Anticancer Effect of Cold Atmospheric Plasma in Syngeneic Mouse Models of Melanoma and Colon Cancer

Abstract

Cold atmospheric plasma (CAP) may have applications in treating various types of malignant tumors. This study assessed the anticancer effects of CAP using melanoma and colon cancer cell lines. CAP treatment significantly reduced the in vitro viability of melanoma and colon cancer cell lines and had a negligible effect on the viability of normal human melanocytes. Additionally, CAP and epidermal growth factor receptor (EGFR) inhibitor had an additive anticancer effect in a CAP-resistant melanoma cell line. Reactive oxygen and nitrogen species known to be generated by CAP enhanced the anticancer effects of CAP and EGFR inhibitors. The in vivo anticancer activities of CAP were evaluated by testing its effects against syngeneic tumors induced in mice by melanoma and colon cancer cells. CAP treatment reduced tumor volume and weight in both cancer models, with the extent of tumor reduction dependent on the duration and number of CAP treatments. Histologic examination also revealed the tumoricidal effects of CAP in both tumor models. In conclusion, CAP inhibits the growth of mouse melanoma and colon cancer cell lines in vitro and shows tumoricidal effects against mouse models of melanoma and colon cancer in vivo.

Keywords: cancer treatment; cold atmospheric plasma; colon cancer; melanoma; syngeneic mouse model.

Figure 1

(a) Effects of cold atmospheric plasma (CAP) on the viability of B16F10 murine melanoma cells, A375 human melanoma cells, A2058 human melanoma cells, MC38 murine colon cancer cells, and normal human melanocytes. Cell viability was measured 24 h after treatment with CAP for the indicated times (30, 60, 90, 120, or 150 s). Exposure to CAP effectively reduced cancer cell viability in a treatment-duration-dependent manner. Data represent the mean ± standard deviation of three separate experiments. * p < 0.05, ** p < 0.01 compared with untreated control cells. (b) Western blot analysis of the expression levels of Bcl-2, cleaved caspase 3, heme oxygenase-1 (HO-1), Nrf2, and β-actin 24 h after treatment of B16F10 and MC38 cells with cold atmospheric plasma (CAP) for the indicated times. Levels of protein expression were quantified by densitometry after normalization to the optical density of β-actin. Treatment with CAP upregulated the expression level of HO-1 in B16F10 and MC38 cancer cells, while the expression level of cleaved caspase-3 was increased by CAP in B16F10 cancer cells.

Figure 2

Additive antimelanoma effects of AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR), and cold atmospheric plasma (CAP). (a) Relative expression levels of EGFR mRNA in A375, B16F10, A2058, and MC38 cells. (b) Viability of A375 cells 24 h after treatment with AG1478 at indicated concentrations. (c) A375 cells were pretreated with AG1478 at indicated concentrations 1 h before CAP treatment for 120 sec, and cell viability was assessed 24 h after the CAP treatment. (d) Fluorescence intensity for reactive oxygen species was detected using a microplate reader. The intensity increased as the treatment duration increased to 3 min. (e,g) Viability of A375 cells 24h after treatment with H₂O₂ or SNP at indicated concentrations. (f,h) A375 cells were pretreated with AG1478 at indicated concentrations (5 μM or 10 μM) 1 h before treatment with H₂O₂ (200 μM) or SNP (400 μM), with cell viability assessed 24 h after treatment. Data represent the mean ± standard deviation of three separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Additive antimelanoma effects of AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR), and cold atmospheric plasma (CAP). (a) Relative expression levels of EGFR mRNA in A375, B16F10, A2058, and MC38 cells. (b) Viability of A375 cells 24 h after treatment with AG1478 at indicated concentrations. (c) A375 cells were pretreated with AG1478 at indicated concentrations 1 h before CAP treatment for 120 sec, and cell viability was assessed 24 h after the CAP treatment. (d) Fluorescence intensity for reactive oxygen species was detected using a microplate reader. The intensity increased as the treatment duration increased to 3 min. (e,g) Viability of A375 cells 24h after treatment with H₂O₂ or SNP at indicated concentrations. (f,h) A375 cells were pretreated with AG1478 at indicated concentrations (5 μM or 10 μM) 1 h before treatment with H₂O₂ (200 μM) or SNP (400 μM), with cell viability assessed 24 h after treatment. Data represent the mean ± standard deviation of three separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

