Abstract
BackgroundRecent studies claimed the important role of cold atmospheric plasma (CAP) with nanotechnology in cancer treatments. In this study, silymarin nanoemulsion (SN) was used along with air CAP as therapeutic agent to counter human melanoma.MethodsIn this study, we examined the combined treatment of CAP and SN on G-361 human melanoma cells by evaluating cellular toxicity levels, reactive oxygen and nitrogen species (RONS) levels, DNA damage, melanoma-specific markers, apoptosis, caspases and poly ADP-ribose polymerase-1 (PARP-1) levels using flow cytometer. Dual-treatment effects on the epithelial–mesenchymal transition (EMT), Hepatocyte growth factor (HGF/c-MET) pathway, sphere formation and the reversal of EMT were also assessed using western blotting and microscopy respectively. SN and plasma-activated medium (PAM) were applied on tumor growth and body weight and melanoma-specific markers and the mesenchymal markers in the tumor xenograft nude mice model were checked.ResultsCo-treatment of SN and air CAP increased the cellular toxicity in a time-dependent manner and shows maximum toxicity at 200 nM in 24 h. Intracellular RONS showed significant generation of ROS (< 3 times) and RNS (< 2.5 times) in dual-treated samples compared to control. DNA damage studies were assessed by estimating the level of γ-H2AX (1.8 times), PD-1 (> 2 times) and DNMT and showed damage in G-361 cells. Increase in Caspase 8,9,3/7 (> 1.5 times), PARP level (2.5 times) and apoptotic genes level were also observed in dual treated group and hence blocking HGF/c-MET pathway. Decrease in EMT markers (E-cadherin, YKL-40, N-cadherin, SNAI1) were seen with simultaneously decline in melanoma cells (BRAF, NAMPT) and stem cells (CD133, ABCB5) markers. In vivo results showed significant reduction in SN with PAM with reduction in tumor weight and size.ConclusionsThe use of air CAP using μ-DBD and the SN can minimize the malignancy effects of melanoma cells by describing HGF/c-MET molecular mechanism of acting on G-361 human melanoma cells and in mice xenografts, possibly leading to suitable targets for innovative anti-melanoma approaches in the future.
Highlights
Recent studies claimed the important role of cold atmospheric plasma (CAP) with nanotechnology in cancer treatments
Characterization of CAP and silymarin nanoemulsion (SN) The μ-dielectric barrier discharge (μ-Dielectric barrier discharge (DBD)) CAP used here with air gas at a flow rate of 1.5 lpm (Fig. 1a) with custom-built inverter operated in “dimming mode” which interacts with the liquid culture media to produce reactive species
Inhibition of the hepatocyte growth factor (HGF)/cell surface-mesenchymal-epithelial transition (c-MET) pathway signaling axis activates p53-mediated apoptosis in melanoma cells We proposed a combinational approach using CAP and SN to target the HGF/c-Met pathway in G-361 cells at effectively doses and checked the phosphorylation status of c-Met and its downstream axis (Fig. 5a)
Summary
Recent studies claimed the important role of cold atmospheric plasma (CAP) with nanotechnology in cancer treatments. Silymarin nanoemulsion (SN) was used along with air CAP as therapeutic agent to counter human melanoma. Previous studies have shown that CAP can kill cancer cells and significantly minimize solid tumor sizes with minimal damage to normal cells. In present melanoma reduction studies, a nanoemulsion was prepared from a well-known herb known as silymarin (SN) which use worldwide as a hepatoprotective agent and shows applications in cancer therapies. It has a natural hydrophobic structure with low water solubility and bioavailability. The combination of CAP and nanoparticles/nanoemulsion could contribute to improving the selective permeability of membranes by CAP-generated RONS species, leading to the intracellular diffusion of nanoparticles towards diseased sites within tissues [13]
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