Breast cancer is the result of genetic alterations in carcinoma of the breast which may either be associated with alteration of the chromosome or deficiency of tumor suppressor function and proliferation of oncogenes. Gene therapy as a treatment for breast cancer is relatively a new approach, which is developed in lieu with the discovery that breast cancer can arise as a result of a genetic disorder. Gene therapy involves the manipulation of genetic material to achieve therapeutic ends.
The use of gene therapy for the treatment of breast cancer has recently captured the attention of many researchers. In line with this, this study aims to analyze gene therapy as a novel approach to treatment of breasts cancer.
Inactivation of the tumor suppressor gene (TSG) may trigger the onset of malignancy. It has been observed in breast cancer that it may arise as a result of loss of heterozygosity (LOH) among somatic cells (Osborne et al., 2004). LOH may not only cause the inactivation of TSG but may alter expression and transcription of TSG. Among the most studied TSGs related with the formation of breast cancer are Rb and mutated p53 (p53). The presence of Rb was found out to cause the amplification of the breast carcinogenesis. The function of p53 on the other hand is important in maintaining regulation of cell cycle, DNA repair, apoptosis of abnormal cells and angiogenesis inhibition (Takahashi et al., 1989) hence mutation of these genes could lead to loss of these functions. Among the other known occurrence of mutation in TSGs which are linked to breast carcinoma are p16, p27, p21, p53, mda7, BRCA-1, BRCA-2, Maspin, and Testin (Stoff-Khalili et al., 2006).
One of the methods employed in gene therapy is through the aid of tumor suppressor genes (TSG). Studies have been performed to replace p53 with viral wild-type p53 in cancer cells of humans and results have shown to combat proliferation of cancer cells as well as to cause their apoptosis. It must be noted that the p53 functions to inhibit proliferation of cells hence it may act as an inhibitory to the growth of normal and malignant cells hence it functions as well to inhibit the proliferation of tumor cells in the breast. Moreover, p53 is also known to act in conjunction with bystander effect hence causing the death not only of the p53-transfecrted cells but also of the neighboring cells. This in turn is important in gene therapy for it significantly lowers the transduction level which is necessary for the success of gene therapy (Stoff- Khalili et al., 2006). In a study conducted to determine the antiproliferative effect of wild-type (wt) p53 mediated through nanoparticles on breast cancer cells, it was shown that the transfection of breast cancer cells led to the sustained counteracting of the proliferation of the cancer cells (Prabha and Lahasetwar, 2004). Significant inhibition of growth of tumor was similarly noted by Nielsen et al., (1997) in models of mouse xenograft breast cancer.
Another tumor suppressor gene which is noted for its antiproliferative effect on the growth of breast cancer cells is BRCA1. According to Obermiller et al., (2000) BRCA1 may also be involved in DNA repair and it is observed to be ever expressed in the presence of cancer cells in breast and ovary but under normal conditions BRCA1 is minimally expressed in these organs. It was also said that BRCA1 has been observed to inhibit the growth of tumor under in vivo and in vitro condition but the specific mechanism as to how it operates remains to be uncertain. However it is hypothesized that it may either stimulate apoptosis or it may interact with WAFI/CIPI, p53 and Rb.
Suppression of proliferation of breast cancer cells is also known to be the effect of expression of Rb. In support with this, a study has shown that restoration of the function of ones mutated Rb was able to decrease the proliferation activity of cancer cells in the breast of mice (Wang et al., 1993).
According to the National Institute of Health, there are three registered TSG which are used for clinical procedures for the suppression of proliferation of cancer cells in breast and these are Rb, mada7, and p53.
