Wpływ alliiny na reorganizację cytoszkieletu aktynowego komórek linii H1299 w obecności niskich stężeń doksorubicyny

Alliin, garlic amino acid, has been thought to be pharmacologically inactive. However, several studies carried out on the cell cultures and the mammalian models demonstrated antioxidative and anticancer properties of alliin. Doxorubicin (DOX) is a well-known cytostatic agent, but  often presented the side effects. Therefore, lowering a dose of DOX may reduce a negative influence of this drug on patients. The combined effect of DOX and alliin on the induction of cell death and the reorganization of actin cytoskeleton have not been yet tested in the human non-small cell lung cancer H1299 cells, thus the aim of the study was to evaluate the effects of DOX and alliin on the induction of cell death, morphological and ultrastructural alterations, as well as the reorganization of F-actin in the H1299 cells, using image cytometry and light, fluorescence and transmission electron microscopy. The experimental results presented here indicate that alliin enhances DOX impact on H1299 cells in a dose-dependent manner by increasing the percentages of apoptotic cells and reducing the cell viability. It also led to F-actin reorganization and both morphological and ultrastructural alternations. Alliin can support the tumoricidal effect of DOX but this potential therapeutic use should be examined in further studies.Alliina, aminokwas pochodzący z czosnku, przez lata była uważana za związek farmakologicznie nieaktywny. Jednakże badania prowadzona na kulturach komórkowych oraz modelach ssaczych dowiodły, że ma ona właściwości antyoksydacyjne i przeciwnowotworowe. Stosowanie cytostatyku doksorubicyny (DOX) w walce z nowotworami wiąże się z wieloma działaniami niepożądanymi, tak więc obniżenie dawki leku może stanowić sposób na zredukowanie toksyczności DOX. Dotychczas nie badano połączonych aktywności DOX i alliiny w indukcji apoptozy oraz reorganizacji cytoszkieletu aktynowego w komórkach niedrobnokomórkowego raka płuca linii H1299. Celem badania było określenie wpływu mieszaniny DOX i alliiny na indukcję apoptozy, a także na zmiany w morfologii, ultrastrukturze oraz organizacji F-aktyny w komórkach linii H1299 poprzez cytometrię obrazową, a także mikroskopie świetlną, fluorescencyjną oraz transmisyjną mikroskopię elektronową. Zaprezentowane w pracy wyniki wskazują, że alliina wzmacnia działanie doksorubicyny na komórki H1299 w zależności od wykorzystanej dawki, poprzez wzrost odsetka komórek apoptotycznych i redukcję przeżywalności komórek. Oba związki prowadzą do reorganizacji F-aktyny oraz zmian morfologicznych i ultrastrukturalnych. Alliina wspiera tym samym działanie antykancerogenne DOX, jednak jej potencjał terapeutyczny powinien zostać przedmiotem dokładniejszych badań

THE INFLUENCE OF DOXORUBICIN ON NUCLEAR AND CYTOPLASMIC POOL OF F-ACTIN IN THE A549 CELL LINE

 The cytoskeleton as an intracellular system plays an important role in the proper functioning of the cell. F-actin is one of the components, which built this structure. Microfilaments are involved in cell shape maintenance, polarity and cell motility. The aim of the present study was to determine the effect of doxorubicin on the actin reorganization and type of induced cell death in A549 cell line. In order to examine F-actin, the material was evaluated by the confocal and classical fluorescence microscope. Furthermore, changes in morphology and ultrastructure were analyzed by a light and transmission electron microscopy. The obtained data showed that doxorubicin causes a dosedependent decrease in A549 cell viability. Moreover, the treatment with doxorubicin resulted in the reorganization of F-actin as well as the induction of apoptosis and mitotic catastrophe in the non-small lung cancer cells. In addition, the existence of actin in the nucleus was confirmed.Cytoszkielet stanowi międzykomórkowy system, odgrywający istotną rolę w prawidłowym funkcjonowaniu każdej komórki. Jednym z jego komponentów jest F-aktyna, zaangażowana w zmiany kształtu, polarność, a także ruch komórek. Celem przedstawionej pracy było określenie wpływu doksorubicyny na reorganizację cytoszkieletu aktynowego oraz rodzaj indukowanej śmierci w komórkach linii A549. Wyniki badań oceniano przy użyciu klasycznego oraz konfokalnego mikroskopu fluorescencyjnego, a także z wykorzystaniem mikroskopii świetlnej oraz transmisyjnej mikroskopii elektronowej. W toku badań wykazano dawkozależną wrażliwość komórek linii A549 na doksorubicynę. Wraz ze wzrostem cytostatyku, wzrastał odsetek komórek martwych. Ponadto stwierdzono, że doksorubicyna powoduje zmiany w reorganizacji F-aktyny w komórkach niedrobnokomórkowego raka płuca, a także może indukować apoptozę oraz katastrofę mitotyczną. Dodatkowo potwierdzono występowanie aktyny na terenie jądra komórkowego

WPŁYW DOKSORUBICYNY NA JĄDROWĄ I CYTOPLAZMATYCZNĄ PULĘ F-AKTYNY W LINII KOMÓRKOWEJ A549

 The cytoskeleton as an intracellular system plays an important role in the proper functioning of the cell. F-actin is one of the components, which built this structure. Microfilaments are involved in cell shape maintenance, polarity and cell motility. The aim of the present study was to determine the effect of doxorubicin on the actin reorganization and type of induced cell death in A549 cell line. In order to examine F-actin, the material was evaluated by the confocal and classical fluorescence microscope. Furthermore, changes in morphology and ultrastructure were analyzed by a light and transmission electron microscopy. The obtained data showed that doxorubicin causes a dosedependent decrease in A549 cell viability. Moreover, the treatment with doxorubicin resulted in the reorganization of F-actin as well as the induction of apoptosis and mitotic catastrophe in the non-small lung cancer cells. In addition, the existence of actin in the nucleus was confirmed.Cytoszkielet stanowi międzykomórkowy system, odgrywający istotną rolę w prawidłowym funkcjonowaniu każdej komórki. Jednym z jego komponentów jest F-aktyna, zaangażowana w zmiany kształtu, polarność, a także ruch komórek. Celem przedstawionej pracy było określenie wpływu doksorubicyny na reorganizację cytoszkieletu aktynowego oraz rodzaj indukowanej śmierci w komórkach linii A549. Wyniki badań oceniano przy użyciu klasycznego oraz konfokalnego mikroskopu fluorescencyjnego, a także z wykorzystaniem mikroskopii świetlnej oraz transmisyjnej mikroskopii elektronowej. W toku badań wykazano dawkozależną wrażliwość komórek linii A549 na doksorubicynę. Wraz ze wzrostem cytostatyku, wzrastał odsetek komórek martwych. Ponadto stwierdzono, że doksorubicyna powoduje zmiany w reorganizacji F-aktyny w komórkach niedrobnokomórkowego raka płuca, a także może indukować apoptozę oraz katastrofę mitotyczną. Dodatkowo potwierdzono występowanie aktyny na terenie jądra komórkowego

Possibilities in the application of solid lipid nanoparticles in combination with 5-fluorouracil to overcome the drugresistance of non-small cell lung cancer cell line A549

Introduction: Multidrug resistance of non-small cell lung cancer cells is associated with a high percentageof therapeutic failures. The aim of this study was to assess the ability of solid lipid nanoparticles as atransporter of the conventionally used cytostatic (5-fluorouracil) to overcome the resistance of A549 cells.Material and methods: MTT assay was used to assess the differences in viability of cells treated with5-fluorouracil alone or in combination with different types of solid lipid nanoparticles. Type of cell deathand distribution of cell cycle phases were evaluated using flow cytometry.Results: The use of nanoparticles as a 5-fluorouracil transporter reduced the viability of A549 cells to agreater extent than the cytostatic alone. This was mainly due to the increase in apoptosis, but also necrosisand cell cycle arrest.Conclusion: Our results indicate the great potential of nanotechnology in the treatment of non-small celllung cancer. By using nanoparticles, it is possible to sensitise tumour cells to cytostatics to which theyare normally resistant. In addition, literature data confirm the safety of solid lipid nanoparticle application

The effect of sanguinarine on the RPMI-7951 and A375 melanoma cell lines

Background: Considering the resistance of melanoma to standard treatment protocols, the possibility of metastasis and the high mortality risk, the selection of new alternatives seems to be necessary. Compounds of natural origin are a promising option in anti-cancer therapy. One of them — sanguinarine has a wide spectrum of pro-health properties. Thus, the study aimed to assess the effect of the alkaloid on selected melanoma cellular models. Material and methods: Two types of melanoma cell lines were used in the study — A375 and RPMI-7951. The cells were treated with sanguinarine at concentrations ranging from 0.1 to 2 μM for 24 and 48 h. The influence of the alkaloid on such processes as cell death, cell cycle, organization of the main cytoskeletal proteins and migration potential was assessed. In addition, the sensitivity of selected cell lines to sanguinarine was evaluated based on the MTT assay. Results: The results showed that sanguinarine caused a dose-dependent decrease in cell survival compared to the untreated control. Further studies confirmed that it resulted from the pro-apoptotic and anti-proliferative action of the alkaloid. There were also significant changes in the organization of cytoskeletal proteins and the number of cells visible after fluorescent labelling. Moreover, in A375 cells, characteristics of entosis and mitotic catastrophe were noted. Sanguinarine-induced impaired cell migration was also confirmed. Conclusion: According to the authors’ knowledge, they are the first to present the influence of sanguinarine on the basic life processes of the RPMI-7951 cell line and supplement the knowledge regarding the A375 melanoma cells. The present results confirm the anticancer properties of the alkaloid (cytotoxicity, anti-migratory and pro-apoptotic effect)

Synergistic effect of oxymatrine and 5-fluorouracil on the migratory potential in A549 non-small cell lung cancer cells

Introduction: An interesting research direction is the development of new therapies to reduce metastasis, especially in highly invasive cancer such as lung cancer. One of the commonly used anti-cancer drugs is 5-fluorouracil. Oxymatrine is a natural alkaloid with a wide range of effects. Combined with a cytostatic, it may enhance its action and protect normal cells. Therefore, the study aimed to analyse the effect of oxymatrine and 5-fluorouracil on non-small lung cancer cell line A549. Material and methods: The study was based on the assessment of the interaction between drugs, cell death, cell cycle phase distribution, fluorescent labelling of F-actin and b-catenin, as well as wound healing and transwell migration assay. Results: The combined treatment with oxymatrine and the cytostatic in a 1:1 ratio resulted in synergism. Incubation of cells with both substances induced changes in the life processes of A549 cells. In turn, the reorganization of F-actin and b-catenin contributed to the limitation of lung cancer cell migration compared to individual treatment with compounds. Conclusions: This study demonstrated that the combination of oxymatrine and 5-fluorouracil in the 1:1 ratio may limit the migratory potential of A549 cells. In summary, oxymatrine can support the anti-cancer effect of 5-fluorouracil, but its potential application should be examined in further studies

The influence of house dust extract on normal lung cell Mrc5 and non-small lung carcinoma A549

Introduction. Desensitisation is a therapeutic method of allergic disease treatment, but its impact on the cellular level remains to be elucidated. The purpose of this study was to identify the influence of house dust extract on two cell lines types: non-small lung cancer A549 and non-cancerous lung fibroblast Mrc5. Furthermore, we analysed cell viability, type of cell death, and reorganisation of mainly cytoskeletal proteins such as vimentin, F-actin, and b-tubulin. Material and methods. To determine the cell viability, the MTT test was used. The type of cell death was analysed using double staining of annexin V and iodide propidium. The reorganisation of cytoskeletal proteins was evaluated by fluorescent staining and microscopy observation. Results. Our data presented non-statistical differences in a population of live, apoptotic, and necrotic cells. We did not observe significant abnormalities in cytoskeletal reorganisation. Moreover, Mrc5 cell line exhibited a lower sensitivity for house dust extract in comparison to A549 cell line. Conclusions. Our study suggests that desensitisation has no significant influence on survival and cytoskeleton in both cell lines, which correlate with potential use of this method in cancer patients. Obviously, the choice of these kinds of treatment should be used carefully in consultation with a specialist. Additionally, to our knowledge, it was the first presentation of the influence of house dust extract on cells in the context of the main cytoskeletal reorganisations.

THE EFFECTS OF CAFFEINE ON CYTOSKELETON IN CHO AA8 CELL LINE

The caffeine is one of the most widely consumed alkaloids, which is found in energy drinks, coffee, tea, cocoa and chocolate. This neuroactive substance is a component of the daily diet of millions people in the world. For many years, the attention of researches focused on the effects of caffeine on the living organism, especially in the context of physiological processes. Today, the mechanism of the action of caffeine is the object of the studies. The cytoskeletal proteins play an important role in cell death. In our work, we analyzed changes in vimentin, F-actin and β-tubulin cytoskeleton. Furthermore, alternations in morphology and ultrastructure were also evaluated. We used fluorescence and light microscopy and transmission electron microscopy as well. Our data showed that the treatment with caffeine resulted in decreased in the survival of CHO cells. Moreover, we noticed two types of cell morphology: giant, multinucleated cells and shrunken cells with chromatin condensation. This investigation suggested that caffeine may induce apoptosis and mitotic catastrophe cell death.Kofeina to jeden z najczęściej spożywanych alkaloidów, spotykany między innymi w napojach energetyzujących, kawie, herbacie czy czekoladzie, przez co stanowi ona składnik codziennej diety milionów ludzi. Przez wiele lat uwaga naukowców skupiała się na działaniu kofeiny na organizmy żywe, w tym procesy fizjologiczne. Od niedawna, przedmiotem badań jest mechanizm działania kofeiny. Ze względu na zaangażowanie białek cytoszkieletu w procesy śmierci komórkowej, za zasadne uznano ocenę indukowanych przez alkaloid zmian w obrębie wimentyny, F-aktyny oraz β-tubuliny. Ponadto analizowano morfologię i ultrastrukturę komórek badanej linii. Podczas realizacji tematu wykorzystano metodykę z zakresu mikroskopii świetlnej, klasycznej mikroskopii fluorescencyjnej, a także transmisyjnej mikroskopii elektronowej. Uzyskane wyniki wskazują, iż kofeina wpływa istotnie statystycznie na przeżycie komórek linii CHO AA8. Obserwowane dwa typy morfologii komórek – obkurczonych, ze skondensowaną chromatyną, a także olbrzymich z widocznymi mikrojądrami, sugeruje, iż alkaloid może indukować zarówno apoptozę, jak i katastrofę mitotyczną

WPŁYW KOFEINY NA CYTOSZKIELET LINII KOMÓRKOWEJ CHO AA8

The caffeine is one of the most widely consumed alkaloids, which is found in energy drinks, coffee, tea, cocoa and chocolate. This neuroactive substance is a component of the daily diet of millions people in the world. For many years, the attention of researches focused on the effects of caffeine on the living organism, especially in the context of physiological processes. Today, the mechanism of the action of caffeine is the object of the studies. The cytoskeletal proteins play an important role in cell death. In our work, we analyzed changes in vimentin, F-actin and β-tubulin cytoskeleton. Furthermore, alternations in morphology and ultrastructure were also evaluated. We used fluorescence and light microscopy and transmission electron microscopy as well. Our data showed that the treatment with caffeine resulted in decreased in the survival of CHO cells. Moreover, we noticed two types of cell morphology: giant, multinucleated cells and shrunken cells with chromatin condensation. This investigation suggested that caffeine may induce apoptosis and mitotic catastrophe cell death.Kofeina to jeden z najczęściej spożywanych alkaloidów, spotykany między innymi w napojach energetyzujących, kawie, herbacie czy czekoladzie, przez co stanowi ona składnik codziennej diety milionów ludzi. Przez wiele lat uwaga naukowców skupiała się na działaniu kofeiny na organizmy żywe, w tym procesy fizjologiczne. Od niedawna, przedmiotem badań jest mechanizm działania kofeiny. Ze względu na zaangażowanie białek cytoszkieletu w procesy śmierci komórkowej, za zasadne uznano ocenę indukowanych przez alkaloid zmian w obrębie wimentyny, F-aktyny oraz β-tubuliny. Ponadto analizowano morfologię i ultrastrukturę komórek badanej linii. Podczas realizacji tematu wykorzystano metodykę z zakresu mikroskopii świetlnej, klasycznej mikroskopii fluorescencyjnej, a także transmisyjnej mikroskopii elektronowej. Uzyskane wyniki wskazują, iż kofeina wpływa istotnie statystycznie na przeżycie komórek linii CHO AA8. Obserwowane dwa typy morfologii komórek – obkurczonych, ze skondensowaną chromatyną, a także olbrzymich z widocznymi mikrojądrami, sugeruje, iż alkaloid może indukować zarówno apoptozę, jak i katastrofę mitotyczną

Effect of combined action of doxorubicin and calcifediol on MCF-7 breast cancer cells

Introduction: Breast cancer is one of the most common cancers in women. Current recommendations for combination therapy in patients with breast cancer are still being developed and new therapies with greater success are sought. A relatively new approach is the administration of cytostatics in combination with vitamins. Hence, the study aimed to clarify whether the combination of calcifediol [25(OH)D3] with doxorubicin affects the response of the MCF-7 breast cancer cell line to the cytostatic. Material and methods: In the MCF-7 cell line, the authors assessed cytotoxicity using the MTT assay, analysed the cell cycle and cell death mechanism using flow cytometry, and examined the structure of the cytoskeleton and cell morphology. Results: The results showed that doxorubicin in combination with calcifediol in a 1:1 ratio showed a synergistic effect resulting in a dose-dependent decrease in cell survival. Further studies have shown that this is due to the pro-apoptotic and necrotic effects of the combination of these compounds. There were also changes in the organization of the cytoskeleton and cell morphology. In addition, features of entosis were noted in MCF-7 cells. Conclusion: The synergistic effect of doxorubicin and calcifediol significantly reduced the viability of MCF-7 breast cancer cells. Inducing the desired effect by lowering the cytostatic dose is of great clinical importance, taking into account the cardiotoxicity of doxorubicin. Another very interesting aspect is the entosis process induced in the present research, which may have a dual nature