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  3. Investigating the toxicity effects of single-walled carbon nanotubes on breast cancer cells in vitro

Investigating the toxicity effects of single-walled carbon nanotubes on breast cancer cells in vitro

Number of pages: 106 File Format: word File Code: 32021
Year: 2014 University Degree: Master's degree Category: Medical Sciences
Tags/Keywords: breast cancer - Breast cancer cells - Cancer cells - Carbon nanotube - Cytotoxicity - Dehydrogenase - Lactate release - MCF7 cell line
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  • Summary of Investigating the toxicity effects of single-walled carbon nanotubes on breast cancer cells in vitro

    ((M.Sc)) Thesis for Master's Degree

    Abstract

    This study was conducted to investigate the toxic effects of functionalized single-wall carbon nanotubes on three cell lines MCF7, HUVEC, PC12. The cells were treated with different concentrations of two single-walled carbon nanotubes with different functional groups (carboxyl and cyclic functional groups) and incubated for 24, 48 and 72 hours. Then MTT, lactate dehydrogenase and apoptosis assays were performed to evaluate the inhibitory effect of nanomaterials on cell growth. The results of the experiments on MCF7 cell line showed that the decrease in cell growth and the increase in the release of lactate dehydrogenase into the culture medium occurred simultaneously with the increase in the dose and incubation time, but there was an exception in the case of 72 hours because the inhibitory effect on cell growth was slightly reduced from the dose of 6 to 10 ?g/ml, and the release of lactate dehydrogenase was slightly reduced. Also, in two other cell lines, increasing the dose in 24 hours led to a decrease in cell growth and an increase in the release of lactate dehydrogenase, but in the case of 48 and 72 hours, the decrease in cell growth and the increase in the release of lactate dehydrogenase were inhibited compared to 24 hours, in other words, the resistance of the cells increases. At the end of the experiment, apoptosis was performed and observation with a fluorescence microscope indicated the induction of cell death through the apoptosis pathway. All the results of this study indicate the cytotoxic effect of functionalized single-walled carbon nanotubes on cell lines. But the toxicity of carboxylated carbon nanotubes was slightly higher than that of nanotubes with a cyclic functional group, and the toxicity effect was slightly higher for PC12 than for breast cancer cells.

    Key words: functionalized single-walled carbon nanotubes, breast cancer, MCF7 cell line, cytotoxicity, lactate dehydrogenase release

    Introduction

    For the first time, the Greek philosopher Hippocritus used the word carcinos or carcinoma to refer to tumors. Probably, the reason for that was the appearance of cancerous lumps like crabs. In normal tissues of the body, after a certain number of cell divisions, feedback control stops proliferation and starts differentiation. Cancer or neoplasm consists of tissues that have abnormal division. The origin of the disease is genetic and includes two simultaneous events: the constant proliferation of a cell due to genetic imbalance, as well as gene mutation and inactivation of natural cell death pathways. With the disruption of the normal pathways of cell proliferation and death, a group of cells divides uncontrollably and at the same time as they multiply excessively and form a mass of cells, they begin to release chemicals that stimulate the growth of abnormal capillaries within the mass of cells. This complex is known as a malignant mass that can severely damage the surrounding tissues by consuming too much food. Finally, in some cases, when the tumor is large enough, some cells enter the blood circulation and form masses in other parts of the body. This phenomenon is called metastasis or cancerous tumor invasion (36). In women, it is 45-55 years.

    This disease affects about one million people worldwide every year; Of these, 320,000 people live in Europe and 200,000 in America, which make up more than half of the total number of patients (10). This disease accounts for three to five percent of deaths in the western world and one to three percent of deaths in developing countries, but it rarely occurs in Japan (55). But recently, the annual death rate due to this cancer in America has been reported as 2.3%, which is attributed to the use of advanced screening methods and better and newer treatment options.

