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Soypeptide lunasin in cytokine immunotherapy for lymphoma.

Chang HC, Lewis D, Tung CY, Han L, Henriquez SM, Voiles L, Lupov IP, Pelloso D, Sinn AL, Pollok KE, de Lumen BO, Li F, Blum JS, Srivastava S, Robertson MJ - Cancer Immunol. Immunother. (2013)

Bottom Line: Adding lunasin to IL-12- or IL-2-stimulated NK cells demonstrated synergistic effects in the induction of IFNG and GZMB involved in cytotoxicity.In addition, NK cells stimulated with lunasin plus cytokines displayed higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models.The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, School of Science, Indiana University-Purdue University Indianapolis, 723 West Michigan Street, SL310, Indianapolis, IN, 46202, USA, huchang@iupui.edu.

ABSTRACT
Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that post-transplant lymphoma patients have an acquired deficiency of signal transducer and activator of transcription 4, which results in defective IFNγ production during clinical immunotherapy. With the goal of further improving cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that synergistically works with cytokines on natural killer (NK) cells. Peripheral blood mononuclear cells of healthy donors and post-transplant lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-stimulated NK cells demonstrated synergistic effects in the induction of IFNG and GZMB involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNγ production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines displayed higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.

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In vivo effects of lunasin. a Lunasin enhances the secretion of IFNγ in the serum. BALB/c mice received single intraperitoneal (IP) injection with PBS (−), IL-2 (1 × 105 U/mouse) without (−) or with (+) lunasin (0.4 mg/kg body weight), or lunasin alone as indicated. Mice were killed 18 h following injection, and blood samples were collected by cardiac puncture. The serum levels of IFNγ were analyzed using ELISA. Data are presented as mean ± SD from 3 mice per group. ND, not detectable. b, c NK activation in vivo following short-term treatment. BALB/c mice received single daily IP injection for 3 consecutive days as indicated. The following day mice were killed, and spleens were collected for analysis of NK activation using flow cytometry. NK cells gated on CD3− NKp46+ populations (b) were analyzed for surface expression of activation marker CD69 (c, upper panel) and intracellular staining for granzyme B (c, middle panel). The production of IFNγ was analyzed from splenocytes that were incubated with GolgiPlug (Brefeldin A) for 4 h in vitro, and NK populations gated in b were analyzed for intracellular IFNγ expression using flow cytometry (c, bottom panel). Data are presented as mean ± SD averaged from 5 mice per group. d–e NK activation in vivo following long-term treatment. BALB/c mice received single daily IP injection for 5 consecutive days per week for a total of 8 weeks as indicated. Three days after the last injection, mice were killed, and spleens were collected for analysis of NK activation (d) using the same parameters as described in c. Blood samples were collected for analysis of serum IFNγ using ELISA (e). Data are presented as mean ± SD averaged from 5 mice per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001
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Fig5: In vivo effects of lunasin. a Lunasin enhances the secretion of IFNγ in the serum. BALB/c mice received single intraperitoneal (IP) injection with PBS (−), IL-2 (1 × 105 U/mouse) without (−) or with (+) lunasin (0.4 mg/kg body weight), or lunasin alone as indicated. Mice were killed 18 h following injection, and blood samples were collected by cardiac puncture. The serum levels of IFNγ were analyzed using ELISA. Data are presented as mean ± SD from 3 mice per group. ND, not detectable. b, c NK activation in vivo following short-term treatment. BALB/c mice received single daily IP injection for 3 consecutive days as indicated. The following day mice were killed, and spleens were collected for analysis of NK activation using flow cytometry. NK cells gated on CD3− NKp46+ populations (b) were analyzed for surface expression of activation marker CD69 (c, upper panel) and intracellular staining for granzyme B (c, middle panel). The production of IFNγ was analyzed from splenocytes that were incubated with GolgiPlug (Brefeldin A) for 4 h in vitro, and NK populations gated in b were analyzed for intracellular IFNγ expression using flow cytometry (c, bottom panel). Data are presented as mean ± SD averaged from 5 mice per group. d–e NK activation in vivo following long-term treatment. BALB/c mice received single daily IP injection for 5 consecutive days per week for a total of 8 weeks as indicated. Three days after the last injection, mice were killed, and spleens were collected for analysis of NK activation (d) using the same parameters as described in c. Blood samples were collected for analysis of serum IFNγ using ELISA (e). Data are presented as mean ± SD averaged from 5 mice per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001

Mentions: To assess whether lunasin could enhance IFNγ production in vivo, BALB/c mice were given single IP injection with IL-2 in the presence and absence of lunasin as indicated. Eighteen hours following the treatment, we were unable to detect serum IFNγ levels from mice receiving PBS, IL-2, or lunasin. In contrast, the combination of IL-2 and lunasin resulted in the secretion of IFNγ in the serum using lunasin at the dose at 0.4 mg/kg body weight (Fig. 5a).Fig. 5


Soypeptide lunasin in cytokine immunotherapy for lymphoma.

