scholarly journals Hepatoblastoma: glutamine depletion hinders cell viability in the embryonal subtype but high GLUL expression is associated with better overall survival

2021 ◽  
Author(s):  
Andreas Schmidt ◽  
Angela Armento ◽  
Ovidio Bussolati ◽  
Martina Chiu ◽  
Verena Ellerkamp ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Overall Survival ◽  
Growth Inhibition ◽  
Cell Viability ◽  
Cell Lines ◽  
Prognostic Significance ◽  
Asparagine Synthetase ◽  
Inhibition Of Proliferation ◽  
Its Gene ◽  
Glutamine Synthesis

Abstract Purpose Glutamine plays an important role in cell viability and growth of various tumors. For the fetal subtype of hepatoblastoma, growth inhibition through glutamine depletion was shown. We studied glutamine depletion in embryonal cell lines of hepatoblastoma carrying different mutations. Since asparagine synthetase was identified as a prognostic factor and potential therapeutic target in adult hepatocellular carcinoma, we investigated the expression of its gene ASNS and of the gene GLUL, encoding for glutamine synthetase, in hepatoblastoma specimens and cell lines and investigated the correlation with overall survival. Methods We correlated GLUL and ASNS expression with overall survival using publicly available microarray and clinical data. We examined GLUL and ASNS expression by RT-qPCR and by Western blot analysis in the embryonal cell lines Huh-6 and HepT1, and in five hepatoblastoma specimens. In the same cell lines, we investigated the effects of glutamine depletion. Hepatoblastoma biopsies were examined for histology and CTNNB1 mutations. Results High GLUL expression was associated with a higher median survival time. Independent of mutations and histology, hepatoblastoma samples showed strong GLUL expression and glutamine synthesis. Glutamine depletion resulted in the inhibition of proliferation and of cell viability in both embryonal hepatoblastoma cell lines. ASNS expression did not correlate with overall survival. Conclusion Growth inhibition resulting from glutamine depletion, as described for the hepatoblastoma fetal subtype, is also detected in established embryonal hepatoblastoma cell lines carrying different mutations. At variance with adult hepatocellular carcinoma, in hepatoblastoma asparagine synthetase has no prognostic significance.

Effect of 5'-fluoro-2'-deoxycytidine and sodium butyrate on the genes of the intrinsic apoptotic pathway, p21, p53, cell viability, and apoptosis in human hepatocellular carcinoma cell lines

2021 ◽  
Author(s):  
Masumeh Sanaei ◽  
Fraidoon Kavoosi ◽  
Mohammad Amin Moezzi
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Cell Viability ◽  
Cell Lines ◽  
Sodium Butyrate ◽  
Cell Apoptosis ◽  
Chromatin Organization ◽  
Expression Level ◽  
Intrinsic Apoptotic Pathway ◽  
Apoptotic Pathway

Backgrounds: Epigenetic regulation such as DNA methylation plays a major role in chromatin organization and gene transcription. Additionally, histone modification is an epigenetic regulator of chromatin structure and influences chromatin organization and gene expression. The relationship between DNA methyltransferase (DNMTs) expression and promoter methylation of the tumor suppressor genes (TSGs) has been reported in various cancers. Previously, the effect of 5-aza-2'-deoxycytidine (5-AZA-CdR), trichostatin A (TSA), and valproic acid (VPA) was shown on various cancers. This study aimed to investigate the effect of 5'-fluoro-2'-deoxycytidine (FdCyd) and sodium butyrate on the genes of the intrinsic apoptotic pathway, p21, p53, cell viability, and apoptosis in human hepatocellular carcinoma SNU449, SNU475, and SNU368 cell lines. Materials and Methods: In this lab trial study, the SNU449, SNU475, and SNU368 cells were cultured and treated with 5'-fluoro-2'-deoxycytidine and sodium butyrate. To determine cell viability, cell apoptosis, and the relative gene expression level, MTT assay, flow cytometry assay, and qRT-PCR were done respectively. Results: 5'-fluoro-2'-deoxycytidine and sodium butyrate changed the expression level of the BAX, BAK, APAF1, Bcl-2, Bcl-xL, p21, and p53 gene (P<0.0001) by which induced cell apoptosis and inhibit cell growth in all three cell lines, SNU449, SNU475, and SNU368.  Conclusion: Both compounds played their roles through the intrinsic apoptotic pathway to induce cell apoptosis.

scholarly journals A Nomogram Based on a Three-Gene Signature Derived from AATF Coexpressed Genes Predicts Overall Survival of Hepatocellular Carcinoma Patients

