Limits...
Polyclonal rabbit anti-murine plasmacytoma cell globulins induce myeloma cells apoptosis and inhibit tumour growth in mice.

Mu B, Yang JL, Gou LT, Yao YQ, Zhou Y, Cheng ZH, Shi HS, Li ZY, Wen Y, Leng F, Cui FY, Ma TT, Wei YQ - Apoptosis (2011)

Bottom Line: Multiple myelomas (MMs) are etiologically heterogeneous and there are limited treatment options; indeed, current monoclonal antibody therapies have had limited success, so more effective antibodies are urgently needed.The cytotoxicity of PAb on MPC-11 cell lines was both dose-dependent and time-dependent; PAb exerted a 50% inhibitory effect on MPC-11 cell viability at a concentration of 200 µg/ml in 48 h.Serial intravenous or intraperitoneal injections of PAb inhibited tumour growth and prolonged survival in mice bearing murine plasmacytoma, while TUNEL assay demonstrated that PAb induced statistically significant apoptosis (P < 0.05) compared to control treatments.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biotherapy, West China Hospital and School of Lifesciences, Sichuan University, Keyuan Road 4, Chengdu, Sichuan, People's Republic of China.

ABSTRACT
Multiple myelomas (MMs) are etiologically heterogeneous and there are limited treatment options; indeed, current monoclonal antibody therapies have had limited success, so more effective antibodies are urgently needed. Polyclonal antibodies are a possible alternative because they target multiple antigens simultaneously. In this study, we produced polyclonal rabbit anti-murine plasmacytoma cell immunoglobulin (PAb) by immunizing rabbits with the murine plasmacytoma cell line MPC-11. The isolated PAb bound to plasma surface antigens in several MM cell lines, inhibited their proliferation as revealed by MTT assay, and induce apoptosis as indicated by flow cytometry, microscopic observation of apoptotic changes in morphology, and DNA fragmentation on agarose gels. The cytotoxicity of PAb on MPC-11 cell lines was both dose-dependent and time-dependent; PAb exerted a 50% inhibitory effect on MPC-11 cell viability at a concentration of 200 µg/ml in 48 h. Flow cytometry demonstrated that PAb treatment significantly increased the number of apoptotic cells (48.1%) compared with control IgG (8.3%). Apoptosis triggered by PAb was confirmed by activation of caspase-3, -8, and -9. Serial intravenous or intraperitoneal injections of PAb inhibited tumour growth and prolonged survival in mice bearing murine plasmacytoma, while TUNEL assay demonstrated that PAb induced statistically significant apoptosis (P < 0.05) compared to control treatments. We conclude that PAb is an effective agent for in vitro and in vivo induction of apoptosis in multiple myeloma and that exploratory clinical trials may be warranted.

Show MeSH

Related in: MedlinePlus

PAb-induced apoptosis in myeloma cell lines. a–c The MPC-11 was cultured in the presence of NS, control IgG, or PAb (200 µg/ml) in microtiter plates for 48 h before photography. d MPC-11 cells were incubated with PAb (200 µg/ml) in culture medium at 37°C. At 48 h, 106 cells were removed and DNA was isolated. M marker, NS normal saline group, C control IgG, S PAb group. e Flow cytometric analysis revealed the proportion of sub-G1 cells (apoptotic cells) to be 7.0% in the NS group (left panel), 8.3% in the control IgG group (middle panel), and 48.1% in PAb-treated cells (right panel). f PAb-induced activity of caspase-3, -8, and -9 in MPC-11 cells. MPC-11 cells were treated with 200 µg/ml PAb (right lane), the same concentration of control IgG (middle lane), or NS (left lane) for 48 h and then lysed as described in “Materials and methods”. Protein extracts were immunoblotted to monitor the activation of caspase-3, -8, and -9. β-actin was used as the loading control. g Myeloma cells were pretreated with the caspase inhibitor zVAD-fmk at 100 µM for 1 h before treatment with PAb at 200 µg/ml for 48 h. Cell viability was assessed by MTT assay. *Represents the significant difference in cell viability in presence of zVAD-fmk versus PAb treatment alone. Bar graph indicates the mean ± SD of 3 independent experiments (P < 0.05)
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3066393&req=5

