Immunohistochemical analysis of SHH, SMO and GLI-1 proteins in epithelial odontogenic lesions

Abstract The present study analyzed the expression of proteins involved in the sonic hedgehog signaling pathway (SHH, SMO, and GLI-1) in benign epithelial odontogenic lesions (odontogenic keratocyst - OKC, ameloblastoma - AB, and adenomatoid odontogenic tumor - AOT) in order to identify the role of these proteins in the pathogenesis of these lesions. The sample consisted of 20 OKCs, 20 ABs, and 10 AOTs. The Kruskal-Wallis, Mann-Whitney U, and Spearman's (r) tests were used for statistical analysis, with the level of significance set at 5% (p < 0.05). The membrane/cytoplasmic expression of SHH was significantly higher in AB compared to AOT (p = 0.022) and OKC (p = 0.02). No differences were found in the membrane/cytoplasmic expression of SMO between the lesions studied. Regarding GLI-1, significant differences were observed at the nuclear level for AB and OKC compared to AOT (p < 0.0001). In addition, significant positive correlations were found between cytoplasmic and nuclear GLI-1 in AB (r = 0.482; p = 0.031) and OKC (r = 0.865; p < 0.0001), and between membrane/cytoplasmic SMO and cytoplasmic GLI-1 in AOT (r = 0.667; p = 0.035) and OKC (r = 0.535; p = 0.015). The results of this study confirm the participation of the sonic hedgehog signaling pathway in the pathogenesis of the lesions studied. Overexpression of SHH in ABs and nuclear expression of GLI-1 in ABs and OKCs indicate that these proteins contribute to the more aggressive behavior of these two lesions when compared to AOT.

Resumo O presente estudo analisou a expressão de proteínas envolvidas na via de sinalização Sonic Hedgehog (SHH, SMO e GLI-1) em lesões benignas do epitélio odontogênico de comportamento biológico distintos, tais como ceratocistos odontogênicos (CO), ameloblastomas (AMB) e tumores odontogênicos adenomatoides (TOA), com o intuito de identificar o papel destas proteínas na patogênese destas lesões. A amostra foi constituída de 20 CO, 20 AMB e 10 TOA, analisada pela técnica da imuno-histoquímica de forma semiquantitativa por compartimento celular, onde foi feita uma análise da membrana e citoplasma das células nas proteínas SHH e SMO, enquanto que para a proteína GLI-1, foi feita uma análise nuclear e/ou citoplasmática. Para análise estatística, foram utilizados os testes de Kruskal-Wallis (KW), Mann-Whitney (U) e Spearman (r), com o nível de significância estabelecido em 5% (p < 0,05). Ao analisar a proteína SHH, observou-se que o AMB demonstrou expressão membranar/citoplasmática significativamente maior em comparação ao TOA (p = 0,022) e CO (p = 0,020). Com relação à análise membranar/citoplasmática da SMO, não foram identificadas diferenças entre as lesões estudadas. Para a proteína GLI-1, foram constatadas diferenças estatisticamente significativas, em nível nuclear, para o AMB e CO em comparação ao TOA (p< 0,0001). Além disso, foram observadas correlações positivas com significância estatística entre GLI-1 citoplasmático e GLI-1 nuclear para o AMB (r = 0,482; p = 0,031) e CO (r = 0,865; p< 0,0001), e entre o SMO membranar/citoplasmático e o GLI-1 citoplasmático para o TOA (r = 0,667; p = 0,035) e o CO (r = 0,535; p = 0,015). Os resultados deste estudo confirmam a participação da via de sinalização Sonic Hedgehog na patogênese das lesões estudadas e, a superexpressão de SHH em AMBs e GLI-1 nuclear em AMBs e COs, indica que estas proteínas contribuem com o comportamento biológico mais agressivo destas duas lesões quando comparado ao TOA.

Expression and significance of GLI1 and Shh in the malignant transformation of ovarian endometriosis / 中华妇产科杂志

