Prostate cancer (PCa) progression has been demonstrated to depend on a variety of signalling pathways such as mitogen-activated protein kinases (MAPKs), phosphatidylinositol 3-kinase PI3K/Akt pathway, phosphorylation of glycogen synthase kinase 3-beta (GSK-3β) and Proto-oncogene tyrosine-protein kinase Src signalling (c-Src or Src). β-endorphins are the primary agonists of μ-opioid receptors (MOR) and are secreted by the anterior pituitary gland via the hypothalamus after the cleavage of pro-opiomelanocortin (POMC). It is present throughout the body, including the brain and immune cells, and regulates several systems. Furthermore, β-endorphins may have therapeutic value in cancer prevention and general health improvement by inhibiting the inflammatory response of bovine endometrial epithelial and stromal cells via the μ-opioid receptor. Moreover, it can inhibit tumour cells and prevents them from transforming into metastatic cancer cells by decreasing body stress, maintaining an active immune system, and balancing the levels of pro-inflammatory and anti-inflammatory cytokines such as IL-1β, IL-8 and IL-12. The PI3K-Akt pathway is one of the most frequently over-activated intracellular pathways which has become a vital tumour-related pathway by regulating cell life processes including apoptosis and proliferation. Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase that plays a significant role in the progression of PCa that promotes cell proliferation and survival. The expression of GSK-3β can promote PCa and its inhibition reduces prostate cancer cell proliferation, in part by reducing androgen receptor (AR) signalling. In this study, we aimed at exploring the potential therapeutic effect of β-endorphin against prostate cancer through suppression of the malignancy-associated tissue inflammation using an in vitro model.

PCa cells (PC3) were treated with various concentrations of morphine (0.5-2.0  μmol/L), and an MTT assay was used to explore the cytotoxic effect of various concentrations of morphine (0.5-2.5 μmol/L), naloxone, combined naloxone and morphine, and β-endorphin (10-60 pg/ml) on the PC3 cells at 24, 48, and 72-hour incubation periods. In addition, an ELISA assay was used to assess the release of the pro-inflammatory cytokines IL-6, IL-8 and IL-1β by the PC3 cells upon their incubation with TNF-α (10 ng/ml) for 24 hours with or without pre-incubation with morphine. Finally, the associated intracellular signalling events were assessed using a specific Proteome Profiler Human Phospho-Kinase Array Kit.

Interestingly, various concentrations of β-endorphin indicated a significant cytotoxic effect on PC3 cells, which was maximal at 30 pg/mL concentration (Figure 1(A)). Moreover, 24-hour incubation with TNF-α induced significant release of the cytokines IL-1β, IL-6 and IL-8 by PC3 cells. Such release was significantly inhibited by pre-incubation with 30 pg/mL of β-endorphin (Figure 1(B1-B3)). Furthermore, upon 2 hours of incubation, TNF-α induced a significant elevation in the phosphorylation levels of the protein kinases GSK-3α/β (S21/S9), Src (Y419), WNK1 (T60), Akt 1/2/3 (Ser473), p53 (S15), PRAS40 (T246) and WNK1 (T60) (Figure 2).

Interestingly, various concentrations of β-endorphin exerted a significant cytotoxic effect on PC3 cells, which was maximal at 30 pg/mL concentration. Moreover, 24-hour incubation with TNF-α induced significant release of the cytokines IL-1β, IL-6 and IL-8 by PC3 cells. In addition, at 2 and 4 hours of incubation, TNF-α induced a significant increase in the phosphorylation levels of the protein kinases GSK-3α/β (S21/S9), Src (Y419), WNK1 (T60), Akt 1/2/3 (Ser473), p53 (S15), PRAS40 (T246) and WNK1 (T60).

Interestingly, β-endorphin demonstrated a high potency in preventing the TNF-α induced tissue inflammation through suppression of the pro-inflammatory cytokine release by PC3 cells. Such was accompanied by modulation of the TNF-α-intracellular signalling events in PC3 cells. The current study demonstrates preliminary evidence suggesting that β-endorphin could be used as a novel therapeutic approach to PCa patients.