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Introduction

Cresols are organic compounds, also known as methylphenols, which have various biological effects: Dinitro-o-cresol induces cell death but does not change oxidase expression in soybean cells (1) and causes cytotoxicity via apoptosis in LNCaP prostate cancer cells (2). Additionally, 3-methyl-4-nitrophenol induces toxic effects in rat renal tubular epithelial cells (3). Para (p)-cresol is found in various essential oils from plants such as cloves, cinnamon and basil, contributing to their unique scent (4,5). p-Cresol has been shown to affect proliferation, viability, differentiation and glucose uptake in 3T3-L1 adipocytes (6) and induce cytotoxicity in polymorphonuclear cells (7). Furthermore, p-cresol is rapidly absorbed and excreted, primarily into urine as conjugate metabolites in rats (8). However, it is still unclear how p-cresol affects physiology in human glioblastoma.

Changes in concentration of calcium ions (Ca2+) inside cells, known as intracellular Ca2+ concentration ([Ca2+]i), play a crucial role in regulating various cell processes associated with cell death (9,10). Cells use several mechanisms to regulate [Ca2+]i both globally and at the subcellular level. One of the mechanisms involves G-protein-coupled receptors, which activate phospholipase C (PLC) to release Ca2+ from intracellular stores and influence Ca2+ entry across the plasma membrane (9,10). Understanding the mechanisms underlying compound-induced increases in intracellular Ca2+ is crucial to comprehend the biological effects on the cell.

To the best of our knowledge, there is a scarcity of literature that discusses the effect of cresol-related compounds in Ca2+ signaling. 4-chloro-m-cresol is the most studied cresol and is commonly used as an inhibitor of sarcoendoplasmic reticulum calcium ATPase Ca2+ pumps (11,12). Non-specific effects of 4-chloro-m-cresol may cause Ca2+ flux and respiratory burst in human neutrophils (13). Additionally, 4-chloro-m-cresol increases myoplasmic free Ca2+ concentration and force of contraction in mouse skeletal muscle (14) and inhibits voltage-gated potassium (K+) channels at the rat calyx of Held (15). Since p-cresol has a similar structure to 4-chloro-m-cresol, p-cresol may affect Ca2+ homeostasis in cell models.

p-Cresol has been implicated in various pathological conditions due to its cytotoxic effects on different cell types, such as 3T3-L1 adipocytes (6) and polymorphonuclear cells (7). To the best of our knowledge, however, no studies have examined its impact on the cytotoxicity in human glioma cells. Glioma, particularly glioblastoma multiforme (GBM), is known for its aggressive nature and poor prognosis (16-18). The 5-year survival rate for GBM is less than 10%, with a median survival time of approximately 15 months following diagnosis (16-18). The incidence of GBM varies by region, but it is generally reported at 3-4 cases/100,000 individuals annually (16-18). This data highlights the urgent need for effective therapeutic strategies to improve outcomes for patients. Understanding the molecular and cellular mechanisms underlying glioma pathophysiology has driven extensive research (16-18). Exploring environmental and metabolic factors influencing glioma progression is key for identifying novel therapeutic targets.

Understanding how p-cresol influences glioma cell biology may reveal novel insights into the environmental factors contributing to glioma progression, as well as potential biomarkers for glioma prognosis and therapeutic targets. Prevalence of glioma has gradually increased, with current incidence rates reported at ~6 cases per 100,000 individuals annually in North America and Europe over the past decade (16-18). Despite advancements in surgical techniques, radiation therapy and chemotherapy, the overall efficacy of current treatment options remains limited, particularly for aggressive forms such as GBM (16-18). Given these challenges, exploring novel therapeutic avenues, including the impact of essential oil components such as p-cresol, could have practical applications in improving glioma treatment outcomes. This may contribute to developing more effective strategies for managing glioma progression and improving patient outcomes.

