Contributions
Abstract: PB2066
Type: Publication Only
Background
Bendamustine hydrochloride (BH) has been one of the most promising genotoxic moieties for mantle cell lymphoma (MCL), however, its mechanisms of action and the mechanisms for the acquisition of resistance to BH have not been fully clarified.
Aims
We tried to identify the underlying mechanisms for BH resistance to develop the strategy to overcome BH resistance.
Methods
This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. Patient’s sample was obtained along with the written informed consent. We firstly established a novel MCL-derived cell line, KPUM-YY1, from circulating lymphoma cells of a 77-year-old male patient with MCL. A BH-resistant subline of KPUM-YY1 (KPUM-YY1R) was established by continuous exposure to BH with gradual escalation of its concentration from 5 μM up to 50 μM for about 8 months. Cytogenetic analysis was performed by double color-fluorescence in situ hybridization and spectral karyotyping (SKY). The comparative gene expression profile (GEP) and the ingenuity canonical signal pathway analyses between of KPUM-YY1 and KUPM-YY1R were performed to identify the differential gene expression pattern along with the acquisition of BH resistance. Cell viability was evaluated by a modified MTT assay.
Results
SKY analysis revealed that both primary tumor cells and KPUM-YY1 had complex karyotype including three-way translocation t(8;14;11)(q24;q32;q13), involving the rearrangement of cyclin D1. The growth inhibitory IC50 to BH was 20 μM in KPUM-YY1 cells, while the cell proliferation was not inhibited by up to 60 μM of BH in KPUM-YY1R cells. When compared with the parental KPUM-YY1 cells, KPUM-YY1R cells showed the partial cross-resistance against doxorubicin, mafosfamide, melphalan, and vincristine. By GEP analyses, total of 472 genes were differentially expressed in KPUM-YY1R compared with KPUM-YY1 cells, including 312 upregulated more than 1.5-folds and 160 downregulated less than 0.67-folds in KPUM-YY1R cells. The ingenuity canonical signal pathway analysis based on the GEP results suggested that KPUM-YY1R cells harbored the distinct gene expression patterns in MDR1, a gene for p-glycoprotein (P-gp) of drug transporter molecule, MGST1, a member of glutathione S-transferase (GST) families, and argininosuccinate synthetase 1 (ASS1), a rate-limiting enzyme for arginine biosynthesis. The upregulation of MDR1 (P-gp) and MGST1 were confirmed by Western blot or RT-PCR analysis in KPUM-YY1R compared with KPUM-YY1. Importantly, the addition of P-gp inhibitors, such as cyclosporine A, or GST inhibitors, such as ethacrynic acid, at least partly restored the sensitivity to BH in KPUM-YY1R cells, indicating the functional significance of the upregulation of MDR1 and MGST1 in the development of BH resistance in MCL. In addition, BH-resistance cells were also found to express decreased mRNA level of ASS1 which has been reported to play tumor suppressor roles and its loss has been associated with clinical aggressiveness in various cancers.
Conclusion
This study revealed that the multiple molecular mechanisms overlappingly underlie the development of BH resistance, therefore, the acquisition of BH resistance potentially leads multidrug resistance in MCL cells. The newly developed KPUM-YY1 cells and KPUM-YY1R cells deserve the identification of multiplex mechanisms underlying BH activity/resistance and the future development of strategy which overcomes the treatment refractoriness in MCL.
Session topic: 18. Non-Hodgkin & Hodgkin lymphoma - Biology
Keyword(s): MDR1, Mantle cell lymphoma, bendamustine
Abstract: PB2066
Type: Publication Only
Background
Bendamustine hydrochloride (BH) has been one of the most promising genotoxic moieties for mantle cell lymphoma (MCL), however, its mechanisms of action and the mechanisms for the acquisition of resistance to BH have not been fully clarified.
Aims
We tried to identify the underlying mechanisms for BH resistance to develop the strategy to overcome BH resistance.
Methods
This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. Patient’s sample was obtained along with the written informed consent. We firstly established a novel MCL-derived cell line, KPUM-YY1, from circulating lymphoma cells of a 77-year-old male patient with MCL. A BH-resistant subline of KPUM-YY1 (KPUM-YY1R) was established by continuous exposure to BH with gradual escalation of its concentration from 5 μM up to 50 μM for about 8 months. Cytogenetic analysis was performed by double color-fluorescence in situ hybridization and spectral karyotyping (SKY). The comparative gene expression profile (GEP) and the ingenuity canonical signal pathway analyses between of KPUM-YY1 and KUPM-YY1R were performed to identify the differential gene expression pattern along with the acquisition of BH resistance. Cell viability was evaluated by a modified MTT assay.
Results
SKY analysis revealed that both primary tumor cells and KPUM-YY1 had complex karyotype including three-way translocation t(8;14;11)(q24;q32;q13), involving the rearrangement of cyclin D1. The growth inhibitory IC50 to BH was 20 μM in KPUM-YY1 cells, while the cell proliferation was not inhibited by up to 60 μM of BH in KPUM-YY1R cells. When compared with the parental KPUM-YY1 cells, KPUM-YY1R cells showed the partial cross-resistance against doxorubicin, mafosfamide, melphalan, and vincristine. By GEP analyses, total of 472 genes were differentially expressed in KPUM-YY1R compared with KPUM-YY1 cells, including 312 upregulated more than 1.5-folds and 160 downregulated less than 0.67-folds in KPUM-YY1R cells. The ingenuity canonical signal pathway analysis based on the GEP results suggested that KPUM-YY1R cells harbored the distinct gene expression patterns in MDR1, a gene for p-glycoprotein (P-gp) of drug transporter molecule, MGST1, a member of glutathione S-transferase (GST) families, and argininosuccinate synthetase 1 (ASS1), a rate-limiting enzyme for arginine biosynthesis. The upregulation of MDR1 (P-gp) and MGST1 were confirmed by Western blot or RT-PCR analysis in KPUM-YY1R compared with KPUM-YY1. Importantly, the addition of P-gp inhibitors, such as cyclosporine A, or GST inhibitors, such as ethacrynic acid, at least partly restored the sensitivity to BH in KPUM-YY1R cells, indicating the functional significance of the upregulation of MDR1 and MGST1 in the development of BH resistance in MCL. In addition, BH-resistance cells were also found to express decreased mRNA level of ASS1 which has been reported to play tumor suppressor roles and its loss has been associated with clinical aggressiveness in various cancers.
Conclusion
This study revealed that the multiple molecular mechanisms overlappingly underlie the development of BH resistance, therefore, the acquisition of BH resistance potentially leads multidrug resistance in MCL cells. The newly developed KPUM-YY1 cells and KPUM-YY1R cells deserve the identification of multiplex mechanisms underlying BH activity/resistance and the future development of strategy which overcomes the treatment refractoriness in MCL.
Session topic: 18. Non-Hodgkin & Hodgkin lymphoma - Biology
Keyword(s): MDR1, Mantle cell lymphoma, bendamustine