Gene therapy for gastric cancer: A review

Abstract

Gastric cancer is common in China, and its early diagnosis and treatment are difficult. In recent years great progress has been achieved in gene therapy, and a wide array of gene therapy systems for gastric cancer has been investigated. The present article deals with the general principles of gene therapy and then focuses on how these principles may be applied to gastric cancer.

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INTRODUCTION

Enormous progress has been seen in molecular genetics over the past few decades. It has given us insights at the molecular level, into vital progresses in living organisms, such as embryonic development, growth regulation, differentiation, pathogenesis and carcinogenesis. Insights into the mechanism of pathologic progresses such as developmental disorders and carcinogenesis, have stimulated efforts to develop therapeutic approaches to prevent or correct these processes. Techniques to directly change the genetic information of a cell have greatly improved expectations of the therapeutic potential of genetic manipulation. These developments have raised hopes that diseases appearing to be incurable can soon be cured. Especially for cancer such as gastric cancer, which is common in China. It is well established that most cancers result from a series of accumulated, acquired genetic lesions in somatic cells that are faithfully reproduced until a malignant clone is created, which is ultimately able to destroy the host. Gene therapy has emerged as a new method of therapeutic and possibly preventive intervention against cancer at the level of cellular gene expression[1-14]. Generally speaking, gene therapy can be defined as the introduction and expression of an exogenous gene into human cells for therapeutic benefit, and is conventionally restricted to human diseases associated with single gene defects[15].

In oncology, it can be defined as the introduction of DNA into cells (either neoplastic or normal) in order to shrink or eliminate a malignant tumor. This may be achieved by means of directly inducing malignant cell death, modulating immune response to tumors or reversing the malignant process by correcting genetic abnormalities. It may also be possible to enhance a tumor's responsiveness to conventional treatments such as chemotherapy and radiotherapy, and to protect normal tissue by introduction of genetic materials that confers resistance to the toxic effects of such treatment[16,17]. A number of strategies have been developed to accomplish cancer gene therapy. These approaches included cytotoxic gene therapy, antisense therapy, and immunotherapy[18-22]. However, despite progress in the field, wide clinical applications and success have not been achieved[23,24]. As with all forms of gene therapy in cancer, the main problems to overcome will be optimizing delivery in order to maximize the proportion of successfully transduced cancer cells, In treatment of human malignant tumours, several obstacles explain the limitations of currently available treatments for achieving definitive cures in most cases of advanced disease. There are some problems in gene therapy. A combination of new chemotherapy drugs, higher doses of drugs, novel cytokines, improved regimens of radiotherapy, and more sophisticated surgery can achieve incremental improvements in cancer treatment. But these therapies do not address critical biological obstacles, and thus, probably will not bring about the much-needed radical advances in the implementation and results of cancer treatment. In contrast, gene therapy offers the potential for overcoming some of these fundamental barriers.

APPROACHES TO GENE THERAPY

Vector

One of the major problems in gene therapy is difficulty in delivering appropriate nucleic acid sequences to target cells.Various strategies have been evolved, which can be essentially divided into two types. One use viral vectors while the other uses non-viral vectors.

Viruses vector The main viruses which have been studied as potential vectors for transducing genes into cancer cells are retrovirus and adenovirus. Retroviruses are single-stranded RNA viruses, and after deletion of one or more structural genes, a foreign gene can be incorporated forming a "recombinant" retrovirus. This could then be used to infect a cell so that it integrates into the host cell's genome which then expresses viral genes as well as the "therapeutic" gene[25,26]. Adenoviruses consist of a core containing double-stranded DNA surrounded by a protein capsid. When an adenovirus vector is created, E1 genes are deleted in order to render the virus incapable of replication, thereby obviating the risk of transforming healthy host cells[27]. Adenoviruses have a greater potential than retroviruses in that they have a higher efficiency of infection and it is possible to incorporate larger segments of DNA. Weber et al[28] thought that oncoretrovirus-based vector was a safe and reliable vector system that could achieve permanent integration of delivered transgenes. Successful application of these vectors for gene therapy has proven difficult due to their relatively low transduction efficiency. However, cumulative improvements in methodology have recently yielded promising clinical results. Furthermore, significant improvements in basic retrovirus vector technology now can revitalize the field. But they do induce antiviral immune responses that may compromise the ability to treat an immunocompetent host on more than one occasions. Other viruses which have been used in this context include herpes virus, vaccinia virus and adenovirus-associated virus. Pieroni et al[29] found the use of baculovirus vectors for gene expression in mammalian cells was in continuous expansion. These vectors do not replicate in mammalian cells, do not cause a cytopathic effect upon infection and are able to carry large DNA inserts. Baculovirus vectors have been shown to transduce various cell types in vitro and in vivo with significant efficiency leading to stable gene expression. This review focuses on recent the developments in baculovirus vector that highlight its potential use for new gene therapy strategies. Okada et al[30]. have done something about adeno-associated viral vector-mediated gene therapy for ischemia-induced neuronal death.

