Relationship between RFC gene expression and intracellular drug concentration in methotrexate-resistant osteosarcoma cells

Wang, J J; Li, G J

doi:10.4238/2014.july.24.10

Cited by 11 publications

(10 citation statements)

References 17 publications

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“…1 ); however, the IC 50 value obtained for both of these cell lines was within a similar range of concentrations, i.e., 10 −8 M MTX. These results are in accordance with those obtained by other research groups ( 5 , 13 ). The negative effect of MTX on cell proliferation was clearly evident at day 6 of treatment ( Fig.…”

Section: Discussionsupporting

confidence: 94%

“…Nevertheless, there is a fear in clinical practice that HD-MTX chemotherapy can induce drug resistance, resulting in a reduced treatment effect ( 5 ). The primary and the most frequent mechanism of resistance to MTX is caused by defects in reduced folate carrier (RCF)-mediated transport, which are caused by mutations in the RCF gene or by the downregulation of its expression ( 6 ).…”

Section: Introductionmentioning

confidence: 99%

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DHFR-mediated effects of methotrexate in medulloblastoma and osteosarcoma cells: The same outcome of treatment with different doses in sensitive cell lines

et al. 2015

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Although methotrexate (MTX) is the most well-known antifolate included in many standard therapeutic regimens, substantial toxicity limits its wider use, particularly in pediatric oncology. Our study focused on a detailed analysis of MTX effects in cell lines derived from two types of pediatric solid tumors: medulloblastoma and osteosarcoma. The main aim of this study was to analyze the effects of treatment with MTX at concentrations comparable to MTX plasma levels in patients treated with high-dose or low-dose MTX. The results showed that treatment with MTX significantly decreased proliferation activity, inhibited the cell cycle at S-phase and induced apoptosis in Daoy and Saos-2 reference cell lines, which were found to be MTX-sensitive. Furthermore, no difference in these effects was observed following treatment with various doses of MTX ranging from 1 to 40 μM. These findings suggest the possibility of achieving the same outcome with the application of low-dose MTX, an extremely important result, particularly for clinical practice. Another important aspect of treatment with high-dose MTX in clinical practice is the administration of leucovorin (LV) as an antidote to reduce MTX toxicity in normal cells. For this reason, the combined application of MTX and LV was also included in our experiments; however, this application of MTX together with LV did not elicit any detectable effect. The expression analysis of genes involved in the mechanisms of resistance to MTX was a final component of our study, and the results helped us to elucidate the mechanisms of the various responses to MTX among the cell lines included in our study.

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Section: Discussionsupporting

confidence: 94%

Section: Introductionmentioning

confidence: 99%

DHFR-mediated effects of methotrexate in medulloblastoma and osteosarcoma cells: The same outcome of treatment with different doses in sensitive cell lines

et al. 2015

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“…The MTT assay indicated that Saos-2 cells were very sensitive to MTX treatment, which showed a strong cytotoxic effect in these cells at 0.1 μM. This observation is in full accordance with our previous study [ 22 ] and with results obtained by other research groups studying the sensitivity of Saos-2 cells to MTX [ 23 ]. All five OSA cell lines, which were derived from diagnostic biopsies of primary tumors without any previous neoadjuvant chemotherapy, were significantly more resistant to the DHFR-mediated effect of MTX than Saos-2 cell line.…”

Section: Discussionsupporting

confidence: 92%

Non-DHFR-mediated effects of methotrexate in osteosarcoma cell lines: epigenetic alterations and enhanced cell differentiation

