The Rate of Cellular Energy Production of Muscle Cells Is Attenuated By Carnitine Intracellular Deficiency Caused By Imatinib Treatment

Abstract Introduction: Previous works identified that imatinib intake through the carnitine-specific OCTN2 (SLC22A5) transporter resulted in a significant decrease of carnitine intracellular concentrations in chronic myeloid leukemia (CML) and muscle cell lines. On contrary, even high doses of carnitine in preincubation did not influence imatinib cell intake capacity. Specifically performed inhibition of OCTN2 activity by vinorelbine resulted in block of carnitine cell intake, while imatinib intake was only slightly reduced (13-30%). This observation is in line with the knowledge that imatinib is transported also through other known SLC transporters. OCTN2 transporter is the major transporter for carnitine, an essential compound in cell energy metabolism. Presented work follows a hypothesis that non-equal competition between imatinib and carnitine intake through OCTN2 can lead to the carnitine intracellular deficiency, which can be in CML patients manifested by a disruption of skeletal muscle mitochondrial density and cause side effects like fatigue, muscle pain and cramps reported up to 80% of patients treated with imatinib (Kekale et al., 2015). Methods: Muscle cell HTB-153 (human rhabdomyosarcoma, ATCC HS 729), CML cell line KCL-22 (DSMZ ACC 519) were used for in vitro experiments. Intracellular concentration of imatinib, carnitine and metabolites were measured by chromatographic separation using XBridge Amide column (50x2.1mm, 3.5µm; Waters, Milford (MA), USA) and ZIC-pHILIC column (50x2.1mm, 5 µm; Merck, Darmstadt, Germany) coupled to tandem mass spectrometer (QTRAP 4000; Sciex, USA). Results: Carnitine, resp. L-carnitine transports long-chain fatty acids to mitochondria and its high rate is required especially in energetically demanding tissues such as skeletal and cardiac muscles. The concentrations of citric acid cycle (CAC) metabolites (citrate, malate, alpha-ketoglutarate, succinate, fumarate, 2-hydroxyglutarate, cis-aconitate), glycolysis (phosphoenolpyruvate, 3- phosphoglycerate, lactate), production of ATP, ADP and AMP were measured in HTB-153 cells 3 and 24 hours after imatinib treatment in vitro. The significant decrease of malate (CAC), lactate (glycolysis) and ATP levels were found at both time points after imatinib treatment compared to baseline. The same observations were found in KCL-22, which was used for comparison as BCR-ABL1 positive cell line. Additionally, significant decrease of succinate and 2-hydroxyglutarate (CAC) was detected in KCL-22 after imatinib treatment. Next, HTB-153 was incubated with imatinib (1-8 µM) for 24 hours and carnitine (8 µM) was supplied for last 3 hours of incubation, i.e., after 21 hours of imatinib treatment start. No significant changes were found in any metabolites of CAC and glycolysis. Production of ATP, ADP and AMP was not changed as well. Conclusions: Imatinib treatment of muscle (rhabdomyosarcoma) and CML cell lines caused a significant decrease of intracellular concentrations of carnitine. Significant decrease of ATP levels and of certain metabolites of CAC and glycolysis outlined that cells struggle from attenuated mitochondria energy production after imatinib treatment. This has not happened, if carnitine was supplied to the culture for final 3 hours of 24 hours incubation with imatinib. Observed data strongly support the hypothesis that decreased carnitine intake to the muscle cells due to competition with imatinib transport through OCTN2 attenuated mitochondria energy production. Interestingly, the clinical trial NCT03426722 (Chae H et al. 2019) showed that L-carnitine could effectively relieve imatinib-related muscle cramps and significantly increase QoL in patients with advanced gastrointestinal stromal tumor. Supported by GACR18-18407S, MZCR00023736 Disclosures No relevant conflicts of interest to declare.

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Heat Shock Protein 70 Inhibitor, Alone or in Combination with Bortezomib, Prevented Plasmacytoma Development in Immunodeficient Mice Transplanted with Myeloma Cell Lines

Blood ◽

10.1182/blood.v128.22.5658.5658 ◽

2016 ◽

Vol 128 (22) ◽

pp. 5658-5658

Author(s):

Mariana Bleker de Oliveira ◽

Angela Isabel Eugenio ◽

Veruska Lia Fook Alves ◽

Daniela Zanatta ◽

Mihoko Yamamoto ◽

...

