A Novel Pyrazole Pyrimidine Derivative MBP346 Induces Cell Death via ROS-Mediated Mitochondrial Damage in Human Head and Neck Squamous Cell Carcinoma

Abstract

Background: Head and neck squamous cell carcinoma (HNSCC) represents almost 95% of head and neck cancer cases and ranks as the sixth most prevalent malignant tumor globally. Several treatment strategies, such as surgery, radiation, and chemotherapy, are implemented to boost the outcomes for patients with HNSCC. However, the overall survival rate for patients with HNSCC has remained poor. MBP346 is a novel pyrazole pyrimidine compound that is cytotoxic to HNSCC cells. Therefore, this study aims to investigate its effect on HNSCC and to explore its possible molecular mechanism. Methods: Cell viability of HNSCC (Cal33 and Scc15) cells and normal NOK cells treated with MBP346 was determined by Methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Colony formation assay and Edu assay were used to detect cell proliferation. Cell cycle and apoptosis were analyzed by flow cytometry. Western blot was used for detecting cell cycle-related and cell apoptotic-related proteins. Immunofluorescence assay was performed to analyze the effect of MBP346 on reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Results: MBP346 significantly inhibited the proliferation of Cal33 and Scc15 cells, with half inhibitory concentrations of 1.56 ± 0.13 μmol·L⁻¹ and 4.41 ± 0.28 μmol·L⁻¹, respectively. The cell cycle-related proteins CyclinD1, CyclinA2, and CDK2 were downregulated, and P21 was upregulated in Cal33 and Scc15 cells treated with MBP346, which blocked the cell cycle in the S phase. MBP346 induced cell apoptosis in Cal33 and Scc15 cells by inducing ROS production. In addition, the elevated ROS decreased MMP to accelerate apoptosis. N-acetylcysteine (NAC), an ROS inhibitor, suppressed MBP346-induced cell apoptosis. Conclusions: MBP346 may serve as a therapeutic agent in HNSCC by inducing cell death. It achieves this by halting cell proliferation through cell cycle arrest and enhancing apoptosis due to increased ROS, which results in mitochondrial dysfunction.

1. Introduction

Head and neck cancer represents a highly invasive and heterogeneous category of malignant tumors, with head and neck squamous cell carcinoma (HNSCC) comprising over 95% of these cases [1]. The invasive nature and heterogeneity of HNSCC contribute to the challenges associated with early-stage diagnosis. Research indicates that the development of HNSCC is linked to various factors, including tobacco use, excessive alcohol consumption, and infection with human papillomavirus (HPV) [2]. Furthermore, the heterogeneity of HNSCC is evident not only in its pathophysiological characteristics but also in its molecular biological features, which further complicates therapeutic interventions. Despite the availability of diverse treatment modalities for HNSCC, including surgical intervention, radiotherapy, chemotherapy, and targeted therapy, the therapeutic outcomes frequently remain suboptimal [3]. This is primarily attributed to the tumor’s inherent heterogeneity and invasive characteristics. Consequently, there is a pressing necessity to develop novel pharmacological agents for the effective management of HNSCC.

Research indicates that elevated levels of reactive oxygen species (ROS) in cancer cells not only promote tumor progression but also increase cancer cells’ susceptibility to ROS-mediated interventions, thereby positioning ROS as a critical target in anticancer therapies [4]. In cancer therapy, ROS serve as essential cellular signaling molecules that initiate apoptosis in tumor cells by mitochondrial dysfunction [5]. For example, the light-activated ROS generator, TBTP, compromises mitochondrial integrity by inducing bursts of ROS within mitochondria, resulting in reduced ATP levels, diminished mitochondrial membrane potential, and the activation of Caspase-3 and Caspase-9, ultimately leading to apoptosis [6]. Similarly, Cyclovirobuxine D (CVBD) triggers apoptosis and mitochondrial damage in glioblastoma cells through ROS-mediated mitochondrial translocation. Additionally, the study found that compounds such as Shikonin and Curcumin promote apoptosis in tumor cells by enhancing intracellular ROS production, which subsequently depletes mitochondrial membrane potential and causes DNA damage [7].

