Plasma medicine is a rapidly expanding new field of interdisciplinary research that combines physics, chemistry, biology, and medicine. Non-thermal atmospheric pressure plasma (NTAPP) has recently been applied to living cells and tissues, and has emerged as a novel technology for medical applications, such as sterilization, wound healing, blood coagulation, and cancer treatment. NTAPP was found to affect cells indirectly through the treatment of cells with previously prepared medium irradiated by NTAPP, termed plasma-activated medium (PAM). We found that PAM triggered a spiral apoptotic cascade in the mitochondrial-nuclear-membrane network in A549 cancer cells. However, difficulties are associated with applying PAM to the clinical phase because culture media cannot be used for medical treatments. The antitumor activity of NTAPP-activated acetate Ringer’s solution (PAA) was significantly stronger than that of PAM. PAA maintained its ability for at least 1 week stored at any temperature tested, whereas PAM was stable only at -80 ℃. At the end, we herein demonstrated the advantages of the combined application of PAA and hyperthermia, a heat treatment at 42 ℃, for A549 cancer cell death with increases in intracellular calcium ([Ca²⁺]i). The activation of transient receptor potential melastatin 2 (TRPM2) may enhance cell death because the addition of TRPM2 inhibitors and knockdown of TRPM2 significantly abrogated the above phenomena. TRPM2 is a temperature-sensitive, Ca²⁺-permeable, non-selective cation channel, and hydrogen peroxide (H₂O₂) and ADP-ribose are its main agonists. PAA functioned as a donor of reactive oxygen species, mainly H₂O₂, and a treatment of A549 cells with PAA under hyperthermia enhanced both mitochondrial and nuclear damage with DNA breaks. Although further studies are needed, the results of our studies provide evidence for the antitumor effects of the synergism between NTP-irradiated liquid and hyperthermia as well as its potential for clinical applications.