Objective:To investigate the expression levels and clinical significance of glioma-associated oncogene homolog 1 (GLI1) and sonic hedgehog signaling molecule (Shh) in the malignant transformation of ovarian endometriosis (EM).Methods:The expressions of GLI1 and Shh were detected by real-time reverse transcription (RT)-polymerase chain reaction (PCR) and EnVision method in 50 cases of ovarian EM tissues, 35 cases of atypical endometriosis (aEM) and 50 cases of endometriosis-associated ovarian cancer (EAOC). The expression differences of two molecular markers in the malignant transformation of ovarian EM were compared, and the relationships between two molecular markers and the clinicopathological features and prognosis of EAOC were analyzed.Results:(1) RT-PCR showed that the expression levels of GLI1 mRNA in EM, aEM and EAOC group were 1.77±0.40, 3.54±0.44, and 7.80±0.24, respectively. The expression levels of Shh mRNA were 0.95±0.21, 3.14±0.35, and 5.41±0.31, respectively. GLI1 and Shh mRNA in EAOC group were significantly higher than those in EM and aEM group (all P<0.01), and there were statistically significant differences between EM and aEM group (all P<0.01). The percentages of GLI1 in ovarian EM, aEM and EAOC were 32% (16/50), 57% (20/35), and 66% (33/50), respectively, meanwhile, the positive expression rates of Shh were 20% (10/50), 49% (17/35), and 54% (27/50), respectively (all P<0.01). GLI1 mRNA expression was positively correlated with Shh mRNA expression in EAOC tissues ( r=0.721, P<0.01). The expressions of GLI1 protein were proportionated to Shh protein in EAOC tissues ( r=0.608, P=0.001). (2) The expression of GLI1 was significantly related to the International Federation of Gynecology and Obstetrics (FIGO) stage, cancer antigen 125 (CA 125) levels, lymph node metastasis, and Platinum resistance in EAOC patients (all P<0.05). The expression of Shh were related to FIGO stage and lymph node metastasis in EAOC patients (all P<0.05). Logistic regression analysis showed that GLI1 expression was an independent risk factor for poor prognosis in EAOC patients ( P<0.05). Kaplan-meier survival analysis showed that the overall survival rate of EAOC patients with high GLI1 expression and low GLI1 expression was 12.1% and 35.3%, respectively, with statistical significance ( χ2=10.73, P<0.01). The overall survival rate of EAOC patients with high and low expression of Shh protein was 11.1% and 30.4%, in which there was statistically significant difference ( χ2=3.96, P=0.047). Conclusion:GLI1 and Shh are highly associated with the malignant transformation of ovarian EM, which may play a role in promoting malignant degeneration of ovarian EM, and the high expression of GLI1 and Shh indicates a poor prognosis in EAOC patients.

Immunohistochemical Evaluation of the Hedgehog Route as a Potential Prognostic Factor in Hepatocellular Carcinoma / Avaliação imuno-histoquímica da rota Hedgehog como potencial fator prognóstico no carcinoma hepatocelular

ABSTRACT Introduction Hepatocellular carcinoma (HCC) is the most common primary malignant neoplasm in the liver. HCC develops gradually from multiple stages that control proliferation and apoptosis. In hepatocarcinogenesis, multiple signaling pathways were already described, such as the Hedgehog pathway (Hh). However, few studies have investigated the expression of Hh proteins as a potential prognostic factor in human HCC. This study aimed to investigate the expression of the Shh protein in HCC and to correlate with clinical and morphological prognostic characteristics of the tumor. Methods Immunohistochemical expression of Shh protein in tumor and cirrhotic parenchyma was performed in 36 HCC samples from patients who underwent liver transplantation at Clinical Hospital - UFMG. Correlation between the Shh tumor expression and etiology, number of nodules, size of the nodules, levels of alpha-fetus-protein (AFP), MELD score, tumor differentiation, and vascular invasion were performed. Results In our study, Shh protein labeling gradually increased from the normal to the cirrhotic and neoplastic parenchyma. Degree of tumor differentiation and vascular invasion were correlated with high Shh protein expression (p = 0.014 and p = 0.003, respectively). The other variables did not show a statistically significant correlation with Shh labeling. Conclusion Hedgehog pathway has importance in hepatocarcinogenesis. The immunohistochemical study of the Hh signaling pathway may have a promising role as a prognostic factor for HCC, especially due to the positive correlation between the Shh expression and the degree of tumor differentiation and invasion vascular.

RESUMO Introdução O carcinoma hepatocelular (CHC) é a neoplasia maligna primária mais comum no fígado. O CHC se desenvolve gradualmente a partir de múltiplos estágios que controlam a proliferação e a apoptose. Na hepatocarcinogênese, múltiplas vias de sinalização já foram descritas, como a via Hedgehog (Hh). No entanto, poucos estudos investigaram a expressão de proteínas Hh como um potencial fator prognóstico no CHC humano. Este estudo teve como objetivo investigar a expressão da proteína Shh no CHC e correlacionar com características prognósticas clínicas e morfológicas do tumor. Métodos A expressão imuno-histoquímica da proteína Shh em tumor e parênquima cirrótico foi realizada em 36 amostras de CHC de pacientes submetidos a transplante hepático no Hospital das Clínicas - UFMG. Correlação entre a expressão e etiologia do tumor Shh, número de nódulos, tamanho dos nódulos, níveis de proteína alfa-feto (AFP), pontuação MELD, diferenciação tumoral e invasão vascular foram realizadas. Resultados Em nosso estudo, a marcação da proteína Shh aumentou gradualmente do parênquima normal para o cirrótico e neoplásico. Grau de diferenciação tumoral e invasão vascular foram correlacionados com alta expressão da proteína Shh (p = 0,014 ep = 0,003, respectivamente). As demais variáveis não apresentaram correlação estatisticamente significativa com a marcação de Shh. Conclusão A via Hedgehog tem importância na hepatocarcinogênese. O estudo imuno-histoquímico da via de sinalização Hh pode ter um papel promissor como fator prognóstico para CHC, principalmente devido à correlação positiva entre a expressão de Shh e o grau de diferenciação tumoral e invasão vascular.

