Impacts of potential anticancer agents based on pillar[5]arene for head and neck squamous cell carcinoma cells

This study was conducted to investigate impacts of potential anticancer (associated with apoptosis and caspase pathways) of two newly synthesized derivatives of pillar[5]arene, named as d-Q-P5 and p-Q-P5, on Squamous cell carcinomas of the head and neck (HNSCC) cells. The MTT method was used to determine the IC50 doses of the derivatives on HNSCC cells, and the changes in gene expression were analyzed by real-time polymerase chain reaction (qPCR). The apoptosis change was confirmed by flow cytometry analysis. The results showed that the d-Q-P5 and p-Q-P5 effectively inhibited the proliferation of the cells by upregulating proapoptotic genes (Bax, Bad, p53, Bak, and Apaf-1) and genes involved in the caspase pathway (Casp2, Casp3, and Casp9), while downregulating the antiapoptotic gene (Bcl-2). This study is the first to demonstrate the potential anticancer effects of these two agents on HNSCC cells by positively regulating apoptosis gene expression.


Introduction
Cancer has become an important public health problem with its incidence increasing every year. One of these cancers is squamous cell carcinoma of the head and neck (HNSCC), which consists of a group of tumors originating from the epithelium of the oral cavity and pharynx (Solomon et al. 2018). HNSCCs are the sixth most common cancer worldwide, and their incidence is estimated to rise by 30% with about 1.08 million new cases per year by 2030 (Ferlay et al. 2019;Johnson et al. 2020), with varying prevalence across different regions (Bray et al. 2018). In Southeast Asia and Australia, the high prevalence of HNSCCs is associated with exposure to carcinogenic products (Hashibe et al. 2007), while in the USA and Western Europe, the increasing incidence is linked to human papillomavirus (HPV) infections (Mehanna et al. 2013;Michaud et al. 2014). The International Agency for Research on Cancer (IARC) of the World Health Organization (WHO) has identified various risk factors in the development of HNSCCs, including tobacco and tobacco products, alcohol consumption, environmental pollution, and HPV (Human Papilloma virus) and EBV (Epstein-Barr virus) infections (Thun et al. 2010). The molecular characterization of cancer cells, such as HNSCC, has led to the development of better treatment options including chemotherapy, immunotherapy, and cancer vaccines (Sarode et al. 2022;Wang et al. 2021). However, progress in treating HPV-negative HNSCC has been limited over the past decade. As a result, it is necessary to investigate new candidate agents for treating HNSCC. Although several treatment options exist for localized HNSCCs, such as surgery, radiotherapy, chemotherapy, immunotherapy, and radiotherapy plus immunotherapy (Bonner et al. 2006;De Felice and Bossi 2022;de Sousa and Ferrarotto 2021;Johnson et al. 2020), the increasing incidence of HNSCCs underscores the need for new therapeutic agents.
Cancer drug research in recent years is targeted research and is aimed at eliminating specific cancer cells. It is thought that one of these candidates has pillar[n]arenes derivatives. Pillar[n]arenes have emerged as a new and important class of macrocyclic compounds since their discovery in 2008, with potential applications in pharmacology, biochemistry, and medicine (Bastug et al. 2020;Kursunlu et al. 2019Kursunlu et al. , 2020Ogoshi et al. 2008). It is known that the functionalization of macrocyclic compounds with organic groups with pharmacological functions plays a very important role in the anticancer activity of these substances (Gunes et al. 2020). Quinolines and their derivatives are known to exhibit various biological activities, including antimalarial, analgesic, antibacterial, anti-inflammatory, anticancer, antineoplastic, antifungal, and anthelmintic activity. These compounds are found in several natural products and are used as building blocks in general synthesis, as well as in agrochemical applications (Kursunlu et al. 2017). Therefore, in this study, we synthesized quinoline-derived pillar[5]arene molecules. Previous research has examined the cytotoxic impact on the apoptotic process of various forms of pillar[5]arene on cancer cell lines such as LnCap, Caco-2, MDA-MB-231, and HepG2 (Gunes et al. 2020;Liman et al. 2022). However, no studies have yet been conducted on the effects of pillar[5]arene derivatives on HNSCC cell lines. Therefore, it is essential to investigate these compounds as potential new anticancer agents. The goal of this study is to explore the effects of two synthesized potential anticancer agents, d-Q-P5 and p-Q-P5 pillar[5]arene derivatives, on the processes of apoptosis and caspase pathways in HNSCC cancer cell lines by examining changes in gene expression.

