CC BY
14
Гуля А. П.1, Тодераш И. К.2, Гудумак В. С.3, Цапков В. И.4, Гарбуз О. С.5, Рошков Е. В.6, Сардарь В. В.7, Тагадюк О. К.8
:ORCID 0000-0003-2010-7959, доктор химических наук, профессор, академик АН, Кишиневский Государственный
Университет, Кишинёв, Молдова 2ORCID 0000-0003-1599-838X, доктор биологических наук, профессор, академик АН, Институт зоологии академии наук Молдовы 3ORCID 0000-0001-9773-1878, доктор медицинских наук, профессор, Государственный университет медицины и фармации им. Н. Тестемицану, Кишинёв, Молдова 4ORCID 0000-0003-1732-3116, доктор химических наук, доцент, Кишиневский Государственный Университет, Кишинёв, Молдова 5ORCID 0000-0001-8783-892X, аспирант; Молдавский Государственный Университет, Кишинёв, Молдова 6ORCID 0000-0001-7565-2845, кандидат биологических наук, Институт зоологии академии наук Молдовы 7ORCID 0000-0002-1047-9145, кандидат медицинских наук;
8ORCID 0000-0002-5503-8052, доктор медицинских наук, '^Государственный университет медицины и фармации им. Н. Тестемицану, Кишинёв, Молдова
ПРОТИВОРАКОВАЯ И ТОКСИЧЕСКАЯ АКТИВНОСТИ НОВОГО СИНТЕЗИРОВАННОГО ВЕЩЕСТВА
Аннотация
В данной работе представлен ряд сравнительных биологических исследований нового синтезированного вещества CMT-122.Антипролиферативная активность этого вещества тестировалась на двух клеточных линиях. Установлено, что CMT-122 проявляет цитотоксичность в отношении RD (рабдомиосаркома) с IC50 - 1,1±0.1 ßmol/L и HeLa (аденокарцинома шейки матки) с IC50 - 8,3±2,0 ¡imol/L. Сравнительное изучение CMT-122 и доксорубицина в отношении раковых клеточных линий показало, что CMT-122 сильнее ингибирует пролиферацию раковых клеток, чем DOX. Дополнительный эксперимент, направленный на оценку цитотоксического эффекта с использованием нормальной клеточной линии MDCK (Madin Darby Canine Kidney), показал, что CMT-122 практически не ингибирует пролиферацию и не вызывает гибель этой линии клеток. Токсичность вещества определяли спектрофотометрическим биоанализом на тест-объектах Paramecium caudatum. Установлено, что LC50 для CMT-122 в 5 раз меньше, чем у DOX.
Ключевые слова: противораковая активность, токсичность.
Gulea А. P.1, Toderas I. K.2, Gudumac V. S.3, Tapcov V. I.4, Garbuz О. S.5, Roscov E. V.6, Sardari V. V. 7, Tagadiuc O.C. 8
1ORCID 0000-0003-2010-7959, PhD in Chemistry, Professor, Academician ASM, Moldova; Moldova State University, 2ORCID 0000-0003-1599-838X, PhD in Biology, Professor, Academician ASM, Moldova; Academy of Sciences of Moldova Institute of Zoology 3ORCID 0000-0001-9773-1878, MD, Professor, State University of Medicine and Pharmacy "Nicolae Testemitanu", Chisinau, Moldova
4
6
ORCID 0000-0003-1732-3116, PhD in Chemistry, Associate professor, Moldova State University, Chisinau, Moldova ORCID 0000-0001 -8783 -892X, Postgraduate student, Moldova State University, Chisinau, Moldova ORCID 0000-0001-7565-2845, PhD in Biology, Academy of Sciences of Moldova Institute of Zoology
7ORCID 0000-0002-1047-9145, MD; 8ORCID 0000-0002-5503-8052, MD,
7,8
State University of Medicine and Pharmacy "Nicolae Testemitanu", Chisinau, Moldova;
ANTICANCER AND TOXICITY ACTIVITIES OF NEW SYNTHESIZED COMPOUND
Abstract
This work represents a series of comparative biological studies of the new synthesized compound CMT-122, exhibiting selective cytotoxicity. The antiproliferative effect of this compound was tested on two cell lines. It was established that CMT-122 exhibited cytotoxic activity against cell lines RD (rhabdomyosarcoma), HeLa (cervix carcinoma) with IC50 values of 1,1±0.1; 8,3±2,0 fimol/L, respectively. Comparative study between test compound and doxorubicin in regard to cancer cell lines was showed that CMT-122 exhibits stronger inhibitory activity on cancer cells proliferation than DOX. An additional experiment aiming on the evaluation of the citotoxic effect on MDCK (Madin Darby Canine Kidney) normal cells of line revealed that compound CMT-122 does not inhibit proliferation through induction of cell death. Toxicological testing method of compounds was performed by Paramecium caudatum colorimetric bioassay. It was founded, that the LC50 for compound CMT-122 is 5 times less than DOX.
