PTCH1 isoform 1b is the major transcript in the development of basal cell nevus syndrome.

Basal cell nevus syndrome (BCNS) is an autosomal dominant disorder most commonly caused by a germline mutation in the PTCH1 gene. PTCH1 is known to have different isoforms with different functional properties and expression patterns among tissues. We detected a novel, pathogenic de novo mutation in PTCH1 isoform 1b (c.114delG) in a BCNS patient. Furthermore, we elucidated the specific expression pattern of PTCH1 isoforms in normal skin, BCC and peripheral blood by studying expression of different PTCH1 isoforms. Human skin showed expression of isoforms 1b and 1d, while peripheral blood additionally showed 1a and 1e expression. BCCs showed expression of all isoforms. Here we report a patient with a novel, isoform 1b specific mutation in PTCH1 and thereby distinguish PTCH1 isoform 1b as the major transcript in the development of BCNS.

Anticancer drugs and the regulation of Hedgehog genes GLI1 and PTCH1, a comparative study in nonmelanoma skin cancer cell lines.

Nonmelanoma skin cancer is the most common cancer in humans, comprising mainly basal cell carcinoma (BCC) and squamous cell carcinoma (SCC). BCC proliferation is highly dependent on the Hedgehog signaling pathway. We aimed to investigate a panel of anticancer drugs with known activity against skin cancer for their therapeutic potential in localized, enhanced topical treatment of SCC and BCC. Cytotoxicity profiles for vismodegib, 5-fluorouracil (5-FU), methotrexate (MTX), cisplatin, bleomycin, and vorinostat were established in terms of half maximal inhibitory concentration values in a panel of immortalized keratinocytes (HaCaT), BCC (UWBCC1 and BCC77015), and SCC (A431 and SCC25) cell lines. The impact of treatment on the regulation of Hedgehog pathway target genes (GLI1 and PTCH1), measured by real-time PCR, was compared between UWBCC1 and HaCaT. Varying cell line sensitivity profiles to the examined anticancer drugs were observed. Generally, 24-h drug exposure was sufficient to reduce cell viability. We found that 5-FU, MTX, and cisplatin significantly downregulated the expression of two genes controlled by the Hedgehog pathway (≤25-, 2.9-, and 12.5-fold, respectively, for GLI1 in UWBCC1 cells at 48 h, P<0.0001). The gene regulation showed clear concentration dependence and correlated with cytotoxicity for both 5-FU and MTX. We find a potential for the use of anticancer drugs in localized and enhanced topical treatment of nonmelanoma skin cancer. Of importance in the clinical setting, 24-h drug exposure may be sufficient for significant cytotoxicity for vismodegib, 5-FU, cisplatin, and bleomycin. MTX, 5-FU, and cisplatin may offer particular promise through combined cytotoxicity and downregulation of Hedgehog pathway genes GLI1 and PTCH1.

Alcohol Reduces Arterial Remodeling by Inhibiting Sonic Hedgehog-Stimulated Stem Cell Antigen-1 Positive Progenitor Stem Cell Expansion.

BACKGROUND: Cell and molecular mechanisms mediating the cardiovascular effects of alcohol are not fully understood. Our aim was to determine the effect of moderate ethanol (EtOH) on sonic hedgehog (SHh) signaling in regulating possible stem cell antigen-1 positive (Sca1+ ) progenitor stem cell involvement during pathologic arterial remodeling. METHODS: Partial ligation or sham operation of the left carotid artery was performed in transgenic Sca1-enhanced green fluorescent protein (eGFP) mice gavaged with or without "daily moderate" EtOH. RESULTS: The EtOH group had reduced adventitial thickening and less neointimal formation, compared to ligated controls. There was expansion of eGFP-expressing (i.e., Sca1+ ) cells in remodeled vessels postligation (day 14), especially in the neo intima. EtOH treatment reduced the number of Sca1+ cells in ligated vessel cross-sections concomitant with diminished remodeling, compared to control ligated vessels. Moreover, EtOH attenuated SHh signaling in injured carotids as determined by immunohistochemical analysis of the target genes patched 1 and Gli2, and RT-PCR of whole-vessel Gli2 mRNA levels. Intraperitoneal injection of ligated Sca1-eGFP mice with the SHh signaling inhibitor cyclopamine diminished SHh target gene expression, reduced the number of Sca1+ cells, and ameliorated carotid remodeling. EtOH treatment of purified Sca1+ adventitial progenitor stem cells in vitro inhibited SHh signaling, and their rSHh-induced differentiation to vascular smooth muscle cells. CONCLUSIONS: EtOH reduces SHh-responsive Sca1+ progenitor cell myogenic differentiation/expansion in vitro and during arterial remodeling in response to ligation injury in vivo. Regulation of vascular Sca1+ progenitor cells in this way may be an important novel mechanism contributing to alcohol's cardiovascular protective effects.

Cryptolepine derivative-6h inhibits liver fibrosis in TGF-ß1-induced HSC-T6 cells by targeting the Shh pathway.