Chang HC, Lewis D, Tung CY, Han L, Henriquez SM, Voiles L, Lupov IP, Pelloso D, Sinn AL, Pollok KE, de Lumen BO, Li F, Blum JS, Srivastava S, Robertson MJ - Cancer Immunol. Immunother. (2013)

In vivo effects of lunasin. a Lunasin enhances the secretion of IFNγ in the serum. BALB/c mice received single intraperitoneal (IP) injection with PBS (−), IL-2 (1 × 105 U/mouse) without (−) or with (+) lunasin (0.4 mg/kg body weight), or lunasin alone as indicated. Mice were killed 18 h following injection, and blood samples were collected by cardiac puncture. The serum levels of IFNγ were analyzed using ELISA. Data are presented as mean ± SD from 3 mice per group. ND, not detectable. b, c NK activation in vivo following short-term treatment. BALB/c mice received single daily IP injection for 3 consecutive days as indicated. The following day mice were killed, and spleens were collected for analysis of NK activation using flow cytometry. NK cells gated on CD3− NKp46+ populations (b) were analyzed for surface expression of activation marker CD69 (c, upper panel) and intracellular staining for granzyme B (c, middle panel). The production of IFNγ was analyzed from splenocytes that were incubated with GolgiPlug (Brefeldin A) for 4 h in vitro, and NK populations gated in b were analyzed for intracellular IFNγ expression using flow cytometry (c, bottom panel). Data are presented as mean ± SD averaged from 5 mice per group. d–e NK activation in vivo following long-term treatment. BALB/c mice received single daily IP injection for 5 consecutive days per week for a total of 8 weeks as indicated. Three days after the last injection, mice were killed, and spleens were collected for analysis of NK activation (d) using the same parameters as described in c. Blood samples were collected for analysis of serum IFNγ using ELISA (e). Data are presented as mean ± SD averaged from 5 mice per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001
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Related In: Results  -  Collection

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Fig5: In vivo effects of lunasin. a Lunasin enhances the secretion of IFNγ in the serum. BALB/c mice received single intraperitoneal (IP) injection with PBS (−), IL-2 (1 × 105 U/mouse) without (−) or with (+) lunasin (0.4 mg/kg body weight), or lunasin alone as indicated. Mice were killed 18 h following injection, and blood samples were collected by cardiac puncture. The serum levels of IFNγ were analyzed using ELISA. Data are presented as mean ± SD from 3 mice per group. ND, not detectable. b, c NK activation in vivo following short-term treatment. BALB/c mice received single daily IP injection for 3 consecutive days as indicated. The following day mice were killed, and spleens were collected for analysis of NK activation using flow cytometry. NK cells gated on CD3− NKp46+ populations (b) were analyzed for surface expression of activation marker CD69 (c, upper panel) and intracellular staining for granzyme B (c, middle panel). The production of IFNγ was analyzed from splenocytes that were incubated with GolgiPlug (Brefeldin A) for 4 h in vitro, and NK populations gated in b were analyzed for intracellular IFNγ expression using flow cytometry (c, bottom panel). Data are presented as mean ± SD averaged from 5 mice per group. d–e NK activation in vivo following long-term treatment. BALB/c mice received single daily IP injection for 5 consecutive days per week for a total of 8 weeks as indicated. Three days after the last injection, mice were killed, and spleens were collected for analysis of NK activation (d) using the same parameters as described in c. Blood samples were collected for analysis of serum IFNγ using ELISA (e). Data are presented as mean ± SD averaged from 5 mice per group. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001
Mentions: To assess whether lunasin could enhance IFNγ production in vivo, BALB/c mice were given single IP injection with IL-2 in the presence and absence of lunasin as indicated. Eighteen hours following the treatment, we were unable to detect serum IFNγ levels from mice receiving PBS, IL-2, or lunasin. In contrast, the combination of IL-2 and lunasin resulted in the secretion of IFNγ in the serum using lunasin at the dose at 0.4 mg/kg body weight (Fig. 5a).Fig. 5

Bottom Line: Adding lunasin to IL-12- or IL-2-stimulated NK cells demonstrated synergistic effects in the induction of IFNG and GZMB involved in cytotoxicity.In addition, NK cells stimulated with lunasin plus cytokines displayed higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models.The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, School of Science, Indiana University-Purdue University Indianapolis, 723 West Michigan Street, SL310, Indianapolis, IN, 46202, USA, huchang@iupui.edu.

ABSTRACT
Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that post-transplant lymphoma patients have an acquired deficiency of signal transducer and activator of transcription 4, which results in defective IFNγ production during clinical immunotherapy. With the goal of further improving cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that synergistically works with cytokines on natural killer (NK) cells. Peripheral blood mononuclear cells of healthy donors and post-transplant lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-stimulated NK cells demonstrated synergistic effects in the induction of IFNG and GZMB involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNγ production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines displayed higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.

Show MeSH
Related in: MedlinePlus