论文总字数：53501字

2020 届毕业设计（论文）

题 目：Dual gas nanoparticles for tumor cells treatment in vitro

专 业：Pharmacy

班 级： 1601 Pharmacy

姓 名：Issa Assem

指导老师：任浩

起讫日期：2020.02-2020.05

年 月

Abstract

The initiation and progression of cancer is characterized by the activity of cancer cells per se and the growth of tumor tissues, both modulated by crosstalks between cancer cells and the microenvironment around them. Advances in cancer research have highlighted the significance of constant evolution of the tumor microenvironment, leading to tumor formation, metastasis and refractoriness to therapy.

Tissue hypoxia results from an inadequate supply of oxygen (O₂) that compromises biologic functions.There is growing evidence from laboratory and clinical research that hypoxia plays a fundamental role in solid tumors. Hypoxia in tumors is primarily a pathophysiologic consequence of structurally and functionally disturbed microcirculation and the deterioration of diffusion conditions.Tumor hypoxia tends to be closely associated with tumor growth, malignant development, and treatment resistance, and has thus become a central concern in tumor physiology and cancer therapy. Biochemists and clinicians (as well as physiologists) define hypoxia differently; biochemists define it as O₂-limited electron transport, and physiologists and clinicians define it as a state of reduced O₂ availability or decreased O₂ partial pressure that restricts or even abolishes functions of organs, tissues, or cells. Because malignant tumors no longer execute functions necessary for homeostasis (such as the production of adequate amounts of adenosine triphosphate), the physiology-based definitions of the term “hypoxia” are not necessarily valid for malignant tumors.

Instead, alternative definitions based on clinical, biologic, and molecular effects that are observed at O₂ partial pressures below a critical level have to be applied. Traditionally, tumor hypoxia has been considered a potential therapeutic problem because it renders solid tumors more resistant to sparsely ionizing radiation. More recent experimental and clinical studies reviewed in suggest that intratumoral oxygen levels may influence a series of biologic parameters that also affect the malignant potential of a neoplasm. Sustained hypoxia in a growing tumor may cause cellular changes that can result in a more clinically aggressive phenotype. During the process of hypoxia-driven malignant progression, tumors may develop an increased potential for local invasive growth, perifocal tumor cell spreading, and regional and distant tumor cell spreading. Likewise, intrinsic resistance to radiation and other cancer treatments may be enhanced.

Tumors are usually hypoxic, which limits the efficacy of current tumor therapies, especially photodynamic therapy (PDT) in which oxygen is essential to promote singlet oxygen-induced cell damage. So in this study, the perfluorocarbon was used as oxygen carrier to deliver oxygen to tumor. It can release oxygen to overcome hypoxia. Also due to the high concentration of H₂O₂ in tumor microenvironment, MnO2 could catalyze H2O2 to produce oxygen to reverse hypoxia. NO is similar as oxygen, which could inhibit tumor respiration to realize oxygen enhancement. With these two gas delivery or generation, the hypoxia will reverse and the efficacy of PDT will be enhanced.

Table of contents

1. Introduction 1

2. Current status of cancer 5

1. 3.Cancer treatments 6

3.1. Surgical treatments 6

3.2. Radiation therapy 8

3.3. Drug therapy 10

4. Tumor microenvironment 11

5. Oxygen and NO delivery for cancer therapy 16

References 21

Introduction

Recent advances in cancer therapeutics have yielded several new-generation therapeutic modalities, and cancer prognoses have improved significantly. However, further advances are required before major improvements are achievable, especially in patients with advanced and/or intractable cancers, which are refractory to conventional therapeutic options. Although cancer therapies ideally seek eradication of all cancer cells in tumor tissues, recent work has emphasized that targeting of cancer cells is not equivalent to targeting of tumor tissues. The research suggests that the initiation and progression of cancer is determined not only by the activity of cancer cells per se but also by tumor tissue growth, mediated in effect by crosstalks between cancer cells and the surrounding microenvironment. Recent cancer research has emphasized the significance of constant evolution of the tumor microenvironment, facilitating tumor formation, metastasis and development of refractoriness to cancer therapy.

Tumor microenvironments are highly heterogeneous, comprising various types of cells including fibroblasts, endothelial cells, pericytes, immune cells and stromal stem and progenitor cells originating from local and bone marrow, and an extracellular matrix ( ECM) surrounding them.These cells originate from normal cells but become altered during tumor development. For example, many types of solid tumor are accompanied by variable extents of stromal cell infiltration and ECM deposition, termed desmoplasia. Such cancer tissue remodeling allows tumor cells to grow and disseminate and contributes to an increase in interstitial fluid pressure, which can impede delivery of cancer drugs. Cancer-associated fibroblasts (CAFs) make versatile contribution to these responses.

