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. 2011 Oct;60(10):1503-7.
doi: 10.1007/s00262-011-1074-7. Epub 2011 Jul 22.

The n3-polyunsaturated fatty acid docosahexaenoic acid induces immunogenic cell death in human cancer cell lines via pre-apoptotic calreticulin exposure

Romina Molinari  1 , Donatella D'Eliseo, Laura Manzi, Lello Zolla, Francesca Velotti, Nicolò Merendino
Affiliations

The n3-polyunsaturated fatty acid docosahexaenoic acid induces immunogenic cell death in human cancer cell lines via pre-apoptotic calreticulin exposure

Romina Molinari et al. Cancer Immunol Immunother. 2011 Oct.

Abstract

Some anticancer chemotherapeutics, such as anthracyclines and oxaliplatin, elicit immunogenic apoptosis, meaning that dying cancer cells are engulfed by dendritic cells and tumor antigens are efficiently presented to CD8+ T cells, which control residual tumor cells. Immunogenic apoptosis is characterized by pre-apoptotic cell surface exposure of calreticulin (CRT), which usually resides into the endoplasmic reticulum. We investigated the ability of the n3-polyunsaturated fatty acid docosahexaenoic acid (22:6n3, DHA) to induce pre-apoptotic CRT exposure on the surface of the human PaCa-44 pancreatic and EJ bladder cancer cell lines. Cells were treated with 150 μM DHA for different time periods, and, by immunoblot and immunofluorescence, we showed that DHA induced CRT exposure, before the apoptosis-associated phosphatidylserine exposure. As for the known immunogenic compounds, CRT exposure was inhibited by the antioxidant GSH, the pan-caspase zVAD-FMK, and caspase-8 IETD-FMK inhibitor. We provide the first evidence that DHA induces CRT exposure, representing thus a novel potential anticancer immunogenic chemotherapeutic agent.

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Conflict of interest statement

We do not have any financial/commercial conflict of interest.

Figures

Fig. 1
Fig. 1
DHA induces pre-apoptotic CRT exposure. PaCa-44 and EJ cells were incubated with 150 μM DHA or, as negative control, with 10 μM CDDP for the indicated period, followed by: a immunofluorescence staining of PaCa-44 cells using annexin V-FITC (AV)/iodure propide (IP) and cytofluorimetry; results are expressed as the mean ± SD of three experiments; b immunoblot using anti-CRT antibody in total cell lysates (T) or surface proteins (S) of PaCa-44 cells; β-actin was used as both loading and intracellular protein control; representative experiment out of three; c immunofluorescence and flow cytometry on viable PaCa-44 cells using anti-CRT or isotype control antibody; numbers indicate mean fluorescence intensity (MFI) ratio, calculated as the ratio between MFI of positive cells and MFI of control; representative experiment out of three; d immunofluorescence staining of EJ cells using AV/IP and cytofluorimetry; results are expressed as the mean ± SD of three experiments; e immunofluorescence and flow cytometry on viable EJ cells using anti-CRT or isotype control antibody; numbers indicate MFI ratio; representative experiment out of three. NT not treated, CTR control for CDDP: PBS alone, CDDP CDDP dissolved in PBS, Ctr control for DHA: ethanol solution alone, DHA DHA dissolved in ethanol solution. *P < 0.05
Fig. 2
Fig. 2
DHA-induced CRT exposure is ROS- and caspase-dependent. PaCa-44 cells, treated with 150 μM DHA for the indicated period, were pre-incubated with antioxidant GSH, pan-caspase zVAD-FMK or caspase-8 IETD-FMK inhibitor, and immunofluorescence and flow cytometry was performed using: a anti-CRT or isotype control antibody; numbers indicate MFI ratio; representative experiment out of three; b annexin V-FITC (AV) and iodure propide (IP); results are expressed as the mean ± SD of three experiments. NT not treated, Ctr control: ethanol solution alone, DHA DHA dissolved in ethanol solution. *P < 0.05
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