scholarly journals Targeting the Mitotic Checkpoint in Myeloma with OSU-13, a Novel Mps1/Ttk Inhibitor

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2660-2660
Author(s):  
Larissa Valle Guilhen Longo ◽  
Betina McNeil-Laidley ◽  
Francesca Cottini ◽  
Tiffany Hughes ◽  
Gerald Hilinski ◽  
...  
Keyword(s):  
Cell Death ◽  
High Risk ◽  
Tumor Growth ◽  
Cell Lines ◽  
Annexin V ◽  
Fold Increase ◽  
Mitotic Checkpoint ◽  
Genetic Alterations ◽  
P Value ◽  
Daily Dose

Abstract Despite recent advances, there is still a lack of treatment options for patients with high-risk multiple myeloma (MM), warranting the need for novel therapeutic targets. Monopolar spindle 1 (Mps1), also known as TTK protein kinase, is localized at the kinetochores and centromeres essential for the mitotic spindle checkpoint and centrosome duplication. In cancer cells, high levels of Mps1 help to support aneuploidy, a common malignant trait. In fact, high MPS1 expression correlates to unfavorable prognosis in colon and breast cancers. Here, we report the importance of Mps1 in MM and perform functional characterization of a novel Mps1 inhibitor, OSU-13. To assess the relevance of Mps1 in MM prognosis, we analyzed the CoMMpass database and examined the association of MPS1 expression with clinical outcome and genetic alterations in MM patients (n=769). Elevated MPS1 expression correlates with decreased overall survival (p-value = 0.0001) and decreased event-free survival (p-value < 0.0001). In addition, MPS1 expression is higher in high-risk MM with specific genetic alterations, such as deletion of 17p (p-value = 0.0003), Myc translocation t(8;14) (p-value = 0.02), and gain of 1q21 (p-value = 0.0001). We therefore compared MPS1 expression in eight different MM cell lines and primary CD138 - and CD138 + cells isolated from bone marrow (BM) of four recently diagnosed, untreated MM patients. Gene expression levels assessed by quantitative qPCR were normalized to 18S mRNA internal control, and relative quantification was performed using the ΔΔCt method. MPS1 expression was higher in all MM cell lines compared to the average expression level in primary CD138 + BM plasma cells, with a minimum of 3.2-fold increase in L363 and a maximum of 16.2-fold increase in MM1S. In addition, MPS1 expression was 2-fold higher in CD138 + MM cells compared to CD138 - counterparts from the BM of the same patient, suggesting its selectivity as a target. Next, we characterized the effects of OSU-13, a novel Mps1 inhibitor. In an Mps1 target engagement assay (NanoBRET™) in HEK293 cells, we measured the relative levels of OSU-13-mediated inhibition of Mps1-NanoLuc binding to a fluorescent tracer in comparison to MM clinical candidates. OSU-13 showed EC 50=10 nM, almost 10-fold lower than other agents tested. Endpoint measurement by MTT (3-[4,5- dimethylthiazol- 2-yl]-2,5-diphenyltetrazolium bromide) assay was used to evaluate cytotoxicity of OSU-13. MM cell lines were treated with increasing doses of OSU-13 for 72h and cell viability was measured. OPM-2 was the most sensitive cell line (IC 50 = 610 nM), followed by H929 (IC 50 = 1440 nM), RPMI-8226 (IC 50 = 4196 nM), and U266 (IC 50 = 5071 nM). To investigate the kinetics and the mechanisms used by OSU-13 to cause cell death, we treated H929 and OPM-2 cell lines with 500 nM of OSU-13 for 24h, 48h, 72h, 96h, and 120h and assessed both Annexin V-FITC and Zombie-Aqua staining by flow cytometry. Treatment with OSU-13 caused an increase in Annexin V staining as early as 24h in H929 (2.1-fold) and OPM-2 (1.6-fold). Maximum increase of apoptotic cells (Annexin V single positive) was observed after 72h, with a 5-fold increase in OPM-2 and 8.6-fold increase in H929 cell line. Cell death peaked at 120h, with Zombie staining 74% of OPM-2 and 60% of H929 cells. The activity of caspases 3/7 was further assessed in H929 myeloma cells at five time points after treatment with different concentrations of OSU-13. Caspases 3/7 activation started after 48h with 500nM OSU-13, and peaked after 72h post-treatment with the same dose, in agreement with the Annexin staining kinetics results. Finally, we evaluated the in vivo effect of OSU-13 in tumor growth in a H929 MM subcutaneous xenograft model in CB.17 SCID mice. Mice were treated with 5 mg/kg or 10 mg/kg daily dose of OSU-13 per oral gavage for 21 days. Treatment with 10 mg/kg daily dose of OSU-13 produced a 22% tumor growth delay (p<0.05). Collectively, we show here that elevated MPS1/TTK expression correlates with poorer prognosis in MM, and OSU-13, a novel Mps1/TTK inhibitor, induces apoptosis and cell death in MM cell lines, and decreases tumor growth in mice containing H929 cell xenografts. Together, our findings reveal that targeting the mitotic checkpoint using OSU-13 is a potential novel strategy, particularly for high risk MM. Disclosures No relevant conflicts of interest to declare.