Non-viral vector The most widely studied non-viral vector is liposome. Liposome is a positively charged lipid membrane which can be complexed with DNA, and fusion of liposome-DNA complex with a negatively charged membrane leads to transfer of DNA into cells. Lasic et al[31] reviewed stabilized liposomes in cancer therapy and gene delivery. Unfortunately, the efficiency of gene transduction using liposomes was currently much lower than that achieved by viral vectors[32].

Other approach

Another approach is to use direct injection of plasmid DNA, but this technique can only transfect cells immediately adjacent to the injection site so that only a small number of cells can be treated[33].Vanden et al[34] found that oncoretroviral vectors and lentiviral vectors offered the potential for long-term gene expression by virtue of their stable chromosomal integration and lack of viral gene expression. Gomez et al[35] analyzed conditionally replicative adenoviral vectors. Liu et al[19] described that the successful transformation of C. sporogenes, a clostridial strain with the highest reported tumor colonization efficiency, with E. coli cytosine deaminase (CD) gene and showed that systemically injected spores of these bacteria expressed CD only in the tumor.

It is hoped, however, that by complexing adenovirus and plasmid DNA with protein ligands which bind to specific receptors, enhanced gene transfer into specific cellular targets might be achieved[36]. An example of this is the argenine-glycine-aspartic acid motif that targets integrin receptors[37]. Aside from different approaches to introducing genetic material into cells, gene therapy can be classified according to the different end results. The main aims in this respect are gene replacement, antisense therapy, cytotoxic gene therapy, immunotherapy and drug resistance transfer.

Cytotoxic gene therapy

One of the most promising strategies for gene therapy against various types of cancer is the introduction of a suicide gene, which is transduction of a gene that transforms a non- toxic "pro-drug" into a toxic substance. One approach to this general concept is the transfer of the gene for HSV thymidine kinase (HSV-tk), as this phosphorylates nucleoside analogues such as acyclovir and ganciclovir which are then incorporated into DNA as it replicates[38]. Floeth et al[39] analyzed the mechanisms of the "bystander effect" in VPC-mediated HSV-Tk/GCV gene therapy. Thus, these compounds are only toxic to cells expressing HSV-tk, although the bystander effect has also been noted in this type of gene therapy[40]. This is presumably due to release of toxic metabolites produced by the prodrug-activating enzymes which then kill surrounding non-transduced cells. A similar type of cytotoxic gene therapy involves an adenovirus carrying cDNA for cytosine deaminase enzyme of E.coli and prodrug 5-fluorocytosine. The prodrug is given orally and converted to 5-fluorouracil in the cells containing cytosine deaminase[41,42].

Antisense therapy

When oligonucleotides bind to their complementary RNA or DNA, they prevent translation or transcription, respectively. This process, known as "anti-sense", is a theoretically attractive method for inactivating oncogenes which are overexpressed in tumors[43,44]. Tang et al[45] amplified the 200 VEGF cDNA fragment and inserted it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165 and VEGF121 mRNA. Their conclusion was expression of antisense VEGF RNA in SMMC-7721 cells could decrease tumorigenicity and antisense-VEGF gene therapy might be an adjuvant treatment for hepatoma. Like gene replacement therapy, however, it would seem that all cells in tumors would have to be transduced, and oligonucleotides would have to last long enough to down-regulate the appropriate genes. Nonetheless, this approach did seem to be effective in certain animal models[46-48]. Kumai et al[49] investigated the effect of antisense oligodeoxynucleotides (AS ODN) against tyrosine hydroxylase (TH) on hypertension and sympathetic nervous system activity in spontaneously hypertensive rats (SHR). Systolic blood pressure (SBP) in SHR treated with TH AS ODN (50, 200 mg/rat, i.v.) was significantly lower than that in control SHR. Epinephrine and norepinephrine levels, TH activity, and TH protein levels in adrenal medulla of SHR were reduced concomitantly with TH AS ODN treatment-induced changes in SBP. In contrast, TH AS ODN (200 mg/rat) had no effect on SBP in Wistar-Kyoto rats (WKY), though catecholamine levels, TH activity, and TH protein levels were significantly decreased. These findings suggest that peripheral systemic injection of TH AS ODN may be effective as hypotensive therapy in SHR. Marchand et al[50] found the use of miniosmotic pumps, phosphate-buffered saline, VEGF, or VEGF combined with AS-Flk-1, AS-Flt-1, or AS-scrambled oligonucleotides were released in mouse testis for 14 d. VEGF (1, 2.5, and 5 mg) increased the formation of new capillary blood vessels by 236%, 246%, and 287%, respectively. The combination of AS-Flk-1 or AS-Flt-1 (200 mg) to VEGF (2.5 mg) reduced by 87% and 85% of new blood vessel formation, respectively, and the expression of their corresponding proteins. These data demonstrate the therapeutical potential of AS-Flk-1 or AS-Flt-1 to prevent VEGF-mediated angiogenesis in vivo.