Vismodegib for treatment of periocular basal cell carcinoma - 6-year experience from a tertiary cancer center

Abstract Background: The treatment of advanced periocular basal cell carcinomas becomes a challenge as surgery may involve highly mutilating procedures. Vismodegib is the first selective hedgehog inhibitor approved for the treatment of locally advanced tumors or metastatic disease. Objective: Analyze the results of treatment with vismodegib for advanced periocular basal cell carcinomas in a real-life setting of a reference center between 2014 and 2020. Methods: Retrospective longitudinal study. The patient's demographic profile, comorbidities, tumor characteristics, and treatment outcomes were analyzed. Results: A total of 13 patients were included. Median follow-up and treatment duration were 15.9 and 10.5 months, respectively. Objective clinical response rate was 76.9%: 30.8% had a complete response and 46.2% a partial response. The median duration of response was 13 months. Progressive disease was observed in 38.5% of cases, with a median of 19 months after the beginning of treatment. Eighty-four percent of the patients had at least one adverse event, and 61.54% needed to interrupt treatment temporarily or permanently to increase tolerability. Study limitations: Being a retrospective study in a real-life setting, the evaluation of objective clinical response was subjective to physician appreciation. Conclusion: Vismodegib is a safe and effective treatment for locally advanced basal cell carcinoma. To prevent recurrences, the drug should be used continually when tolerated. The role of neoadjuvant vismodegib before surgery is being investigated and might add an important step in searching for a definitive treatment for these cases.

Experiencia en el tratamiento del carcinoma basocelular localmente avanzado o metastásico con Vismodegib en el instituto nacional de cancerología entre los años 2014 y 2015 / Treatment of locally advanced or metastatic basal cell carcinoma with Vismodegib: Experience at the national Institute of Cancerology during 2014 -2015

RESUMEN Introducción: El carcinoma basocelular es el cáncer de piel más frecuente, y en su patogenía se ha descrito la activación de la vía Hedgehog. El Vismodegib, un inhibidor selectivo de esta vía ha demostrado ser efectivo en el tratamiento de la enfermedad localmente avanzada irresecable o metastásica. El presente estudio describe la respuesta clínica al tratamiento con Vismodegib en una población de Colombia. Materiales y métodos: Presentamos una serie de casos entre enero de 2014-noviembre de 2015 del Instituto Nacional de Cancerología (Bogotá Colombia). El tratamiento establecido fue Vismodegib (cápsulas de 150 mg vía oral diario), en ciclos cada 28 días hasta progresión o toxicidad limitante. El análisis estadístico se basó en el cálculo de proporciones en variables cualitativas, medidas de tendencia central y dispersión para las cuantitativas. Se realizó análisis de supervivencia descriptiva (Kaplan-Meier). El análisis estadístico se realizó por el software estadístico STATA 11.0. Resultados: 33 pacientes fueron incluidos, de los cuales el 73% recibieron al menos 3 ciclos de tratamiento. De estos el 63,6% tuvieron respuesta parcial y el 12,1% presentaron respuesta completa. La media de supervivencia global y libre de progresión fue de 21,7 meses (IC 95% 18,9 a 24,4 meses) y 22,3 meses (IC 95% 20,6 a 23,9 meses), respectivamente. Los eventos adversos más frecuentes fueron: espasmos musculares (35,2%), disgeusia (24,7%) y alopecia (15%). Conclusiones: En esta serie de casos, a pesar de las irregularidades en el inicio y continuidad del manejo con Vismodegib, se obtuvieron tasas de respuesta similares a las previamente descritas.

Abstract Introduction: Basal cell carcinoma is the most frequent skin cancer, and the activation of the Hedgehog pathway has been described in its pathogenesis. Vismodegib is a selective inhibitor of this pathway, that has shown to be effective in the treatment of locally unresectable or metastatic advanced disease. The present study describes the clinical response to treatment with vismodegib in a Colombian population. Methods: We present a case series, carried out between January 2014 and November 2015 at the National Institute of Cancerology (Bogotá, Colombia). The established treatment was Vismodegib (capsules of 150 mg orally daily), in cycles every 28 days until progression or limiting toxicity. The statistical analysis was based on the calculation of proportions in qualitative variables, measures of central tendency and dispersion for quantitative ones. Descriptive survival analysis (Kaplan-Meier) was performed. The statistical analysis was performed by the statistical software STATA 11.0. Results: 33 patients were included, of which 73% received at least 3 treatment cycles. Of these, 63.6% had a partial response and 12.1% had a complete response. The mean of progression-free and overall survival was 21.7 months (95% CI 18.9 to 24.4 months) and 22.3 months (95% CI 20.6 to 23.9 months), respectively. The most frequent adverse events were: muscle spasms (35.2%), dysgeusia (24.7%) and alopecia (15%). Conclusion: In this series of cases, despite the irregularities in the initiation and continuity of management with Vismodegib, response rates were similar to those described in the literature.