In recent years, pyrazole derivatives have been proved to have anti-anxiety [8], anti-inflammatory [9], anti-bacterial [10] and anti-tumor activities [11]. Based on this, our research team synthesized methyl 2-(4-(3-bromopyrazolo[1,5-a]pyrimidin-6-yl)phenyl)acetate (MBP346) compound (Figure 1A), which significantly inhibited the proliferation of HNSCC, but its anti-tumor mechanism is still unclear. Therefore, the antitumor activity of MBP346 on human tongue squamous carcinoma cell lines Cal33 and Scc15 and the molecular mechanism of apoptosis induced by ROS-mediated mitochondrial damage were investigated.

2. Results and Discussion

2.1. MBP346 Suppressed Proliferation and Clonogenic Survival of Head and Neck Squamous Cell Carcinoma

The anti-proliferative properties of the pyrazole derivative MBP346 were assessed in two human head and neck squamous cell carcinoma cell lines, Cal33 and Scc15. MTT assays conducted after a 48 h exposure period demonstrated a dose-dependent reduction in cell viability, with half-maximal inhibitory concentrations (IC50) determined to be 1.56 ± 0.13 μmol·L⁻¹ for Cal33 cells and 4.41 ± 0.28 μmol·L⁻¹ for Scc15 cells (Figure 1B,C). Importantly, MBP346 did not exhibit significant cytotoxicity towards normal oral keratinocytes (NOK) at similar concentrations (Figure 1D), indicating a selective effect on malignant cells. This selectivity was corroborated by colony formation assays, which showed a significant, dose-dependent decrease in both the number and size of colonies (p < 0.01; Figure 1E–G). Furthermore, EdU incorporation assays confirmed the anti-proliferative activity of compound MBP346, revealing a significant and dose-dependent reduction in the percentage of proliferating (EdU-positive) cells (p < 0.01). Specifically, in Cal33 cells, the EdU/DAPI percentage decreased from 59.20% ± 7.64% in the DMSO control group to 25.43% ± 3.82% following treatment with 5 μmol·L⁻¹ MBP346. A comparable inhibitory effect was noted in Scc15 cells, with a reduction from 39.93% ± 3.85% to 15.15% ± 3.04% (Figure 1H–K). Collectively, these findings indicate that MBP346 effectively and selectively suppresses the proliferation and clonogenic capacity of HNCC cells.

2.2. MBP346 Induced S-Phase Cell Cycle Arrest Through Modulation of Key Regulatory Proteins

Given the potent antiproliferative effects observed, we examined the influence of MBP346 on cell cycle progression. Flow cytometric analysis revealed that treatment with MBP346 resulted in a significant, dose-dependent accumulation of cells in the S phase. Specifically, in Cal33 cells, the S-phase population increased from 22.32% ± 0.44% with DMSO to 65.42% ± 2.01% with 5 μmol·L⁻¹ MBP346 (p < 0.001). A similar pattern was observed in Scc15 cells, where the S-phase population rose from 13.87% ± 0.18% to 31.22% ± 2.52% (p < 0.001; Figure 2A–D). To elucidate the molecular mechanisms underlying this arrest, we assessed the expression levels of key cell cycle regulatory proteins. Western blot analysis indicated that MBP346 treatment resulted in the downregulation of Cyclin D1, Cyclin A2, and CDK2 protein levels, while simultaneously upregulating the cyclin-dependent kinase inhibitor P21 (p < 0.01; Figure 2E,F). The dysregulation of the cell cycle constitutes a critical factor in tumorigenesis and tumor progression, as it results in uncontrolled cellular proliferation, a defining characteristic of cancer. The suppression of cyclin D1, cyclin A2, and CDK2 expression by various compounds has been demonstrated to induce apoptosis in cancer cells, underscoring their potential as therapeutic targets in oncology. CDK2 plays a pivotal role in cell cycle regulation, and its inhibition can result in cell cycle arrest and decreased cellular proliferation. For instance, RNA interference targeting CDK2 has been shown to effectively down-regulate its expression, leading to cell cycle arrest at the G0/G1 phases and subsequent inhibition of cancer cell proliferation [12]. The compound 6,7,4-trihydroxyisoflavone has been shown to target CDK2, suppressing its activity and leading to cell cycle arrest in colon cancer cells. This compound effectively inhibits CDK2 activity in vitro and in vivo, demonstrating its potential as a therapeutic agent against colon cancer [13]. Additionally, the overexpression of Neuronal Pentraxin 1 (NPTX1) in colon cancer cells results in the down-regulation of cyclin A2 and CDK2, thereby impeding cell growth and indicating a potential therapeutic application for NPTX1 in colon cancer [14]. In this study, we demonstrated that the inhibition of cyclin D1, cyclin A2, and CDK2 expression by MBP346 exhibits significant potential in inducing Cal33 and Scc15 cell apoptosis and augmenting the efficacy of existing cancer therapies. These findings underscored that MBP346 impeded the progression of human tongue squamous carcinoma cells by arresting the cell cycle in the S phase and inhibiting DNA synthesis, thereby exerting its anti-tumor effects.

