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Non-Hodgkin's lymphoma cells begin to grow when lymphocytes such as B-cells turn into abnormal cells. These abnormal cells divide to make copies of themselves, continue dividing and do not die when they are supposed to. Due to their abnormality they lose their function to protect the body via the immune system and the uncontrolled division of the B-cells start to form a tumour. As B-cells are found in the lymphatic system, the tumour can grow almost anywhere in the body. However, the tumour growth usually starts in the lymph nodes.
These cells are resistant to chemotherapeutic agents that are available for cancer treatment today and so are the major well-known downside in the treatment due to their complete or long-term loss of response to chemotherapy. However, some cells may respond to the treatment for a short time and lead to progression after the treatment. Cancer cells avoid the cytotoxic effects of chemotherapy by the mechanism known as the multi-drug resistance (MDR), on observation of these cancer cells the MDR mechanism is due to their decreased sensitivity to many different drugs. Multi-drug resistance can occur due to the structural and functional changes of a certain cell organelles that are drug receptors which are mutated.
Multi-drug resistance effected cells express a 170-180kDa energy-dependant efflux pump called P-glycoprotein (P-gp) which plays a significant role in regulating the concentration of chemotherapeutic agents inside cytoplasm of normal or cancerous cells as they are able to interact with many drugs including the chemotherapeutic drugs. Nevertheless, there are drugs such as Verapamil that are calcium channel blockers that reverse the action of P-gp and so the cancer cells remain resistant to the chemotherapeutic drugs. A substrate for P-gp is an important drug in the standard regimen, which is anthracycline, hydroxy daunorubicin hydrochloride and therefore used for treatment for chemoresistant patients. As drugs such as Cyclophosphamide, Hydroxydaunorubiun, Oncovin and Prednisone (CHOP) in standard regimen has shown resistance in some NHL patients.
Most information regarding mechanisms of chemresistance derives from in vitro models of cells selected by exposure to extremely high levels of drugs, which cannot be achieved clinically. These studies have shown that there are many mechanisms of chemoresistance to anthrocyclines and that resistance is often multi-factorial (Nooter & Sonneveld, 1994). Human cancers such as leukaemia and malignant lymphoma are chemoresistant due to the presence of P-gp expression. The major cause of death of cancer patients is that they are initially sensitive to CHOP chemotherapy, but the treatment seems to have failed as there is an existence or development of a resistant disease and treatment related toxicity. All NHL patients have been found to have the P-gp expression at detectable levels in the histological types of NHL. The clinical outcome and drug sensitivity show a relationship with P-glycoprotein in NHL patients but there are some studies that have found no relationship between drug response and P-gp expression. Over-expression of the p53 mutant protein in the in vivo chemoresistance of NHL does not show any relationship. Therefore, in vitro studies suggest that abnormalities of the p53 gene providing increased levels of p53 mutant protein may increase chemoresistance in NHL patients (Koduru et al., 1997). P-gp promoter activity is repressed by the wild type p53 protein while P-gp promoter activity is enhanced by the mutant forms of p53 protein. It has been demonstrated that mutant p53 does not activate the multi-drug resistance-related protein, MRP1, promoter or the endogenous gene. In contrast, mutant p53 strongly up-regulated the MDR1 promoter and expression of the endogenous MDR1 gene (Chin et al., 1992).
The response of DNA in tumour cells by combining a chemotherapeutic drug such as doxorubicin with verapamil, a P-gp antagonist in p53 mutant protein over-expressing cells has not been investigated and therefore the study carried out was aimed to reverse the action of P-gp in vitro with verapamil to try and mimic chemoresistance observed in vivo in doxorubicin based treatments in patients with the over-expression of p53 mutant protein. By using the SCGE assay it was seen that p53 is frequently mutated in the Burkitt's lymphoma derived Raji lymphoblastoid cell sublines i.e. thymidine kinase proficient (TK+) and thymidine kinase deficient(TK-)(Farrell et al., 1991). Raji cells possess numerous receptors for certain complements and is therefore suitable for use in detection of immune complexes. They express certain complement receptors as well as Fc receptors from IgG.
Inhibiting DNA synthesis or DNA damage mediates the cytotoxicity of most chemotherapeutic agents, so it is very important for an effective treatment of malignancies such as lymphoma, to have a control on the concentration of chemotherapeutic agents in the cytostatic/cytotoxic range in the cell cytoplasm after passing through the cell membrane. SCGE assay measures the effects of the drugs.TK+ and TK- lymphoma cell sublines with over-expressed p53 mutant protein either by using or not using a chemosynthesizer investigated the altered DNA damage.
Raji cells are similar to the cells that NHL patients with over-expressing p53 mutant protein have and of those cells that are chemoresistant. The Raji TK- cells were more resistant compared to the TK+ cells following treatment with 5-FU.The cross-resistance between 5-FU and doxorubicin showed no effect in TK- cell lines but it showed a greater response to only doxorubicin compared to the response with only 5-FU.
5-Fluorouracil (5-FU) is not a P-gp substrate and so it is not effective in treating lymphomas but it is used in treating colon cancer.TK- resistant cells are sensitive to doxorubicin however the NHL patients with over-expressing p53 mutant protein are resistant to it as they use verapamil as one of the cancer cells defensive mechanism by increasing the efflux of cytotoxic compounds out the cell due to the over-expression of P-gp which is a membrane transport protein.
In this study, it was found that there was an increase in DNA damage for all clinical samples after the addition of verapamil compared to the addition of doxorubicin, hence sample show resistance to doxorubicin. However, after the addition of P-gp antagonist in the cells, the clinical samples DNA damage decreased.
Non-cytotoxic compounds such as verapamil act as substrates for P-gp , blocking the cellular extrusion of anthracyclines and this stops the exit of the drug from the cell. Therefore, verapamil may prevent the exit of doxorubicin out the cell and so over-coming chemoresistance in the NHL patients that over-express p53 mutant protein.
In conclusion, the study carried out could be useful in the NHL bio-monitoring but it may also be applicable for all types of chemoresistant cancer cells and so making it flexible for treatment and results into an improved chemotherapy for cancer patients.