Another approach used in gene therapy is through the use of antisense oligodeoxynucleotides. As to the function these short ssDNA molecules alter expression of the gene through the inhabiting the translation of the genetic information to protein (Dias and Stein, 2002). The antisense oligodeoxynucleotide has the ability to disrupt the translation of mRNA inside the cell. Its mode of disruption may be at the translation or transcription level, or it may also interfere during splicing, or it may inhibit RNase-mediated mRNA cleavage (Stoff-Khalili, 2006). In a study conducted by Fan et al. (2003), the p21 oncogene was tested for gene therapy of the breast carcinoma through the use of p21 antisense oligodeoxynucleotide. In the study, eight breast tumors from human subjects were obtained and it was observed that three of the eight samples manifested an increase in levels of p21 signifying the presence of breast carcinoma while the rest were normal. Two human breast carcinoma lines were particularly observed namely T47D (ductal carcinoma) and MCF7 (adenocarcinoma) for the reaction with p21 antisense oligodeoxynucleotide. This in turn will be used to decipher if targeting of the p21 oncogene through the use of p21 antisense oligodeoxynucleotide will significantly inhibit the proliferation of the cancer cells. Results of the study show that inhibition of the p21 indeed resulted into a decreased proliferation of the cancer cells both in T47D and MCF7 human breast cancer lines. Hence it was concluded that the use of p21 antisense oligodeoxynucleotide may be used to treat human breast cancer whether in conjunction with chemotherapy or not, but this remains to be subjected to further clinical trials and research.
Aside from p21 other oncogenes, other oncogenes which have been found out to be acted by antisense oligodeoxynucleotide include c-myc, p2, PKC- α, Bcl-2, MTHFR, p21, αV integrin, c-erbB-2, c-fos and IGF-IR (Stoff-Khalili et al., 2006). Thus the interaction of oncogenes with antisense oligodeoxynucleotides could possibly inhibit the advance of human breast cancer. In addition, it could be used in conjunction with chemotherapy to even intensify its anti-cancer effects.
Suicide gene therapy is also one of the commonly used approaches to gene therapy. The ontogeny of this approach in gene therapy started in 1986 upon the use of diphtera toxin A chain which was used as a suicide gene and in turn it was used for destroying cancer cells (Vassaux and Lemoine, 2000). There are two classification of suicide gene therapy which are the toxin gene therapy and the enzyme-prodrug therapy.
In toxin gene therapy, toxic molecules are synthesized as a product of transfected genes that codes for it. In contrast, the enzyme-prodrug activation therapy results into the synthesis of enzymes that are specifically assigned to activate particular prodrugs which also is a product of the transfected genes that codes for it. The enzyme-prodrug activation therapy is also known as gene prodrug activation therapy (GPAT), gene-directed enzyme prodrug therapy (GDEPT) and virally directed enzyme prodrug therapy (VDEPT) (Niculescu-Duvaz and Springer, 2005).
The enzyme-prodrug activation therapy first involves the transduction of the gene encoding for the enzyme into the breast cancer cell and followed by the introduction of the nontoxic prodrug. The enzymes used in the process are classified either as foreign enzymes which are nonmammlian in origin and human origin enzymes. Examples of foreign enzymes include viral TK, bacterial cytosine deaminase (CD) and carboxypeptidase G2 (CPG2) while human origin enzymes include P450 isophorms (Stoff-Khalili et al., 2006).
It has been observed in a certain study that when breast cancer cells in mice if injected with the cytokine genes granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-2 (IL-2) in addition to HSV-1 thymidine kinase (HSV-TK) results into the a decreased growth proliferation of the tumor cells, as when compared with HSV-TK alone. This suggests the possible toxicity of GM-CSF and IL-2 to the tumor cells in the breast (Vrionis et al., 1997).
In another study demonstrating enzyme-prodrug activation therapy, the retroviral vector MetXia-P450 was introduced to patients with either breast cancer or melanoma. MetXia-P450 is the one responsible for encoding the cytochrome P450 type 2B6 gene (CYP2B6) which encodes the needed enzyme cytochrome P450. Cytochrome P450 enzymes function for the conversion of the prodrug cyclophosphamide to its active form phosphoramide mustard and acrolein. Results revealed a probable association for the antitumor activity of the gene MetXia-P450 hence it was recommended that further clinical investigation regarding this retroviral vector should be conducted (Braybrooke et al., 2005).
In addition to the abovementioned example, thymidine kinase is also commonly used to convert ganciclovir into its toxic form. Another well tested example is the enzyme cytosine deaminase which is also used to convert 5-fluorocutosine into its toxic form (Stoff-Khalili et al., 2006).
With the discovery of the specific genes involved in gene therapy, greater knowledge paved the way for the advancement of the use of genetic vaccines. Genetic vaccines require the use of genes only hence the creation and production of antigens is deemed unnecessary. Probable target site for the activity of genetic vaccines in breast carcinoma are tumor cell-associated extracellular matrix metalloproteinase inducer (EMMPRIN), Fos-related antigen 1 (Fra-1), carcinoembryonic antigen (CEA), B7-H4, hTERT, MAGE-1 and MUC-1 (Stoff-Khalili et al., 2006).