2.3. MBP346 Triggered Apoptosis in HNCC Cells via the Mitochondrial Pathway

We next assessed whether the growth inhibition was accompanied by the induction of apoptosis. Flow cytometric analysis after Annexin V/PI staining revealed a dramatic, dose-dependent increase in apoptosis upon MBP346 treatment. The apoptosis rate in Cal33 cells rose from 8.27% ± 0.91% (DMSO) to 63.46% ± 2.33% (5 μmol·L⁻¹ MBP346). Similarly, in Scc15 cells, the rate increased from 6.12% ± 0.63% to 56.71% ± 4.96% (p < 0.01; Figure 3A–D). Consistent with this, Western blot analysis showed a marked upregulation of pro-apoptotic proteins, including cleaved forms of PARP, Caspase-3, and Caspase-8, as well as Bax. Conversely, the levels of anti-apoptotic proteins Bcl-2 and MCL-1 were significantly downregulated (p < 0.01; Figure 3E,F). The cleavage of PARP and Caspases, along with the shift in the Bax/Bcl-2 ratio, strongly indicates the activation of both intrinsic (mitochondrial) and extrinsic apoptosis pathways.

2.4. MBP346 Elevated Intracellular ROS Levels and Depleted Mitochondrial Membrane Potential

Reactive oxygen species (ROS) play a complex, context-dependent role in cancer, where excessive accumulation can induce oxidative stress and apoptosis. Using the DCFH-DA probe, we found that MBP346 treatment significantly and dose-dependently increased intracellular ROS levels in both cell lines. In Cal33 cells, relative DCF fluorescence intensity increased from 1.23 ± 0.21 (DMSO) to 20.28 ± 1.24 (5 μmol·L⁻¹ MBP346). A comparable increase was observed in Scc15 cells (Figure 4A–D). Numerous studies have demonstrated that excessive levels of ROS exerted cytotoxic effects on cancer cells, resulting in oxidative damage and subsequent cell death. Wahi et al. elucidated the dual role of ROS in non-small cell lung cancer (NSCLC) through the action of Emodin, a natural compound. The study demonstrated that Emodin inhibits the redox-protective protein MTH1, resulting in elevated ROS levels and subsequent DNA damage within cancer cells. This elevation in ROS was associated with impaired cancer cell proliferation and the induction of cellular senescence, thereby underscoring the potential of ROS as a therapeutic target in oncological interventions [15]. Similarly, a study on ROS-based nanomaterials for cancer therapy explores the strategic design of nanomaterials to selectively generate ROS within tumor cells. This targeted approach aims to disrupt the redox equilibrium of cancer cells, thereby inducing cytotoxicity and enhancing the efficacy of cancer treatments [16]. Nevertheless, the dual role of ROS can lead to the development of chemoresistance, as cancer cells may adapt to oxidative stress by augmenting their antioxidant defenses. This underscores the complexity of targeting ROS in cancer therapy, given the simultaneous involvement of both pro-survival and pro-apoptotic mechanisms [17]. The role of ROS in tumors underscores the complex interplay between ROS and the tumor microenvironment. Modulating ROS levels may exert anti-tumor effects by inducing apoptosis, inhibiting angiogenesis, and enhancing drug resistance, thereby highlighting the potential of ROS modulation as a therapeutic strategy in cancer treatment [18]. Since ROS overproduction often leads to mitochondrial dysfunction, we evaluated the mitochondrial membrane potential (ΔΨm) using the JC-1 dye. With increasing concentrations of MBP346, a progressive shift from red JC-1 aggregates (high ΔΨm) to green monomers (low ΔΨm) was observed, indicating ΔΨm dissipation (Figure 5A–D). For instance, the percentage of green fluorescence in Cal33 cells increased from 4.69% ± 0.11% (DMSO) to 31.04% ± 4.36% (5 μmol·L⁻¹ MBP346). The role of ROS in the induction of apoptosis in tumor cells is a well-documented phenomenon, particularly through the mechanism of mitochondrial membrane potential (MMP) depolarization. Jiang et al. found that Furanodienone, a sesquiterpene from Rhizome Curcumae, induced mitochondrial dysfunction by promoting the decrease in mitochondrial membrane potential, thus inducing apoptosis of HepG2 cells [19]. Huang et al. Reported that 4-imidazolidinone derivatives promote the production of ROS in colorectal cancer cells and then cause the decrease in mitochondrial membrane potential to play an anti-colorectal cancer activity [20]. This process is crucial in the pathway leading to apoptosis, as it facilitates the release of pro-apoptotic factors such as cytochrome c from the mitochondria into the cytosol, thereby activating the caspase cascade. Du et al. demonstrated Tephrosin, a natural rotenoid isoflavonoid, induced apoptosis in pancreatic cancer cells through the generation of ROS. This generation of ROS resulted in the depolarization of the mitochondrial membrane potential, followed by the release of cytochrome c [21]. Mitochondrial depolarization is a critical early event in the intrinsic apoptotic pathway, facilitating the release of cytochrome c and other pro-apoptotic factors. Our data suggest that MBP346-induced ROS generation precedes and likely contributes to mitochondrial damage and subsequent apoptosis, a mechanism reported for other anti-cancer agents.

