Abstract
The major drawback of current anti-angiogenic therapies is drug resistance, mainly caused by overexpression of the transcription factor, hypoxia-inducible factor 1α (HIF-1α) as a result of treatment-induced hypoxia, which stimulates cancer cells to develop aggressive and immunosuppressive phenotypes. Moreover, the cancer cell resistance to anti-angiogenic therapies is deeply mediated by the communication between tumor cells and tumor-associated macrophages (TAMs)—the most important microenvironmental cells for the coordination of all supportive processes in tumor development. Thus, simultaneous targeting of TAMs and cancer cells could improve the outcome of the anti-angiogenic therapies. Since our previous studies proved that simvastatin (SIM) exerts strong antiproliferative actions on B16.F10 murine melanoma cells via reduction of TAMs-mediated oxidative stress and inhibition of intratumor production of HIF-1α, we investigated whether the antitumor efficacy of the anti-angiogenic agent—5,6-dimethylxanthenone-4-acetic acid (DMXAA) could be improved by its co-administration with the lipophilic statin. Our results provide confirmatory evidence for the ability of the combined treatment to suppress the aggressive phenotype of the B16.F10 melanoma cells co-cultured with TAMs under hypoxia-mimicking conditions in vitro. Thus, proliferation and migration capacity of the melanoma cells were strongly decelerated after the co-administration of SIM and DMXAA. Moreover, our data suggested that the anti-oxidant action of the combined treatment, as a result of melanogenesis stimulation, might be the principal cause for the simultaneous suppression of key molecules involved in melanoma cell aggressiveness, present in melanoma cells (HIF-1α) as well as in TAMs (arginase-1). Finally, the concomitant suppression of these proteins might have contributed to a very strong inhibition of the angiogenic capacity of the cell co-culture microenvironment.
Highlights
Melanoma cells, as well as normal melanocytes are under persistent oxidative stress due to their specialized function of melanin synthesis that generates reactive oxygen species (ROS) mainly due to the catalytic activity of tyrosinase, the rate-limiting enzyme of melanogenesis [1,2,3]
Since 3.5 μM was the lowest concentration of SIM which administered in combination with 100 μM dimethylxanthenone-4-acetic acid (DMXAA) exerted strong inhibitory effects on B16.F10 cell proliferation (Fig 1B), this combination was used throughout the experiments to further investigate the molecular mechanisms of the synergistic antitumor efficacy of both drugs on melanoma microenvironment model in vitro
Our results showed that oxidative stress was notably suppressed by all treatments, but the strongest inhibition was caused by the combined treatment, which almost completely reduced the MDA levels in cell co-culture lysates (Fig 4)
Summary
As well as normal melanocytes are under persistent oxidative stress due to their specialized function of melanin synthesis that generates reactive oxygen species (ROS) mainly due to the catalytic activity of tyrosinase, the rate-limiting enzyme of melanogenesis [1,2,3]. In melanoma cells, melanogenesis can regulate the expression of the transcription factor, hypoxia-inducible factor 1α (HIF-1α), the key molecule for the cancer cell resistance to anti-angiogenic therapies, decreasing the effectiveness of the treatments and the survival rates during stages III and IV of melanoma [4,5,6,7]. Efforts to target melanoma angiogenesis suggested that the remaining cancer cells developed scavenger mechanisms, such as overexpression of HIF-1α, as a result of hypoxia and oxidative stress generated by these treatments. These mechanisms lead to enhanced invasive capacity of the tumor cells [8]. The perspective of synergistic therapies targeting multiple microenvironment stromal cells has led to the emergence of the widely accepted “targeted microenvironment therapy” concept, rooted in the ideea that an overall “healthy” state of the microenvironment can protect against tumorigenesis and invasion [10]
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