The trend for the use of vaccines to treat cancer has been explored along with the use cytokines and costimulatory molecules. GM-CSF is an example of costimulatory molecule used to enhance the function of antigen in breast cancer (Chang et al., 2004).
Another antigen-presenting cell called dendritic cell (DC) plays an important role in stimulating immune responses. DCs are also known to encode costimulatory molecules as well as cytokines that are needed for the development of immune response. Also DCs have been the major focus in formulation of antitumor vaccines. This is done by attempting to enhance the ability of DCs to present tumor antigens to the immune system.
In a study conducted by Yu et al. (2006), a therapeutic vaccine for ovarian cancer was created through fusion of bone marrow-derived tumor cell line and modified with the aid of a suicide gene. The live vaccine created showed a greater cytotoxic response of T lymphocyte to ovarian tumor. Also it was able to initiate an in vivo immunopreventive and immunotherapeutic effects in response to primary tumor cells. Hence the study provided an insight for the use of suicide gene altered live vaccine in treatment of cancer of the ovary. In line with breast cancer, DC vaccines altered by HER-2/neu which are nonfunctional tumor antigens being altered by adenoviruses showed a decrease in the progress of breast cancer among mice that are BALB-neuT (Sakai et al., 2004).
The method of gene transfer remains to be an underdeveloped aspect of gene therapy. Up to date there are two categories by which the methods of gene delivery could fall into. First is with the group of viral vectors while the other is with the group of nonviral vectors. It was mentioned in The Journal of Medicine that as of the year 2000, 42.3% of the studies conducted in gene therapy employed the use of retroviruses to transduct the foreign gene while about 20% used adenoviruses.
Retroviruses work by translating their RNA genome into DNA in the infected host cell. Among the advantages in terms of using retroviruses include efficient transfection, stability of the transuded genetic material which confer its expression for a long time and does not leave immunogenic viral proteins in host cells. However, it is not capable of infecting non-dividing cells; contain only a small fraction of genetic information; probable initiation of the transformation of infected cells into malignant form; and possible event of recombination which could lead to formation of replication-competent viruses (Rochlitz, 2001). Adenoviruses are group of viruses that have the ability to infect both dividing and non-dividing cells. Compared with retroviruses adenoviruses have greater capacity to carry genetic material, low recombination activity and able to transduct non-dividing cells. However, immunogenic property poses a problem for repetitive application and the lack of ability to integrate into the host cell genome resulting into elimination of the genetic material after several successive cell divisions (Rochlitz, 2001). Other viral vectors used are adeno-associated viral vectors and lentiviral vectors. None of these however have been described to be a perfect vector for genes transfected in gene therapy.
Nonviral vectors include liposomes, plasmid DNA, protein DNA complexes, calcium-phosphate-precipitation, electroporation and many others. Among these the most commonly used is the liposome which accounts for about 18% of the recorded clinical studies dealing with gene therapy as of the year 2000. It can be used for the in situ genetic alteration of tumors.
In conclusion the use of the different approaches to gene therapy has a long way to go with respect to development and enhancement of the therapeutic effects. With respect to gene therapy through the aid of tumor suppressor genes, only a slim fraction showed a positive response to therapeutic effects and the same is true with gene therapy through the use of antisense oligodeoxynucleotide molecules and through suicide genes. Much of the slim success reported in most studies could be attributed to the unresolved problem in the employment of the most efficient method for the delivery of the transuded gene to the host system.
With respect to the development of genetic vaccines, much has still to be discovered on the area of new tumor antigens including sufficient knowledge about the nature of these tumor antigens. Also antigenic markers or determinants need as well to be further discovered in order to facilitate observation of antitumor immune responses.
Moreover, it has been established through the studies conducted on gene therapy up to date that gene therapy is indeed safe and possible to accomplish only that the obstacle with regards to gene transfer vectors still remains. Nevertheless, the abovementioned approaches to gene therapy offer a future for improvement of breast cancer treatment. All of these also offer a clinical significance which may prove to be beneficial in treatment of breast cancer regardless of the stage and may also offer the possibility of an even better and faster recovery from breast cancer, when used in conjunction with other treatments of breast cancer such as chemotherapy.
Thus, gene therapy is worth the painstaking task to tie all its loose ends.
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