2020 ◽  
Vol 2020 ◽  
pp. 1-17
Author(s):  
Jun Liu ◽  
Jianjun Lu ◽  
Zhanzhong Ma ◽  
Wenli Li
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Overall Survival ◽  
Clinical Practice ◽  
Correlation Coefficient ◽  
Cox Regression ◽  
Prognostic Significance ◽  
Gene Signature ◽  
Cancer Genome ◽  
Survival Prediction

Background. Hepatocellular carcinoma (HCC) is a common cancer with an extremely high mortality rate. Therefore, there is an urgent need in screening key biomarkers of HCC to predict the prognosis and develop more individual treatments. Recently, AATF is reported to be an important factor contributing to HCC. Methods. We aimed to establish a gene signature to predict overall survival of HCC patients. Firstly, we examined the expression level of AATF in the Gene Expression Omnibus (GEO), the Cancer Genome Atlas (TCGA), and the International Union of Cancer Genome (ICGC) databases. Genes coexpressed with AATF were identified in the TCGA dataset by the Poisson correlation coefficient and used to establish a gene signature for survival prediction. The prognostic significance of this gene signature was then validated in the ICGC dataset and used to build a combined prognostic model for clinical practice. Results. Gene expression data and clinical information of 2521 HCC patients were downloaded from three public databases. AATF expression in HCC tissue was higher than that in matched normal liver tissues. 644 genes coexpressed with AATF were identified by the Poisson correlation coefficient and used to establish a three-gene signature (KIF20A, UCK2, and SLC41A3) by the univariate and multivariate least absolute shrinkage and selection operator Cox regression analyses. This three-gene signature was then used to build a combined nomogram for clinical practice. Conclusion. This integrated nomogram based on the three-gene signature can predict overall survival for HCC patients well. The three-gene signature may be a potential therapeutic target in HCC.

scholarly journals Synthesis and Cytotoxic Activity of Chiral Sulfonamides Based on the 2-Azabicycloalkane Skeleton

Molecules ◽  
2020 ◽  
Vol 25 (10) ◽  
pp. 2355 ◽  
Author(s):  
Mahzeiar Samadaei ◽  
Matthias Pinter ◽  
Daniel Senfter ◽  
Sibylle Madlener ◽  
Nataliya Rohr-Udilova ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Cell Viability ◽  
Cytotoxic Activity ◽  
Cell Lines ◽  
Diels Alder ◽  
Human Hepatocellular Carcinoma ◽  
Sulfonyl Chlorides ◽  
Reduce Cell Viability

A series of chiral sulfonamides containing the 2-azabicycloalkane scaffold were prepared from aza-Diels–Alder cycloadducts through their conversion to amines based on 2-azanorbornane or the bridged azepane skeleton, followed by the reaction with sulfonyl chlorides. The cytotoxic activity of the obtained bicyclic derivatives was evaluated using human hepatocellular carcinoma (HCC), medulloblastoma (MB), and glioblastoma (GBM) cell lines. Chosen compounds were shown to notably reduce cell viability as compared to nonmalignant cells.

Selective HDAC6 Inhibition and Antileukemic Activity of the Novel HDAC Inhibitor ST80 in Myeloid Leukemia Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4808-4808
Author(s):  
Bjorn W. Hackanson ◽  
Leander Rimmele ◽  
Manfred Jung ◽  
Michael Lübbert
Keyword(s):  
Growth Inhibition ◽  
Cell Viability ◽  
Cell Lines ◽  
Hydroxamic Acid ◽  
Myeloid Cell ◽  
The Novel ◽  
Antileukemic Activity ◽  
Alpha Tubulin ◽  
Tubulin Acetylation ◽  
Acid Derivate