scholarly journals Comparative Analysis on the Anti-Tumor Activity of Venetoclax and Obinutuzumab (VO) Versus Venetoclax and Rituximab (VR) in Primary CLL Cells, Ex Vivo

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5558-5558
Author(s):  
Madiha Iqbal ◽  
Aarushi Sharma ◽  
Alak Manna ◽  
Sharoon Akhtar ◽  
Taimur Sher ◽  
...  
Keyword(s):  
Cell Death ◽  
High Risk ◽  
Cell Line ◽  
Tumor Cells ◽  
Cell Lines ◽  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Single Agent ◽  
Annexin V ◽  
Intermediate Risk

Abstract INTRODUCTION: Treatment of chronic lymphocytic leukemia (CLL) has expanded significantly with the approval of multiple small molecule inhibitors. This is of great significance for patients with adverse cytogenetic features who tend to respond poorly to standard chemo-immunotherapy (CIT). While single agent ibrutinib and venetoclax (V) have shown high rates of overall response, complete remission and minimal residual disease (MRD) eradication rates remain low. This argues for further testing and development of various combination strategies. The MURANO trial compared venetoclax and rituximab (VR) versus bendamustine and rituximab (BR) in patients with relapsed/refractory CLL reporting clear superiority of VR over BR. MRD rates in the bone marrow were reported to be 27.3% for VR versus 1.5% for BR. Given much higher rates of MRD eradication with combination of small molecule inhibitors and monoclonal antibodies (mAb) compared to standard CIT, we performed a comparative investigation into the direct and immune-mediated cytolytic effects of VR versus V + Obinutuzumab (O, type II anti-CD20 mAb) in primary CLL cells and B-lymphoid cell lines. METHODS: CD19+ B-cells were isolated from PBMCs of CLL patients (N=3). For all experiments using primary CLL cells, concentration of VR and VO was 3nM (V) and 10ug /ml (R, O), respectively. For cell lines, VR and VO was used at 5uM (V) and 10ug /ml (R, O), respectively. Apoptosis was determined by annexin-V/PI staining followed by flow cytometry. Antibody-dependent cell-mediated cytotoxicity (ADCC) induced by VR and VO was assessed in Calcein AM labeled CLL cells or cell lines co-cultured with healthy donor PBMCs (E:T ratio, 40:1); complement-dependent cytotoxicity (CDC) was measured using 10% serum from a healthy human donor. RESULTS: We assessed the ability of V+/-O or V+/-R to induce apoptotic cell death in the CD20+ BCWM.1 cell line (Waldenström's macroglobulinemia [WM] phenotype) and the MEC-1 cell line (B-PLL phenotype); with CD20- RPCI-WM1 (WM cells, negative control). Notably, Bcl-2 protein is expressed in all the aforementioned cell lines. We observed that single agent V, O and R induced ~30%, 61% and 13.64% annexin V/PI positivity in BCWM.1 cells, respectively. However, a significant degree of cell death was noted in VO-treated cells (~74%) compared to VR-treated cells (~40%) (p<0.01). Next, we examined for apoptosis in MEC1 cells and noted a similar trend; where the VO combination induced markedly more cell death (~71%) than VR (~57%). Contrastingly, in RPCI-WM1 cells neither single agent O or R could elicit >12% annexin V/PI positivity and where the addition of V increased apoptosis by only 3 - 4%. We also examined the apoptotic potential of VO or VR in tumor cells from low, intermediate and high-risk CLL patients. In low and intermediate-risk CLL cells from low and intermediate-risk patients, V alone induced ~30% cell death, which increased significantly with the addition of O (VO) to between 48 - 52%. Contrastingly, the combination of VR did not induce more than 29 - 32% apoptosis. In CLL cells from high-risk patient, we noted that exposure to single agent V induced ~ 28% cell death and in VO-treated cells, this number increased to 47%. We also examined for ADCC and CDC in the same cell lines and primary CLL cells. Despite considerable variability, single agent O and VO treatment of tumor cells resulted in greater ADCC than VR treatment. By contrast, in single agent R or VR-treated cells, more CDC was observed. CONCLUSION: Our preliminary investigation in VR- and VO-treated cell lines and primary CLL cells suggests the VO combination may be superior to VR in induction of direct tumor cell death. Mechanistic experiments underway will provide further insight and can aid in design of future VO-based clinical studies in CLL. Disclosures Ailawadhi: Pharmacyclics: Research Funding; Takeda: Consultancy; Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy.

