In vitro investigation of antitumor effect and mechanism of action of the chalcone analogues on human tumor cells

Uvod: Halkoni su heterociklična jedinjenja koja predstavljaju prekursore u biosintezi flavonoida i drugih biološki aktivnih heterocikličnih jedinjenja. Hemijska struktura halkona, koju karakterišu dva aromatična prstena povezana α, β – nezasićenim karbonilnim sistemom sa tri ugljenikova atoma, omogućava širok spektar bioloških aktivnosti od kojih su najznačajnija antitumorska, antiinflamatorna i antiokidativna dejstva. Cilj: Cilj ovog istraživanja bio je ispitivanje potencijalnog antitumorskog efekta i mehanizma delovanja sedam analoga halkona (H1, H2, H3, H4, H5, H6, H7) na tri različite vrste humanih tumorskih ćelija (HeLa, HCT-116 i MDA-MB-231 ćelijske linije) i na zdrave fibroblaste kao kontrolne ćelije (MRC-5). Kao kontrolne supstance korišćene su cisplatina (cisPt) i dehidrozingeron (DHZ). Metode: Citotoksični efekat analiziran je upotrebom MTT testa nakon 24 i 48 časovne inkubacije tumorskih ćelija različitim koncentracijama ispitivanih halkona H1-H7 i referentnih supstanci (cisPt i DHZ). Tip ćelijske smrti određen je korišćenjem protočne citometrije (flow) korišćenjem bojenja Annexin V-FITC/7-AAD, dok je ekpresija, aktivacija i lokalizacija regulatornih proteina apoptoze određena korišćenjem metoda protočne citometrije i imunofluorescentne mikroskopije. Kao deo ekperimenta, metodom protočne citometrije analiziran je uticaj autofagije na promenu procenta citotoksičnosti ispitivanih halkona. Rezultati: Ispitivani halkoni H1-H7 pokazuju snažnu antitumorsku aktivnost koja je u većem broju parametara efikasnija u odnosu cisplatinu. Takođe, ispitivani halkoni indukuju apoptozu tumorskih ćelija putem aktivacije mitohondrijalnog apoptotskog puta, pri čemu halkoni rezultuju povećanje ekspresije regulatornih apoptotskih proteina u tretiranim tumorskim ćelijama u odnosu na netretirane, kontrolne ćelije. Ispitivani halkoni u određenoj meri indukuju autofagiju u tumorskim ćelijama što doprinosi njihovoj citotoksičnosti.Introduction: Chalcones represents precursors in the biosynthesis of flavonoids, isoflavonoids and other biologically active heterocyclic compounds. The chemical structure of the chalcones is characterized by two aromatic rings connected by α, β – unsaturated carbonyl system with three carbon atoms. Chemical structure of chalcones is responsible for their antitumor, antiinflammatory and antioxidant effects. Aim: The main goal of this study was examine cytotoxic and apoptotic effect of the seven chalcone analogues (Х1, Х2, Х3, Х4, Х5, Х6, Х7) on three different types of human tumor cells (HeLa - human cervical cancer, HCT-116 - human colon cancer and MDA-MB-231 - human breast adenocarcinoma) and healthy fibroblasts as control cells (MRC-5). Method: The cytotoxic effect was analyzed using the MTT assay after 24 and 48 hour incubation of tumor cells at different concentrations of the tested Х1-Х7 chalcones and reference substances (cisPt and ДХЗ). Cell death type (relative ratio of apoptotic and necrotic cells) was determined using flow cytometry using Annexin V-FITC / 7-AAD staining, while expression, activation and localization of apoptosis regulatory proteins were determined using flow cytometry and immunofluorescence microscopy. As part of the experiment, by flow cytometry was analyzed the contribution of autophagy in the process of apoptosis induced by the tested chalcone analogues was determined. Results: The examined chalcones Х1-Х7 show strong antitumor activity which is more effective compared to cisplatin. Also, the examined chalcones Х1-Х7 induce cell death via activation of apoptosis in the tumor cells. The investigated chalcones achieve an effective apoptotic effect by activating the mitochondrial apoptotic pathway, resulting increased in expression of regulatory apoptotic proteins in treated tumor cells compared to untreated, control cells. Also, the examined chalcones to induce autophagy in tumor cells, which contributes to their cytotoxicity

In vitro investigation of antitumor effect and mechanism of action of the chalcone analogues on human tumor cells

Uvod: Halkoni su heterociklična jedinjenja koja predstavljaju prekursore u biosintezi flavonoida i drugih biološki aktivnih heterocikličnih jedinjenja. Hemijska struktura halkona, koju karakterišu dva aromatična prstena povezana α, β – nezasićenim karbonilnim sistemom sa tri ugljenikova atoma, omogućava širok spektar bioloških aktivnosti od kojih su najznačajnija antitumorska, antiinflamatorna i antiokidativna dejstva. Cilj: Cilj ovog istraživanja bio je ispitivanje potencijalnog antitumorskog efekta i mehanizma delovanja sedam analoga halkona (H1, H2, H3, H4, H5, H6, H7) na tri različite vrste humanih tumorskih ćelija (HeLa, HCT-116 i MDA-MB-231 ćelijske linije) i na zdrave fibroblaste kao kontrolne ćelije (MRC-5). Kao kontrolne supstance korišćene su cisplatina (cisPt) i dehidrozingeron (DHZ). Metode: Citotoksični efekat analiziran je upotrebom MTT testa nakon 24 i 48 časovne inkubacije tumorskih ćelija različitim koncentracijama ispitivanih halkona H1-H7 i referentnih supstanci (cisPt i DHZ). Tip ćelijske smrti određen je korišćenjem protočne citometrije (flow) korišćenjem bojenja Annexin V-FITC/7-AAD, dok je ekpresija, aktivacija i lokalizacija regulatornih proteina apoptoze određena korišćenjem metoda protočne citometrije i imunofluorescentne mikroskopije. Kao deo ekperimenta, metodom protočne citometrije analiziran je uticaj autofagije na promenu procenta citotoksičnosti ispitivanih halkona. Rezultati: Ispitivani halkoni H1-H7 pokazuju snažnu antitumorsku aktivnost koja je u većem broju parametara efikasnija u odnosu cisplatinu. Takođe, ispitivani halkoni indukuju apoptozu tumorskih ćelija putem aktivacije mitohondrijalnog apoptotskog puta, pri čemu halkoni rezultuju povećanje ekspresije regulatornih apoptotskih proteina u tretiranim tumorskim ćelijama u odnosu na netretirane, kontrolne ćelije. Ispitivani halkoni u određenoj meri indukuju autofagiju u tumorskim ćelijama što doprinosi njihovoj citotoksičnosti.Introduction: Chalcones represents precursors in the biosynthesis of flavonoids, isoflavonoids and other biologically active heterocyclic compounds. The chemical structure of the chalcones is characterized by two aromatic rings connected by α, β – unsaturated carbonyl system with three carbon atoms. Chemical structure of chalcones is responsible for their antitumor, antiinflammatory and antioxidant effects. Aim: The main goal of this study was examine cytotoxic and apoptotic effect of the seven chalcone analogues (Х1, Х2, Х3, Х4, Х5, Х6, Х7) on three different types of human tumor cells (HeLa - human cervical cancer, HCT-116 - human colon cancer and MDA-MB-231 - human breast adenocarcinoma) and healthy fibroblasts as control cells (MRC-5). Method: The cytotoxic effect was analyzed using the MTT assay after 24 and 48 hour incubation of tumor cells at different concentrations of the tested Х1-Х7 chalcones and reference substances (cisPt and ДХЗ). Cell death type (relative ratio of apoptotic and necrotic cells) was determined using flow cytometry using Annexin V-FITC / 7-AAD staining, while expression, activation and localization of apoptosis regulatory proteins were determined using flow cytometry and immunofluorescence microscopy. As part of the experiment, by flow cytometry was analyzed the contribution of autophagy in the process of apoptosis induced by the tested chalcone analogues was determined. Results: The examined chalcones Х1-Х7 show strong antitumor activity which is more effective compared to cisplatin. Also, the examined chalcones Х1-Х7 induce cell death via activation of apoptosis in the tumor cells. The investigated chalcones achieve an effective apoptotic effect by activating the mitochondrial apoptotic pathway, resulting increased in expression of regulatory apoptotic proteins in treated tumor cells compared to untreated, control cells. Also, the examined chalcones to induce autophagy in tumor cells, which contributes to their cytotoxicity

