Date Published: April , 2010
Publisher: A.I. Gordeyev
Author(s): O.A. Patutina, N.L. Mironova, E.I. Ryabchikova, N.A. Popova, V.P. Nikolin, V.I. Kaledin, V.V. Vlassov, M.A. Zenkova.
In our work the antitumor and antimetastatic activities of RNase A and DNase I were studied
using two murine models of pulmonary (Lewis lung carcinoma) and liver (hepatoma A–1)
metastases. We found that intramuscular administration of RNase A at the dose range of
0.1–50 µ g/kg retarded the primary tumor growth by 20–40%, and this effect
disappeared with the increase in RNase A dose over 0.5 mg/kg. DNase I showed no effect on the
primary tumor growth. The intramuscular administration of RNase A (0.35–7 µ g/kg) or
DNase I (0.02–2.3 mg/kg) resulted in a considerable decrease in the metastasis number
into the lungs of animals with Lewis lung carcinoma and a decrease of the hepatic index of
animals with hepatoma 1A. A histological analysis of the organs occupied by metastases revealed
that the administration of RNase A and DNase I induced metastasis pathomorphism as manifested
by the destruction of oncocytes, an increase in necrosis and apoptosis foci in metastases, and
mononuclear infiltration. Our data indicated that RNase A and DNase I are highly promising as
supplementary therapeutics for the treatment of metastasizing tumors.
Recent data on the implication of small noncoding RNAs in tumorigenesis [1–3] and tumor–derived DNAs
in metastasis progression (genometastasis hypothesis) 
gave a new initiative to the study of enzymes cleaving nucleic acids as potential antitumor and
RNase A (mol. wt 13,700) and DNase I (2.155 kU/mg) from bovine pancreas were
purchased from Sigma (United States); [ γ
–32P]ATP) (3,000 Ci/mmole) was purchased from Biosan (Russia), and T4
polynucleotide kinase was purchased from Fermentas (Lithuania). The pHIV–2 plasmid was
kindly provided by Prof. Hans J. Gross (University of Wuerzburg, Wuerzburg, Germany).
Choice of Dose Ranges for RNase A and DNase I Used in Experiments In
Vivo. Since the enzymatic activities of RNase A and DNase I were assumed to be
essential for the antitumor effect of these enzymes, concentrations which provide a 50%
cleavage of substrates in a relatively short time were determined in experiments in
Intramuscular administration of RNase A to LLC–bearing C57Bl/6J mice. The
effect of RNase A on the primary tumor growth was examined in experiments with
LLC–bearing C57Bl/6J mice. On day 4 after tumor transplantation, the animals began
receiving daily intramuscular injections of a saline (control) or RNase solution ranging in
concentration from 0.1 µg to 10 mg per kg of body weight (experiment).
The antimetastatic activities of RNase A and DNase I (their capability to decrease the number
of metastases in target organs) were estimated from (1) a histological analysis of target
organs (the lungs for LLC and liver for HA–1), (2) a
microscopic examination of the metastasis number in the lungs of LLC–bearing animals, and
(3) the liver weight alteration (hepatic index) in animals with HA–1.
Metastasis formation in the pulmonary tissue is a characteristic feature of LLC. Distinct
metastases and multiple groups of tumor cells were observed in the lungs of the control mice
(Fig. 2A1, 2A2).
Metastases of different sizes and irregular shapes were predominantly localized in the
subpleural area. Some signs of mononuclear infiltration were observed in large metastases
extending over several bronchi and large vessels (Fig.
2A1). Surface metastases were composed of two or three layers of tumor cells expanding
along the pleura.
The administration of RNase A or DNase I to animals
with LLC induced dystrophic changes in metastases in the lungs (Fig. 2B). The morphologic parameters of these changes were
identical in all groups irrespective of the dose: an increase in the number of necroses and
apoptoses, a dystrophic transformation of oncocytes, and a considerable mononuclear
infiltration of tumor extravasates and metastases (Fig.
A microscopic examination of metastases on the surface of the LLC–bearing mouse lungs
has shown that treating these animals with enzymes leads to a significant decrease in the
metastasis number. The average number of metastases in groups of LLC–bearing mice treated
with RNase A at doses of 0.5 µg/kg, 0.7 µg/kg, and 10 mg/kg were 14 ± 3, 15 ± 4, and 18 ± 4,
respectively. The average number of metastases in groups of LLC–bearing mice treated with
DNase I at doses of 0.02, 0.12, and 2.3 mg/kg were 10 ± 4, 16 ± 7, and 18 ± 4, respectively,
whereas in the untreated animal group this amount was 30 ± 5. Thus, the observed amount of
metastases in groups of LLC–bearing mice treated with the enzymes was two– to
threefold less than in the control.
The diffuse boundaries of metastatic foci in hepatic parenchyma made it impossible to use
microscopy for counting metastases in the liver of animals bearing HA–1. Since the liver
increases in weight during the metastasis development, we used the hepatic index (HI)
reflecting disease severity and calculated as HI = (liver weight/body weight) × 100% to
estimate the antimetastatic effects of the enzymes: the relative HI reduction in a group of
treated animals compared to the control group served as the criterion of the therapeutic
efficacy (TE). The data on the average liver increment (ALI) of animals
with HA–1 compared to that of the healthy ones were used to estimate TE
(Table 1). A noticeable decrease in HI in HA–1–bearing
animals treated with the enzymes was observed relatively to the control. The TE value varied from 30% to 42% in
HA–1–bearing animals treated with RNase A and from 40% to 53% in those treated with DNase I.
As was mentioned in the Introduction, the largest representative of the RNase A family,
pancreatic RNase A, demonstrated weak antitumor activity at high doses (above 10 mg/kg) [14, 15] and DNase I was
capable of metastasis growth suppression [21, 22].
We have shown that the intramuscular administration of RNase A or DNase I has a systemic
effect on malignant tumors, which is manifested as a retardation of tumor growth (RNase A), a
decrease in the amount and area of metastases, and destructive changes in metastatic foci (both
enzymes). The most effective antimetastatic doses of the enzymes had no toxic effect on
animals. Our data make it possible to recommend using RNase A and DNase I in the supplementary
therapy of metastasizing tumors.