Darinaparsin in T-Cell Lymphoma (TCL) and Hodgkin Lymphoma (HL) Cells Lines and SCID Xenograft Mouse Models: Single-Agent Activity and Synergistic Cell Death Combined with a PI3K/mTOR Inhibitor

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1646-1646
Author(s):  
Ravi Dashnamoorthy ◽  
Savita Bhalla ◽  
Jennifer Crombie ◽  
Irawati Kandela ◽  
Andrew Mazar ◽  
...  
Keyword(s):  
Cell Death ◽  
T Cell ◽  
Tumor Growth ◽  
Cell Lines ◽  
Kinase Inhibitor ◽  
Cell Lymphoma ◽  
Single Agent ◽  
Annexin V ◽  
Dose Dependent

Abstract Abstract 1646 Background: Darinaparsin is a novel organic arsenical compound with potent anti-neoplastic activity. Preliminary clinical studies reported encouraging activity in patients with relapsed/refractory TCL and HL. We investigated the molecular mechanisms of action of darinaparsin in HL and TCL cell lines, the degree and method of cell death, and we investigated rational drug combinations for enhancement of therapeutic activity. Methods: TCL (Jurkat, Hut78, HH) and HL cell lines (L428, L540, L1236) were treated with darinaparsin at increasing time and concentrations. MAPK, AKT/PI3K, mTOR and relevant pathways were analyzed by Western blot. Cell viability was assessed by MTT and apoptosis with Annexin V/PI flow cytometric analysis; this was confirmed by Western for caspase activation and PARP cleavage. In vivo tumor growth inhibition and survival of SCID mice was determined using xenografts derived from Jurkat (TCL) or L540 (HL) cell lines. The in vivo studies started with 7–8 mice for each control and treatment group. Once tumor volume average reached 100–250 mm3, treatment groups were injected (5 days/week) with darinaparsin SQ daily for 3 weeks. For drug combinations, varied novel agents were rationally combined with darinaparsin (BEZ-235 [PI3K/mTOR dual kinase inhibitor], PXD101 [HDAC inhibitor], olaparib [PARP inhibitor], MG132 [proteasome inhibitor], MK-2206 [pan-AKT inhibitor], SP600125 [JNK inhibitor], SB203580 [p38 MAPK inhibitor] and PD98059 (anti-MEK small molecule). We also interrogated pertinent signaling pathways with shRNA stable knock outs (KO) +/− darinaparsin. Results: Treatment with 1–5μM Darinaparsin for 72 hours resulted in time- and dose-dependent cytotoxicity in all TCL and HL cell lines. IC50 observed with 72-hour treatment in the TCL lines Jurkat, HH, and Hut87 were 2.7μM, 3.2μM, and 6.7μM, respectively, and for the HL clines L540, L1236, and L428 were 1.3μM, 2.8μM, and 7.2 μM, respectively. Darinaparsin treatment also resulted in a dose dependent increase in apoptosis, detected by Annexin-V positivity and cleavage of PARP and caspases 3, 8 and 9 in all TCL and HL cell lines. In vivo experiments with lymphoma tumor xenografts derived from Jurkat showed significant inhibition of tumor growth (P<0.001) and improved survival (P<0.001) in darinaparsin-treated SCID mice compared with untreated control (see Figure); identical tumor reduction and survival data was noted in L540 xenografts. Next, we investigated relevant signaling pathways that were up- or down-regulated following darinaparsin exposure. We identified activation of the PI3K, MAPK, and mTOR pathways in all TCL and HD lines, expect for lack of MAPK response in L540; activation of pAKT was observed only in TCL cell lines (Jurkat and HH). Additionally, a concomitant decrease in levels of the inhibitory phosphatases (PTEN, SHP1) associated with AKT and MEK/ERK pathways were apparent in all TCL and HL cell lines treated with darinaparsin. Next, we tested cytotoxicity of darinaparsin (by MTT) in MEK and ERK KO using stably transfected shRNA in L540, Hut78 and Jurkat lines. There was minimal effect of these KOs in Jurkat, while there was increased cytotoxic effect with ERK shRNA in L540 and MEK shRNA in Hut78. Finally, we performed combination studies in L540 and Jurkat cell lines using the aforementioned novel targeted agents combined with darinaparsin in order to identify potential syngerism. Interestingly, the most notable combination was the PI3K/mTOR dual kinase inhibitor, BEZ-235, combined with darinaparsin; this resulted in synergistic cell death in Jurkat TCL cells (combination indices [CI]=0.5) and L428 HL cells (CI=0.5) cells as confirmed by MTT, Annexin-V and cleavage of PARP and caspases. Conclusions: Collectively, these data show that the novel organic arsenical, darinparsin, induced significant cell death in TLC and HL cell lines and in lymphoma SCID xenograft models. Cell death with darinaparsin appeared to be dependent in part on the MAPK pathway, however this was cell line dependent. Additionally, we identified synergistic cell death in TCL and HL cell lines when darinaparsin was combined with a PI3K/mTOR dual inhibitor. Continued study of darinaparsin in TCL and HL is warranted either as a single agent or through co-targeting with PI3K/mTOR-based therapy. Disclosures: Off Label Use: Darinaparsin for the treatment of T cell lymphoma and Hodgkin's disease.