Ca2+ signaling serves a key role in various cellular processes, including proliferation, migration and apoptosis. Because these processes contribute to tumor growth, invasion, and resistance to treatment, they are particularly relevant in cancer biology (9,10). In glioblastoma, dysregulated Ca2+ signaling is implicated in tumor progression and resistance to therapy. Elevated intracellular Ca2+ levels activate various downstream pathways, leading to changes in gene expression, metabolic activity and cell behavior (9,10). PLC is a key enzyme in the Ca2+ signaling pathway, catalyzing hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to generate inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to its receptors on the endoplasmic reticulum, leading to release of Ca2+ into the cytoplasm. When agonists activate cells, PLC is stimulated, leading to the breakdown of PIP2 into IP3 and DAG (9). Increased DAG concentration activates protein kinase C (PKC), while IP3 binds IP3 receptor in the endoplasmic reticulum, causing Ca2+ release from internal stores (9). This increase in [Ca2+]i triggers downstream signaling pathways that contribute to glioblastoma cell survival and proliferation (9,10). Understanding the specific roles of PLC isoforms in Ca2+ signaling may reveal novel therapeutic targets for glioblastoma. Targeting dysregulated PLC-mediated pathways may enhance the efficacy of existing treatments or lead to the development of novel therapeutic strategies (9,10).

Although p-cresol has been shown to promote blood-brain barrier (BBB) integrity and cross BBB in vivo (19), it is unknown how p-cresol affects [Ca2+]i in human glioblastoma. The present study aimed to investigate this effect using DBTRG-05MG human glioblastoma cells, a commonly used model for glioblastoma research (20-22). Fluorescent Ca2+-sensitive dye fura-2-AMwas used to measure changes in [Ca2+]i in response to p-cresol. Additionally, the study explored the effect of p-cresol on cell viability.

Materials and methods

Chemicals

p-Cresol (Fig. 1A), WST-1, nifedipine (voltage-gated Ca2+ channel blocker), SKF96365 (store-operated Ca2+ entry modulator), GF109203X (a PKC inhibitor), thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump), ATP, and U73122 (a PLC inhibitor) were obtained from Sigma-Aldrich (Merck KGaA). Fura-2-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N'N'-tetraacetic acid (BAPTA-AM), and 2-aminoethoxydiphenyl borate (2-APB) were obtained from Molecular Probes. Finally, the reagents for cell culture, including RPMI-1640 medium, fetal bovine serum (FBS), penicillin, and streptomycin, were obtained from Gibco (Thermo Fisher Scientific, Inc.).

Figure 1 Effect of p-cresol on [Ca2+]i. (A) Structure of p-cresol. p-cresol was added in Ca2+ (B) -containing and (C) -free medium. (D) Concentration-response plots of p-cresol-induced [Ca2+]i rises. *P<0.05 vs. Ca2+-free. p-, para.

Cell culture

DBTRG-05MG human glioblastoma cells from Bioresource Collection and Research Center (Taiwan) were cultured in RPMI-1640 medium with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin, all obtained from Gibco (Thermo Fisher Scientific, Inc.), at 37˚C in a humidified atmosphere containing 5% carbon dioxide (CO2).

Solutions used in [Ca2+]i measurements

The Ca2+-containing medium at pH 7.4 contained the following components and concentrations: NaCl (140 mM), KCl (5 mM), MgCl2 (1 mM), CaCl2 (2 mM), HEPES (10 mM), and glucose (10 mM). The Ca2+-free medium had the same components, with CaCl2 replaced by 2 mM EGTA. p-Cresol was dissolved in ethanol to make a 0.1 M stock solution. Other chemicals were dissolved in water, ethanol or DMSO. The impact of organic solvent concentration (≤0.1%) on cell viability and basal [Ca2+]i was determined through control experiments. Cells were exposed to the same concentration of the solvent (without p-cresol or other treatments).

