Selective Death of Leukemia Initiating Cells Induced By Deferasirox

Abstract Despite high remission rate after therapy, only 30-40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. The main cause of treatment failure is thought to be insufficient eradication of leukemia initiating cells (LIC). Identifying drugs that can efficiently eradicate LICs is therefore imperative. HIF1α is essential for LIC maintenance and targeting HIF1α selectively eliminates LIC. Deferasirox is an iron chelator used to reduce chronic iron overload in patients receiving long-term blood transfusions. Our aim is to study the ability ofdeferasirox to induce apoptosis and to target HIF1α in AML LICs. CD34+CD38- LIC-like cells, sorted from the KG1a cell line, exhibited increased sensitivity to deferasirox with an IC50 of 1.3μM compared to 8.9μM calculated for the more mature CD34+CD38+ cells. The LIC-like cells, which were more sensitive to deferasirox, were less sensitive to ARA-C compared to the CD34+CD38+ cells. Deferasirox was >2-fold more efficient in inducing apoptosis in the CD34+CD38− cells, compared to the CD34+CD38+ cells (74±7.2% & 32±6.2% apoptosis, respectively). Deferasirox demonstrated a synergistic cytotoxic effect with ARA-C on both the CD34+CD38− and CD34+CD38+ KG1a cell fractions. Similar results were observed with CD34+CD38-CD123+ LICs and CD34+CD38+ CD123+ progenitor cells isolated from AML patients. As shown with the cell line, AML patient LICs were 2-fold more sensitive to deferasirox treatment showing a 62±15% induction of cell death compared to only a 34±9% induction in leukemic progenitor cell death (p<0.01). The increase in cell death was accompanied by an increment of reactive oxygen species (ROS) levels which was more prominent in the LICs compared to the progenitor cells. Furthermore, deferasirox enhanced the cytotoxic effects of ARA-C on both the LSCs and the leukemic progenitor cells. These data indicate that deferasirox is highly cytotoxic to AML cells; however, it is significantly more specific to AML initiating cells. In addition, deferasirox exhibited significant inhibitory effect on the proliferation of both the CD34+CD38− and CD34+CD38+ KG1a cell fractions accompanying by an S-phase arrest. The inhibition of proliferation was also demonstrated using proliferation dye and colony assay on both the LIC-like cells and the LICs isolated from AML patient (p<0.001). Since deferasirox is an NFkB inhibitor which regulates HIF1α levels, and since HIF1α is selectively activated in AML stem cells and is essential for maintenance of these cells, we studied the effect of deferasirox on this pathway. We found that NFkB translocation to the nucleus was reduced by over 80% and as a result the binding of NFkB to the HIF1α promoter was decreased in the LIC-like cells treated with deferasirox. HIF1α was downregulated transcriptionally (~45% reduction) and translationally (~55% reduction) in the LIC-like cells while it was upregulated in the CD34+CD38+KG1a cells following deferasirox treatment. In order to understand which of deferasirox's properties is crucial for its apoptotic effect, we used the iron chelator, deferipron, which does not effect on NFkB or HIF1α and siHIF1α. Each one of these treatments alone did not lead to an apoptotic effect in LIC-like cells or in the CD34+CD38+ kg1a cells. Conversely, the combination of the two lead to an increase in apoptosis in the LIC-like cells suggesting that only a combination of iron chelation and HIF1α reduction is sufficient to result in an apoptotic response. The apoptotic effect wasn't observed in the CD34+CD38+ fraction. These data hint that AML-LICs are selectively targeted by deferasirox. We describe a novel and unique anti-LIC property of deferasirox, which was originally developed as an iron chelator. We found clinically relevant concentrations of deferasirox to be cytotoxic in vitro to AML progenitor cells but even more potent against LICs. We believe that deferasirox exerts its cytotoxic effect, at least in part, by downregulating HIF1α levels in the LIC population. Pending further characterization, deferasirox can be considered as a potential therapeutic agent for eradicating LICs. Disclosures No relevant conflicts of interest to declare.

