toxicity of artificial ribonucleases
TRANSCRIPT
-
8/8/2019 Toxicity of Artificial Ribonucleases
1/29
A MECHANISM OF THE TOXICITYOF ARTIFICIAL RIBONUCLEASES
FOR HUMAN CANCER CELLS.
E. B. LOGASHENKO, I. L. KUZNETSOVA,
E. I. RYABCHIKOVA, V. V. VLASSOV AND
M. A. ZENKOVA
INSTITUTE OF CHEMICAL BIOLOGY AND FUNDAMENTAL
MEDICINE, PR. LAVRENTIEVA 8, NOVOSIBIRSK, 630090RUSSIA.
PUBLISHED IN BIOMEDITSINSKAYA KHIMIYA
-
8/8/2019 Toxicity of Artificial Ribonucleases
2/29
-
8/8/2019 Toxicity of Artificial Ribonucleases
3/29
INTRODUCTION :-
Malignant tumors are one of the main problems of modern
medicine and oncology because very frequently it is not
possible to achieve complete recovery of patients and
maintain corresponding quality of their lives even in the
case of favourable course of this disease.
Proposed schemes of cancer cure employ surgical
methods and high dose chemotherapy, which has suchserious drawback as high toxicity to vitally important
organs and systems of the body.
Solution of the problem of cancer cure requires use of new
approaches, having high selectiveness and effectiveness of
treatment.
-
8/8/2019 Toxicity of Artificial Ribonucleases
4/29
Natural RNase exhibit cytotoxicity that can beincreased by their modification with cationic groups.
Recently, various compounds performing effective.RNA
cleavage and known as the artificial Rnases (aRNases),
have attracted much attention.
Like natural enzymes, these compounds catalyze the
transesterification reaction and cause irreversible RNA
degradation under physiological conditions.
Example: aRNases based on conjugates of 1, 4
diazabicyclo[2.2.2]octane and imi dazole effectively cleave
RNA under physiological conditions in vitro at
concentration of 10^510^4 M . Within 6-12 hours total
cleavage of RNA was observed.
Thus, it was interesting to elucidate, whether thesecompounds are cytotoxic for human cancer cells and
whether mechanism responsible for cell death involves
mRNA cleavage or other reasons account for toxic effect
of these aRNases.
-
8/8/2019 Toxicity of Artificial Ribonucleases
5/29
MATERIALS AND METHODS
The following cell lines have been used in
experiments: Human epidermoid carcinoma KB31 cells, human HeLa
cervical epithelial adenocarcinoma cells, human
neuroblastoma SKNMC cells, human embryonic kidney
(HEK293) cells, and also canine kidney epithelial MDCK
cells. Cells were cultivated in the IMDM medium containing 10%
embryonic calf serum, antibiotics(100 U/ml penicillin and
0.1 mg/ml streptomycin), and amphotericin (0.25 g/ml)
in an atmosphere of 5% CO2 at 37C.
Cells were seeded to a 96well plate (3000 cells in 100 l of themedium per well) or in a 24well plate (10000 cells in 500 l of themedium per well) and cultivated for 24 h, after which aRNaseswere added.
-
8/8/2019 Toxicity of Artificial Ribonucleases
6/29
Total cell RNA was isolated from the cultivated KB31 cells.
Cells (105106) were centrifugated at 200g for10 min at 4C.
After supernatant removal cells were lysed by 1% SDS. The mixture was treated with 1/10 volume of 2 M sodium
acetate, pH 4.5, and 2 volumes of phenol equilibrated withwater under careful stirring.
The aqueous and organic phases were separated by
centrifugation (1000g, 10 min) and remaining proteins wereextracted by an equal volume of the phenol chloroform mixture(1 : 1, v/v).
RNA was precipitated from the aqueous phase with 2.53volumes of ethanol in the presence of 0.3 M sodium acetate, pH
4.5, the precipitate was formed during incubation at 20C for12 h.
-
8/8/2019 Toxicity of Artificial Ribonucleases
7/29
The RNA precipitate was separated by centrifugation (12000
g, 4C, 15 min), washed with 75% ethanol, dried, dissolved in
Milli Q water and kept at 20C.
RNA concentration was determined spectrophotometrically
by measuring the absorbance at 260 nm.
The quality of the isolated RNA was evaluated by
electrophoresis in 1.5% agarose gel.