The synthesis of d-Q-P5 and p-Q-P5
The compounds were synthesized with a known procedure (Karaselek et al. 2023). The synthesis steps of d-Q-P5 and p-Q-P5 macrocyclic compounds have been shown in Fig. 1. In 1 H-NMR of target compounds, the proton peaks of d-Q-P5 and p-Q-P5 agents appeared in the expected ppm values. The aromatic region protons on the main skeleton of the molecules and quinoline units appeared around 6.99-8.55 ppm while five protons of bridge-CH 2 fragments were observed at 3.85-3.80 ppm. These results were also supported with the 13 C-NMR spectra of the compounds.

Cell culture
The HNSCC cancer cell lines were procured from the American Type Culture Collection (ATCC, Rockville, Maryland 20,852, USA). These cell lines were cultivated in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS, Gibco, Thermo Fisher) and 1% penicillin/streptomycin antibiotic solution (Sigma-Aldrich, St. Louis) at 37 °C and 5% CO 2 . The HNSCC cancer cell lines were treated with solutions of d-Q-P5 and p-Q-P5 at determined concentrations (15.62, 31.25, 62.5, 125, 250, and 500 µM) for 24, 48, and 72 h. In the control group where d-Q-P5 and p-Q-P5 were not administered, at least five wells were used for each dose.

Cytotoxicity assay
The MTT assay was carry out to measure the cytotoxicity of d-Q-P5 and p-Q-P5 in the HNSCC cell lines at different fixed times (24 h, 48 h, and 72 h). The proportion of live (viable) was determined using an absorbance value of 480 nm in 1 × PBS. The IC 50 values for d-Q-P5 and p-Q-P5 were calculated after the initial 24 h measurement. Thermo Scientific equipment was used for the measurements.

Isolation of RNA and cDNA synthesis
QIAzol (Qiagen, India) was used to extract total RNA from cells and then the cDNA synthesis was carried out using the Revertaid First Strand cDNA Synthesis Kit according to established procedures.
The qPCR reaction was carried out using the HibriGen 2X SYBR Green Master Mix. For each gene, a reaction was prepared at 10 µL containing 5 µL of the SYBR green mix, 5 pmol of primary pairwise, and 2 µL of cDNA. Initial activation from the PCR steps was set at 95 °C for 10 s in denaturation, then each step of 40 cycles followed by denaturation of 95 °C for 15 s, annealing for 30 s at 57 °C.

Apoptosis analysis by flow cytometry
The impact of apoptosis with d-Q-P5 or p-Q-P5 in HNSCC cells was evaluated using the APC Annexin V Apoptosis Detection Kit with Propidium Iodide (PI) from BioLegend Inc., San Diego. After determining the appropriate application time and dose, the cells were washed out of cold cell staining buffer at 1 × 10 6 cells/mL, and the resulting pellet was suspended in 100 µL of Annexin V binding buffer. APC Annexin V/PI was added (vol 1:2) to the suspension and the cells were incubated for 15-20 min/25 °C in a room without light. After the incubation, 400 µL of Annexin V conjugate/ binding buffer was added to each sample. The cell count was performed using a Becton-Dickinson Canto II flow cytometry device and by the FACSDiva software v 6.1.3.

Statistical analysis
The reference gene, GAPDH, was utilized for normalization in this study. To determine relative gene expression, the comparative Livak's ΔΔCT method was employed (Livak and Schmittgen 2001). Cancerous (treated with p-Q-P5 and d-Q-P5) and control group comparisons were made using the student's t-test in the Statistical Package for Social Sciences software, version 21 (IBM SPSS Corp.; Armonk, NY, USA). ΔCt values were utilized in the analyses after normalization, and p < 0.05 value was considered as the statistical significance. Volcano plots were generated using VolcaNoseR (Goedhart and Luijsterburg 2020).