Immunotherapy

The main principle of genetic immunotherapy is to improve the host's immune response to a particular tumor. One approach is to employ intramuscular injection of DNA which encodes a tumor-associated antigen such as CEA either directly or in form of a viral vaccine[51]. Cheng et al[52] have developed a new strategy to enhance nucleic acid vaccine potency by linking VP22, a herpes simplex virus type 1 (HSV-1) tegument protein, to a model antigen. This strategy facilitated the spread of linked E7 antigen to neighboring cells. In their study, they created a recombinant Sindbis virus (SIN)-based replicon particle encoding VP22 linked to a model tumor antigen, human papillomavirus type 16 (HPV-16) E7, using a stable SIN PCL. The linkage of VP22 to E7 in these SIN replicon particles resulted in a significant increase in the number of E7-specific CD8 (+) T cell precursors and a strong antitumor effect against E7-expressing tumors in vaccinated C57BL/6 mice relative to wild-type E7 SIN replicon particles. Furthermore, a head-to-head comparison of VP22-E7-containing naked DNA, naked RNA replicons, or RNA replicon particle vaccines indicated that SINrep5-VP22/E7 replicon particles generated the most potent therapeutic antitumor effect. By leading to an active immune response, this was thought to be more effective than passive immunisation using specific antibodies against the antigen in question. Another way was to enhance immunity by using genes for cytokines such as interleukins (IL) which could recruit and stimulate appropriate effector cells[53,54]. Nishioka et al[55] have done something about genetic modification of dendritic cells and its application to cancer immunotherapy. Although the results in the experimental systems were promising, the clinical application of gene-modified DCs had several problems such as the standardization of methods of manipulation and gene-transduction of DCs. Approaches to solve them require further studies. Takemura et al[56] have previously produced an anti-MUC1 x anti-CD3 diabody (Mx3 diabody) in an Escherichia coli (E. coli) expression system, other approaches have been found, for instance, Vonderheide et al[57] applied telomerase as a universal tumor-associated antigen. Schadendorf et al[58] reviewed the use of histamine in cancer immunotherapy.

APPLICATION OF GNEE THERAPY FOR GASTRIC CANCER

p53 gene

About 60% of human gastric cancers carry point mutations of p53 gene, and because of its central role, this nuclear protein is believed to play a role in the regulation of cellular response to DNA damage. Wild-type p53 replacement therapy is an attractive concept in this disease. The responses of human gastric cancer cell lines to recombinant adenovirus encoding wild-type p53 gene have been analysed in vitro and in vivo[59]. In that study, growth inhibition was observed in cell lines expressing p53 mutations, but not in lines with wild-type p53. Furthermore, the mechanism of cell killing was found to be apoptosis. Thus, it seems that p53 replacement therapy has potential as a therapeutic strategy for human gastric cancer.

Antisense therapy

Antisense therapy has also been used in gastric cancer cell line. Proliferating cell nuclear antigen (PCNA) has been shown to stimulate DNA synthesis by DNA polymerase delta, and to be strongly expressed by gastric cancer cells with a high proliferative activity. Antisense oligonucleotides specific for PCNA mRNA have been shown to inhibit the growth of all gastric cancer cell lines tested, whereas random sequence oligonucleotides had no effect.