Fig3: PAb-induced apoptosis in myeloma cell lines. a–c The MPC-11 was cultured in the presence of NS, control IgG, or PAb (200 µg/ml) in microtiter plates for 48 h before photography. d MPC-11 cells were incubated with PAb (200 µg/ml) in culture medium at 37°C. At 48 h, 106 cells were removed and DNA was isolated. M marker, NS normal saline group, C control IgG, S PAb group. e Flow cytometric analysis revealed the proportion of sub-G1 cells (apoptotic cells) to be 7.0% in the NS group (left panel), 8.3% in the control IgG group (middle panel), and 48.1% in PAb-treated cells (right panel). f PAb-induced activity of caspase-3, -8, and -9 in MPC-11 cells. MPC-11 cells were treated with 200 µg/ml PAb (right lane), the same concentration of control IgG (middle lane), or NS (left lane) for 48 h and then lysed as described in “Materials and methods”. Protein extracts were immunoblotted to monitor the activation of caspase-3, -8, and -9. β-actin was used as the loading control. g Myeloma cells were pretreated with the caspase inhibitor zVAD-fmk at 100 µM for 1 h before treatment with PAb at 200 µg/ml for 48 h. Cell viability was assessed by MTT assay. *Represents the significant difference in cell viability in presence of zVAD-fmk versus PAb treatment alone. Bar graph indicates the mean ± SD of 3 independent experiments (P < 0.05)

Mentions: A decrease in myeloma cell proliferation may also result from the induction of apoptosis, so we determined whether PAb induced apoptosis using light microscopy, flow cytometry, and DNA fragmentation analysis. The PAb-treated MM cells appeared morphological distinct from controls, with condensation of the cytoplasm and membrane blebbing (Fig. 3a–c). In addition, agarose gel electrophoresis demonstrated that PAb-treated cells exhibited a ladder-like pattern of DNA fragments consisting of multiples of approximately 180–200 bp, consistent with endonuclease-induced DNA fragmentation (Fig. 3d). Furthermore, flow cytometry indicated that PAb treatment significantly increased the number of apoptotic cells compared with the control treatments (PAb-treated, 48.1%; NS-treated, 7.0%; control Rabbit IgG-treated, 8.3%) (Fig. 3E). To confirm that PAb induced apoptosis of myeloma cells, we examined the activation of caspases-3, -8, and -9 by Western blotting (Fig. 3f). Indeed, treatment with PAb led to increased enzyme activities of these caspases at 48 h post-treatment and ultimately causing apoptosis by DNA fragmentation. In addition, the broad-spectrum caspase inhibitor of z-VAD-fmk inhibited PAb-induced MPC-11 cell death (Fig. 3g), confirming that caspases are activated following PAb treatment and are necessary for apoptosis in MPC-11cells. Thus, PAb inhibited proliferation and induced apoptosis in a myeloma cells in vitro. Fig. 3


Polyclonal rabbit anti-murine plasmacytoma cell globulins induce myeloma cells apoptosis and inhibit tumour growth in mice.

Mu B, Yang JL, Gou LT, Yao YQ, Zhou Y, Cheng ZH, Shi HS, Li ZY, Wen Y, Leng F, Cui FY, Ma TT, Wei YQ - Apoptosis (2011)

PAb-induced apoptosis in myeloma cell lines. a–c The MPC-11 was cultured in the presence of NS, control IgG, or PAb (200 µg/ml) in microtiter plates for 48 h before photography. d MPC-11 cells were incubated with PAb (200 µg/ml) in culture medium at 37°C. At 48 h, 106 cells were removed and DNA was isolated. M marker, NS normal saline group, C control IgG, S PAb group. e Flow cytometric analysis revealed the proportion of sub-G1 cells (apoptotic cells) to be 7.0% in the NS group (left panel), 8.3% in the control IgG group (middle panel), and 48.1% in PAb-treated cells (right panel). f PAb-induced activity of caspase-3, -8, and -9 in MPC-11 cells. MPC-11 cells were treated with 200 µg/ml PAb (right lane), the same concentration of control IgG (middle lane), or NS (left lane) for 48 h and then lysed as described in “Materials and methods”. Protein extracts were immunoblotted to monitor the activation of caspase-3, -8, and -9. β-actin was used as the loading control. g Myeloma cells were pretreated with the caspase inhibitor zVAD-fmk at 100 µM for 1 h before treatment with PAb at 200 µg/ml for 48 h. Cell viability was assessed by MTT assay. *Represents the significant difference in cell viability in presence of zVAD-fmk versus PAb treatment alone. Bar graph indicates the mean ± SD of 3 independent experiments (P < 0.05)
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3066393&req=5