For more than eight decades, it has been known that cancer cells exhibit altered metabolism when compared to their normal counterparts. These alterations include increased rates of glycolysis and pentose phosphate cycle activity along with slightly reduced mitochondrial respiration. Originally these metabolic abnormalities were thought to result from some impairment or damage to the cancer cell’s ability to undergo respiration but the underlying mechanisms responsible for these mebolic abnormalities are still not well understood.

In normal mammalian cells, mitochondria represent the major cellular organelle responsible for respiration. Electron transport chains (ETCs) in the inner mitochondrial membrane are believed to be responsible for the majority of cellular O₂ consumption as well as being hypothesized to be a source of reactive oxygen species (ROS) during metabolism. In normal cells, as much as 1 % of the electrons flowing through ETCs are thought to undergo one-electron reductions of O₂ to form superoxide (O₂^•−), which can then react to form other ROS such as hydrogen peroxide (H₂O₂) and organic hydroperoxides However, studies directly comparing steady-state levels of O₂^•− in cancer vs. normal epithelial cells are lacking.

Studies of cancer cell mitochondria have noted many structural abnormalities and epithelial cancers from colon and breast have also demonstrated higher rates of mutations in mitochondrial DNA . These data have lead to the speculation that cancer cells may have mitochondrial ETC defects that could contribute to increased steady-state levels of O₂^•− and H₂O₂ in human tumor cells when compared to normal cells but direct evidence supporting this speculation is lacking In addition, previous studies using glucose deprivation have suggested that increases in glucose metabolism in cancer cells, relative to normal cells, could be necessary to provide reducing equivalents in the form of NADPH and pyruvate for the detoxification of ROS. However, there is no direct evidence linking increased steady-state levels of O₂^•− and H₂O₂ to the differential susceptibility of epithelial cancer vs. normal cells to glucose deprivation-induced cytotoxicity and oxidative stress.

In the current study carcinoma cells from human colon and breast epithelial tissue are shown to have increased steady-state levels of endogenous O₂^•− and ROS compared to normal cells derived from the same tissues as well as consuming more glucose, having higher activities of Pentose Phosphate Pathway enzymes associated with NADPH regeneration, and being more sensitive to oxidative stress and cell killing induced by glucose deprivation or treatment with an inhibitor of glucose metabolism (2-DG). Furthermore, glucose deprivation or 2DG-induced cytotoxicity as well as parameters indicative of oxidative stress could be inhibited by co-over expression of mitochondrially targeted superoxide dismutase (MnSOD) and mitochondrially targeted catalase (mitoCAT), that scavenge O₂^•− and H₂O₂, respectively. The results demonstrate that metabolic oxidative stress mediated by O₂^•− and H₂O₂ significantly contributes to the differential susceptibility of cancer vs. normal epithelial cells to glucose deprivation or 2DG-induced cytotoxicity. These results support the hypothesis that cancer cells exhibit increased glucose metabolism to compensate for excess metabolic production of ROS as well as the hypothesis that inhibition of glucose and hydroperoxide metabolism may provide a biochemical target for selectively enhancing cytotoxicity and oxidative stress in human cancer cells.

Nitric Oxide (NO) is a reactive nitrogen species (RNS), which makes it highly reactive with metal ions and biomacromolecules. As such, it is often involved in the deactivation of proteins, lipids, and deoxyribonucleic acid (DNA) within cells. NO can also act as a signaling molecule to regulate the development of body systems such as the vascular and the nervous systems. As a free radical gas, NO is highly reactive with reactive oxygen species (ROS), particularly during periods of oxidative stress. Free NO mediates numerous biological processes, within the human body. In biological systems, nitric oxide is produced by enzymes known as nitric oxide synthases, which exist in three isoforms: inducible NOS (iNOS), neuronal NOS (nNOS), and endothelial NOS (eNOS) [3]. iNOS generates large amounts of NO in tumors, which suppresses the growth of tumors sensitive to NO but promotes the growth of tumors which are resistant to NO, often depending on the concentration of NO existing in the cellular environment. eNOS generates NO that stimulates angiogenesis, the formation of blood vessels. NO and NOSs are found in abundance in cancerous tumors, where depending on their concentration, can promote or suppress tumor progression. This quality of NO where it can both promote and repress the proliferation of cancer cells based primarily on its concentration is put under consideration in many studies. The exact concentrations of NO can vary based on other factors such as the donor or delivery method, but in general, the concentration of NO that is needed to influence cancer cell proliferation is very small. Lower concentrations tend to promote the proliferation of cancer cells, while higher concentrations tend to inhibit the proliferation of cancer cells. One study demonstrated that low concentrations of SNAP/Deta- NONOate, NO donor, around 20-2000 nM, promoted the growth of cancer, while high concentrations of greater than 20 μM of the NO donor suppressed the growth of the cancer. Studies have shown that NO promotes tumor growth in ovarian cancer cells by regulating the Warburg effect, which increases glycolysis and decreases mitochondrial activity, allowing the cancer cells to sustain their ATP production under hypoxic condition of the tumor cell. Additionally, NO degrades the effectiveness of leukocytes and promotes hypoxia inducible factors, which keep tumor cells from invading blood vessels and promote tumor growth respectively.

