A Caesalpinia spinosa fraction with antitumor activity induces calreticulin surface expression involving vesicles and autophagy during immunogenic cell death
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1
Pontificia Universidad Javeriana, Microbiología, Colombia
A plant fraction from Caesalpinia spinosa named P2Et has been standardized and proven cytotoxic activities over human (MCF-7) and murine (4T1) breast cancer cells lines and murine melanoma (B16) cancer cell line. The in vivo studies show that treatment with P2Et reduces tumor size, metastasis, and IL-6 secretion, a cytokine associated with bad prognosis on murine 4T1 (BALB/c) breast model, and reduces tumor size in B16 (C57BL/6) melanoma model. Results on 4T1 model suggest that immune response is implicated in metastasis reduction. Vaccination with 4T1 cells previously treated with P2Et induced multifunctional memory of T lymphocytes producing IFNγ, TNFα and IL-2. Treatment with P2Et fraction induces immunogenic cell death (ICD) on B16 cells characterized by calreticulin (CRT) cell surface expression, HMGB-1 translocation and ATP release. CRT has been postulated as a danger signal, which promotes antigen transference to dendritic cells via CD91. Nonetheless, how this interaction is carry out is not yet clear. Autophagy is a stress response mechanism associated with the ATP release during ICD. Since preliminary results showed that P2Et fraction induces autophagy in cancer cell line MCF-7, we hypothesized that cell stress induced by the treatment modulates CRT exposure through autophagy and is associated to tumor material transference into dendritic cells.
Autophagy and CRT cell surface expression were evaluated on MCF-7, 4T1 and B-16 cells using different concentrations of P2Et fraction and Doxorubicin (Doxo) as ICD inductor. Fraction and Doxo IC50 was determined by MTT. Autophagy assessment was done by confocal microscopy using monodansylcadaverine (MDC) fluorescent probe. CRT surface expression was estimated by flow cytometry using an anti-CRT antibody from Abcam (Ab2907) and a secondary antibody conjugated with Alexa-Fluor 488. Simultaneous visualization of autophagy and CRT cell surface expression was evaluated by confocal microscopy using MDC and anti-CRT antibody and a secondary antibody conjugated with Alexa-Fluor 647. Transference of tumor cell material into dendritic cells was evaluated on B16 cells CFSE labeled co-cultured with bone marrow-derived dendritic cells (BMDC) from C57BL/6 mice. For CRT cell surface expression cells were stained with CRT antibody (as described before) and anti-CD45-PE, and observed by confocal microscopy. For FACs analysis, cells were labeled with anti-CD45-PECy5, anti-CD86-PECy7 and anti-I-Ab-BV421.
The present study showed that P2Et treatment induced autophagy in MCF-7 and B-16 tumor cells. In addition P2Et increased CRT cell surface expression in 4T1 and B16 cells but not in MCF-7. Analysis of simultaneous presence of autophagy and CRT cell surface expression revealed a kind of vesicles exposed on the plasma membrane and labeled with MDC and CRT. Flow cytometry phagocytosis experiments showed transference of CFSE from tumor cells to dendritic cells when tumor cells were previously treated with P2Et and Doxo. Additionally, a possible participation of surface CRT in the interaction of B16 cell and BMDC was revealed by confocal microscopy. Herein, we found that CRT surface cell exposure comprises vesicles and can be an antigen transfer mechanism. Currently, we are assessing if the differences in CRT expression between different cell lines treated may be related to cellular stress differential response and possible associated with mechanisms such as autophagy and ICD.
Acknowledgements
The authors thank Pontificia Universidad Javeriana for their support and the Departamento Administrativo de Ciencia, Tecnología e Innovación COLCIENCIAS (grant number 120113I0101103 and 120113L0101103) Bogotá, Colombia, for financial support and to Colombian Environmental Ministry for allowing use of genetic resources and derived products (agreement number 0454 of 15/05/2013).
Keywords:
tumor immunity,
Immunogenic cell death,
Autophagy,
Melanoma,
B16 cells,
P2Et
Conference:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología, Medellin, Colombia, 13 Oct - 16 Oct, 2015.
Presentation Type:
Poster Presentation
Topic:
Tumor immunology
Citation:
Prieto
K,
Gomez
A,
Tovar
MC,
Urueña
CP,
Fiorentino
S and
Barreto
A
(2015). A Caesalpinia spinosa fraction with antitumor activity induces calreticulin surface expression involving vesicles and autophagy during immunogenic cell death.
Front. Immunol.
Conference Abstract:
IMMUNOCOLOMBIA2015 - 11th Congress of the Latin American Association of Immunology - 10o. Congreso de la Asociación Colombiana de Alergia, Asma e Inmunología.
doi: 10.3389/conf.fimmu.2015.05.00069
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Received:
15 May 2015;
Published Online:
14 Sep 2015.
*
Correspondence:
PhD. Susana Fiorentino, Pontificia Universidad Javeriana, Microbiología, Bogotá, Colombia, susana.fiorentino@javeriana.edu.co
PhD. Alfonso Barreto, Pontificia Universidad Javeriana, Microbiología, Bogotá, Colombia, alfonso.barreto@javeriana.edu.co