Downregulation of VEGF-C expression in lung and colon cancer cells decelerates tumor growth and inhibits metastasis via multiple mechanisms.

Experimental and clinical studies positively correlate expression of vascular endothelial growth factor (VEGF)-C in cancer cells with accelerated tumor progression and/or unfavorable clinical outcome. However, many aspects of tumor-promoting activity of VEGF-C and consequences of its downregulation for tumor progression remain poorly understood. To clarify these points, we created a set of VEGF receptor 3-positive lung carcinoma A549 and colon carcinoma HCT116 cell sublines with stable repression of VEGF-C synthesis. Analysis of the behavior of these cells revealed multiple effects of VEGF-C downregulation, which, in addition to deceleration of cell proliferation and invasion in vitro and inhibition of lymphangiogenesis in tumor and surrounding tissues observed earlier, included previously undescribed effects, in particular, partial restoration of epithelial phenotype, reduction in the percentage of tumor-initiating cells (cancer stem cells) in the cell population and inhibition of metastasis of orthotopic lung cancer xenografts to other lung lobes. These results are consistent with the idea of high potentiality of VEGF-C as a cancer drug target.

p53 hot-spot mutants increase tumor vascularization via ROS-mediated activation of the HIF1/VEGF-A pathway.

The function of p53 tumor suppressor is often altered in various human tumors predominantly through missense-mutations resulting in accumulation of mutant proteins. We revealed that expression of p53 proteins with amino-acid substitutions at codons 175 (R175H), 248 (R248W), and 273 (R273H), representing the hot-spots of mutations in various human tumors, increased the number of vessels in HCT116 human colon carcinoma xenografts and, as a result, accelerated their growth. Stimulation of tumor angiogenesis was connected with about 2-fold increase in intracellular level of reactive oxygen species (ROS). Antioxidant N-acetyl-l-aspartate (NAC) decreased vessels number in tumors formed by cells with inactivated p53 and inhibited their growth. Effect of ROS on angiogenesis in tumors expressing hot-spot p53 mutants was correlated with their ability to increase a content of HIF1 transcriptional factor responsible for up-regulation of VEGF-A mRNAs.

Mitochondria-targeted plastoquinone derivatives as tools to interrupt execution of the aging program. 3. Inhibitory effect of SkQ1 on tumor development from p53-deficient cells.

It was proposed that increased level of mitochondrial reactive oxygen species (ROS), mediating execution of the aging program of an organism, could also be critical for neoplastic transformation and tumorigenesis. This proposal was addressed using new mitochondria-targeted antioxidant SkQ1 (10-(6'-plastoquinonyl) decyltriphenylphosphonium) that scavenges ROS in mitochondria at nanomolar concentrations. We found that diet supplementation with SkQ1 (5 nmol/kg per day) suppressed spontaneous development of tumors (predominantly lymphomas) in p53(-/-) mice. The same dose of SkQ1 inhibited the growth of human colon carcinoma HCT116/p53(-/-) xenografts in athymic mice. Growth of tumor xenografts of human HPV-16-associated cervical carcinoma SiHa was affected by SkQ1 only slightly, but survival of tumor-bearing animals was increased. It was also shown that SkQ1 inhibited the tumor cell proliferation, which was demonstrated for HCT116 p53(-/-) and SiHa cells in culture. Moreover, SkQ1 induced differentiation of various tumor cells in vitro. Coordinated SkQ1-initiated changes in cell shape, cytoskeleton organization, and E-cadherin-positive intercellular contacts were observed in epithelial tumor cells. In Ras- and SV40-transformed fibroblasts, SkQ1 was found to initiate reversal of morphological transformation of a malignant type, restoring actin stress fibers and focal adhesion contacts. SkQ1 suppressed angiogenesis in Matrigel implants, indicating that mitochondrial ROS could be important for tumor angiogenesis. This effect, however, was less pronounced in HCT116/p53(-/-) tumor xenografts. We have also shown that SkQ1 and related positively charged antioxidants are substrates of the P-glycoprotein multidrug resistance pump. The lower anti-tumor effect and decreased intracellular accumulation of SkQ1, found in the case of HCT116 xenografts bearing mutant forms of p53, could be related to a higher level of P-glycoprotein. The effects of traditional antioxidant N-acetyl-L-cysteine (NAC) on tumor growth and tumor cell phenotype were similar to the effects of SkQ1 but more than 1,000,000 times higher doses of NAC than those of SkQ1 were required. Extremely high efficiency of SkQ1, related to its accumulation in the mitochondrial membrane, indicates that mitochondrial ROS production is critical for tumorigenesis at least in some animal models.