Keywords: anticancer activity, toxicity.
I traduction
According to a recent report by the World Health Organization, there are now more than 10 million cases of cancer
per year worldwide. Cancer refers to a diversity of diseases, characterized by the uncontrolled proliferation of cells into a
different form, against the normal complement of the organism. The continuous proliferation of cancer cells develops into
tumor tissues and may spread across to other organs. The principal need in the chemoprevention of cancer remains the
discovery of new effective and safe agents, since the therapeutic application of antiproliferative drugs are restricted due to their
toxic potentials, resistance and genotoxicity [1, P. 1659].
This work represents the research results of the new synthesized compound CMT-122 exhibiting selective cytotoxicity [2, P. 774], [3, P. 59]. The antiproliferative effect [4, P. 5189], [5, P. 78], [6, P. 650] of compound was determined using subsequent human cancer cells of lines: HeLa (cervix carcinoma) and RD (rhabdomyosarcoma).
Considering that drugs are primarily metabolized in the liver and excreted by the kidneys, renal impairment can ultimately affect the treatment outcome and toxicity. Based on this, we exploited MDCK (Madin Darby Canine Kidney) normal cells of line for selective cytotoxicity evaluation. The antiproliferative activity [7, P. 674] of compound CMT-122 was determined by alamar blue dye, which is one of the indicators of mitochondrial metabolic activity.
To estimate the results on the in vitro cytoxicity of the cancer and normal cell lines, the tested compound CMT-122 was compared to doxorubicin (DOX) as a positive control, which is used in the clinical management of a wide range of cancers [8, P. 806].
The direct toxic evaluation of o the tested compound CMT-122 was studied, by the colorimetric neutral red bioassay, using as test-objects the protozoan Paramecium caudatum, which is one of the most commonly used test-objects in laboratory research aimed at directly determining the toxicity of chemical compounds, which are used in toxicological medicine.
Experimental
Cell Culture
HeLa, RD and MDCK cells of line in this study were used. All cells of lines were grown cultured in T-75 cell culture flasks using Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/F12) supplemented with HEPES, L-Glutamine, 10% (FBS) fetal bovine serum and 100 U/ml of penicillin-streptomycin. Cells were maintained at 37oC in a humidified 2% CO2 atmosphere.
Alamar blue in vitro proliferation analysis
Cells viability was measured using the Alamar blue assay with each data point measured in triplicate. The absorbance was read by Hybrid reader H1 (Bio Tek) with 570 nm and 600 nm filters.
The percentage inhibition was calculated according to the formula:
Inhibition (%) = 100 - ((Abs570nmsmp. - Abs600nmsmp.) / (Abs570nmcon. - Abs600nmcon.) x 100).
Neutral red in vivo toxicity analysis
Direct toxic evaluation of compound CMT-122 was studied by colorimetric neutral red assay [9] of the quantification of the membrane permeability and lysosomal activity of Paramecium caudatum [10, P. 445]. The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture.