Abstract Abstract 4808 The antileukemic activity of histone deacetylase inhibitors (HDACi) has driven the search for epigenetic drugs with higher substrate specificity. Most of the currently used HDACi target class I, II and IV, with some bearing a class preference but only very few being selective in inhibiting specific HDACs, such as the HDAC6-selective inhibitor tubacin. HDAC6 inhibition leads to acetylation of non-histone proteins such as Hsp90 and alpha-tubulin. As it was recently demonstrated that HDAC6 is overexpressed in AML cells, we sought to investigate the effect of selective HDAC6 inhibition by the novel hydroxamic acid derivate ST80 in myeloid cell lines. Methods ST80 (selective HDAC6 inhibitor, 30-fold higher enzyme inhibition as compared to HDAC1) and the non-selective novel hydroxamic acid derivates ST13 (pan-HDACi) and ST34 (pan-HDACi with preference for class I) had previously been tested in enzymatic assays for their HDAC inhibitory potential (Scott et al. Mol Cancer Res. 6:1250-8, 2008). Cell lines HL60, Kasumi-1, NB4, THP1, U937 and K562 were treated with 10 nM to 30 μM of these three drugs. Viability and growth inhibition were determined using trypan blue staining. Acetylation of histone H3, H4 and alpha-tubulin and HDAC6 expression were determined by Western blot and quantified by densitometry. Tubulin-selective acetylation was calculated as the ratio of tubulin acetylation vs. H4 acetylation (ac-tubulin:ac-H4 quotient). Results At 1 μm, ST80, ST13 and ST34 all acetylated tubulin (8-, 8- and 2-fold in NB4, 11-, 14- and 3.4-fold in HL60, respectively, after a 12 h treatment). However, the calculated ac-tubulin:ac-H4 ratio of ST80 was 15- and 8-fold higher in NB4 and 9-fold higher in HL60 when compared to ST13 and ST34. The inhibitory concentration (IC) 50 (cell growth) of ST80 in the six myeloid cell lines ranged from 2.8 μM (NB4) to 5.1 μM (Kasumi-1) after 48 h treatment. Median cell viability of all 6 cell lines at 48 h was 93.7 % (range: 87.0 - 96.8 %) at 1 μm and 90.3 % (65.7 - 95.7 %) at 5 μm of ST80. HDAC6 protein levels were strongly variable between cell lines; however, growth inhibition by ST80 was independent of HDAC6 expression. Conclusion The novel hydroxamic acid derivate ST80 shows antileukemic activity in myeloid cell lines at low micromolar concentrations, which affect cell viability only modestly. The degree of relative tubulin acetylation by ST80 indicates a selective HDAC6 inhibitory activity in myeloid leukemias. The favorable ratio of ST80 growth inhibition vs. cytotoxicity warrants combination studies of this drug with other compounds. Disclosures: No relevant conflicts of interest to declare.

A Novel Small Molecule Inhibitor Of Deubiquitylating Enzyme USP14 and UCHL5 Induces Apoptosis In Myeloma Cells and Overcomes Bortezomib Resistance

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1923-1923 ◽  
Author(s):  
Ze Tian ◽  
Padraig D'Arcy ◽  
Xin Wang ◽  
Arghya Ray ◽  
Yu-Tzu Tai ◽  
...  
Keyword(s):  
Cell Viability ◽  
Er Stress ◽  
Board Of Directors ◽  
Cell Lines ◽  
Mammalian Cells ◽  
Ubiquitin Ligases ◽  
Advisory Board ◽  
In Vivo Studies ◽  
Advisory Committees ◽  
Inhibition Of Proliferation

