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Abstract
BackgroundDendritic cells (DCs) are unique antigen presenting cells (APC) which play a pivotal role in immunotherapy and induction of an effective immune response against tumors. In the present study, 80% ethanol extract of Phyllanthus amarus was used to generate tumor lysate (TLY) derived from HCT 116 and MCF-7 cancer cell lines via induction of apoptosis. Monocyte-derived DCs were generated ex vivo from the adherent population of peripheral blood mononuclear cells (PBMCs). The generated TLY were used to impulse DCs to investigate its effect on their cellular immune functions including antigen presentation capacity, phagocytic activity, chemotaxis capacity, T-cell proliferation and cytokines release.MethodsThe effect of P. amarus-generated TLY on DCs maturation was evaluated by determination of MHC class I, II and CD 11c expression as well as the co-stimulatory molecules CD 83 and 86 by using flow cytometry. The phagocytic capacity of TLY-pulsed DCs was investigated through FITC-dextran uptake by using flow cytometry. The effect on the cytokines release including IL-12, IL-6 and IL-10 was elucidated by using ELISA. The migration capacity and T cell proliferation activity of pulsed DCs were measured. The relative gene expression levels of cytokines were determined by using qRT-PCR. The major constituents of P. amarus extract were qualitatively and quantitatively analyzed by using validated reversed-phase high performance liquid chromatography (HPLC) methods.ResultsP. amarus-generated TLY significantly up-regulated the expression levels of MHC class I, CD 11 c, CD 83 and 86 in pulsed DCs. The release of interleukin IL-12 and IL-6 was enhanced by TLY-DCs at a ratio of 1 DC: 3 tumor apoptotic bodies (APO), however, the release of IL-10 was suppressed. The migration ability as well as allogeneic T-cell proliferation activities of loaded DCs were significantly enhanced, but their phagocytic capacity was highly attenuated. The gene expression profiles for IL-12 and IL-6 of DCs showed increase in their mRNA gene expression in TLY pulsed DCs versus unloaded and LPS-treated only DCs.ConclusionThe effect of P. amarus-generated TLY on the immune effector mechanisms of DCs verified its potential to induce an in vitro anti-tumor immune response against the recognized tumor antigen.
Highlights
Dendritic cells (DCs) are unique antigen presenting cells (APC) which play a pivotal role in immunotherapy and induction of an effective immune response against tumors
Generation and pulsing of immature DCs with tumor lysate The induction of apoptosis after treatment with P. amarus in HCT 116 and MCF-7 at 1000 μg/mL for 24 h was determined by Annexin V-FITC/PI dual staining assay (Fig. 2a)
At day 6, immature dendritic cells were co-cultured with tumor lysate that was generated by five freeze and thaw cycles from P. amarus-treated HCT 116 human colon cells and MCF-7 adenocarcinoma breast cancer cells
Summary
Dendritic cells (DCs) are unique antigen presenting cells (APC) which play a pivotal role in immunotherapy and induction of an effective immune response against tumors. Cancer immunotherapy represents a novel approach that destroys the existing tumor cells as well as develops a long-lasting immunity which will prevent tumor relapse [1] It aims to induce the immune system to target the tumor antigens and proteins that are expressed by the tumor cells. Mature DCs are characterized by the high expression level of MHC as well as co-stimulatory molecules such as CD 80, CD 83 and CD 86 that stabilize the interaction between DCs and naïve T lymphocytes for induction of antitumor immune response [4] This is in addition to the release of pro-inflammatory cytokines such as interferon (IFN)-γ and interleukin (IL)-12 that activate Th1 response, which catalyzes the activation of cytotoxic T lymphocyte (CTL) [5]. DCs possess the ability to induce NK cells and B cells This dual role in both the innate and adaptive immune systems derived into the possibility of using DCs in immunotherapy in combination with alternative treatment modalities. The protein fragments derived from phagocytosed cells are well presented more than the processed by MHC II products of DCs [9]
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