Sonic Hedgehog Pathway as the Prognostic Marker in Patients with Extensive Stage Small Cell Lung Cancer

PURPOSE: Sonic hedgehog (Shh) signaling pathway is known to play a crucial role in carcinogenesis in various malignancies, including lung cancer regarding tumorigenesis, angiogenesis, and cellular differentiation. The aim of this study was to investigate the value of components of Shh pathway as a prognostic marker in extensive stage small cell lung cancer (ES-SCLC) patients. MATERIALS AND METHODS: We retrospectively analyzed data of 36 patients who were diagnosed with ES-SCLC between 2008 and 2012 at a single center. We performed immuo-histochemistry for glioma-associated oncogene homolog zinc finger protein 1 (Gli1), patched, Shh, and Ptch-mediated repression of smoothened (Smo) proteins using formalin-fixed, paraffin-embedded tissue derived from primary tumors. We then conducted survival analysis to evaluate the prognostic impact of these markers. RESULTS: All 36 patients received platinum-based doublet chemotherapy. The median progression free survival and median overall survival were 6.9 months [95% confidence interval (CI), 6.5–7.3] and 11.7 months (95% CI, 9.1–14.3), respectively. The overall response rate was 84%. Of the 36 tissue specimens examined, over-expression of Gli1, Patched, Shh, and Smo was found in 12 (33.3%), five (13.9%), five (13.9%), and six (16.7%) cases, respectively. We found that high expression of Shh was associated with worse progression free survival (6.3 vs. 7.6 months, p=0.005) and overall survival (9.2 vs. 12.0 months, p=0.039) by both univariate and multivariate analyses, whereas other markers were not related to patient prognosis. CONCLUSION: A high proportion of small cell lung cancer tumors express proteins related to Shh pathway, and over-expression of Shh is correlated with poor prognosis.

Effects of isoflurane postconditioning on angiogenesis during cerebral ischemia-reperfusion in rats and the role of Shh signaling pathway / 中华麻醉学杂志

Objective To evaluate the effects of isoflurane postconditioning on angiogenesis during cerebral ischemia-reperfusion ( I∕R) in rats and the role of Shh signaling pathway. Methods Thirty-two clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 220-280 g, were divided into 4 groups ( n=8 each) by a random number table method:sham operation group ( Sham group) , I∕R group, isoflurane postconditioning group ( ISO group) , and isoflurane postconditioning plus Shh signaling pathway specific inhibitor cyclopamine group ( ISO+CYC group) . Cerebral ischemia was produced by inserting a 3-0 nylon thread with a rounded tip into the internal jugular vein. The nylon thread was threaded cranially until resistance was met. Occlusion was maintained for 1. 5 h followed by 24 h reperfusion. Neurological deficit was scored at 24 h of reperfusion. Rats were then sacrificed, and brains were removed for determination of cerebral infarct volume ( by TTC) and expression of glioma-associated oncogene homolog 1 ( Gli1) , vascu-lar endothelial growth factor ( VEGF) and transmembrane phosphoglycoprotein protein ( CD34) in cerebral cortex (by Western blot) and for examination of the pathological changes (by Nissl staining). Results Compared with Sham group, the neurological deficit score and cerebral infarct volume were significantly in-creased, and the expression of Gli1, VEGF and CD34 in the cerebral cortex was up-regulated in I∕R and ISO groups ( P <0. 05) . Compared with I∕R group, the neurological deficit score and cerebral infarct vol-ume were significantly decreased, and the expression of Gli1, VEGF and CD34 in the cerebral cortex was up-regulated ( P<0. 05) , and the pathological changes of brain tissues were significantly attenuated in ISO group, and no significant change was found in the parameters mentioned above in ISO + CYC group ( P>0. 05) . Compared with ISO group, the neurological deficit score and cerebral infarct volume were signifi-cantly increased, and the expression of Gli1, VEGF and CD34 in the cerebral cortex was down-regulated in ISO+CYC group ( P<0. 05) . Conclusion The mechanism by which isoflurane post-conditioning attenuates cerebral I∕R injury is related to activating Shh signaling pathway and promoting angiogenesis in rats.

Relationship between spinal Sonic Hedgehog signaling pathway and inflammatory responses in rats with neuropathic pain / 中华麻醉学杂志

Objective To evaluate the relationship between spinal Sonic hedgehog (Shh) signaling pathway and inflammatory responses in rats with neuropathic pain (NP).Methods Forty-eight cleangrade healthy male Sprague-Dawley rats,aged 8 weeks,weighing 250-300 g,were divided into 4 groups (n=12 each) using a random number table method:sham operation group (group S),group NP,dimethyl sulfoxide (DMSO) group (group D) and specific Shh signaling pathway inhibitor cyclopamine group (group CP).Spared nerve injury was produced by exposing the sciatic nerve and branches followed by ligation and transection of tibial and common fibular nerves in anesthetized rats.The mechanical paw withdrawal threshold (MWT) was measured at 1 day before establishing the model and 1 and 7 days after establishing the model.,The animals were sacrificed after measurement of pain threshold at 7 days after operation,and the lumbar segment L4-6 of the spinal cord was obtained for determination of the expression of Shh,Patched homolog (Ptch),Smoothened (Smo) and zinc finger-containing transcription factors 1 (Gli1) (by Western blot) and contents of tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) (by enzyme-linked immunosorbent assay).Results Compared with group S,the MWT was significantly decreased,the expression of Shh,Patched,Smo and Gli1 was up-regulated,and the contents of TNF-α and IL-1β were increased in NP,D and CP groups (P＜0.05).Compared with group NP,the MWT was significantly increased,the expression of Shh,Patched,Smo and Gli1 was down-regulated,and the contents of TNF-α and IL-1β were decreased in group CP (P＜0.05),and no significant change was found in the parameters mentioned above in group D (P＜0.05).Conclusion The mechanism by which Shh signaling pathway is involved in the development and mainterance of inflammatory responses is related to inhibiting inflammatory responses of the spinal cord in rats with NP.