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Anti-mutagenic and synergistic cytotoxic effect of cisplatin and Honey Bee venom on 4T1 invasive mammary carcinoma cell line

10.1101/168542 ◽

2017 ◽

Author(s):

Faranak Shiassi Arani ◽

Latifeh Karimzadeh ◽

Seyed Mohammad Ghafoori ◽

Mohammad Nabiuni

Keyword(s):

Cell Death ◽

Cell Line ◽

Honey Bee ◽

Mtt Assay ◽

Sodium Azide ◽

Mammary Carcinoma ◽

Ames Test ◽

Cytotoxic Effect ◽

Mutagenic Activity ◽

Bee Venom

ABSTRACTHoney Bee Venom has various biological activities such as inhibitory effect on several types of cancer. Cisplatin is an old and potent drug to treat the most of cancer. Our aims in this study were determination of the anti-mutagenic and cytotoxic effects of HBV on mammary carcinoma, lonely and in combination with cisplatin. In this study 4T1 cell line were cultured and incubated at 37 C in humidified CO2-incubator. The cell viabilities were examined by MTT assay. Also HBV was screened for its anti-mutagenic activity against sodium azide by Ames test. The result showed that 6μg/ml HBV, 20μg/ml cisplatin and 6μg/ml HBV with 10μg/ml cisplatin can induce an approximately 50% 4T1 cell death. 7mg/ml HBV with the inhibition of 62.76% sodium azide showed high potential in decreasing the mutagenic agents. MTT assay demonstrated that HBV and cisplatin can cause cell death in a dose-dependent manner. The cytotoxic effect of cisplatin is also promoted by HBV. Ames test results indicated that HBV can inhibit sodium azide as a mutagenic agent. Anti-mutagenic activity of HBV was increased significantly in presence of S9 mix. Hence, our findings reveal that HBV can enhance the cytotoxic effect of cisplatin drug and it has cancer preventing effects.

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Antimutagenic and Synergistic Cytotoxic Effect of Cisplatin and Honey Bee Venom on 4T1 Invasive Mammary Carcinoma Cell Line

Advances in Pharmacological Sciences ◽

10.1155/2019/7581318 ◽

2019 ◽

Vol 2019 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Faranak Shiassi Arani ◽

Latifeh Karimzadeh ◽

Seyed Mohammad Ghafoori ◽

Mohammad Nabiuni

Keyword(s):

Cell Death ◽

Cell Line ◽

Honey Bee ◽

Mtt Assay ◽

Mammary Carcinoma ◽

Ames Test ◽

Cytotoxic Effect ◽

Bee Venom ◽

Antimutagenic Activity ◽

Dependent Manner

Introduction. Honey bee venom (HBV) has various biological activities such as the inhibitory effect on several types of cancer. Cisplatin is an old and potent drug to treat most of the cancers. Our aim in the present study was to determine antimutagenic and cytotoxic effects of HBV on mammary carcinoma, exclusively and in combination with cisplatin. Methods. In this study, 4T1 cell line was cultured in RPMI-1640 with 10% fetal bovine serum (FBS), at 37°C in humidified CO2 incubator. The cell viabilities were examined by the MTT assay. Also, HBV was screened‏ for its antimutagenic activity via the Ames test. The results were assessed by SPSS software version 19 and one-way ANOVA method considering p<0.05 level of significance. Results. The results showed that 6 mg/ml of HBV, 20 μg/ml of cisplatin, and 6 mg/ml HBV with 10 μg/ml cisplatin could induce approximately 50% of 4T1 cell death. The concentration 7 mg/ml of HBV with of 62.76% inhibitory rate showed the highest antimutagenic activity in comparison with other treatment groups. Conclusions. The MTT assay demonstrated that HBV and cisplatin could cause cell death in a dose-dependent manner. The cytotoxic effect of cisplatin also promoted by HBV. Ames test outcomes indicated that HBV could act as a significant mutagenic agent. The antimutagenic activity of HBV was increased considerably in the presence of S9 mix. Therefore, our findings have revealed that HBV can enhance the cytotoxic effect of cisplatin drug and its cancer-preventing effects.

