7‐Epitaxol induces apoptosis in cisplatin‐resistant head and neck squamous cell carcinoma via suppression of AKT and MAPK signalling

Abstract Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. Although cisplatin‐based chemotherapy is commonly used in HNSCC, frequent development of cisplatin resistance is a potential cause of poor HNSCC prognosis. In the present study, we investigated the anticancer efficacy of a major paclitaxel metabolite namely 7‐Epitaxol in cisplatin‐resistant HNSCC. The findings revealed that 7‐Epitaxol exerts cytotoxic effects in cisplatin‐resistant HNSCC cell lines by inducing cell cycle arrest and intrinsic and extrinsic apoptotic pathways. Specifically, 7‐Epitaxol increased Fas, TNF‐R1, DR5, DcR3 and DcR2 expressions, reduced Bcl‐2 and Bcl‐XL (anti‐apoptotic proteins) expressions, and increased Bid and Bim L/S (pre‐apoptotic proteins) expressions, leading to activation of caspase‐mediated cancer cell apoptosis. At the upstream cell signalling level, 7‐Epitaxol reduced the phosphorylation of AKT, ERK1/2 and p38 to trigger apoptosis. In vivo results showed that animals treated with 7‐Epitaxol show antitumor growth compared to control animals. Taken together, the study demonstrates the potential anticancer efficacy of 7‐Epitaxol in inducing apoptosis of cisplatin‐resistant HNSCC cells through the suppression of AKT and MAPK signalling pathways.


| INTRODUC TI ON
Head and neck squamous cell carcinoma (HNSCC) refers to a group of cancers that originate primarily from the mucosal epithelium in the oral cavity, pharynx and larynx. These types of cancers can occur due to excessive consumption of tobacco and alcohol, or due to infection with human papilloma virus (HPV). Regarding prevalence, HNSCC is considered to be the 6th most common cancer worldwide, with 890,000 new cases and 450,000 deaths in 2018. 1 Although the 5-year survival rate of HPV-positive cancers has improved considerably over the past three decades, 2 no such improvement has been observed in older patients and those with HPV-negative cancers. 3 Surgery along with chemotherapy or radiation therapy is considered to be the gold-standard for treating HNSCC patients. 4 In patients with recurrent or metastatic HNSCC, treatment with monoclonal antibodies targeting epidermal growth factor receptor (EGFR), such as cetuximab, has shown promising outcomes when used in combination with radiation therapy. 5 However, studies have identified cetuximab as a less potent radiosensitizer than cisplatin in treating HPV-positive cancer patients. 6 Cisplatin-based chemotherapy is considered as the standard of care treatment for patients with HNSCC. 7 However, in locally advanced HNSCC, cisplatin treatment has been found to associate with limited efficacy and severe acute and late persistent toxicity. 8 Another disadvantage of cisplatin-based chemotherapy is frequent development of drug resistance, which is associated with reduced treatment efficacy and poor disease prognosis. 9,10 For cisplatin-refractory recurrent or metastatic HNSCC, immune checkpoint inhibitors pembrolizumab and nivolumab have shown promising outcomes in clinical trials. [11][12][13] Despite advancement in therapeutic interventions, no significant change in overall prognosis has been observed particularly for HPV-negative HNSCC. 4 Moreover, because of high treatment expenses, most of the targeted therapies, including immunotherapy, remain unavailable or inaccessible in many countries across the world. 8 Plant-derived bioactive compounds, also known as phytochemicals, have opened a new path towards prophylactic and therapeutic management of various cancer types, including HNSCC. 14 Paclitaxel, also known as Taxol, is a widely used natural chemotherapeutic agent derived from the evergreen tree, Taxus brevifolia. 15 This tricyclic diterpenoid compound has been found to exert anticancer effects in various types of cancer, including breast, ovarian, lung and brain cancers. [16][17][18][19] 7-Epitaxol is the major bioactive metabolite of paclitaxel, which is more stable and cytotoxic to cancer cells than paclitaxel. 20 In this context, one recent study has shown that the treatment with 7-Epitaxol induces apoptosis and autophagy in HNSCC via ERK signalling pathway. 21 Another study investigating the efficacy of mesenchymal stem cells (MSCs) for targeted drug delivery has revealed that MSCs incorporated with paclitaxel are capable of producing and releasing 7-Epitaxol together with paclitaxel without changing their biological activity. 22 These findings highlight that targeted delivery of 7-Epitaxol as an anticancer agent is possible via MSCs.
Despite being a superior anticancer agent than paclitaxel, anticancer properties of 7-Epitaxol have not been studied widely. In the present, we investigated the anticancer effect as well as mode of action of 7-Epitaxol in cisplatin-resistant HNSCC.
The final concentration of DMSO in the working solutions was less than 0.2%. Other chemical reagents used in the study including 3- propidium iodide (PI), RNase A, DAPI dye, protease inhibitor cocktail and phosphatase inhibitor cocktail were obtained from Sigma-Aldrich (St Louis, MO). The primary antibodies were purchased from Cell Signaling Technology (Danvers, MA). Specific inhibitor for AKT (LY294002), ERK1/2 (U0126), p38 (SB203580) and JNK1/2 (SP600125) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).