Hedgehog pathway antagonist-induced oromandibular limb hypogenesis in mouse / 中华口腔医学杂志

Objective@#To analysis teratogenic effect of GDC-0449 to fetus and set up the animal model of GDC-0449 induced oromandibular limb hypogenesis in mouse for further research of its pathogenesis.@*Methods@#Twenty-seven pregnant Institute of Cancer Research (ICR) mice were randomly divided into: control group, embryonic day 8.5 (E8.5) exposed groups, E9.5 exposed groups, E10.5 exposed groups, E11.5 exposed groups, E12.5 exposed groups, E13.5 exposed groups, E14.5 exposed groups and E15.5 exposed groups. Each group had 3 mice. Exposed groups were treated with the Hedgehog pathway antagonist GDC-0449 at a single dose 150 mg/kg by oral gavage from E8.5 to E15.5. At E16.5, embryonic phenotypes were analyzed in detail by stereo microscope and histology. After establish an optimal dysmorphogenic concentration, 6 pregnant ICR mice were randomly divided into control group and the optimal group, embryonic phenotypes were analyzed by whole-mount skeletal staining and micro-computed tomography at E18.5.@*Results@#The mice were exposed to GDC-0449 on E11.5 and E12.5 had a high incidence of cleft palate. GDC-0449 exposed between E9.5 and E10.5 caused craniofacial and limb dysmorphology, including micrognathia, microglossia, ectrodactylia, partial anodontia and cleft palate. Most interestingly, these are extremely similar to oromandibular limb hypogenesis syndrome.@*Conclusions@#The results of this study indicate that GDC-0449 can be used to induce micrognathia, microglossia, ectrodactylia, partial anodontia and cleft palate. This work established a novel mouse model for oromandibular limb hypogenesis.

The relationship between the expression of sonic hedgehog and vascular endothelial growth factor in hypoxic human retinal pigment epithelial cells / 中华眼底病杂志

Objective To study the relationship between the expression of sonic hedgehog (Shh) and vascular endothelial growth factor (VEGF) in hypoxic human retinal pigment epithelial (hRPE) cells.Methods Cultured hRPE-19 cells (3rd-6th generations) were used in this experiment.hRPE-19 cells were divided into three groups including the control group,the hypoxia experimental group (100 μmol/L CoCl2) and the inhibition group (pretreatment with 20 μmol/L cyclopamine 1 hour before hypoxia).After culturing for 4,8,12 and 24 hours,the mRNA level of Shh and VEGF genes in these cells were measured by fluorescence quantitative polymerase chain reaction,and the protein level of Shh and VEGF in the supematants were measure by enzymelinked immunosorbent assay.The relationship between the expression of Shh and VEGF was analyzed by Pearson correlation analysis.Results The control group expressed low levels of Shh and VEGF mRNA/protein.The expression of Shh and VEGF mRNA/protein in the hypoxia experimental group was significantly higher than that in the control group (F=178.364,183.732,77.456,91.572;P＜0.01).The expression of Shh and VEGF mRNA in the inhibition group was significantly lower than that in the hypoxia experimental group (F=68.745,121.834;P＜0.01).In the hypoxia experimental group,the expression of VEGF protein was positively correlated with the expression of Shh protein (r=0.942,P＜ 0.05);and the expression of VEGF and Shh mRNA was positively correlated (r=0.970,P＜0.01).However,there was no significant correlation in the expression of VEGF and Shh mRNA in the inhibition group (r=0.915,P＞ 0.05).Conclusion There is a positive correlation between the expression of Shh and VEGF in hypoxic hRPE cells.

Síndrome de carcinoma de células basales nevoides (Síndrome Gorlin): reporte de dos casos y revisión de la literatura / Nevoid basal-cell carcinoma syndrome (Gorlin Syndrome): report of two cases and review of the literature

RESUMEN El síndrome Gorlin (SG) es una condición genética, con patrón de herencia autosómico dominante, con penetrancia completa y expresividad variable, debida a mutaciones germinales en los genes PTCH1 o SUFU, los cuales son componentes de la vía molecular Sonic hedgehog. El SG se caracteriza por la presencia de múltiples carcinomas de células basales nevoides, quistes odontogénicos, calcificación de la hoz del cerebro y lesiones en sacabocado en palmas y plantas. Este es el primer reporte de casos en el Perú sobre pacientes con SG, que cuentan con evaluación y asesoría genética. Presentamos dos casos de SG que cumplen criterios clínicos del síndrome y una revisión de la literatura.