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Second Mitochondria-Derived Activator of Caspases Mimetic Compounds (Smac) Enhance Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) Pro-Apoptotic Effect On Human Myeloid Leukemia Cell Lines.

Blood ◽

10.1182/blood.v114.22.2756.2756 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2756-2756

Author(s):

Federica Servida ◽

Cinzia Scavullo ◽

Daniele Lecis ◽

Pierfausto Seneci ◽

Carmelo Drago ◽

...

Keyword(s):

Progenitor Cells ◽

Myeloid Leukemia ◽

Cytotoxic Effect ◽

Hematological Malignancies ◽

Leukemia Cell ◽

Hematopoietic Progenitor ◽

Apoptotic Effect ◽

Smac Mimetics ◽

Smac Mimetic ◽

Necrosis Factor

Abstract Abstract 2756 Poster Board II-732 The Inhibitor of Apoptosis Proteins (IAP) are important regulators of programmed cell death. Among them, XIAP, which is characterized by 3 tandem BIR domains selectively blocking caspases 3, 7 and 9, is the most potent and is over-expressed in several hematological malignancies. Its activity is antagonized by Second Mitochondria-derived Activator of Caspases (Smac) and also by small molecules mimicking Smac able to induce apoptosis in tumor cells, alone or in combination with other drugs. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a pro-apoptotic cytokine, is capable of triggering programmed death in cancer cells, where it synergizes with chemotherapeutic agents and/or radiation by several malignant cell-type specific mechanisms, and it is currently under evaluation in clinical trials for its strong pro-apoptotic activity. Here we describe the pro-apoptotic effect of newly synthesized monovalent and bivalent Smac-mimetic compounds tested for cytotoxicity on a cohort of human leukemic cell lines over-expressing XIAP (HL60, K562 and Jurkat) as well as on normal CD34+ hematopoietic progenitor cells, alone or in combination with TRAIL. The Smac-mimetics, designed and produced by the University of Milan Center for biomolecular Interdisciplinary Studies and Industrial applications, were dissolved in dimethylsulfoxide (DMSO) to obtain a 10 mM solution and stored at −20°C. Drug stocks were diluted with phosphate buffer (PBS) prior to their use. The cells were treated with 0.1 nM – 50 μM doses for up to 72 hours. TRAIL was kindly provided by Prof. Henning Walczak (Imperial College, London, UK). In combined treatments, Smac-mimetics and TRAIL were used simultaneously. The cytotoxic effect was evaluated by a colorimetric assay for the quantification of cell proliferation and viability based on the cleavage of the WST-8 tetrazolium salt by mitochondrial dehydrogenases. The effect on fresh human CD34+ hematopoietic normal progenitor cells selected from healthy donors' peripheral blood stem cells (PBSC) was assayed by myeloid colony (CFU-GM) formation. Apoptosis was determined by flow cytometric analysis of DNA fragmentation and by Western blot analysis of caspases activation and PARP cleavage. The combination of some of our Smac-mimetics (both monomers and dimers) with TRAIL highly enhanced the inhibition of proliferation of the chronic myelogenous leukemia cell line K562, which was shown to be resistant to TRAIL single agent. In particular, a synergistic effect was observed in combined treatment with Smac012 10 μM and TRAIL 50 ng/ml (R Kern index = 6.2). Our Smac-mimetics, particularly the dimeric ones, were capable of inducing apoptosis, as demonstrated by DNA fragmentation and accumulation of cleaved PARP, caspase 8 and caspase 3, especially when administrated in combination with TRAIL. No cytotoxic effect was observed on normal CD34+ progenitor cells by Smac mimetics at doses ranging from 40 μM to 70 μM, even normal controls when were treated simultaneously with TRAIL. As many hematological malignancies are resistant to TRAIL alone, thus limiting its therapeutic effectiveness, the observed strong synergistic effect with Smac mimetic compounds not affecting the normal hematopoietic progenitor cell compartment might be of great consequence for the development of innovative potent pro-apoptotic drug combinations in myeloid leukemia treatment. Disclosures: No relevant conflicts of interest to declare.

