Inosine Strongly Enhances Proliferation of Human C32 Melanoma Cells through PLC‐PKC‐MEK1/2‐ERK1/2 and PI3K Pathways

Abstract

Malignant melanoma is the most deadly type of skin cancer. The lack of effective pharmacological approaches for this tumour can be related to the incomplete understanding of the pathophysiological mechanisms involved in melanoma cell proliferation. Adenosine has growth-promoting and growth inhibitory effects on tumour cells. We aimed to investigate effects of adenosine and its metabolic product, inosine, on human C32 melanoma cells and the signalling pathways involved. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and bromodeoxyuridine (BrdU) proliferation assays were used to evaluate adenosine, adenosine deaminase and inosine effects, in the absence or presence of adenosine receptor (AR), A 3 AR and P2Y 1 R antagonists and PLC, PKC, MEK1/2 and PI3K inhibitors. ERK1/2 levels were determined using an ELISA kit. Adenosine and inosine levels were quantified using an enzyme-coupled assay. Adenosine caused cell proliferation through AR activation. Adenosine deaminase increased inosine levels (nanomolar concentrations) on the extracellular space, in a time-dependent manner, inducing proliferation through A 3 AR activation. Micromolar concentrations of inosine enhanced proliferation through A 3 AR activation, causing an increase in ERK1/2 levels, and P2Y 1 R activation via ENT-dependent mechanisms. We propose the simultaneous activation of PLC-PKC-MEK1/2-ERK1/2 and PI3K pathways as the main mechanism responsible for the proliferative effect elicited by inosine and its significant role in melanoma cancer progression.

Inosine Released from Dying or Dead Cells Stimulates Cell Proliferation via Adenosine Receptors

“…We could only verify the stimulating capacity of adenosine in our system, and it seems to be limited by the concentration of the adenosine vehicle ammonia. It has already been reported that inosine, and not adenosine, exerts potent proliferation-stimulatory actions on melanoma cells, mainly through the engagement of A3 adenosine receptor (40). Since we have not inhibited the catabolism of adenosine to inosine in the SNs, it is likely that the stimulation of proliferation of melanoma cells is ultimately supported by inosine.…”

Targeting Adenosine Receptor Signaling in Cancer Immunotherapy

“…Indeed, the A3R agonists CL-IBMECA and CF102 have shown promise in vitro by inducing apoptosis in multiple tumor types through the suppression of PKA, ERK and AKT pathways [80,[97][98][99]. Conversely, it has also been reported that inosine-mediated activation of the A3R can enhance melanoma cell proliferation through activation of the ERK pathway [100] and a cytoprotective role for the A3R has also been described [101]. Similarly, activation of the A3R has been suggested to enhance the migration or invasion of tumor cells [73,102,103].…”

Identification of melanoma biomarkers based on network modules by integrating the human signaling network with microarrays

“…Hence, PKC enzymes play important roles in several signal transduction cascades. Soares et al [26] proposed that the simultaneous activation of the phospholipase C (PLC)-PKC-MEK1/2-ERK1/2 and phosphatidylinositol-3-kinase (PI3K) pathways is the principal mechanism responsible for the proliferative effect elicited by inosine and its significant role in melanoma cancer progression. Moreover, Halder et al [27] observed a marked variation in the expression of PKCα and PKCδ isotypes in B16F10 melanoma tumor cells compared with normal melanocytes and suggested the presence of a reciprocal PKC signaling pathway.…”