Background Information: Breast cancer is the most common type of non-skin malignancy among women worldwide. It has been reported that the incidence and mortality of breast cancer have increased during the last two decades (Evrik et al., 2013). Breast cancer constitutes a major public health issue globally with over 400,000 annual deaths and about 4.4 million women living with the disease (Elima et al., 2012). In Nigeria, about 10,000 cancer deaths are recorded annually while 250,000 new cases are recorded yearly (Maria et al., 2012). It is predicted that estimated 232,670 new cases of breast cancer and 40,000 breast cancer related deaths will occur among women in 2014 worldwide (Jemal et al., 2011). N-Methyl-N-Nitrosourea has been studied in mutagenicity and genetics studies as a cancer causative agent. It is a highly potent carcinogen which acts directly as an alkylating agent that interacts with DNA to generate mammary tumors in female rats and does not require metabolic activatioin (Macejova and Brtko, 2001; Saminathan et al., 2014).
Although many treatment cancer therapies are currently used such as surgery, radiotherapy, biological therapy, hormone therapy, and chemotherapy, these therapies are less effective and relapsing is still an issue in breast cancer management. Due to the adverse effects of these therapies to normal cells, and aggressive behavior of the tumors, there is the need to develop alternate treatment option (Connor and Attai, 2013). In spite of improvements in the use of hormonal and adjuvant cytotoxic therapies for breast cancer patients, there has been no considerable reduction in breast cancer mortality till present (Winer et al., 2011). Another setback to therapeutic approach towards breast cancer treatment is patient’s resistance to widely used and standard chemotherapeutic agents, such as 5-fluorouracil, taxol, doxorubicin, cisplatin, campothecin, paclitaxel and topo-tecan (Sarkar et al., 2007). Due to this resistance to cancer drugs, it is important to search for new anti-tumor agents as a novel anticancer drug that can circumvent the existing resistance mechanisms. Natural plant products have become interesting sources for this purpose.
Kolaviron (KV), isolated from seeds of Garcinia kola has been shown to possess wide pharmacological properties (Olaleye et al., 2010). The beneficial effects of kolaviron, a natural biflavonoid have been attributed to its antioxidant and anti-inflammatory effects (Adaramoye et al., 2014). As a result, it is proposed that the anti-oxidative, anti-genotoxic, and anti-inflammatory properties of this biflavonoid can qualifies it as a potential candidate in cancer prevention by inhibiting certain stages of mammary gland carcinogenesis. Therefore, this proposal is to investigate and to provide insight into underlying mechanisms of chemopreventive effects of kolaviron in N-Methyl-N-Nitrosourea (NMU)-induced mammary gland carcinoma in female rats as well as in cancer cell lines.
Hypothesis to be tested:
The hypothesis to be tested in this study is that KV may possess anti-inflammatory and antigenotoxic properties and may therefore elicit antiproliferative and chemopreventive effect in N-Methyl-N-Nitrosourea-induced mammary tumor in female rats.
3.0 Broad Objective
Kolaviron (KV) may serve as a new class of drug to treat breast cancer. This alternative treatment may be less toxic than commonly used anti-neoplastic drugs. Overall, mortality rate from human breast cancer may be reduced.
3.1 Specific Objective
- To establish mammary gland carcinogenesis in rats model by determining the expression of proteins by immuno histochemstry and histology of tissue
- To investigate the impact of KV on NMU-induced mammary tumor on selected biochemical and oxidative stress biomarkers such as Lipid peroxidation, Reduced glutathione, Glutathione peroxidase, Glutathione –S- Transferase and Catalase
• To determine the effects of KV on apoptosis pathway using indices such as expression of caspases- 3 and -9, Bax, Bcl2 and cytochrome c release - To examine the effects of KV on cell cycle in NMU-induced mammary tumor
- To elucidate the effect of KV on inflammation by measuring Interleukin 6 , Interleukin 1β and Tumor necrosis factor-alpha
- To assess immune-histochemical expression of ER+, PGR+ and HER in NMU-induced mammary gland tumor of rats
In the letter of intent, the use of two established breast cancer cell lines (MCF and MDA-MB-231) were stated for the proper understanding of biochemical mechanism underlying the anti-proliferative effect of kolaviron on breast cancer cell lines in vitro. However, due to budget constraint I have decided to deal with only the in vivo part of the study.
Selected Chemicals
- N-Methyl-N-Nitrosourea, sulphosalicyclic acid, 5!, 5!-dithiobis-(2-nitrobenzoate) (DTNB), Tris base, potassium chloride, Thiobarbituric acid (TBA), 30% Trichloroacetic acid (TCA) will be purchased from Sigma aldrich and all other selected chemicals will be purchased from appropriate vendors.
Preparation of sample for biochemical assays
Preparation of Serum. Blood will be collected into 5.0 mL heparinized tubes and plasma will be separated by centrifugation at 800 × g for 5 min at 4°C in a Beckman bench centrifuge. The clear supernatant will be used for the estimation of serum enzymes and other biochemical indices.
Preparation of tissue homogenate: The mammary tumor tissue will be excise immediately and wash with chilled isotonic saline. The tissue homogenate will be prepared in ice cold 0.1M Tris-HCL buffer at pH 7.2. The homogenate will be centrifuged and the supernatant will be used for assays.
Determination of Protein
Determination of Lipid peroxidation Activity
An Aliquot of 0.4ml of
Determination of GSH Activity
This will be determined by the method of Beutler et al., 1963 and the absorbance will be measured at 412nm.
Determination of Catalase activity of samples
This will be determined according to the method of Sinha, 1971 and the absorbance will be measured at 570nm.
Determination of TNF-α activity, Caspase -3 and Caspase -9 activities
This will be determined using
Histopathological analysis
Organs and
Statistical Analysis:
Expected Duration of Experiments: Eleven (11) months