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Antioxidant and Proapoptotic Activities ofSclerocarya birrea[(A. Rich.) Hochst.] Methanolic Root Extract on the Hepatocellular Carcinoma Cell Line HepG2

BioMed Research International ◽

10.1155/2015/561589 ◽

2015 ◽

Vol 2015 ◽

pp. 1-11 ◽

Cited By ~ 21

Author(s):

Maria Francesca Armentano ◽

Faustino Bisaccia ◽

Rocchina Miglionico ◽

Daniela Russo ◽

Nicoletta Nolfi ◽

...

Keyword(s):

Cell Death ◽

Cell Line ◽

Hepg2 Cells ◽

Cytotoxic Effect ◽

Antioxidant Activities ◽

Dermal Fibroblasts ◽

Root Extract ◽

Dependent Manner ◽

Mitochondrial Membrane Depolarization

The main goal of this study was to characterize thein vitroantioxidant activity and the apoptotic potential ofS. birreamethanolic root extract (MRE). Among four tested extracts, obtained with different solvents, MRE showed the highest content of polyphenols, flavonoids, and tannins together with antioxidant activities tested with superoxide, nitric oxide, ABTS, and beta-carotene bleaching assays. Moreover, the cytotoxic effect of MRE was evaluated on the hepatocarcinoma cell line HepG2. In these cells, MRE treatment induced apoptosis and generated reactive oxygen species (ROS) in dose-dependent manner. The cytotoxic effect promoted by MRE was prevented by pretreatment of HepG2 cells with N-acetyl-L-cysteine (NAC), suggesting that oxidative stress was pivotal in MRE-mediated cell death. Moreover, we showed that the MRE treatment induced the mitochondrial membrane depolarization and the cytochromecrelease from mitochondria into the cytosol. It suggests that the apoptosis occurred in a mitochondrial-dependent pathway. Interestingly, MRE showed a sensibly lower cytotoxicity, associated with a low increase of ROS, in normal human dermal fibroblasts compared to HepG2 cells. It is suggested that the methanolic root extract ofS. Birreais able to selectively increase intracellular ROS levels in cancer cells, promoting cell death.

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In vitro Cytotoxicity of Bisphenol A in Monocytes Cell Line

Blood Purification ◽

10.1159/000437039 ◽

2015 ◽

Vol 40 (2) ◽

pp. 180-186 ◽

Cited By ~ 11

Author(s):

Mauro Neri ◽

Grazia Maria Virzì ◽

Alessandra Brocca ◽

Francesco Garzotto ◽

Jeong Chul Kim ◽

...

Keyword(s):

Bisphenol A ◽

Cell Line ◽

Direct Contact ◽

Cytotoxic Effect ◽

In Vitro Cytotoxicity ◽

Maximum Value ◽

In Vitro Investigation ◽

Apoptotic Effect ◽

Monocyte Cell

Background/Aim: Bisphenol A (BPA) is used in the production of many plastics, which are used to build biomaterials that sometimes are in direct contact with blood. It is believed that the release of BPA into bloodstream may give rise to cytotoxic events for blood components. The aim of the present study was to perform an in vitro investigation of the observable cytotoxic effect of BPA, at increasing concentrations, on the monocyte cell line. Methods: We incubated in vitro monocyte cells (U937) for 24 h in cell line medium samples (RPMI 1640) at different concentrations of BPA. We then generated curves to evaluate viability, necrosis and apoptosis of monocytes against increasing concentrations of BPA. Results: The percentage values of concentrations of BPA corresponding to 50% of the viability and necrosis of the monocytes were 1.39 and 1.48 ng/ml, respectively. Based on our observations, we reported an increasing cytotoxic effect for higher concentrations. The apoptotic effect reached the maximum value at BPA concentration of 1.5 ng/ml; at still higher concentrations, we observed a predominantly necrotic cell death. Conclusion: Viability, necrosis and apoptosis of monocytes are strongly and positively correlated with BPA concentration. A direct contact of such compound with biological components of blood may lead to high levels of cytotoxicity, and require us to evaluate additional factors while judging the bio-incompatibility of BPA.

