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Anticancer Research Mar 2021Breast cancer (BC) may be affected by diabetes and anti-diabetic medication, as well as its therapeutic agents. Low-dose metronomic chemotherapy (LDMC) is an available...
BACKGROUND/AIM
Breast cancer (BC) may be affected by diabetes and anti-diabetic medication, as well as its therapeutic agents. Low-dose metronomic chemotherapy (LDMC) is an available treatment option in BC. We investigated the impact of insulin on low-dose metronomic vinorelbine and mafosfamide in BC cell lines.
MATERIALS AND METHODS
Human BC cell lines T-47D, MCF-7, MDA-MB-231, BT-549 and non-tumorigenic breast cell line MCF-10A were exposed to 0.01 μg/ml and 10 μg/ml insulin in combination with low-dose metronomic vinorelbine or mafosfamide. The cell viability was determined after 24-72 hours using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
RESULTS
Insulin, especially at a concentration of 10 μg/ml, seemed to increase viability of vinorelbine-treated hormone receptor-positive BC cells, whereas low-dose mafosfamide treatment tended to be potentiated by insulin in triple-negative cells.
CONCLUSION
Our findings suggest that insulin may influence the cytotoxic activity of LDMC depending on insulin concentration, type of cytotoxic drug used and BC cell line.
Topics: Administration, Metronomic; Antineoplastic Agents; Breast Neoplasms; Cell Line, Tumor; Cell Survival; Cyclophosphamide; Female; Humans; Insulin; Vinorelbine
PubMed: 33788715
DOI: 10.21873/anticanres.14881 -
Cancer Cell International Sep 2020The genetic variants of the ARID5B gene have recently been reported to be associated with disease susceptibility and treatment outcome in childhood acute lymphoblastic...
BACKGROUND
The genetic variants of the ARID5B gene have recently been reported to be associated with disease susceptibility and treatment outcome in childhood acute lymphoblastic leukemia (ALL). However, few studies have explored the association of ARID5B with sensitivities to chemotherapeutic agents.
METHODS
We genotyped susceptibility-linked rs7923074 and rs10821936 as well as relapse-linked rs4948488, rs2893881, and rs6479778 of ARDI5B by direct sequencing of polymerase chain reaction (PCR) products in 72 B-cell precursor-ALL (BCP-ALL) cell lines established from Japanese patients. We also quantified their ARID5B expression levels by real-time reverse transcription PCR, and determined their 50% inhibitory concentration (IC50) values by alamarBlue assays in nine representative chemotherapeutic agents used for ALL treatment.
RESULTS
No significant associations were observed in genotypes of the susceptibility-linked single nucleotide polymorphisms (SNPs) and the relapsed-linked SNPs with ARID5B gene expression levels. Of note, IC50 values of vincristine (VCR) (median IC50: 39.6 ng/ml) in 12 cell lines with homozygous genotype of risk allele (C) in the relapse-linked rs4948488 were significantly higher (p = 0.031 in Mann-Whitney U test) than those (1.04 ng/ml) in 60 cell lines with heterozygous or homozygous genotypes of the non-risk allele (T). Furthermore, the IC50 values of mafosfamide [Maf; active metabolite of cyclophosphamide (CY)] and cytarabine (AraC) tended to be associated with the genotype of rs4948488. Similar associations were observed in genotypes of the relapse-linked rs2893881 and rs6479778, but not in those of the susceptibility-linked rs7923074 and rs10821936. In addition, the IC50 values of methotrexate (MTX) were significantly higher (p = 0.023) in 36 cell lines with lower ARID5B gene expression (median IC50: 37.1 ng/ml) than those in the other 36 cell lines with higher expression (16.9 ng/ml).
CONCLUSION
These observations in 72 BCP-ALL cell lines suggested that the risk allele of the relapse-linked SNPs of ARID5B may be involved in a higher relapse rate because of resistance to chemotherapeutic agents such as VCR, CY, and AraC. In addition, lower ARID5B gene expression may be associated with MTX resistance.
PubMed: 33499894
DOI: 10.1186/s12935-020-01524-0 -
Cells Jun 2020The concept of immunogenic cell death (ICD) has emerged as a cornerstone of therapy-induced anti-tumor immunity. To this end, the following chemotherapies were evaluated...
