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Autophagy plays a critical role in ChLym-1-induced cytotoxicity of non-hodgkin's lymphoma cells.

Fan J, Zeng X, Li Y, Wang S, Wang Z, Sun Y, Gao H, Zhang G, Feng M, Ju D - PLoS ONE (2013)

Bottom Line: In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin's Lymphoma (NHL) cells.Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1.Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2).

View Article: PubMed Central - PubMed

Affiliation: Department of Biosynthesis, School of Pharmacy, Fudan University, Shanghai, China.

ABSTRACT
Autophagy is a critical mechanism in both cancer therapy resistance and tumor suppression. Monoclonal antibodies have been documented to kill tumor cells via apoptosis, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin's Lymphoma (NHL) cells. Interestingly, inhibition of autophagy by pharmacological inhibitors (3-methyladenine and NH4Cl) or genetic approaches (siRNA targeting Atg5) suppresses chLym-1-induced growth inhibition, apoptosis, ADCC and CDC in Raji cells, while induction of autophagy could accelerate cytotoxic effects of chLym-1 on Raji cells. Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1. Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2). These results elucidate the critical role of autophagy in cytotoxicity of chLym-1 antibody and suggest a potential therapeutic strategy of NHL therapy by monoclonal antibody chLym-1 in combination with autophagy inducer.

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Autophagy could be significantly induced by chLym-1 in Raji cells.A: Autophagy-related protein LC3-II is significantly up-regulated in Raji cells treated with chLym-1. Raji cells were treated with 10 µg/ml of chLym-1 for 24 h as described, while vehicles were treated with complete medium. Statistics was applied to detect relative intensities of LC3-II (LC3-II/actin). B: ChLym-1 induces autophagosomes accumulation (arrows) in Raji cells. Raji cells were treated with/without 10 µg/ml of chLym-1 for 24 h, and then prepared for transmission electron microscope. N = Nuclear. C: Appearance of autophagosome membrane-associated LC3-II in Raji cells treated with chLym-1. Raji cells were treated with/without chLym-1 for 24 h, and vehicles were treated with RMPI1640 supplemented with 10% fetal bovine serum (FBS). Raji cells treated with 50 nM of Rapamycin for 6 h were used as positive control. Spots were quantified by IQuantTL (GE Health Care). D: chLym-1 induces autophagy via autophagosomes accumulation but not via inhibition of autophagosomes degradation. Raji cell were treated with chLym-1 and/or 10 mM of Chloroquine (CQ) for 24 h.
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pone-0072478-g001: Autophagy could be significantly induced by chLym-1 in Raji cells.A: Autophagy-related protein LC3-II is significantly up-regulated in Raji cells treated with chLym-1. Raji cells were treated with 10 µg/ml of chLym-1 for 24 h as described, while vehicles were treated with complete medium. Statistics was applied to detect relative intensities of LC3-II (LC3-II/actin). B: ChLym-1 induces autophagosomes accumulation (arrows) in Raji cells. Raji cells were treated with/without 10 µg/ml of chLym-1 for 24 h, and then prepared for transmission electron microscope. N = Nuclear. C: Appearance of autophagosome membrane-associated LC3-II in Raji cells treated with chLym-1. Raji cells were treated with/without chLym-1 for 24 h, and vehicles were treated with RMPI1640 supplemented with 10% fetal bovine serum (FBS). Raji cells treated with 50 nM of Rapamycin for 6 h were used as positive control. Spots were quantified by IQuantTL (GE Health Care). D: chLym-1 induces autophagy via autophagosomes accumulation but not via inhibition of autophagosomes degradation. Raji cell were treated with chLym-1 and/or 10 mM of Chloroquine (CQ) for 24 h.

Mentions: Autophagy can be induced upon chLym-1 treatment in Raji lymphoma cells. The expression of autophagy related protein LC3-II (16 KD) significantly increases in chLym-1-treated Raji cells but not in Daudi cells, which does not combine to chLym-1 (p<0.01, Figure 1A, Figure S1,S2). Transmission electron microscopy studies reveal that autophagosomes accumulation in Raji cells after chLym-1 treatment for 24 h, while autophagosomes are scarce in non-treated control cells and chLym-1-treated Daudi cells (Figure 1B, Figure S3). When stained by Cyto-ID® Autophagy Detection Kit, as Rapamycin-treated Raji cells (positive control), cells treated with chLym-1 (10 µg/ml) for 24 h display more punctuate fluorescence (LC3-II) than non-treated cells which show minimal punctuate fluorescence under immunofluorescence confocal microscopy (Figure 1C). Moreover, blocking autophagy by CQ, an autolysosome inhibitor, can additionally enhance the expression of LC3-II in chLym-1-treated Raji cells (Figure 1D), suggesting that chLym-1 induces autophagy via autophagosomes accumulation, but not via inhibition of autophagosomes degradation. Together, our results strongly suggest that chLym-1 can induce autophagy in Raji lymphoma cells.


Autophagy plays a critical role in ChLym-1-induced cytotoxicity of non-hodgkin's lymphoma cells.