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Establishment of a Therapeutic Anti-Pan HLA-Class II Monoclonal Antibody That Directly Induces Lymphoma Cell Death Via Large Pore Formation

Blood ◽

10.1182/blood.v124.21.5488.5488 ◽

2014 ◽

Vol 124 (21) ◽

pp. 5488-5488

Author(s):

Shuji Matsuoka ◽

Yasuyuki Ishii ◽

Atsuhito Nakao ◽

Hiroshi Masutani ◽

Satoshi Takahashi ◽

...

Keyword(s):

Monoclonal Antibody ◽

Cell Death ◽

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

Class Ii ◽

Hla Class Ii ◽

Lymphoma Cell ◽

Death Process

Abstract To develop a new therapeutic monoclonal Antibody (mAb) for Hodgkin lymphoma (HL), we immunized a BALB/c mouse with live HL cell lines, alternating between two HL cell lines. After hybridization, we screened the hybridoma clones by assessing direct cytotoxicity against a HL cell line not used for immunization. We developed this strategy for establishing mAb to reduce the risk of obtaining clonotypic mAb specific for single HL cell line. A newly established mouse anti-human mAb (4713) triggered cytoskeleton-dependent, but complement- and caspase-independent, cell death in HL cell lines, Burkitt lymphoma cell lines, and advanced adult T-cell leukemia cell lines. Intravenous injection of mAb 4713 in tumor-bearing SCID mice improved survival significantly. mAb 4713 was revealed to be a mouse anti-human pan-HLA class II mAb. Treatment with this mAb induced the formation of large pores on the surface of target lymphoma cells within 30 min. This finding suggests that the cell death process induced by this anti-pan HLA-class II mAb may involve the same death signals stimulated by a cytolytic anti-pan MHC class I mAb that also induces large pores formation. This multifaceted study supports the therapeutic potential of mAb 4713 for various forms of lymphoma. Disclosures No relevant conflicts of interest to declare.

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MJ05, a Novel p53 Activating Compound, Effectively and Selectively Eliminates Human CML Stem/Progenitor Cells

Blood ◽

10.1182/blood.v122.21.1464.1464 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1464-1464

Author(s):

Su Chu ◽

Catherine Drummond ◽

Ling Li ◽

YinWei Ho ◽

Sonia Lain ◽

...

Keyword(s):

