Vasily I. Kaledin

Inhibition of metastasis development by daily administration of ultralow doses of RNase A and DNase I

Recent data on the involvement of miRNA and circulating tumor-derived DNA in regulation of tumorigenesis showed a great prospect for these molecules as a novel class of therapeutic targets and gave a new start for the study of enzymes cleaving nucleic acids as potential antitumor and antimetastatic agents. In the present paper using two murine tumor models with pulmonary or liver metastases we studied the antimetastatic potential of RNase A and DNase I and performed a search for possible molecular targets of the enzymes. Herein, we show for the first time that daily administration of ultralow doses of RNase A (0.5-50 μg/kg) and DNase I (0.02-2.3 mg/kg) inhibits the development of metastasis to 60-90% and RNase A exerts 30% retardation of tumor growth. Remarkably, the increase in RNase A dose from 50 μg/kg to 10mg/kg leads to a disappearance of antitumor and antimetastatic effects. Simultaneous treatment of tumor-bearing animals with RNase A and DNase I leads to an additive effect and results in almost total absence of metastases. The use of RNase A as an adjuvant in conjunction with conventional cytostatic cyclophosphamide results in a reliable enhancement of antitumor and antimetastatic effect of the therapy compared with the use of these agents individually. The search for possible molecular mechanism of antimetastatic effect of nucleases showed that daily administration of the enzymes reduced the pathologically increased level of extracellular nucleic acids and increased nuclease activity of the blood plasma of tumor-bearing mice back to the level of healthy animals. Thus, we unequivocally show that the proposed protocol of treatment of tumor-bearing animals with RNase A and DNase I has a general systemic and immunomodulatory effect, leads to a drastic suppression of metastasis development, and in perspective may become an effective component of intensive complex therapy of cancer.

Involvement of Transcription Factor HNF3γ in the Effect of o-Aminoazotoluene on Glucocorticoid Induction of Tyrosine Aminotransferase in Mice Sensitive to its Hepatocarcinogenic Action

In the rodent liver, hepatocarcinogens inhibit the glucocorticoid induction of several liver‐specific genes, including tyrosine aminotransferase (TAT). A distinct positive correlation exists in mice between the extent of inhibition of TAT induction after acute administration of o‐aminoazotoluene (OAT) and the frequency of liver tumors after chronic exposure to the carcinogen. To elucidate the mechanism of the carcinogenic action, the effects of OAT on the DNA‐binding activity of several transcription factors participating in the glucocorticoid regulation of TAT gene expression were studied. The experimental inbred male mice were sensitive (A/He and SWR/J, tumor induction frequency of 75–100%, TAT induction inhibition of 35–50%) and resistant (CC57BR/Mv and AKR/J, 0–6% and 10–15%, respectively) to OAT. Gel retardation experiments showed that hepatocyte nuclear factor 3 (HNF3)γ DNA‐binding activity was strongly reduced in nuclear extracts from the livers of OAT‐treated A/He and SWR/J mice but only slightly reduced in CC57Br/Mv and AKR/J mice. The DNA‐binding activities of Ets, AP1 family members, and GME binding proteins were unaffected. HNF3γ DNA‐binding activity was reduced by 1 h after OAT administration and remained low for 1 mo, as did inhibition of TAT induction in the liver. These results suggested that the inhibitory effect of OAT on the glucocorticoid induction of TAT is mediated by reduced HNF3γ DNA‐binding activity.

The siRNA targeted to mdr1b and mdr1a mRNAs in vivo sensitizes murine lymphosarcoma to chemotherapy

BackgroundOne of the main obstacles for successful cancer polychemotherapy is multiple drug resistance phenotype (MDR) acquired by tumor cells. Currently, RNA interference represents a perspective strategy to overcome MDR via silencing the genes involved in development of this deleterious phenotype (genes of ABC transporters, antiapoptotic genes, etc.).MethodsIn this study, we used the siRNAs targeted to mdr1b, mdr1a, and bcl-2 mRNAs to reverse the MDR of tumors and increase tumor sensitivity to chemotherapeutics. The therapy consisting in ex vivo or in vivo application of mdr1b/1a siRNA followed by cyclophosphamide administration was studied in the mice bearing RLS40 lymphosarcoma, displaying high resistance to a wide range of cytostatics.ResultsOur data show that a single application of mdr1b/1a siRNA followed by treatment with conventionally used cytostatics results in more than threefold decrease in tumor size as compared with the control animals receiving only cytostatics.ConclusionsIn perspective, mdr1b/1a siRNA may become a well-reasoned adjuvant tool in the therapy of MDR malignancies.