ABSTRACT Gorlin syndrome (GS) is a genetic disorder with an autosomal dominant inheritance pattern, with complete penetrance and variable expressivity. GS is caused by germline mutations in the genes PTCH1 or SUFU, which are components of the Sonic hedgehog molecular pathway. GS is characterized by the presence of multiple nevoid basal cell carcinomas, odontogenic cysts, calcification of the brain sickle, and lesions in the palms and soles. This study is the first to report cases in Peru of patients with GS who underwent genetic evaluation and counseling. We present two GS cases that meet the clinical criteria for the syndrome and review the literature

The role of Hedgehog protein and gene methylation in the pathogenesis of esophageal cancer / 中国基层医药

Objective To explore the role of the Hedgehog singaling in the pathogenesis of esophageal cancer.Methods Tissue samples of normal esophageal mucosa,low-grade dysplasia,high-grade dysplasia and esophageal cancer were collected.Immunohistochemical stain,Western-blot and RT-PCR were employed to detect theexpression of Hedgehog protein and gene,methylation special PCR was used to detect the methylation status of Hedgehog gene.Results There was no difference in the expression of SHh protein among nornaml esophageal and low-grade dysplasia,high-grade dysplasia and esophageal cancer(LGD vs.N:t=1.96,P=0.67;HGD vs.EC:t=1.59,P=0.53).However,the expression of SHh protein in high-grade dysplasia and esophageal cancer was higher than that in normal esophageal mucosa and low-grade dysplasia(HGD vs.N:t=0.593,P=0.004;HGD vs.LGD:t=1.308,P=0.325;EC vs.N:t=0.292,P=0.000;EC vs.LGD:t=0.734,P=0.004).The expression of Hedgehog gene in esophageal cancer was higher than that in normal esophageal mucosa,low-grade dysplasia and high-grade dysplasia(EC vs.N:t=0.909,P=0.019,EC vs.LGD:t=0.398,P=0.007;EC vs.HGD:t=0.843,P=0.012).The gene methylation in esophageal cancer was lower than that in normal esophageal mucosa,low-grade dysplasia and high-grade dysplasia(EC vs.N:t=0.0340,P=0.000;EC vs.LGD:t=0.102,P=0.000;EC vs.HGD:t=0.367,P=0.018).Conclusion Hedgehog signaling may play a role in the pathogenesis and development of esophageal cancer,however,demethylation of Hedgehog gene may be one of the mechanism that cause the activity of oncogene in esophageal cancer.

Role of sonic hedgehog signaling pathway in spinal neurons in morphine tolerance in mice / 中华麻醉学杂志

Objective To evaluate the role of sonic hedgehog (SHH) signaling pathway in spinal neurons in morphine tolerance (MT) in mice.Methods Pathogen-free healthy female Kunming mice,weighing 20-25 g,aged 8-10 weeks,were used in the study.MT was induced with morphine 10 mg/kg injected subcutaneously twice a day for 7 consecutive days.The experiment was performed in two parts.Experiment Ⅰ Forty-eight mice were randomly assigned into 2 groups:control group (group C,n =8) and MT group (group M,n=40).The thermal pain threshold (TPT) was measured at 1 day before morphine injection and 1,3,5,7 and 14 days after the end of injection.Eight mice in each group were sacrificed at 2 h after measurement of TPT at each time point after the end of injection in group M or at 2 h after the last measurement of TPT in group C,and the lumbar segment (L4-6) of the spinal cord was removed.Experiment Ⅱ Forty-eight mice were randomly assigned into 6 groups (n=8 each):SHH inhibitor cyclopamine plus MT group (group CP+M),cyclopamine solvent plus MT group (group D1 +M),SHH agonist SAG plus MT group (group SAG+M),SAG solvent plus MT group (group D2+M),MT plus cyclopamine group (group M+CP) and morphine plus cyelopamine solvent group (group M+D1).At 15 min before morphine injection,cyclopamine 10 mg/kg was injected subcutaneously in group CP+M,and SAG 5 mg/kg was injected subcutaneously in group SAG+M.Cyclopamine 10 mg/kg was injected subcutaneously once a day during the 1-3 days after the end of morphine injection in group M+CP.The TPT was measured before injection of morphine,at 30 min after the first injection of morphine every day and at 1-3 days after the end of morphine injection.The animals were sacrificed at 2 h after the last measurement of TPT,and the lumbar segment (L4-6) of the spinal cord was removed for determination of the expression of SHH signaling pathway-related proteins SHH,ptch1,smo,gli1 and gli3 using Western blot.Results Experiment Ⅰ Compared with group C,the TPT was significantly decreased at 1 and 3 days after the end of morphine injection (P＜0.05),no significant change was found in TPT at 5-14 days after the end of morphine injection (P＞0.05),and the expression of SHH,smo and glil at 1-5 days after the end of morphine injection,of ptchl at 1 and 3 days after the end of morphine injection and of gli3 at 7 days after the end of morphine injection was up-regulated in group M (P＜0.05).Experiment Ⅱ Compared with group D1+M,the TPT was significantly increased,the expression of SHH,ptchl,smo and glil was down-regulated,and gli3 expression was up-regulated in group C P+M (P＜0.05).Compared with group D2+M,the TPT was significantly decreased,the expression of SHH,ptch1,smo and glil was up-regulated,and gli3 expression was down-regulated in group SAG+M (P＜0.05).There was no significant difference in the parameters mentioned above between group M+CP and group M+D1 (P＞0.05).The TPT was significantly lower on 3rd-7th days after beginning of morphine injection and 1-3 days after the end of morphine injection than at 30 min after the first injection of morphine in group CP+M (P＜0.05).Conclusion The mechanism underlying the development of MT is partially related to activation of SHH signaling pathway in spinal neurons of mice,however,the maintenance mechanism has no marked relationship with it.