| Cell culture
Two HNSCC cell lines including SCC-9 and SAS (Japanese Collection of Research Bioresources Cell Bank, JCRB Cell Bank) were cultured in Dulbecco's Modified Eagle Medium-F12 (DMEM; Life Technologies, Grand Island, NY) supplemented with 10% foetal bovine serum, 0.1 mM nonessential amino acids, 1 mM glutamine, 1% penicillin/ streptomycin (10,000 U/ml penicillin and 10 mg/ml streptomycin), 1.5 g/L sodium bicarbonate and 1 mM sodium pyruvate. The cells were maintained at 37°C in a humidified atmosphere of 5% CO 2. The cisplatin-resistant cell line was established by continuous culturing of the cells with increasing concentrations of cisplatin (1-1000 nM) for 6 months. The culture medium of cells was supplemented with 1 μM cisplatin to maintain drug resistance.

| Cell cytotoxicity
The cells were cultured in 96-well plates at a density of 1 × 10 4 cells/ formed were dissolved in DMSO, and the absorbance was measured at 595 nm using spectrophotometry. The entire procedure was repeated three times using the same conditions to obtain three independent experimental replicates.

| Colony formation assay
The cell lines were seeded onto 6-well plates at a density of 5 × 10 3 cells/well and cultured overnight, followed by incubation with different concentrations of 7-Epitaxol (0, 25, 50 and 100 nM). The incubation medium was changed every 3 days. After 2 weeks, the colonies were fixed with 4% paraformaldehyde and then stained with 0.3% crystal violet solution. The stained colonies were dissolved in DMSO and counted by a stereomicroscope as previously described. 21

| Cell cycle analysis
The cell lines were seeded onto 6-well plates at a density of 5 × 10 5 cells/well and cultured overnight. The cells were next incubated with different concentrations of 7-Epitaxol for 24 h. Afterwards, the cells were collected, fixed in 70% ice-cold ethanol overnight and stained with PI buffer (4 mg/ml PI, 1% Triton X-100, 0.5 mg/ml RNase A in PBS) for 30 min in the dark at room temperature. The cell cycle distribution was analysed by BD Accuri C6 Plus flow cytometry (BD Biosciences, San Jose, CA), and the data were analysed using BD CSampler Plus software.

| DAPI staining
The cells were cultured in an 8-well glass chamber slide at a density of 1 × 10 4 cells/well overnight, followed by treatment with different concentrations of 7-Epitaxol for 24 h. Afterwards, the cells were collected, fixed in 4% formaldehyde for 30 min and stained with DAPI dye (50 ug/ml) for 15 min in the dark. The nuclear morphological changes were assessed in at least 500 cells and photographed using Olympus FluoView FV1200 Confocal Microscope (Olympus Corporation, Shinjuku, Tokyo).

| Annexin V/PI double staining assay
As previously described, 21

| Mitochondrial membrane potential measurement
As previously described, 21

| Western blot analysis
The cells were first treated with different concentrations of 7-Epitaxol for 24 h, followed by lysis with RIPA buffer containing protease/phosphatase inhibitor cocktails to obtain cellular proteins.
After measuring protein concentrations using BCA (Thermo Fisher Scientific) assay, the samples were separated using SDS-PAGE and transferred to PVDF membranes (Millipore, Bedford, MA). The membranes were then blocked with 5% nonfat milk in TBST for 1 h, followed by incubation with appropriate primary and secondary antibodies (dilution ratio 1:1000) overnight at 4°C. The protein bands were visualized using enhanced chemiluminescence with an HRP substrate (Millipore).

| In vivo antitumor growth effects of 7-Epitaxol on xenograft transplantation
As previously described. 23 For the experimental study of xenograft growth inhibition, 5-6-week-old male C57BL/6 mice The mean weight of the mice at the start of the study and at the end of the study did not differ significantly between the groups. All animal procedures were conducted according to the institutional animal welfare guidelines of the Institutional Animal Care and Use Committee (IACUC) of Changhua Christian Hospital.

| Statistical analysis
The experimental data are expressed as means ± standard deviation. Each experiment was replicated at least three times. The statistical analyses were conducted by anova, Tukey's post hoc test and Student's t-test. In all cases, a p value of < 0.05 was considered statistically significant. All statistical analyses were performed using Sigma-Stat 2.0 (Jandel Scientific, San Rafael, CA).

| Cytotoxic effects of 7-Epitaxol in cisplatinresistant HNSCC
Cytotoxicity of 7-Epitaxol ( Figure 1A As observed in Figure 1B, C, the treatment with 7-Epitaxol caused a dose-dependent reduction in cell viability in both cisplatin-resistant cell lines compared to that in untreated control cells. Similar effects were also observed in cells without resistance ( Figure 1D, E).
These findings highlight the cytotoxic effects of 7-Epitaxol in both cisplatin-resistant and non-resistant HNSCC cells.

