The effect of low doses of doxorubicin on the rat glioma C6 cells in the context of the proteins involved in intercellular interactions

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Abstract

The aim of this investigation was to determine the effect of doxorubicin on F–actin rearrangement and β–catenin and cofilin–1 in a rat glioma C6 cell line in combination with changes in their morphology and ultrastructure. The experimental material constituted rat glioma C6 cell line. The cells were incubated with sublethal doses of doxorubicin in the concentration of 50, 100 and 200 nM. The blue trypan dye method was used to determine the number of dead cells. Morphological and ultrastructural changes in the cells were evaluated using light and transmission electron microscope, respectively. In order to determine the rearrangements and level of expression of F–actin, β–catenin and cofilin–1 they were analyzed using a fluorecence microscope. In turn, cell death and cell cycle were evaluated by Guava 6HT–2 L Cytometer. The performed experiments showed a dose–dependent decrease in the survival of C6 cells after treatment with doxorubicin. The analysis of cell death showed a dose–dependent increase in the population of apoptotic and necrotic cells. These results were confirmed by microscopy observation. The changes in morphology, ultrastructure, and rearrangements of F–actin, β–catenin and cofilin–1 were also observed. The results obtained in the study showed that sublethal concentrations of doxorubicin influenced the structure of F–actin and other proteins involved in cell–cell interactions. Moreover, mitotic catastrophe may preceding apoptosis, what suggest the cytotoxic effect of low dose of doxorubicin. Furthermore, our results confirmed the multi–dimensional mechanism of DOX action in tumor cells.

Introduction

In recent years, the attention of many researchers has focused on understanding the carcinogenicity and nature of cancer cells. Despite great advances in medicine and cancer treatment, some of them are still a big mystery. These cancers types include glioblastoma, which is the most common and aggressive primary brain tumor (Birk et al., 2016). Glioma cells are characterized by aggressive invasive growth, mutations in tumor suppressor genes and oncogenes as well as resistance to therapies. It results in the median survival of approximately 14–15 months (Gieryng et al., 2017; Hatoum et al., 2019; Zhang et al., 2019). Nowadays, research on animal models and cell lines provide a lot of information on the mechanisms of glioma development and therapy.

Rat glioblastoma C6 cell line is often used to analyze the biological and biochemical properties of a brain tumor as well as in experimental neuro–oncology, especially to evaluate the therapeutic effects of various cancer treatments. Furthermore, C6 glioblastoma cells administered to Wistar rats are widely used as a model for glioblastoma. As presented in studies by Gieryng et al. (2017) C6 gliomas demonstrate almost the same gene expression profiles and histopathological characteristics as human glioblasomas (Gieryng et al., 2017). Furthermore, the C6 cells are characterized by a high similarity in the structure of proteins associated with cancer development like the Ras proteins family (Giakoumettis et al., 2018).

Doxorubicin (DOX) is an anti–cancer drug produced by Streptomyces peucetius that is characterized by the wide scope of effects. It is used in the treatment of many cancer types, including breast, lung, and brain tumors (Zhao et al., 2018). As it has been shown in many studies, the different concentrations of doxorubicin may induce the various pathways of cellular death or senescence (Hu and Zhang, 2019). One of the main problems with the treatment of brain tumors with DOX is its low capability to pass the brain–blood barrier (BBB) (Zou et al., 2017). However, new drug delivery strategies using liposomes or nanoparticles generally solve the problem which makes them an effective tool when using DOX in glioma therapy (Chen et al., 2011; Sun et al., 2014). Additionally, as shown in the studies presented by Villodre et al. low doses of doxorubicin increase the effect of Temozolomide in glioblastoma cells (Villodre et al., 2018). It is known, that DOX is an inhibitor of topoisomerase II (Ganapathi and Ganapathi, 2013). On the other hand, the cytostatic induces reactive oxygen species (ROS) production (Asensio-López et al., 2017). Hence, the universal mechanism of DOX action remains elusive.