Changes in mR
NAlevels of
GAPDH, 18S rRN
A, actin,microglobulin, (2m) and cmyc genes inKB31 cells were
performed by reverse transcription polymerase chain
reaction (RTPCR).
For synthesis of cDNA we used total RNA isolated from the
KB31 cells as described above. The reac- tion of cDNA
synthesis was performed at 42C for 1h using the reaction
mixture, containing 50 mM Tris HCl, pH 8.3, 3 mM MgCl2, 5
mM DTT, 75 mM KCl, 0.05 g/ml of total RNA, 5 M primer
d(T)15, 0.5 mM each deoxynucleoside triphosphate, and 0.5
U/l of MMuLV RNAdependent DNA polymerase.
-
8/8/2019 Toxicity of Artificial Ribonucleases
8/29
PCR was performed in a reaction mixture containing 250 M
deoxynucleoside triphosphates, 0.25 Mof forward andreverse primers, cDNA obtained from50 ng of total cell RNA,
and 2 U of Taq DNA polymerase in the buffer containing 10
mM TrisHCl,pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01%
Tween-20.
Amplification products were analyzed by electrophoresis in
1.5% agarose gel under standard conditions. Gel was stained
with ethidium bromide(0.002%) for 15 min and
photographed.
-
8/8/2019 Toxicity of Artificial Ribonucleases
9/29
FOR TOXIC EFFECT :
The toxic effect of compounds Dp12 and ABL3C3on cell cultures were evaluated by the MTT assay.
Cells were seeded into 96well plates as described
above and treated with increasing concentrations
of these compounds (from 107 to 103 M).
A solution of MTT(3(4,5dimethylthiazol2yl)2,
5diphenyl tetrazolium bromide) (5 mg/ml, final
concentration 0.5 mg/ml) was added in phosphate
buffered saline to cells.
After incubation for 3 h under the same conditions
the medium was removed.
-
8/8/2019 Toxicity of Artificial Ribonucleases
10/29
Cells were treated with 100 l of DMSO causing dissolution
of formazan crystals formed in cells during incubation.
Optical density was registered using a multichannel
spectrophotometer at 570 and 630 nm (A570 is formazan
absorption and A630 is back ground absorbance of cells).
Data represent the percentage of survived cells versus
control. The number of control cells incubated in the
absence of aRNases was defined as 100%.
-
8/8/2019 Toxicity of Artificial Ribonucleases
11/29
FOR MEMBRANOTROPIC ACTIVITY
Membranotropic activity of compounds Dp12 and
ABL4C3 was determined by means of cell staining
with Trypan blue.
Data represent the percentage of living (Trypan
blue unstained) cells versus cells with damaged
membrane (and stained with the dye).
The ratio of stained to unstained cells in control
(cells incubated in the absence of aRNAses and
detergent) defined as 100%.
-
8/8/2019 Toxicity of Artificial Ribonucleases
12/29
FOR ULTRASTRUCTURAL CHANGES
Study of ultrastructural changes of KB31 cells induced
byABL3C3 was performed using electron microscopy. Cells grown on Millicell plate membranes were treated
with 20 and 50M ABL3C3 aRNase for 30 min, 2.5, 6and 24 h, and fixed with 4% paraform aldehyde at 4C.
After washing with Hanks solution membranes with
cells were fixed with 1% osmic acid and dehydrated byincreasing concentration of ethanol and acetone usingthe standard method and then embedded in theeponaraldite resin.
Ultrathin sections were prepared in the plane
perpendicular to the surface of the supportingmembrane using aReichert ultratome (Reichert,Austria), contrasted with uranyl acetate and citratelead solutions and analyzed using a Hitachi H600electron microscope.
-
8/8/2019 Toxicity of Artificial Ribonucleases
13/29
RESULTS AND DISCUSSIONS
It was demonstrated that various compoundsimitating active sites of natural RNases effectivelycleave RNA under physiological conditions (pH 7.0,37C).
ABL3C3, a conjugate of 1,4diazabicyclo[2.2.2]octane and imidazole is one of the mosteffective compounds of this type; ABL3C3 has anoligomethylene fragment at quaternary nitrogen
atom and histidine. At the concentrations(15)104 M this compound caused total cleavage ofRNA within 612 h at 37C.
