Title Page 1

Abstract 8

Contents 12

List of Abbreviations 31

CHAPTER 1. General introduction 33

1.1. Cancer and its microenvironment 34

1.2. Cancer cell metabolism and apoptosis 39

1.3. What is plasma? 41

1.3.1. Thermal and nonthermal plasmas 41

1.3.2. Reactive species generation in nonthermal plasma discharge 42

1.3.3. NTAPP for cancer therapy 44

1.4. An overview of pulsed high-power microwaves 46

1.4.1. Biological interactions with electromagnetic radiation 47

1.4.2. Key parameters and the unexplored effects of pulsed HPM 49

1.5. Objective of Studies 50

1.5.1. Objective of Study 1 50

1.5.2. Objective of Study 2 50

1.5.3. Objective of Study 3 51

1.5.4. Objective of Study 4 51

CHAPTER 2. Unveiling the therapeutic potential of soft plasma jet and nitric-oxide enriched plasma activated water (NO-PAW) on oral cancer YD-10B cells: A comprehensive investigation of direct and indirect treatments 52

2.1. Introduction 53

2.2. Materials and methods 55

2.2.1. NTAPP devices and treatment method 55

2.2.2. Cell culture method 55

2.2.3. Metabolic viability 56

2.2.4. ATP measurement 56

2.2.5. Flow cytometric analysis for cell death 56

2.2.6. Intracellular ROS/RNS detection 57

2.2.7. DNA damage confirmation using γ-H2AX antibody 57

2.2.8. Quantitative real-time polymerase chain reaction (qRT-PCR) 57

2.2.9. Western blotting analysis 58

2.2.10. Statistical analysis 58

2.3. Results 58

2.3.1. Characteristic of SPJ and cDBD 58

2.3.2. Effect of NTAPP on the cell viability 61

2.3.3. Effect of NTAPP on ATP levels using direct and indirect treatments 61

2.3.4. NTAPP induced cell death in oral cancer (YD-10B) cells 64

2.3.5. NTAPP treatment induce a higher level of intracellular ROS/RNS 66

2.3.6. Induction of apoptosis in YD-10B oral cancer cells 66

2.3.7. The induction of apoptosis in oral cancer YD-10B cells via direct and indirect NTAPP treatments 68

2.3.8. Role of reactive species in inducing apoptosis in oral cancer 69

2.4. Discussions 71

2.5. Conclusions 74

CHAPTER 3. ROS production in response to high-power microwave pulses induces P53 activation and DNA damage in brain cells: Radiosensitivity and biological dosimetry evaluation 76

3.1. Introduction 77

3.2. Materials and methods 79

3.2.1. HPM generator: experimental setup and methodology 79

3.2.2. Reactive species in cell medium after HPM exposure 80

3.2.3. Cell culture 81

3.2.4. Cell cytotoxicity analysis 81

3.2.5. Intracellular and extracellular ATP measurement 81

3.2.6. Flow cytometric analysis for cell death 81

3.2.7. Intracellular ROS detection 82

3.2.8. Molecular genes analysis 82

3.2.9. Protein analysis 82

3.3. Results 83

3.3.1. Properties of 3.5 GHz pulsed HPM 83

3.3.2. Impact of high-power microwave on human brain cells 85

3.3.3. Effect of 3.5 GHz-HPM on the mitochondrial ATP levels 87

3.3.4. Extracellular ATP levels 88

3.3.5. Cell death analysis after HPM exposure 89

3.3.6. Intracellular ROS levels 90

3.3.7. HPM-induced ROS production upregulates apoptotic markers 91

3.3.8. Pulsed 3.5 GHz HPM induced oxidative DNA damage 93

3.4. Discussion 95

3.5. Conclusions 98

CHAPTER 4. Formation of reactive species via high power microwave induced DNA damage and promoted intrinsic pathway-mediated apoptosis in lung cancer cells: an in vitro investigation 100

