Cellular stress responses as modulators of drug cytotoxicity in pharmacotherapy of glioblastoma.

Despite the extensive efforts to find effective therapeutic strategies, glioblastoma (GBM) remains a therapeutic challenge with dismal prognosis of survival. Over the last decade the role of stress responses in GBM therapy has gained a great deal of attention, since depending on the duration and intensity of these cellular programs they can be cytoprotective or promote cancer cell death. As such, initiation of the UPR, autophagy or oxidative stress may either impede or facilitate drug-mediated cell killing. In this review, we summarize the mechanisms that regulate ER stress, autophagy, and oxidative stress during GBM development and progression to later discuss the involvement of these stress pathways in the response to different treatments. We also discuss how a precise understanding of the molecular mechanisms regulating stress responses evoked by different pharmacological agents could decisively contribute to the design of novel and more effective combinational treatments against brain malignancies.

RabGAP AS160/TBC1D4 deficiency increases long-chain fatty acid transport but has little additional effect on obesity and metabolic syndrome in ADMSCs-derived adipocytes of morbidly obese women.

The Akt substrate of 160 kDa (AS160), also known as TBC1 domain family member 4 (TBC1D4), represents a crucial regulator of insulin-stimulated glucose uptake in skeletal muscle and adipose tissue. Recent evidence suggests that AS160/TBC1D4 may also control the cellular entry of long-chain fatty acids (LCFAs), resulting in changes to the lipid profile of muscles and fat cells in lean subjects. However, there are virtually no data on AS160/TBC1D4 expression and its modulatory role in lipid metabolism in the adipocytes from morbidly obese individuals of different metabolic status. In this study, we evaluated the effect of the three main factors, i.e., AS160 silencing, obesity, and metabolic syndrome on lipid uptake and profile in fully differentiated adipocytes derived from mesenchymal stem cells (ADMSCs) of lean and obese (with/without metabolic syndrome) postmenopausal women. Additionally, we tested possible interactions between the explanatory variables. In general, obesity translated into a greater content of fatty acid transporters (especially CD36/SR-B2 and SLC27A4/FATP4) and boosted accumulation of all the examined lipid fractions, i.e., triacylglycerols (TAGs), diacylglycerols (DAGs), and free fatty acids (FFAs). The aforementioned were further enhanced by metabolic syndrome. Moreover, AS160 deficiency also increased the abundance of SLC27A4/FATP4 and CD36/SR-B2, especially on the cell surface of the adipocytes derived from ADMSCs of subcutaneous deposit. This was further accompanied by increased LCFA (palmitic acid) uptake. Despite the aforementioned, AS160 silencing seemed unable to significantly affect the phenotype of the adipocytes stemming from obese patients with respect to their cellular lipid profile as we observed virtually no changes in TAG, DAG, and FFA contents when compared to cells with the reference level of proteins. Nevertheless, knockdown of AS160 stimulated fatty acid oxidation, which may indicate that adaptive mechanisms counteract excessive lipid accumulation. At the same time, adipocytes of visceral origin were rather insensitive to the applied intervention.

The Phenotype of the Adipocytes Derived from Subcutaneous and Visceral ADMSCs Is Altered When They Originate from Morbidly Obese Women: Is There a Memory Effect?

Adipose tissue is an abundant source of mesenchymal stem cells (ADMSCs). Evidence has suggested that depot-specific ADMSCs (obtained from subcutaneous or visceral adipose tissue-subADMSCs or visADMSCs, respectively) account for differential responses of each depot to metabolic challenges. However, little is known about the phenotype and changes in metabolism of the adipocytes derived from ADMSCs of obese individuals. Therefore, we investigated the phenotypic and metabolic characteristics, particularly the lipid profile, of fully differentiated adipocytes derived from ADMSCs of lean and obese (with/without metabolic syndrome) postmenopausal women. We observed a depot-specific pattern, with more pronounced changes present in the adipocytes obtained from subADMSCs. Namely, chronic oversupply of fatty acids (present in morbid obesity) triggered an increase in CD36/SR-B2 and FATP4 protein content (total and cell surface), which translated to an increased LCFA influx (3H-palmitate uptake). This was associated with the accumulation of TAG and DAG in these cells. Furthermore, we observed that the adipocytes of visADMSCs origin were larger and showed smaller granularity than their counterparts of subADMSCs descent. Although ADMSCs were cultured in vitro, in a fatty acids-deprived environment, obesity significantly influenced the functionality of the progenitor adipocytes, suggesting the existence of a memory effect.