Wound healing - A literature review

Abstract: Regeneration and tissue repair processes consist of a sequence of molecular and cellular events which occur after the onset of a tissue lesion in order to restore the damaged tissue. The exsudative, proliferative, and extracellular matrix remodeling phases are sequential events that occur through the integration of dynamic processes involving soluble mediators, blood cells, and parenchymal cells. Exsudative phenomena that take place after injury contribute to the development of tissue edema. The proliferative stage seeks to reduce the area of tissue injury by contracting myofibroblasts and fibroplasia. At this stage, angiogenesis and reepithelialization processes can still be observed. Endothelial cells are able to differentiate into mesenchymal components, and this difference appears to be finely orchestrated by a set of signaling proteins that have been studied in the literature. This pathway is known as Hedgehog. The purpose of this review is to describe the various cellular and molecular aspects involved in the skin healing process.

Role of Sonic hedgehog signaling pathway in spinal cord in neuropathic pain in rats / 中华麻醉学杂志

Objective To evaluate the role of Sonic hedgehog (Shh) signaling pathway in the spinal cord in neuropathic pain (NP) in the rats.Methods Seventy-two male Sprague-Dawley rats,aged 8 weeks,weighing 250-300 g,were randomly divided into 2 groups (n=36 each) using a random number table:sham operation group (S group) and NP group.Spared nerve injury was produced by exposing the sciatic nerve and its branches and ligation and transection of tibial nerve and common fibular nerve in anesthetized rats.The mechanical paw withdrawal threshold (MWT) was measured before operation and at 1,4,7,14 and 21 days after operation.After measurement of the pain threshold at 1,4,7,14 and 21 days after operation,the animals were then sacrificed,and the lumbar segment (L46) of the spinal cord was obtained for determination of Shh,Patched (Ptch),Gli1 and glial fibrillary acidic protein (GFAP)expression (by Western blot),Shh,Ptch and Gli1 mRNA expression (by fluorescent quantitative real-time reverse transcriptase-polymerase chain reaction),and Shh and GFAP expression (by immunohistochemistry).Results Compared with group S,the MWT was significantly decreased,and the expression of Shh,Ptch and Gli1 protein and mRNA and GFAP in spinal cord tissues was up-regulated in group NP (P＜ 0.05).Shh was mainly expressed in the cytoplasm of spinal dorsal horn neurons and in the gap around glial cells.Conclusion Shh signaling pathway in spinal cord is involved in the development and maintenance of NP in the rats.

Research advances in Hedgehog signaling pathway in hepatocellular carcinoma / 临床肝胆病杂志

Hedgehog (Hh) signaling pathway is present in many animals and plays an important role in regulating embryonic development and differentiation. Aberrant activation of Hh signaling contributes to the pathogenesis of many malignancies. Recent studies have shown that dysregulated Hh signaling pathway participates in the tumorigenesis, tumor invasion, and metastasis of hepatocellular carcinoma (HCC). Investigation of the relationship between Hh signaling pathway and HCC will help elucidate the molecular mechanism of pathogenesis of HCC and provide a new insight into the development of novel anticancer therapy and therapeutic target.

Estudo imunoistoquímico de componentes da Via Sonic Hedgehog, Stat3 e Mcm3 em tumores de glândula salivar / Immunohistochemical study components of Via Sonic Hedgehog, Stat3 and MCM3 in salivary gland tumors

O adenoma pleomórfico (AP), o carcinoma mucoepidermóide (CME) e o carcinoma adenóide cístico (CAC) representam tumores frequentes em glândula salivar. A via de sinalização Sonic Hedgehog (Hh) e o Transdutor de sinal e ativador da transcrição 3 (STAT3) desempenham funções importantes na proliferação celular, favorecendo o desenvolvimento tumoral e a proteína MCM3 tem sido considerada uma nova classe de marcadores de proliferação celular. Portanto, o presente trabalho propõe-se a estudar componentes da via Hh, bem como o STAT3 e o MCM3 em neoplasias de glândula salivar, na tentativa de adicionar informações sobre as características biológicas dessas neoplasias. Foram utilizados 9 casos de AP, 17 casos de CAC e 20 casos de CME e, por meio da técnica imunoistoquímica, realizou-se a detecção das seguintes proteínas: SHH, GLI1, SUFU, HHIP, STAT3 e MCM3. No AP, observou-se alta expressão citoplasmática de SHH e SUFU, e baixa expressão de STAT3 e MCM3. No CAC, observou-se alta expressão de GLI1, HHIP e STAT3 e baixa expressão de SHH, SUFU e MCM3. No CME, observou-se alta expressão de SHH, GLI1, SUFU e HHIP e baixa expressão de STAT3 e MCM3. Quando comparado entre os tipos tumorais, observou-se diferença estatisticamente significante para expressão de SHH (p=0.0064), STAT3 (p=0.0003) e MCM3 (p=0.0257)...

