Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - Anticancer-PAM (Anticancer activity of plasma activated medium and its underlying mechanisms: Combined experimental and computational study)

Teaser

Cold atmospheric plasma (CAP) is attracting a lot of attention due to its vast biomedical potential. Specifically, the emerging field of the role of CAP in cancer treatment is of high importance. CAP generates a variety of molecular, ionic and radical reactive oxygen and...

Summary

Cold atmospheric plasma (CAP) is attracting a lot of attention due to its vast biomedical potential. Specifically, the emerging field of the role of CAP in cancer treatment is of high importance. CAP generates a variety of molecular, ionic and radical reactive oxygen and nitrogen species (RONS) upon interaction with ambient gas and aqueous environment of biological substrates. In-vitro studies have shown that CAP has a promising anticancer activity against more than 20 cancer types. However, CAP treatment has certain limitations in the treatment of internal organs of the body. For these circumstances, CAP-irradiated solutions or plasma treated media (PTM) might be a promising alternative. However, the anticancer potential of CAP or PTM (also referred as PAM in our project title) is only scarcely explored, mainly because the underlying mechanisms are still largely unknown.
By elucidating and explaining the effects of CAP on various proteins, we can open a long-term perspective for a new class of patient-tailored personalised cancer treatment, contributing to a healthier society.

Work performed

To validate the interaction of RONS produced in direct CAP or PTM with proteins, I have performed both wet laboratory experiments, as well as computational simulations. Firstly, I learned the basics of non-reactive molecular dynamics (MD) simulations, using the GROMACS package, implementing the CHARMM27 all-atom force field. Using the GROMACS package, I have developed the force fields for oxidized amino-acids based on literature. To understand the fluid dynamics of reactive species generated by plasma, I became familiar with 3D fluid dynamics modeling. After learning the MD simulations, I checked the effect of plasma on various proteins.
Firstly, we track the plasma jet delivery of RONS into a tissue model target. Our results imply that the flux of UV photons from plasma jets is important for delivering RONS through seemingly impenetrable barriers such as skin.
Secondly, we explored the importance of RNS in PTW for their bactericidal effect. Interestingly, we observed that with increasing RNS content in PTW, more deactivation of bacteria was observed. Further, we investigated the CAP effect on the deactivation of thermophilic bacteria to understand the CAP potential in the food industry. We treated thermophilic bacteria protein with dielectric barrier discharge (DBD) plasma operating in air. The structural changes of MTH were analysed upon CAP treatment. Additionally, we performed MD simulations to determine the stability of both the native and oxidised protein.
We also studied the effect of plasma treated media (PTM) and plasma treated water (PTW) on two different pancreatic ductal adenocarcinomas (MiaPaca-2, BxPc3) and pancreatic stellate cells (PSCs) (hPSC128-SV). Our study revealed that PTM and PTW have a similar efficacy to kill pancreatic cancer cells, while PTW is slightly more effective in killing PSCs as compared to PTM. In another study, we used PTW to treat non-small lung carcinoma (NSCLC) through oral administration. PTW shows favorable anticancer effect on chemoresistance xenograft mice.
We also investigated the effect of RONS on the NOX and Bacteriorhodopsin (BR)G protein-coupled receptor (GPCR)proteins.
Finally, we studied the role of catalase protein in CAP treatment.Our study concludes that the enzymatic activity of catalase decreases after plasma treatment, but this decrease is not strong enough that it can completely inhibit the catalase cellular function (to metabolize H2O2) in cancer cells.

Throughout the project, I used different dissemination channels for my research. The non-industrial nature of the project implied that the main dissemination method was published in scientific journals and participation in conferences. 4 paper has been published SCI indexed journals, while 3 manuscripts are currently under preparation. We succeeded in publishing both in specific journals targeting the plasma community (Journal of Physics D: Applied Physics), and in journals of wider scientific interest (Scientific Reports, and RSC Advances). The aim of this was to bring more attention to the state-of-the-art of plasma research from different fields of science, including wide chemistry, biology and engineering readership. My results were also presented to the scientific community in the form of 7 conference contributions at the major domestic and international conferences in the plasma domain as invited lectures, tutorial lectures, oral presentation and posters.

Final results

This work is the first attempt to obtain detailed insight in the anti-cancer and anti-bacterial activity of CAP and PTM, focusing on the structural analysis of catalase, lysozyme, MTH1880, NOX1 and Bacteriorhodopsin (BR) GPCR proteins upon CAP treatment. Catalase plays an important role in overcoming oxidative stress in cancer cells. Lysozyme protein shows a promising role in sterilisation and cancer treatments. MTH1880 is a thermophilic protein which protects thermophilic bacteria against thermal and chemical degradation. Finally, GPCRs can detect molecules outside the cell and activate internal signal transduction pathways. Furthermore, the combination of experiments and MD simulations provides full insight to better understand the anti-cancer activity of PTM or CAP.
We provided the first evidence of denaturation of the thermophilic protein by RONS generated from CAP. This study is very important as thermophilic bacteria are resistant to temperature and chemical denaturation. Therefore, an alternative method is required to kill the thermophilic bacteria. Additionally, CAP action on the Bacteriorhodopsin protein destroyed its structure in the presence and absence of co-solvents. This is the first study that revealed the CAP action on GPCRs.
The research on catalase opens new dimensions in the field of cancer research. We observed that inhibition of catalase increases the anti-cancer activity of PTM and CAP. In addition, CAP can alter the structure of catalase in cell-free condition and also decrease its activity, but inside the cell the decreases in activity of catalase is not significant. Hence, we need catalase inhibition to increase the PTM or CAP effect on the cancer cells.
Moreover, for the first time we observed that the NOX1 structure is affected by CAP treatment that resulted in a decrease of the cell proliferation pathways. This study opens new possibilities of the use of CAP in cancer treatment. Finally, my study on the changes in lysozyme structure is groundbreaking in the field of anti-cancer and anti-bacterial research.
Overall, our work within the anticancer PAM project presents a great interest for both the general public and the scientific community. It provides many important insights into the fundamental mechanism of cold plasma for cancer treatment and made it understandable for the lay audience. Cold plasma therapy is one of the promising novel techniques to fight cancer, which can improve the overall health status in Europe and the world.

Website & more info

More info: http://nano.uantwerpen.be/nanorefs/search.php.