Keyword(s):

Cell Line ◽

Tumor Cells ◽

Cell Lines ◽

Proteasome Inhibitor ◽

Conflicts Of Interest ◽

Control Group ◽

Immunodeficient Mice ◽

Late Apoptosis

Abstract Introduction: HSP70 has an integrative role in protein degradation due to the interaction with many pathways, such as ubiquitin proteasome (UPS), unfolded protein response (UPR) and autophagy. In multiple myeloma (MM) HSP70 is overexpressed and helps to prevent proteotoxic stress and cell death caused by overload of unfolded/misfolded proteins produced by tumor cells. Aims: To explore the role of HSP70 inhibition, isolated or in association with proteasome inhibitor, as therapeutic strategy for MM through in vitro and in vivo analyses. Methods: RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines were treated with HSP70 inhibitor (VER155008- 50 μM or 80μM) and proteasome inhibitor (bortezomib 100nM) for evaluation of apoptosis induction by flow cytometry using annexin V and propidium iodide. NOD.Cg-rkdcscid Il2rgtm1Wjl/SzJ immunodeficient mice were used for plasmacytoma xenograft model and treated with intravenous VER155008 (40mg/kg) and bortezomib (1mg/kg), immediately after transplant of RPMI8226-LUC-PURO and U266-LUC-PURO bioluminescent cell lines (N=3 for each group, including controls, bortezomib, VER155008, and combination of bortezomib and VER155008). Bioluminescence was measured in IVIS Kinetic (Capiler Life Science) once a day for seven days. Results: Bortezomib used as single treatment was able to induce apoptosis in RPMI8226-LUC-PURO cell line: the best result for in vitro studies RPMI8226-LUC-PURO was 65% of late apoptosis after treatment with bortezomib. On the other hand, U266-LUC-PURO cell line presented higher percentage of apoptosis when treated with bortezomib and VER155008 combination: U266-LUC-PURO cell line presented more than 60% of late apoptosis after VER155008 (80μM) combined with bortezomib, showing that inhibition of HSP70 could overcome U266-LUC-PURO resistance to bortezomib alone. Mice treated with VER155008, alone or in combination with bortezomib, showed complete inhibition of tumor growth (absence of bioluminescence) for both cell lines when compared with control group after one week of treatment (p<0.001, Two-way ANOVA). Therefore, in vivo studies using mice treated with VER155008, alone or in combination with bortezomib, prevented tumor development after one week of treatment, independent of the cell line used in the xenotransplant. Conclusion: Our study shows that HSP70 and proteasome inhibitors combination induced apoptosis in tumor cells in vivo for both MM cell lines. Since HSP70 is overexpressed in MM and connects several signaling pathways that maintain cell survival, such as UPS, UPR and autophagy, it can represent a key role to establish a new approach for the treatment of MM. Financial support: FAPESP 2010/17668-6 and CNPq (155272/2013-6). UNIFESP Ethics Committee (0219/12). Disclosures No relevant conflicts of interest to declare.

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In Vitro Efficacy of a Novel Liposomal Formulation of a Protein Kinase C Inhibitor In the Treatment of Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v116.21.3282.3282 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3282-3282

Author(s):

Kuan Boone Tan ◽

Leong Uung Ling ◽

Gigi Ngar Chee Chiu

Keyword(s):