Additive antimelanoma effects of AG1478, a selective inhibitor of epidermal growth factor receptor (EGFR), and cold atmospheric plasma (CAP). (a) Relative expression levels of EGFR mRNA in A375, B16F10, A2058, and MC38 cells. (b) Viability of A375 cells 24 h after treatment with AG1478 at indicated concentrations. (c) A375 cells were pretreated with AG1478 at indicated concentrations 1 h before CAP treatment for 120 sec, and cell viability was assessed 24 h after the CAP treatment. (d) Fluorescence intensity for reactive oxygen species was detected using a microplate reader. The intensity increased as the treatment duration increased to 3 min. (e,g) Viability of A375 cells 24h after treatment with H₂O₂ or SNP at indicated concentrations. (f,h) A375 cells were pretreated with AG1478 at indicated concentrations (5 μM or 10 μM) 1 h before treatment with H₂O₂ (200 μM) or SNP (400 μM), with cell viability assessed 24 h after treatment. Data represent the mean ± standard deviation of three separate experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 3

Anticancer effect of cold atmospheric plasma (CAP) in a syngeneic mouse model with B16F10 melanoma cells. After the growth of tumors, five mice were each treated five times, once every other day (i.e., on days 0, 2, 4, 6, and 8), with CAP for 2 min, 5 min, or 15 min, whereas five untreated mice served as controls. (a) Tumor volumes on days 0, 3, 5, 7, and 10. Tumor sizes were measured using calipers, and tumor volumes were calculated as (length × width²)/2. (b) Tumor weights were measured on day 10. (c) Representative images of immunohistochemical labeling of Ki-67 in control and 15 min plasma treatment groups on day 10 (Ki-67, ×400). The Ki-67 indices of left and mid panel were assessed as 38.1% and 24.4%, respectively. The mean value of Ki-67 index ± standard deviation in CAP-treated mice relative to control is shown in the right panel. * p < 0.05. (d) Representative images of immunohistochemical labeling of caspase-3 in control and 15 min plasma treatment groups on day 10 (caspase-3, ×400). The mean expression level of caspase-3 ± standard deviation in CAP-treated mice relative to control is shown in the right panel. * p < 0.05, ** p < 0.01, *** p < 0.001. (e) Representative histological images of B16F10 melanomas on day 10 from a control mouse and from mice treated with CAP for 2 min, 5 min, and 15 min (hematoxylin and eosin, ×100). Arrows indicate preserved normal structures overlying the tumors.

Figure 4

Anticancer effects of cold atmospheric plasma (CAP) in a syngeneic mouse model with MC38 colon cancer cells. After the growth of tumors, six mice were each treated five times, once every other day (i.e., on days 0, 2, 4, 6, and 8), with CAP for 2 min or 5 min, whereas six untreated mice served as controls. (a) Tumors were measured on days 0, 3, 5, 7, and 10. Tumor sizes were measured using calipers, and tumor volumes were calculated as (length × width²)/2. (b) Tumor weights were measured on day 10. (c) Representative histological images of MC38 colon tumors on day 10 from a control mouse and from mice treated with CAP for 2 min and 5 min (hematoxylin and eosin, ×40). Arrows indicate preserved normal structures overlying the tumors.

Figure 4

Anticancer effects of cold atmospheric plasma (CAP) in a syngeneic mouse model with MC38 colon cancer cells. After the growth of tumors, six mice were each treated five times, once every other day (i.e., on days 0, 2, 4, 6, and 8), with CAP for 2 min or 5 min, whereas six untreated mice served as controls. (a) Tumors were measured on days 0, 3, 5, 7, and 10. Tumor sizes were measured using calipers, and tumor volumes were calculated as (length × width²)/2. (b) Tumor weights were measured on day 10. (c) Representative histological images of MC38 colon tumors on day 10 from a control mouse and from mice treated with CAP for 2 min and 5 min (hematoxylin and eosin, ×40). Arrows indicate preserved normal structures overlying the tumors.

Figure 5

Schematic diagram of the cold atmospheric plasma (CAP) device placed just over the culture medium.