Cytotoxic gene therapy

Gastrointestinal cancer is the most important clinical target of gene therapy. Suicide gene therapy with herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, has been shown to exert antitumor efficacy in various cancer models in vitro. A modificiation of this approach has been madee to insert carcinoembryonic antigen (CEA) promoter into the viral vector to increase the efficiency of transfection of HSV-tk into cells expressing CEA. When compared with transduction of HSV-tk with a ubiquitous promoter, the use of CEA promoter enhanced the killing effect of ganciclovir in CEA producing cells. While in colorectal cancer, about 40% of gastric cancer expressed CEA, and CEA producing gastric cancer cell lines were susceptible to this treatment. Okino et al[60] described the sequential histopathological changes after suicide gene therapy of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced gastric cancer in rats. Gastric tumors were induced by MNNG in 38/73 (52%) of Wistar strain rats. The suicide gene therapy group (14 rats) was subjected to in situ gene transfer with a recombinant adenovirus vector carrying the HSV-TK gene driven by CAG promoter (Ad.CAGHSV-TK) in gastric tumor, followed by the antiviral drug ganciclovir (GCV). They observed the histopathological changes at various times after HSV-TK/GCV gene therapy, groups of animals were sacrificed at 3, 8, and 30 d after gene transfer. Apoptosis in gastric tumors was detected by the TUNEL method to assess the efficacy of HSV-TK/GCV gene therapy, and it was markable in the 8- and 30-day treatment groups compared to the sham operation controls (P < 0.001). Various histopathological changes, degeneration of cancer tissue and fibrosis after necrosis and apoptosis were significantly greater in the 30-day treatment group. The HSV-TK gene was detectable in peripheral blood by PCR until 30 d after gene transfer. These results might be useful in devising a method of suicide gene therapy for humans.

Other forms of cytotoxic gene therapy which have been used with success in gastric cancer cell lines include transfection of E.coli phosphoribosyltransferase (UPRT) which could catalyse the synthesis of UMP from uracil and 5-phosphoribosyl-alpha-1-diphosphate, thereby sensitising the cell to 5-fluorouracil (5-FU). This has been shown to enhance the cell killing effect of 5-FU in gastric cell lines both in vitro and in vivo. Shimizu et al[61] have generated a recombinant adenovirus encoding the UP gene (AxCA.UP) which has been applied in gastric cancer gene therapy to sensitize cancer cells to lower concentrations of 5-FU.

Immunotherapy

Genetic immunotherapy is another area of active research, and work with severe combined immunodeficiency (SCID) mice given human peripheral lymphocytes and autologous human tumour cells from patients with gastric cancer has yielded interesting results. In one study, administration of an adenovirus vector expressing IL-6 cDNA-induced CD8+cytotoxic T-lymphocytes specific for tumour cells from the precursor human T-lymphocytes in vivo, inhibited growth and metastasis of autologous human tumours. In another study, SCID mice reconstituted with peripheral blood cells containing CD34+ cells were inoculated with human gastric cancer cell lines transduced with cytokine genes including IL-2 and IL-6. It was found that the tumourigenicity of IL-2 producing tumour cells was significantly reduced in the CD34+ reconstituted but not in the non-reconstituted mice, whereas transduction of IL-6 did not affect tumourigenicity, irrespective of the reconstitution status of the mice. This system could provide a model for investigating the utility of transfecting tumours with individual cytokines. Yu et al[62] described the bioactivity of MG7 scFv for its application as a targeting mediator in gene therapy of gastric cancer. Two positive recombinant phage clones have been found to contain the exogenous scFv gene. ELISA showed that MG7 scFv had a strong antigen-binding affinity. Immunodotting assay showed that transfected E.coli HB2151 could successfully produce soluble MG7scFv with a high yield via induction by IPTG. The molecular mass of MG7 scFv was 30 kDa by Western blot. DNA sequencing demonstrated that VH and VL genes of MG7 scFv were 363 bp and 321 bp, respectively.

PROSPECT

With development of the genomic research, more and more individual patients have benefited from the revolution so far. Thus, despite a paucity of clinical information, gene therapy for gastric cancer is on the horizon. As with all forms of gene therapy in cancer, the main problems are to optimize delivery in order to maximize the proportion of successfully transduced cancer cells, and to choose the most appropriate targets for an individual tumor. There is no doubt that human cancers are heterogeneous in terms of genetic abnormality, and a better understanding of the mutational spectrum associated with a cancer type along with the ability to obtain mutation profiles for individual tumors is an important step to successful gene replacement and antisense therapy[63-68].

One factor critical to successful human gene therapy is the development of efficient gene delivery systems. Although numerous vector systems for gene transfer have been developed, a perfect vector system has not yet been constructed. Difficulties of in vivo gene transfer appear to be due to resistance of living cells to invasion by foreign materials and interference of cellular functions. We should analyze what barriers in tissues affect in vivo gene transfection and focus on how to solve these problems for gene therapy[68-71].