Toxicity of Artificial Ribonucleases

8/8/2019 Toxicity of Artificial Ribonucleases

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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


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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.


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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.


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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.


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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.


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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 reaction 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.


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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.


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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.


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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%.


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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%.


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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.


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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.


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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.


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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.


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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:


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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.


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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.


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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.


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Similar results have also been obtained in

experiments with Dp12.

The membranotropic activity of these aRNases issignificantly lower than in detergents.


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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


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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.


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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.


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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


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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


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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.


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Toxicity of Artificial Ribonucleases

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