Neutral red (3-Amino-7-dimethylamino-2-methylphenazine hydrochloride) was used for four hours, which is a weak cationic dye that easily penetrates the cell membrane and accumulates intracellularly in lysosomes, where it binds with anionic sites to the lysosomal matrix. The quantity of neutral red dye incorporated into cells was measured in 96-well plates by Hybrid reader H1 (Bio Tek), by spectrometry at 540 nm and 690 nm. All data about total toxicity activity are the averages of triplicate measurements.
Results and discussion
The antiproliferative activity of compound CMT-122 on two cell lines was tested using the Alamar blue method. Comparative study and concentration ranges identification of cytotoxic activity of CMT-122 and DOX in regard to RD, HeLa cancer cell lines and MDCK normal cell line are shown in Fig. 1.
It was found, that the tested compound CMT-122 exhibited in vitro cytotoxic activity against RD, HeLa lines, with IC50 values of 1,1±0,1; 8,3±2,0; ^mol/L, respectively. In addition, compound CMT-122 showed low citotoxic activity against MDCK line, with IC50 values of >100 ^mol/L. DOX, a positive control, exhibited cytotoxic activity against cell lines, RD,
(A) inhibition of cell proliferation on RD line; (B) inhibition of cell proliferation on HeLa line; (C) inhibition of proliferation
on MDCK line
Ultimately, our results have demonstrated that compound CMT-122 exhibits stronger inhibitory activity on RD, HeLa cancer lines proliferation than DOX. Concomitant was found, that the cytotoxic activity of compound CMT-122 on MDCK normal cells of line is significantly lower than that exerted on the cancer cells, and lower than that exerted by DOX.
The toxicity activity of the tested compound and DOX was performed by Paramecium bioassay. The effect of compound CMT-122 and DOX at a single concentration 100 ^mol/L on the locomotor behavior of Paramecium caudatum was investigated for 30 min by inverted microscope (LOMO) with camera, and compared with control where organisms P. caudatum without treatment (Fig. 1). In culture with compound CMT-122 and DOX the following changes were observed: ciliates actively moved, almost did not form a cluster, there were single fixed specimens, also cellular volume initially increased followed by disintegration of protoplasm and internal membranes (Fig. 2.). The DOX was found more potent for lysis of Paramecium caudatum among compound CMT-122.
After 24 hrs, P. caudatum were in the bottom of wells. The light microscope revealed that most of the protozoa P. slowly moved. Some organisms in this period were motionless, contractile vacuoles were ruptured and their contents were thoroughly mixed up with protoplasm, appears as coagulation of proteins.
Fig. 2 - Effects of (A) compound CMT-122 and (B) DOX on Paramecium caudatum exposed the high concentration 100 ^mol/L after 30 min; (C) organisms without treatment. Arrows indicating rupture of outer membrane
Percent of viability was determined after 24 hrs treatment for compound CMT-122 and DOX, it is graphically indicated in Fig. 3. It was founded, that the LC50 (lethal concentration) value is 4,9±1,5 ^mol/L for CMT-122 and is 1±0,4 ^mol/L for DOX.
so
и <U
E
(О ^
(О
Q. ^
о >
■Q
(О
Parametium toxicity activity
1 1,5
log C (^mol/L)
♦ DOX
y = -25,235x + 44,298 R2 = 0,848 LC50=1±0,4 ^mol/L
■ CMT-122
y = -32,6x + 79,033
R2 = 0,778 LC50=4,9±1,5 ^mol/L
Fig. 3 - Effect of compound CMT-122 and DOX on population growth of Paramecium caudatum
Thus, our results have demonstrated that CMT-122 is lower toxicity than that exerted by doxorubicin.
Conclusion
In summary, these results suggest that the tested compound CMT-122 is of great interest due to their possibility for use as less toxic and more effective anticancer drug. The obtained data will serve as a basis to determine further investigations ways to elucidate pathogenic intimate and detailed mechanisms that can certainly be used to optimize and improve the effectiveness of the cancer treatment.