[Progress in understanding moleculr mechanisms of oncogenesis, and novel methods of tumor growth control].

Malignant tumor develop from cells with distorted signaling pathways controlling proliferation, migration, viability, differentiation, and genome integrity, as well as their influence on microenvironment. Progress in understanding molecular mechanisms of such alterations has led to the elaboration of new methods of anti-tumor therapy based on the modulation of the activity of molecules playing a key role in tumor development (so-called "target therapy"). The paper describes basic mechanisms of the development of cell features determining malignant phenotype and new possibilities for its correction. In particular, recent finding concerning the role of reactive oxygen species in oncogenesis and anti-tumor therapy are considered.

[Genome instability and oncogenesis].

Molecular alterations leading to genetic instability play a key role in tumor development. The basic reasons of genetic instability of tumor cells, i.e. up-regulation of intracellular level of endogenous mutagens, in particular reactive oxygen spesies (ROS); decreased fidelity of DNA replication and chromosome segregation in mitosis; defects in DNA repair systems; and inactivation of cell cycle checkpoints preventing proliferation of abnormal cells are reviewed. In addition, tissue-specificity of tumorigenesis connected with genetic instability and development of new therapeutic approaches based on diminishing genetic instability or selective killing of neoplastic cells showing such defects are discussed.

Restoration of p53 tumor-suppressor activity in human tumor cells in vitro and in their xenografts in vivo by recombinant avian adenovirus CELO-p53.

Human adenovirus (Ad) vectors are extensively used as gene transfer vehicles. However, a serious obstacle for the use of these vectors in clinical applications is due to pre-existing immunity to human Ads affecting the efficacy of gene transfer. One of the approaches to circumvent host immune response could be the development of vectors based on non-human Ads that are able to transduce genes into human cells. In this study, we explored the possibility of using avian Ad CELO vectors as gene-transfer vehicles. For this purpose, we constructed a set of recombinant CELO viruses and demonstrated that they are able to deliver transgenes into various organs on the background of pre-existing immunity to human Ad5. The created CELO-p53 vector restored the function of the p53 tumor suppressor both in cultured human tumor cells in vitro and in their xenografts in nude mice in vivo. The latter effect was accompanied by inhibition of tumor growth. Noteworthily, the delivery of CELO-p53 led to activation of p53 target genes in cells showing inactivation of endogenous p53 by three different mechanisms, that is, in the human epidermoid carcinoma A431, lung adenocarcinoma H1299, and cervical carcinoma HeLa.

[The protective role of p53 in Ras-induced transformation of REF52 cells]. / Zashchitnaia funktsiia p53 pri ras-indutsirovannoi transformatsii kletok REF52.

A study was made of the effect of activated oncogene N-RAS on the function of tumor suppressor p53 and the proliferating ability of rat embryo fibroblasts REF52. The proliferation rate and the portion of S-phase cells increased in the first three days of N-RAS expression. After 5-7 days, the p53 function was enhanced, as manifest in increased p53 lifespan and nuclear content and induced transcription of p53-responsive genes. In particular, Cdk2 p21WAF1/CIP1, an inhibitor of cyclin-dependent kinase 2, was produced to a higher level and arrested the cell cycle in G1. Cells with abrogated or dramatically inhibited N-RAS expression were generated at this stage. Having a selective advantage, these cells gradually displaced N-RAS-expressing cells arrested in G1, so that one month after oncogene induction the culture mostly consisted of morphologically normal, actively proliferating Res-negative cells. Neither cell cycle arrest nor reversion to the normal phenotype were observed in N-RAS expressing cells devoid of the p53 function. Thus, p53 prevented stable N-RAS-induced transformation of REF52 cells, arresting the cell cycle and expediting revertant selection.