4.1. Introduction 101

4.2. Materials and methods 103

4.2.1. Cell culture 103

4.2.2. Metabolic viability/cell cytotoxicity assay 103

4.2.3. Intracellular and extracellular ATP measurement 103

4.2.4. Flow cytometric analysis for cell death 104

4.2.5. Intracellular ROS/RNS detection 104

4.2.6. Apoptosis detection by DAPI and PI staining 104

4.2.7. Migration assay 104

4.2.8. Quantitative real-time polymerase chain reaction (qRT-PCR) 105

4.2.9. Western blotting analysis 105

4.2.10. High-power microwave system "Chundoong" and exposure method 105

4.3. Results 106

4.3.1. HPM characteristics and their influence on inducing physiological changes in RPMI after irradiation 106

4.3.2. Effects of HPM on the cytotoxicity of lung cancer and normal cells 108

4.3.3. Pulsed HPM irradiation induces cell death via elevated ROS/RNS 109

4.3.4. Cell membrane and DNA damage following HPM exposure 112

4.3.5. HPM exposure inhibits cell migration in lung cancer 113

4.3.6. Pulsed HPM irradiation elicits upregulation of apoptotic markers 114

4.3.7. Protein analysis validates DNA damage and apoptosis 116

4.3.8. ROS/RNS production primarily responsible for cellular effects 118

4.4. Discussions 123

4.5. Conclusion 126

CHAPTER 5. Harnessing the synergy of nanosecond high-power microwave pulses and cisplatin to increase the induction of apoptosis in cancer cells through the activation of ATR/ATM and intrinsic pathways 127

5.1. Introduction 128

5.2. Material and methods 130

5.2.1. Cell culture 130

5.2.2. Cell viability assessment 130

5.2.3. Assessment of intracellular adenosine triphosphate (ATP) levels 130

5.2.4. Cell death analysis 130

5.2.5. Detection of intracellular reactive oxygen species (ROS) 131

5.2.6. Cell membrane integrity assessment via PI staining 131

5.2.7. Molecular analysis 131

5.2.8. Western blotting for protein analysis 131

5.2.9. DNA damage confirmation using γ-H2AX antibody 132

5.3. Results 132

5.3.1. Properties of nanosecond pulsed HPM 132

5.3.2. Cell viability and intracellular ATP levels: synergistic effects of HPM60 and cisplatin 135

5.3.3. Nanosecond pulsed HPM60 and cisplatin combination treatment increases cell death via membrane damage and elevated intracellular ROS levels 137

5.3.4. The combined treatment of HPM60 and cisplatin enhances DNA damage and promotes the upregulation of apoptotic markers in cancer cells 139

5.3.5. The confirmation of DNA damage and apoptosis via protein analysis 141

5.4. Discussions 143

5.5. Conclusions 146

CHAPTER 6. Summary and future scope 147

6.1. Summary and findings 148

6.1.1. NTAPP for Oral Cancer Treatment 148

6.1.2. Exposure of HPM on brain cells and dosimetry evaluation 150

6.1.3. Exposure of HPM on non-small cell lung cancers (NSCLC) 150

6.1.4. Harnessing the synergy of nanosecond high-power microwave pulses and cisplatin 152

6.2. Future scope 153

6.2.1. Optimization of NTAPP parameters and delivery systems 153

6.2.2. Expanding the range of cancer types treated with NTAPP 153

6.2.3. Long-term effects and safety of NTAPP 154

6.2.4. Mechanistic Insights and dosimetry of HPM treatments 154

6.2.5. Combination therapies with HPM 154

6.2.6. Development of HPM delivery Systems 155

6.2.7. Clinical translation and trials 155

6.2.8. Exploring NTAPP and HPM for adjunctive and multimodal therapies 155

6.2.9. Personalized medicine approaches 156

6.2.10. Enhancing understanding of tumor microenvironment interactions 156

Reference 159

Appendix 180

Table 1. List of primers used in these studies 157

 Nonthermal atmospheric pressure plasma (NTAPP) and pulsed high-power microwaves (HPM) have garnered interest for their potential therapeutic efficacy against cancer. NTAPP, a partially ionized gas, selectively induces apoptosis in cancer cells due to differential electrical properties and membrane structures compared to normal cells. HPM delivers targeted electromagnetic radiation energy, causing thermal ablation or disruption of essential cellular functions in tumor cells. Both modalities influence the tumor microenvironment by modulating reactive species levels, creating an unfavorable milieu for cancer cell survival. Furthermore, they stimulate immune responses by enhancing cytokine release and immune cell activation against cancer cells. Despite promising preliminary results, challenges persist. Long-term safety profiles, potential risks of repeated exposures, and interactions with other treatments require further investigation. Optimization of delivery methods for precise tumor targeting and the identification of reliable biomarkers for treatment monitoring are essential for clinical translation. In summary, while NTAPP and HPM offer targeted and non-invasive cancer treatment potential, comprehensive research is needed to address safety, delivery, and integration challenges.