Список литературы / References
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DOI: https://doi.org/10.23670/IRJ.2017.66.044 Дадабаев В.К.1, Алексеев Р.К.2 :к.м.н. доцент кафедры судебной медицины c курсом правоведения ФГБОУ ВО Тверской ГМУ Минздрава России
2врач-нейрохирург ГБУЗ МО Солнечногорская ЦРБ МУЛЬТИСПИРАЛЬНЫЙ МЕТОД КОМПЬЮТЕРНОЙ ТОМОГРАФИИ В ПРОИЗВОДСТВЕ СУДЕБНО-МЕДИЦИНСКИХ ЭКСПЕРТИЗ ПРИ НЕЙРОХИРУРГИЧЕСКОЙ ПАТОЛОГИИ
Аннотация
Статья посвящена возможностям применения методов исследования РКТ и МСКТ в клинической практике с целью диагностики, лечения, а в судебной медицине при решении вопросов причин диагностических и лечебных ошибок на этапах оказания медицинской помощи. В статье представлен случай из практики, где обследование пациента при помощи МСКТ позволило точно установить клинический диагноз, четко локализовать повреждения головного мозга и инородные тела, наметить план оперативного вмешательства, помогло судебно-медицинскому эксперту ответить на многие вопросы следствия, не дожидаясь патологоанатомического исследования трупа.
Ключевые слова: томографические методы исследования (РКТ, МСКТ, МРТ), вещественные доказательства, огнестрельное ранение головы.
Dadabaev V.K.1, Alekseev R.K.2 :MD, Associate professor of the Department of Forensic Medicine with a course in jurisprudence of FSBEI of HE Tver State Medical University of the Ministry of Health of the Russian Federation Neurosurgeon Budgetary Public Health Facility, Solnechnogorsk CDH MULTISPIRAL METHOD OF COMPUTER TOMOGRAPHY IN THE PRODUCTION OF FORENSIC MEDICAL
EXPERTISE IN NEUROSURGERIC PATHOLOGY
Abstract
The paper is devoted to the possibilities of the use of RCT and MSCT diagnostics in clinical practice for diagnostics, treatment, and in forensic medicine in solving the causes of diagnostic and medical errors at the stages of medical care. The article also describes the case from practice where the examination of the patient with the help of MSCT allowed to establish the clinical diagnosis accurately, clearly localize brain damage and debrides, outline the plan of operative intervention, helped the forensic expert to answer numerous questions of the investigation without waiting for the pathologicoanatomic study of the corpse.
Keywords: tomographic research methods (RCT, MSCT, MRI), material evidence, gunshot head wound.
Введение
Использование в судебно-медицинских и медико-криминалистических экспертизах современных методов исследования, в частности компьютерные томографы (РКТ), мультиспиральный компьютерный томограф (МСКТ), позволяет экспертам объективно и научно обосновано отвечать на вынесенные вопросы экспертизы, а при необходимости, повторно проводить исследование по имеющимся данным [1, С. 22 ].
Актуальность
Применение современных инновационных технологий в судебно-медицинской экспертной практике становятся неотъемлемой частью технологического обеспечения их повседневной деятельности. В судебно-медицинской экспертной деятельности отдается предпочтение и приоритет методам, обладающим следующими критериями: простота использования, достоверность и объективность, возможность сопоставления и повторного проведения исследования без потери полученных данных, не изменяющий свойств исследования объекта и дополнительного времени. Такими критериями обладают хорошо зарекомендовавшие себя и уже используемые в практической медицине рентгенологические методы: компьютерная томография (РКТ), магнитно-резонансная томография (МРТ) и мультиспиральная компьютерная томография (МСКТ).
Применение вышеперечисленных методов в судебно-медицинской экспертной деятельности позволяют улучшить процессы качественного изменения в области переоснащения высокоэффективной диагностической медицинской рентгенологической техникой в системе здравоохранения Российской Федерации (Национальный проект здоровья).