The concept of immunogenic cell death (ICD) has emerged as a cornerstone of therapy-induced anti-tumor immunity. To this end, the following chemotherapies were evaluated for their ability to induce ICD in non-small cell lung cancer (NSCLC) cell lines: docetaxel, carboplatin, cisplatin, oxaliplatin and mafosfamide. The ICD hallmarks ATP, ecto-calreticulin, HMGB1, phagocytosis and maturation status of dendritic cells (DCs) were assessed in vitro. Furthermore, an in vivo vaccination assay on C57BL/6J mice was performed to validate our in vitro results. Docetaxel and the combination of docetaxel with carboplatin or cisplatin demonstrated the highest levels of ATP, ecto-calreticulin and HMGB1 in three out of four NSCLC cell lines. In addition, these regimens resulted in phagocytosis of treated NSCLC cells and maturation of DCs. Along similar lines, all mice vaccinated with NSCLC cells treated with docetaxel and cisplatin remained tumor-free after challenge. However, this was not the case for docetaxel, despite its induction of the ICD-related molecules in vitro, as it failed to reject tumor growth at the challenge site in 60% of the mice. Moreover, our in vitro and in vivo data show the inability of oxaliplatin to induce ICD in NSCLC cells. Overall with this study we demonstrate that clinically relevant chemotherapeutic regimens in NSCLC patients have the ability to induce ICD.
Topics: Animals; Calreticulin; Carcinoma, Non-Small-Cell Lung; Cell Death; Cell Line, Tumor; Drug Therapy; Humans; Immunogenic Cell Death; Lung Neoplasms; Mice; Phagocytosis
PubMed: 32560232
DOI: 10.3390/cells9061474 -
Molecular Medicine Reports May 2019Chronic lymphocytic leukemia (CLL) treatment is improving; however, some patients do not respond to therapy. Due to the high heterogeneity in disease development, there...
Dose and drug changes in chronic lymphocytic leukemia cell response in vitro: A comparison of standard therapy regimens with two novel cyclin‑dependent kinase inhibitors.
Chronic lymphocytic leukemia (CLL) treatment is improving; however, some patients do not respond to therapy. Due to the high heterogeneity in disease development, there is an urgent need for personalization of therapy. In the present study, the response of leukemic mononuclear cells to anticancer drugs used for CLL treatment (cladribine + mafosfamide; CM or CM combined with rituximab; RCM) was compared with the response to new cyclin‑dependent kinase (CDK) inhibitors: BP14 and BP30. Viable apoptotic and necrotic cells were quantified by flow cytometry using propidium iodide and Yo‑Pro stains. CDK inhibitors were studied in several doses to determine the reduction of necrosis and simultaneous increase of apoptosis in leukemic cell incubations with anticancer agents. The distinct cell response to applied doses/anticancer agents was observed. Results obtained in the current manuscript confirmed that modulation of doses is important. This was particularly indicated in results obtained at 24 h of cells incubation with anticancer agent. While an important time for analysis of anticancer response efficacy (monitoring of apoptosis induction potential) seems to be 48 h of cells exposition to anticancer agents. High variability in response to the drugs revealed that both the nature and the dose of the anticancer agents could be important in the final effect of the therapy. The present findings support the thesis that personalized medicine, before drug administration in the clinic, could be important to avoid the application of ineffective therapy.
Topics: Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cell Line, Tumor; Cell Survival; Cyclin-Dependent Kinases; Dose-Response Relationship, Drug; Female; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Protein Kinase Inhibitors; Tumor Cells, Cultured
PubMed: 30864706
DOI: 10.3892/mmr.2019.10007 -
Methods in Molecular Biology (Clifton,... 2019A cell's genomic integrity is at risk when DNA-damaging stress, evoked by mitogenic oncogenes or genotoxic treatment modalities such as radiation or chemotherapy, apply....
