Resistance of muscle to tumor metastases: a role for a3 adenosine receptor agonists

Abstract

Tumor metastases are extremely rare in striated muscles. Lately, we have found that muscle cell conditioned medium (MCM) inhibits the proliferation of various tumor cells while maintaining the growth of normal murine bone marrow cells. This dual activity was confirmed in vivo when the MCM was administered orally, i.e., it inhibited the development of tumor growth in mice and prevented the myelotoxic effects of chemotherapy. Adenosine was found to be one of the active components of MCM, inhibiting tumor cell growth while maintaining bone marrow cell proliferation in vitro. Adenosine is known to act as an important regulatory molecule through its binding to specific G-protein-associated A1, A(2a), A(2b) and A3 cell surface receptors. In distinction from MCM, adenosine did not suppress tumor development in mice and was not active as a chemoprotective agent when administered orally or intravenously. Thus, the in vivo activity of MCM could not be attributed to adenosine. In this study, MCM from which adenosine was enzymatically removed still retained its dual activity that was also found to be mediated through the A3 adenosine receptor (A3AR). This result led to the conclusion that natural agonists to A3AR were responsible for the activity of MCM. We further tested synthetic agonist to the A3AR and demonstrated that it possessed the same in vitro and in vivo activity profile as MCM. Taken together, muscle cells, in addition to adenosine, secrete natural agonists to A3AR. These agonists are stable nondegradable molecules and may contribute to the systemic anticancer and chemoprotective activity exerted by MCM. This group of molecules may account for the rarity of tumor metastases in muscle.

Figure 1

The effect of adenosine, MCM, and MCM+ADA on the development of lung metastases in mice inoculated with B16-F10 melanoma cells. The number of melanoma foci was inhibited (P<0.001) following treatment with 4 activity units of MCM or MCM+ADA. All the preparations were administered to mice through an oral route. Adenosine failed to inhibit tumor growth.

Figure 2

The in vivo effect of adenosine, MCM, and MCM+ADA on the number of WBC and percentage of neutrophils in mice treated with 50 mg/kg body weight cyclophosphamide. Chemotherapy alone decreased the number of WBC and percentage of neutrophils. MCM or MCM+ADA, administered after chemotherapy, increased the number of WBC (a) and percentage of neutrophils (b) to almost normal values.

Figure 3

MCM and to a lesser extent MCM+ADA induced the inhibitory effect on the growth of B16-F10 melanoma cells (a) or stimulatory effect on the proliferation of murine bone marrow cells (b). Cell proliferation was measured by [³H]thymidine incorporation assay. The introduction of MRS-1220, canceled most of the inhibitory (a) or stimulatory (b) effect that was exerted by MCM+ADA.

Figure 4

IB-MECA, a synthetic A3AR agonist, at different concentrations, inhibitedthe proliferation of B16-F10 melanoma cells (a) and stimulated bone marrow cell proliferation (b), measured by [³H]thymidine incorporation assay.

Figure 5

The effect of IB-MECA on the development of lung metastases in mice inoculated with B16-F10 melanoma cells. IB-MECA was administered orally daily at a dosage of 3 or 6 µg/kg body weight. A dose-dependent inhibition of lung metastatic foci is depicted.

Figure 6

The effect of IB-MECA on the growth of HCT-116 human colon carcinoma cells in nude mice. Tumor cells (1.2x10⁶) were subcutaneously injected to the flank of nude mice. One group was treated every other day with 6 µg/kg IB-MECA and the other, treated with vehicle only, served as control. Tumor size was measured every 4 days. Tumor size in the IB-MECA treated mice was smaller than controls.

Figure 7

The effect of IB-MECA on the myeloid system in mice treated with 50 mg/kg body weight cyclophosphamide. Chemotherapy alone decreased the number of WBC and percentage of neutrophils. IB-MECA, administered 48 and 72 hours after chemotherapy, increased the number of total WBC (a) and restored the percentage of neutrophils (b).

Figure 8

Metabolization of adenosine to inosine occurred rapidly in vivo mainly by the enzyme adenosine deaminase. The A3AR agonist, IB-MECA, resists degradation by adenosine deaminase, due to substitution, at the 2-position of adenosine, combined with modifications at N⁶ and 5′-positions [26,27].