Cyclophosphamide Therapy For Cancer Patient

Published Date: 02 Nov 2017

CHAPTER 1

INTRODUCTION

Cyclophosphamide therapy for cancer patient causes various negative side effects including gonadal toxicity which later on lead to infertility problem. It happens as Cyclophosphamide (CPA) is an alkylating agent that shows cytostatic effect by formation of DNA adduct. It causes single strand breaks in DNA. It acts upon rapidly dividing cells which not only to the cancerous cells but other cells that have the same character such as reproductive cells.

Therefore, the use of Nigella Sativa Extract (NSE) along with anticancer agent, Cyclophosphamide (CPA) is hoped to reduce the infertility problem in male cancer patients as the plant and its extract is used as fertility promoting agent . It has also shown beneficial therapeutic effects, including anti-oxidant, anti-inflammatory, anti-cancer, anti-microbial, and immunomodulatory effects .

8-Oxoguanine glycosylase gene (OGG1) is a gene that encodes DNA glycosylase enzyme. OGG1 is a DNA repair enzyme that incises DNA at 8-oxoG residues. It excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (FAPY) from damaged DNA. It also has a beta-lyase activity that nicks DNA 3' to the lesion.OGG1 is involved in Base Excision Repair (BER) .BER is a cellular mechanism that repairs damaged DNA throughout the cell cycle. This mechanism is very essential for removing damaged bases that could otherwise cause mutations by mispairing or lead to breaks in DNA during replication. Defect in BER genes can lead to the development of cancer.

Therefore, in this experiment, it is hoped that Cyclophosphamide (CPA) exposed mice treated with Nigella Sativa Extract (NSE) shows a significant result by decreasing the expression of OGG1 as the antioxidant component in NSE will reduced the cell damage due to the effect of CPA.

CHAPTER 2

LITERATURE REVIEW

2.1 Cyclophosphamide

Cyclophosphamide is a bioactivated metabolite and alkylating agent that shows cytostatic effects by forming covalent DNA adducts. (Elangovan et al,2005). It is derived from the group of oxazophorines. The cytotoxicity of Cyclophosphamide is mediated by alkylation of DNA at the N7 position of guanine and the formation of DNA–DNAcross-links, DNA–protein cross-links, and single-strand breaks ,according to Hemminki and Kallama(cited by Elangovan et al. 2005).Elangovan et al ,(2005) also cited from Dollery(1999) that Cyclophosphamide is extensively used as anticancer treatment, as well as an immunosuppressive agent for organ transplantation, leukemia, Hodgkin’s disease, etc. The cytotoxic effect of Cyclophosphamide targets rapidly dividing cells and reproductive cell is one of the good targets for damaging effects. Cyclophosphamide therapy is also commonly used to treat variety of glomerular diseases. In spite of its therapeutic importance, this drug leads to gonadal toxicity in prolonged usage. Hence, this issue often has a critical role in deciding whether or not to undergo treatment with Cyclophosphamide as the consequences of infertility can have great physical and emotional impact on both sexes. A wide range of adverse effects including reproductive toxicity has been demonstrated following Cyclophosphamide (CPA) treatment in patients and in experimental animals. Trasler et al. (1986) said there were decrease in weight of reproductive organ, impaired fertility, growth and development of next generation was also observed in low dose cylophosphamide treated Sprague-Dawley male rats. The cancer male patients treated with cyclophosphamide exhibited an increased incidence of oligospermia and azoospermia (Charak et al., 1990)