A cell's genomic integrity is at risk when DNA-damaging stress, evoked by mitogenic oncogenes or genotoxic treatment modalities such as radiation or chemotherapy, apply. If the DNA repair machinery fails to fix the damaged site during a temporary cell-cycle arrest, or if massive genotoxic stress overwhelmed the repair capacity, cellular failsafe programs such as apoptosis or senescence will be triggered to limit aberrant propagation of these damaged and potentially harmful cells. After decades of scientific focusing on apoptosis, cellular senescence is increasingly recognized as an equally important but biologically and fundamentally different type of ultimate cell-cycle exit program, because of its lastingly persistent nature and cell-intrinsic and extrinsic roles within the tissue and tumor microenvironment. We established primary apoptosis-compromised, Bcl2-expressing Eμ-myc transgenic mouse lymphomas as a versatile and clinically relevant model system to study therapy-induced senescence (TIS). Given the lack of a single specific senescence-defining marker, we previously exploited co-staining of senescence-associated β-galactosidase (SA-β-gal) activity with immunohistochemical detection of trimethylated histone H3 lysine 9 (H3K9me3), an established S-phase gene expression-controlling, repressive chromatin mark, and the proliferation marker Ki67. This biomarker panel is instrumental to characterize cells as senescent via their high SA-β-gal activity, strong nuclear H3K9me3 expression and Ki67-negative profile. In this chapter, we demonstrate the detection of viable senescent cells by novel methods based on a fluorescent version of the SA-β-gal (fSA-β-gal) assay, combined with immuno-fluoroscence staining of H3K9me3 or Ki67, or analysis of the DNA replication status by incorporating 5-ethynyl-2'-deoxyuridine (EdU) detection into the protocol. Notably, while most senescence markers, irrespective of their specificity and sensitivity, may only be assessed in endpoint assays, we would like to emphasize here the strength of viable fSA-β-gal to track single-cell fate in senescent populations over time.
Topics: Animals; Apoptosis; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cellular Senescence; Cyclophosphamide; DNA Damage; Fluorescence; Humans; Lymphoma; Mice; Mice, Transgenic; beta-Galactosidase
PubMed: 30474843
DOI: 10.1007/978-1-4939-8931-7_10 -
Cell Death & Disease Oct 2018Activation of T cells, a major fraction of peripheral blood lymphocytes (PBLCS), is essential for the immune response. Genotoxic stress resulting from ionizing radiation...
Sensitivity of CD3/CD28-stimulated versus non-stimulated lymphocytes to ionizing radiation and genotoxic anticancer drugs: key role of ATM in the differential radiation response.
Activation of T cells, a major fraction of peripheral blood lymphocytes (PBLCS), is essential for the immune response. Genotoxic stress resulting from ionizing radiation (IR) and chemical agents, including anticancer drugs, has serious impact on T cells and, therefore, on the immune status. Here we compared the sensitivity of non-stimulated (non-proliferating) vs. CD3/CD28-stimulated (proliferating) PBLC to IR. PBLCs were highly sensitive to IR and, surprisingly, stimulation to proliferation resulted in resistance to IR. Radioprotection following CD3/CD28 activation was observed in different T-cell subsets, whereas stimulated CD34+ progenitor cells did not become resistant to IR. Following stimulation, PBLCs showed no significant differences in the repair of IR-induced DNA damage compared with unstimulated cells. Interestingly, ATM is expressed at high level in resting PBLCs and CD3/CD28 stimulation leads to transcriptional downregulation and reduced ATM phosphorylation following IR, indicating ATM to be key regulator of the high radiosensitivity of resting PBLCs. In line with this, pharmacological inhibition of ATM caused radioresistance of unstimulated, but not stimulated, PBLCs. Radioprotection was also achieved by inhibition of MRE11 and CHK1/CHK2, supporting the notion that downregulation of the MRN-ATM-CHK pathway following CD3/CD28 activation results in radioprotection of proliferating PBLCs. Interestingly, the crosslinking anticancer drug mafosfamide induced, like IR, more death in unstimulated than in stimulated PBLCs. In contrast, the bacterial toxin CDT, damaging DNA through inherent DNase activity, and the DNA methylating anticancer drug temozolomide induced more death in CD3/CD28-stimulated than in unstimulated PBLCs. Thus, the sensitivity of stimulated vs. non-stimulated lymphocytes to genotoxins strongly depends on the kind of DNA damage induced. This is the first study in which the killing response of non-proliferating vs. proliferating T cells was comparatively determined. The data provide insights on how immunotherapeutic strategies resting on T-cell activation can be impacted by differential cytotoxic effects resulting from radiation and chemotherapy.