Identification of the TAK1-NF-κB Axis As Critical Regulator of AML Stem and Progenitor Cell Survival.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2982-2982
Author(s):  
Matthieu C.J. Bosman ◽  
Jan J. Schuringa ◽  
Wim J. Quax ◽  
Edo Vellenga
Keyword(s):  
Stem Cells ◽  
Cell Death ◽  
Cell Survival ◽  
Cell Lines ◽  
Combined Treatment ◽  
Annexin V ◽  
Fold Increase ◽  
Dependent Manner ◽  
Leukemic Stem Cells ◽  
Induced Apoptosis

Abstract Abstract 2982 A small population of leukemic stem cells is resistant to chemotherapy and is responsible for the leukemic out-growth and relapse in acute myeloid leukemia (AML) patients. Evasion of apoptosis might be one of the essential mechanisms involved in this process. In order to gain more insight into the differences in the apoptotic programming between normal and leukemic (stem) cells, we recently performed gene array analysis by comparing CD34+ AML cells versus CD34+ normal bone marrow (NBM) cells. Gene ontology (GO) analysis of the differentially expressed genes between AML and NBM cells revealed differences in GO terms metabolic processes and apoptosis. In order to characterize differences in apoptotic programming in more detail 429 apoptotic related genes were selected and cluster analysis showed that CD34+ AML and CD34+ NBM cells could be separated into two distinct groups. In particular TGF-β activated kinase 1 (TAK1)/MAP3K7 was one of the apoptosis-related genes that was significantly higher expressed in CD34+ AML cells compared to CD34+ NBM cells (p = 1.8e−7). This increased expression of TAK1 could be confirmed by Q-PCR, showing an increase of on average 5.8 fold in TAK1 expression in the studied CD34+ AML cells. In mice it has been demonstrated that TAK1 is required for the survival of hematopoietic cells which is largely dependent on TNFR1 and TNFR2. In accordance with these data, we showed that TAK1 is also necessary in human hematopoiesis. Colony-forming cell (CFC) assays showed that inhibition of TAK1 in human cord blood CD34+cells, either by shRNAs targeting TAK1 or the TAK1 inhibitor 5z–7-oxozeaenol, resulted in a 2 fold reduction in CFU-GM and BFU-E frequencies compared to control cells. The efficacy was strongly further enhanced by the addition of TNFα, which resulted in a 9.4 fold decrease in CFC colonies upon TAK1 inhibition. Subsequently, we questioned whether TAK1 inhibition would affect CD34+ AML cell survival. Treatment of the AML cell lines MOLM13, OCI-M3 and HL60 with the TAK1 inhibitor 5z–7-oxozeaenol alone only induced modest effects, but in combination with TNFα for 24 hrs a strong induction of apoptosis was observed (IC50 respectively = 23nM, 215nM and 60 nM). Comparable results were observed in HL60 cells transduced with shRNAs targeting TAK1 whereby a downmodulation of TAK1 resulted in a 5.4 fold increase in Annexin V+ cells upon TNFα addition. In accordance with previous data, Western blot analysis showed that TAK1 inhibition reduced the levels of p-IκBα, p-p38, p-ERK and p-C-JUN. To test which of these pathways would be important for cell survival, AML cell lines were treated with either the p38 inhibitor SB203580, MEK/ERK inhibitor U0126, JNK inhibitor SP600125 and the NF-κB inhibitor BMS-345541, alone or in combination with TNFα. Addition of the NF-κB inhibitor BMS-345541 induced apoptosis in OCI-M3 and MOLM13 which was significantly increased in combination with TNFα (2.4 fold, p = 0.02). In contrast, inhibition of p38, MEK/ERK and JNK, either alone or in combination with TNFα, did not induce cell death in the AML cell lines. These data suggest that cell death induced by TAK1 inhibition is mainly due to inhibition of the NF-κB pathway. To determine the effects of TAK1 inhibition on primary AML cells, long-term expansion of the leukemic stem cell enriched CD34+ AML cell fraction was evaluated in MS5 stromal co-cultures in the absence or presence of TAK1 inhibitor and/or TNFα. Combined treatment for a period of 2 weeks completely abrogated the out-growth of CD34+ AML cells, indicating that both leukemic progenitors as well as leukemic stem cells were targeted. In contrast, addition of the single agents did not efficiently reduce cell growth. Similarly, downmodulation of TAK1 using shRNAs strongly sensitized primary CD34+ AML cells for TNFα-induced apoptosis, showing a 6 fold increase in Annexin V+ cells compared to control cells. Results on the in vivo efficacy of TAK1 inhibition on primary AML cells are in progress. In conclusion, our results show that TAK1 is frequently overexpressed in CD34+ AML cells, and that inhibition of TAK1 in combination with TNFα is highly efficient in inducing apoptosis of leukemic stem/progenitor cells in a NF-κB-dependent manner. Disclosures: No relevant conflicts of interest to declare.