Acute Myeloid Leukemia ◽

Cell Line ◽

Cell Lines ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Drug Product ◽

Myelogenous Leukemia ◽

Liposome Encapsulation ◽

Acute Myeloid

Abstract Abstract 3282 The prognosis of patients with acute myeloid leukemia (AML) remains poor, despite the use of the first-line, anthracycline- and cytarabine-based induction chemotherapy aiming to induce complete remission in patients. Given the recent findings that intensive chemotherapy may not benefit older leukemia patients who are not candidates for stem cell transplantation (Kantarjian, H. et al, Blood, 2010; DOI: 10.1182/blood-2010-03-276485) and that the monoclonal antibody-based cytotoxic agent, gemtuzumab ozogamicin, has been voluntarily withdrawn from the market, there is a pressing need to find effective treatment for recurrent AML patients who are >60 years. Safingol [(2S, 3S)-2-amino-1,3-octadecanediol] is a potential anti-cancer bioactive lipid that induces apoptosis through PKC inhibition in leukemia cells and other cancer types. Owing to its poor solubility, safingol is administered as an oil-based emulsion; however, this formulation suffers from severe hemolysis as the dose-limiting toxicity in pre-clinical models, and its toxicity profile is yet to be determined from an ongoing Phase I clinical trial for advanced solid tumors. Liposome is a commonly used drug delivery system to solubilize hydrophobic drugs. It is anticipated that liposome encapsulation of safingol would yield a viable injectable drug product without the need of toxic vehicle such as ethanol or Cremophor-EL, and would substantially reduce the hemolytic toxicity of safingol. In this study, our intention is to develop a suitable liposome formulation of safingol and to test its therapeutic efficacy using human AML cell lines and primary patient samples. Safingol could be formulated into stable liposomes using distearyolphosphatidylcholine and cholesterol with encapsulation efficiency of ∼100%. Safingol was released from the liposomes with a sustained release profile, mainly by a diffusion-controlled mechanism. The extent of hemolysis of 0.5 mM safingol could be significantly reduced from 76% to 14% through liposome encapsulation, as determined by an in vitro hemolysis assay. The cytotoxicity of liposomal safingol was tested with MTT assay on various AML cell lines representing different subtypes, including KG-1 (M1), HL-60 (M2), NB4 (M3), U937 (M5), MV4-11 (M5) and HEL (M6), as well as K562, a cell line of blast crisis of chronic myelogenous leukemia (BC-CML). All cell lines tested responded well to the treatment of liposomal safingol, with IC50 values ranging from 1.5–14 μM. Among the various AML subtypes, NB4 was found to be the most sensitive cell line with the lowest IC50 value of 1.5±0.2 μM. Importantly, liposome encapsulation of safingol did not compromise the ability of the drug to induce apoptosis as compared to the free drug, which was mediated possibly through a mechanism dependent on the generation of reactive oxygen species and caspase activation. Liposomal safingol was further tested in 10 leukemic patient samples, and the formulation was able to induce complete loss of viability of the primary cell samples at 20 μM after 72 h of treatment. Taken together, our results demonstrated the therapeutic potential of liposomal safingol for the treatment of various AML subtypes. Further evaluation of the pharmacokinetics and the efficacy of the formulation in animal models is warranted. Disclosures: No relevant conflicts of interest to declare.

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Biological Impact of Monoallelic and Biallelic BIRC3 Loss in Del(11q) Chronic Lymphocytic Leukemia Progression

Blood ◽

10.1182/blood-2020-142658 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 4-4

Author(s):

Miguel Quijada Álamo ◽

Maria Hernandez-Sanchez ◽

Ana E. Rodriguez ◽

Claudia Pérez Carretero ◽

Marta Martín Izquierdo ◽

...

Keyword(s):