-
8/8/2019 Toxicity of Artificial Ribonucleases
14/29
The second compound used in this study, Dp12, is a
ribonuclease that consists of two1,4diazabicyclo[2.2.2]octane residues carryingdodecamethylene substituents at quaternary nitrogenatoms attached in theparaposition of the benzene ring.
-
8/8/2019 Toxicity of Artificial Ribonucleases
15/29
RESULTS FOR CYTOTOXICITY EFFECT:
Cytotoxicity of artificial ribonucleases was investigatedby the MTT assay, which allows to performspectrophorometric quantification of alive cells.
Cells were incubated with increasing concentrations ofaRNases (from 107 to 103 M) for 24 h and then the
number of survived cells versus control cells incubatedwithout aRNases was evaluated.
For all cell used in this study, the concentrationdependent toxicity curves had a classic S shape.
In the range of concentrations from 107 to 105 M
ABL3C3 and Dp12 exhibited weak toxicity, whereasincubation of cells with higher concentrations of thesecompounds 10^510^4 M resulted in a sharp decreasein the number of survived cells.
-
8/8/2019 Toxicity of Artificial Ribonucleases
16/29
-
8/8/2019 Toxicity of Artificial Ribonucleases
17/29
The IC50 values (concentration required for death of 50% of cells)were 610 M for Dp12, and the highest rate of RNA cleavage in
vitro was observed at 10 M concentration.
Similar results were obtained for ABL3C3: optimal concentration ofthe compound required for RNA cleavage varied from 0.1 to 0.5mM and the IC50 values of 1060 M (in dependence of cell type)were in the same concentration range.
THE IC 50 VALUES OF DIFFERENT CELL LINES ARE:
-
8/8/2019 Toxicity of Artificial Ribonucleases
18/29
Thus, results of these experiments indicate that at concentrations105 M the artificial ribonucleases Dp12 and ABL3C3 are toxic for
tumor cells and it is possible that these compounds damage vitallyimportant cell RNAs.
KINETICS OF ACTION OF DP12 AND ABL3C3:
In these experiments cells were incubated with ABL3C3concentration close to the IC50 value and the number of survivedcells was quantified during 2 days after certain intervals using theMTT assay.
Kinetics of ABL3C3 aRNase toxicity for HeLa cells. Similar results
were also obtained for Dp12 toxicity with respect to all cell linesused in this study.
-
8/8/2019 Toxicity of Artificial Ribonucleases
19/29
A biphasic mode of kinetics of cell death induced by aRNases. Thehighest rate of cell death was observed during the first hours ofincubation with aRNases.
The RNases concentrations influenced only the percentage of deadcells but not the rate of the cells death.
During the first period (6 h) kinetics of cell death induced byABL3C3 and Dp12 did not correlate with kinetics of RNA cleavage
by these compounds, however, in the time interval 648 h thebehaviour of the cell death kinetic curve coincided with kinetics ofRNA cleavage.
-
8/8/2019 Toxicity of Artificial Ribonucleases
20/29
Results of toxic effect of aRNases:
In order to evaluate possible interrelationship between thetoxic effect of Dp12 and ABL3C3 and their amphiphilicproperties (i.e. membranotropic activity) we have usedstaining of aRNasetreated cells with Trypan Blue.
The detergent NP40 demonstrating high membranotropicactivity was used as a control.
This experiment has shown that the toxic effect of ABL3C3 ismuch weaker than that of NP40, however, after 30 min about1020% of cells were stained by Trypan Blue.
After treatment for 2.5 h 0.05% NP40 caused death of almostall cells, whereas proportion of stained cells after the sameincubation with the ABL3C3 aRNase remained basicallyunchanged.
-
8/8/2019 Toxicity of Artificial Ribonucleases
21/29
Similar results have also been obtained in
experiments with Dp12.
The membranotropic activity of these aRNases issignificantly lower than in detergents.
-
8/8/2019 Toxicity of Artificial Ribonucleases
22/29
To evaluate the effect of aRNases on expression levelof housekeeping genes:
In these experiments KB31 cells were incubated with 20and 50 M ABL3C3 for 024 h and then total RNA wasisolated and gene expression was evaluated by RTPCR.Cells incubated without ABL3C3 were used as control.
The toxic aRNase effect was evaluated by comparingmRNA levels of GAPDH, 18S rRNA, actin,microglobulin (2m), and cmyc genes in aRNasetreated anduntreated KB31 cells.
