Selective effects of non-thermal atmospheric plasma on triple-negative breast normal and carcinoma cells through different cell signaling pathways

Abstract

Non-thermal atmospheric plasma (NTP) has shown its selective anticancer effects in many types of tumors in vitro and one of the main mechanisms is that the different increase of intracellular ROS in cancer and homologous normal cells. In this study, we report that NTP treatment reduces the proliferation in triple negative breast cancer (TNBC) and normal cell lines. Simultaneously, STAT3 pathway is inhibited by NTP effects. However, it is observed that normal cells MCF10A are more sensitive to ROS toxicity induced by NTP than cancer cells MDA-MB-231. When 5 mM of ROS inhibitor N-acetyl cysteine (NAC) is employed in NTP treatments, the proliferation of normal breast cells MCF10A recovers. Meanwhile, NTP effects remain significant inhibition of MDA-MB-231 cells. Our results further reveal that NTP can induce apoptosis in MDA-MB-231 cells through inhibiting interleukin-6 receptor (IL-6R) pathway. Moreover, the mechanism of NTP anti-cancer selectivity relates to constantly HER2/Akt activation induced by NTP especially in MCF10A cells but not in MDA-MB-231 cells. Therefore, these two different cell signaling pathways induced by NTP treatments in TNBC and homologous normal cells make NTP becoming a potential tool in future therapy.

Figure 1

NTP treatment reduces proliferation of MDA-MB-453, MDA-MB-231 and MCF10A cells in a time dose dependent inhibition. (A) Schematic of the non-thermal atmospheric plasma set-up utilised in this study, and a photograph of the air plasma. All cells were exposed to NTP for 0, 60, 90 or 120 s. Forty-eight hours later, cell images were captured with 100 × magnification (B) and cell numbers of each dish were counted by an automatic analyzer CountStar (C). All experiments were replicated a minimum of three times. Data are presented as means ± S.D. and statistical analysis was carried out using one-way ANOVA with Tukey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001 versus control).

Figure 2

Effects of NTP treatment with or without ROS inhibitor NAC on MDA-MB-453, MDA-MB-231 and MCF10A cells proliferation. Cells were treated with (PRE NAC and POST NAC) or without NAC (NTP) exposed to NTP for 0, 60, 90 or 120 s. Forty-eight hours later, cell numbers of each dish were counted by an automatic analyzer CountStar. ROS fluorescence was analyzed by FACSVerse flow cytometer, 1 h after 120 s NTP treatment. Cells were treated with: Negative Control (NC), 120 s NTP treatment (NTP), pretreat with 5 mM NAC before 120 s NTP treatment (NTP + PRE NAC), post-treat with 5 mM NAC immediately after 120 s NTP treatment (NTP + POST NAC). For improved illustration, a guidance ratio (compared with NC group) has been indicated through histograms. All experiments were replicated a minimum of three times.

Figure 3

Effects of NTP on the expression of pSTAT3 and tSTAT3 in MDA-MB-231 and MCF10A cells. All cells were exposed to NTP for 0, 60, 90 or 120 s. After forty-eight hours post-treated with 5 mM N-acetyl cysteine (NAC+) or without (NAC−), proteins from total cell lysates were harvested. The expressions of pSTAT3 and STAT3 in MDA-MB-231 (A) and MCF10A (B) cells were detected by Western blot analysis. GAPDH was taken as a loading control throughout. The gels/blots were processed under the same experimental condition. Cropped gels/blots are displayed and full-length blots/gels are presented in Supplementary Figure 1.

Figure 4

Effects of 120 s NTP treatments with extracellular and intracellular ROS inhibitor on cells’ proliferation. MDA-MB-453 (A), MDA-MB-231 (B) and MCF10A (C) cells were treated: Negative Control (NC), 120 s NTP treatment (NTP) without or with 500 U/ml catalase (NTP + Catalase). MDA-MB-231 cells (D) were treated: exposed to NTP for 0 and 120 s, pretreat with 5, 6, 10 and 12 mM NAC before 120 s NTP treatment. After forty-eight hours, cell numbers of each dish were counted by an automatic analyzer CountStar. All experiments were replicated a minimum of three times. Data are presented as means ± S.D. and statistical analysis was carried out using one-way ANOVA with Tukey’s multiple comparison test (NS = No significance, *p < 0.05, **p < 0.01, ***p < 0.001).

Figure 5

Effects of H₂O₂ on the proliferation of MDA-MB-231 and MCF10A cells. MCF10A (A) and MDA-MB-231 (B) cells were incubated with multi-dose H₂O₂. Forty-eight hours later, cell images were captured with 100 × magnification and cell numbers of each dish were counted by an automatic analyzer CountStar. All experiments were replicated a minimum of three times. Data are presented as means ± S.D. and statistical analysis was carried out using one-way ANOVA with Tukey’s multiple comparison test. MDA-MB-231 cells were post-treated with 5 mM NAC and 0/50 μM H₂O₂ immediately after 120 s NTP treatment (C). Cell numbers of each dish were counted by an automatic analyzer CountStar and proteins from total cell lysates were harvested after forty-eight hours. Data are presented as means ± S.D. for three independent experiments and analyzed by Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001 versus control. The expressions of IL-6R, pSTAT3, tSTAT3, PTEN, pAkt and tAkt were detected by Western blot analysis. GAPDH was taken as a loading control throughout. The gels/blots were processed under the same experimental condition. Cropped gels/blots are displayed and full-length blots/gels are presented in Supplementary Figure 4.

Figure 6

Effects of NTP on the IL-6R/pSTAT3 and HER2/pAkt pathway in MDA-MB-231 and MCF10A cells. MDA-MB-231 (A) and MCF10A (B) cells were exposed to NTP for 0, 60, 90 or 120 s. After forty-eight hours post-treated with 5 mM N-acetyl cysteine (NAC+) or without (NAC−), proteins from total cell lysates were harvested. The expressions of HER2, L-6R, pSTAT3, tSTAT3, PTEN, pAkt and tAkt were detected by Western blot analysis. GAPDH was taken as a loading control throughout. The gels/blots were processed under the same experimental condition. Cropped gels/blots are displayed and full-length blots/gels are presented in Supplementary Figure 5.

Figure 7

A general survey of NTP treatment affection on triple-negative breast carcinoma and normal cells through different cellular pathways.(A) NTP treatment inhibits proliferation of MDA-MB-453 and MCF10A cells through ROS-dependent cellular pathway (not discussed in this article). (B) NTP treatment effects on reduction of proliferation through IL-6R/pSTAT3 pathway in MDA-MB-231 and MCF10A cells. (C) NTP treatment effects on recovering the proliferation especially in normal breast MCF10A cells with post-treated 5 mM NAC.