[Ca2+]i measurement

The cells were grown to 80-90% confluence on 6 cm dishes, trypsinized, and suspended in Ca2+-containing or free medium at a concentration of 1x106 cells/ml. The seeding density for subsequent experiments was 1x105 cells/well in 24-well plates or 5x105 cells/well in 6-well plates. Following addition of 2 µM fura-2-AM for 30 min at 25˚C, cells were washed twice with Ca2+-containing medium before being suspended in Ca2+-containing medium at a concentration of 1x107 cells/ml. Fura-2-AM fluorescence measurements were performed in a water-jacketed cuvette at 25˚C. The cuvette contained 1 ml medium and 5x105 cells. The fluorescence was monitored with a Shimadzu RF-5301PC spectrofluorophotometer. For calibration of [Ca2+]i, Triton X-100 (0.1%) and CaCl2 (5 mM) were added to obtain the maximal fura-2 fluorescence. Ca2+ chelator EGTA (10 mM) was added to chelate Ca2+ to obtain the minimal fura-2 fluorescence. The fura-2-loaded cells were excited alternately at 340 and 380 nm and emission was recorded at 510 nm. The fluorescence ratio (F340/F380) was used to estimate [Ca2+]i. The fluorescence ratio was calibrated using standard solutions with known Ca2+ concentrations and ionophore treatments to determine minimum and maximum fluorescence ratios (23). EC50 (half-maximal effective concentration) was calculated. Hill equation was used to describe dose-response relationships.

SKF96365 (5 µM) and 2-APB (20 µM), store-operated Ca2+ entry modulators, and GF109203X (2 µM), a PKC inhibitor, were applied at 37˚C throughout the experiment assessing p-cresol-induced changes in [Ca2+]i. Nifedipine, a voltage-gated Ca2+ channel blocker, was used at a concentration of 10 µM, Thapsigargin (1 µM), ATP (10 µM) as a PLC agonist, U73122 (2 µM) as a PLC inhibitor, and U73343 (2 µM), an inactive analog of U73122, were all included in the experiments to evaluate their effects on intracellular Ca2+ dynamics. All treatments were conducted at a temperature of 37˚C and the duration of each treatment was 30 sec.

Mn2+ measurement

The experiment involved quenching of fura-2 fluorescence by Mn2+ in medium containing Ca2+ and 50 µM MnCl2. MnCl2 was added to the cell suspension in the cuvette 30 sec before fluorescence recording. Data was recorded at an excitation signal of 360 nm (Ca2+-insensitive) and an emission signal of 510 nm at 1-sec intervals, as previously described (24).

Cell viability assay

Cell viability assay was conducted following the manufacturer's instructions. Following treatment with p-cresol at 50, 100, 150, 200 and 250 µM at 37˚C for 24 h, WST-1 (10 µM) was added and cells were incubated at 37˚C for 30 min. Cells were treated with 5 µM BAPTA-AM for 1 h at 37˚C before incubating with p-cresol. The absorbance was determined using an ELISA reader at a wavelength of 450 nm, with a reference wavelength of 620 nm for background correction. In the control, cells treated with the vehicle (organic solvent at ≤0.1%) without p-cresol or BAPTA-AM were used as the baseline for normalization.

Statistical analysis

Data were analyzed using GraphPad Prism version 9.0 (GraphPad Software, Inc.; Dotmatics). Statistical significance was determined using one-way ANOVA followed by Tukey's multiple comparison post hoc test. Tests for normality (Shapiro-Wilk test) and homogeneity of variances (Levene's test) were performed to ensure the assumptions of ANOVA were met. Data are presented as the mean ± SD of three independent experiments. P<0.05 was considered to indicate a statistically significant difference.

Results

p-Cresol increases [Ca2+]i

In Ca2+-containing and -free medium, p-cresol at concentrations between 50 and 150 µM increased [Ca2+]i in a concentration-dependent manner (Fig. 1B and C). The cells were found to have a viability >95% after 20 min. The cell viability of >95% was determined using the trypan blue exclusion assay. Control experiments confirmed that the organic solvent at a concentration ≤0.1% did not impact cell viability or basal [Ca2+]i (data not shown). EC50 value was 70±2 in a Ca2+-containing and 70±3 µM in Ca2+-free medium by fitting to a Hill equation (Fig. 1D).