[Dominant-negative inactivation of p53: the effect of the proportion between trans-dominant inhibitor and its target]. / Dominantno-negativnaia inaktivatsiia p53: vliianie kolichestvennykh sootnoshenii transdominantnogo ingibitora i ego misheni.

Dominant-negative mutations of the p53 tumor suppressor gene and oligomerization of the mutant and wild-type p53 are considered responsible for functional inactivation of the p53 tetramer. Although dominant-negative inactivation of p53 is well reproducible in experimental systems, its contribution to processes occurring in tumor cells heterozygous at p53 is still unclear. To study the effect of dominant-negative inhibitor GSE22 on the p53 activity, cultures coexpressing GSE22 and tetracycline-suppressible p53 were derived from p53-negative cell lines. Transcriptional activity and expression of p53 proved to depend on the proportion between p53 and GSE22. The dominant-negative effect was observed only when GSE22 was in a multifold excess to p53. GSE22 was shown to be suitable for complete reversible inactivation of p53.

[Construction of chimeric tumor suppressor p53 resistant to the dominant-negative interaction with p53 mutants]. / Sozdanie khimernoi molekuly opukholevogo supressora p53, ustoichivoi k dominantno-negatvnym vzaimodeistviiam s mutantnymi formami belka p53.

A chimeric p53 cDNA was constructed so that the fragment coding for 39 residues of the chicken p53 tetramerization domain replaced the corresponding region of human p53. The chimeric cDNA substantially inhibited the colony-forming ability of transfected human and mouse cells, suggesting a suppressory potential for its product. The chimeric p53 activated promoters containing p53-responsive elements. In contrast to wild-type human p53, the chimeric p53 remained capable of transcription activation in the presence of dominant-negative mutant p53-His175. This makes the chimeric p53 a convenient model for elaborating gene therapy protocols for tumors with dominant-negative p53 forms. The chimeric p53 may be used to study the role of transdominance of p53 mutants in carcinogenesis and the interactions of p53 with related transcription factors (p73, p63).

p53 activation in response to microtubule disruption is mediated by integrin-Erk signaling.

The p53 tumor suppressor is activated in response to various stresses driving the cells into growth arrest or apoptosis. We have addressed the question of how disintegration of microtubule system induces activation of p53. Depolymerization of microtubules by colcemid in rat and human quiescent fibroblasts resulted in accumulation of transcriptionally active p53 that caused cell-cycle arrest at the G1/S boundary. The p53 activation correlated with prominent activation of Erk1/2 MAP kinases that resulted from colcemid-stimulated development of focal adhesions. Inhibition of focal contacts development by plating of cells onto poly-L-lysine abrogated both Erk1/2 and p53 activations in colcemid-treated cells, while plating of cells onto fibronectin caused transient up-regulation of p53 even in the absence of colcemid. Pre-treatment of cells with the specific MEK1 inhibitor PD098059 also attenuated colcemid-induced p53 activation and G1 cell cycle arrest. Cell types which either failed to develop focal adhesions in response to colcemid treatment (human MCF-7 epithelial cells), or lacked colcemid-induced sustained Erk activation (primary mouse embryo fibroblasts and 12(1) cells) showed virtually no p53 up-regulation in response to disruption of microtubules during G0/G1. Our results indicate that p53 activation is not triggered by disintegration of microtubule system by itself, but rather originates from some of the consequences of such disintegration, in particular, from the development of focal adhesions leading to activation of Erk signaling pathway.