2.5. The ROS Scavenger NAC Attenuated MBP346-Induced Effects

To establish a causal link between ROS generation and the observed apoptotic effects, we employed the antioxidant N-acetylcysteine (NAC). Pre-treatment with NAC significantly attenuated MBP346-induced apoptosis in both Cal33 and Scc15 cells (Figure 6A–D). Concurrently, NAC effectively quenched the MBP346-triggered ROS surge (Figure 6E,F) and partially restored the mitochondrial membrane potential, as evidenced by an increased JC-1 aggregate/monomer ratio (Figure 6G,H). These rescue experiments confirm that ROS induction is a primary upstream event in MBP346’s mechanism of action. The role of ROS as a double-edged sword in cancer therapy is well-documented [15,16,17,18]. While basal ROS levels can promote tumorigenesis, pharmacological elevation beyond a cytotoxic threshold, as achieved by MBP346, represents a viable therapeutic strategy. NAC is well-known for inhibiting ROS production, as supported by many studies. Its capacity to reduce oxidative stress is crucial for its therapeutic use, especially in conditions with high ROS levels and oxidative damage. In the context of UVB-induced skin aging, NAC has been shown to significantly attenuate the effects of DUOX2 overexpression, which is linked to increased ROS production and the activation of NF-κB signaling pathways. By inhibiting ROS production, NAC effectively mitigates the cellular inflammatory response and the associated processes of skin aging [22]. Moreover, NAC’s capacity to inhibit ROS production has been demonstrated to reduce the activation of the NLRP3 inflammasome, a critical component in inflammatory responses, thereby diminishing inflammation and pyroptosis across various cellular models [23,24]. In tumor cells, NAC functions as an effective ROS scavenger, capable of inhibiting ROS overexpression and thereby reversing tumor cell death. In non-small cell lung cancer (NSCLC), NAC inhibited the production of ROS, thereby preventing the degradation of nuclear factor erythroid 2-related factor 2 (Nrf2) induced by dihydrodanshenone I, which subsequently suppresses tumor cell proliferation and survival [25]. Similarly, in ovarian cancer cells, NAC counteracts cell proliferation inhibition and apoptosis induced by the isothiocyanate Iberin by inhibiting ROS accumulation [26]. Moreover, NAC has exhibited comparable protective effects in other cancer types. In esophageal squamous cell carcinoma, NAC attenuates the downregulation of specificity protein 1 (SP1) and human telomerase reverse transcriptase (hTERT) expression induced by dihydroartemisinin