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Ibragimov Shavkat Narzikulovich, Enikeeva Zulfiya Makhmudovna, Abdukhalilov Majid Makhamatkulovich, Saidkulov Bunyod Suyunovich, Narzikulova Kamola Shavkatovna, Republican Specialized Scientific-Practical Medical Center of Oncology and Radiology, Tashkent, Uzbekistan Samarkand medical Institute, Uzbekistan E-mail: [email protected]
THE STUDY OF MITOTIC ACTIVITY, ALKYLATING ACTION AND EFFECT ON TOPOISOMERASE PREPARATIONS K-19, DECOVIN AND K-20, OBTAINED ON THE BASIS OF COLCHICINE AND COLCHAMINE
Abstract: New drugs derived from colchicine K-19, decovin and K-20, have high antitumor activity and the ability to potentiate the effects of radiation. The elements of the mechanism of action of new drugs: mitotic activity, alkylating action, effects on topoisomerases I and II were studied.
Keywords: derivatives of colchicine K-19, decovin, K-20, mitotic activity, alkylating action, topoisomerase.
Introduction. The pronounced general toxic effect, the rapidly developing resistance of the organism to the drugs used, as well as the variety of forms of oncological diseases dictate the need to expand the arsenal of active drugs. A large group of new substances based on tropolinic alkaloids colchicine and colchamine with activity equal to or exceeding the activity of the known antitumor drugs (vincristine, taxol, etoposide, cyclophosphamide, 5-fluorouracil and xeloda) has been developed in the Republican Specialized Scientific and Practical Medical Center of Oncology and Radiology (RSSPMCOR) of the Ministry of Health of the Republic of Uzbekistan [6; 7]. For 3 drugs of this series K-19, decovin and K-20 studies were conducted to their toxic properties and antitumor activity, both in vitro (NCI), as well as in vivo in strains of 6-8 tumors [1; 3; 4; 6; 7]. It was necessary to find out the elements of the mechanism of action of these substances.
The purpose of this study was to study mitotic activity, alkylating action, effects on topoisomerases I and II.
Research methods: Mitotic activity has been studied in the crypts of the intestine of intact mice, which were in-traperitoneally injected with the study drug at a dose of 1/2 LD16. After 30, 60 minutes and every hour during the day the animals were slaughtered with decapitation. For histologi-cal examination the material was taken-1 cm 12 duodenum, which was fixed in a saturated solution of the Bowen mixture, then poured into paraffin with the preparation of histological preparations with hematoxylin-eosin staining. After that, microscopy was performed with counting the number of cells in crypts and in division, to calculate the mitotic index /MI/ and mitotic activity /MA/ [12].
The effect on the synthesis of DNA and RNA was studied using the spectrophotometric method [9]. The effect on internucleosomal DNA degradation and topoisomerase I and II activity was studied on tumor cells of ACATOL. DNA was isolated by the method (Maniatis TF, 1984) [9]. The DNA preparations were analyzed by electrophoresis in 1.5% agarose gel, 60 V, 4 h. The results were determined by evaluation of DNA degradation in comparison with control variants, as well as with doxorubicin and etoposide [8].
The effect of radiation therapy was studied in mice on a THERATRON device with a power of 1.25 MeV, a Co60 source in a sublethal dose equal to 6 Gy on the 9th day after irradiation [5; 14]. Statistical processing was carried out using the program Statistica, version 6.0. The level of statistical significance was assumed to be p < 0.05.
Results
As can be seen from picture 1, all the studied substances cause an increase in the mitotic index within 1 hour after administration (mainly 2 times compared to the control and 1.1-15 times compared to colchicine). The number of mitoses increased for 3 hours and reached the maximum values for almost all substances after 6 hours and remained so for another 6 hours, then by 18 hours it decreased to the level of control and remained unchanged after 24 hours.
The values of the mitotic index (MI) of the epithelium of the intestinal crypt at various times after the administration of preparations in K-19 turned out to be the highest MI - 38.97 + 0.33%, higher than that of colchicine and colchamine. The values of MI decovin (29.68 + 0.33%) and
K-20 (25.18 + 0.87%) were lower than those of colchicine, but 19-20% higher compared to the control.
It is known that the amine derivatives of colchamine have, in comparison with colchicine, generally a smaller MI on normal cells (crypts of the intestine, bone marrow cells) [2; 12], but their MI is higher than that ofcolchicine on the tumor [13].
The effect of the studied preparations of decovin, K-19 and K-20 and Etoposide on the synthesis of DNA, RNA and internucleosomal DNA degradation was carried out on the
cells of the tumor strain of ACATOL. The results of the experiment are shown in picture 2: at an exposure of 48 h, decovin inhibits DNA synthesis by 81.5%, K-19 by 83.7%, K-20 by 79.3%, and etoposide by 80.4%. Under the influence of these preparations, the synthesis of RNA was suppressed by 63.7-66.5%, under the effect of etoposide by 62.4%. It can be seen that the preparations of decovin, K-19 and K-20 are not inferior in activity to the inhibitory effect of etoposide when exposed to the synthesis of DNA and RNA.
Figure 1. Mitotic index of the epithelium of the crypt of the small intestine (in percent) when animals are administered drugs
Figure 2. Effect of Decovin, K-19 and K-20 on the synthesis of DNA and RNA of an intact tumor of Acatol in comparison with etoposide (VP-16)
Figure 3. Electrophoregram of DNA degradation of intact tumor Akatol under the influence of preparations of Dekovin, K-19, K-20 and etoposide
Tracks: M-DNA ladder 100 kb; X - native phage DNA; N - intact DNA of tumor cells; 3-10 - DNA isolated from the tumor after application of antitumor drugs. Tracks: 3,4 - Decovin; 5.6 - K-19; 7.8 - K-20; 9.10 - VP-16 (etoposide)
thesis of RNA (from 64 to 67%) in tumors and are alkylating agents. Often, their alkylating effect is higher than that of etoposide and 40-50% of the initial colchicine and
Figure 3 shows the high natality of DNA isolated from an intact tumor of Akatol, which is not degraded and has no plume (lane N). When using the drugs under study, DNA fragmentation is observed in all variants in the range of 90% in the form of a plume.
With internucleosomal degradation, DNA is fragmented as a "ladder", which is associated with proteolytic cleavage of the specific protein of topoisomerase II [11]. In this connection, we link data on internucleosomal DNA degradation and changes in the activity of topoisomerases. The investigated drugs decovin, K-19, K-20, as well as etoposide (topoisomerase II inhibitor) promote high DNA degradation, which results from the suppression of topoisomerase II activity.
Conclusion
All 3 new drugs have mitotic activity, actively inhibit both the synthesis of DNA (from 79 to 84%) and the syn-
colchamine [2]. All drugs suppressed the activity of topoisomerase II, which leads to a decrease in DNA repair under their influence. The mechanism of action of the drugs under study consists of a pronounced cytotoxic effect, consisting of mitotic, highly alkylating activity and internucleosomal degradation and DNA fragmentation, suppression of topoi-somerase II.
Colchicine in the literature [10] is attributed to radiomi-metics, substances similar in effect to irradiation. It should be noted that both K-19 and some other derivatives due to their structural features due to the introduction of alkylat-ing fragments are stronger radiomimetics than the original alkaloids.
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