Eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells

Cancer Epidemiology 37 (2013) 1014–1019

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Eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells Elena M. Glinka * Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia

A R T I C L E I N F O

A B S T R A C T

Article history: Received 26 June 2013 Received in revised form 10 September 2013 Accepted 20 September 2013 Available online 21 October 2013

Background: Gene therapy has attracted attention for its potential to specifically and efficiently target cancer cells with minimal toxicity to normal cells. At present, it offers a promising direction for the treatment of cancer patients. Numerous vectors have been engineered for the sole purpose of killing cancer cells, and some have successfully suppressed malignant tumours. Many plant proteins have anticancer properties; consequently, genes encoding some of these proteins are being used to design constructs for the inhibition of multiplying cancer cells. Results: Data addressing the function of vectors harbouring genes specifically encoding ricin, saporin, lunasin, linamarase, and tomato thymidine kinase 1 under the control of different promoters are summarised here. Constructs employing genes to encode cytotoxic proteins as well as constructs employing genes of enzymes that convert a nontoxic prodrug into a toxic drug are considered here. Conclusion: Generation of eukaryotic expression vectors containing genes encoding plant proteins for killing of cancer cells may permit the broadening of cancer gene therapy strategy, particularly because of the specific mode of action of anticancer plant proteins. ß 2013 Elsevier Ltd. All rights reserved.

Keywords: Cell death Cancer gene therapy Toxicity Toxic proteins Cancer Enzyme/prodrug combination Plant proteins Gene expression Therapeutic genes

1. Introduction Cancer is one of the most dangerous diseases; it causes people great suffering and often leads to death. Despite substantial progress that has been made towards understanding the molecular basis, diagnosis, and treatment of cancer, it remains a major health concern. Cancer gene therapy strategy allows for the specific and efficient targeting of cancer cells without harming healthy cells. At the end of the last century, cancer gene therapy actively emerged as a potential therapeutic regimen; presently, it offers one of the most attractive and promising strategies for the suppression of cancer cells and the eradication of malignant tumours [1–6]. Different strategies have been used to increase the potency of this approach to cancer treatment. These methods may include refining viral and non-viral methods of delivery of therapeutic genes to cancer cells and improving tissue/tumour specific promoters. Additionally, the identification of new genes that encode therapeutic proteins is necessary. Presently, genes encoding proteins from different organisms are incorporated into constructs

* Correspondence to: Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Miklukho-Maklaya, 16/10, Moscow 117997, Russia. Tel.: +7 495 779 23 66; fax: +7 495 335 71 03. E-mail address: [email protected] 1877-7821/$ – see front matter ß 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canep.2013.09.013

as therapeutic regimens [7–11]. The first demonstration of the use of the controlled expression of an exogenous gene encoding a toxin to eliminate cancer cells was described in 1986 [1]. In this same period, enzyme-activating prodrug therapy was suggested [12]. Later, this approach became known as gene-directed enzyme prodrug therapy (GDEPT) or virus-directed enzyme prodrug therapy (VDEPT) [13]. The most popular enzyme-activating prodrug system is the herpes simplex virus thymidine kinase/ ganciclovir (HSV-TK/GCV) system [14]. This treatment combination has entered clinical trials, in which both its safety and its partial efficacy have been demonstrated [15–20]. In 1998, plant genes began to be used in the engineering of cancer gene therapy constructs [21]. Historically, many biologically active substances derived from plants have been utilised in folk medicine for cancer treatment. In the last 50 years of the past century, plant extracts began to be actively exploited for cancer chemotherapy [22,23]. Currently, extracts from different plants are both undergoing preclinical and clinical studies [24–27]. In the 1980s, plant proteins, such as ricin, which is a ribosomeinactivating protein (RIP), entered into clinical trials [28]. Other promising approaches have included the use of plant-derived anticancer proteins in the design of immunotoxins. For example, RIPs, such as ricin A, saporin, bouganin, gelonin, momordin and others, have been used to produce immunotoxins [29–33]. RIPcontaining conjugates have been used in many experimental

E.M. Glinka / Cancer Epidemiology 37 (2013) 1014–1019

strategies to target cancer cells, often showing great efficacy in clinical trials [34,35]. Many new plant proteins that have been tested for their ability to kill cancer cells showed cytotoxic effects, including the following: nucleolytic proteins from Corydalis cava tubers [36], potato (Solanum tuberosum) aspartic proteases [37], Solanum nigrum Linn (SNL) glycoprotein [38], and pectinesterase inhibitor from jelly fig (Ficus awkeotsang Makino) [39]. At the present time, identification of new therapeutic genes is under way. The therapeutic genes are selected by taking into consideration the molecular biology of the cancer and the complex interactions between tumour cells and the immune system. The choice of therapeutic transgenes and gene therapy strategies are rapidly evolving with advances in the identification of new genes and new targets, and the improvement of vectors and expression systems. The choice of the transgene usually determines the therapeutic strategy [40]. Use of genes from plant proteins in cancer gene therapy may open new possibilities for the development of different therapy strategies, as there are many plant proteins with various modes of actions that are able to suppress the growth of cancer cells. Genes of some plant proteins with anticancer properties are currently used in cancer gene therapy constructs. This review summarises the data on the available eukaryotic expression vectors that contain genes encoding plant proteins for the selective elimination of cancer cells, which use two specific approaches: the induction of cell death after the expression of genes encoding toxic proteins, and the induction of cell death after the expression of genes encoding enzymes that convert a nontoxic prodrug into a toxic drug. Constructs containing genes of ricin, saporin, lunasin, linamarase, tomato thymidine kinase1 are considered. 2. Vectors containing genes encoding toxic proteins and genes encoding enzymes activating prodrugs 2.1. Expression of ribosome-inactivating proteins and chromatinbinding peptide Ribosome-inactivating proteins (RIPs) are enzymes that belong to the polynucleotide adenosine glycosidase class of plant enzymes [41–44]. RIPs depurinate large ribosomal RNA that result in damage to the ribosome in an irreversible manner, which leads to the inability of the ribosome to bind elongation factor-2, causing the arrest of protein synthesis and eventually cell death [45–48]. RIPs are divided into two main types: type 1 RIPs, which are singlechain proteins, and type 2 RIPs, which are proteins consisting of an active A chain, similar to the type 1 RIP, linked to a B chain with lectin properties [29,31,34,49,50]. Some RIPs are potent toxins. It has been shown that there are differences in the cytotoxicity of RIPs and, consequently, in their toxicity within animals. RIPs cause apoptotic and necrotic lesions and induce production of cytokines, causing inflammation [31]. Ricin, which comes from the seeds of the castor oil plant Ricinus communis, is a highly toxic protein classified as a type 2 RIP [51– 55]. It has been used in cancer gene therapy constructs. A retroviral construct (retro-1.3MBPp-ri-toxin), containing the A chain of the ricin gene under the thyroid hormone (T3) regulatable promoter of the rat myelin basic protein (MBPp), was created for gliomaspecific transcription initiation. It is worth noting that retroviruses are ideally suited for gene therapy of malignant gliomas in the CNS, where the normal cells are already mature and are not actively dividing. Consequently, glioma cells can be specifically targeted with a recombinant retrovirus, as rapidly growing normal cells are rare in the adult CNS. A 50% reduction in the incorporation of [3H] thymidine into DNA was observed after T3 treatment in the human glioblastoma cell line U-373-MG infected with the retro-1.3MBPpri-toxin. In vivo, retroviruses bearing the toxin gene are capable of

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eradicating experimentally induced brain tumours in Wistar rats. It is also noteworthy that rejection of tumours is more efficient in the brain of immunocompetent Wistar rats than in SCID mice flanks. For this reason an immunomediated bystander effect was suggested in glioblastoma treated by retro-1.3MBPp-ri-toxin construct [56]. Developed as an approach for the suppression of cancer cells, the strategy employed the co-application of the ricin A chain with the recombinant adenovirus expressing the ricin B chain. The ricin A chain (RTA) was expressed in Escherichia coli in the form of a 6XHis-tagged fusion protein and purified. Additionally, a replication-deficient ricin B chain (RTB)-expression adenovirus green fluorescence protein (AdGFP-RTB) was constructed. RTA and AdGFP-RTB were tested for cytotoxicity either individually or in combination in human cell lines HEK293, human cervical carcinoma cell line HeLa, and human liver cancer cell lines SMMC7721 and HL7702. Significant cell death or loss of cell viability was observed in all of the cell lines tested, when RTA and AdGFP-RTB were applied together, which resulted in approximately 60% cell mortality [57]. Similarly, the ricin toxin A chain cDNA was cloned into the mammalian expression vector, pcDNA3.1, where gene expression is under the control of the constitutive cytomegalovirus (CMV) promoter, to generate the pcDNA3-ricin construct. Transfection of HeLa cells with pcDNA3ricin construct demonstrated that direct cytoplasmic expression of ricin in cells induced cell death [58]. Saporin, from the soapwort plant Saponaria officinalis, which is similar to ricin, is a highly toxic protein. It is classified as a type 1 RIP [35,59]. A plasmid (pCI-SAP) expressing the cytosolic saporin (SAP) gene was generated by placing the region encoding the mature plant toxin under the control of the CMV promoter. The ability of the pCI-SAP to inhibit protein synthesis was tested in cultured tumour cells co-transfected with a luciferase reporter gene. SAP expression, when driven by the CMV promoter, demonstrated that only 10 ng of plasmid DNA per 1.6  104 B16 melanoma cells was required to drastically reduce luciferase reporter activity to only 18% when compared to control cells. Furthermore, the effects of the pCI-SAP construct exceeded that of the pSfiSV19-SAP construct by 15- to 700-fold, where SAP expression was driven by the SV40 promoter. Notably, the effect of transfecting pCI-SAP was particularly prominent in B16 cells. Direct intra-tumoural injections of pCI-SAP in B16 melanomabearing mice caused a significant suppression of tumour growth [60,61]. Lunasin, an acid peptide, is the antimitotic agent isolated from soybean (Glycine max) seeds. It is known for its anticancer properties [62]. A chimeric construct (lunasin pEGFP-C1) encoding the small-subunit peptide of Gm2S-1 tagged with the green fluorescent protein (GFP) has been generated. The Gm2S-1 cDNA encodes lunasin as a small- subunit component of posttranslationally processed 2S albumin. It was shown that this chimeric gene GFP-lunasin arrested cell division, causing abnormal spindle fibre elongation, chromosomal fragmentation, and cell lysis when transiently transfected into murine embryo fibroblast, murine hepatoma, and human breast cancer cells. Thus, the results suggested that the binding of lunasin to chromatin prevents the normal formation of the kinetochore complex in the centromere, thereby leading to the disruption of mitosis and eventually to cell death [63]. Therefore, constructs containing genes encoding type 1 RIP (saporin), type 2 RIP (ricin) and chromosome-binding peptide (lunasin) have the potential to suppress cancer cells (Table 1). Expression of genes encoding these toxic proteins in cancer cells leads to the disruption of important cell processes and induces cell death. It should be noted that toxin genes, in particular RIP genes, act in a cell cycle independent way, therefore they can target quiescent and rapidly dividing tumour cells [60]. Importantly,

E.M. Glinka / Cancer Epidemiology 37 (2013) 1014–1019

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Table 1 Constructs expressing genes encoding plant cytotoxic proteins. Name of construct

Promoter

Anticancer agent

Cell lines which showed growth inhibition after transfection with construct

Target

References

Retro-1.3MBPp-ri-toxinb

Ricin

Human glioblastoma cell line U-373-MG

Glioblastoma

56

AdGFP-RTBa and RTA

MBPp (thyroid hormone (T3) regulatable promoter of the rat myelin basic protein) CMV (human cytomegalovirus)

Ricin

HEK293; human cervical carcinoma cell line HeLa, human liver cancer cell lines SMMC7721, and HL7702

Various cancers

57

pcDNA3-ricin pCI-SAP

CMV CMV

Ricin Saporin

Various cancers Various cancers

58 60, 61

lunasin pEGFP-C1

CMV

Lunasin

HeLa B16F1 murine melanoma cell line; African green monkey kidney Vero cell line Human breast cancer cells MCF-7; murine embryo fibroblast cells C3H 10T1/2; murine hepatoma Hepa 1c1c7

Various cancers

63

a b

Adenoviral gene delivery. Retroviral gene delivery.

significant reduction of tumour size by constructs bearing genes encoding ricin A chain and saporin has already been shown. Few constructs harbouring genes of plant cytotoxic proteins have been generated, as compared with the number of vectors exploiting genes of other toxic proteins, such as the diphtheria toxin and the Pseudomonas exotoxin A genes. These are the most commonly used toxins for the production of cancer gene therapy vectors and have shown great potential in preclinical and clinical trials. However, different factors may influence the effectiveness of a therapeutic gene construct on the suppression of cancer growth upon their introduction. For example, comparison of the efficacy of the ricin toxin construct with that of Pseudomonas aeruginosa exotoxin A revealed that the ricin toxin was less potent than Pseudomonas aeruginosa exotoxin A, which had been cloned with the eukaryotic receptor recognition domain. These results suggest that the success of this gene therapy system in vivo may depend, in part, on the choice of the toxic gene as well as the orientation of the gene in the retroviral construct [56]. 2.2. Suicide gene therapy systems The suicide gene therapy approach relies upon the intracellular conversion of a relatively nontoxic prodrug into a toxic drug by an enzyme of xenobiotic origin [64–66]. Although suicide gene therapy has been successfully used in a large number of in vitro and in vivo studies, its application to cancer patients has not reached the desirable clinical significance. However, recent reports on pre-clinical cancer models demonstrate the great potential of this method when used in combination with new therapeutic approaches [67]. The success of suicide gene therapy relies on the comprehensive catalytic activity of the enzyme encoded by the suicide gene, the targeted gene delivery system, a suitable prodrug with adequate access into the tumour, sufficient transgene expression, and an efficient bystander effect [68]. One strategy for cancer gene therapy that has been developed utilises the linamarase/linamarin (lis/lin) suicide gene therapy system [21]. Both linamarase and linamarin are found in the cassava plant Manihot esculenta Crantz. Linamarase, a b-glucosidase, is capable of hydrolysing the cyanogenic glucoside substrate, linamarin, into glucose, acetone and hydrogen cyanide [69]. Cyanide inhibits the cytochrome c oxidase of the mitochondrial respiratory chain, causing the block of oxidative phosphorylation [70,71]. The cyanide ion can diffuse freely and hydrogen cyanide is a gas; therefore, cyanide release is toxic to the neighbours of a linamarase-containing cell. As a result, the mode of cyanide action does not require cell-to-cell contact or gap junctions for its bystander effect. Thus, the type of bystander

effect in this system differs from the more commonly described induction of cell death of adjacent unmodified cells, which is dependent on connexin expression and cell communication via gap junctions. This retroviral construct carries the (pLlinSp) linamarase gene. The use of a retrovirus in brain cancer gene therapy has the advantage of targeting only dividing cells in a quiescent background of neurons. It has been demonstrated that transduction of mouse, rat and human cells using retroviral vectors that express the linamarase gene drastically increases the sensitivity of these cells to the cytotoxic effects of linamarin in vitro. Neighbouring cells may potentially be affected by the cyanide released, thereby amplifying the killing effect. The sensitivity of the system was also evaluated in vivo using an intracerebral tumour model. It was shown that the system could eradicate very large intracerebral gliomas in vivo, aided by a cyanide bystander effect [21]. Additionally, it was shown that the bystander effect associated with the lis/lin gene system is mediated by the production of the cyanide ion. A population of rat glioblastoma (C6) cells that carried and regularly expressed the linamarase gene (C6lis) was obtained using the retoviral plasmid pLlinSp. C6lis cells, mixed with naive C6 cells and exposed to linamarin, induced generalised cell death. As few as 10% C6lispositive cells were sufficient to eliminate the entire cell culture in 96 h. This type of bystander effect, which is not dependent on gapjunctional communication, provides an advantage in the treatment of malignant gliomas because gap junctions are rarely observed in the membranes of higher-grade gliomas. Moreover, it was shown that this bystander mechanism does not preferentially kill toxic metabolite producer cells compared with bystander cells, thus allowing for the production of sufficient cyanide to cause tumour regression [72]. Suicide gene therapy to treat malignant gliomas, using the lis/lin gene system, has been improved. It was shown that the combination of lis/lin with the nontoxic level of Aspergillus niger glucose oxidase (GO) accelerated cell death and enhanced the therapeutic potential. GO produces hydrogen peroxide, which can diffuse freely through the cellular membranes, inducing oxidative damage and increasing cellular stress. A canine glioblastoma cell line (Wodinsky and Walker cell line) stably transfected with a plasmid carrying linamarase gene was shown to be sensitive to linamarin; cell death was accompanied by mitochondrial fission and ATP depletion. Cell death occurred 48 h earlier than in absence of GO. It was shown that adenoviral vector (adenolis) containing the linamarase gene could eliminate the entire cell culture in 96 h, confirming the potent bystander effect associated with the system, affecting not only the enzyme producer cells but also their neighbouring ones. It was shown that the lis/lin/GO treatment is also very efficient in vivo against canine malignant brain tumours,

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Table 2 Constructs for suicide gene therapy systems (lis/lin and toTK1/AZT combinations). Name of construct

Promoter

Enzyme/prodrug combination

Cell lines which showed growth inhibition after transfection with construct

Target

References

pLlinSpb

5’ LTR (long terminal repeat) promoter

lis/lin

Intracerebral gliomas

21

pLlinSpb adenolisa adenolisa

5’ LTR CMV CMV

lis/lin lis/lin lis/lin

Glioblastoma Glioma Malignant tumours

72 73 74

pWW315c

PhEF1a (human elongation factor 1a promoter)

lis/lin

Malignant tumours

76

Ad-lisa

CMV

lis/lin

Hepatocellular carcinoma

77

ZG59b

CMV

toTK1/AZT

Rat glioblastoma cell lines C6 and L9; human glioblastoma U373MG; human glioma Hs683 Rat glioblastoma C6 cells Wodinsky and Walker cell line (W&W) Human glioma cell line U-87 MG; human breast cancer cell line MCF7 MCF7 cells; human fibrosarcoma HT-1080 cells; Chinese hamster ovary cells CHO-K1; mouse mammary gland cells 4T1 Human hepatocellular carcinoma cell lines HepG2 and HuH-7 Human glioblastoma cell line U87MG

Malignant gliomas

68

a b c

Adenoviral gene delivery. Retroviral gene delivery. Protein-transducing lentiviral nanoparticles.

where death is mediated by autophagy. It was surmised that the mitochondrial fragmentation observed is primarily due to the block of oxidative phosphorylation caused by the cyanide produced by the lis/lin system and that the presence of GO increases swelling and clusters the mitochondria to a perinuclear pattern. The lis/lin system induced necrosis [73]. In addition, the efficacy of the lis/lin/GO treatment was evaluated in vivo by using two xenograft models: the human glioma cell line U-87 MG and the human breast cancer cell line MCF7, genetically modified to express the lis gene (MCF7lis). A significant reduction of the treated tumour compared to the untreated one was clearly observed in MCF7lis. Furthermore, the potential of adenolis has been studied in animals inoculated with U-87 MG cells. It was found that cyanide production in situ, combined with oxidative stress, acts as a potent mitophagy inducer that severely reduces human tumour growth in xenotransplanted models [74]. It was noted that any successful in vitro procedure requires a satisfactory performance in an animal model before reaching a clinical trial. When the entire system was reproduced in the brain, new aspects of the complex process of tumour growth and drug delivery emerged [75]. The pWW315 plasmid, containing the linamarase gene, was used for the production of protein-transducing lentiviral nanoparticles (PTNs) to deliver linamarase into tumour cell lines. PTNs delivering linamarase into rodent or human tumour cell lines resulted in spheroids that mediated the hydrolysis of linamarin to cyanide and induced efficient cell death. Following linamarin injection into nude mice, linamarase-transducing nanoparticles perturbed solid tumour development via the bystander effect of cyanide. With the demonstration of the production and functional delivery of the lis/lin prodrug system to eliminate tumour cells both in vitro and in vivo, generic protein transduction systems have the potential to provide affordable, safe and efficient medicines in future [76]. The lis/lin system may provide a potential strategy for hepatocellular carcinoma (HCC) treatment. Recombinant adenovirus Ad-lis (pAd/CMV/V5-DEST) carrying linamarase cDNA was constructed to provide a treatment for HCC using gene-directed enzyme prodrug therapy. Application of linamarin resulted in the effective killing of HepG2 and HuH-7 cells infected with Ad-lis, but not cells infected with Ad-EGFP. It was shown that necrosis is the major mechanism of cell death observed in response to Ad-lis/lin treatment. Indeed, in a mixture of cells containing only 10% HepG2/lis cells, most cells were killed, demonstrating the powerful bystander effect of this system. Administration of Ad-lis and

linamarin directly into the HepG2 tumour foci resulted in suppression of tumour growth relative to the control group [77]. Plant thymidine kinase 1 from the tomato (Lycopersicon esculentum) plant (toTK1) was used in suicide gene-prodrug system. ToTK1 is highly specific to the nucleoside analogue prodrug zidovudine (azidothymidine, AZT), which is known to penetrate the blood–brain barrier. An important feature of toTK1 is that it efficiently phosphorylates its substrate AZT not only to AZT monophosphate but also to AZT diphosphate. The retrovirus vector ZG59 expressing toTK1 under the control of the CMV promoter was constructed. Transduction of U87MG cells with toTK1 and HSV-tk was performed using the retroviral vectors. U87MG cells transduced with toTK1 or HSV-tk were treated with AZT or GCV, respectively. Treatment with toTK1 dramatically increased the sensitivity of the human glioblastoma (GBM) cells to AZT, whereas HSV-tk increased the sensitivity to GCV modestly. In vitro testing of toTK1/AZT showed that the toTK1/AZT combination eradicated GBM cells efficiently and was found to be superior to the HSV-tk/ GCV system. Moreover, 10%–20% of toTK1-expressing cells in the culture were sufficient to exert a substantial bystander effect upon exposure to AZT. In addition, when neural progenitor cells were used as delivery vectors for toTK1 in intracranial GBM xenografts in nude rats, substantial attenuation of tumour growth was achieved in animals exposed to AZT, and survival of the animals was significantly improved compared with controls [68]. Information on the suicide gene therapy systems is summarised in Table 2. The lis/lin and toTK1/AZT systems have advantages over the well-known HSV-TK/GCV system. Unlike the HSV-TK/GCV system, the lis/lin system produces cyanide ion that diffuses freely across membranes. To have an effect, cyanide action does not require cell-to-cell contact or gap junctions for its bystander effect. This type of bystander effect offers an advantage for the treatment of malignant gliomas. Similarly, it was shown that the adenovirus-mediated lis/lin suicide system might provide a feasible and effective form of treatment for HCC. The toTK1/AZT system exhibits specificity, unlike the HSV-TK/GCV system, as a result of the ability of AZT to penetrate the blood– brain barrier better than GCV. Additional evidence of the promise of incorporating genes encoding anticancer plant proteins in gene therapy vectors includes the enzyme/prodrug combination, consisting of horseradish peroxidase (HRP) and the plant hormone indole-3-acetic acid (IAA) [78–87], which is not considered in this review. Interestingly, the HRP/IAA-induced cell death was effective in both normoxic and anoxic conditions. This finding could suggest a therapeutic advantage because hypoxia is common to solid tumours and presents an adverse

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E.M. Glinka / Cancer Epidemiology 37 (2013) 1014–1019

prognostic indicator. Importantly, the activated drug is able to cross cell membranes, and cell-to cell contact is not required for a bystander effect to take place [79]. 3. Conclusion Various gene therapy approaches have been developed and some of them hold great promise. They include the development of viral and non-viral methods of therapeutic gene delivery to target cells, the creation of suitable promoters, and the identification of new genes with anticancer properties. Presently, significant progress has been made towards the development of a strategy to efficiently deliver therapeutic genes to target cells and to achieve controlled, high-level expression of therapeutic genes to selectively inhibit tumour growth without harming normal cells. Success was also obtained in the use of various anticancer genes as therapeutic tools to specifically kill cancer cells, arrest angiogenesis, and suppress the growth of different tumours. However, the identification of new genes encoding therapeutic proteins and new targets is still essential. Historically, genes encoding proteins from bacteria have been used most often in the creation of constructs over genes encoding proteins from plants. While few have been created with genes from plants, in some cases, they have shown greater potential than genes of proteins from other organisms. These particular constructs contain both genes encoding cytotoxic proteins and genes encoding enzymes activating prodrugs. Fewer constructs containing plant toxic genes have been created in comparison to constructs containing genes of prodrug-activating enzymes. However, constructs containing genes of RIPs types 1 (saporin), type 2 (ricin), and chromatin-binding peptide (lunasin) have shown promising results. Sometimes GDEPT-bearing plant protein genes have advantages over the well-known HSV-TK/GCV system. Thus, the identification of new genes that code for therapeutic proteins is an important and promising cancer gene therapy approach, offering the possibility of increasing the efficacy of this strategy. Including genes encoding plant proteins within constructs for the suppression of cancer cells may permit the broadening of cancer gene therapy strategy, particularly because of the specific mode of action of anticancer plant proteins. Conflict of interest I wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. I confirm that there are no other persons who satisfied the criteria for authorship but are not listed. Acknowledgments The author would like to thank the library workers of IBCH RAS (Moscow, Russia) for their kindness. This study was supported in part by the Russian Foundation for Basic Researches (Grant 06-0800556_a). References [1] Maxwell IF, Maxwell F, Glode LM. Regulated expression of a diphtheria toxin A-chain gene transfected into human cells: possible strategy for inducing cancer cell suicide. Cancer Res 1986;46(9):4660–4. [2] Robson T, Hirst DG. Transcriptional targeting in cancer gene therapy. J Biomed Biotechnol 2003;2003(2):110–37. [3] Seth P. Vector-mediated cancer gene therapy: an overview. Cancer Biol Ther 2005;4(5):512–7. [4] Dorer DE, Nettelbeck DM. Targeting cancer by transcriptional control in cancer gene therapy and viral oncolysis. Adv Drug Deliv Rev 2009;61(7–8):554–71.

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