et al. 2016

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BackgroundMethotrexate is an important chemotherapeutic drug widely known as an inhibitor of dihydrofolate reductase (DHFR) which inhibits the reduction of folic acid. DHFR-mediated effects are apparently responsible for its primary antineoplastic action. However, other non-DHFR-mediated effects of methotrexate have been recently discovered, which might be very useful in the development of new strategies for the treatment of pediatric malignancies. The principal goal of this study was to analyze the possible impact of clinically achievable methotrexate levels on cell proliferation, mechanisms of epigenetic regulation (DNA methylation and histone acetylation), induced differentiation and the expression of differentiation-related genes in six osteosarcoma cell lines.MethodsThe Saos-2 reference cell line and five other patient-derived osteosarcoma cell lines were chosen for this study. The MTT assay was used to assess cell proliferation, DNA methylation and histone acetylation were detected using ELISA, and western blotting was used for a detailed analysis of histone acetylation. The expression of differentiation-related genes was quantified using RT-qPCR and the course of cell differentiation was evaluated using Alizarin Red S staining, which detects the level of extracellular matrix mineralization.ResultsMethotrexate significantly decreased the proliferation of Saos-2 cells exclusively, suggesting that this reference cell line was sensitive to the DHFR-mediated effects of methotrexate. In contrast, other results indicated non-DHFR-mediated effects in patient-derived cell lines. Methotrexate-induced DNA demethylation was detected in almost all of them; methotrexate was able to lower the level of 5-methylcytosine in treated cells, and this effect was similar to the effect of 5-aza-2′-deoxycytidine. Furthermore, methotrexate increased the level of acetylated histone H3 in the OSA-06 cell line. Methotrexate also enhanced all-trans retinoic acid-induced cell differentiation in three patient-derived osteosarcoma cell lines, and the modulation of expression of the differentiation-related genes was also shown.ConclusionsOverall non-DHFR-mediated effects of methotrexate were detected in the patient-derived osteosarcoma cell lines. Methotrexate acts as an epigenetic modifier and has a potential impact on cell differentiation and the expression of related genes. Furthermore, the combination of methotrexate and all-trans retinoic acid can be effective as a differentiation therapy for osteosarcoma.

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“…In the present study, shock treatment and a gradually increasing dose of MTX were used to investigate acquired resistance in the MTX-resistant osteosarcoma cell line, Saos-2/MTX4.4. Our previous study demonstrated that MTX induces resistance in MTX-resistant cell lines ( 11 ). This resistance may be associated with the downregulation of folate carrier gene expression levels, as well as the reduced cellular influx of MTX, reducing the ability of MTX to competitively inhibit tumor cell DNA synthesis ( 12 – 15 ).…”

Section: Introductionmentioning

confidence: 99%

Mechanisms of methotrexate resistance in osteosarcoma cell lines and strategies for overcoming this resistance

Wang

2014

Oncology Letters

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The aim of the present study was to investigate the underlying mechanisms of methotrexate (MTX) resistance in the human osteosarcoma cell line, Saos-2/MTX4.4, and to evaluate various methods of overcoming the resistance to this chemotherapeutic agent. MMT assays were performed to determine the resistance of the primary (Saos-2) and resistant (Saos-2/MTX4.4) cell lines to MTX, cisplatin [cis-diamminedichloroplatinum II (DDP)], ifosfamide (IFO), Adriamycin (ADM), epirubicin (EPI) and theprubicin (THP). The Saos-2/MTX4.4 cells exhibited a low resistance to IFO, ADM, EPI and THP; however, no resistance to DDP was identified. Overall, the Saos-2/MTX4.4 cells exhibited a greater resistance to all the chemotherapeutic agents investigated compared with the Saos-2 cells. Rhodamine 123 (R123) fluorescence was measured in the Saos-2/MTX4.4 and Saos-2 cells 30 and 60 min after the addition of R123, and R123 plus verapamil (VER). VER administration increased the intracellular accumulation of R123. In addition, reverse transcription-quantitative polymerase chain reaction was performed to determine the mRNA expression levels of multidrug resistance gene 1 (MDR1) in the two cell lines. Although the Saos-2/MTX4.4 cells were more resistant to the chemotherapeutic agents than the Saos-2 cells, no significant difference was identified between the relative mRNA expression levels of MDR1 in the Saos-2/MTX4.4 and Saos-2 cells (0.4350±0.0354 vs. 0.3886±0.0456; P>0.05).

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Relationship between RFC gene expression and intracellular drug concentration in methotrexate-resistant osteosarcoma cells

Cited by 11 publications

References 17 publications

DHFR-mediated effects of methotrexate in medulloblastoma and osteosarcoma cells: The same outcome of treatment with different doses in sensitive cell lines

DHFR-mediated effects of methotrexate in medulloblastoma and osteosarcoma cells: The same outcome of treatment with different doses in sensitive cell lines

Non-DHFR-mediated effects of methotrexate in osteosarcoma cell lines: epigenetic alterations and enhanced cell differentiation

Mechanisms of methotrexate resistance in osteosarcoma cell lines and strategies for overcoming this resistance

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