Chronic Lymphocytic Leukemia ◽

Cell Line ◽

Cell Lines ◽

Nuclear Translocation ◽

Conflicts Of Interest ◽

Lymphocytic Leukemia ◽

Negative Regulator ◽

Depth Analysis ◽

The Impact

Chronic lymphocytic leukemia (CLL) patients harboring 11q22.3 deletion, del(11q), are characterized by a rapid disease progression. One of the suggested genes to be involved in the pathogenesis of this deletion is BIRC3, a negative regulator of NF-κB, which is monoallelically deleted in ~80% of del(11q) CLL cases. In addition, truncating mutations in the remaining allele of this gene can lead to BIRC3 biallelic inactivation, which accounts for marked reduced survival in CLL. Nevertheless, the biological mechanisms by which monoallelic or biallelic BIRC3 lesions could contribute to del(11q) CLL pathogenesis, progression and therapy response are partially unexplored. We used the CRISPR/Cas9 system to model monoallelic and biallelic BIRC3 loss in vitro. First, we generated an isogenic HG3 CLL cell line harboring monoallelic del(11q) - HG3-del(11q) - by the introduction of 2 guide RNAs targeting 11q22.1 and 11q23.3 (~17 Mb). Loss-of-function BIRC3 mutations (MUT) were introduced in the remaining allele, generating 3 HG3-del(11q) BIRC3MUT clones. In addition, single BIRC3MUT were introduced in HG3 and MEC1 CLL-derived cells for experimental validation (n = 3 clones/cell line). We first questioned whether monoallelic and biallelic BIRC3 loss had an impact in the DNA-binding activity of NF-κB transcription factors. Interestingly, HG3-del(11q) had higher p52 and RelB (non-canonical NF-κB signaling) activity than HG3WT cells (P = 0.005; P = 0.007), being this activity further increased in HG3-del(11q) BIRC3MUT cells (P < 0.001; P < 0.001). In depth analysis of the non-canonical signaling components by immunoblot revealed that HG3-del(11q) and, to a greater extent, HG3-del(11q) BIRC3MUT cells presented NF-κB-inducing kinase (NIK) cytoplasmic stabilization, high p-IKKα levels and p52-RelB nuclear translocation. Besides, HG3-del(11q) BIRC3MUT cells showed increased levels of the anti-apoptotic proteins BCL2 and BCL-xL. We next assessed this pathway ex vivo in stroma and CpG-stimulated primary CLL cells with or without BIRC3 deletion (n = 22; 11 each group). Remarkably, stimulated BIRC3-deleted primary cells showed higher p52 and RelB activity than BIRC3WT cases (P = 0.01; P = 0.07), and the percentage of BIRC3-deleted cells correlated with p52 activity in del(11q) cases (P = 0.04). We further performed western blot analyses in a homogenous cohort of del(11q) cases including (n = 4) or not including (n = 3) BIRC3 within the deleted region. Interestingly, del(11q)/BIRC3 deleted cases presented high levels of stabilized NIK, which correlated with higher p52 processing (P = 0.003). These patients also showed higher BCL2 levels than those del(11q)/BIRC3 undeleted, and we could further observe a correlation between p52 and BCL2 levels (P = 0.01). Given this p52-dependent BCL2 upregulation, we treated the CRISPR/Cas9 edited clones with venetoclax, demonstrating that HG3-del(11q) BIRC3MUT cells were more sensitive upon BCL2 inhibition than HG3WT clones (mean IC50 3.5 vs. 5.75 μM; P = 0.005). In vitro proliferation assays were performed to interrogate the impact of BIRC3 loss in CLL cell growth, revealing that HG3 BIRC3MUT cell lines had higher growth rates than BIRC3WT cells (P = 0.001). HG3-del(11q) BIRC3MUT cells also showed enhanced proliferation in comparison to HG3-del(11q) clones (P = 0.009). We further determined the clonal dynamics of del(11q) and/or BIRC3MUT cell lines in clonal competition experiments, showing that HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells progressively outgrew HG3WT and HG3-del(11q) cells, respectively, overtime (P = 0.02; P = 0.006). Furthermore, we injected these edited cell lines into NSG mice (n = 20) in vivo, showing that mice xenografted with HG3 BIRC3MUT and HG3-del(11q) BIRC3MUT cells presented, by flow cytometry, an increase of human CD45+ cells in spleen 14 days after injection, compared to HG3WT and HG3-del(11q) cells (P = 0.02; P = 0.015). In summary, this work demonstrates that biallelic BIRC3 deletion through del(11q) and mutation triggers non-canonical NF-κB signaling, driving BCL2 overexpression and conferring clonal advantage, which could account for the negative predictive impact of BIRC3 biallelic inactivation in CLL. Taken together, our results suggest that del(11q) CLL patients harboring BIRC3 mutations should be considered as a CLL subgroup at a high risk of progression that might benefit from venetoclax-based therapies. Funding: PI18/01500 Disclosures No relevant conflicts of interest to declare.