The influence of the pH on the incorporation of caffeic acid into biomimetic membranes and cancer cells.

Caffeic acid (CA) is a phenolic compound synthesized by all plant species. It constitutes the main hydroxycinnamic acid found in human diet and presents a variety of beneficial effects including anticancer activity. Current data suggests essential role of the interplay between anticancer drugs and the cell membrane. Given this, biophysical interactions between CA and cancer cells or biomimetic membranes were investigated. Glioblastoma cell line U118MG and colorectal adenocarcinoma cell line DLD-1, as well as lipid bilayers and liposomes, were used as in vitro models. Electrophoretic light scattering was used to assess the effect of CA on the surface charge of cancer cells and liposomal membranes. Electrochemical impedance spectroscopy was chosen to evaluate CA-dependent modulatory effect on the electrical capacitance and electrical resistance of the bilayers. Our results suggest that CA fulfills physicochemical criteria determining drug-like properties of chemical compounds, and may serve as a potential cytostatic agent in cancer treatment.

A Preliminary Study of the Effect of Quercetin on Cytotoxicity, Apoptosis, and Stress Responses in Glioblastoma Cell Lines.

A growing body of evidence indicates that dietary polyphenols show protective effects against various cancers. However, little is known yet about their activity in brain tumors. Here we investigated the interaction of dietary flavonoid quercetin (QCT) with the human glioblastoma A172 and LBC3 cell lines. We demonstrated that QCT evoked cytotoxic effect in both tested cell lines. Microscopic observations, Annexin V-FITC/PI staining, and elevated expression and activity of caspase 3/7 showed that QCT caused predominantly apoptotic death of A172 cells. Further analyses confirmed enhanced ROS generation, deregulated expression of SOD1 and SOD2, depletion of ATP levels, and an overexpression of CHOP, suggesting the activation of oxidative stress and ER stress upon QCT exposure. Finally, elevated expression and activity of caspase 9, indicative of a mitochondrial pathway of apoptosis, was detected. Conversely, in LBC3 cells the pro-apoptotic effect was observed only after 24 h incubation with QCT, and a shift towards necrotic cell death was observed after 48 h of treatment. Altogether, our data indicate that exposure to QCT evoked cell death via activation of intrinsic pathway of apoptosis in A172 cells. These findings suggest that QCT is worth further investigation as a potential pharmacological agent in therapy of brain tumors.

GPR18-Mediated Relaxation of Human Isolated Pulmonary Arteries.

GPR18 receptor protein was detected in the heart and vasculature and appears to play a functional role in the cardiovascular system. We investigated the effects of the new GPR18 agonists PSB-MZ-1415 and PSB-MZ-1440 and the new GPR18 antagonist PSB-CB-27 on isolated human pulmonary arteries (hPAs) and compared their effects with the previously proposed, but unconfirmed, GPR18 ligands NAGly, Abn-CBD (agonists) and O-1918 (antagonist). GPR18 expression in hPAs was shown at the mRNA level. PSB-MZ-1415, PSB-MZ-1440, NAGly and Abn-CBD fully relaxed endothelium-intact hPAs precontracted with the thromboxane A2 analog U46619. PSB-CB-27 shifted the concentration-response curves (CRCs) of PSB-MZ-1415, PSB-MZ-1440, NAGly and Abn-CBD to the right; O-1918 caused rightward shifts of the CRCs of PSB-MZ-1415 and NAGly. Endothelium removal diminished the potency and the maximum effect of PSB-MZ-1415. The potency of PSB-MZ-1415 or NAGly was reduced in male patients, smokers and patients with hypercholesterolemia. In conclusion, the novel GPR18 agonists, PSB-MZ-1415 and PSB-MZ-1440, relax hPAs and the effect is inhibited by the new GPR18 antagonist PSB-CB-27. GPR18, which appears to exhibit lower activity in hPAs from male, smoking or hypercholesterolemic patients, may become a new target for the treatment of pulmonary arterial hypertension.

Vasoprotective Endothelial Effects of Chronic Cannabidiol Treatment and Its Influence on the Endocannabinoid System in Rats with Primary and Secondary Hypertension.

Our study aimed to examine the endothelium (vascular)-protecting effects of chronic cannabidiol (CBD) administration (10 mg/kg once daily for 2 weeks) in aortas and small mesenteric (G3) arteries isolated from deoxycorticosterone-induced hypertensive (DOCA-salt) rats and spontaneously hypertensive rats (SHR). CBD reduced hypertrophy and improved the endothelium-dependent vasodilation in response to acetylcholine in the aortas and G3 of DOCA-salt rats and SHR. The enhancement of vasorelaxation was prevented by the inhibition of nitric oxide (NO) with L-NAME and/or the inhibition of cyclooxygenase (COX) with indomethacin in the aortas and G3 of DOCA-salt and SHR, respectively. The mechanism of the CBD-mediated improvement of endothelial function in hypertensive vessels depends on the vessel diameter and may be associated with its NO-, the intermediate-conductance calcium-activated potassium channel- or NO-, COX-, the intermediate and the small-conductance calcium-activated potassium channels-dependent effect in aortas and G3, respectively. CBD increased the vascular expression of the cannabinoid CB1 and CB2 receptors and aortic levels of endocannabinoids with vasorelaxant properties e.g., anandamide, 2-arachidonoylglycerol and palmitoyl ethanolamide in aortas of DOCA-salt and/or SHR. In conclusion, CBD treatment has vasoprotective effects in hypertensive rats, in a vessel-size- and hypertension-model-independent manner, at least partly via inducing local vascular changes in the endocannabinoid system.

The Pro-Apoptotic Effect of Silica Nanoparticles Depends on Their Size and Dose, as Well as the Type of Glioblastoma Cells.

Despite intensive investigations, nanoparticle-induced cellular damage is an important problem that has not been fully elucidated yet. Here, we report that silica nanoparticles (SiNPs) demonstrated anticancer influence on glioblastoma cells by the induction of apoptosis or necrosis. These effects are highly cell type-specific, as well as dependent on the size and dose of applied nanoparticles. Exposure of LN-18 and LBC3 cells to different sizes of SiNPs-7 nm, 5-15 nm, or 10-20 nm-at dosages, ranging from 12.5 to 1000 µg/mL, for 24 and 48 h reduced the viability of these cells. Treatment of LN-18 and LBC3 cells with 7 nm or 10-20 nm SiNPs at doses ≥50 µg/mL caused a strong induction of apoptosis, which is connected with an increase of intracellular reactive oxygen species (ROS) production. The 5-15 nm SiNPs exhibited distinct behavior comparing to silica nanoparticles of other studied sizes. In contrast to LBC3, in LN-18 cells exposed to 5-15 nm SiNPs we did not observe any effect on apoptosis. These nanoparticles exerted only strong necrosis, which was connected with a reduction in ROS generation. This suggests that SiNPs can trigger different cellular/molecular effects, depending on the exposure conditions, the size and dose of nanoparticles, and cell type of glioblastoma.

Molecular and Cellular Effects of Chemical Chaperone-TUDCA on ER-Stressed NHAC-kn Human Articular Chondrocytes Cultured in Normoxic and Hypoxic Conditions.

Osteoarthritis (OA) is considered one of the most common arthritic diseases characterized by progressive degradation and abnormal remodeling of articular cartilage. Potential therapeutics for OA aim at restoring proper chondrocyte functioning and inhibiting apoptosis. Previous studies have demonstrated that tauroursodeoxycholic acid (TUDCA) showed anti-inflammatory and anti-apoptotic activity in many models of various diseases, acting mainly via alleviation of endoplasmic reticulum (ER) stress. However, little is known about cytoprotective effects of TUDCA on chondrocyte cells. The present study was designed to evaluate potential effects of TUDCA on interleukin-1ß (IL-1ß) and tunicamycin (TNC)-stimulated NHAC-kn chondrocytes cultured in normoxic and hypoxic conditions. Our results showed that TUDCA alleviated ER stress in TNC-treated chondrocytes, as demonstrated by reduced CHOP expression; however, it was not effective enough to prevent apoptosis of NHAC-kn cells in either normoxia nor hypoxia. However, co-treatment with TUDCA alleviated inflammatory response induced by IL-1ß, as shown by down regulation of Il-1ß, Il-6, Il-8 and Cox2, and increased the expression of antioxidant enzyme Sod2. Additionally, TUDCA enhanced Col IIα expression in IL-1ß- and TNC-stimulated cells, but only in normoxic conditions. Altogether, these results suggest that although TUDCA may display chondoprotective potential in ER-stressed cells, further analyses are still necessary to fully confirm its possible recommendation as potential candidate in OA therapy.

Electrophoretic Light Scattering and Electrochemical Impedance Spectroscopy Studies of Lipid Bilayers Modified by Cinnamic Acid and Its Hydroxyl Derivatives.

Pharmacological efficiency of active compounds is largely determined by their membrane permeability. Thus, identification of drug-membrane interactions seems to be a crucial element determining drug-like properties of chemical agents. Yet, knowledge of this issue is still lacking. Since chemoprevention based on natural compounds such as cinnamic acid (CinA), p-coumaric acid (p-CoA) and ferulic (FA) is becoming a strong trend in modern oncopharmacology, determination of physicochemical properties of these anticancer compounds is highly important. Here, electrophoretic light scattering and impedance spectroscopy were applied to study the effects of these phenolic acids on electrical properties of bilayers formed from 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-diacyl-sn-glycero-3-phospho-l-serine (PS) or DOPC-PS mixture. After phenolic acid treatment, the negative charge of membranes increased in alkaline pH solutions, but not in acidic ones. The impedance data showed elevated values of both the electrical capacitance and the electrical resistance. We concluded that at acidic pH all tested compounds were able to solubilize into the membrane and permeate it. At neutral and alkaline pH, the CinA could be partially inserted into the bilayers, whereas p-CoA and FA could be anchored at the bilayer surface. Our results indicate that the electrochemical methods might be crucial for predicting pharmacological activity and bioavailability of phenolic acids.

Monitoring of the Surface Charge Density Changes of Human Glioblastoma Cell Membranes upon Cinnamic and Ferulic Acids Treatment.

Cinnamic acid (CA) and ferulic acid (FA) are naturally occurring phenolic acids claimed to exert beneficial effects against disorders related to oxidative stress, including cancer. One such malignancy that still remains a therapeutic challenge mainly due to its heterogeneity and inaccessibility to therapeutic agents is Glioblastoma multiforme (GBM). Here, the influence of CA and FA on the surface charge density of human GBM cell line LN-229 was studied using the electrophoretic light scattering technique. Also, the cytotoxicity of both phenolic acids was determined by metabolic activity-assessing tetrazolium test (MTT) analysis after exposure to CA and FA for 24 h and 48 h. Results showed that both compounds reduced cell viability of LN-229 cells, with more pronounced effect evoked by CA as reflected in IC50 values. Further analyses demonstrated that, after treatment with both phenolic acids, the negative charge of membranes decreased at high pH values and the positive charge of the membranes increased at low pH values compared to the data obtained for untreated cells. Afterward, a four-equilibrium model was applied to estimate the total surface concentrations of both acidic and basic functional groups and their association constants with solution ions in order to calculate theoretical values of membrane surface charge densities. Then, the theoretical data were compared to the experimental data in order to verify the mathematical model. As such, our results indicate that application of electrochemical methods to determine specific drug-membrane interactions might be crucial for predicting their pharmacological activity and bioavailability.

Vasodilatory effects of cannabidiol in human pulmonary and rat small mesenteric arteries: modification by hypertension and the potential pharmacological opportunities.

OBJECTIVE: Cannabidiol (CBD) has been suggested as a potential antihypertensive drug. The aim of our study was to investigate its vasodilatory effect in isolated human pulmonary arteries (hPAs) and rat small mesenteric arteries (sMAs). METHODS: Vascular effects of CBD were examined in hPAs obtained from patients during resection of lung carcinoma and sMAs isolated from spontaneously hypertensive (SHR); 11-deoxycorticosterone acetate (DOCA-salt) hypertensive rats or their appropriate normotensive controls using organ bath and wire myography, respectively. RESULTS: CBD induced almost full concentration-dependent vasorelaxation in hPAs and rat sMAs. In hPAs, it was insensitive to antagonists of CB1 (AM251) and CB2 (AM630) receptors but it was reduced by endothelium denudation, cyclooxygenase inhibitors (indomethacin and nimesulide), antagonists of prostanoid EP4 (L161982), IP (Cay10441), vanilloid TRPV1 (capsazepine) receptors and was less potent under KCl-induced tone and calcium-activated potassium channel (KCa) inhibitors (iberiotoxin, UCL1684 and TRAM-34) and in hypertensive, overweight and hypercholesteremic patients. The time-dependent effect of CBD was sensitive to the PPARγ receptor antagonist GW9662. In rats, the CBD potency was enhanced in DOCA-salt and attenuated in SHR. The CBD-induced relaxation was inhibited in SHR and DOCA-salt by AM251 and only in DOCA-salt by AM630 and endothelium denudation. CONCLUSION: The CBD-induced relaxation in hPAs that was reduced in hypertensive, obese and hypercholesteremic patients was endothelium-dependent and mediated via KCa and IP, EP4, TRPV1 receptors. The CBD effect in rats was CB1-sensitive and dependent on the hypertension model. Thus, modification of CBD-mediated responses in disease should be considered when CBD is used for therapeutic purposes.

Tauroursodeoxycholate-Bile Acid with Chaperoning Activity: Molecular and Cellular Effects and Therapeutic Perspectives.

Tauroursodeoxycholic acid (TUDCA) is a naturally occurring hydrophilic bile acid that has been used for centuries in Chinese medicine. Chemically, TUDCA is a taurine conjugate of ursodeoxycholic acid (UDCA), which in contemporary pharmacology is approved by Food and Drug Administration (FDA) for treatment of primary biliary cholangitis. Interestingly, numerous recent studies demonstrate that mechanisms of TUDCA functioning extend beyond hepatobiliary disorders. Thus, TUDCA has been demonstrated to display potential therapeutic benefits in various models of many diseases such as diabetes, obesity, and neurodegenerative diseases, mostly due to its cytoprotective effect. The mechanisms underlying this cytoprotective activity have been mainly attributed to alleviation of endoplasmic reticulum (ER) stress and stabilization of the unfolded protein response (UPR), which contributed to naming TUDCA as a chemical chaperone. Apart from that, TUDCA has also been found to reduce oxidative stress, suppress apoptosis, and decrease inflammation in many in-vitro and in-vivo models of various diseases. The latest research suggests that TUDCA can also play a role as an epigenetic modulator and act as therapeutic agent in certain types of cancer. Nevertheless, despite the massive amount of evidence demonstrating positive effects of TUDCA in pre-clinical studies, there are certain limitations restraining its wide use in patients. Here, molecular and cellular modes of action of TUDCA are described and therapeutic opportunities and limitations of this bile acid are discussed.

The Modulating Effect of p-Coumaric Acid on The Surface Charge Density of Human Glioblastoma Cell Membranes.

p-Coumaric acid (p-CoA), a phenolic acid belonging to the hydroxycinnamic acids family, is a compound with tentative anticancer potential. Microelectrophoretic mobility measurements conducted at various pH values of electrolyte solution were applied to study p-CoA effects on electrical properties of human glioblastoma cell membranes. The obtained results demonstrated that after the p-CoA treatment, the surface charge density of cancer cells changed in alkaline pH solutions, while no noticeable changes were observed in cell membranes incubated with p-CoA compared to control at acidic pH solutions. A four-equilibrium model was used to describe the phenomena occurring on the cell membrane surface. The total surface concentrations of both acidic and basic functional groups and their association constants with solution ions were calculated and used to define theoretical curves of membrane surface charge density versus pH. The resulting theoretical curves and the experimental data were compared to verify the reliability and validity of the adopted model. The deviation of both kinds of data obtained at a higher pH may be caused by disregarding interactions between the functional groups of cancer cells. Processes occurring in the cell membranes after their incubation with p-CoA can lead to disorders of existing equilibria, which result in changes in values of the parameters describing these equilibria.

The modulating effect of lipid bilayer/p-coumaric acid interactions on electrical properties of model lipid membranes and human glioblastoma cells.

Biological membranes are one of the most important elements of living cells determining their permeability to the active compounds. Still, little is known about the drug-membrane interactions in terms of pharmacological properties of potential drugs. Chemoprevention based on natural compounds is becoming a strong trend in modern oncopharmacology, and p-coumaric acid (p-CoA) is one such compound with tentative anticancer activity. The microelectrophoretic mobility measurements and electrochemical impedance spectroscopy were applied to study the effects of p-CoA on electrical properties of liposomes, spherical bilayers, and human glioblastoma cell membranes. Our results demonstrated that after treatment with p-CoA, the surface charge of LBC3, LN-229 and LN-18 cell lines was significantly changed in alkaline pH solutions, but not in acidic pH solutions. In contrast, no changes in surface charge density values were registered for phosphatidylethanolamine liposomal membranes and A172 cell membranes after p-CoA treatment. The impedance data showed an increase in values of both the electrical capacitance and the electrical resistance, indicating that p-CoA can be partially inserted into the phosphatidylcholine bilayers. The MTT assay showed cell line-dependent cytotoxic effect of p-CoA. Further molecular analyses revealed the ATP depletion and gene transcription modulation, which might indicate organelle membrane-crossing potential of p-CoA. These results suggest, that changes in surface charge of membranes of living cells not only might be potential predictor of membrane permeability, but also indicate differential composition of cell membranes in various cell lines. Thus further multidirectional analyses are required to implement electrochemical methods as standard testing procedures during drug development process.

Modulation of Cardiovascular Function in Primary Hypertension in Rat by SKA-31, an Activator of KCa2.x and KCa3.1 Channels.

The aim of this study was to investigate the hemodynamic effects of SKA-31, an activator of the small (KCa2.x) and intermediate (KCa3.1) conductance calcium-activated potassium channels, and to evaluate its influence on endothelium-derived hyperpolarization (EDH)-KCa2.3/KCa3.1 type relaxation in isolated endothelium-intact small mesenteric arteries (sMAs) from spontaneously hypertensive rats (SHRs). Functional in vivo and in vitro experiments were performed on SHRs or their normotensive controls, Wistar-Kyoto rats (WKY). SKA-31 (1, 3 and 10 mg/kg) caused a brief decrease in blood pressure and bradycardia in both SHR and WKY rats. In phenylephrine-pre-constricted sMAs of SHRs, SKA-31 (0.01-10 µM)-mediated relaxation was reduced and SKA-31 potentiated acetylcholine-evoked endothelium-dependent relaxation. Endothelium denudation and inhibition of nitric oxide synthase (eNOS) and cyclooxygenase (COX) by the respective inhibitors l-NAME or indomethacin, attenuated SKA-31-mediated vasorelaxation. The inhibition of KCa3.1, KCa2.3, KIR and Na+/K+-ATPase by TRAM-34, UCL1684, Ba2+ and ouabain, respectively, reduced the potency and efficacy of the EDH-response evoked by SKA-31. The mRNA expression of eNOS, prostacyclin synthase, KCa2.3, KCa3.1 and KIR were decreased, while Na+/K+-ATPase expression was increased. Collectively, SKA-31 promoted hypotension and vasodilatation, potentiated agonist-stimulated vasodilation, and maintained KCa2.3/KCa3.1-EDH-response in sMAs of SHR with downstream signaling that involved KIR and Na+/K+-ATPase channels. In view of the importance of the dysfunction of endothelium-mediated vasodilatation in the mechanism of hypertension, application of activators of KCa2.3/KCa3.1 channels such as SKA-31 seem to be a promising avenue in pharmacotherapy of hypertension.

The effect of quercetin on the electrical properties of model lipid membranes and human glioblastoma cells.

Quercetin is a naturally-occurring flavonoid claimed to exert many beneficial health effects. In this report, the influence of quercetin on the surface charge of phosphatidylcholine liposomes and human glioblastoma LN-229 and LN-18 cells was studied using microelectrophoretic mobility measurements. The effect of quercetin on the electrical resistance and capacitance of bilayer lipid membranes was analyzed via electrochemical impedance spectroscopy. The results showed that after flavonoid treatment, the cell lines demonstrated changes in surface charge only in alkaline pH solutions, whereas there were no significant alterations in quercetin-treated vs. control cells in acidic pH solutions. The same tendency was found for liposomal membranes proving that quercetin insertion into membranes is strongly pH-dependent. Capacitance and resistance measurements conducted in acidic electrolyte solutions demonstrated an increase in both electrical parameters, indicating an increased amount of quercetin inserted into the bilayers. Moreover, the cytotoxic effect of quercetin confirms that the flavonoid enters the cells and perturbs the proliferation of LN-229 and LN-18 glioblastoma cell lines. As such, our results indicate that the specific localization of quercetin, membrane-bound or cell-entering, might be crucial for its pharmacological activity. However, further studies are necessary prior to applying these physicochemical measurements as standard methods of evaluating drug activity.

Silica nanoparticle-induced oxidative stress and mitochondrial damage is followed by activation of intrinsic apoptosis pathway in glioblastoma cells.

INTRODUCTION: Recently, the focus of oncological research has been on the optimization of therapeutic strategies targeted at malignant diseases. Nanomedicine utilizing silicon dioxide nanoparticles (SiNPs) is one such strategy and is rapidly developing as a promising tool for cancer diagnosis, imaging, and treatment. Nevertheless, little is known about the mechanisms of action of SiNPs in brain tumors. MATERIALS AND METHODS: Here, we explored the effects of 5-15 nm SiNPs in the human glioblastoma cell line LN229. In this respect, MTT assays, microscopic observations, flow cytometry analyses, and luminescent assays were performed. Moreover, RT-qPCR and Western blot analyses were done to determine gene and protein expressions. RESULTS: We demonstrated that SiNPs triggered evident cytotoxicity, with microscopic observations of the nuclei, annexin V-fluorescein isothiocyanate/propidium iodide staining, and elevated caspase 3/7 activity, suggesting that SiNPs predominantly induced apoptotic death in LN229 cells. We further showed the occurrence of oxidative stress induced by enhanced reactive oxygen-species generation. This effect was followed by deregulated expression of genes encoding the antioxidant enzymes SOD1, SOD2, and CAT, and impaired mitochondria function. SiNP- induced mitochondrial dysfunction was characterized by membrane-potential collapse, ATP depletion, elevated expression of BAX, PUMA, and NOXA with simultaneous downregulation of BCL2/BCL2L1, and activation of caspase 9. Moreover, RT-qPCR and Western blot analyses demonstrated increased levels of the endoplasmic reticulum stress markers GRP78, GRP94, and DDIT3, as well as strongly increased expressions of the IL1B and COX2 genes, suggesting activation of endoplasmic reticulum stress and a proinflammatory response. CONCLUSIONS: Altogether, our data indicate that in LN229 cells, SiNPs evoke cell death via activation of the intrinsic apoptosis pathway and suggest that other aspects of cellular function may also be affected. As such, SiNPs represent a potentially promising agent for facilitating further progress in brain cancer therapy. However, further exploration of SiNP long-term toxicity and molecular effects is necessary prior to their widespread application.