2.2 Nigella Sativa Extract

People in different cultures and places have used medicinal plant to treat certain medical problems. A larger number of these plants and their extract have shown beneficial therapeutic effects, including anti-oxidant, anti-inflammatory, anti-cancer, anti-microbial, and immunomodulatory effects. Among the promising medicinal plants, Nigella sativa, a dicotyledonous of the Ranunculaceae family, is an amazing herb with rich historical and religious background. According to Mukhallad,Mohammad & Drakha (2009),Nigella sativa is mainly found in southern Europe, northern Africa, and Asia Minor. It is a bushy, self-branching plant with white or pale to dark blue flowers. The seeds of Nigella sativa are the source of the active ingredients of this plant. It is the black seed referred to by the prophet Mohammed (pbuh) as having healing powers.In Arabic, it is known as "Habbatus Sauddah". Black seed is also identified as the curative black cumin in the Holy Bible and is described as the Melanthion of Hippocrates and Discroides and as the Gith of Pliny. Historically, it has been recorded that Ancient Egyptian and Greek physicians use Nigella Sativa oil to treat headache, nasal congestion, toothache and intestinal worms, diuretic and to increase milk production. Present investigation done by Mukhallad,Mohammad & Drakha (2009) also proved that oral administration of Nigella Sativa increased fertility in male albino rats. Their weights of reproductive organs were markedly increased. They also found that there were significant increase in the sperm motility and sperm density in Nigella Sativa treated rats as compared to the controls.

2.3 8-Oxoguanine glycosylase

8-Oxoguanine glycosylase gene (OGG1) is a gene that encodes DNA glycosylase enzyme. According to Genecards, (2012) OGG1 is a DNA repair enzyme that incises DNA at 8-oxoG residues. It excises 7,8-dihydro-8-oxoguanine and 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (FAPY) from damaged DNA. It also has a beta-lyase activity that nicks DNA 3' to the lesion. OGG1 is involved in Base Excision Repair (BER) .BER is one of the cellular mechanism that repairs damaged DNA throughout the cell cycle besides Nucleotide Excision Repair (NER), Double Strands Break (DSB) repair and mismatch repair. This mechanism is very essential for removing damaged bases that could otherwise cause mutations by mispairing or lead to breaks in DNA during replication. Defect or deletions in BER genes can lead to the development of cancer. It can be caused by exposure to the X-Ray, Oxygen radicals, alkylating agents and spontaneous reactions.(Hoijmakers ,Jackson,Jiricny,Kanaar & Krokan,2009)

2.4 QUANTITATIVE REAL-TIME POLYMERASE CHAIN REACTION (QRT-PCR)

Real-time PCR is a variant of the conventional PCR method. It is highly sensitive technique that enables amplification and quantification of a specific nucleic acid sequence with detection of the PCR product in real time. DNA, cDNA and RNA targets can be easily quantified by determining the cycle when the PCR product can first be detected. This technique is highly compatible for a wide range of application such as gene expression analysis, determination of viral load, detection of genetically modified organisms, SNP genotyping, and allelic discrimination.DNA template can be used to quantify genomic DNA and qualitative analysis such as single nucleotide polymorphism(SNP) detection. RNA template is used for analysis of gene expression levels and viral load of RNA viruses.

2.5 SYBR GREEN

In order to detect PCR products in real-time, fluorescent signal is measured during each cycle. The amount of fluorescence is proportional to the amount of PCR product. SYBR GREEN is the fluorescence dye that binds all double stranded DNA molecules. It emits a fluorescent signal of a defined wavelength on binding. The maximum excitation and emission of SYBR Green are at 494nm and 521 nm, allowing use of the dye with any real-time cycler. Detection takes place in the extension step of real-time qPCR. The accumulation of PCR products due to increasing cycle increases the signal intensity. It enables analysis of many different targets without having to synthesize target-specific labelled probes. A high PCR specificity is required when using SYBR Green because non-specific PCR products and primer-dimers will also contribute to the fluorescent signal.

2.6 TWO-STEP RT-PCR

In two-step RNA, the RNA is initially reverse transcribed into cDNA using oligo-dT primers, random oligomers, or gene specific primers. An aliquot of the reverse transcriptase reaction is later added to the real-time PCR. The use of oligo-dT primers or random oligomers for reverse transcription means that several different transcripts can be analyzed by PCR from single RT reaction. RNA samples are transcribed into more stable cDNA for long term storage.

2.7 ONE-STEP RT-PCR

It is also known as one-tube RT-PCR. In this procedure, both reverse transcription and real-time PCR take place in the same tube with reverse transcription preceding PCR. It enables rapid processing of multiple samples and is easy to automate. it is easy to handle and minimized the risk of contamination as less manipulation is required. It also results in highr reproducibility from sample to sample. (Qiagen,2009)