Species‐specific effects of the hepatocarcinogens 3′‐methyl‐4‐dimethyl‐aminoazobenzene and ortho‐aminoazotoluene in mouse and rat liver

The effects of rat‐specific hepatocarcinogen 3′‐methyl‐4‐dimethylaminoazobenzene (3′‐MeDAB), mouse‐specific hepatocarcinogen ortho‐aminoazotoluene (OAT), non‐species‐specific hepatocarcinogen diethylnitrosamine (DENA), and non‐carcinogenic 4′‐methyl‐4‐dimethylaminoazobenzene (4′‐MeDAB) on glucocorticoid induction of tyrosine aminotransferase (TAT) and DNA‐binding activity of hepatocyte nuclear factor 3 (HNF3) family of transcription factors were investigated with carcinogen‐susceptible and ‐resistant animals. Species‐specific hepatocarcinogens 3′‐MeDAB and OAT strongly inhibited glucocorticoid induction of TAT in the liver of susceptible but not resistant animals. DENA, which is highly carcinogenic for the liver of both rats and mice inhibited glucocorticoid induction of TAT in both species, while non‐carcinogenic 4′‐MeDAB was absolutely ineffective both in rats and mice. The inhibition of TAT activity by the carcinogens was due to reduced levels of TAT mRNA, which is most likely to be a result of the reduced rate of transcription initiation of the TAT gene. In all cases, the TAT inhibition was accompanied by significant reduction of DNA‐binding activity of the HNF3 transcription factor, which is known to be critical to glucocorticoid regulation of TAT gene. We also demonstrated that the described species‐specific effects of OAT and of 3′‐MeDAB on HNF3 DNA‐binding activity may be initiated not only by administration in vivo, but also by their direct administration to homogenate, intact nuclei or nuclear lysate, but not to nuclear extract fraction, obtained by precipitation with 0.32 g/mL of ammonium sulfate (Fraction I). We showed, that a factor responsible for this effect might be precipitated in 0.32–0.47g/mL interval of ammonium sulfate concentration. In contrast, non‐specific hepatocarcinogen DENA was effective upon being added directly to Fraction I, implying a different mechanism of its action.

A novel pro-apoptotic effector lactaptin inhibits tumor growth in mice models

Lactaptin, a human milk-derived protein, induces apoptosis in cultured tumor cells. We designed a recombinant analog of lactaptin (RL2) and tested its antitumor activity. The sensitivity of hepatocarcinoma A-1 (HA-1), Lewis lung carcinoma, and Ehrlich carcinoma to RL2 were tested to determine the most reliable in vitro animal model. HA-1 cells, which had the highest sensitivity to RL2, were transplanted into A/Sn mice to investigate RL2 antitumor activity in vivo. Investigation of the molecular effects of RL2 shows that RL2 induces apoptotic transformation of HA-1 cells in vitro: phosphatidylserine translocation from inner side of the lipid bilayer to the outer one and dissipation of the mitochondrial membrane potential. Repetitive injections of RL2 (5-50 mg/kg) for 3-5 days effectively inhibited ascites and solid tumor transplant growth when administered intravenously or intraperitoneally, without obvious side effects. The solid tumor inhibitory effect of RL2 (5 i.v. injections, cumulative dose 125 mg/kg) was comparable with that of cyclophosphamide at a therapeutic dose (5 i.v. injections, cumulative dose 150 mg/kg). In combination therapy with cyclophosphamide, RL2 had an additive antitumor effect for ascites-producing tumors. Histomorphometric analysis indicated a three-fold reduction of spontaneous metastases in the liver of RL2-treated mice with solid tumor transplants in comparison with control animals. Repeated RL2 treatment substantially prolonged the lifespan of mice with intravenously injected tumor cells. Recombinant analog of lactaptin effectively induced apoptosis of tumor cells in vitro and suppressed the growth of sensitive tumors and metastases in vivo.

Effects of o-aminoazotoluene on liver regeneration and p53 activation in mice susceptible and resistant to hepatocarcinogenesis

The susceptibility to hepatocellular carcinoma (HCC) varies greatly within human populations in response to environmental risk agents. The mechanisms underlying differential susceptibility are still largely unknown and need to be clarified to improve HCC chemoprevention and therapeutic treatment. Inbred rodent strains with established predispositions for hepatocarcinogenesis offer the opportunity to identify intrinsic susceptibility and resistance factors. Previously, we have characterized mouse strains showing differential susceptibility to o-aminoazotoluene (OAT) and established that susceptibility does not result from OAT metabolism or genotoxicity in the livers of resistant and susceptible mice. In this study we have found that OAT differently affects hepatocyte proliferation in mice after partial hepatectomy (PH). OAT inhibited hepatocyte proliferation by 60-80% in the livers of susceptible mice, whereas resistant mice showed less than 15% inhibition. The inhibition resulted in significant delay of hepatic mass recovery in susceptible mice. OAT induced p53 stabilization and transcriptional activation in response to carcinogen treatment to the same degree in both, susceptible and resistant mice. Taken together, our data support inhibition of hepatocyte proliferation as a major cause for increased mouse susceptibility to hepatocarcinogenesis, and acceleration of functional liver recovery may offer a way to increase resistance to hepatic neoplasms. These results may have relevance to clinical observations of HCCs and implications for HCC chemoprevention and treatment.

