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Osf Preprints OSF PREPRINTS Review Paper Title: -To study the selective potentiality and anti-tumor activity of CAP in oral cancer treatment. Swapnil Dey(1) , Rachana Phadke (2) , Dattatreya Mukherjee*(3) , Debraj Mukhopadhyay (4) 1. Dept of Zoology, Cotton University, Guwahati, India 2. Indira Gandhi Government Medical College, Nagpur, India 3. Jinan University, Guangzhou, P.R China 4. Department of Public Health, School of Allied Health Sciences, Delhi Pharmaceutical Sciences and Research University (DPSRU), Govt. of N.C.T Delhi, New Delhi, India Corresponding Author: Dattatreya Mukherjee, [email protected] Non funded project and Authors have no Conflict of Interest Abstract: - Oral Cancer is one of the leading causes of death in India, specifically in its Northeastern region. India holds about one third cases of the total oral cancer in the world. According to a report (2018), India holds 1,19,992 new cases of oral cancer and 72,616 deaths. Current approaches in cancer treatment mainly include surgery, chemotherapy and radiation therapy. These treatments often give a morbid outcome and reduce survival rate. This is mainly due to their non-selective nature to kill the tumor cells along with the normal cells. Recently the potential of Cold Atmospheric Plasma (CAP) in cancer treatment is gaining great interest among scientists and researchers. This is mainly due to the enhanced selective nature of the CAP in targeting and killing the tumor cells without affecting the surrounding tissue and causing minimal side effects. Reactive Oxygen and Nitrogen species (RONS) are the main contributors of the efficacy of CAP in killing cancer cells. Although many attempts have been made to understand the underlying mechanism of CAP in cancer treatment, there is a lack of detailed studies with respect to different treatment conditions. The use of CAP can be a potential innovative therapy in the upcoming days and more clinical trials should be encouraged to prove its efficacy and relevance in clinical purpose. Introduction and Background Cancer is one of the biggest health care issues faced by mankind of this century. According to National Institute of Cancer Prevention and Research (NICPR), estimated number of people living with disease in India is around 2.25 million[1].Oral cancer is one of the leading causes of death in cancer patients. Although distributed globally, this cancer type is more prevalent in South Central Asia. India contributes to about one-third of the total global cases. The death-toll of the disease in 2018 was 72,616. Statistically, this is a very devastating number.[2] 1 | P a g e OSF PREPRINTS Oral cancers are a group of neoplasms mainly affecting the oral cavity, salivary glands and some parts of pharyngeal region. The term “oral cancer” often tends to get replaced with oral squamous cell carcinoma (OSCC) which is most frequent among all other oral neoplasms. It is reported that around >90% of all oral neoplasms are OSCC [3]. The most common and recent types of cancer treatments include surgery, chemotherapy and radiation therapy. Surgery is the most common approach in oral cancer followed by chemotherapy and rarely radiotherapy. Despite the many advances in therapeutic approaches and the availability of several interventions, the morbidity and mortality rates seem to be unchanged. This is mainly due to the non-selective nature of these therapeutic approaches, killing the tumor cells as well as healthy cells, reducing the patient survival rate to a great extent [4]. Thus, the current situation demands a proactive strategy to develop an effective cure with minimal side effects. Recently, the fourth state of matter i.e. plasma has been used in a wide range of applications in biomedical field. More precisely, non-thermal plasma (NTP) has gained much popularity in recent times in the domain of cancer therapy by using cold plasma due to its selective nature to kill tumor cells [4]. It has become a novel treatment method for various cancers including oral cancer, lung cancer, melanoma and colorectal cancer. NTPs are actually a cocktail of ionized gas with electrons, ions, neutral atoms, radicals, and UV photons. NTPs mainly work by inducing apoptosis via the signaling pathway, inhibiting cell invasion and migration, or by arresting the cell cycle. Devices such as plasma jets are used to treat the tumor cells effectively. [5-6] However, there still is not much promising clinical evidences about cold plasma therapy in oral cancer but still this unique synergistic approach of physical science with that of biological domain is believed to add a major contribution in the field of cancer treatments in near future with minimal side effects, low cost and less time consumed. Thus, it is necessary to get a comprehensive idea about the mechanism of action and effectiveness of NTPs in oral cancer tissues. Therefore, in this review we discuss the mechanism of action and potential efficacy of NTPs in treating oral cancer tissues specifically along with its side effects and clinical significance. 