The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC) and the adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands. The Sonic Hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The MCM3 protein has been considered as a novel class of cell proliferation markers. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 and MCM3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC and 20 cases of MEC. Using immunohistochemistry, were investigated: SHH, GLI1, Sufu, HHIP, STAT3 and MCM3. In PA, there was high expression of cytoplasmic SHH and Sufu, and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP and STAT3 and low expression of SHH, SUFU and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p=0.0064), STAT3 (p=0.0003) and MCM3 (p=0.0257) when all tumors were compared...

Hedgehog signal regulates the chondrogenesis from bone marrow mesenchymal stem cells:controlling methods and cross-talking relationship with other signals need further studies / 中国组织工程研究

BACKGROUND:The hedgehog pathway has paid an important role in the progress of chondrogenesis from bone marrow mesenchymal stem cells. However, the definite signal transduction pathway and cross-talking relationship with other common signal pathways are stil poorly understood and the researches related to this field is to continue as a hotspot in the future study. OBJECTIVE:To investigate the research progress of hedgehog signal pathway on the regulation of the chondrogenesis from bone marrow mesenchymal stem cells and the relationship between hedgehog and other signal pathways in the process. METHODS:A computer-based online search in CNKI, PubMed and Google Scholar databases was performed using key words of“Hedgehog, IHH, SHH, bone marrow mesenchymal stem cells, cartilage, chondrogenesis”in English and Chinese, respectively. Literatures related to the process of chondrogenesis from bone marrow mesenchymal stem cells were included and 36 articles were extensively summarized for review. RESULTS AND CONCLUSION:Bone marrow mesenchymal stem cells are currently accepted optimal cellseeds for the cartilage tissue engineering, and hedgehog is a critical signal molecule in the development of skeletal system. The IHH and SHH in hedgehog signal closely participate in control ing the processes of bone marrow mesenchymal stem cellproliferation and chondrogenesis, chondrocyte phenotype maintenance and cooperation with other common single pathways. However, the specific signal transduction mechanism and cross-talking contact with other signal pathways stil need to be further studied, and it stands for the future research directions.

ResveratroI attenuates renaI interstitiaI fibrosis by inhibiting activationof sonic hedgehog signaIing in rats with uniIateraI ureteraI obstruction / 中国药理学与毒理学杂志

OBJECTIVE To investigate the molecular mechanisms of resveratrol( Res)in renal interstitial fibrosis(RlF)in rats with unilateral ureteral obstruction(UUO). METHODS Forty-eight Spra-gur-Dawley rats were randomly divided into UUO( normal saline,n = 16),UUO with Res treatment (Res,20 mg·kg-1 ,n=16),and sham-operation(sham,n=16)models. The kidneys were excised on the 7th and 14th day. The deposition of collagen fiber in the kidney was detected with HE and Masson staining. The levels of sonic hedgehog(SHH,an inducer of SHH pathway)in kidney tissues were deter-mined by ELlSA. lmmunohistochemical analysis was performed to evaluate the protein expression of SHH signaling-related molecules,including SHH,smoothened(Smo),patched-1(Ptch1),and Gli1, proliferating cell nuclear antigen(PCNA)and matrix component typeⅢ collagen. The mRNA expression levels of Smo,Ptch1 and Gli1 were detected by real-time RT-PCR. RESULTS The degree of RlF observed with HE and Masson staining was obviously increased in UUO kidneys,but decreased in Res-treated kidneys. Enhanced expression levels of typeⅢ collagen and PCNA in UUO rats were suppressed by Res treatment(P﹤0.05). Res administration decreased the expression levels of SHH,Smo,and Gli1 (P﹤0.05),but increased the expression of Ptch1(P﹤0.05),suggesting that Res inhibit the obstruction-induced activation of SHH signaling. CONCLUSION Res can attenuate RlF in UUO rats,and the possi-ble mechanism is that Res down-regulates the activity of SHH signaling and inhibits cellular proliferation, resulting in inhibition of matrix accumulation in renal interstitium of UUO rats.

Estudo do perfil de expressão de componentes da via de sinalização sonic hedgehog em displasia epitelial oral / Study of the expression profile of components of sonic hedgehog signaling pathway in oral epithelial dysplasia

A displasia epitelial oral (DEO) é um aspecto histológico descrito em lesões potencialmente malignas, cujos mecanismos relacionados a patogênese e potencial de transformação são pouco conhecidos. Sabendo-se que a via de sinalização Sonic Hedgehog(SHH)tem participação no desenvolvimento do carcinoma escamocelular de boca(CEB) e que as proteínas relacionadas a esta via de sinalização estão envolvidas nos mecanismos biológicos relacionados a iniciação e progressão de tumores humanos. O objetivo deste trabalho foi estudar a expressão das proteínas da via de sinalização SHH em DEO, a fim de contribuir para o conhecimento do perfil biológico desta lesão...

Oral epithelial dysplasia (OED) is a histological aspect described in premalignant lesions and themechanisms related to the pathogenesis and malignant progression are poorly understood. It is knownthat Sonic Hedgehog (SHH) signaling pathway participates in the development of oral squamous cellcarcinoma, and proteins related to this cascade are involved in biological mechanisms related to theinitiation and progression of human tumors. The aim of this study was to investigate SHH signalingmolecules in OED, in order to contribute to the knowledge of the biological profile of this lesion...