Cytotoxicity analysis
To evaluate the impact of d-Q-P5 and p-Q-P5 on HNSCC cell line proliferation, an MTT assay was performed. The IC 50 values for d-Q-P5 and p-Q-P5 were identified as 500 μM and 62.5 μM, respectively, for a 24-h period, based on the MTT cell viability analysis (as shown in Fig. 2).

qPCR results of apoptotic and caspase pathway analysis
The mRNA expressions of all the genes were evaluated with analysis results of qPCR in the HNSCC cell lines after treatment with d-Q-P5 and p-Q-P5. The qPCR data are presented in Fig. 3. Expression results of qPCR showed that treatment with p-Q-P5 increased the proapoptotic genes such as Bax, Bad, Bak, p53, and Apaf-1 by 1.42-fold, 5.41-fold, 3.90fold, 7.04-fold, and 3.04-fold, respectively, while decreasing the antiapoptotic gene Bcl-2 by 1.88-fold when compared to the non-treated group. Although the increase in Bax and Bcl-XL genes was not significant (p > 0.05), the increase in Bad, Bak, p53, Apaf-1, or the decrease in Bcl-2 was significant (p < 0.05).
When comparing the expression of caspase pathways between the p-Q-P5-treated and non-treated groups, it was found that p-Q-P5 treatment increased the expressions of Casp2 (3.85-fold), Casp3A (1.23-fold), and Casp9 (3.16fold), while causing a decrease in the Casp6 (1.38-fold) and Casp12 (1.02-fold). The increase in caspases was significant (p < 0.05), but the decrease in Casp6 and Casp12 was not significant (p > 0.05) as shown in Fig. 3.

Apoptosis analysis
The impact on the apoptotic process of d-Q-P5 and p-Q-P5 on HNSCC cell lines was assessed using Annexin V/PI flow cytometry. The cells that entered early apoptosis were described as Annexin V + , and the cells that entered late apoptosis were described as Annexin V + and PI + cells. In addition to apoptotic cells, necrotic cells were described as the PI + group. It was gained from the flow cytometry results that the rate of early apoptosis was 8.9% and 10.2%, the rate of late apoptosis was 0.1% and 2.1%, and the rate of necrotic cells was 0.1% and 1.9% for each control group of d-QP5 and p-QP5, respectively (Fig. 5). In contrast, in the group treated with p-Q-P5 at a concentration of 62.5 µM, the rate of early apoptosis was 66.7%, the rate of late apoptosis was 22.1%, and the rate of necrotic cells was 2.5%. In the group treated with d-Q-P5 at a concentration of 62.5 µM, the rate of early apoptosis was 47.6%, the rate of late apoptosis was 29.9%, and the rate of necrotic cells was 1.1% (Fig. 5).