scholarly journals EXPERIMENTAL MODELING OF APOPTOSIS UNDER CONDITIONS OF STABLE STRONTIUM EXPOSURE

2019 ◽  
Vol 21 (1) ◽  
pp. 137-140
Author(s):  
O. V. Dolgikh ◽  
N. V. Zaitseva ◽  
D. G. Dianova ◽  
A. V. Krivtsov ◽  
K. D. Starkova ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Death ◽  
Programmed Cell Death ◽  
Annexin V ◽  
Fold Increase ◽  
Apoptosis Markers ◽  
Stable Strontium ◽  
Cell Death Mechanisms ◽  
Apoptotic Pathways ◽  
And Control

Apoptosis is defined as a highly regulated form of programmed cell death with typical morphological and biochemical features. A variety of factors, including heavy metals, may influence the intensity of programmed cell death. The aim of the work was to simulate apoptosis in an in vitrosystem under the conditions of stable strontium exposure. The children’s population consuming drinking water with high strontium (Sr2+) content (n = 49) was observed. The level of lymphocyte apoptosis was determined with flow cytometry technique, by means of labeled annexin V-FITC conjugate (AnnV-FITC) and propidium iodide (PI) staining. AnnV-FITC+PI- cells were regarded as early apoptotic forms, whereas late apoptotic and/or necrotic cells were AnnV-FITC+PI+. The isolated leukocytes were incubated with Sr2+ at a concentration of 7.0 mg/l, the maximal permitted concentration (MPC) for water of aqueous objects, for 4 hours at 37 ºC. Expression of CD95 and p53 apoptosis markers was performed by flow cytometry using labeled monoclonal antibodies.In vitroexposure to strontium was associated with significantly decreased expression of apoptosisregulating factors, i.e., membrane marker CD95 and intracellular transcription protein p53, 1.56- and 1.68-fold, respectively. Meanwhile, we revealed a significantly (4.68-fold) decreased amounts of AnnV-FITC+PI--cells, as well as a statistically significant (1.35-fold) increase of the AnnV-FITC+PI+-cells. Moreover, the amounts of AnnV-FITC+ PI--lymphocytes in all samples were below the physiological ranges and control values. The number of samples with higher contents of AnnV-FITC+PI+-lymphocyte exceeding the established standards and control values, was 30.8%. Thus, it has been experimentally proven that strontium, at a concentration corresponding to MPC for water objects may significantly inhibit cell death along apoptotic pathways, with switching to necrotic cell death mechanisms, according to phosphatidylserine contents, as detected by annexin V binding test. The data have revealed an ability of strontium to have a significant effect upon the parameters of regulation and maintenance of cellular homeostasis, by influencing the apoptosis intensity, due to shifting a balance towards necrosis and reducing expression of apoptosis-regulating factors. The results of this study may be used in order to identify some marker indexes of immune disorders potentially induced by external influence of strontium upon human health under specific environmental factors.

scholarly journals Anticancer effects of mifepristone on human uveal melanoma cells

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Prisca Bustamante Alvarez ◽  
Alexander Laskaris ◽  
Alicia A. Goyeneche ◽  
Yunxi Chen ◽  
Carlos M. Telleria ◽  
...  
Keyword(s):  
Cell Death ◽  
Cell Growth ◽  
Progesterone Receptor ◽  
Dna Fragmentation ◽  
Cell Lines ◽  
Caspase 3 ◽  
Annexin V ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Free Dna

Abstract Background Uveal melanoma (UM), the most prevalent intraocular tumor in adults, is a highly metastatic and drug resistant lesion. Recent studies have demonstrated cytotoxic and anti-metastatic effects of the antiprogestin and antiglucocorticoid mifepristone (MF) in vitro and in clinical trials involving meningioma, colon, breast, and ovarian cancers. Drug repurposing is a cost-effective approach to bring approved drugs with good safety profiles to the clinic. This current study assessed the cytotoxic effects of MF in human UM cell lines of different genetic backgrounds. Methods The effects of incremental concentrations of MF (0, 5, 10, 20, or 40 μM) on a panel of human UM primary (MEL270, 92.1, MP41, and MP46) and metastatic (OMM2.5) cells were evaluated. Cells were incubated with MF for up to 72 h before subsequent assays were conducted. Cellular functionality and viability were assessed by Cell Counting Kit-8, trypan blue exclusion assay, and quantitative label-free IncuCyte live-cell analysis. Cell death was analyzed by binding of Annexin V-FITC and/or PI, caspase-3/7 activity, and DNA fragmentation. Additionally, the release of cell-free DNA was assessed by droplet digital PCR, while the expression of progesterone and glucocorticoid receptors was determined by quantitative real-time reverse transcriptase PCR. Results MF treatment reduced cellular proliferation and viability of all UM cell lines studied in a concentration-dependent manner. A reduction in cell growth was observed at lower concentrations of MF, with evidence of cell death at higher concentrations. A significant increase in Annexin V-FITC and PI double positive cells, caspase-3/7 activity, DNA fragmentation, and cell-free DNA release suggests potent cytotoxicity of MF. None of the tested human UM cells expressed the classical progesterone receptor in the absence or presence of MF treatment, suggesting a mechanism independent of the modulation of the cognate nuclear progesterone receptor. In turn, all cells expressed non-classical progesterone receptors and the glucocorticoid receptor. Conclusion This study demonstrates that MF impedes the proliferation of UM cells in a concentration-dependent manner. We report that MF treatment at lower concentrations results in cell growth arrest, while increasing the concentration leads to lethality. MF, which has a good safety profile, could be a reliable adjuvant of a repurposing therapy against UM.