    One ??out of every nine women in British and American societies will get this disease during their lifetime; In general, the prevalence of this disease in Western countries is more than in Africa, South America and Asia, where several pathological factors are involved in the development of this disease (2).

    The factors affecting the occurrence of this disease are: age, genetics, family history, diet, alcohol consumption, obesity, lifestyles, lack of physical activity and endocrine factors (endogenous and exogenous). Other factors related to breast cancer include mammographic density and history of benign disease; However, it is still not clear which of the mentioned factors, compared to other factors, plays a more prominent role in causing breast cancer.

    1-2- Epidemiology

    Breast cancer is the most common cancer in adult women all over the world and after lung cancer, it is the second leading cause of death (10,4). The prevalence of this cancer is high in Western countries such that one out of every 12 women in Wales and England will suffer from it during their lifetime (55).

    Some researchers believe that this number is even higher and in American and English women's societies, it sometimes reaches one ninth (4). In total, this cancer includes a quarter of women's cancers in Western countries, which indicates a high prevalence in those countries. Worldwide, breast cancer accounts for 22% of all women's cancers (excluding non-melanoma skin cancers) so that in 2008 alone, this disease caused 458,503 deaths worldwide (13.7% of all women's deaths). 

    ABSTRACT

    This study was conducted to investigate the potential cytotoxicity of F-SWCNTs using three different cell lines including MCF7, HUVEC and PC12. All of these cell lines were treated and incubated with different concentrations of two kinds of F-SWCNTs for 24, 48, and 72 h. After that MTT assay, LDH assay and apoptosis assay were conducted. The result was shown that: about MCF7 cell line decrease in cell growth and increase in LDH release were occur parallel to increase of doses and times of treating except, in 72h that the LDH release and inhibited cell growth were decreased in doses of 6-10 µg/ml. Also in two other cell lines decrease in cell viability and increase in LDH release were observed in a dose dependent manner after 24h. While after 48 and 72h, decrease in cell viability and increase in LDH release were inhibited when compared to 24h. In conclusion, apoptosis assay was conducted and observation under the fluorescence microscopy indicating the occurrence of cell death via apoptosis pathway. Altogether these results emphasize on cytotoxicity of F-SWCNTs in above mentioned cell lines. And the toxicity effect of the COOH-SWNT was more than that which was seen about one other F-SWNT and was more than for PC12 when compared to MCF7.

  • Contents & References of Investigating the toxicity effects of single-walled carbon nanotubes on breast cancer cells in vitro

    List:

