Hesperidin and cisplatin, the MTT cell viability kit (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), trypsin–EDTA solution, and phosphate-buffered saline (PBS) were sourced from Sigma Aldrich, Merck KGaA (Darmstadt, Germany). Eagle’s Minimum Essential Medium (EMEM) and foetal bovine serum (FBS) were obtained from ATCC (American Type Cell Collection, Lomianki, Poland). Paraformaldehyde 4% was supplied by Santa Cruz Biotechnology (Dallas, TX, USA). Hoechst 33342 dye and MitoTracker™ Red CMXRos were procured from Thermo Fisher Scientific (Waltham, MA, USA). Penicillin 100 U/mL–streptomycin 100 µg/mL and dimethyl sulfoxide (DMSO) were purchased from PanBiotech (Aidenbach, Germany). Cytation 5, Lionheart FX, and Gen5™ Microplate Data Collection and Analysis Software (v3.14) were purchased from BioTek Instruments Inc. (Winooski, VT, USA).

The cells used in the study, Detroit-562 (pharyngeal carcinoma), were cultured in EMEM supplemented with 10% foetal bovine serum (FBS) and 1% penicillin–streptomycin. The cell line was kept in a humidified incubator at 37 °C with 5% CO₂ and checked daily under a microscope. HSP was prepared in DMSO, making sure the final DMSO concentration did not go over 0.5%. The CIS stock solution was prepared by dissolving the compound in ultrapure water.

As a first step, to evaluate the impact of the samples in vitro, the MTT colorimetric technique was utilised to measure cell viability. Following the 24 h treatment period of Detroit 562 cells with HSP 5 µM, 10 µM, 25 µM, 50 µM, 75 µM, and 100 µM; CIS 1 µM, 2 µM, 5 µM, 10 µM, 15 µM, and 20 µM; and the combinatorial treatment HSP 5 µM + CIS 1 µM, HSP 10 µM + CIS 2 µM, HSP 25 µM + CIS 5µM, HSP 50 µM + CIS 10 µM, HSP 75 µM + CIS 15 µM, and HSP 100 µM + CIS 20 µM, 10 µL/well of MTT kit 1 was administered for 3 h while the plates were incubated. After this period, 100 µL/well of MTT solubilising solution was added for 30 min at room temperature. Utilising Cytation 5, the absorbance at 570 nm and 630 nm was determined.

To verify changes in cell structure after treating Detroit 562 cells with the samples of interest (HSP 5 µM, 10 µM, 25 µM, 50 µM, 75 µM, 100 µM; CIS 1 µM, 2 µM, 5 µM, 10 µM, 15 µM, 20 µM; and the combinatorial treatment HSP 5 µM + CIS 1 µM, HSP 10 µM + CIS 2 µM, HSP 25 µM + CIS 5 µM, HSP 50 µM + CIS 10 µM, HSP 75 µM+CIS 15 µM, HSP 100 µM+ CIS 20 µM) for 24 h, they were analysed using a Lionheart FX microscope and the Cell Analysis Tool included in the Gen5™ Microplate Data Collection and Analysis software (version 3.14) at 20× magnification in bright field.

To analyse mitochondrial and nuclear morphology, Detroit 562 cells were exposed to HSP, CIS, and the HSP+CIS association for 24 h. Following this treatment period, to perform the evaluation, the MitoTracker dye stock solution (1 mM) in DMSO was diluted in the specific culture medium to a final concentration of 300 nM, and after a 30 min incubation with the staining solution under standard culture conditions, the cells were rinsed to remove excess dye with culture medium. Afterwards, following MitoTracker staining, to preserve cellular structures, the cells were fixed for 10 min with 4% paraformaldehyde at room temperature, followed by thorough washing with PBS.

To examine nuclear alteration induced by the test samples, staining with Hoechst 33342 was performed. After the fixation process, Hoechst 33342 solution (diluted 1:2000 in PBS) was added to each well and incubated in the dark for 5–10 min. Then, the staining solution was removed, and the wells were washed three times with PBS.

Representative images were collected using the Lionheart FX automated microscope at 20× magnification. Image analysis was performed using Gen5™ Microplate Data Collection and Analysis software (version 3.14; BioTek Instruments Inc., Winooski, VT, USA). The entire protocol was performed in accordance with Talpos et al. [3].

Statistical analysis was performed using GraphPad Prism version 10.2.3 (GraphPad Software, San Diego, CA, USA, www.graphpad.com). The differences between the cells treated with HSP, CIS, and the HSP+CIS associative treatment groups, and the control were determined using one-way ANOVA and Dunnett’s multiple-comparison test. All statistically significant results were noted with “*”: *** p < 0.001; **** p < 0.0001.

To observe the cytotoxic potential of the samples, the first step was the determination of cell viability after 24 h of treatment using the MTT technique. The MTT test is one of the most widely used methods. It is based on the principle of transforming yellow MTT tetrazolium salt into purple formazan crystals. The colour change from yellow to purple gives a visual signal for cell viability. It is an indispensable tool for assessing metabolic activity and is used in areas such as toxicological research, cancer research, new drug discovery, and many others [19]. Initially, as illustrated in Figure 1, individual compounds were tested. Both HSP (5–100 μM) and CIS (1–20 μM) caused a dose-dependent decrease in viable cells. HSP at 5 µM reduced cell viability to 78%, while at the highest concentration tested (100 µM), viability decreased further to 57.29%. CIS, at the highest concentrations tested, 15 and 20 µM, significantly reduced the viability to 25% and 24%, respectively.

Subsequently, the combination of the two compounds was evaluated under the same conditions. When HSP 5 µM and CIS 1 µM, the lowest concentrations tested, were combined, cell viability reached almost 63%, followed by percentages such as 56%, 54%, 49%, and 29%, and at the highest concentrations tested in combination, represented by HSP 100 µM and CIS 20 µM, the cell viability percentage was reduced to 16%, as depicted in Figure 2.

The next step was to evaluate the morphology of the cells 24 h after treatment of Detroit 562 cells. Microscopic examination of the cell morphology can collect important information regarding potential mechanisms of action and the specific models of cell death caused by the compounds under investigation [20].

As can be observed in Figure 3, treatment of cells with HSP decreases cell confluence in a dose-dependent manner. In the case of CIS, cell confluence also gradually decreases, but cellular dysmorphologies (cell shrinkage, cell rounding) appear upon application of 10 µM CIS, which are accentuated up to a dose of 20 µM. However, the most notable and frequent cellular structure abnormalities can be observed with the application of the combined HSP + CIS treatment, where, in addition to the massive, dose-dependent decrease in confluence, dysmorphologies such as cell rounding, cell shrinkage, and detachment from the plate are evident starting with the lowest concentrations of HSP 5 µM + CIS 1 µM. The combination treatment led to the most significant morphological changes compared to the control.

The final step in the evaluation of the cytotoxic impact of HSP, CIS, and the combination HSP + CIS in Detroit 526 cells consisted of nuclear and mitochondrial assessments. The Hoechst method provides information on nuclear structure and behaviour, offering economic advantages, while MitoTracker tracks mitochondria [21]. Being the essential source of intracellular reactive oxygen species, mitochondria provide an important function in energy metabolism and control of stress responses. According to its multiple roles, altered mitochondrial function is associated with metabolic diseases, cancer, and other disorders associated with dysregulated cell death [20,21]. The nucleus provides a necessary regulatory and control centre within the cell. Due to its capacity to guide many aspects of cellular functions, including DNA replication, transcription, and RNA processing, any deviation in nuclear morphology and structure can significantly affect cellular activities [22].

Thus, the mitochondrial and nuclear appearance after 24 h of treatment of Detroit 562 cells is illustrated in Figure 4. As can be seen, HSP did not induce notable signs of cytotoxicity at the nuclear level up to a concentration of 25 µM. Once this concentration was exceeded, the 50–100 µM range caused nuclear condensation and slight mitochondrial condensation. In the case of CIS, condensation can be observed in the nuclei and mitochondria starting at 2 µM, with a gradual decrease in confluence. The strongest signs of cytotoxicity are observed with the combined treatment of HSP + CIS, where alterations can be observed starting at the lowest dose applied and gradually progressing. Finally, at the highest concentrations, the nuclei are small and condensed, and the mitochondria have an altered appearance.

Chemotherapy is currently the first line of treatment for cancer. Nevertheless, a significant challenge is posed by cancer resistance to chemotherapy, which affects patient well-being and treatment success. Given these considerations, identifying agents that sensitise tumour cells to chemotherapy is a developing and active area of research. Plant-based compounds have emerged as notable alternatives in this direction, helping to reduce side effects and sensitise tumours [23].

HSP is a botanical constituent that has repeatedly been shown to have anticancer properties and to improve results when combined with various chemotherapeutic agents. Along with CIS, through a delivery system, HSP improved the anti-cancer effect, increased apoptosis, and limited adverse effects in MDA-MB-231 cancer cells [24]. Korga et al. showed that both HSP and apigenin have the ability to enhance the toxic effects of doxorubicin, another chemotherapy agent, in HepG2 [25]. Dziedzic et al. investigated several flavonoids, including HSP (5–100 µM), in Detroit 562 cells. The results indicated that cell viability decreases with the applied dose. Furthermore, the same study showed that HSP stimulates cell death [26]. Another study identified that HSP suppresses the growth of oral cancer cells through apoptosis and mechanisms mediated by inflammatory signalling. It was found that treatment with HSP led to significantly lower levels of TNF-α, interleukin-1 beta (IL-1-β), IL-6, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and B-cell lymphoma 2 (Bcl-2), emphasising its inhibitory function in cell proliferation, migration, and inflammation processes. In parallel, hesperidin promoted BAX mRNA expression, suggesting an increase in cell death in the KB cell line [27]. Fatima Bijani and co-workers evaluated CIS together with crocin, a natural carotenoid, to examine the response to treatment in the HN5 cell line of human oral squamous cell carcinoma. According to the group’s research, it was found that smaller doses of crocin, used in combination with cisplatin, along with the observed anticancer effect, can reduce the toxicity of cisplatin in healthy tissue [28]. Similarly, in epidermoid cancer cells, A431 HSP, CIS, and the combination of the two were examined. According to cell viability results, after 48 h of treatment, the combination of the two compounds produced the most notable effects, with the lowest percentage of viable cells. It was also observed that the combination improved and promoted apoptosis through mitochondrial-mediated intrinsic pathways, which suggested that HSP acts as a potential adjuvant for CIS by amplifying its pro-apoptotic effects and reducing its resistance in vitro [29].

Overall, in the present study, HSP and CIS individually showed dose-dependent cytotoxic effects on the Detroit 562 cell line. Notably, when the two compounds were combined, the results indicated that the cellular response was enhanced, with cytotoxicity being much more prominent. However, future research directions should include the elucidation of the exact molecular mechanism of action of the combination treatment, as well as examination in other experimental models specific to OPSCC. Furthermore, this treatment option requires an assessment of biosafety in normal cell lines, as well as its potential irritant effect on tissues. By achieving these objectives in future lines of research, this type of combinatorial strategy may have the chance to be translated into clinical studies, based on proven preclinical results.