Primary cold atmospheric plasma combined with low dose cisplatin as a possible adjuvant combination therapy for HNSCC cells-an in-vitro study

Abstract

Background: The aim of the present study was to examine the cytostatic effects of cold atmospheric plasma (CAP) on different head and neck squamous carcinoma (HNSCC) cell lines either in isolation or in combination with low dose cisplatin. The effect of CAP treatment was investigated by using three different HNSCC cell lines (chemo-resistant Cal 27, chemo-sensitive FaDu and OSC 19).

Materials and method: Cell lines were exposed to CAP treatment for 30, 60, 90, 120 and 180 s (s). Cisplatin was added concurrently (cc) or 24 h after CAP application (cs). Cell viability, DNA damage and apoptosis was evaluated by dye exclusion, MTT, alkaline microgel electrophoresis assay and Annexin V-Fit-C/PI respectively.

Results: In all cell lines, 120 s of CAP exposure resulted in a significant reduction of cell viability. DNA damage significantly increased after 60 s. Combined treatment of cells with CAP and low dose cisplatin showed additive effects. A possible sensitivity to cisplatin could be restored in Cal 27 cells by CAP application.

Conclusion: CAP shows strong cytostatic effects in HNSCC cell lines that can be increased by concurrent cisplatin treatment, suggesting that CAP may enhance the therapeutic efficacy of low dose cisplatin.

Keywords: Adjuvant combination therapy; Cold atmospheric plasma; Head and neck cancer.

Fig. 1

Overview of the characteristics of MiniFlatPlaSter® [17]

Fig. 2

Photograph of the SMD device (MiniFlatPlaSter®) used in this study [17]

Fig. 3

Schematic illustration of the plasma treatment on the different cell types

Fig. 4

Cisplatin dose–response curve for Cal 27, FaDu and OSC 19 cells, IC₅₀ and used concentration of 2.5 µM are displayed

Fig. 5

An example of fluorescence images demonstrating the development of tail DNA after CAP treatment (a) intact DNA and (b) degraded DNA, (horizontal scale bar 100 μm)

Fig. 6

(a-b) An exemplary fluorescence microscopy image of Annexin V/PI stained cells (a) green fluorescence showing early apoptotic cells, (b) red fluorescence showing late apoptotic/necrotic cells) (horizontal scale bar 50 µm)

Fig. 7

Detection of cell viability (%) with an MTT assay for all three cell types at different CAP treatment times. Line graph is used to present the results (mean and error bars 95% CI is shown for all three cell lines, (for each treatment time per cell type n = 15)

Fig. 8

Detection of DNA Damage (comet assay) for all three cell types. Line graph is used to present the results (mean and error bars 95% CI is shown for all three cell lines, (for each treatment time per cell type n = 15)

Fig. 9

Detection of apoptotic cells for each cell line (Fig. 9a: Cal 27, Fig. 9b: FaDu, Fig. 9c: OSC 19) after the different CAP exposure times (for each treatment time per cell type n = 15)

Fig. 10

Detection of cell viability (%) with an MTT assay for all three cell types after cisplatin treatment. Mean and error bars 95% CI is shown for all three cell lines, (for each treatment time per cell type n = 15)

Fig. 11

Detection of cell viability (%) with an MTT assay for the different treatment types and times for each of the individual cell types and the cisplatin reference value, (Fig. 11a: Cal 27, Fig. 11b: FaDu, Fig. 11c: OSC 19). Line graph is used to present the results (mean and error bars 95% CI is shown for all three cell lines, (for each treatment time per cell type n = 15)