Stem Cells ◽

Progenitor Cells ◽

Kinase Inhibitors ◽

Conflicts Of Interest ◽

P53 Protein ◽

Wild Type ◽

Inhibition Of Proliferation ◽

Protein Levels ◽

Cd34 Cells

Abstract Tyrosine kinase inhibitors (TKI) are the mainstay of CML treatment but fail to eliminate leukemia stem cells (LSC), leading to high risk of disease recurrence when treatment is stopped. There is considerable interest in developing new strategies to target CML LSC. We have previously shown that p53 activation following SIRT1 deacetylase inhibition can inhibit growth and survival of TKI-treated CML LSC (Cancer Cell 2012, 21:266). We are therefore interested in investigating other strategies to activate p53 as potential approaches to target CML LSC. While conducting a screen of 20,000 small molecules for ability to activate p53-dependent transcription in TP53 wild-type ARN8 human melanoma cells we identified MJ05 amongst the top-ranking compounds. Importantly MJ05 did not activate p53-dependent transcription in T22 fibroblast cells. Increased p53 protein levels in ARN8 cells were seen within 6 hours and were accompanied by an increase in p21, pig3 and mdm2 mRNA and protein levels. Dependency on p53 was confirmed using p53-null and wild-type H1299 cells. Activation of p53 occurred without concurrent increase in DNA damage evidence by g-H2AX labeling, increased p53 Ser 15-phosphorylation, or inhibition of p53-HDM2 interaction. MJ05 treatment inhibited S-Phase progression of ARN8 cells without inhibition of ATM, ATR or DNA-PK phosphorylation, suggesting a unique mechanism of action. We tested the effect of MJ05 on primary normal or CML CD34+ cell by itself and in combination with TKI inhibitor Nilotinib, and compared with effects on Nutlin, a well studied inhibitor of p53-HDM2 interactions. Apoptosis was assessed by Annexin V labeling, proliferation by CFSE labeling, and colony forming cell (CFC) frequency, in methylcellulose progenitor assays. Treatment with MJ05 (5 and 10μM), with or without Nilotinib (1μM), for 72 hours in the presence of low concentrations of growth factor significantly and selectively increased apoptosis, inhibited proliferation and reduced colony CFC frequency in CML CD34+ cells compared to normal CD34+ cells. Combination of MJ05 with Nilotinib (1μM) resulted in significant increase in apoptosis of CML but not normal CD34+ cells. In contrast treatment with Nutlin (2 and 5 μM) resulted in similar increase in apoptosis in CML and normal CD34+ cells. We next evaluated the effect of treatment with MJ05 (10μM), Nilotinib and the combination for 72 hours on purified CML and normal CD34+CD38- stem/primitive progenitor cells and CD34+CD38+ committed progenitor cells. MJ05 significantly enhanced apoptosis of CML but not normal CD34+CD38- cells and CD34+CD38+ cells. Apoptosis was further enhanced by combination with Nilotinib (Table). MJ05 also resulted in significant reduction of proliferation in CML CD34+38+ and CD34+38- cells, with significantly less inhibition of proliferation of normal cells. MJ05 treatment markedly reduced CFU-GM and BFU-E generation from CML compared to normal CD34+CD38- and CD34+CD38+ cells. The combination with Nilotinib resulted in almost complete abrogation of CML CFC (Table). MJ05 resulted in significantly less inhibition of normal CFC, with greater effect on normal BFU-E compared to CFU-GM. Ongoing xenograft experiments are testing the effect of in vitro treatment with MJ05, Nilotinib or the combination on engraftment of CML and normal stem cells in NSG mice. Our studies indicate that MJ05, a unique, potent and selective p53 activating compound, is remarkably effective in inducing apoptosis and inhibiting growth of primitive CML stem/progenitor cells by itself and to an even greater extent in combination with Nilotinib. MJ05 treatment has significantly lesser effects on normal stem cells, and may offer a promising approach to selectively target CML LSC in combination with Nilotinib.Table 1NormalCMLUntreatedMj05+NilMj05+NilUntreatedMj05NilMj05+NilCD34+38+% Apoptosis4.5±0.19.0±1.14.13±018.8±7.17.5±0.815.9±4.412.3±1.723±7.4CFU-GM86±757.3±4.191±3.852±278.7±19.95±336±141±0BFU-E166±15.315±3.2155.7±9.314±1.595±19.30.7±0.721.3±5.50.3±03CD34+38-% Apoptosis4.2±0.26.1±0.34.2±0.15.3±0.423.0±1.230±4.114.6±3.850.1±7.2CFU-GM67.3±15.941.7±5.549±4.730.3±4.9192.7±50.66±2.372.67±48.70.7±0.7BFU-E58.7±20.211±2.526.3±2.97.6±0.9150.3±58.71±0.672±440.3±0.3 Disclosures: No relevant conflicts of interest to declare.