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FDC Like Cell Line HK With IL-2, IL-4 and OX40 Ligand Supports The Growth Of ATLL Cells

Blood ◽

10.1182/blood.v122.21.4319.4319 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4319-4319

Author(s):

Yoshitoyo Kagami ◽

Dai Chihara ◽

Harumi Kato ◽

Noriaki Yoshida ◽

Tomohiro Kinoshita ◽

...

Keyword(s):

Cell Line ◽

Tumor Cells ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cancer Center ◽

Human Umbilical Cord ◽

Feeder Cells ◽

Atll Patient ◽

Ox40 Ligand

Abstract Adult T-cell leukemia/lymphoma (ATLL) is an aggressive neoplasm derived from CD4+ T-cells with HTLV-I infection, and its mechanisms of tumorigenesis still remain to be elucidated. The fact that tumor cells rarely proliferate in vitro is one of the most important problems to be solved. The establishment of cell line from ATLL patient samples has been difficult even in the presence of interleukins. Previously we established one cell line (HU-ATTAK) from acute or lymphoma types of 10 ATLL cases which did not proliferate in the presence of IL-2 and/or IL-4. HU-ATTAK is critically dependent on IL-2 and human umbilical cord vein endothelial cells (HUVEC) as feeder cells. In HU-ATTAK, adding anti-OX40 ligand antibody into the culture system completely inhibited its proliferation. So, OX40 ligand as well as L-2 and/or IL-4 is suggested be necessary for the proliferation of ATLL cells, and feeder cells may also confer the favorable environment. As the substitute of HUVEC, follicular dendritic cell like cell line HK which expresses OX40 ligand was used by introducing human OX40 ligand cDNA (HK-OX40L). When 9 ATLL patient samples were co-cultured with HK-OX40L in the presence of L-2 and/or IL-4, two ATLL cells proliferate vigorously only in the presence of both IL-2 and IL-4 simultaneously. These cell lines were confirmed to be derived from original tumor cells by array CGH analysis, and continued the growth for more than a year. Depletion of IL-2 and IL-4 made these cell lines stop growing within 6 days even on HK-OX40L. In the presence of IL-2 and IL-4, the conditions such as HK alone without OX40 ligand or OX40 ligand alone without HK made the cell lines growing for three months at most. In the presence of IL-2 and IL-4 without HK-OX40L, these cell lines vigorously proliferated for more than three months but finally stopped growing. These data suggested that for the growth of these two cell lines, the cell division is dependent on IL-2 and IL-4, and the maintenance of immortalization is dependent on OX40 ligand and HK cells. This culture analysis would provide important factors for cell growth of ATLL which will explore new targets for ATLL treatment. *HK cells are kindly provided by Dr. Young S Choi at Ochsner Cancer Center, New Orleans. Disclosures: No relevant conflicts of interest to declare.

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Targeting Myeloma Stem Cells through Simultaneous Inhibition of Wnt and Hedgehog (Hh) Signaling Pathways

Blood ◽

10.1182/blood.v116.21.615.615 ◽

2010 ◽

Vol 116 (21) ◽

pp. 615-615

Author(s):

Guido J. Tricot ◽

Ye Yang ◽

Chujiao Xu ◽

Hongwei Xu ◽

Ming Zeng ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Signaling Pathways ◽