    Abstract. 1

    Introduction. 2

    Chapter One: Generalities

    1-1- Breast cancer. 4

    1-2- Epidemiology. 5

    1-3- Etiology factors. 5

    1-3-1- overweight. 6

    1-3-2- Lack of physical activity. 7

    1-3-3-alcohol. 7

    1-3-4- diet. 8

    1-3-5- Cigarettes 9

    1-3-6- steroid hormones and their receptors. 9

    1-3-6-1- steroid hormones. 9

    1-3-6-2- receptors of steroid hormones. 10

    1-3-6-3- History of pregnancy and breastfeeding. 11

    1-3-6-4- estrogen. 12

    1-3-6-5- hormone replacement therapy. 12

    1-3-7- Using oral contraceptives. 13

    1-3-8- Working at night and environmental factors. 14

    1-3-9- age. 14

    1-3-10- Gender. 15

    1-3-11- History of being sexually active. 15

    1-3-12- Family history. 16

    1-3-13- Personal history. 16

    1-3-14- breast cell changes, wound and breast injury. 17

    1-3-15- consumption of diethyl acetylbestrol. 17

    1-3-16- History of benign breast disease. 17

    1-3-17- High-risk genes 18

    1-3-18- High-risk syndromes 19

    1-3-19- The role of HER2/neu antigen. 20

    1-3-20- density of mammography. 20

    1-4- Hypotheses of the formation and origin of tumors 20

    1-5- Breast cancer heterogeneity. 21

    1-6- types of breast cancer. 22

    1-6-1-luminal breast cancer A and B. 22

    1-6-2- HER2+ breast cancer: 22

    1-6-3- pseudo-basal. 22

    1-7- Staging of cancer. 22

    1-7-1- zero stage 23

    1-7-2- first stage. 23

    1-7-3- The second stage. 23

    1-7-4- The third stage. 24

    1-7-5- the fourth step. 24

    1-8- Nano in cancer treatment and prevention. 24

    1-9- Carbon nanotubes. 26

    1-9-1- Properties of carbon nanotubes. 26

    1-9-2-Use as a drug carrier. 28

    1-9-3- dispersing the carbon nanotube in the solvent. 28

    1-9-4- carbon nanotube functionalization. 29

    1-9-4-1- Kovalan method. 29

    1-9-4-2- non-covalent method. 30

    1-9-4-3-Pegylation to improve properties in biological applications. 31

    Chapter Two: A Review of Past Texts

    2-1- Administration, absorption and transfer of CNT. 33

    2-2- intravenous administration. 34

    2-3- Application of single-wall nanocarbon tube and biological safety. 35

    2-3-1- Biosafety of single-walled nanotube as drug carrier. 36

    2-4- Application in targeted treatment. 37

    2-5- Application in gene delivery. 38

    2-6- Application in targeted drug delivery systems for cancer treatment. 38

    Chapter three: Materials and methods

    3-1- Materials and methods 42

    3-1-1- Materials 42

    3-1-2- Properties of carbon nanoparticles. 43

    3-2- Methods 45

    3-2-1- Cell culture and treatment. 45

    3-2-1-1- Cell lines used 45

    General characteristics of the cell. 46

    Name of the cell line. 46

    3-2-1-2- Defreeze frozen cells. 47

    3-2-1-3- Freezing cells 47

    3-2-1-4- Required solutions and buffers 48

    3-2-1-4-1- Salt phosphate buffer (PBS) 48

    3-2-1-4-2- Complete culture medium. 48

    3-2-1-5-Testing cells 49

    3-2-1-5-1- Counting cells 49

    3-2-1-5-3-Checking the viability of cells using the MTT method. 50

    3-2-1-5-4- Determination of IC-50. 51

    3-2-1-5-5- measuring the release of lactate dehydrogenase enzyme into the culture medium. 52

    3-2-1-5-6- Staining with 33258 Hoechst 52

    Chapter Four: Results

    4-1- Toxicity induced by single-walled nanotube with cyclic functional group on HUV-EC-C, MCF-7 and PC12 cells 54

    4-2- Investigating the effect of single-walled nanotube with functional group Cyclic on the fate of MCF-7 cells by MTT assay 55 4-3- Investigating the effect of single-walled nanotube with cyclic functional group on the fate of MCF-7 cells by LDH assay 59 4-4- Investigating the effect of single-walled nanotube with cyclic functional group on the fate of HUVEC cells by MTT assay 62 4-5- Investigating the effect of single-walled nanotube with functional group Cyclic on the fate of HUVEC cells by LDH assay 65 4-6- Investigating the effect of single-walled nanotubes with cyclic functional group on PC12 cells by MTT assay. 69

    4-6- Review. 69

    4-6- Investigating the effect of single-walled nanotubes with a cyclic functional group on PC12 cells by measuring LDH. 71

    4-7- Toxicity induced by carboxylated single-walled nanotubes on HUV-EC-C, MCF-7 and PC12 cells. 75

    4-8- Investigating the effect of carboxylated single-walled nanotubes on the fate of MCF-7 cells by MTT assay. 76

    4-9- Investigating the effect of carboxylated single-walled nanotubes on the fate of MCF-7 cells by measuring LDH. 77

    4-100- Investigating the effect of carboxylated single-walled nanotubes on the fate of HUVEC cells by MTT assay. 78