Fan J, Zeng X, Li Y, Wang S, Wang Z, Sun Y, Gao H, Zhang G, Feng M, Ju D - PLoS ONE (2013)

Autophagy could be significantly induced by chLym-1 in Raji cells.A: Autophagy-related protein LC3-II is significantly up-regulated in Raji cells treated with chLym-1. Raji cells were treated with 10 µg/ml of chLym-1 for 24 h as described, while vehicles were treated with complete medium. Statistics was applied to detect relative intensities of LC3-II (LC3-II/actin). B: ChLym-1 induces autophagosomes accumulation (arrows) in Raji cells. Raji cells were treated with/without 10 µg/ml of chLym-1 for 24 h, and then prepared for transmission electron microscope. N = Nuclear. C: Appearance of autophagosome membrane-associated LC3-II in Raji cells treated with chLym-1. Raji cells were treated with/without chLym-1 for 24 h, and vehicles were treated with RMPI1640 supplemented with 10% fetal bovine serum (FBS). Raji cells treated with 50 nM of Rapamycin for 6 h were used as positive control. Spots were quantified by IQuantTL (GE Health Care). D: chLym-1 induces autophagy via autophagosomes accumulation but not via inhibition of autophagosomes degradation. Raji cell were treated with chLym-1 and/or 10 mM of Chloroquine (CQ) for 24 h.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756084&req=5

pone-0072478-g001: Autophagy could be significantly induced by chLym-1 in Raji cells.A: Autophagy-related protein LC3-II is significantly up-regulated in Raji cells treated with chLym-1. Raji cells were treated with 10 µg/ml of chLym-1 for 24 h as described, while vehicles were treated with complete medium. Statistics was applied to detect relative intensities of LC3-II (LC3-II/actin). B: ChLym-1 induces autophagosomes accumulation (arrows) in Raji cells. Raji cells were treated with/without 10 µg/ml of chLym-1 for 24 h, and then prepared for transmission electron microscope. N = Nuclear. C: Appearance of autophagosome membrane-associated LC3-II in Raji cells treated with chLym-1. Raji cells were treated with/without chLym-1 for 24 h, and vehicles were treated with RMPI1640 supplemented with 10% fetal bovine serum (FBS). Raji cells treated with 50 nM of Rapamycin for 6 h were used as positive control. Spots were quantified by IQuantTL (GE Health Care). D: chLym-1 induces autophagy via autophagosomes accumulation but not via inhibition of autophagosomes degradation. Raji cell were treated with chLym-1 and/or 10 mM of Chloroquine (CQ) for 24 h.
Mentions: Autophagy can be induced upon chLym-1 treatment in Raji lymphoma cells. The expression of autophagy related protein LC3-II (16 KD) significantly increases in chLym-1-treated Raji cells but not in Daudi cells, which does not combine to chLym-1 (p<0.01, Figure 1A, Figure S1,S2). Transmission electron microscopy studies reveal that autophagosomes accumulation in Raji cells after chLym-1 treatment for 24 h, while autophagosomes are scarce in non-treated control cells and chLym-1-treated Daudi cells (Figure 1B, Figure S3). When stained by Cyto-ID® Autophagy Detection Kit, as Rapamycin-treated Raji cells (positive control), cells treated with chLym-1 (10 µg/ml) for 24 h display more punctuate fluorescence (LC3-II) than non-treated cells which show minimal punctuate fluorescence under immunofluorescence confocal microscopy (Figure 1C). Moreover, blocking autophagy by CQ, an autolysosome inhibitor, can additionally enhance the expression of LC3-II in chLym-1-treated Raji cells (Figure 1D), suggesting that chLym-1 induces autophagy via autophagosomes accumulation, but not via inhibition of autophagosomes degradation. Together, our results strongly suggest that chLym-1 can induce autophagy in Raji lymphoma cells.

Bottom Line: In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin's Lymphoma (NHL) cells.Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1.Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2).

View Article: PubMed Central - PubMed

Affiliation: Department of Biosynthesis, School of Pharmacy, Fudan University, Shanghai, China.

ABSTRACT
Autophagy is a critical mechanism in both cancer therapy resistance and tumor suppression. Monoclonal antibodies have been documented to kill tumor cells via apoptosis, antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). In this study, we report for the first time that chLym-1, a chimeric anti-human HLA-DR monoclonal antibody, induces autophagy in Raji Non-Hodgkin's Lymphoma (NHL) cells. Interestingly, inhibition of autophagy by pharmacological inhibitors (3-methyladenine and NH4Cl) or genetic approaches (siRNA targeting Atg5) suppresses chLym-1-induced growth inhibition, apoptosis, ADCC and CDC in Raji cells, while induction of autophagy could accelerate cytotoxic effects of chLym-1 on Raji cells. Furthermore, chLym-1-induced autophagy can mediate apoptosis through Caspase 9 activation, demonstrating the tumor-suppressing role of autophagy in antilymphoma effects of chLym-1. Moreover, chLym-1 can activate several upstream signaling pathways of autophagy including Akt/mTOR and extracellular signal-regulated kinase 1/2 (Erk1/2). These results elucidate the critical role of autophagy in cytotoxicity of chLym-1 antibody and suggest a potential therapeutic strategy of NHL therapy by monoclonal antibody chLym-1 in combination with autophagy inducer.

Show MeSH
Related in: MedlinePlus