜0.05).Conclusion The expression of main Hedgehog signaling pathway components were detected in HCC,KAAD-cyclopamine specifically inhibited the Hedgehog signaling pathway.
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PURPOSE: The Hedgehog (HH) signaling pathway is important in development. Recently,ectopic activation of this pathway has been implicated in several forms of solid cancer including basal cell carcinoma, pancreatic cancer, colon cancer, and prostate cancer. There are three HH proteins involved in the pathway: Sonic HH, Indiana HH, and Desert HH. Cyclopamine disrupts Sonic HH signaling by inhibition of the seven-transmembrane receptor Smoothened (SMO). Whereas cyclopamine is cytotoxic to several human cancer cells, its effect on thyroid cancer cellsis unknown. We therefore investigated the effect of cyclopamine on cell proliferation in human thyroid cancer cell lines. METHODS: We used fivethyroid cancer cell lines: TPC-1 (papillary), FTC-133, FTC-236, FTC-238 (follicular), and XTC-1 (Hurthle cell). The MTT assay and cell cycle analysis were used to evaluate anti-proliferative effects. Tomatidine, a structural analogue of cyclopamine, was used as a control agent. Statistical significance was tested by ANOVA. RESULTS: After 4 days of treatment, the percent inhibition of growth with a concentration of 5, 10, and 20 M cyclopamine in the cell lines were 23.6±4.9%, 66.4±4.7% and 69.3±1.3% in TPC-1 7.5±2.8%, 10.7±3.2% and 49.6±6.4% in FTC-133, 19.2±9.5%, 50.4±4.8% and 60.4±2.0% in FTC- 236 22.8±4.2%, 53.4±5.5% and 63.7±4.8% in FTC- 238 7.6±5.8%, 16.6±2.2%, 24.0±4.3% in XTC-1. Treatment with tomatidine at the same concentrations did not significantly affect cell growth. Exposure to cyclopamine, however, did not affect the cell cycle significantly CONCLUSION: Cyclopamine inhibits cancer cell proliferation in a dose dependent manner in thyroid cancer cell lines. The Hh signaling pathway might be a useful therapeutic target for thyroid cancer.
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Humans , Carcinoma, Basal Cell , Cell Cycle , Cell Line , Cell Proliferation , Colonic Neoplasms , Hedgehogs , Indiana , Pancreatic Neoplasms , Prostatic Neoplasms , Thyroid Gland , Thyroid NeoplasmsABSTRACT
Objective:To investigate the effect of cyclopamine on growth and proliferation of human mammary carcinoma cell line Bcap-37.Methods:The Bcap-37 cells were incubated with cyclopamine,which is the specific inhibitor of Hedgehog signaling pathway.Then the inverted microscope was used to observe the morphologic changes of the cells,and MTT assay,BrdU incorporation and cell cycle analysis were used to examine the influence of cyclopamine on Bcap-37 cell growth and DNA synthesis.Results:Compared with the control group,after the cells were incubated with cyclopamine,obvious morphologic changes of Bcap-37 cells were observed;the growth of Bcap-37 cell was inhibited in a dose and time dependent manner;the DNA synthesis of Bcap-37 cells was obviously inhibited;the number of Bcap-37 cells in S phase decreased and the cells were blocked in G2 phase.Conclusion:Cyclopamine can effectually inhibit the growth and proliferation of Bcap-37 cells,which indicates that Hedgehog signaling pathway may be inappropriately activated in breast cancer and inhibiting Hedgehog signaling pathway can be a useful method in breast cancer treatment.
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Objective To investigate the effect and mechanism of cyclopamine,the inhibitor of Sonic hedgehog signaling pathway,on the proliferation and apoptosis of pancreatic cancer cell line SUIT-2.Methods The SUIT-2 cells used in the experiment were cultured in vitro,and the MTT assay was used to examine the(antiproliferative) effect of cyclopamine.Flow cytometry(FCM) was used to examine the effects of cyclopamine on the proliferation index(PI) and apoptosis index(AI) of SUIT-2 cells.Nude mice tumor graft model was used to determine the effect of cyclopamine on growth inhibition of tumor graft.Results Cyclopamine(produced) time and concentration dependent antiproliferative effects on SUIT-2 cells.Cyclopamine could induce apoptosis and block cell cycle at G_0/G_1 phase.In cyclopamin treated SUIT-2 cells,the apoptosis index(AI) was 14.3?0.35,and proliferation index(PI) was 36.1?0.44;and in control group,the AI was 1.3?0.24,the PI was 52.3?0.28(all P
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Objective:To research the effects of HH singaling pathway on proliferation,apoptosis and CyclinD1 expression of breast cancer MDA-MB-231 cells by MDA-MB-231 cells treated with Cyclopamine,a HH singaling pathway inhibitor.Methods:MDA-MB-231 cells were treated with Cyclopamine.Cell proliferation was detected by MTT;Cell cycle distribution and apoptosis were analysed by flow cytometry(FCM).The mRNA levels of GLI1,CyclinD1 were measured by reverse transcription-polymerase chain reaction(RT-PCR);The protein level of CyclinD1 was detected by immunostaining of cell lines.Results:Compared with control group,Cyclopamine could significantly inhibit the proliferation of MDA-MB-231 cells in a dose-and time-dependent manner;By FCM the treatment of MDA-MB-231 cells with Cyclopamine induced a remarkable increase in the proportion of cells in the G0/G1 phase of the cell cycle and after 48 h the sub-G1 peak appeared.The mRNA levels of GLI1,CyclinD1 and the expression of CyclinD1 protein were reduced.Conclusion:The HH signaling pathway was activeted in MDA-MB-231 cells.HH signaling pathway inhibitor,Cyclopamine,could inhibit the proliferation,induce the apoptosis and reduce the expression of CyclinD1 in MDA-MB-231 cells.