| Effects of 7-Epitaxol on cell cycle regulation in HNSCC
Given the significant cytotoxic effects of 7-Epitaxol, we next investigated its impact on cell cycle regulation and proliferation. Firstly, we performed colony formation assay using 7-Epitaxol-treated cisplatin-resistant HNSCC cells to assess its ability to inhibit cancer cell proliferation. Afterwards, we performed flow cytometric analysis to cell cycle distribution of 7-Epitaxol-treated cells. As observed  Figure 2C, D). However, a variation in response to 7-Epitaxol was observed between two cell lines at the S phase ( Figure 2C, D).
Overall, the findings indicate that 7-Epitaxol reduces HNSCC proliferation by inducing cell cycle arrest.

| Effects of 7-Epitaxol on specific apoptotic pathways in HNSCC
Considering the apoptotic-inducing ability of 7-Epitaxol, we next thought of identifying specific apoptotic pathways involved. Given the significant involvement of mitochondria in mediating apoptosis, we firstly determined mitochondrial membrane potential in 7-Epitaxol-treated cells. Afterwards, we performed Western blot analyses to determine whether 7-Epitaxol can alter the expressions of proteins related to both intrinsic and extrinsic apoptotic pathways.
As observed in Figure 4A

| Effects of 7-Epitaxol on AKT and MAPK signalling pathways in HNSCC
To identify the cellular signalling pathways involved in 7-Epitaxol-mediated apoptosis, we determined the expressions of AKT and MAPK pathway components (ERK1/2, p38 and JNK) in 7-Epitaxol-treated cells. As observed in Figure 6A, B, 7-Epitaxol treatment significantly reduced the phosphorylation of AKT, ERK1/2 and p38 in both cell lines. In contrast, a significantly increased phosphorylation of JNK was observed when the cells were treated with higher concentration of 7-Epitaxol ( Figure 6A, B). For further validations, the cells were pretreated with specific inhibitors of ERK1/2 (U0126), AKT (LY294002), p38 (SB203580) and JNK (SP600125), followed by treatment with 7-Epitaxol. In these co-treated cells, we determined the expressions of PARP and caspases 3, 8 and 9.
Interestingly, we observed that the cotreatment with AKT, ERK1/2 and p38 inhibitors further increased the expressions of cleaved PARP and caspases 3, 8 and 9, as compared to 7-Epitaxol treatment alone ( Figure 6C-F). These findings confirm that 7-Epitaxol activates caspase-mediated apoptotic signalling pathways by suppressing the activation of AKT, ERK1/2 and p38.

| Significant anti-proliferative effects of 7-Epitaxol in the orthotopic graft model of HNSCC resistant to cisplatin
To test the effect of 7-epitaxol on tumour growth, the in vivo antitumor effect of 7-epitaxol was evaluated. Tumour volumes were determined by calliper measurements every 3 days. The control group of animals that transplanted Cis-SAS cancer cells showed a progressive increase in their tumour volumes. In mice treated with 7-epitaxol receiving 0.2 mg/kg with cisplatin, the mean tumour volume ( Figure 7A, B) and tumour weight ( Figure 7C) were significantly inhibited compared to vehicles treated. As illustrated in Figure 7D, no significant differences in body weight were detected among these groups. These results showed that animals treated with 7-Epitaxol show antitumor growth compared to control animals.

| DISCUSS ION
The present study has been designed to investigate the anticancer effects and mode of action of 7-Epitaxol, a major metabolite of paclitaxel with higher stability and cytotoxicity, in cisplatin-resistant HNSCC. There are many studies on the effect of paclitaxel, but research on 7-epitaxol in HNSCC is still limited. Paclitaxel is an agent that stabilizes microtubules and prevents cell mitosis in the G2/M phase. It is widely used to treat many cancers including breast cancer, ovarian cancer, endometrial cancer, lung cancer, bladder cancer and cervical cancer. 24  Aberrant activation of the AKT/PI3K and MAPK pathways is a major hallmark in many cancers. 31,32 Given the significance of these pathways in modulating cancer cell proliferation, survival, angiogenesis and metastasis, 33,34 we investigated whether 7-Epitaxol treatment alters the expressions of AKT/PI3K and MAPK pathway components. We observed a significant reduction in AKT, ERK1/2 and p38 phosphorylation ( Figure 6). Moreover, the treatment of cells with respective inhibitors of these components along with 7-Epitaxol caused a further activation of caspases and PARP compared to that caused by 7-Epitaxol treatment alone ( Figure 6).
In line with our findings, a previous study has shown that 7-Epitaxol induces apoptosis of HNSCC cells by reducing the phosphorylation of ERK1/2 and AKT. 21  F I G U R E 6 7-Epitaxol induces apoptosis by affecting the AKT, ERK1/2 and p38 MAPK pathway in Cis-SCC9 and Cis-SAS cells. Cell lines were pre-treatment with specific inhibitors (U0126, LY294002, SB203580, SP600125) or without for 1 h, then treated 7-E for 24 h. