Cancer Epidemiology 37 (2013) 1014–1019

Eukaryotic expression vectors containing genes encoding plant proteins for killingof 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

Article history:

Received 26 June 2013

Received in revised form 10 September 2013

Accepted 20 September 2013

Available online 21 October 2013

Keywords:

Cell death

Cancer gene therapy

Toxicity

Toxic proteins

Cancer

Enzyme/prodrug combination

Plant proteins

Gene expression

Therapeutic genes

A B S T R A C T

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.

� 2013 Elsevier Ltd. All rights reserved.

Contents lists available at ScienceDirect

Cancer EpidemiologyThe International Journal of Cancer Epidemiology, Detection, and Prevention

jou r nal h o mep age: w ww.c an cer ep idem io log y.n et

1. Introduction

Cancer is one of the most dangerous diseases; it causes peoplegreat suffering and often leads to death. Despite substantialprogress that has been made towards understanding the molecularbasis, diagnosis, and treatment of cancer, it remains a major healthconcern. Cancer gene therapy strategy allows for the specific andefficient targeting of cancer cells without harming healthy cells. Atthe end of the last century, cancer gene therapy actively emergedas a potential therapeutic regimen; presently, it offers one of themost attractive and promising strategies for the suppression ofcancer cells and the eradication of malignant tumours [1–6].Different strategies have been used to increase the potency of thisapproach to cancer treatment. These methods may include refiningviral and non-viral methods of delivery of therapeutic genes tocancer cells and improving tissue/tumour specific promoters.Additionally, the identification of new genes that encodetherapeutic proteins is necessary. Presently, genes encodingproteins 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: em_glinka@mail.ru

1877-7821/$ – see front matter � 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 useof the controlled expression of an exogenous gene encoding a toxinto eliminate cancer cells was described in 1986 [1]. In this sameperiod, enzyme-activating prodrug therapy was suggested [12].Later, this approach became known as gene-directed enzymeprodrug therapy (GDEPT) or virus-directed enzyme prodrugtherapy (VDEPT) [13]. The most popular enzyme-activatingprodrug system is the herpes simplex virus thymidine kinase/ganciclovir (HSV-TK/GCV) system [14]. This treatment combina-tion has entered clinical trials, in which both its safety and itspartial efficacy have been demonstrated [15–20].

In 1998, plant genes began to be used in the engineering ofcancer gene therapy constructs [21]. Historically, many biologi-cally active substances derived from plants have been utilised infolk medicine for cancer treatment. In the last 50 years of the pastcentury, plant extracts began to be actively exploited for cancerchemotherapy [22,23]. Currently, extracts from different plants areboth undergoing preclinical and clinical studies [24–27]. In the1980s, plant proteins, such as ricin, which is a ribosome-inactivating protein (RIP), entered into clinical trials [28]. Otherpromising approaches have included the use of plant-derivedanticancer proteins in the design of immunotoxins. For example,RIPs, such as ricin A, saporin, bouganin, gelonin, momordin andothers, have been used to produce immunotoxins [29–33]. RIP-containing conjugates have been used in many experimental

E.M. Glinka / Cancer Epidemiology 37 (2013) 1014–1019 1015

strategies to target cancer cells, often showing great efficacy inclinical trials [34,35]. Many new plant proteins that have beentested for their ability to kill cancer cells showed cytotoxic effects,including the following: nucleolytic proteins from Corydalis cavatubers [36], potato (Solanum tuberosum) aspartic proteases [37],Solanum nigrum Linn (SNL) glycoprotein [38], and pectinesteraseinhibitor from jelly fig (Ficus awkeotsang Makino) [39]. At thepresent time, identification of new therapeutic genes is under way.The therapeutic genes are selected by taking into consideration themolecular biology of the cancer and the complex interactionsbetween tumour cells and the immune system. The choice oftherapeutic transgenes and gene therapy strategies are rapidlyevolving with advances in the identification of new genes and newtargets, and the improvement of vectors and expression systems.The choice of the transgene usually determines the therapeuticstrategy [40]. Use of genes from plant proteins in cancer genetherapy may open new possibilities for the development ofdifferent therapy strategies, as there are many plant proteins withvarious modes of actions that are able to suppress the growth ofcancer cells. Genes of some plant proteins with anticancerproperties are currently used in cancer gene therapy constructs.

This review summarises the data on the available eukaryoticexpression vectors that contain genes encoding plant proteins forthe selective elimination of cancer cells, which use two specificapproaches: the induction of cell death after the expression ofgenes encoding toxic proteins, and the induction of cell death afterthe expression of genes encoding enzymes that convert a nontoxicprodrug into a toxic drug. Constructs containing genes of ricin,saporin, lunasin, linamarase, tomato thymidine kinase1 areconsidered.

2. Vectors containing genes encoding toxic proteins and genesencoding enzymes activating prodrugs

2.1. Expression of ribosome-inactivating proteins and chromatin-

binding peptide

Ribosome-inactivating proteins (RIPs) are enzymes that belongto the polynucleotide adenosine glycosidase class of plant enzymes[41–44]. RIPs depurinate large ribosomal RNA that result indamage to the ribosome in an irreversible manner, which leads tothe inability of the ribosome to bind elongation factor-2, causingthe arrest of protein synthesis and eventually cell death [45–48].RIPs are divided into two main types: type 1 RIPs, which are single-chain proteins, and type 2 RIPs, which are proteins consisting of anactive A chain, similar to the type 1 RIP, linked to a B chain withlectin properties [29,31,34,49,50]. Some RIPs are potent toxins. Ithas been shown that there are differences in the cytotoxicity ofRIPs and, consequently, in their toxicity within animals. RIPs causeapoptotic and necrotic lesions and induce production of cytokines,causing inflammation [31].

Ricin, which comes from the seeds of the castor oil plant Ricinuscommunis, is a highly toxic protein classified as a type 2 RIP [51–55]. It has been used in cancer gene therapy constructs. A retroviralconstruct (retro-1.3MBPp-ri-toxin), containing the A chain of thericin gene under the thyroid hormone (T3) regulatable promoter ofthe rat myelin basic protein (MBPp), was created for glioma-specific transcription initiation. It is worth noting that retrovirusesare ideally suited for gene therapy of malignant gliomas in the CNS,where the normal cells are already mature and are not activelydividing. Consequently, glioma cells can be specifically targetedwith a recombinant retrovirus, as rapidly growing normal cells arerare in the adult CNS. A 50% reduction in the incorporation of [3H]thymidine into DNA was observed after T3 treatment in the humanglioblastoma cell line U-373-MG infected with the retro-1.3MBPp-ri-toxin. In vivo, retroviruses bearing the toxin gene are capable of

eradicating experimentally induced brain tumours in Wistar rats.It is also noteworthy that rejection of tumours is more efficient inthe brain of immunocompetent Wistar rats than in SCID miceflanks. For this reason an immunomediated bystander effect wassuggested in glioblastoma treated by retro-1.3MBPp-ri-toxinconstruct [56]. Developed as an approach for the suppression ofcancer cells, the strategy employed the co-application of the ricin Achain with the recombinant adenovirus expressing the ricin Bchain. The ricin A chain (RTA) was expressed in Escherichia coli inthe form of a 6XHis-tagged fusion protein and purified. Addition-ally, a replication-deficient ricin B chain (RTB)-expression adeno-virus green fluorescence protein (AdGFP-RTB) was constructed.RTA and AdGFP-RTB were tested for cytotoxicity either individu-ally or in combination in human cell lines HEK293, human cervicalcarcinoma cell line HeLa, and human liver cancer cell linesSMMC7721 and HL7702. Significant cell death or loss of cellviability was observed in all of the cell lines tested, when RTA andAdGFP-RTB were applied together, which resulted in approxi-mately 60% cell mortality [57]. Similarly, the ricin toxin A chaincDNA was cloned into the mammalian expression vector,pcDNA3.1, where gene expression is under the control of theconstitutive cytomegalovirus (CMV) promoter, to generate thepcDNA3-ricin construct. Transfection of HeLa cells with pcDNA3-ricin construct demonstrated that direct cytoplasmic expression ofricin in cells induced cell death [58].

Saporin, from the soapwort plant Saponaria officinalis, which issimilar to ricin, is a highly toxic protein. It is classified as a type 1RIP [35,59]. A plasmid (pCI-SAP) expressing the cytosolic saporin(SAP) gene was generated by placing the region encoding themature plant toxin under the control of the CMV promoter. Theability of the pCI-SAP to inhibit protein synthesis was tested incultured tumour cells co-transfected with a luciferase reportergene. SAP expression, when driven by the CMV promoter,demonstrated that only 10 ng of plasmid DNA per 1.6 � 104 B16melanoma cells was required to drastically reduce luciferasereporter activity to only 18% when compared to control cells.Furthermore, the effects of the pCI-SAP construct exceeded that ofthe pSfiSV19-SAP construct by 15- to 700-fold, where SAPexpression was driven by the SV40 promoter. Notably, the effectof transfecting pCI-SAP was particularly prominent in B16 cells.Direct intra-tumoural injections of pCI-SAP in B16 melanoma-bearing mice caused a significant suppression of tumour growth[60,61].

Lunasin, an acid peptide, is the antimitotic agent isolated fromsoybean (Glycine max) seeds. It is known for its anticancerproperties [62]. A chimeric construct (lunasin pEGFP-C1) encod-ing the small-subunit peptide of Gm2S-1 tagged with the greenfluorescent protein (GFP) has been generated. The Gm2S-1 cDNAencodes lunasin as a small- subunit component of posttransla-tionally processed 2S albumin. It was shown that this chimericgene GFP-lunasin arrested cell division, causing abnormal spindlefibre elongation, chromosomal fragmentation, and cell lysis whentransiently transfected into murine embryo fibroblast, murinehepatoma, and human breast cancer cells. Thus, the resultssuggested that the binding of lunasin to chromatin prevents thenormal formation of the kinetochore complex in the centromere,thereby leading to the disruption of mitosis and eventually to celldeath [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 cellsleads to the disruption of important cell processes and induces celldeath. It should be noted that toxin genes, in particular RIP genes,act in a cell cycle independent way, therefore they can targetquiescent and rapidly dividing tumour cells [60]. Importantly,

Table 1Constructs 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 MBPp (thyroid hormone

(T3) regulatable promoter

of the rat myelin basic protein)

Ricin Human glioblastoma cell line U-373-MG Glioblastoma 56

AdGFP-RTBa and RTA CMV (human cytomegalovirus) Ricin HEK293; human cervical carcinoma cell

line HeLa, human liver cancer cell lines

SMMC7721, and HL7702

Various cancers 57

pcDNA3-ricin CMV Ricin HeLa Various cancers 58

pCI-SAP CMV Saporin B16F1 murine melanoma cell line;

African green monkey kidney Vero cell line

Various cancers 60, 61

lunasin pEGFP-C1 CMV Lunasin Human breast cancer cells MCF-7;

murine embryo fibroblast cells C3H 10T1/2;

murine hepatoma Hepa 1c1c7

Various cancers 63

a Adenoviral gene delivery.b Retroviral gene delivery.

E.M. Glinka / Cancer Epidemiology 37 (2013) 1014–10191016

significant reduction of tumour size by constructs bearing genesencoding ricin A chain and saporin has already been shown. Fewconstructs harbouring genes of plant cytotoxic proteins have beengenerated, as compared with the number of vectors exploitinggenes of other toxic proteins, such as the diphtheria toxin and thePseudomonas exotoxin A genes. These are the most commonlyused toxins for the production of cancer gene therapy vectors andhave shown great potential in preclinical and clinical trials.However, different factors may influence the effectiveness of atherapeutic gene construct on the suppression of cancer growthupon their introduction. For example, comparison of the efficacy ofthe ricin toxin construct with that of Pseudomonas aeruginosaexotoxin A revealed that the ricin toxin was less potent thanPseudomonas aeruginosa exotoxin A, which had been cloned withthe eukaryotic receptor recognition domain. These results suggestthat the success of this gene therapy system in vivo may depend, inpart, on the choice of the toxic gene as well as the orientation of thegene in the retroviral construct [56].

2.2. Suicide gene therapy systems

The suicide gene therapy approach relies upon the intracellularconversion of a relatively nontoxic prodrug into a toxic drug by anenzyme of xenobiotic origin [64–66]. Although suicide genetherapy has been successfully used in a large number of in vitroand in vivo studies, its application to cancer patients has notreached the desirable clinical significance. However, recent reportson pre-clinical cancer models demonstrate the great potential ofthis method when used in combination with new therapeuticapproaches [67].

The success of suicide gene therapy relies on the comprehensivecatalytic activity of the enzyme encoded by the suicide gene, thetargeted gene delivery system, a suitable prodrug with adequateaccess into the tumour, sufficient transgene expression, and anefficient bystander effect [68]. One strategy for cancer genetherapy that has been developed utilises the linamarase/linamarin(lis/lin) suicide gene therapy system [21]. Both linamarase andlinamarin are found in the cassava plant Manihot esculenta Crantz.Linamarase, a b-glucosidase, is capable of hydrolysing thecyanogenic glucoside substrate, linamarin, into glucose, acetoneand hydrogen cyanide [69]. Cyanide inhibits the cytochrome coxidase of the mitochondrial respiratory chain, causing the block ofoxidative phosphorylation [70,71]. The cyanide ion can diffusefreely and hydrogen cyanide is a gas; therefore, cyanide release istoxic to the neighbours of a linamarase-containing cell. As a result,the mode of cyanide action does not require cell-to-cell contact orgap junctions for its bystander effect. Thus, the type of bystander

effect in this system differs from the more commonly describedinduction of cell death of adjacent unmodified cells, which isdependent on connexin expression and cell communication viagap junctions. This retroviral construct carries the (pLlinSp)linamarase gene. The use of a retrovirus in brain cancer genetherapy has the advantage of targeting only dividing cells in aquiescent background of neurons. It has been demonstrated thattransduction of mouse, rat and human cells using retroviral vectorsthat express the linamarase gene drastically increases thesensitivity of these cells to the cytotoxic effects of linamarin invitro. Neighbouring cells may potentially be affected by thecyanide released, thereby amplifying the killing effect. Thesensitivity of the system was also evaluated in vivo using anintracerebral tumour model. It was shown that the system coulderadicate very large intracerebral gliomas in vivo, aided by acyanide bystander effect [21]. Additionally, it was shown that thebystander effect associated with the lis/lin gene system ismediated by the production of the cyanide ion. A population ofrat glioblastoma (C6) cells that carried and regularly expressed thelinamarase gene (C6lis) was obtained using the retoviral plasmidpLlinSp. C6lis cells, mixed with naive C6 cells and exposed tolinamarin, induced generalised cell death. As few as 10% C6lis-positive cells were sufficient to eliminate the entire cell culture in96 h. This type of bystander effect, which is not dependent on gap-junctional communication, provides an advantage in the treatmentof malignant gliomas because gap junctions are rarely observed inthe membranes of higher-grade gliomas. Moreover, it was shownthat this bystander mechanism does not preferentially kill toxicmetabolite producer cells compared with bystander cells, thusallowing for the production of sufficient cyanide to cause tumourregression [72]. Suicide gene therapy to treat malignant gliomas,using the lis/lin gene system, has been improved. It was shown thatthe combination of lis/lin with the nontoxic level of Aspergillusniger glucose oxidase (GO) accelerated cell death and enhanced thetherapeutic potential. GO produces hydrogen peroxide, which candiffuse freely through the cellular membranes, inducing oxidativedamage and increasing cellular stress. A canine glioblastoma cellline (Wodinsky and Walker cell line) stably transfected with aplasmid carrying linamarase gene was shown to be sensitive tolinamarin; cell death was accompanied by mitochondrial fissionand ATP depletion. Cell death occurred 48 h earlier than in absenceof GO. It was shown that adenoviral vector (adenolis) containingthe 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 theirneighbouring ones. It was shown that the lis/lin/GO treatment isalso very efficient in vivo against canine malignant brain tumours,

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

Name of construct Promoter Enzyme/prodrug

combination

Cell lines which showed growth inhibi-

tion after transfection with construct

Target References

pLlinSpb 5’ LTR (long terminal

repeat) promoter

lis/lin Rat glioblastoma cell lines C6 and L9;

human glioblastoma U373MG;

human glioma Hs683

Intracerebral gliomas 21

pLlinSpb 5’ LTR lis/lin Rat glioblastoma C6 cells Glioblastoma 72

adenolisa CMV lis/lin Wodinsky and Walker cell line (W&W) Glioma 73

adenolisa CMV lis/lin Human glioma cell line U-87 MG;

human breast cancer cell line MCF7

Malignant tumours 74

pWW315c PhEF1a (human elongation

factor 1a promoter)

lis/lin MCF7 cells; human fibrosarcoma HT-1080 cells;

Chinese hamster ovary cells CHO-K1;

mouse mammary gland cells 4T1

Malignant tumours 76

Ad-lisa CMV lis/lin Human hepatocellular carcinoma cell

lines HepG2 and HuH-7

Hepatocellular carcinoma 77

ZG59b CMV toTK1/AZT Human glioblastoma cell line U87MG Malignant gliomas 68

a Adenoviral gene delivery.b Retroviral gene delivery.c Protein-transducing lentiviral nanoparticles.

E.M. Glinka / Cancer Epidemiology 37 (2013) 1014–1019 1017

where death is mediated by autophagy. It was surmised thatthe mitochondrial fragmentation observed is primarily due tothe block of oxidative phosphorylation caused by the cyanideproduced by the lis/lin system and that the presence of GOincreases swelling and clusters the mitochondria to a peri-nuclear pattern. The lis/lin system induced necrosis [73]. Inaddition, the efficacy of the lis/lin/GO treatment was evaluatedin vivo by using two xenograft models: the human glioma cellline U-87 MG and the human breast cancer cell line MCF7,genetically modified to express the lis gene (MCF7lis). Asignificant reduction of the treated tumour compared to theuntreated one was clearly observed in MCF7lis. Furthermore,the potential of adenolis has been studied in animalsinoculated with U-87 MG cells. It was found that cyanideproduction in situ, combined with oxidative stress, acts as apotent mitophagy inducer that severely reduces humantumour growth in xenotransplanted models [74]. It was notedthat any successful in vitro procedure requires a satisfactoryperformance in an animal model before reaching a clinical trial.When the entire system was reproduced in the brain, newaspects of the complex process of tumour growth and drugdelivery emerged [75].

The pWW315 plasmid, containing the linamarase gene, wasused for the production of protein-transducing lentiviral nano-particles (PTNs) to deliver linamarase into tumour cell lines. PTNsdelivering linamarase into rodent or human tumour cell linesresulted in spheroids that mediated the hydrolysis of linamarin tocyanide and induced efficient cell death. Following linamarininjection into nude mice, linamarase-transducing nanoparticlesperturbed solid tumour development via the bystander effect ofcyanide. With the demonstration of the production and functionaldelivery of the lis/lin prodrug system to eliminate tumour cellsboth in vitro and in vivo, generic protein transduction systemshave the potential to provide affordable, safe and efficientmedicines in future [76].

The lis/lin system may provide a potential strategy forhepatocellular carcinoma (HCC) treatment. Recombinant adeno-virus Ad-lis (pAd/CMV/V5-DEST) carrying linamarase cDNA wasconstructed to provide a treatment for HCC using gene-directedenzyme prodrug therapy. Application of linamarin resulted in theeffective killing of HepG2 and HuH-7 cells infected with Ad-lis, butnot cells infected with Ad-EGFP. It was shown that necrosis is themajor mechanism of cell death observed in response to Ad-lis/lintreatment. Indeed, in a mixture of cells containing only 10%HepG2/lis cells, most cells were killed, demonstrating the powerfulbystander effect of this system. Administration of Ad-lis and

linamarin directly into the HepG2 tumour foci resulted insuppression of tumour growth relative to the control group [77].

Plant thymidine kinase 1 from the tomato (Lycopersiconesculentum) plant (toTK1) was used in suicide gene-prodrugsystem. ToTK1 is highly specific to the nucleoside analogueprodrug zidovudine (azidothymidine, AZT), which is known topenetrate the blood–brain barrier. An important feature of toTK1 isthat it efficiently phosphorylates its substrate AZT not only to AZTmonophosphate but also to AZT diphosphate. The retrovirus vectorZG59 expressing toTK1 under the control of the CMV promoter wasconstructed. Transduction of U87MG cells with toTK1 and HSV-tkwas performed using the retroviral vectors. U87MG cellstransduced with toTK1 or HSV-tk were treated with AZT or GCV,respectively. Treatment with toTK1 dramatically increased thesensitivity of the human glioblastoma (GBM) cells to AZT, whereasHSV-tk increased the sensitivity to GCV modestly. In vitro testingof toTK1/AZT showed that the toTK1/AZT combination eradicatedGBM cells efficiently and was found to be superior to the HSV-tk/GCV system. Moreover, 10%–20% of toTK1-expressing cells in theculture were sufficient to exert a substantial bystander effect uponexposure to AZT. In addition, when neural progenitor cells wereused as delivery vectors for toTK1 in intracranial GBM xenograftsin nude rats, substantial attenuation of tumour growth wasachieved in animals exposed to AZT, and survival of the animalswas significantly improved compared with controls [68].

Information on the suicide gene therapy systems is sum-marised in Table 2. The lis/lin and toTK1/AZT systems haveadvantages over the well-known HSV-TK/GCV system. Unlike theHSV-TK/GCV system, the lis/lin system produces cyanide ion thatdiffuses freely across membranes. To have an effect, cyanideaction does not require cell-to-cell contact or gap junctions for itsbystander effect. This type of bystander effect offers an advantagefor the treatment of malignant gliomas. Similarly, it was shownthat the adenovirus-mediated lis/lin suicide system mightprovide a feasible and effective form of treatment for HCC. ThetoTK1/AZT system exhibits specificity, unlike the HSV-TK/GCVsystem, as a result of the ability of AZT to penetrate the blood–brain barrier better than GCV. Additional evidence of the promiseof incorporating genes encoding anticancer plant proteins in genetherapy vectors includes the enzyme/prodrug combination,consisting of horseradish peroxidase (HRP) and the planthormone indole-3-acetic acid (IAA) [78–87], which is notconsidered in this review. Interestingly, the HRP/IAA-inducedcell death was effective in both normoxic and anoxic conditions.This finding could suggest a therapeutic advantage becausehypoxia is common to solid tumours and presents an adverse

E.M. Glinka / Cancer Epidemiology 37 (2013) 1014–10191018

prognostic indicator. Importantly, the activated drug is able tocross cell membranes, and cell-to cell contact is not required for abystander effect to take place [79].

3. Conclusion

Various gene therapy approaches have been developed andsome of them hold great promise. They include the development ofviral and non-viral methods of therapeutic gene delivery to targetcells, the creation of suitable promoters, and the identification ofnew genes with anticancer properties. Presently, significantprogress has been made towards the development of a strategyto efficiently deliver therapeutic genes to target cells and toachieve controlled, high-level expression of therapeutic genes toselectively inhibit tumour growth without harming normal cells.Success was also obtained in the use of various anticancer genes astherapeutic tools to specifically kill cancer cells, arrest angiogene-sis, and suppress the growth of different tumours. However, theidentification of new genes encoding therapeutic proteins and newtargets is still essential. Historically, genes encoding proteins frombacteria have been used most often in the creation of constructsover genes encoding proteins from plants. While few have beencreated with genes from plants, in some cases, they have showngreater potential than genes of proteins from other organisms.These particular constructs contain both genes encoding cytotoxicproteins and genes encoding enzymes activating prodrugs. Fewerconstructs containing plant toxic genes have been created incomparison to constructs containing genes of prodrug-activatingenzymes. However, constructs containing genes of RIPs types 1(saporin), type 2 (ricin), and chromatin-binding peptide (lunasin)have shown promising results. Sometimes GDEPT-bearing plantprotein genes have advantages over the well-known HSV-TK/GCVsystem.

Thus, the identification of new genes that code for therapeuticproteins is an important and promising cancer gene therapyapproach, offering the possibility of increasing the efficacy of thisstrategy. Including genes encoding plant proteins within con-structs for the suppression of cancer cells may permit thebroadening of cancer gene therapy strategy, particularly becauseof the specific mode of action of anticancer plant proteins.

Conflict of interest

I wish to confirm that there are no known conflicts of interestassociated with this publication and there has been no significantfinancial support for this work that could have influenced itsoutcome.

I confirm that there are no other persons who satisfied thecriteria 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 inpart by the Russian Foundation for Basic Researches (Grant 06-08-00556_a).

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