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Cyclin D3 ◽

Expression Levels ◽

Gli1 Expression

Abstract Abstract 615 Cancer stem cells (CSC) have been identified in a variety of tumor types, including multiple myeloma (MM). Different signaling pathways, such as Wnt, Hedgehog (Hh), Notch and Bmi, are up-regulated in CSC. MM stem cells (MMSC) are enriched in the side population and have increased aldehyde dehydrogenase 1 A1 (ALDH1A1) activity. They are non-cycling and resistant to drugs typically used to treat MM. In this study, MMSC were isolated from 10 MM cell lines (CD138− fraction) and 4 primary MM patients by selecting the CD19+ CD138− cells from bone marrows heavily involved with MM. We confirmed that the CD19+/CD138− cells were part of the malignant clone by comparing expression levels of spiked genes, such as MAF-B and Cyclin D3, in MMSC and CD138+ MM cells. We also verified that CD138− from MM cell lines and the primary CD138−CD19+ had stem cell characteristics by clonogenic in vitro assays and tumor formation in NOD/SCID mice. GEP and real-time PCR analyses indicated that RARa2 levels were significantly higher levels in MM cell line-derived and primary MMSC than in CD138+ bulk MM cells. We also showed that over-expression of RARα2 in low-expressing MM cell lines resulted in increased Wnt and Hh activity, as evidenced by higher levels of nuclear β-catenin, Cyclin D1, TCF4, LEF1, Smo and GLI1. Expression levels of these proteins were also higher in MMSC than in CD138+ MM cells. We subsequently evaluated the efficacy of inhibition of Wnt (CAY10404, a COX-2 inhibitor) and Hh (cyclopamine and itraconazole) signaling on MMSC in vitro and also in vivo using the 5TGM1 mouse model. These drugs given separately induced potent cell death and growth inhibition in MMSC. Quantitative PCR and western blot revealed that CAY10404 decreased nuclear β-catenin and Gli1 expression, while cyclopamine and itraconazole inhibited nuclear GLI1 expression. The efficacy of CAY10404 and cyclopamine was further evaluated in the 5TGM1 mouse model. The CD138− fraction of this cell line has many of the same characteristics as human CD138− MMSC. After injection of 0.5 M cells into the C57BL/KaLwRij mice through the tail vein and allowing growth for 1 week, CAY10404 (20mg/kg, I.P.) and cyclopamine (20mg/kg, I.P) were given separately three times per week. Interestingly, we observed that the mice injected with untreated CD138+ cells survived longer than untreated mice receiving CD138− cells (P < 0.05). Treatment with CAY10404 and cyclopamine significantly reduced the tumor burden measured by idiotype IgG2 protein levels and dramatically prolonged survival (P < 0.05). We conclude that activation of Wnt and Hh pathway maintains the “stemness” features of MMSC. In MMSC, these pathways are activated by increased RARα2 expression, which we have shown to be related to drug resistance. MMSC can be eradicated by a combination of Wnt and Hh inhibitors. Disclosures: No relevant conflicts of interest to declare.

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Lncrna RP1-68D18.2 Promotes K562 Cell Line Resistance to Imatinib through CD44 and Its Downstream Signal Molecules

Blood ◽

10.1182/blood-2018-99-115865 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5149-5149

Author(s):

Tianyou Yan ◽

Yuping Gong

Keyword(s):

Cell Line ◽

Cell Lines ◽

Chronic Myelogenous Leukemia ◽

Conflicts Of Interest ◽

Leukemia Cell ◽

Myelogenous Leukemia ◽

Leukemia Cell Line ◽

Signal Molecules ◽

Imatinib Treatment ◽

Imatinib Resistance

Abstract As the first-generation tyrosine kinase inhibitor specifically targeting the BCR-ABL oncoprotein, imatinib has been successfully used in the therapy for chronic myelogenous leukemia (CML) with a significant response and good compliance. However, imatinib resistance becomes the main reason for failure of the treatment of CML. In addition to the common BCR-ABL dependence of imatinib resistance, e.g. BCR-ABL kinase domain mutations and BCR-ABL gene amplification, BCR-ABL independence also plays important roles, which involves alteration of dynamics of drug import and efflux, persistent or compensatory activation of signaling pathways etc. Recently, several studies have reported the correlation of lncRNA with resistance of chemotherapeutic drugs in many cancers. Here, we report, for the first time, discernible overexpression of lncRNARP1-68D18.2 and CD44 mRNA in the imatinib resistant chronic myelogenous leukemia cell line K562R, using lncRNA and mRNA microarray, and further verify it using qPCR. Knockdown of RP1-68D18.2 in K562R cell lines increased their sensitivity to imatinib, and simultaneously caused a decrease in CD44 and c-Myc protein levels. Correspondingly, overexpression of RP1-68D18.2 in K562, a sensitive cell line, led to a slightly decrease in imatinib sensitivity and partially reverse downregulation of c-Myc, p-ERK and p-MEK caused by imatinib treatment. Analysis of correlations revealed positive correlation between RP1-68D18.2 and CD44 mRNA expression in the primary CML blasts and hematological malignant cell lines. We conclude that overexpression of RP1-68D18.2 could promote imatinib resistance in the K562R cell line through the regulation of CD44 and its downstream signal molecules. Disclosures No relevant conflicts of interest to declare.