Liver fibrosis is a worldwide problem with a significant morbidity and mortality. Cryptolepis sanguinolenta (family Periplocaceae) is widely used in West African countries for the treatment of malaria, as well as for some other diseases. However, the role of C. sanguinolenta in hepatic fibrosis is still unknown. It has been reported that Methyl-CpG binding protein 2 (MeCP2) had a high expression in liver fibrosis and played a central role in its pathobiology. Interestingly, we found that a cryptolepine derivative (HZ-6h) could inhibit liver fibrosis by reducing MeCP2 expression, as evidenced by the dramatic downregulation of α-smooth muscle actin (α-SMA) and type I collagen alpha-1 (Col1α1) in protein levels in vitro. Meanwhile, we also found that HZ-6h could reduce the cell viability and promote apoptosis of hepatic stellate cells (HSCs) treated with transforming growth factor beta 1(TGF-ß1). Then, we investigated the potential molecular mechanisms and found that HZ-6h blocked Shh signaling in HSC-T6 cells, resulting in the decreased protein expression of Patched-1 (PTCH-1), Sonic hedgehog (Shh), and glioma-associated oncogene homolog 1 (GLI1). In short, these results indicate that HZ-6h inhibits liver fibrosis by downregulating MeCP2 through the Shh pathway in TGF-ß1-induced HSC-T6 cells.

The Effect of PTCH1 on Ovarian Cancer Cell Proliferation and Apoptosis.

OBJECTIVE: Patched (PTCH1) is an important receptor protein in the Hedgehog pathway and plays a tumor suppressor role in a variety of tumors. This study was to detect the expression of PTCH1 in ovarian cancer (OC) tissues to analyze the relationship between expression of PTCH1 and prognosis, and to explore its role in regulating OC cell proliferation and apoptosis. MATERIALS AND METHODS: OC tissues and normal ovarian tissues were collected to detect PTCH1 expression. SKOV3, A2780, Caov3, and IOSE80 cells were cultured in vitro to test PTCH1 expression. pIRES2-Scramble and pIRES2-PTCH1 were transfected into SKOV3 and A2780 cells, respectively. PTCH1 and Gli1 expressions were detected by western blot. Cell apoptosis was determined by flow cytometry. Cell proliferation was assessed by EdU staining. RESULTS: PTCH1 expression was significantly decreased in OC tissue compared with normal ovarian tissue and was associated with tumor size, TNM stage, and pathological grade (p < 0.05). The prognosis of patients with low PTCH1 expression was obviously worse than that of patients with high PTCH1 expression. The expression of PTCH1 in OC SKOV3, A2780, and Caov3 cells was markedly lower than that in normal ovarian epithelial IOSE80 cells. Transfection of pIRES2-PTCH1 apparently upregulated PTCH1 level, inhibited GLI1 expression and cell proliferation, and promoted apoptosis of SKOV3 and A2780 cells. CONCLUSION: PTCH1 level in OC was abnormally decreased and related to prognosis. Overexpression of PTCH1 inhibited GLI1 expression, attenuated OC cell proliferation, and induced apoptosis, suggesting that manipulation of PTCH1 expression might be a novel approach for the treatment of OC.

In vitro and in vivo inhibition of breast cancer cell growth by targeting the Hedgehog/GLI pathway with SMO (GDC-0449) or GLI (GANT-61) inhibitors.

Aberrant Hedgehog (Hh)/glioma-associated oncogene (GLI) signaling has been implicated in cancer progression. Here, we analyzed GLI1, Sonic Hedgehog (Shh) and NF-κB expression in 51 breast cancer (ductal carcinoma) tissues using immunohistochemistry. We found a positive correlation between nuclear GLI1 expression and tumor grade in ductal carcinoma cases. Cytoplasmic Shh staining significantly correlated with a lower tumor grade. Next, the in vitro effects of two Hh signaling pathway inhibitors on breast cancer cell lines were evaluated using the Smoothened (SMO) antagonist GDC-0449 and the direct GLI1 inhibitor GANT-61. GDC-0449 and GANT-61 exhibited the following effects: a) inhibited breast cancer cell survival; b) induced apoptosis; c) inhibited Hh pathway activity by decreasing the mRNA expression levels of GLI1 and Ptch and inhibiting the nuclear translocation of GLI1; d) increased/decreased EGFR and ErbB2 protein expression, reduced p21-Ras and ERK1/ERK2 MAPK activities and inhibited AKT activation; and e) decreased the nuclear translocation of NF-κB. However, GANT-61 exerted these effects more effectively than GDC-0449. The in vivo antitumor activities of GDC-0449 and GANT-61 were analyzed in BALB/c mice that were subcutaneously inoculated with mouse breast cancer (TUBO) cells. GDC-0449 and GANT-61 suppressed tumor growth of TUBO cells in BALB/c mice to different extents. These findings suggest that targeting the Hh pathway using antagonists that act downstream of SMO is a more efficient strategy than using antagonists that act upstream of SMO for interrupting Hh signaling in breast cancer.