Effect of BAPTA-AM on reversing p-cresol-induced cell death

Following exposure of DBTRG-05MG cells to p-cresol, a significant and long-lasting increase in [Ca2+]i was observed (Fig. 1). Since unregulated [Ca2+]i can impact cell viability (9,10), the effect of p-cresol on cell viability was investigated. There was a decrease in cell viability in the presence of 50-250 µM p-cresol (Fig. 2). The intracellular Ca2+ chelator BAPTA-AM (25) was used to prevent [Ca2+]i increases during p-cresol treatment. Treatment with 5 µM BAPTA-AM effectively prevented increases in cytosolic Ca2+ levels induced by 50-150 µM p-cresol, indicating successful chelation of intracellular Ca2+. Additionally, 5 µM BAPTA-AM did not alter the baseline cell viability, demonstrating its specific action on Ca2+ signaling without affecting overall cell health. BAPTA-AM reversed p-cresol-induced decreases in cell viability (Fig. 2).

Figure 2 Effect of P on cell viability. *P<0.05 vs. control. #P<0.05 vs. B. P, para-cresol; B, BAPTA-AM.

p-Cresol induces Mn2+ influx

Experiments were conducted to confirm that increases in [Ca2+]i in response to p-cresol involved influx of Ca2+. Mn2+ enters cells using mechanisms similar to those of Ca2+ but quenches fluorescence of the dye fura-2 at all excitation wavelengths (24). Therefore, quenching of fura-2 fluorescence when excited at the Ca2+-insensitive excitation wavelength of 360 nm by Mn2+ suggests involvement of Ca2+ influx. As the Ca2+ response induced by p-cresol peaked at 150 µM, subsequent experiments used 150 µM p-cresol as a control; 150 µM p-cresol triggered an immediate decrease in the 360 nm excitation signal, reaching a maximum value of 62±2 arbitrary units at 100 sec (Fig. 3). This indicated participation of Ca2+ influx in p-cresol-induced increases in [Ca2+]i.

Figure 3 Effect of p-cresol on Ca2+ influx using Mn2+ quenching of fura-2 fluorescence. MnCl2 (50 µM) was added at 60 sec. p-, para.

Pathways of p-cresol-induced Ca2+ entry

Experiments were carried out to investigate the Ca2+ entry pathway underlying the p-cresol-induced increases in [Ca2+]i. SKF96365 (5 µM) and 2-APB (20 µM), store-operated Ca2+ entry modulators (26,27) and GF109203X (2 µM), a PKC inhibitor (28), inhibited p-cresol-induced increases in [Ca2+]i by 65-70%, but nifedipine (a voltage-gated Ca2+ channel blocker, 10 µM) (29) did not (Fig. 4).

Figure 4 Effect of Ca2+ channel modulators on p-cresol-induced [Ca2+]i rises. *P<0.05 vs. p-cresol. p-, para; 2-APB, 2-aminoethoxydiphenyl borate.

Source of p-cresol-induced Ca2+ release

The endoplasmic reticulum is the primary Ca2+ store in most types of cell, including DBTRG-05MG cells (9,10). Therefore, the present study investigated the role of the endoplasmic reticulum in p-cresol-induced Ca2+ release in DBTRG-05MG cells in Ca2+-free medium to eliminate the involvement of Ca2+ influx. Addition of 1 µM thapsigargin (30), an inhibitor of the endoplasmic reticulum Ca2+ pump, resulted in [Ca2+]i rises of 75±2 nM (Fig. 5A). Subsequent addition of 150 µM p-cresol induced [Ca2+]i rises of 10±2 nM. Following p-cresol-induced [Ca2+]i rises, addition of 1 µM thapsigargin at 500 sec failed to induce further [Ca2+]i rises (Fig. 5B).

Figure 5 Effect of thapsigargin on p-cresol-induced Ca2+ release. (A) 1 µM TG and (B) p-cresol (150 µM) were added. TG, thapsigargin; p-, para.

Role of PLC in p-cresol-induced [Ca2+]i rises

The enzyme PLC serves a key role in regulating release of Ca2+ from Ca2+ stores (9,10). PLC inhibitor U73122(31) was applied to investigate if activation of this enzyme was necessary for p-cresol-induced Ca2+ release. ATP induced [Ca2+]i rise of 55±2 nM (Fig. 6A). Since ATP is a PLC-dependent agonist that induces [Ca2+]i rises in most types of cell (32), it was used to study the inhibitory effect of U73122 on PLC activity. Incubation with 2 µM U73122 did not affect basal [Ca2+]i but eliminated ATP-induced [Ca2+]i rises, suggesting effective suppression of PLC activity (Fig. 6B). Additionally, incubation with 2 µM U73122 did not alter basal [Ca2+]i but abolished 150 µM p-cresol-induced [Ca2+]i rises. However, U73343 (negative control), an analog of U73122, did not cause inhibition (data not shown).

Figure 6 Effect of U73122 on p-cresol-induced Ca2+ release. (A) ATP (10 µM) and (B) U73122 (2 µM) and p-cresol (150 µM) were added. *P<0.05 vs. p-cresol. p-, para.

Discussion

Ca2+ signaling is key in regulating physiological processes in human cells, including glioblastoma cells (9,10). Previous studies have shown that the cresol-related compound 4-chloro-m-cresol affects Ca2+ homeostasis in different cell models, such as human neutrophils (13) and mouse skeletal muscle (14). To the best of our knowledge, however, the present study is the first to demonstrate that p-cresol causes [Ca2+]i rises in human glioblastoma cells. p-Cresol between 50 and 150 µM induced a concentration-dependent increase in [Ca2+]i. The mechanism underlying this increase may involve depleting intracellular Ca2+ stores and causing Ca2+ influx from the extracellular environment. p-Cresol-induced [Ca2+]i rises are significantly reduced in the absence of extracellular Ca2+, which suggests that Ca2+ influx occurred continuously throughout the stimulation period. This indicates that p-cresol triggers Ca2+ influx from extracellular sources, likely through mechanisms such as store-operated Ca2+ entry or other channels sensitive to changes in membrane potential or receptor activation.

p-cresol is toxic to glioblastoma cells. Excessive Ca2+ in cells can lead to detrimental changes in cell viability, including apoptosis, necrosis, and impaired cellular functions. These effects occur due to disrupted Ca2+ homeostasis, which can activate enzymes like proteases and phosphatases, destabilize mitochondrial function, and induce oxidative stress, ultimately contributing to cell death or dysfunction (9,10). Ca2+ mobilization causes Ca2+ influx across the plasma membrane via store-operated Ca2+ entry (9,10). If elevation in [Ca2+]i is prolonged, or regulation of [Ca2+]i is abnormal, it can lead to cell death. The present data suggest that p-cresol-induced cell death depended on the rise in [Ca2+]i. Furthermore, increased [Ca2+]i levels may affect Ca2+-dependent downstream responses, leading to changes in cell physiology (for example, activation of calmodulin-dependent enzymes, modulation of ion channel activity, regulation of gene expression through Ca2+-responsive transcription factors like NFAT (nuclear factor of activated T-cells), and triggering of apoptotic pathways via activation of caspases (9,10). These responses highlight the critical role of Ca2+ signaling in regulating cellular processes such as metabolism, proliferation, and cell fate determination (9,10).

p-Cresol causes entry of Ca2+ by stimulating store-operated Ca2+ entry. The depletion of intracellular Ca2+ stores induces this entry (9,10). p-cresol-induced [Ca2+]i rises were inhibited by specific compounds, namely SKF96365 and 2-APB, which are often used as modulators of store-operated Ca2+ entry in various types of cell, such as cortical neurons, myofibroblasts, T lymphocytes, and rat sensory neurons (26,27,33,34). The present data showed that these modulators inhibited p-cresol-induced [Ca2+]i rises. On the other hand, nifedipine, a voltage-gated Ca2+ channel blocker (20), did not inhibit p-cresol-induced [Ca2+]i rises, which suggested that p-cresol-induced Ca2+ entry occurred via a store-operated Ca2+ pathway.

Ca2+ stores in thapsigargin-sensitive endoplasmic reticulum serve a dominant role in p-cresol-induced Ca2+ release. PLC produces IP3 and DAG when activated, activating PKC (9,10). To examine the effect of modulation of PKC activity on p-cresol-induced [Ca2+]i rises, PKC inhibitor GF109203X was used. PKC is involved in signaling pathways that regulate Ca2+ dynamics; inhibiting PKC with GF109203X attenuated or blocked the increase in [Ca2+]i typically triggered by p-cresol Additionally, the release of Ca2+ was PLC-dependent, as shown by the abolition of release when PLC activity was decreased by the inhibitor U73122.

The present results showed a significant increase in [Ca2+]i following p-cresol treatment. This suggested that p-cresol disrupted Ca2+ homeostasis, potentially by enhancing Ca2+ influx or inhibiting Ca2+ efflux mechanisms. The increase in Ca2+ levels may be attributed to the activation of store-operated Ca2+ entry channels or inhibition of Ca2+-ATPases on the endoplasmic reticulum (9,10). There was a dose-dependent decrease in cell viability with p-cresol treatment, which aligned with disruptions in Ca2+ signaling. Dysregulated Ca2+ signaling can lead to mitochondrial dysfunction, triggering cell death pathways (9,10). The decrease in cell viability supports this mechanistic pathway, where p-cresol-induced Ca2+ dysregulation results in cell death. The present results provide more robust and comprehensive understanding of how p-cresol affects Ca2+ signaling in glioma cells.

Exposing cells to 100 µM p-cresol for 24 h can induce cell death due to its cytotoxic effects. This concentration exceeds physiological levels typically found in plasma, which are 20-40 µM for cresol compounds in vivo (8,35). Elevated plasma levels of cresol compounds, especially in patients with liver or kidney disorders (8,35), may further increase the concentration of p-cresol beyond normal physiological ranges, exacerbating its cytotoxic effects. Although the present study does not focus on liver or kidney disorders, this information is relevant to understanding the potential cytotoxicity and biological effects of p-cresol at concentrations that may exceed physiological levels observed in healthy individuals. This broader context helps interpret the impact of p-cresol on cellular systems and provides insights into its potential health implications under different physiological conditions. The potential use of p-cresol or its derivatives in treating human glioblastoma requires further exploration. The effect of p-cresol should be studied in vivo. Furthermore, since increases in [Ca2+]i levels affect numerous Ca2+-coupled cellular processes, these include activation of calmodulin-dependent enzymes involved in signal transduction, modulation of ion channel activity influencing membrane potential and neurotransmitter release, and initiation of apoptotic pathways through activation of caspases, highlighting the pivotal role of Ca2+ signaling in regulating diverse cellular functions (9,10). The effect of p-cresol-induced [Ca2+]i rises on other cellular responses requires further exploration.

The present study had a limited sample size, which may decrease statistical power and generalizability. A small sample size increases risk of errors, potentially overlooking subtle but biologically significant effects of p-cresol. Future studies should increase the sample size to validate the present findings. Furthermore, the present study utilized in vitro models (human glioblastoma cell line) to investigate the effects of p-cresol. In vitro models do not fully replicate the complexity of in vivo tumor microenvironments, such as presence of immune cells, extracellular matrix components and dynamic interactions with other types of cell. While the present study primarily addressed the short-term impacts, it is necessary to understand how p-cresol influences cell behavior over extended periods. Additional experiments should assess long-term effects of p-cresol on glioblastoma cell survival and proliferation.