by inhibiting ROS generation, thus safeguarding cells from apoptosis [26]. In hepatocellular carcinoma, NAC diminished apoptosis induced by the combination of dichloroacetic acid and sorafenib through inhibition of the ROS-c-Jun N-terminal kinase (JNK) pathway [27]. Our findings are consistent with studies where NAC reverses apoptosis induced by various compounds through ROS scavenging, underscoring the central role of oxidative stress in MBP346-mediated cell death.

3. Materials and Methods

3.1. Cell Culture

Human tongue squamous cancer cell lines Cal33, Scc15 and normal oral keratinocyte NOK were purchased from Nanjing Cobioer Biotechnology Co., Ltd., Nanjing, China and were cultured in DMEM medium (PM150210, Procell, Wuhan, China) with 10% fetal bovine serum (NTC-HK022, Natocor, Villa Carlos Paz, Argentina), 1% penicillin-streptomycin (C0222, Beyotime, Nantong, Jiangsu, China) at 37 °C and 5% CO₂ incubator (Thermo Model 3111, Shanghai, China).

3.2. MTT Assay

Cal33, Scc15, and NOK cells in the logarithmic growth phase were subjected to trypsin digestion, and the cell concentration was adjusted to 5 × 10⁴ cells/mL. Subsequently, 5 × 10³ cells per well were seeded into a 96-well cell culture plate and incubated overnight in an incubator at 37 °C and 5% CO₂. MBP346 was administered at final concentrations of 0, 0.05, 0.1, 0.5, 1, 2, 5, and 10 μmol·L⁻¹ to each well and incubated for 48 h. Following this, 10 μL of MTT reagent (ST1537, Beyotime, Nantong, Jiangsu, China) was added to each well, and the incubation was continued for an additional 4 h. The supernatant was then removed, 200 μL of DMSO was added to each well, and the plate was agitated at 80 rpm for 30 min. Optical density (OD) was measured at 570 nm using a multifunctional microplate reader (BioTek Cytation 5, Winooski, VT, USA). Cell viability was calculated using the formula: Cell Viability (%) = [(OD_ MBP346 − OD_blank)/(OD_DMSO − OD_blank)] × 100%.

3.3. Clone Formation Assay

Cal33 and Scc15 cells were seeded into a 6-well cell culture plate at a density of 1000 cells per well. The cells were maintained in a CO₂ incubator for 14 days, with the cell culture medium supplemented with 0, 1, 2.5, and 5 μmol·L⁻¹ of MBP346, respectively. Following incubation, the supernatant was removed, and the cells were fixed using 4% paraformaldehyde. Subsequently, the cells were washed with PBS and stained with 2 mL of crystal violet solution at room temperature for 30 min. The stain solution was then removed, and the cells were washed five times with PBS. Images were captured, and the number of colonies was analyzed.