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ANTITUMOR EFFECT OF SUNITINIB AND BORTEZOMIB ON BREAST CANCER CELL LINE IN VITRO

Problems in oncology ◽

10.37469/0507-3758-2017-63-1-141-145 ◽

2017 ◽

Vol 63 (1) ◽

pp. 141-145

Author(s):

Yuliya Khochenkova ◽

Eliso Solomko ◽

Oksana Ryabaya ◽

Yevgeniya Stepanova ◽

Dmitriy Khochenkov

Keyword(s):

Breast Cancer ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Antitumor Effect ◽

Cancer Cell Lines ◽

Breast Cancer Cell Lines ◽

Actual Problem

The discovery for effective combinations of anticancer drugs for treatment for breast cancer is the actual problem in the experimental chemotherapy. In this paper we conducted a study of antitumor effect of the combination of sunitinib and bortezomib against MDA-MB-231 and SKBR-3 breast cancer cell lines in vitro. We found that bortezomib in non-toxic concentrations can potentiate the antitumor activity of sunitinib. MDA-MB-231 cell line has showed great sensitivity to the combination of bortezomib and sunitinib in vitro. Bortezomib and sunitinib caused reduced expression of receptor tyrosine kinases VEGFR1, VEGFR2, PDGFRa, PDGFRß and c-Kit on HER2- and HER2+ breast cancer cell lines

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Design, Synthesis, Molecular Docking, and Anticancer Evaluation of Pyrazole Linked Pyrazoline Derivatives with Carbothioamide Tail as EGFR Kinase Inhibitors

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200727093613 ◽

2020 ◽

Vol 21 (1) ◽

pp. 42-60

Author(s):

Farah Nawaz ◽

Ozair Alam ◽

Ahmad Perwez ◽

Moshahid A. Rizvi ◽

Mohd. Javed Naim ◽

...

Keyword(s):

Molecular Docking ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Cancer Cell Line ◽

Cancer Cell Lines ◽

Kinase Assay ◽

Cervix Uteri ◽

Egfr Kinase

Background: The Epidermal Growth Factor Receptor (known as EGFR) induces cell differentiation and proliferation upon activation through the binding of its ligands. Since EGFR is thought to be involved in the development of cancer, the identification of new target inhibitors is the most viable approach, which recently gained momentum as a potential anticancer therapy. Objective: To assess various pyrazole linked pyrazoline derivatives with carbothioamide for EGFR kinase inhibitory as well as anti-proliferative activity against human cancer cell lines viz. A549 (non-small cell lung tumor), MCF-7 (breast cancer cell line), SiHa (cancerous tissues of the cervix uteri), and HCT-116 (colon cancer cell line). Methods: In vitro EGFR kinase assay, in vitro MTT assay, Lactate dehydrogenase release, nuclear staining (DAPI), and flow cytometry cell analysis. Results: Compounds 6h and 6j inhibited EGFR kinase at concentrations of 1.66μM and 1.9μM, respectively. Furthermore, compounds 6h and 6j showed the most potent anti-proliferative results against the A549 KRAS mutation cell line (IC50 = 9.3 & 10.2μM). Through DAPI staining and phase contrast microscopy, it was established that compounds 6h and 6j also induced apoptotic activity in A549 cells. This activity was further confirmed by FACS using Annexin-V-FITC and Propidium Iodide (PI) labeling. Molecular docking studies performed on 6h and 6j suggested that the compounds can bind to the hinge region of ATP binding site of EGFR tyrosine kinase in a similar pose as that of the standard drug gefitinib. Conclusion: The potential anticancer activity of compounds 6h and 6j was confirmed and need further exploration in cancer cell lines of different tissue origin and signaling pathways, as well as in animal models of cancer development.