Nitric oxide mediates the oxidation of lipids and lipoproteins in many oxidative pathways. NO also induces cell death by activating the ASK1/JNK1 (apoptosis signal-regulating kinase 1/c-Jun N-terminal kinase 1) pathway, which mediates the degradation of the anti-apoptotic protein, MCL-1 (induced myeloid leukemia cell differentiation protein), and activates BAK and BAX, leading to intrinsic cell apoptosis. The high reactivity of NO with reactive oxygen species (ROS) often triggers cytotoxic pathways with higher concentration of ROS being more susceptible to NO mediated cell death. Nitric oxide reacts with ROS to produce peroxynitrite, which is highly toxic to cells. Whether a cancer cell undergoes NO-mediated cell death depends on whether the concentration of NO is high enough. However, it has also been shown that the cancer cell’s position in the cell cycle is important. A study showed that apoptotic colon cancer cells exposed to NO accumulated in the G2-M phase. Nitric oxide production has been shown to activate cytotoxic bone marrow-derived dendritic cells as well, which has cancer killing properties. Therefore, cytotoxicity of NO towards various cancerous cells makes NO the subject of much study in the medical treatment of cancer.

2. Current status of cancer

Cancer is the second leading cause of death globally, and is responsible for an estimated 9.6 million deaths in 2018. Globally, about 1 in 6 deaths is due to cancer.

In low- and middle-income countries, approximately 70 per cent of cancer deaths occur.

About one third of cancer deaths are attributable to the five major behavioral and dietary risks: high body mass index, low intake of fruit and vegetables, lack of physical activity, use of tobacco, and alcohol use.

Tobacco use is the most significant risk factor for cancer and accounts for around 22 percent of cancer deaths.

Cancer causing infections, such as hepatitis and human papilloma virus (HPV), are responsible for up to 25% of cancer cases in low- and middle-income countries.

Late-stage presentation and inaccessible diagnosis and treatment are common. In 2017, only 26% of low-income countries reported having pathology services generally available in the public sector. More than 90% of high-income countries reported treatment services are available compared to less than 30% of low-income countries.

The economic impact of cancer is significant and is increasing.The total annual cancer economic cost was estimated at about US$ 1.16 trillion in 2010.

Just 1 in five low- and middle-income countries has the data needed to guide cancer policy. Cancer is a generic term for a large group of diseases which can affect any body part. Other terms used include neoplasms and malignant tumors.One defining feature of cancer is the rapid development of abnormal cells that grow beyond their usual boundaries and can then invade adjacent parts of the body and spread to other organs, the latter being called metastasizing. Metastases represent a major cause of cancer death.

Cancer is a leading cause of death worldwide, with an estimated death toll of 9.6 million in 2018.

The most common cancers are:

Lung (2.09 million cases)

Breast (2.09 million cases)

Colorectal (1.80 million cases)

Prostate (1.28 million cases)

Skin cancer (non-melanoma) (1.04 million cases)

Stomach (1.03 million cases)

The most common causes of cancer death are cancers of:

Lung (1.76 million deaths)

Colorectal (862 000 deaths)

Stomach (783 000 deaths)

Liver (782 000 deaths)

Breast (627 000 deaths)

Cancer results from the transformation of normal cells into tumor cells in a multi-stage cycle that generally progresses from a pre-cancerous lesion to a malignant tumor. These changes are the result of the interaction between a person's genetic factors and 3 categories of external agents, including:

physical carcinogens, such as ultraviolet and ionizing radiation;

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