3.4. Edu Staining Assay

Cal33 and Scc15 cells were seeded in a 12-well plate and treated with different MBP346 concentrations (0, 1, 2.5, 5 μmol·L⁻¹) for 24 h. Edu (KTA2031, Abbkine Scientific Co., Ltd., Wuhan, China) was then added to a concentration at 10 μmol·L⁻¹ for 2 h. Cells were fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with a Click-It reaction solution for 30 min. After DAPI staining, cells were imaged with a fluorescence microscope (OLYMPUS IX73, Nagano-ken, Japan), and Edu expression positive rates were calculated from five random fields per group.

3.5. Cell Cycle and Apoptosis Assay

Flow cytometry was used to analyze the cell cycle and apoptosis. For cell cycle analysis, 5 × 10⁵ Cal33 and Scc15 cells were seeded in 6-well plates and treated with 0, 1, or 5 μmol·L⁻¹ MBP346 for 24 h. Cells were then fixed with ethanol at 4 °C overnight, stained with a cell cycle detection kit (C548, DOJINDO), and analyzed by flow cytometry (Accuri C6, BD Biosciences, Milpitas, CA, USA). For apoptosis analysis, the same number of cells were treated similarly for 48 h, stained with an Annexin V-FITC/PI kit (C1062, Beyotime, China), and analyzed by flow cytometry.

3.6. Reactive Oxygen Species Assay

A total of 5 × 10⁴ Cal33 and Scc15 cells were seeded into a 24-well cell culture plate. Following a 24 h treatment with 0, 1, 2.5, and 5 μmol·L⁻¹ MBP346, the cell culture medium was aspirated. Subsequently, the cells were incubated with DCFH-DA probe and Hoechst 33,342 staining solution for 20 min. The samples were then imaged and analyzed using a fluorescence microscope (OLYMPUS IX73, Nagano-ken, Japan).

3.7. Mitochondrial Membrane Potential Assay

A total of 2 × 10⁵ Cal33 and Scc15 cells were seeded into a 6-well cell culture plate. Following a 24 h treatment with MBP346 at concentrations of 0, 1, 2.5, and 5 μmol·L⁻¹, the culture medium was refreshed. JC-1 staining solution was added and thoroughly mixed. The cells were incubated at 37 °C for 20 min, after which the supernatant was removed, and the cells were washed twice with JC-1 staining buffer. JC-1 fluorescence levels were analyzed by flow cytometry to quantitate MMP levels.

3.8. Western Blot

Cal33 and Scc15 cells were cultured in a 6cm dish, treated with varying MBP346 concentrations for 24 h, and washed with cold PBS. Total protein was extracted using 200 μL RIPA lysate with protease inhibitor. After quantifying the protein, 20 μg of total protein was subjected to 10-15% SDS-PAGE, transferred to a 0.22 μm PVDF membrane, and blocked with 5% skim milk for 2 h. Proteins including Cyclin D1 (55506, Cell signaling technology, CST, Danvers, MA, USA), Cyclin A2 (R24022, Zen BIO, Chengdu, China), CDK2 (10122-1-AP, Proteintech, Wuhan, China), P21 (2947, CST, Danvers, MA, USA), PARP (9532, CST, Danvers, MA, USA), Cleaved-Caspase 3 (9664, CST, Danvers, MA, USA), Cleaved-Caspase 8 (9496, CST, Danvers, MA, USA), MCL1 (220062, Zen BIO, Chengdu, China), Bcl2 (12789-1-AP, Proteintech, Wuhan, China), Bax (2774, CST, Danvers, MA, USA), α-Tubulin (3873, CST, Danvers, MA, USA) were incubated overnight at 4 °C. After washing with TBST, a 1:10000 diluted IRDye secondary antibody was applied for 1 h at room temperature, followed by TBST washes. Protein bands were detected using an Odyssey^® CLx imaging system (LI-COR, Lincoln, NE, USA).

3.9. Statistical Analysis

Statistical analyses were conducted utilizing GraphPad Prism 9.0 software, with experimental procedures replicated three times. Data are presented as $\overline{x}\pm s$, and intergroup comparisons were performed using ANOVA analysis. p < 0.05 was determined to be significant for statistical comparisons.