Enhancing Doxorubicin-Induced MCA-Fibrosarcoma Cytotoxicity by an Eriobotrya japonica Hydrophilic Butanol-Treated Extract through Natural Killer Cells Activation

Abstract

The combination of cytotoxic drugs with immunotherapy should be more effective than monotherapy alone since both therapeutic modalities may target different mechanisms. In addition, combination therapy may reduce adverse events associated with cytotoxic drugs. Eriobotrya japonica hydrophilic butanol-treated extract (EJWR) was found to modulate cytokines by enhancing IL-12, IFN-γ and TNF-α in vitro and in vivo and within tumor microenvironment. This was associated with enhancing survival time of mice bearing intra-peritoneal MCA fibrosarcoma (MCA FS).In the present work, we evaluated the combination of EJWR with doxorubicin (Dox) on MCA FS cytotoxicity using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay in the absence and presence of spleen cells or Natural Killer (NK) lymphocytes from tumor bearing mice. The results showed that Dox exhibited mild cytotoxicity to healthy spleen cells and EJWR reversed such cytotoxicity. In addition, increasing concentrations of Dox induced 40% (p<0.01) MCA FS cytotoxicity. This percent increased significantly to 60% at Dox 5 µM when co-cultured with NK cells from tumor bearing mice and increased further to 80% (p<0.01) when Dox was combined with EJWR. The latter increase in cytotoxicity was significantly (p<0.01) higher than each agent alone. This enhancement was associated with significant production of TNF-α and retaining IFN-γ levels from NK cells lysates. This concluded that the immunomodulator, EJWR, mediates NK activation and enhances Dox-induced MCA FS cytotoxicity.

Eriobotrya japonica Water Extract Characterization: An Inducer of Interferon-Gamma Production Mainly by the JAK-STAT Pathway

“…Extraction of HE by n-butanol to yield EJ water residue (WP) was previously performed to concentrate polysaccharides, high molecular weight oligomeric procyanidins and related polyphenols and to exclude low molecular weight compounds [12,16]. This WP extract was proven to induce the release of IFN-γ from mouse spleen cells and NK cells [16,29]. In the current investigation, however, further fractionation of WP was performed using methanol:acetone to yield the MAU fraction and MAL.…”

“…The anti-inflammatory action is mediated mainly by the triterpene acids and epicatechin isolated from Eriobotrya leaves [6,7,9,14,31]. However, studies by us and another group showed that EJ leaves polarize T helper 1 cytokines over T helper 2 cytokines [15,16,17,18,29]. Therefore, to identify the compounds or group of compounds that are responsible for the induction of IFN-γ, further sub-fractionation of MAU fraction was conducted by column chromatography using eluents with different polarities.…”

Dichloroacetate modulates cytokines toward T helper 1 function via induction of the interleukin-12&ndash;interferon-&gamma; pathway

“…Four hours later 50 μL sodium dodecyl sulfate was added. When spleen was cocultured with the adherent MCA fibrosarcoma, and before adding the MTT reagent, the supernatant content of each well was carefully removed leaving the adherent MCA-fibrosarcoma in the wells 19. Absorbance was read at 570 nm by GloMax®-Multi Detection System (Promega, Madison, WI, USA).…”

“…Production of IL-12 is a major inducer of T helper 1(Th1) and natural killer (NK) lymphocytes and their cytokines. These produced cytokines polarize macrophages, enhance dendritic cell maturation, and lower regulatory T-cell function by inhibiting regulatory cytokines such as transforming growth factor (TGF)-β and IL-10 16–19. Since it has been shown that DCA inhibits PDK and facilitates signaling pathways that lead to the inhibition of transcription factor nuclear factor of activated T-cells,2 we were encouraged to study DCA immunomodulatory activity.…”

“…These produced cytokines polarize macrophages, enhance dendritic cell maturation, and lower regulatory T-cell function by inhibiting regulatory cytokines such as transforming growth factor (TGF)-β and IL-10. 16 – 19 Since it has been shown that DCA inhibits PDK and facilitates signaling pathways that lead to the inhibition of transcription factor nuclear factor of activated T-cells, 2 we were encouraged to study DCA immunomodulatory activity. The present study was first based on whether DCA antiproliferative activity and cytotoxicity on 3-methylcholanthrene (MCA) fibrosarcoma could be enhanced in the presence of immune cells, and then determining if this phenomenon is related to DCA’s ability to modulate cytokines in vitro and in vivo.…”