In the first study, Oral cancer ranks sixth among global cancer cases, comprising about 3% of all cancer diagnoses. Nonthermal atmospheric pressure plasma (NTAPP) has demonstrated efficacy against various cancers, yet its impact on YD-10B oral cancer cells remains unexplored. This study investigated NTAPP's potential using both direct and indirect approaches. Direct treatment employed a soft plasma jet (SPJ) on cells, while indirect treatment utilized a cylindrical dielectric barrier discharge (cDBD) to produce nitric oxide plasma-activated water (NO-PAW) applied subsequently. While oral normal cells (HGF) viability remained unchanged, a significant decrease was observed in YD-10B cancer cells following SPJ and NO-PAW treatments. SPJ exposure elevated intracellular reactive oxygen and nitrogen species (ROS/RNS) levels, whereas NO-PAW increased RNS levels exclusively. Markers indicative of DNA damage and apoptosis were upregulated in YD-10B cells with both treatments. Inhibitor studies using NAC and cPTIO confirmed that reactive species primarily induced DNA damage and apoptosis in YD-10B cells. In vivo analysis revealed that NO-PAW15 notably inhibited tumor growth and volume in a mouse model. This research underscores SPJ and NO-PAW as promising approaches for targeted oral cancer treatments, offering molecular insights to guide future investigations.

In the second study Pulsed high-power microwave (HPM) technology, with its expanding applications, necessitates thorough investigation into its biological effects and safety levels. This study aimed to elucidate the impact of 3.5 GHz pulsed HPM on brain astrocytes and U87 MG cancer cells, along with the underlying mechanisms. Utilizing an axial virtual cathode oscillator on the pulsed power generator "Chundoong," cells were exposed to varying HPM pulse doses. Results showed that HPM irradiation induced a strong electric field, leading to reactive oxygen species (ROS) production, impacting cell viability, mitochondrial activity, and cell death rates in both cell types. Notably, 25 pulses exhibited therapeutic effects on U87 cells through p53, Bax, and Caspase-3 activation, while 60 pulses were detrimental to normal brain cells. This study highlights the apoptosis-related mechanisms and establishes safety thresholds for HPM exposure, offering valuable insights for future HPM research and its potential therapeutic applications.

In third study Lung cancer remains a leading cause of cancer-related mortality globally, emphasizing the need for innovative treatment strategies, particularly for non-small cell lung cancer (NSCLC). This study explored, for the first time, the effects of pulsed high-power microwave (HPM) on NSCLC cell lines H460 and A549, as well as the underlying mechanisms. Cells were exposed to varying HPM pulse doses, and effects were assessed at different time points. HPM exposure generated a strong electric field, elevating intracellular reactive species levels, which significantly impacted NSCLC cell viability, mitochondrial activity, and survival rates. The study revealed DNA damage pathways involving upregulation of ATR/ATM, Chk1/Chk2, and P53, alongside increased apoptotic marker expression. Inhibition studies confirmed that reactive species predominantly mediated these cellular effects. With a calculated skin depth of 30 mm ensuring practical applicability, these findings elucidate the cellular mechanisms of HPM-induced NSCLC cell death, offering potential therapeutic advancements and suggesting HPM as a potential adjunct to non-surgical cancer treatments.

Further study including Cisplatin, an effective anticancer drug, is limited in therapeutic application due to its toxicity to normal cells, suggesting the potential for combination treatments to enhance its efficacy at lower doses. This study investigated the synergistic effects of nanosecond pulsed high-power microwave (HPM) combined with cisplatin on three cancer cell lines (SKOV3, H460, and MDA-MB231) and two normal cell lines (MRC5 and HGF). Combination treatments resulted in a significantly higher reduction in cancer cell viability after 24 hours compared to individual treatments or controls. Increased cell death, membrane permeability, and intracellular reactive oxygen species (ROS) levels were observed with combined HPM60 and cisplatin (0.5 μM) treatments, along withelevated γ-H2AX levels indicating DNA double-strand breaks. Upregulation of DNA damage and apoptosis markers, including ATR/ATM, Chk1/Chk2, P53, caspase 3/8, Bax, PARP, and Bcl2, confirmed the mechanisms leading to apoptosis. Remarkably, the combination treatment resulted in 16 times higher cell death in SKOV3 and H460 cancer cells compared to cisplatin alone, while normal cells showed minimal viability reduction. These findings highlight the potential of combining nanosecond pulsed HPM with cisplatin as a promising strategy for enhancing cancer therapy efficacy while minimizing toxicity to normal cells.