    4-11- Investigating the effect of carboxylated single-walled nanotubes on the fate of HUVEC cells by measuring LDH. 79

    4-12- Investigating the effect of carboxylated single-walled nanotubes on the fate of PC12 cells by MTT assay. 80

    4-13- Investigating the effect of carboxylated single-walled nanotubes on the fate of PC12 cells by LDH assay. 81

    4-14- induction of apoptotic death. 82

    Chapter Five: Discussion and Suggestions

    5-1-Discussion. 85

    5-2-Proposition 94

     

    Resources. 95                      

    English summary. 102

    Appendixes. 103

     

    Source:

     

    1. Abdellatif, KH., Abdelall, E., Abdelgawad, M., Ahmed, M., Bakr, R., 2014, Synthesis and anticancer activity of some new pyrazolo[3,4-d]pyrimidin-4-one derivatives, Molecules, Vol. 19, P.3297-3309.

     

    2. . Aguas, F., Martins, A., Gomes, T., De Sousa, M., Silva, D., 2006, Portuguese enopause Society and Portuguese Gynecology Society, 2005, Prophylaxis approach to a symptomatic post-menopausal women breast cancer, Maturitas, Vol. 52, p. S23-31.

     

    3. Aiello, EJ., Buist, DS., White, E., Porter, PL., 2005, Association between

    mammographic breast density and breast cancer tumor characteristics, Cancer

    Epidemiol Biomarkers Prev,Vol. 14, p. 662-668.

    4. Ali S, Coombes RC, 2002, Endocrine-responsive breast cancer and strategies

    for combating resistance, Nat Rev Cancer, Vol. 2, p.101-112.

    5. Antoniou, A., Pharoah, PD., Narod, S., 2003, Average risks of breast and ovarian cancer

    associated with BRCA1 or BRCA2 mutations detected in case series unselected for family

    history: a combined analysis of 22 studies, Am J Hum Genet, Vol.72, P.1117-30.

    6.Borresen-Dale, AL., 2003, TP53 and breast cancer, Hum Mutat, Vol.21, P.292-300

    7. CHEK2*1100delC and susceptibility to breast cancer: a collaborative analysis involving 10,860 breast cancer cases and 9,065 controls from 10 studies, 2004, Am J Hum Genet, Vol.74, P.1175-82.

    8. Cheng, J., [etal]., 2008, Reversible accumulation of PEGylated single-walled carbon nanotubes in the mammalian nucleus, ACS Nano, Vol. 2, P.2085-2094.

    9. Cole, MP., Jones, CT., Todd, ID., 1971, A new anti-oestrogenic agent in late breast cancer. An early clinical appraisal of ICI46474, Br J Cancer, Vol. 25, p. 270-275.

    10. Dumitrescu RG., Cotarla I., 2005, Understanding breast cancer risk where do

    we stand in 2005? , J Cell Mol Med, Vol 9, P. 208-221.

    11. Easton, DF.,[ et al]., 2007, Genome-wide association study identifies novel breast cancer susceptibility loci, Nature, Vol.447, P.1087-93.

    12. Foldvari, M., Bagonluri, M., 2008, Carbon nanotubes as functional excipients for nanomedicines: I. Pharmaceutical properties, Nanomedicine, Vol.3, P.173-182. 13. Foldvari, M., Bagonluri, M., 2008, Carbon nanotubes as functional excipients for

    nanomedicines: II. Drug delivery and biocompatibility issues, Nanomedicine, Vol. 3, P.183-200.

    14. Georgakilas, V., Tzitzios, V., Gournis, D., Petridis, D., 2005, Attachment of magnetic nanoparticles on carbon nanotubes and their soluble derivatives, Chem Mater, Vol. 17, P.1613-1617.

    15. Ghorab, M., Ragab, F., Alqasoumi, S., Alafeefy, A., Aboulmagd, S.

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