Docosahexaenoic acid (DHA) promotes immunogenic apoptosis in human multiple myeloma cells, induces autophagy and inhibits STAT3 in both tumor and dendritic cells

Abstract

Docosahexaenoic acid (DHA), a ω-3 polyunsaturated fatty acid found in fish oil, is a multi-target agent and exerts anti-inflammatory and anticancer activities alone or in combination with chemotherapies. Combinatorial anticancer therapies, which induce immunogenic apoptosis, autophagy and STAT3 inhibition have been proposed for long-term therapeutic success. Here, we found that DHA promoted immunogenic apoptosis in multiple myeloma (MM) cells, with no toxicity on PBMCs and DCs. Immunogenic apoptosis was shown by the emission of specific DAMPs (CRT, HSP90, HMGB1) by apoptotic MM cells and the activation of their pro-apoptotic autophagy. Moreover, immunogenic apoptosis was directly shown by the activation of DCs by DHA-induced apoptotic MM cells. Furthermore, we provided the first evidence that DHA activated autophagy in PBMCs and DCs, thus potentially acting as immune stimulator and enhancing processing and presentation of tumor antigens by DCs. Finally, we found that DHA inhibited STAT3 in MM cells. STAT3 pathway, essential for MM survival, contributed to cancer cell apoptosis by DHA. We also found that DHA inhibited STAT3 in blood immune cells and counteracted STAT3 activation by tumor cell-released factors in PBMCs and DCs, suggesting the potential enhancement of the anti-tumor function of multiple immune cells and, in particular, that of DCs.

Keywords: STAT3; autophagy; dendritic cells-DCs; docosahexaenoic acid-DHA; immunogenic cell death.

Figure 1. DHA induces apoptosis in MM cells and does not affect PBMC viability

A. DHA decreases viability of MM cell lines in a dose- and time-dependent manner, whereas it does not affect the survival of PBMCs derived from healthy donors. RPMI-8226, OPM-2 and PBMCs were cultured with vehicle (Ctrl) or DHA (μM) and their viability evaluated by trypan blue exclusion assay; mean of the percentage of cell surviaval plus SD of three independent experiments is indicated; B. RPMI-8226 and OPM-2 were cultured with vehicle (Ctrl) or DHA (μM) and apoptosis was assessed by Annexin V-FITC (AV) and propidium iodide (PI) cell staining and flow cytofluorimetry; representative experiments out of three; C. RPMI-8226 and OPM-2 were cultured with vehicle (Ctrl) or 100 μM DHA for 24 hours in the absence or presence of z-VAD-FMK (50 μM) and analyzed for apoptosis by AV and PI cell staining; representative experiments out of three.

Figure 2. DHA triggers the emission of immunogenic DAMPs by MM cells

A. RPMI-8226 and OPM-2 were cultured with vehicle (Ctrl) or 100 μM DHA for 3 and 6 hours, respectively; cell surface immunofluorescence staining using anti-CRT, anti-HSP90 or isotype control antibodies was analyzed by flow cytofluorimetry, while gating on the viable population and excluding dead cells stained with PI; numbers indicate the ratio of the mean fluorescence intensity (MFI) of DHA treated cells/MFI of control cells. B. RPMI-8226 and OPM-2 were cultured with vehicle (Ctrl) or 100 μM DHA for the indicated times; then, tumor cell conditioned media were collected and the presence of HMGB1 was analyzed by Western blot; β-actin was used as intracellular protein control and Ponceau staining as loading control. Representative experiments out of three.

Figure 3. DHA enhances autophagy in MM cells, which contributes to DHA-induced cell death

RPMI-8226 (A) and OPM-2 (B) were cultured with vehicle (Ctrl) or 100 μM DHA for 24 hours in the presence or in the absence of Bafilomycin (Baf) and the expression of the autophagic markers such as LC3I/II and p62 was analyzed by Western blot; β-actin was included as control; numbers indicate band intensities (b.i.) = band volume/area x mean pixel intensity, normalized for β-actin and quantified using Quantity One 1-D analysis software; C. RPMI-8226 cells were cultured for 24 hours with vehicle (Ctrl) or 100 μM DHA in presence or absence of 3-MA (0.3 mM) and their viability assessed by trypan blue exclusion assay (left panel) and cytofluorimetry cell cycle analysis of sub-G1 events, representing apoptotic cells (right panel). Representative experiments out of three.

Figure 4. DHA enhances autophagy in PBMCs and DCs

PBMCs (A) and DCs (B) derived from healthy donors were cultured with vehicle (Ctrl) or 100 μM DHA for 24 hours and the expression of the autophagic markers LC3I/II and p62 was analyzed by Western blot; β-actin was included as control; numbers indicate band intensities (b.i.) = band volume/area x mean pixel intensity, normalized for β-actin and quantified using Quantity One 1-D analysis software; the viability of PBMCs (A) and DCs (B) was assessed by trypan blue exclusion assay. Representative experiment out of three.

Figure 5. DHA-triggered immunogenic apoptosis in MM cells activates DCs

Immature DCs (iDCs), generated from PBMC-derived CD14+ monocytes cultured with GM-CSF and IL-4 for 6 days, were co-cultured with vehicle- (Ctrl) or DHA-treated RPMI-8226 cells for 24 hours and the expression of DC differentiation (CD83) and activation (CD86) markers was analyzed by immunofluorescence and flow cytometry (B). As positive control of DC activation, cells were treated with LPS (100 ng/ml) for the same time (A). Representative experiment out of three.

Figure 6. DHA inhibits STAT3 pathway in MM cells, PBMCs and DCs

A. RPMI-8226 and OPM-2 were cultured with vehicle (Ctrl) or 100 μM DHA for 24 hours in the presence or absence of sodium orthovanadate (OV) (150 μM) and STAT3 tyrosine phosphorylation (p-STAT3) was evaluated by Western blot (left panels); total STAT3 and β-actin were included as control; numbers indicate band intensities (b.i.) = band volume/area x mean pixel intensity, normalized for β-actin and quantified using Quantity One 1-D analysis software; viability of MM cells was assessed by trypan blue exclusion assay (right panels); *p< 0.000; PBMCs from healthy donors (B) and iDCs (C), generated from PBMC-derived CD14+ monocytes cultured with GM-CSF and IL-4 for 6 days, were cultured with vehicle (Ctrl) or 100 μM DHA for 24 hours in the presence or absence of tumor cell conditioned medium (TCCM) or OV (150 μM), and evaluated for STAT3 tyrosine phosphorylation by Western blot (left panels); total STAT3 and β-actin were included as control; numbers indicate band intensities (b.i.) = band volume/area x mean pixel intensity, normalized for β-actin and quantified using Quantity One 1-D analysis software; the viability of PBMCs and DCs was assessed by trypan blue exclusion assay (right panels). Representative experiments out of three.