Topics: Amino Acid Chloromethyl Ketones; Antibodies; Ataxia Telangiectasia Mutated Proteins; CD28 Antigens; CD3 Complex; Caspases; Cell Proliferation; Chromones; DNA-Activated Protein Kinase; Drug Resistance; Gamma Rays; Gene Expression Regulation; Humans; Isoxazoles; Lymphocyte Activation; MRE11 Homologue Protein; Morpholines; Primary Cell Culture; Pyrazines; Pyrones; Radiation Tolerance; Signal Transduction; T-Lymphocytes, Cytotoxic; T-Lymphocytes, Regulatory; Thiophenes; Thioxanthenes
PubMed: 30323167
DOI: 10.1038/s41419-018-1095-7 -
Molecular Medicine Reports Jul 2018The use of chemotherapeutic agents prior to treatment with infusion of cluster of differentiation (CD)19-chimeric antigen receptor (CAR)‑T cells is important for the...
The use of chemotherapeutic agents prior to treatment with infusion of cluster of differentiation (CD)19-chimeric antigen receptor (CAR)‑T cells is important for the efficacy of clinical therapies against hematological malignancies. However, the effect of chemotherapeutic agents on CD19‑CAR‑T cells and the associated underlying mechanisms remain unknown. The first aim of the present study was to determine the effect of chemotherapeutic agents on CAR‑T cells using the in vitro Cell Counting kit 8 assay. The second aim was to evaluate the abilities of fludarabine (FDR) and mafosfamide (MFA; a metabolite of cyclophosphamide) to induce apoptosis of CD19‑CAR‑T cells via the use of Annexin V/propidium iodide double staining. In addition, a JC‑1 fluorescent probe was used to detect alterations in cell membrane potential, and flow cytometry analysis was used to measure concentrations of caspase‑3/7 to identify apoptotic pathways of CD19‑CAR‑T cells. The data of the present study suggested that FDR and MFA inhibit the activities of CD19‑CAR‑T cells. Alterations to the mitochondrial membrane potential and an increase in the concentration of caspase‑3/7 indicated early apoptosis of FDR‑ and MFA‑treated CD19‑CAR‑T cells. The present study laid a theoretical foundation for the development of programs for clinical treatment.
Topics: Antigens, CD19; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Cyclophosphamide; Humans; Membrane Potentials; Receptors, Antigen, T-Cell; Recombinant Fusion Proteins; Vidarabine
PubMed: 29749441
DOI: 10.3892/mmr.2018.8943 -
ACS Biomaterials Science & Engineering Feb 2018Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and...
Improved in vitro models are needed to better understand cancer progression and bridge the gap between in vitro proof-of-concept studies, in vivo validation, and clinical application. Multicellular tumor spheroids (MCTS) are a popular method for three-dimensional (3D) cell culture, because they capture some aspects of the dimensionality, cell-cell contact, and cell-matrix interactions seen in vivo. Many approaches exist to create MCTS from cell lines, and they have been used to study tumor cell invasion, growth, and how cells respond to drugs in physiologically relevant 3D microenvironments. However, there are several discrepancies in the observations made of cell behaviors when comparing between MCTS formation methods. To resolve these inconsistencies, we created and compared the behavior of breast, prostate, and ovarian cancer cells across three MCTS formation methods: in polyNIPAAM gels, in microwells, or in suspension culture. These methods formed MCTS via proliferation from single cells or passive aggregation, and therefore showed differential reliance on genes important for cell-cell or cell-matrix interactions. We also found that the MCTS formation method dictated drug sensitivity, where MCTS formed over longer periods of time via clonal growth were more resistant to treatment. Toward clinical application, we compared an ovarian cancer cell line MCTS formed in polyNIPAAM with cells from patient-derived malignant ascites. The method that relied on clonal growth (PolyNIPAAM gel) was more time and cost intensive, but yielded MCTS that were uniformly spherical, and exhibited the most reproducible drug responses. Conversely, MCTS methods that relied on aggregation were faster, but yielded MCTS with grapelike, lobular structures. These three MCTS formation methods differed in culture time requirements and complexity, and had distinct drug response profiles, suggesting the choice of MCTS formation method should be carefully chosen based on the application required.