Abstract Introduction Proteasome inhibitors have demonstrated that targeting ubiquitin proteasome pathway (UPS) is an effective therapy in multiple myeloma (MM). More recent studies have focused on targeting enzymes modulating protein ubiquitin conjugation/deconjugation upstream of the proteasome rather than the proteasome itself, with the goal of producing more specific, potent, and less toxic therapies targeting UPS. Ubiquitylation is a dynamic reversible process coordinated by many enzymes: ubiquitin ligases attach ubiquitin to proteins allowing for their degradation, whereas deubiquitylating enzymes (DUBs) deconjugate ubiquitin from target proteins, thereby preventing their proteasome-mediated degradation. Importantly, many human diseases are linked to dysfunction of ubiquitin ligases and/or DUBs, suggesting that inhibitors of ubiquitylating or DUBs represent a potential therapeutic strategy.In mammalian cells, three DUBs are associated with the proteasome: USP14, UCHL5/Uch37, and Rpn11. In the present study, we examined the expression of USP14 and UCHL5 in MM by western blot and Immunohistochemistry (IHC). Results Our results show that DUBs USP14 and UCHL5 are more highly expressed in primary MM patient tumor cells and MM cell lines than in normal plasma cells and peripheral blood mononuclear cells (PBMCs). Additionally, USP14 and UCHL5 siRNA knockdown significantly decrease MM cell viability (p < 0.001) in a CellTiter Glo assay. A novel 19S regulatory particle inhibitor b-AP15 selectively blocks deubiquitylating activity of USP14 and UCHL5 without inhibiting activity of other DUBs, proteases and proteasome; Importantly, b-AP15 decreases cell viability in MM cell lines as well as patient MM cells, without markedly affecting PBMCs from normal healthy donors. Moreover, b-AP15 inhibits proliferation of MM cells even in the presence of bone marrow stroma cells and overcomes bortezomib-resistance. Mechanistic studies show that b-AP15 triggers MM cell arrest via downregulation of CDC25C, CDC2 and cyclinB1, followed by caspase-dependent apoptosis through activation of intrinsic and extrinsic apoptotic pathways. b-AP15, like bortezomib, induces ER stress evidenced by the upregulation of ER stress-related proteins p-IRE-alpha, and p-eIF2. In vivo studies using subcutaneous and disseminated human MM xenograft models show that b-AP15 is well tolerated, inhibits tumor growth, and prolongs survival (p < 0.001). In concert with our in vitro study, IHC analysis of tumor tissues showed inhibition of proliferation, induction of apoptosis, and accumulation of ubiquitinated proteins, assessed by staining with ki67, caspase-3, and UB-k48 antibodies, respectively. Finally, combining b-AP15 with SAHA, lenalidomide, or dexamethasone induces synergistic anti-MM activity. Conclusion Our preclinical data showing efficacy of b-AP15 in MM disease models validates targeting DUBs upstream of the proteasome in the ubiquitin proteasomal cascade to overcome proteasome inhibitor resistance, and provides the framework for clinical evaluation of USP14/UCHL5 inhibitors to improve patient outcome in MM. Disclosures: Tai: Onyx: Consultancy. Richardson:Celgen, Inc., Millenium: Membership on an entity’s Board of Directors or advisory committees. Chauhan:Vivolux: Consultancy. Anderson:Oncoprep: Scientific founder:, Scientific founder: Other; Acetylon: Scientific founder, Scientific founder Other; Sonofi Aventis: Advisory board, Advisory board Other; Gilead: Advisory board, Advisory board Other; Onyx: Advisory board, Advisory board Other; Celgene : Membership on an entity’s Board of Directors or advisory committees.

sFRP-1: A functional prognostic marker for gastric cancer?

2011 ◽  
Vol 29 (4_suppl) ◽  
pp. 37-37
Author(s):  
C. J. Moran ◽  
P. S. Ray ◽  
S. P. Bagaria ◽  
Y. Qu ◽  
A. J. Fleisig ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Overall Survival ◽  
Cell Lines ◽  
Clinical Stage ◽  
Univariate Analysis ◽  
Prognostic Significance ◽  
Nuclear Grade ◽  
Functional Markers ◽  
T Stage ◽  
E Cadherin

37 Background: Despite being a leading cause of cancer-related death world wide, gastric adenocarcinoma (GA) lacks distinctive biomarkers and targeted therapies. Underexpression of the E-cadherin gene in GA is associated with an aggressive phenotype and a poor prognosis but the mechanisms of this difference are poorly understood. Developing effective therapies for GA requires identification of critical functional markers and deeper understanding of its pathophysiology. Methods: Unsupervised hierarchical clustering analysis of a publicly available 230-sample GA microarray dataset identified a prominent cluster (21.7%) associated with underexpression of E-cadherin and overexpression of a Wnt-family protein: secreted frizzled-related protein 1 (sFRP-1). Archival GA specimens were then assessed for the expression of sFRP-1 by immunohistochemistry. Prognostic significance was assessed using univariate and multivariate analyses. GA cell lines transfected with sFRP-1 were used to determine the role of sFRP-1 in gastric cancer. Results: 85 patients with GA underwent surgery with curative intent; 39 stained positive for sFRP-1 (46%). In this positive group, sFRP-1 staining was focal; was commonly found on the leading edge of the infiltrating tumor mass; and was not restricted to one histopathologic group, grade, or clinical stage. On univariate analysis T stage, nodal involvement, pathologic stage, nuclear grade, E-cadherin status and sFRP-1 status were predictive of overall survival. In a multivariate model, T stage (p < 0.001), nuclear grade (p < 0.001), E-cadherin status (p = 0.031) and sFRP-1 status (p = 0.0097) were predictive of overall survival. Overexpression of sFRP-1 in GA cell lines induced mesenchymal phenotype, enhanced growth and stem cell-like properties. sFRP-1 also attenuated Wnt signaling and E-cadherin expression, but potentiated Notch and Hedgehog signaling known to be involved in GA progression. These findings suggest a Wnt-independent mechanism mediated by sFRP-1. Conclusions: The aggressive biological subtype of gastric cancer may be linked to overexpression of sFRP-1. Our findings identify sFRP-1 as a functional prognostic biomarker for gastric cancer, which may serve as a potential therapeutic target. No significant financial relationships to disclose.

