Pyruvate Plays a Main Role in the Antitumoral Selectivity of Cold Atmospheric Plasma in Osteosarcoma

Abstract

Osteosarcoma (OS) is the most common primary bone tumor but current therapies still have poor prognosis. Cold Atmospheric Plasma (CAP) and Plasma activated media (PAM) have shown potential to eliminate cancer cells in other tumors. It is thought that Reactive Oxygen and Nitrogen species (RONS) in PAM are key players but cell culture media composition alters treatment outcomes and data interpretation due to scavenging of certain RONS. In this work, an atmospheric pressure plasma jet was employed to obtain PAM in the presence or absence of pyruvate and used to treat the SaOS-2 (OS) cell line or hBM-MSC healthy cells. OS cells show higher sensitivity to PAM treatment than healthy cells, both in medium with and without pyruvate, activating apoptosis, DNA damage and deregulating cellular pathways mediated by c-JUN, AKT, AMPK or STAT3. In line with previous works, lack of pyruvate increases cytotoxic potential of PAM affecting cancer and healthy cells by increasing 10–100 times the concentration of H 2 O 2 without altering that of nitrites and thus decreasing CAP anti-tumor selectivity. Suitable conditions for CAP anti-cancer selectivity can be obtained by modifying plasma process parameters (distance, flow, treatment time) to obtain adequate balance of the different RONS in cell culture media.

Cold Plasma-Treated Ringer’s Saline: A Weapon to Target Osteosarcoma

“…The addition of 10% of FBS after treatment stabilized the pH between 7.8 and 7.1 (Figure 1(Bii)), which allowed isolating the cell effects due only to the concentration and kind of RONS generated in the liquid, as in non-buffered solutions high pH decrease induced by plasma treatment is also reported to severely compromise cell viability [12,48]. It was shown in previous works [36,49] that long treatment time of cell culture medium may produce a lethal concentration of H 2 O 2 which overwhelms cell defense mechanisms and kills both OS and healthy cells [36]. In line with these data, to obtain an equilibrated cocktail of RONS, plasma treatments only up to 5 min were selected ( Figure 1C).…”

“…Despite employing here two jets that use noble gases (Helium in APPJ or Ar in kINPen), an important part of the reactivity of these jets derives from mixing of the plasma gas phase with air. The gas phase of the APPJ during treatment of Ringer's included different nitrogen species, monoatomic oxygen, and hydroxyl radicals, as recorded by optical emission spectroscopy (Figure 1(Ai)), as similarly observed during treatment of cell culture medium [22,36]. Both plasma jets have been extensively characterized in [45,46].…”

“…The highest H 2 O 2 concentration leads, as in kINPen-treated PAR, to a loss of selectivity (hBM-MSC cell death). To obtain an equilibrated cocktail of RONS and avoiding a loss of anti-cancer selectivity [36], for further work we selected APPJ-treated PAR. In order to adjust the production of RONS, plasma parameters like gas flow [36,55], distance of the nozzle [7,36,55], working gas composition [56][57][58][59] and here, device configuration [60,61] are of paramount importance to adjust the composition of RONS generated in the liquid, and the conditions selected here followed previous works of our group [36,62].…”

“…Given the heterogeneity of OS, it is of paramount interest to evaluate OS cell lines with different genomic profiles in views of assessing the real applicability of plasma-activated liquids in OS. Our previous work employing SaOS-2 cells (which are p53 and pRB null), highlighted the importance of an equilibrated cocktail of RONS for anticancer selectivity of the plasma-activated medium and revealed the critical role of H 2 O 2 in these cytotoxic effects [36].…”

“…From another point of view, up to now, cytotoxicity of CAPs or plasma-activated media in OS cell lines have been investigated only in adherent cell cultures [22,23,[29][30][31]36,37]. This kind of in vitro models provides limited clinically relevant information.…”