PubMed: 29527571
DOI: 10.1021/acsbiomaterials.7b00069 -
Oncology Letters Apr 2017There is individual variation in the course of disease development and response to therapy of patients with chronic lymphocytic leukemia (CLL). Novel treatment options...
There is individual variation in the course of disease development and response to therapy of patients with chronic lymphocytic leukemia (CLL). Novel treatment options for CLL include a new generation of purine analogs, antibodies and inhibitors of specific cell signaling pathways, which typically induce apoptosis or necrosis. A prospective analysis of patient blood samples revealed that a combination of four tests allowed the most appropriate and effective type of treatment to be selected prior to drug administration, and for the analysis of leukemic cell sensitivity to anticancer drug(s) during disease development. The comparative analysis of blood from the stable and progressive form of CLL in an individual patient revealed diversity in the response to anticancer agents. CLL peripheral blood mononuclear cells were incubated with cladribine + mafosfamide (CM), fludarabine + mafosfamide, CM + rituximab, rituximab alone (Rit) or kinetin riboside (RK). A combination of cell viability, differential scanning calorimetry (DSC) profiles of nuclear preparations and poly(ADP-ribose) polymerase 1 (PARP-1) protein expression analysis of the leukemic cells was performed to evaluate the anticancer effects of the tested agents during CLL development. The results of the present study indicate that such studies are effective in determining the most appropriate anticancer drug and could monitor disease progression on an individual level. In addition, the results of the current study suggest that CLL progression leads to diversification of the cellular drug response. The most efficient apoptosis inducer for the patient was purine analog RK when the disease was stable, while the CM combination was the most effective agent for the progressive form of disease.
PubMed: 28454364
DOI: 10.3892/ol.2017.5725 -
Oncotarget Apr 2017The study of long noncoding RNAs (lncRNAs) is an emerging area of cancer research, in part due to their ability to serve as disease biomarkers. However, few studies have...
The study of long noncoding RNAs (lncRNAs) is an emerging area of cancer research, in part due to their ability to serve as disease biomarkers. However, few studies have investigated lncRNAs in chronic lymphocytic leukemia (CLL). We have identified one particular lncRNA, treRNA, which is overexpressed in CLL B-cells. We measured transcript expression in 144 CLL patient samples and separated samples into high or low expression of treRNA relative to the overall median. We found that high expression of treRNA is significantly associated with shorter time to treatment. High treRNA also correlates with poor prognostic indicators such as unmutated IGHV and high ZAP70 protein expression. We validated these initial findings in samples collected in a clinical trial comparing the nucleoside analog fludarabine alone or in combination with the alkylating agent cyclophosphamide in untreated CLL samples collected prior to starting therapy (E2997). High expression of treRNA was independently prognostic for shorter progression free survival in patients receiving fludarabine plus cyclophosphamide. Given these results, in order to study the role of treRNA in DNA damage response we generated a model cell line system where treRNA was over-expressed in the human B-CLL cell line OSU-CLL. Relative to the vector control line, there was less cell death in OSU-CLL over-expressing treRNA after exposure to fludarabine and mafosfamide, due in part to a reduction in DNA damage. Therefore, we suggest that treRNA is a novel biomarker in CLL associated with aggressive disease and poor response to chemotherapy through enhanced protection against cytotoxic mediated DNA damage.
Topics: Adult; Aged; Aged, 80 and over; Antineoplastic Combined Chemotherapy Protocols; Biomarkers; DNA Damage; Female; Gene Expression; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Male; Middle Aged; Prognosis; RNA, Long Noncoding; Treatment Outcome; ZAP-70 Protein-Tyrosine Kinase
PubMed: 28412730
DOI: 10.18632/oncotarget.15401