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The effect of β-d-xylosides on the proliferation and proteoglycan biosynthesis of monoblastic U-937 cells

Biochemical Journal ◽

10.1042/bj2650637 ◽

1990 ◽

Vol 265 (3) ◽

pp. 637-645 ◽

Cited By ~ 34

Author(s):

S O Kolset ◽

K Sakurai ◽

I Ivhed ◽

A Overvatn ◽

S Suzuki

Keyword(s):

Cell Line ◽

Phorbol Esters ◽

Chondroitin Sulphate ◽

Inhibitory Effect ◽

Maximum Effect ◽

Gel Chromatography ◽

Parent Cell ◽

Inhibition Of Proliferation ◽

Glycosaminoglycan Synthesis ◽

Cell Fractions

The monoblastic cell line U-937 was cultured in the presence of C-ethyl beta-D-xyloside (E-xyl), hexyl beta-D-thioxyloside (HX-xyl), p-nitrophenyl beta-D-xyloside, phenyl beta-D-xyloside or phenyl alpha-D-xyloside. All of the beta-D-xylosides inhibited proliferation, but HX-xyl was by far the most efficient, and had a maximum effect at 1 mM concentration. The inhibitory effect of HX-xyl could be reversed; after washing, the HX-xyl-treated cells proliferated with a pattern similar to that of control cells. For more detailed analysis of the effects of beta-D-xylosides on cell proliferation and chondroitin sulphate (CS)/chondroitin sulphate proteoglycan (CSPG) structure, a comparison between the effects of E-xyl and HX-xyl was made. Treating the cells with 1 mM-HX-xyl resulted in a large increase in CS synthesis, whereas 1 mM-E-xyl had only minor effects on the rate of PG/glycosaminoglycan synthesis. Sepharose CL-6B gel chromatography of medium and cell fractions from 35S-labelled cells revealed that HX-xyl treatment resulted in the expression of only free CS chains, whereas E-xyl exposure leads to the synthesis of both large and small CSPGs, as well as some free CS chains. The expression of elevated levels of free CS chains was clearly correlated to the inhibition of proliferation. The proliferation of U-937-4, a clone of U-937 synthesizing ten times more CSPG/CS than the parent line, was equally inhibited by HX-xyl treatment. With this clone, however, there was no stimulation of CS synthesis after xyloside exposure, indicating that the elevated level of CS evident after xyloside treatment of the parent cell line is not causing the inhibition of proliferation. Furthermore, the biosynthesis of hyaluronate was shown not to be implicated in the xyloside-induced decrease in proliferation. The inhibition of proliferation observed in the presence of 1 mM-HX-xyl did not lead to differentiation of the cells into macrophage-like cells, as is observed when the cells are cultured in the presence of phorbol esters, agents also known to inhibit proliferation of U-937 cells.

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In Vitro Studies on the Influence of Meloxicam on Cytotoxic Activity Induced by Risedronate Sodium in Canine (D-17) and Human (U-2 OS) Osteosarcoma Cell Lines

Animals ◽

10.3390/ani11113135 ◽

2021 ◽

Vol 11 (11) ◽

pp. 3135

Author(s):

Dominik Poradowski ◽

Izabela Janus ◽

Aleksander Chrószcz ◽

Bożena Obmińska-Mrukowicz

Keyword(s):