4.Objectives and Review of Literature-This review project aims to highlight and discuss the mechanism of CAP treatment in oral cancer cells and to get an insight of the various pathways triggered during the CAPs exposure to target cell lines. The literature of this review can be discussed under the following heads: A) ATMOSPHERIC PRESSURE PLASMA JET DEVICE-Most of the plasma jets are released into the surrounding environment by devices that internally generate non-thermal plasmas (NTP). The plumes are blown out of the device by gases that flow typically at several liters per minute. NTPs are generated with various combinations of electrode geometries and along with a variety of frequencies ranging from DC to Microwave.[6] 2 | P a g e OSF PREPRINTS Typically, a plasma jet consists of a wire electrode (mostly copper), a glass confinement tube with 8mm inner diameter and 10mm outer dimeter(approx.), a Teflon fitting and a pencil-shaped nozzle having 2mm inner diameter at the exit. A sharp tipped tungsten pin wire(0.3 mm diameter) is placed on the tube axis and the nozzle is attached to the glass tube end.[7] Most often, in an attempt to increase the concentration of reactive species for cell treatment, another kind of plasma jet with improved jet source is designed. This plasma jet has a copper wire electrodes(2mm diameter), polyetheretherketone (PEEK) plastic housing and a pencil- shaped grounded ring-electrode(copper, 6-mm inner diameter and 16-mm outer diameter at the exit) covered with dielectric PEEK. The wire is normally inserted coaxially in the housing and covered with a Teflon tube, with 6 mm of the wire exposed to gas and being surrounded by the ground electrode. This small distance between the powered and grounded electrodes aids in the local enhancement of the electric field and reduces breakdown voltage requirement, compared to the plasma jets utilizing annular ring electrode or dielectric barrier discharge jets.[8,9] Usually, the copper tube is connected to the high voltage power supply(preferably,20-25 kHz in DC or AC power supply with the voltage of 5 kV) and the copper ring being connected to the ground .Helium or Argon gas is generally the preferred carrier gas chosen with an approximate flow rate of 3- 4slm.Sometimes a flowmeter is preferred so as to ensure uniform and vigorous mixing of the medium during the plasma treatment. The range of plasma plume length is maintained at (22 ± 1) mm and the distance between the nozzle and the cell surface is around 10-15 mm depending on other physical conditions. 3 | P a g e OSF PREPRINTS FIG1: A)Schematic diagram of a plasma jet. B)Actual picture of a plasma jet.[10] B). SELECTIVE ACTIVITY OF CAP ON TUMOR CELLS Several studies showed the selective nature of NTP treatment between the malignant and the non-malignant cells. Yan et al. in his studies found that 26 of 33 assessed cell lines showed a strong selectivity, 5 of 33 a weak selectivity while only 2 of 33 showed a negative selectivity [11]. However, the cell lines compared in his studies didn't necessarily belong to the same cell type or origin. A recent study by Bishop et al. (2019) showed that the cell type, cancer type and culture conditions significantly influence CAP treatment and hence need to be considered when selective activity of CAP is studied [12]. Furthermore, all these factors are preferably kept identical for a convenient result. The difference between a malignant and a non-malignant cell may explain the CAPs selective activity on cancerous cells.[13]. Normally, the cancer cells are likely to be more sensitive, compared to healthy cells, in terms of oxidative stress. This can be explained taking into account 4 | P a g e OSF PREPRINTS the concept of aquaporins present on the cell membrane. Aquaporins are generally found more in cancer cells [14]. These are considered as water channels that often facilitate the transport of free oxygen and nitrogen species, such as hydrogen peroxide as well as other small molecules including CO2, NO etc. [15,16,17]. The pore size of these channels determines the passage of the reactive species and other molecules. Currently, Aquaporin 1,3 and 8 are known to play role in transport of H2O2 in mammalian cells [19]. Studies show that over-expression of aquaporins increases oxidative stress due to rising intracellular ROS concentration. [18,20] Furthermore, monitoring the intracellular hydrogen peroxide content shows that the tumor cells accumulate H2O2 faster than the non-malignant ones. Besides this, cholesterol, which is the most abundant lipid in plasma membrane of animal cells also plays role in creating oxidative stress. The diffusion of reactive species depends on the amount of the cholesterol in the cell membrane.
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