Proteomic and transcriptomic analyses should be performed to uncover the precise molecular targets of p-cresol. Mass spectrometry-based proteomics can identify protein modifications and changes in expression in response to p-cresol treatment. RNA sequencing can reveal gene expression and signaling pathway alterations. Understanding the specific molecular pathways p-cresol affects may identify novel therapeutic targets for glioblastoma treatment. Furthermore, long-term exposure studies using glioblastoma xenograft models in immunocompromised mice are required. These studies may provide insight into the chronic effects of p-cresol and its potential as a therapeutic agent in vivo, as well as assess the safety and efficacy of p-cresol in a more physiologically relevant context. Future research should determine the detailed mechanism of p-cresol, explore its long-term effects in vivo, investigate combination therapies with existing chemotherapeutic agents or targeted therapies, expand its application to other types of cancer, and develop optimized derivatives to advance understanding and therapeutic potential of p-cresol in cancer treatment.

Phosphoproteomics analysis should be performed to identify signaling pathways activated downstream of Ca2+ influx in response to p-cresol treatment. This approach maps phosphorylation changes in proteins involved in key signaling pathways. Glioblastoma cells treated with p-cresol should be analyzed using mass spectrometry-based phosphoproteomics to identify differentially phosphorylated proteins. Bioinformatics tools should be used to map these proteins to specific signaling pathways. To provide a more holistic view of the biological effects of p-cresol, additional experiments to assess its impact on oxidative stress, autophagy, and mitochondrial function should be performed to provide insights into the mechanisms underlying its cytotoxic effects and uncover potential therapeutic targets for glioblastoma treatment.

PLC serves a key role is in p-cresol-induced Ca2+ signaling. To investigate the involvement of specific PLC isoforms in p-cresol-induced Ca2+ release in glioblastoma cells, molecular biology techniques such as gene expression analysis and siRNA-mediated knockdown will be employed. These methods aim to determine the expression levels and functional significance of different PLC isoforms when glioblastoma cells are treated with p-cresol. Identifying the PLC family and specific isoform(s) involved will provide insights into the molecular mechanisms underlying the dysregulation of p-cresol-induced Ca2+ signaling. Additionally, the functional role of the identified PLC isoforms in p-cresol-induced Ca2+ release and its downstream effects on glioblastoma cell physiology will be assessed. This will involve using pharmacological inhibitors or activators specific to the identified PLC isoform(s) to modulate its activity. The impact on intracellular Ca2+ dynamics, as well as on cell viability, proliferation, and migration, will be evaluated to understand the broader implications of PLC-mediated signaling pathways in p-cresol-treated glioblastoma cells. Functional analysis may elucidate the contribution of PLC isoform(s) to p-cresol-induced Ca2+ signaling alterations and provide mechanistic insights into glioblastoma pathophysiology. This may determine involvement of specific PLC isoforms in p-cresol-induced Ca2+ release and elucidate their functional role in glioblastoma cells. These investigations may provide valuable insight into the molecular mechanisms underlying Ca2+ signaling dysregulation in glioblastoma and uncover novel therapeutic targets for glioblastoma treatment.

In conclusion, p-cresol induced Ca2+ entry in DBTRG-05MG human glioblastoma cells via a PKC-dependent, store-operated mechanism. p-Cresol triggered release of Ca2+ from the endoplasmic reticulum via a PLC-dependent pathway. This led to cell death, which is initiated by a Ca2+ signal. The impact of p-cresol on Ca2+ movement in glioblastoma cells should be investigated in vitro and in vivo.

Acknowledgements

Not applicable.

Funding

Funding: The present study was supported by Department of Pharmacy and Master Program, College of Pharmacy and Health Care, Tajen University.

Availability of data and materials

The data generated in the present study may be requested from the corresponding author.

Authors' contributions

PHC, CLS, SHF, RS and WZL all contributed substantially to the conception and design, acquisition of data, or analysis and interpretation of data. PHC, CLS, SHF and RS confirm the authenticity of all the raw data. WZL wrote the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References