scholarly journals FAM83D is associated with gender, AJCC stage, overall survival and disease-free survival in hepatocellular carcinoma

2019 ◽  
Vol 39 (5) ◽  
Author(s):  
Xuling Liu ◽  
Hong Gao ◽  
Jie Zhang ◽  
Dongying Xue
Keyword(s):  
Hepatocellular Carcinoma ◽  
Overall Survival ◽  
Sequence Similarity ◽  
Prognostic Significance ◽  
Disease Free Survival ◽  
High Expression ◽  
Mrna Levels ◽  
Free Survival ◽  
Ajcc Stage ◽  
Disease Free

AbstractPrognostic significance of family with sequence similarity 83, member D (FAM83D) in hepatocellular carcinoma (HCC) patients has not been well-investigated using Gene Expression Omnibus (GEO) series and TCGA database, we compared FAM83D expression levels between tumor and adjacent tissues, and correlated FAM83D in tumors with outcomes and clinico-pathological features in HCC patients. Validated in GSE33006, GSE45436, GSE84402 and TCGA, FAM83D was significantly overexpressed in tumor tissues than that in adjacent tissues (all P<0.01). FAM83D up-regulation was significantly associated with worse overall survival (OS) and disease-free survival (DFS) in HCC patients (Log rank P=0.00583 and P=4.178E-04, respectively). Cox analysis revealed that FAM83D high expression was significantly associated with OS in HCC patients [hazard ratio (HR) = 1.44, 95% confidence interval (CI) = 1.005–2.063, P=0.047]. Additionally, patients deceased or recurred/progressed had significantly higher FAM83D mRNA levels than those living or disease-free (P=0.0011 and P=0.0238, respectively). FAM83D high expression group had significantly more male patients and advanced American Joint Committee on Cancer (AJCC) stage cases (P=0.048 and P=0.047, respectively). FAM83D mRNA were significantly overexpressed in male (P=0.0193). Compared with patients with AJCC stage I, those with AJCC stage II and stage III–IV had significantly higher FAM83D mRNA levels (P = 0.0346 and P=0.0045, respectively). In conclusion, overexpressed in tumors, FAM83D is associated with gender, AJCC stage, tumor recurrence and survival in HCC.

Role of AXL in metastatic melanoma and impact of TP-0903 as a novel therapeutic option for melanoma brain metastasis.

2020 ◽  
Vol 38 (15_suppl) ◽  
pp. e22021-e22021
Author(s):  
Eemon Tizpa ◽  
Hannah J Young ◽  
Kimberley-Jane C. Bonjoc ◽  
Chou-Wei Chang ◽  
Yilun Liu ◽  
...  
Keyword(s):  
Overall Survival ◽  
Drug Resistance ◽  
Malignant Melanoma ◽  
Cell Viability ◽  
Cell Lines ◽  
Melanoma Cell ◽  
Therapeutic Option ◽  
Poor Survival ◽  
Malignant Melanoma Cell ◽  
Melanoma Cell Lines

e22021 Background: Melanoma brain metastases (MBM) are common with a median overall survival of 4-5 months. Although immunotherapies have improved clinical outcomes and have doubled overall survival in MBM, there is a high incidence rate of relapse caused by drug resistance. AXL, a receptor tyrosine kinase (RTK), is associated with drug resistance and metastasis in many cancers. The activation of AXL via trans-phosphorylation regulates multiple signaling pathways that induce tumor survival, metastasis, drug resistance, and epithelial-to-mesenchymal transition (EMT). In MBM, AXL is upregulated and associated with disease progression, promoting cell invasion and migration. This suggests that targeting AXL can be a novel strategy to overcome treatment-related resistance in MBM. TP-0903, an investigational small molecule inhibitor of AXL, has shown efficacy in reversing the mesenchymal phenotype and re-sensitizing resistant cancer cells to targeted therapies in heme malignancies, pancreatic, and breast cancer. We aim to investigate the efficacy of TP-0903 in MBM. Methods: The Cancer Genome Atlas (TCGA) data was utilized to investigate the signaling pathways downstream of AXL that are upregulated in advanced melanoma. Nine signaling molecules including AKT1, mTOR, and PAK4 were analyzed to identify any correlation between gene expression levels and overall survival. Four metastatic melanoma cell lines were used to evaluate the effect of TP-0903 on cell viability and active AXL downregulation was assessed in vitro through MTS cell viability assays and Immunoblotting. Wound closure assays were executed to understand the functional consequences of AXL downregulation. Results: In all nine genes, high expression levels confer poor survival probability. Cell viability assays of four malignant melanoma cell lines showed that TP-0903 treatment resulted in IC50 values ranging from 32 – 692 nM. Western blot analysis indicated that TP-0903 reduced the levels of phosphorylated AXL in malignant melanoma cell lines. In addition, increasing TP-0903 concentrations reduced the rate of cell migration in these malignant melanoma cell lines. Conclusions: AXL plays a role in EMT, treatment resistance, and metastasis in MBM, resulting in poor survival. Our findings suggest TP-0903 is effective in reducing cell migration, inhibit metastasis, and can be a potential therapeutic option in MBM.

