Cancer can be promoted by several pathogenic bacteria to initiate abnormal cell growth by attacking the immune system or suppressing apoptosis (Mager 2006). However, bacterial toxins can still be used for tumor suppressor and cancer vaccines on immunotoxins of bacterial origin (Patyar et al. 2010). Bacterial colonization of tumors was initially attributed to the hypoxic nature of solid tumors (low O2 levels). It has been proposed that the anaerobic nature of hypoxic/necrotic regions within tumors promotes growth of anaerobic and facultatively anaerobic bacteria. Areas of necrosis may also provide nutrients such as purines to further promote the growth of bacteria. The use of genetically modified and virulence reduced bacteria for destruction of tumors, and bacterial gene-directed enzyme prodrug therapy have shown promising potential in the development of bacteria therapy. (Patyar et al. 2010) Subsequently, it has been realized that anaerobic bacteria can selectively grow in tumors. However, these bacteria were not suitable for cancer therapy because of their high pathogenicity. Later on studies in animal models revealed that obligate anaerobic bacteria such as clostridia species proliferate preferentially in necrotic and therefore anaerobic regions of solid tumors. This actually resulted in tumor regression but was accompanied by acute toxicity and most animals became ill or died.
A wide range of gene therapy strategies exists aimed at inducing malignant cell death, be it directly or indirectly.
Direct cell killing. The most direct gene therapy strategy to treat tumors involves introducing a vector and gene to a malignant cell that directly induces death of that cell. There are a number of mechanisms by which this can be achieved, including the delivery of genes cytotoxic to the cell (pro-apoptotic genes or so-called ‘suicide genes’) or through oncolysis induced by the bacterial vector itself (as is observed with Clostridium and Salmonella).
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Oncolytic vectors. The oncolytic approach uses replication competent bacteria that are capable of spreading through the tumor tissue to infect neighbouring cells, with cancer cells killed as a result of infection. Therapeutic trials employing clostridial species mainly rely on the natural oncolytic activity of the vector to achieve tumor therapeutic responses. Following IV administration, clostridial spores germinate within tumors, killing cancer cells as they replicate, and have been shown to produce significant oncolytic effects in preclinical and clinical studies.
Efforts to refine the process have involved pre-treatment measures to make the tumor environment more hypoxic, combination therapies and more recently, genetic engineering. However, clostridia are typically difficult to manipulate genetically, which has hampered their development in terms of expression or delivery of heterologous genes. Only recently have strains been engineered to encode additional heterologous genes, aimed at enhancing the therapeutic effect (see below). Nonetheless, there is still cause for optimism with this treatment strategy in line with further improvements in genetic technologies for the species.
Cytotoxic genes. Bacterial vectors can mediate expression of agents that are cytotoxic to the host cell. The extracellulardomain of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent apoptotic agent in tumor cells, with minimal toxicity to normal cells. Attenuated S. typhimurium has been used to express TRAIL under the control of the prokaryotic radiation-inducible RecA promoter, with systemic administration of this vector resulting in xenograft tumor reduction. B. longum has also been utilized to express this agent within murine tumors resulting in significant regression. Indirectly cytotoxic genes have also shown promise.
Prodrug activating genes (suicide genes) encode a protein that is capable of directly or indirectly causing cell death. While some suicide genes express products that are directly toxic for the cell, e.g., Diphtheria toxin or Pseudomonas exotoxin, the best known agents encode enzymesthat convert non-toxic pro-drugs into highly toxic metabolites.
Gene-directed enzyme prodrug therapy (GDEPT) is a twostep approach. In the first step, the transgene is delivered into the tumor, while in the second step, a prodrug is administered which is selectively activated by the expressed enzyme. The most widely used system is the thymidine kinase gene of the Herpes Simplex Virus (HSVtk) in combination with the prodrug ganciclovir.
HSVtk phosphorylates ganciclovir to produce a cytotoxic metabolite. Other systems include the cytosine deaminase (CD) gene in conjunction with 5-fluorocytosine. A number of bacterial vectors have been successfully utilized to deliver suicide genes as summarized in Anti-angiogenic therapy. Angiogenesis is the formation of new capillary blood vessels from existing microvessels. For cancer therapy, strategies based on the manipulation of angiogenesis are referred to as anti-angiogenic strategies and seek to prevent new vessel formation or to inactivate pre-existing vessels. Gene-based anti-angiogenic therapy holds the potential to provide long-term anti-angiogenic protein production, and can be readily used in conjunction with other strategies. Endostatin is an endogenous inhibitor of angiogenesis, first discovered in 1997. It suppresses endothelial cell proliferation and acts as a competitor of angiogenic inducers secreted by tumor cells, such as fibroblast growth factor and vascular endothelial growth factor. However, results with administration of recombinant endostatin protein in clinical trials have been disappointing, due to poor solubility of the protein, in addition to the requirement for long-term multiple administrations.
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Upregulating the immune system. Cancer immunotherapy approaches concentrate on killing the tumor cells through direct or indirect intervention of various effector cells of the immune system, which include antibody-producing B cells, CD8+ CTL, CD4+ helper T cells, and NK cells. Gene therapy can be employed to induce tumor or other cells to produce immune upregulating cytokines that can attract and enhance anti-tumor activity of various lymphocytes. S. typhimurium has been used in several murine trials examining immunotherapies, with significant tumor reduction resulting from local bacterial expression or tumor cell expression of the immune-stimulating molecules IL-18, CCL21, LIGHT or Fas ligand.96-99 Preclinical studies have also used bifidobacteria in combination therapy with cytokines such as granulocyte colony-stimulating factor (GCSF), resulting in superior anti-tumor effects. Interestingly, the immune response was primarily directed against tumor cells rather than the bacterial vector cells.