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Survival and Function of Fibroblasts Stored as Stock Cultures at a Non-freezing Temperature

Alternatives to Laboratory Animals ◽

10.1177/026119299302100215 ◽

1993 ◽

Vol 21 (2) ◽

pp. 206-209

Author(s):

Anders H. G. Andrén ◽

Anders P. Wieslander

Keyword(s):

Cell Growth ◽

Cell Line ◽

Cell Lines ◽

Fibroblast Cell ◽

Freezing Temperature ◽

Mouse Fibroblast ◽

Toxic Response ◽

Normal Manner ◽

And Function

Cytotoxicity, measured as inhibition of cell growth of cultured cell lines, is a widely used method for testing the safety of biomaterials and chemicals. One major technical disadvantage with this method is the continuous routine maintenance of the cell lines. We decided to investigate the possibility of storing stock cultures of fibroblasts (L-929) in an ordinary refrigerator as a means of reducing the routine workload. Stock cultures of the mouse fibroblast cell line L-929 were prepared in plastic vials with Eagle's minimum essential medium. The vials were stored in a refrigerator at 4–10°C for periods of 7–31 days. The condition of the cells after storage was determined as cell viability, cell growth and the toxic response to acrylamide, measured as cell growth inhibition. We found that the L-929 cell line can be stored for 2–3, weeks with a viabilty > 90% and a cell growth of about 95%, compared to L-929 cells grown and subcultured in the normal manner. The results also show that the toxic response to acrylamide, using refrigerator stored L-929 cells, corresponds to that of control L-929 cells. We concluded that it is possible to store L-929 cells in a refrigerator for periods of up to 3 weeks and still use the cells for in vitro cytotoxic assays.

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Tissue type-specific expression of intermediate filament proteins in a cultured epithelial cell line from bovine mammary gland

The Journal of Cell Biology ◽

10.1083/jcb.96.1.37 ◽

1983 ◽

Vol 96 (1) ◽

pp. 37-50 ◽

Cited By ~ 93

Author(s):

E Schmid ◽

DL Schiller ◽

C Grund ◽

J Stadler ◽

WW Franke

Keyword(s):

Mammary Gland ◽

Epithelial Cell ◽

Cell Line ◽

Cell Lines ◽

Intermediate Filament ◽

Striking Difference ◽

Intermediate Filament Proteins ◽

Cell Clones ◽

Vimentin Filaments

Different clonal cell lines have been isolated from cultures of mammary gland epithelium of lactating cow's udder and have been grown in culture media containing high concentrations of hydrocortisone, insulin, and prolactin. These cell (BMGE+H), which grow in monolayers of typical epithelial appearance, are not tightly packed, but leave intercellular spaces spanned by desmosomal bridges. The cells contain extended arrays of cytokeratin fibrils, arranged in bundles attached to desmosomes. Gel electophoresis show that they synthesize cytokeratins similar, if not identical, to those found in bovine epidermis and udder, including two large (mol wt 58,500 and 59,000) and basic (pH range: 7-8) and two small (mol wt 45,500 and 50,000) and acidic (pH 5.32 and 5.36) components that also occur in phosphorylated forms. Two further cytokeratins of mol wts 44,000 (approximately pH 5.7) and 53,000 (pH 6.3) are detected as minor cytokeratins in some cell clones. BMGE+H cells do not produce vimentin filaments as determined by immunofluorescence microscopy and gel electrophoresis. By contrast, BMGE-H cells, which have emerged from the same original culture but have been grown without hormones added, are not only morphologically different, but also contain vimentin filaments and a different set of cytokeratins, the most striking difference being the absence of the two acidic cytokeratins of mol wt 50,000 and 45,500. Cells of the BMGE+H line are characterized by an unusual epithelial morphology and represent the first example of a nonmalignant permanent cell line in vitro that produces cytokeratin but not vimentin filaments. The results show that (a) tissue-specific patterns of intermediate filament expression can be maintained in permanent epithelial cell lines in culture, at least under certain growth conditions; (b) loss of expression of relatively large, basic cytokeratins is not an inevitable consequence of growth of epithelial cells in vitro. Our results further show that, during culturing, different cell clones with different cytoskeletal composition can emerge from the same cell population and suggest that the presence of certain hormones may have an influence on the expression of intermediate filament proteins.

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