Hedgehog Pathway as a Potential Therapeutic Target in Multiple Myeloma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 672-672
Author(s):  
Simona Blotta ◽  
Joeseph Negri ◽  
Purushothama Nanjappa ◽  
Anne-Sophie Moreau ◽  
Rao Prabhala ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Proliferation ◽  
Cell Viability ◽  
Cell Lines ◽  
Small Molecule ◽  
Plasma Cells ◽  
Annexin V ◽  
Inhibition Of Proliferation ◽  
Ic50 Values ◽  
Gli 1

Abstract We have previously demonstrated that a consistent feature of malignant plasma cells of multiple myeloma (MM) is the aberrant expression of genes important in patterning and development, such as members of Hedghehog (Hh) pathway (FE Davies et al, Blood 2003). These findings suggest that overexpression of genes of this pathway, already involved in many solid tumors and recently implicated in maintaining a proposed MM stem cell compartment (CD Peacock et al, PNAS 2007), might be one of the mechanism through which Hh-signaling contributes to tumorigenesis in MM. Therefore, several small molecule modulators of Hh-pathway, which work as agonists and antagonists, are currently under development. We evaluated, by microarray analysis, the expression of Hh pathway genes in MM cell lines and primary MM cells vs. plasma cells from healthy donors. We found that primary MM cells overexpress Sonic (Shh), Smoothened (Smo), Patched (Ptc), Gli-1 and Gli-3 (relative expression ratios ranging from +1.8 to +5.0). Overexpression of Patched was also observed in most of the MM cell lines analyzed (+5.0 ratio in 5 of 6 MM cell lines). Additionally, we confirmed the expression of Shh and of Gli-1, by flow cytometry and western blotting respectively, in a large panel of MM cell lines. These data suggest an activation of the Hh-pathway in MM that, in some cell lines, is Shh-dependent. Therefore, we investigated the therapeutic potential of Hh-inhibitors in MM. We assayed the cell viability and proliferation, by MTT and Thymidine uptake respectively, in 8 MM cell lines after 72 hours of treatment with the small molecule Smo-inhibitor CUR-0199691 (Genentech). We observed a reduction in MM cell viability, with IC50 values ranging between 4.5–9.5 μM in these 8 cell lines and an inhibition of MM cell proliferation with IC50 values ranging between 0.5 and 2.5μM in the same cell lines. MM cell sensitivity to this compound appears to be related to the level of expression of Gli-1, since the cell lines with lower level of expression of Gli-1 were more sensitive. The treatment of these MM cell lines with Cyclopamine, another Hh-inhibitor, showed an IC50 between 7.5μM and 10μM after at least 96 hours of treatment in 4 of the MM cell lines tested. CUR-0199691 is also active in primary MM cells, triggering inhibition of proliferation by 50% at 5μM after only 24h of treatment, while cyclopamine reduces MM cell proliferation (normalized to the effect of tomatidine, its inactive analog) by 30% at 20μM after a 48 hour treatment. Annexin V-PI staining of Hh inhibitor-treated KMS11 cells, which are one of the most sensitive MM cell lines, showed induction of apoptosis, evidenced by detection of 12 and 15% of MM cells being Annexin V+/PI- after 48h and 72h respectively with 5μM of CUR-0199691. These results, taken together, show that the Hh-pathway is fuctionally active in MM and that the novel Hh pathway inhibitor CUR-0199691 is 4–5 times more effective than cyclopamine in both MM cell lines and primary MM cells. These studies provide the framework for further preclinical evaluation of CUR-0199691 in MM models towards possible future clinical trials.

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