Hepatocellular carcinoma (HCC) is a major histological subtype of primary liver cancer. Ample evidence suggests that the pathological properties of HCC originate from hepatic cancer stem cells (CSCs), which are responsible for carcinogenesis, recurrence, and drug resistance. Cold atmospheric-pressure plasma (CAP) and plasma-activated medium (PAM) induce apoptosis in cancer cells and represent novel and powerful anti-cancer agents. This study aimed to determine the anti-cancer effect of CAP and PAM in HCC cell lines with CSC characteristics. We showed that the air-based CAP and PAM selectively induced cell death in Hep3B and Huh7 cells with CSC characteristics, but not in the normal liver cell line, MIHA. We observed both caspase-dependent and -independent cell death in the PAM-treated HCC cell lines. Moreover, we determined whether combinatorial PAM therapy with various anti-cancer agents have an additive effect on cell death in Huh7. We found that PAM highly increased the efficacy of the chemotherapeutic agent, cisplatin, while enhanced the anti-cancer effect of doxorubicin and the targeted-therapy drugs, trametinib and sorafenib to a lesser extent. These findings support the application of CAP and PAM as anti-cancer agents to induce selective cell death in cancers containing CSCs, suggesting that the combinatorial use of PAM and some specific anti-cancer agents is complemented mechanistically.

Cold atmospheric pressure plasma (CAP) and plasma-activated medium (PAM) induce cell death in diverse cancer cells and may function as powerful anti-cancer agents. The main components responsible for the selective anti-cancer effects of CAP and PAM remain elusive. CAP or PAM induces selective cell death in hepatocellular carcinoma cell lines Hep3B and Huh7 containing populations with cancer stem cell markers. Here, we investigated the major component(s) of CAP and PAM for mediating the selective anti-proliferative effect on Hep3B and Huh7 cells. The anti-proliferative effect of CAP was mediated through the medium; however, the reactive oxygen species scavenger N-acetyl cysteine did not suppress PAM-induced cell death. Neither high concentrations of nitrite or nitrite/nitrate nor a low concentration of H₂O₂ present in the PAM containing sodium pyruvate affected the viability of Hep3B and Huh7 cells. Inhibitors of singlet oxygen, superoxide anions, and nitric oxide retained the capacity of PAM to induce anti-cancer effects. The anti-cancer effect was largely blocked in the PAM prepared by placing an aluminum metal mesh, but not a dielectric PVC mesh, between the plasma source and the medium. Hence, singlet oxygen, hydrogen peroxide, nitric oxide, and nitrite/nitrate are not the main factors responsible for PAM-mediated selective death in Hep3B and Huh7 cells. Other factors, such as charged particles including various ions in CAP and PAM, may induce selective anti-cancer effects in certain cancer cells.

The spindle position checkpoint (SPOC) of budding yeast delays mitotic exit in response to misaligned spindles to ensure cell survival and the maintenance of genomic stability. The GTPase-activating protein (GAP) complex Bfa1–Bub2, a key SPOC component, inhibits the GTPase Tem1 to induce mitotic arrest in response to DNA and spindle damage, as well as spindle misorientation. However, previous results strongly suggest that Bfa1 exerts a GAP-independent function in blocking mitotic exit in response to misaligned spindles. Thus, the molecular mechanism by which Bfa1 controls mitotic exit in response to misaligned spindles remains unclear. Here, we observed that overexpression of the N-terminal domain of Bfa1 (Bfa1-D16), which lacks GAP activity and cannot localize to the spindle pole body (SPB), induced cell cycle arrest along with hyper-elongation of astral microtubules (aMTs) as Bfa1 overexpression in Δbub2. We found that Δbub2 cells overexpressing Bfa1 or Bfa1-D16 inhibited activation of Mob1, which is responsible for mitotic exit. In anaphase-arrested cells, Bfa1-D16 overexpression inhibited Tem1 binding to the SPB as well as Bfa1 overexpression. Additionally, endogenous levels of Bfa1-D16 showed minor SPOC activity that was not regulated by Kin4. These results suggested that Bfa1-D16 may block mitotic exit through inhibiting Tem1 activity outside of SPBs. Alternatively, Bfa1-D16 dispersed out of SPBs may block Tem1 binding to SPBs by physically interacting with Tem1 as previously reported. Moreover, we observed hyper-elongated aMTs in tem1-3, cdc15-2, and dbf2-2 mutants that induce anaphase arrest and cannot undergo mitotic exit at restrictive temperatures, suggesting that aMT dynamics are closely related to the regulation of mitotic exit. Altogether, these observations suggest that Bfa1 can control the SPOC independent of its GAP activity and SPB localization.