scholarly journals The Comprehensive Roles of ATRANORIN, A Secondary Metabolite from the Antarctic Lichen Stereocaulon caespitosum, in HCC Tumorigenesis

Molecules ◽  
2019 ◽  
Vol 24 (7) ◽  
pp. 1414 ◽  
Author(s):  
Young-Jun Jeon ◽  
Sanghee Kim ◽  
Ji Hee Kim ◽  
Ui Joung Youn ◽  
Sung-Suk Suh
Keyword(s):  
Cell Death ◽  
Cell Lines ◽  
Genetic Diseases ◽  
Annexin V ◽  
Cancer Therapeutics ◽  
Metastatic Potential ◽  
Anti Cancer ◽  
Experimental Approaches ◽  
The Antarctic ◽  
Death Determination

Hepatocellular carcinoma (HCC) is one of the most deadly genetic diseases, but surprisingly chemotherapeutic approaches against HCC are only limited to a few targets. In particular, considering the difficulty of a chemotherapeutic drug development in terms of cost and time enforces searching for surrogates to minimize effort and maximize efficiency in anti-cancer therapy. In spite of the report that approximately one thousand lichen-derived metabolites have been isolated, the knowledge about their functions and consequences in cancer development is relatively limited. Moreover, one of the major second metabolites from lichens, Atranorin has never been studied in HCC. Regarding this, we comprehensively analyze the effect of Atranorin by employing representative HCC cell lines and experimental approaches. Cell proliferation and cell cycle analysis using the compound consistently show the inhibitory effects of Atranorin. Moreover, cell death determination using Annexin-V and (Propidium Iodide) PI staining suggests that it induces cell death through necrosis. Lastly, the metastatic potential of HCC cell lines is significantly inhibited by the drug. Taken these together, we claim a novel functional finding that Atranorin comprehensively suppresses HCC tumorigenesis and metastatic potential, which could provide an important basis for anti-cancer therapeutics.

scholarly journals “Don’t Eat Me” Signals of Neuroblastoma by CD47 for Immune Escape: A Novel Prognostic Biomarker

Proceedings ◽  
2018 ◽  
Vol 2 (25) ◽  
pp. 1538
Author(s):  
Safiye Aktas ◽  
Ayse Pinar Ercetin Ozdemir ◽  
Efe Ozgur Serinan ◽  
Zekiye Altun ◽  
Nur Olgun
Keyword(s):  
High Risk ◽  
Immune Escape ◽  
Prognostic Biomarker ◽  
Immunohistochemical Analysis ◽  
Risk Groups ◽  
Genetic Alterations ◽  
High Risk Group ◽  
Low Risk ◽  
P Value ◽  
Mycn Amplification

Recent studies have shown that cancer cells can deceive phagocytosing macrophage cells through the CD47 protein which gives the message “don’t eat me” or “don’t kill me” to immune components. The efficacy of anti-CD47 treatment approach was shown in cancers such as, non-small cell lung cancer, non-Hodgkin lymphoma, ovarian cancer, and breast cancer. The studies on the immunobiology of neuroblastoma has increased as monoclonal antibody based immunotherapy has shown to be effective in high-risk patients such as anti disialoganglioside. Therefore, the aim of this study was to evaluate the levels of CD47 protein expression among neuroblastoma patients with different risk groups and genetic alterations. This study included paraffin-embedded tumor tissues of 66 neuroblastoma patients (28 girls, 38 boys) with an age range of 0.5 to 108 months with a mean value of 24.9 (±23.5). According to risk classifications 21 were at low risk (31, 8%), 24 were at intermediate risk (36, 4%) and 19 were at high-risk (28, 8%) groups. These samples were evaluated for MYCN amplification, 1p36 LOH, 11q23 deletion and 17q25 gain by real-time PCR. In addition, CD47 expression status (positive or negative) was detected by immunohistochemical analysis. All data was analyzed with Chi-Square and Mann-Whitney U non-parametric tests within SPSS program, version 22.0. p value lower than 0, 5 was found statistically significant. According to the results, patients at low risk did not express CD47, while patients at high-risk group were mostly expressing CD47 (p = 0.049). MYCN amplification positive patients were expressing CD47 protein (p = 0.046). Patients without 17q25 gain were found to be expressing CD47 protein (p = 0.006). In addition, CD47 expression was increasing as age was getting higher in terms of months (p = 0.018). The findings of this study suggest that positive expression pattern of CD47 may be a poor prognostic biomarker especially in high risk 17q gain negative neuroblastoma patients.