Some of the chemotherapeutics manifest their action through direct or indirect effects on proteins of the intercellular junctions. It is known that the interactions between cells are important in the maintenance of cellular homeostasis while their loss is one of the elements of the epithelial–mesenchymal transition (EMT) (Roche, 2018). So far, three main types of cell junctions are known: gap junctions, tight junctions (TJs), and adherens junctions (AJs) (Hartsock and Nelson, 2008). These structures play many different functions. Gap junctions are involved in both contact and transport of many substances including proteins, hormones, vitamins, and ions (Willebrords et al., 2015). TJs serve as a barrier to the diffusion of some molecules and bacteria (Zihni et al., 2016). In turn, AJs protect the endothelium against mechanical damage induced by many factors (Sukriti et al., 2014; Garcia et al., 2018). The stability of those intercellular junctions depends not only on the proteins that make up them but also on the organization of intracellular elements that are strictly connected. Such an element is a cytoskeleton including, among others, actin filaments and actin–binding proteins (Izdebska et al. (2018); Nowotarski and Peifer, 2014).

This study aimed to determine the influence of doxorubicin on intercellular interactions in the context of F–actin rearrangements in the area of cell–cell junctions. We suggest that the destruction of interactions between cells without cell death induction may become a basis for the metastasis process. The presented study may be useful for a better understanding of the mechanism of DOX action in the glioma cells in the context of cell–cell interaction and F–actin reorganization.

Section snippets

Cell culture and treatment

C6 cell line (ATCC, CCL–107, Manassas, VA, USA), was grown as adherent culture in HAM’s F12 medium (LONZA, Basel, Switzerland) supplemented with 10 % (v/v) heat–inactivated fetal bovine serum (FBS, Sigma–Aldrich, Merck KGaA, Darmastadt, Germany) and 50 μg/mL gentamycin (Sigma–Aldrich), in a fully humidified atmosphere of 5% CO2 at 37 °C. After 24 h of culture, the cells were incubated with 50, 100, and 200 nM of DOX (Sigma–Aldrich) for 24 h. Control cells were grown under identical conditions,

Alterations in viability, cell death and cell cycle following doxorubicin exposure

Applied doses of doxorubicin (50, 100, and 200 nM) caused small but statistically significant (p < 0.05) changes in the percentage of trypan blue positive rat glioma C6 cells. Additionally, the percentage of surviving cells decreased along with the increase in doxorubicin dose (Fig. 1).

The next step was the analysis of the alterations in cell death and cell cycle following doxorubicin treatment. During the cell death analysis, we considered that a double–negative signal for AV and IP was

Discussion

Glioma cell lines are commonly used as a model for studying the mechanism of doxorubicin action. C6 rat glioma cell line is a standard model in glioblastoma research (Giakoumettis et al., 2018). Zhang et al. (2013) showed that the survival of the C6 cell line decreased significantly with increasing doses of DOX. Furthermore, they suggested that a combination of doxorubicin and ultrasound may increase the toxicity in the case of the tumor cell line in a synergistic manner (Zhan et al., 2013). In

Conclusion

Although doxorubicin is currently not used in everyday clinical practice in glioma patients, the rapid development of methods allowing the drug to cross the blood-brain barrier may quickly change this. Our results suggest that sublethal doses of doxorubicin induce alterations in the proteins engaged in cell-cell interactions – F-actin, β-catenin. Moreover, cytostatic treatment caused changes in cell morphology and induced varied events related to cell death (apoptosis, mitotic catastrophe,

Author statement

Marta Hałas-Wiśniewska: substantial contributions to the design of the study, prepared the manuscript; substantial contributions in collecting all the data and analyzed the data in the study; critically revised the manuscript for important intellectual content, read and approved the final manuscript.

Magdalena Izdebska: substantial contributions to the design of the study; prepared the manuscript; performed the experiments; critically revised the manuscript for important intellectual content;

CRediT authorship contribution statement

Marta Hałas-Wiśniewska: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editing. Magdalena Izdebska: Conceptualization, Data curation, Funding acquisition, Investigation, Methodology, Visualization, Writing - original draft, Writing - review & editing. Wioletta Zielińska: Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing. Alina Grzanka: Supervision.

Declaration of Competing Interest

The authors report no declarations of interest.

Acknowledgements

This study was supported by research task within the framework of the statutory activities no. 163 (Nicolaus Copernicus University in Toruń, Collegium Medicum in Bydgoszcz, Faculty of Medicine).

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