Incubation of KB31 cells with 20 and 50 M ABL3C3
insignificantly influenced mRNA levels of GAPDH, 18SrRNA, actin, microglobulin, and cmyc genes even afterincubaton for 24 h, when significant cell death wasobserved
-
8/8/2019 Toxicity of Artificial Ribonucleases
23/29
The aRNAse used in this study caused single breaks in these mRNAs,which were not detected due to limited size of the PCR product butinfluenced cell functions in general.
The effect of these RNases involves a mechanism, which does notcause mRNA degradation.
FOR STUDY OF PROCESSES INDUCED BY A RNASES:
Treatment of cells with ABL3C3 for 30 min and 2.5 h caused cleardamage of plasmalemma of KB31 cells.
Defects in apical plasmalemma up to 0.5 m (in crosssections) wereregistered in 10% of total cell number and more than 50% after 30 minand 2.5 h, respectively.
In the defect zone there was a naked cytoplasm lacking any borderstructure over its surface and cytoplasmic matrix directly contactedwith the surrounding medium.
-
8/8/2019 Toxicity of Artificial Ribonucleases
24/29
On the border of naked cytoplasm membrane vesicles, evidentlyrepresenting crosssections of caveolae rafts, were localized.
In cells incubated with ABL3C3 for 2.5 h there was activation of the
Golgi apparatus (the increase of its area, number of smooth andcoated vesicles) and the increase of endosomal structures.Therewere damaged sites of ER membranes, nuclear envelope and Golgiapparatus cisterns.
After incubation for 2.5 h with ABL3C3 there was appearance of
irregular fat inclusions with deposits of an osmiophilic substanceon the periphery thus reflecting activa tion of destructiveprocesses.
Thus, incubation of KB31 cells with ABL3C3 for 30 min caused amarked damaging effect on plasmalemma; this resulted not only in
its detachment and baring of cytoplasmic sites, but also toimpairments in its macromolecular organization.
-
8/8/2019 Toxicity of Artificial Ribonucleases
25/29
Incubation of KB31 cells with 20 and 50 M ABL3C3 for 6 h onlysingle cells with plasmalemma defects were detected.
The main bulk of cells lost characteristic fibroblast shape and
became round; this resulted in organelle redistribution incytoplasm.
The cell morphology suggests intensive endocytosis and membraneutilization: highcontent of endosomes, myelinlike structures,vesicular and lysosomal structures, activation of the Golgi
apparatus. After incubation of KB31 cells with 50 M ABL3C3 for 24 h cells on
the scaffold were basically absent.
There were marked changes reflecting active processes ofutilization of damaged membranes: in cytoplasm there were
numerous endosomes and autophagosomes containing membranefragments of various degradation level, there was thewelldeveloped Golgi apparatus responsible for synthesis ofhydrolases and their delivery to endosomes and autophagosomes
-
8/8/2019 Toxicity of Artificial Ribonucleases
26/29
-
8/8/2019 Toxicity of Artificial Ribonucleases
27/29
CONCLUSION:
In this study we have demonstrated that the aRNases exhibit high
toxicity for all cell cultures used. The toxic effect of aRNases for the culture of canine kidney MDCK
(nontumor) cells was one order of magnitude lower than in thecase of other cell lines. This suggests tumorselective effect ofaRNases.
In addition to the ribonuclease activity these compounds alsoexhibit membranotropic activity promoting their effectivepenetration inside cells.
The toxic effect of these compounds was characterized by thebiphasic kinetics. This suggests that a sharp decrease in cell
viability observed during the first 6 h of incubation with theaRNases may be attributed to their membranotropic activity,whereas subsequent cell death is associated with direct ribonuclease effect
-
8/8/2019 Toxicity of Artificial Ribonucleases
28/29
The electron microscopy analysis of the RNase effect has shownthat during a short period (30 min) artificial ribonucleases causeimpairments of the macromolecular structure of cell membrane and
formation of defects of plasmalemma with baring of cytoplasme.
During prolonged incubation for 2.5 h aRNases damagecytoplasmic organelle membranes in a dose depend
Obtained results suggest that the aRNases have the antitumorpotential and therefore they can be considered as a basis for
therapeutic preparation for treatment of cancer in efficient manner.
-
8/8/2019 Toxicity of Artificial Ribonucleases
29/29