Targeting xIAP Is More Important Than Bcl-Xl in Lymphomas with Constitutive Activation of NF-kB.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2390-2390
Author(s):  
Yanjuan He ◽  
Joan Cain ◽  
Lee Ratner ◽  
Leon Bernal-Mizrachi
Keyword(s):  
Cell Lines ◽  
Annexin V ◽  
Fold Increase ◽  
Daudi Cell ◽  
Xiap Expression ◽  
Apoptotic Effect ◽  
Effective Doses ◽  
Induction Of Apoptosis ◽  
Apoptotic Pathways

Abstract Pathways resulting in resistance to apoptosis are essential to the process of lymphomagenesis. One such pathway, the nuclear factor-kB (NFkB), has been shown to be a key element in coordinating the anti-apoptotic effect of these malignancies. However the mechanisms used by which NFkB prevents apoptosis are not well understood. It has been suggested that NFkB inhibits activation of the intrinsic, extrinsic and common apoptotic pathways. Previous work in our lab using two different virally mediated lymphoma models (Tax/HTLV1 and LMP1/EBV driven tumors) has identified two candidates that could explain these results: X chromosome-linked inhibitor of apoptosis (xIAP) and BCL-xL. Although the current literature extensively demonstrates the role of BCL-xL in lymphomas, little is known about the importance of xIAP in these malignancies. To answer this question we tested the apoptotic effect of etoposide or tumor necrosis factor (TNF) after knocking down bcl-xL and xIAP expression in our lymphoma models (SC and Daudi cell lines) using a lentivirus expressing siRNAs. After 24 hours of treatment with etoposide and TNF, we measured apoptosis by flow cytometry using double staining with Annexin V-Alexa Fluorescense and propidium iodide. Interestingly, xIAP siRNA-expressing cell lines demonstrated 2–4 fold increase in the induction of apoptosis after treatment with etoposide as compared to a nearly 2 fold increase in those expressing Bcl-xL siRNA (see Table below). No synergism was seen after treatment with TNF. Based on this finding, we then tested a novel small molecule, homolog smac, (SHC, kindly provided by Dr. PG Harren) to determine the possible therapeutic effect of xIAP inhibitors. After titration, the two most effective doses were selected (25 μM and 50 μM) to treat Daudi cell lines for 24hrs, with either etoposide or TNF. At doses of 25 μM , we observed a 2 fold increase in the induction of apoptosis produced by etoposide compared to that seen in control (DMSO + etoposide) or SHC alone and no synergism with TNF confirming the siRNA data. More importantly, at doses of 50 μM, SHC alone demonstrated activity with a 5 fold increase in apoptosis and a nearly 10 fold increase as compared to control (DMSO) when etoposide was added. Overall, we have demonstrated that xIAP and bcl-xL are important in mediating NFkB-resistance to apoptosis. However, our findings suggested that xIAP is a more potent anti-apoptotic signal and opens the door for further drug development aimed at testing xIAP-inhibitors in lymphomas. Induction of Apoptosis in xIAP or Bcl-xL siRNA expressing cell lines siRNA/Compound Etoposide TNF Untreated xIAP 43.1 ± 17.6 17.04 ± 1.4 14.3 ± 2 SC Bcl-xL 18.39± 3.7 9.4 ± 0.22 12.5 ± 2.7 Luc/DMSO 14.9 ± 1.8 14.4 ± 5.6 14.03 ± 1.25 xIAP 9.2 ± 3.2 4.7 ± 0.48 4.6 ± 0.44 Bcl-xL 8.9 ± 0.5 5.3 ± 1.7 4.16 ± 0.4 Daudi Luc/DMSO 5.49 ± 1.71 4.28 ± 0.5 6.2 ± 0.9 SHC 25 μM 20.07 ± 4.8 12.8 ± 3.9 12.1 ± 3.2 SHC 50 μM 47.7 ± 14.55 38.3 ± 0.99 32.7 ± 8.99

Histone Deacetylase Inhibitor (HDACi) PCI-24781 and Bortezomib Result in Synergistic Apoptosis in Hodgkin Lymphoma (HL) and Non-Hodgkin’s Lymphoma (NHL) Cell Lines: Investigation of Cell Death and NFKB-Mediated Pathways.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3589-3589 ◽  
Author(s):  
Savita Bhalla ◽  
Kevin David ◽  
Lauren Mauro ◽  
Sheila Prachand ◽  
Mint Sirisawad ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Cell Death ◽  
Cell Lines ◽  
Reactive Oxygen ◽  
Fold Increase ◽  
Oxygen Species ◽  
Minimal Cell ◽  
Histone 3 ◽  
Dichlorofluorescein Diacetate ◽  
20 Nm