Fig3: PAb-induced apoptosis in myeloma cell lines. a–c The MPC-11 was cultured in the presence of NS, control IgG, or PAb (200 µg/ml) in microtiter plates for 48 h before photography. d MPC-11 cells were incubated with PAb (200 µg/ml) in culture medium at 37°C. At 48 h, 106 cells were removed and DNA was isolated. M marker, NS normal saline group, C control IgG, S PAb group. e Flow cytometric analysis revealed the proportion of sub-G1 cells (apoptotic cells) to be 7.0% in the NS group (left panel), 8.3% in the control IgG group (middle panel), and 48.1% in PAb-treated cells (right panel). f PAb-induced activity of caspase-3, -8, and -9 in MPC-11 cells. MPC-11 cells were treated with 200 µg/ml PAb (right lane), the same concentration of control IgG (middle lane), or NS (left lane) for 48 h and then lysed as described in “Materials and methods”. Protein extracts were immunoblotted to monitor the activation of caspase-3, -8, and -9. β-actin was used as the loading control. g Myeloma cells were pretreated with the caspase inhibitor zVAD-fmk at 100 µM for 1 h before treatment with PAb at 200 µg/ml for 48 h. Cell viability was assessed by MTT assay. *Represents the significant difference in cell viability in presence of zVAD-fmk versus PAb treatment alone. Bar graph indicates the mean ± SD of 3 independent experiments (P < 0.05)
Mentions: A decrease in myeloma cell proliferation may also result from the induction of apoptosis, so we determined whether PAb induced apoptosis using light microscopy, flow cytometry, and DNA fragmentation analysis. The PAb-treated MM cells appeared morphological distinct from controls, with condensation of the cytoplasm and membrane blebbing (Fig. 3a–c). In addition, agarose gel electrophoresis demonstrated that PAb-treated cells exhibited a ladder-like pattern of DNA fragments consisting of multiples of approximately 180–200 bp, consistent with endonuclease-induced DNA fragmentation (Fig. 3d). Furthermore, flow cytometry indicated that PAb treatment significantly increased the number of apoptotic cells compared with the control treatments (PAb-treated, 48.1%; NS-treated, 7.0%; control Rabbit IgG-treated, 8.3%) (Fig. 3E). To confirm that PAb induced apoptosis of myeloma cells, we examined the activation of caspases-3, -8, and -9 by Western blotting (Fig. 3f). Indeed, treatment with PAb led to increased enzyme activities of these caspases at 48 h post-treatment and ultimately causing apoptosis by DNA fragmentation. In addition, the broad-spectrum caspase inhibitor of z-VAD-fmk inhibited PAb-induced MPC-11 cell death (Fig. 3g), confirming that caspases are activated following PAb treatment and are necessary for apoptosis in MPC-11cells. Thus, PAb inhibited proliferation and induced apoptosis in a myeloma cells in vitro. Fig. 3

Bottom Line: Multiple myelomas (MMs) are etiologically heterogeneous and there are limited treatment options; indeed, current monoclonal antibody therapies have had limited success, so more effective antibodies are urgently needed.The cytotoxicity of PAb on MPC-11 cell lines was both dose-dependent and time-dependent; PAb exerted a 50% inhibitory effect on MPC-11 cell viability at a concentration of 200 µg/ml in 48 h.Serial intravenous or intraperitoneal injections of PAb inhibited tumour growth and prolonged survival in mice bearing murine plasmacytoma, while TUNEL assay demonstrated that PAb induced statistically significant apoptosis (P < 0.05) compared to control treatments.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Biotherapy, West China Hospital and School of Lifesciences, Sichuan University, Keyuan Road 4, Chengdu, Sichuan, People's Republic of China.

ABSTRACT
Multiple myelomas (MMs) are etiologically heterogeneous and there are limited treatment options; indeed, current monoclonal antibody therapies have had limited success, so more effective antibodies are urgently needed. Polyclonal antibodies are a possible alternative because they target multiple antigens simultaneously. In this study, we produced polyclonal rabbit anti-murine plasmacytoma cell immunoglobulin (PAb) by immunizing rabbits with the murine plasmacytoma cell line MPC-11. The isolated PAb bound to plasma surface antigens in several MM cell lines, inhibited their proliferation as revealed by MTT assay, and induce apoptosis as indicated by flow cytometry, microscopic observation of apoptotic changes in morphology, and DNA fragmentation on agarose gels. The cytotoxicity of PAb on MPC-11 cell lines was both dose-dependent and time-dependent; PAb exerted a 50% inhibitory effect on MPC-11 cell viability at a concentration of 200 µg/ml in 48 h. Flow cytometry demonstrated that PAb treatment significantly increased the number of apoptotic cells (48.1%) compared with control IgG (8.3%). Apoptosis triggered by PAb was confirmed by activation of caspase-3, -8, and -9. Serial intravenous or intraperitoneal injections of PAb inhibited tumour growth and prolonged survival in mice bearing murine plasmacytoma, while TUNEL assay demonstrated that PAb induced statistically significant apoptosis (P < 0.05) compared to control treatments. We conclude that PAb is an effective agent for in vitro and in vivo induction of apoptosis in multiple myeloma and that exploratory clinical trials may be warranted.

Show MeSH
Related in: MedlinePlus