Relationship between the precipitation variability in Montenegro and the Mediterranean oscillation

This study investigates the influence of atmospheric circulation in the Mediterranean region on the precipitation in Montenegro. Nine precipitation parameters have been used in the analysis and the relationship has been investigated by the Mediterranean and West Mediterranean Oscillation change index (MO and WeMO). According to a 60 - year observed period (1951-2010), the research results show that nothing characteristic happens with seasonal and annual precipitation sums because the trend is mainly insignificant. However, precipitation extremes are getting more extreme, which corresponds with a general idea of global warming. Negative consequences of daily intensity increase and frequency of precipitation days above fixed and percentile thresholds have been recorded recently in the form of torrents, floods, intensive erosive processes, etc., but it should be pointed out that human factor is partly a cause of such events. The estimate of the influence of teleconnection patterns primarily related to the Mediterranean Basin has shown that their variability affects the observed precipitation parameters on the territory of Montenegro regarding both seasonal and annual sums and frequency and intensity of extreme events shown by climate indices

Recent extreme air temperature changes in Montenegro

The studies show that the changes in intensity and frequency of the extreme weather events have been registered in many regions of the world. This paper gives an analysis of the change of 12 air temperature parameters, out of which 9 are climate indices. The indices suggested by WMO-CCL/CLIVAR have been used in order to investigate the changes in temperature extremes. The research related to the topic has been carried out by using the data from 23 meteorological stations for the 1951 - 2010 period and calculations have been done on the seasonal level. The results show that the maximum and minimum air temperatures, which have “warmer values”, are becoming more frequent on the territory of Montenegro which corresponds to the general idea of global warming

Recent trends in daily temperature extremes over southern Montenegro (1951-2010)

Montenegro so far has been poorly investigated in terms of climate extremes. The aim of this paper was to analyse the extreme ETCCDI (Expert Team on Climate Change Detection and Indices) temperature indices in the Mediterranean region of Montenegro for the period of 1951-2010. Four stations in the coastal area of Montenegro have been analysed: Herceg Novi, Ulcinj, Budva and Bar. Two periods (before 1980 and after 1980) were separately investigated in this study due to a well-known climate shift that occurred in the late 1970s. Seven indices of temperature extremes have been chosen. The trend was analysed using a Mann-Kendall non-parametric test, while the slope was estimated using Sen's slope estimator. A negative trend has been calculated for cold nights and cold days at almost all stations. The most significant positive trends were obtained for warm conditions. The two separately investigated periods have shown contrasting temperature trends

ANTITUMOR EFFECTS OF VANILLIN BASED CHALCONE ANALOGUES IN VITRO

Chalcones, as a large group of organic compounds, are widely implemented in various types of anti-cancer therapeutics. These plant metabolites are present in fruits, vegetables, spices, and have anti-tumor, anti-inflammation, immunomodulation, antibacterial and anti-oxidative activities, as well as many other pharmacological and biological effects. The aim of the present study was to investigate cytotoxic effects, type of cell death and mechanism of action of the newly synthesized vanillin based chalcone analogues, (CH1) and (CH2) on human colon cancer HCT-116 and noncancerous (control) MRC-5 cell lines. In order to compare effects of vanillin based chalcone analogues on investigated cell lines, as reference substances cisplatin (cisPt) and dehydrozingerone (DHZ) were used. Investigation of antitumor effect of chalcone analogues on HCT-116 cells was carried out by three methods MTT assay, flow cytometry and immunofluorescence analysis. The result of our investigation indicated that newly synthesized vanillin based chalcone analogues expressed powerful antitumor effect on cancer cells (HCT-116 cell line), while their effect on healthy cells (MRC-5 cell line) was not statistically significant. Vanillin based chalcone analogues caused overexpression and activation of mitochondrial Bax protein and caspase-3 in HCT-116 cells, indicating that their mechanism of antitumor action was mediated through activation of inner apoptotic pathway. These results indicate possible usefulness of CH1 and CH2 in antitumor therapy whether through its direct cytotoxic effect or as adjuvant therapy. Our results indicate possible usefulness of CH1 and CH2 vanillin based chalcone analogues in antitumor therapy