Abstract HDACi block cancer cell proliferation by mechanisms that involve epigenetic gene regulation leading to cell growth arrest, differentiation, and apoptosis. Bortezomib inhibits NFKB signaling and induces apoptosis. Furthermore, anti-tumor activity of HDACi and bortezomib both depend in part on reactive oxygen species (ROS)-mediated pathways. Both have activity in NHL. We reasoned that these agents may be synergistic in part due to their dependence on overlapping pathways. We investigated the biology of PCI-24781, a pan-HDACi currently in clinical trials, and bortezomib both alone, and in combination, in HL (L428) and NHL cell lines (HF1, Ramos, & SUDHL4). Cells were incubated with increasing concentrations of PCI-24781 and bortezomib (0.25–2.0μM and 2.5–20nM, respectively) for 24–72 hour (hr). Apoptosis was determined by fluorescence-activated cell sorting (FACS) using AnnexinV-FITC/propidium iodide (AnnexinV+/PI+) staining. Reactive oxygen species (ROS) were measured by oxidation of 2′7′dichlorofluorescein diacetate (H2DCFDA) to DCF and detected by FACS. Downstream targets of NFKB such as NFKB1, Myc and IL-8 were measured in Ramos using quantitative real time polymerase chain reaction (RT-PCR) following 24 hr incubation of cells with PCI-24781 and bortezomib alone, and in combination. Dose-dependent apoptosis was seen with PCI-24781 and bortezomib alone in all HL and NHL cell lines. IC70 (dose to achieve 70% AnnexinV+/PI+) was 1μM for PCI-24781 and 2μM for L428. With bortezomib, the IC50 was 10nM in Ramos, HF1, and SUDHL4 and 20 nM in L428. The combination of PCI-24781 and bortezomib resulted in synergistic apoptosis (combination index &lt;0.2) in all 3 NHL cell lines (IC80=0.25μM PCI-24781/5nM bortezomib) and L428 (IC80=0.5μM PCI-24781/10nM bortezomib) compared with minimal cell death using each agent alone at those concentrations. Furthermore, immunoblots of L428 and Ramos showed enhanced caspase 3 and caspase 8 cleavage with the combination of PCI-24781 and bortezomib compared to either agent alone, suggesting that the synergy seen was in part caspase-dependent. HL and NHL cell lines showed a 3- to 4-fold increase in ROS with PCI-24781 or bortezomib alone and in combination at 24hr. Moreover, we found that hyperacetylation of histone-3 and histone-4 on immunoblots of cells treated with combination PCI-2478/bortezomib was significantly increased compared to PCI-24781 alone. Finally, we found that in Ramos cells PC-24781/bortezomib together resulted in downregulation of NFKB targets NFKB1 and Myc, but not IL-8. We conclude that PCI-24781 and bortezomib are active in lymphoma cell lines and that the combination results in synergistic apoptosis. Apoptosis was accompanied by caspase activation and synergistic downregulation of the NFkB pathway. These data have important clinical implications for NHL and HL.

ITF2357, a Novel Histone Deacetylase Inhibitor, Demonstrates Significant Apoptotic Activity Against Human Multiple Myeloma Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4791-4791
Author(s):  
Michael Kline ◽  
Kathleen A. Donovan ◽  
John A. Lust
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Histone Deacetylase ◽  
Cell Lines ◽  
Hydroxamic Acid ◽  
Stromal Cell ◽  
Hdac Inhibitors ◽  
Annexin V ◽  
Myeloma Cells

Abstract We have evaluated the efficacy of a novel hydroxamic acid-derived histone deacetylase (HDAC) inhibitor, ITF2357, to promote cell death in multiple myeloma (MM) cells. HDAC inhibitors, which promote histone hyperacetylation and increase gene expression, have been evaluated as candidate agents for combating malignancies because they impact the expression of genes related to proliferation, differentiation, and survival. Exposure of MM cell lines to 1 micromolar ITF2357 led to dramatically increased levels of histone acetylation at 4 hours and 8 hours by Western analysis. Sub-micromolar concentrations of ITF2357 promoted time- and concentration-dependent cell death in MM cell lines. Using 500 nM ITF2357, a concentration potentially achievable in vivo, viability of KAS-6/1 IL-6 dependent myeloma cells was reduced to 28% of control at 24 hrs and 2% of control at 48 hours (Figure 1). In contrast, viability of normal PBMCs was 100% at 24 hours and 80% at 48 hours (Figure 2). U266 and 8226 myeloma cells were found to be sensitive to ITF-2357 in a similar fashion with U266 being least sensitive. Cell death proceeded via apoptosis as measured using Annexin V/propidium iodide staining. ITF 2357 was superior to suberoylanilide hydroxamic acid (SAHA) at inhibition of stromal cell IL-6 production. IL-1beta (10 pg/ml) was used to stimulate bone marrow stromal cell IL-6 production (105 ng/ml) after 48 hours. Concentration of ITF2357:Stromal Cell IL-6 production after 48 hours were as follows - 10 nM: 78 ng/ml; 100 nM: 79 ng/ml; 1000 nM; 32 ng/ml. SAHA at similar concentrations showed no significant decrease in stromal cell IL-6 production compared with the no drug control. In summary, ITF2357 induces significant myeloma cell apoptosis and can inhibit stromal cell IL-6 production. It represents an attractive therapeutic candidate for MM clinical trials. Figure Figure Figure Figure

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