Discussion
In this study, we examined two forms of pillar[5]arene, a novel molecule, to determine their effects on gene expressions involved in apoptosis and caspase pathways in HNSCC cell lines. Our findings suggest that the synthesized macrocycles have the potential to be developed into next-generation anticancer drugs by promoting apoptosis and caspase pathway. Pillar[5]arenes are a type of macrocyclic molecule that has gained attention in supramolecular chemistry due to their unique chemical properties (Cragg and Sharma 2012;Fahmy et al. 2021;Guo et al. 2018;Ogoshi et al. 2008;Wang et al. 2022). Recent studies have suggested that they may also have potential as anticancer agents, although research in this area is still limited. One water-soluble form of pillar [5] arene-glutamamide has been found to have minimal impact on cell viability in both normal and cancer cells (Guo et al. 2017), while another study has reported that pillar[5]arene selectively targets cancer cell membranes, leading to their destruction (Chang et al. 2019). Our own research team conducted a study to investigate the cytotoxic and apoptotic process of two different types of pillar[5]arene (asym-P5 and sym-P5) on various cancers (hepatocellular carcinoma, adenocarcinoma, colon carcinoma, and prostate cancer). In that study, it was reported that the ratio of apoptotic cells increased significantly along with the decrease of the cell viability in all cell lines and a dose-dependent manner (Liman et al. 2022). In another study conducted by our study team investigating the anticancer effects of pillar[5] arene on MCF-7 human breast cancer cells, it was reported that it had an anticancer effect by increasing the expression of genes in the pathway of apoptosis (Gunes et al. 2020). Guo et al. reported that the hydrazide-pillar[5]arene complex inhibited the proliferation of hepatoma carcinoma cells. In addition, in the same study, it was shown that the undesirable effects of using hydrazide alone were reduced when used as a hydrazide-pillar[5]arene complex. In that study, it has been shown that pillar[5]arene is a versatile molecule that can be used with other agents in addition to its anticancer effect (Guo et al. 2021). Although there are studies on pillar[5]arene on many cell lines in the literature, there is no study on the HNSCC cancer cell lines. Our study is the first study in this field and revealed the anticancer effect of d-Q-P5 and p-Q-P5 in the HNSCC cancer cell line. The anticancer effects of p-Q-P5 were evaluated by MTT analysis (Liman et al. 2022). IC 50 values for asym-P5 in HepG2, LnCap,and HEK293 cell lines were 22.4,21.57,23.42,38.01,and 26.57 μM, respectively. IC 50 values for sym-P5 in HepG2, MDA-MB-231, Caco-2, LnCap, and HEK293 cell lines were 266.94, 156.41, 181.55, 214.04, and 191.65 respectively (Liman et al. 2022). In our study, the IC 50 dose was determined as 62.5 μM for p-Q-P5 and 500 μM for d-Q-P5 at 24 h. Although the IC 50 dose of p-Q-P5 was similar to other studies in the literature, the IC 50 dose for d-Q-P5 was higher. Since the new drug, p-Q-P5 has a lower IC 50 dose; it is more preferred for HNSCC than the IC 50 dose of phenformin in the HNSCC cancer cell line was found as 1.5 mM and 3 µg/mL in different studies (Kaomongkolgit et al. 2011;Seo et al. 2019).
Both p-Q-P5 and d-Q-P5 showed an excellent anticancer effect on HNSCC cancer cell lines by increasing gene expression. The apoptosis occurs either by the cell's intrinsic or extrinsic pathway. Cytochrome c released from mitochondria is an important intrinsic component and the Bcl-2 protein family has vital importance in this process. In addition, the tumor suppressor protein p53 causes the activation of proapoptotic genes like Bax in cells with DNA damage. Cytochrome-c, which forms a complex with Apaf-1 in the cytosol, cleaves the initiator caspase-9. Then, the executioner caspases 3, 6, and 7 which induce cell death are activated (Wuest et al. 2019). In our study, the expression of Bad, Bak, Bax, p53, Apaf-1, Casp2, Casp3, and Casp9 was upregulated in the p-Q-P5 group compared to the control group, whereas Bcl-2, Caps6, and Casp12 expressions were downregulated. In the group to which d-Q-P5 is added, Bad, p53, Bak, Casp2, Casp3, Caps6, and Casp9 gene expressions were upregulated, while Bcl-2, Apaf-1, and Casp12 were downregulated. It was conspicuous that the derivatives of pillar[5]arene can induce apoptosis by stimulating caspase activation with proapoptotic gene expressions in the HNSCC cancer cell line. However, since the fold increase in these gene expressions was higher in the p-Q-P5 group and it has a lower IC 50 dose, the p-Q-P5 may be considered a better anticancer agent.

Conclusion
The aim of this study was to investigate the impacts of potential anticancer of two different pillar[5]arene molecules, d-Q-P5 and p-Q-P5, in HNSCC cancer cells. The results showed that these derivatives could induce apoptosis and inhibit tumor growth by upregulating the genes in the apoptosis pathway. These findings suggest that d-Q-P5 and p-Q-P5 have the potential to be used as anticancer agents for HNSCC. Nevertheless, further research is necessary to define the mechanisms and effectiveness of these compounds in treating cancer.
Author contributions SK and TD contributed to anticancer activity experiments. MAK contributed to apoptosis experiments, methodology, writing-review & editing. ANK contributed to synthesis, methodology, writing-review & editing. MO contributed to characterization, methodology, writing-review & editing. The final version of the manuscript was read and approved by all the authors.
Funding The study was supported by Necmettin Erbakan University (Project Number: 221218015).

Availability of data and materials
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.