Cell Line ◽

Cell Lines ◽

Cytotoxic Effect ◽

Tissue Level ◽

Negative Control ◽

Terminal Deoxynucleotidyl Transferase ◽

Osteosarcoma Cell ◽

Apoptotic Effect

The study describes the cytotoxic effect against human and canine osteosarcoma (U-2 OS and D-17) cell lines induced by risedronate sodium and meloxicam per se and in combination. Both cell lines were prepared according to standard procedures for cell cultures studies. The cell viability was estimated in both cell lines treated with chosen concentrations of risedronate sodium and meloxicam. The apoptosis assessment was carried out using TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay. EC50 values, computed for risedronate sodium and meloxicam cytotoxicity, showed comparable effects against the canine OS cell line in similar concentration of both drugs. In case of human OS, the stronger cytotoxic effect of risedronate sodium was proved. The EC50 values for meloxicam in both cell lines were, statistically, significantly different (* p < 0.05). Moreover, the cytotoxic effect of a combined administration of meloxicam and risedronate sodium in doses 100 µg/mL, compared with the negative control showed statistically significant differences. The human OS cell line was more resistant to both compounds than the canine OS cell line. The apoptotic effect in canine and human osteosarcoma triggered by risedronate sodium and meloxicam was statistically significant (p < 0.05). The cytotoxic effect induced with 100 µg/mL of risedronate sodium proved statistically significant differences between both tested cell lines compared to negative control. The results obtained with 10 and 100 µg/mL of meloxicam were not statistically significant. The study showed the synergic mechanism of action of risedronate sodium and meloxicam, but the concentrations used in vitro will not be possible to achieve in in vivo. Therefore, our results serve as basis only to design future studies on the tissue level.

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A Deptor Inhibitor Induces Its Degradation with Resulting Anti-Myeloma Cytotoxicity in Vitro and In Vivo

Blood ◽

10.1182/blood-2019-121480 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1821-1821

Author(s):

Mario I Vega ◽

Yijiang Shi ◽

Patrick Frost ◽

Sara Huerta-Yepez ◽

Alan Lichtenstein

Keyword(s):

Multiple Myeloma ◽

Cell Lines ◽

Therapeutic Target ◽

Cytotoxic Effect ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Inhibition Of Proliferation ◽

Induction Of Apoptosis

Multiple myeloma (MM) is a hematological disorder characterized by a proliferation of malignant monoclonal plasma cells in the bone marrow (BM) and / or in extramedullary sites. Despite recent progress in OS rates, MM remains an incurable disease and most patients will relapse and require treatment. Deptor is a component of mTOR complexes and a constitutive inhibitor of their activities. It is known that the inhibition of Deptor results in the inhibition of the proliferation and induction of apoptosis in MM cells. In addition, high levels of Deptor are predictive of a poor response to conventional therapies, indicating that Deptor expression are important as a prognostic marker for patients with myeloma and is a possible therapeutic target. Our group previously identified a drug which prevents mTOR-Deptor binding (NSC126405) and induces cellular cytotoxicity in MM (Shi Y, et al 2016). In this study, we developed a new related chemical inhibitor (43 M) capable of inducing the inhibition of the mTOR / Deptor interaction and results in the negative regulation of Deptor that leads to the inhibition of proliferation and induces apoptosis in several MM cell lines. The cytotoxic effect of 43 M is not dependent of caspase activation and induces the activation of p70 and AKT (T308). This leads to the induction of apoptosis in MM cell lines and tumor cells derived from MM patients. The degradation of Deptor induced by 43 M is dependent on the proteasome complex since it was prevented in the presence of MG132. In vivo, 43 M prevents the expression of Deptor in a xenograft tumor, and delayed tumor growth and interestingly, induces the eradication of tumors in 40% of mice in a murine model of MM, without significant toxic implications. Recent studies show that Deptor expression protects MM cells against Bortezomib treatment, suggesting that anti-Deptor drugs can synergize with proteasome inhibitors (PIs). However, the combination of 43 M + Bortezomib was not synergistic, and was antagonistic in vitro. These results are probably due to the prevention of the proteasomal degradation of Deptor, suggesting a possible use of the 43 M inhibitor in MM in the absence of the current PIs. This study describes for the first time the possible role of Deptor as a therapeutic target using a chemical inhibitor capable of degrading and inducing a cytotoxic effect in MM cell lines. In addition, Deptor is reported as an important therapeutic target in an in vivo MM model. Shi Y, Daniels-Wells TR, Frost P, Lee J, Finn RS, Bardeleben C, Penichet ML, Jung ME, Gera J, Lichtenstein A. Cytotoxic Properties of a DEPTOR-mTOR Inhibitor in Multiple Myeloma Cells. Cancer Res. 2016 Oct 1;76(19):5822-5831 Disclosures No relevant conflicts of interest to declare.

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