DNA vaccination. The goal of cancer vaccines is to break tolerance of the immune system to specific antigens known to be expressed mainly or exclusively by particular tumor cells. DNA vaccines expressing a defined tumor antigen have shown significant promise both preclinically and in clinical trials. This strategy involves delivery of a vector that expresses the gene of interest, and functions to target immune activity in a similar manner to which traditional vaccines work. Vaccination strategies attempt to stimulate immune responses by generatingcytotoxic T lymphocytes and/or antibodies from B cells to break the pre-existing tolerance to specific antigens. Bacteria that target inductive cells of the immune system are highly attractive candidates for vaccine delivery and have been developed as live vehicles for inducing protective responses to a wide variety of antigens. Members of the Salmonella genus have been widely used as antigen carriers and several well-characterized safety attenuated strains are available.105-108 Salmonella is capable of triggering both humoural and antigen-specific T-helper and cytotoxic responses. S. typhimurium vectors deliver transgenes to the body via the monocyte cell population. After oral intake, the bacterial vector cells are phagocytosed by monocytes in the intestine. The monocytes differentiate and migrate to lymph nodes and the spleen. Finally, the attenuated auxotrophic S. typhimurium (unable to replicate in mammals) lyse and release plasmid into the cytoplasm of monocytes, followed by expression of the desired antigen and presentation to the immune system. This delivery platform has shown success in several preclinical tumor models employing various tumor antigens. A number of live attenuated strains of Listeria have been developed expressing a broad range of tumor antigens, such as Her-2/neu (an oncoprotein associated with a wide variety of cancers, Melanoma Associated Antigen (MAGE)117 and prostate specific antigen (PSA).118,119 The cytoplasmic location of L. monocytogenes is significant as this potentiates entry of the antigen into the Class I MHC antigen-processing pathway leading to priming of specific CD8+ T-cell responses. IV administered attenuated L.monocytogenes expressing HPV16 E7 was recently used in phase I clinical trial on patients with metastatic cervical cancer.120 Apart from some flu-like symptoms and fever-related hypertension in some patients, the vector was well tolerated. In addition, 30% tumor reduction was noted with an increase in overall survival, indicating the safety and efficacy of listerial vectors in patients and paving the way for clinical development of this vector strategy.
Much of the current research intended to achieve selective replication within, and lysis of, tumor cells has focused on viruses, but recent observations in murine models with facultative anaerobic bacteria (1), as well as data generated more than 30 years ago with obligate anaerobic bacteria (2), indicate that some bacterial species can also preferentially replicate and accumulate within tumors. In contrast to viruses, the bacteria reside primarily in the extracellular tumor microenvironment (3) and possess certain features that may be advantageous in the treatment of cancer. Thus, bacteria are motile, which facilitates their spread throughout the tumor and can help target systemic disease. Because of their large genome size, bacteria can readily express multiple therapeutic transgenes, such as cytokines or pro–drug-converting enzymes, and their spread can be controlled with antibiotics if necessary.
Bacteria can be used as tumoricidal agents which can suppress or destruct the tumor via direct tumoricidal effects or by delivering the tumoricidal molecules to the tumor site(Patyar et al. 2010). Experimental studies have shown that pathogenic species of the anaerobic clostridia were able to proliferate preferentially within the necrotic (anaerobic) regions of tumors in animals as compared to normal tissues thus resulting in tumour regression but was accompanied by acute toxicity and most animals became ill or died Deletion of two of its genes –msbBandpurI-resulted in its complete attenuation (by preventing toxic shock in animal hosts) and dependence on external sources of purine for survival. Bacteria can also act as vector for gene therapy. Bacteria as carriers of tumoricidal agents.
Bacteria is genetically engineered to express some therapeutic gene(Patyar et al. 2010). Bacterial toxins can cause cell death by alter cellular processes that control proliferation, apoptosis and differentiation(Patyar et al. 2010). Bacterial toxins have to some extent already been tested for cancer treatment. Bacterial toxins can kill cells or at reduced levels alter cellular processes that control proliferation, apoptosis and differentiation. These alterations are associated with carcinogenesis and may either stimulate cellular aberrations or inhibit normal cell controls. Cell-cycle inhibitors, such as cytolethal distending toxins (CDTs) and the cycle inhibiting factor (Cif), block mitosis and are thought to compromise the immune system by inhibiting clonal expansion of lymphocytes. In contrast, cell-cycle stimulators such as the cytotoxic necrotizing factor (CNF) promote cellular proliferation and interfere with cell differentiation. Bacterial toxins binding to tumor surface antigens
Bacterial toxins conjugated to ligands. This process is accomplished by eliminating binding to toxin receptors by conjugating the toxins to cell-binding proteins such as monoclonal antibodies or growth factors. Bacteria also act as immunotherapeutic agents as bacteria can promote the antigenicity of tumor cells and prevent the tumors to escape the immune system even they are weekly immunogenic(Patyar et al. 2010). Thus one of the novel immunotherapeutic strategies employs bacteria to enhance the antigenicity of tumor cells. The majority of all the anaerobic bacteria discussed so far can form highly resistant spores which allow them to survive even in oxygen-rich conditions, although they cannot grow or multiply there. But once they meet favourable conditions, such as the dead areas inside tumors, the spores can germinate and the bacteria thrive, making them ideal to target cancers.
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