Sklias Kyriakos, Santos Sousa João, Girard Pierre-Marie
Université Paris-Saclay, CNRS, Laboratoire de Physique des Gaz et des Plasmas, 91405 Orsay, France.
Institut Curie, PSL Research University, CNRS, INSERM, UMR 3347, 91405 Orsay, France.
Cancers (Basel). 2021 Feb 4;13(4):615. doi: 10.3390/cancers13040615.
(1) Plasma-activated liquids (PAL) have been extensively studied for their anti-cancer properties. Two treatment modalities can be applied to the cells, direct and indirect plasma treatments, which differ by the environment to which the cells are exposed. For direct plasma treatment, the cells covered by a liquid are present during the plasma treatment time (phase I, plasma ON) and the incubation time (phase II, plasma OFF), while for indirect plasma treatment, phase I is cell-free and cells are only exposed to PAL during phase II. The scope of this work was to study these two treatment modalities to bring new insights into the potential use of PAL for cancer treatment. (2) We used two models of head and neck cancer cells, CAL27 and FaDu, and three models of normal cells (1Br3, NHK, and RPE-hTERT). PBS was used as the liquid of interest, and the concentration of plasma-induced HO, NO and NO, as well as pH change, were measured. Cells were exposed to direct plasma treatment, indirect plasma treatment or reconstituted buffer (PBS adjusted with plasma-induced concentrations of HO, NO, NO and pH). Metabolic cell activity, cell viability, lipid peroxidation, intracellular ROS production and caspase 3/7 induction were quantified. (3) If we showed that direct plasma treatment is slightly more efficient than indirect plasma treatment and reconstituted buffer at inducing lipid peroxidation, intracellular increase of ROS and cancer cell death in tumor cells, our data also revealed that reconstituted buffer is equivalent to indirect plasma treatment. In contrast, normal cells are quite insensitive to these two last treatment modalities. However, they are extremely sensitive to direct plasma treatment. Indeed, we found that phase I and phase II act in synergy to trigger cell death in normal cells and are additive concerning tumor cell death. Our data also highlight the presence in plasma-treated PBS of yet unidentified short-lived reactive species that contribute to cell death. (4) In this study, we provide strong evidence that, in vitro, the concentration of RONS (HO, NO and NO) in combination with the acidic pH are the main drivers of plasma-induced PBS toxicity in tumor cells but not in normal cells, which makes ad hoc reconstituted solutions powerful anti-tumor treatments. In marked contrast, direct plasma treatment is deleterious for normal cells in vitro and should be avoided. Based on our results, we discuss the limitations to the use of PAL for cancer treatments.
(1) 血浆激活液（PAL）因其抗癌特性已得到广泛研究。可对细胞应用两种处理方式，即直接血浆处理和间接血浆处理，这两种方式的区别在于细胞所暴露的环境。对于直接血浆处理，在血浆处理时间（第一阶段，血浆开启）和孵育时间（第二阶段，血浆关闭）期间，细胞被液体覆盖；而对于间接血浆处理，第一阶段无细胞，细胞仅在第二阶段暴露于PAL。本研究的范围是研究这两种处理方式，以便为PAL在癌症治疗中的潜在应用带来新的见解。(2) 我们使用了两种头颈部癌细胞模型，CAL27和FaDu，以及三种正常细胞模型（1Br3、NHK和RPE - hTERT）。使用PBS作为感兴趣的液体，并测量血浆诱导的HO、NO和NO₂的浓度以及pH变化。将细胞暴露于直接血浆处理、间接血浆处理或重构缓冲液（用血浆诱导的HO、NO、NO₂浓度和pH调节的PBS）中。对细胞代谢活性、细胞活力、脂质过氧化、细胞内ROS产生和caspase 3/7诱导进行定量分析。(3) 如果我们表明在诱导肿瘤细胞脂质过氧化、细胞内ROS增加和癌细胞死亡方面，直接血浆处理比间接血浆处理和重构缓冲液略有效，那么我们的数据还显示重构缓冲液与间接血浆处理等效。相比之下，正常细胞对这后两种处理方式相当不敏感。然而，它们对直接血浆处理极为敏感。事实上，我们发现第一阶段和第二阶段协同作用触发正常细胞死亡，而对肿瘤细胞死亡则具有累加作用。我们的数据还突出了血浆处理的PBS中存在尚未鉴定的短寿命活性物质，这些物质促成细胞死亡。(4) 在本研究中，我们提供了有力证据，即在体外，活性氧氮类物质（HO、NO和NO₂）的浓度与酸性pH相结合是血浆诱导的PBS对肿瘤细胞毒性的主要驱动因素，但对正常细胞并非如此，这使得特制的重构溶液成为强大的抗肿瘤治疗方法。与之形成鲜明对比的是，直接血浆处理在体外对正常细胞有害，应予以避免。基于我们的结果，我们讨论了使用PAL进行癌症治疗的局限性。