[Pulmonary carcinogenesis susceptibility-associated single-nucleotide polymorphisms in K-ras intron 2 affect the binding of factor Gata-6 but not gene expression].

The sequence of K-ras intron 2, which has been associated with lung tumor susceptibility in inbred mouse strains, was analyzed in susceptible strain GR and in resistant strains PT and UT. In the latter case, the intron had a tandem repeat of a 37-bp sequence with variant GC of its two single-nucleotide polymorphisms (SNPs), as earlier reported for resistant strains AKR, C57BL/c, and C3H/A. Strain GR did not differ in intron structure from susceptible strains A/He and ICR, having one copy of the 37-bp sequence with SNP variant CA. By gel retardation assay, a DNA probe corresponding to “susceptible” allele CA of K-ras region 278–307 formed an additional complex with nuclear proteins extracted from lungs, as compared with probes corresponding to the “resistant” GC and “intermediate” CC alleles. With specific antibodies, the protein binding to the susceptible allele was identified as transcription factor GATA-6. Reverse transcription with subsequent multiplex PCR did not reveal any significant difference in K-ras expression for susceptible and resistant strains. The results suggest that SNPs of K-ras intron 2 do not affect the level of K-ras expression but do control the binding of GATA-6, which plays an important role in lung differentiation.

The increased CAR-dependent metabolism of thyroid hormones in mice with high cancer susceptibility

AIM our aim was to compare activation of the constitutive androstane receptor (CAR), hepatic expression of its target genes, and the serum thyroid hormone levels in C3H/He, C57BL/6J, and CC57BR/Mv mice following phenobarbital treatment. These differences, if present, could help to explain the different susceptibility to phenobarbital-induced liver tumor promotion among these strains of mice. MAIN METHODS CAR DNA-binding activity and CAR content in nuclear protein extracts from mouse livers were assessed using the electrophoretic mobility shift assay and immunoblotting. Serum thyroid hormone concentrations were determined by radioimmunoassay. Real-time PCR was used to measure the hepatic expression level of CAR target genes. KEY FINDINGS we found a 2.3-fold increase of CAR DNA-binding activity in response to phenobarbital in the sensitive C3H/He mice, but no change in the relatively resistant C57BL/6J and CC57BR/Mv mice. Phenobarbital treatment caused a significant decrease in triiodothyronine and free thyroxine concentrations (17% and 40%, respectively) in the sensitive C3H/He mice by the end of 60-day treatment, while in the resistant mice, these changes were not observed. In the sensitive C3H/He mice only, the expression of a CAR target gene encoding sulfotransferase Sult2a1, the thyroid hormone inactivation enzyme, increased by 260-fold after phenobarbital administration. The expression of another CAR target gene, Mdm2, was also increased by phenobarbital treatment in C3H/He mice. SIGNIFICANCE we have shown that phenobarbital activates CAR and increases the expression of its target genes thereby accelerating the metabolism of thyroid hormones only in mice susceptible to liver tumor promotion by phenobarbital, but not in relatively resistant animals.

The mechanisms of endogenous control of hepatocyte proliferation are different in mice of two inbred strains (BALB/c and AKR).

Experiments on the fusion of stimulated fibroblasts of NIH 3T3 mice with hepatocytes obtained from an intact liver of BALB/c mice showed that the hepatocytes only slightly, if at all, suppress the proliferative activity of the fibroblast nuclei in the heterokaryons [1]. Conversely, hepatocytes obtained from a partially recovered liver of mice of the same strain (15 days after partial hepatectomy) suppress the proliferative activity of the fibroblast nuclei in the heterokaryons. A shortterm (for 2 h) preincubation of these hepatocytes with a protein-synthesis inhibitor, cycloheximide, completely eliminated their ability to suppress DNA synthesis in the fibroblast nuclei in the heterokaryons [2]. Based on these results, we assumed that the mechanism of suppression of the proliferative activity of the fibroblasts in a regenerating liver may be connected with the appearance of intracellular inhibitors of proliferation in these cells at the final stages of regeneration. In view of this, the endogenous inhibitors of hepatocyte proliferation should be identified. To solve this problem, we studied the mechanisms of hepatocarcinogenesis and attempted to determine the mechanisms of resistance of mice of different inbred strains to some chemical carcinogens. The basis of these experiments was as follows.

Tempol reduces the therapeutic effect of cyclophosphamide on an experimental tumour model

The nitroxyl radical 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) is reported to elicite some effects on different biological models. This paper studied the influence of Tempol on the therapeutic action of an alkylating agent cyclophosphamide (CP) in the transplantable murine lymphosarcoma LS. When administered exactly before CP, Tempol exerted no influence on efficacy of the tumour therapy but significantly reduced it under a single or multiple preliminary injections 1–3 days before CP. This regimen of Tempol administration is found to elevate the activity of aldehyde dehydrogenase in the liver, the enzyme which is known to reduce the yield of the activated metabolite(s) of CP.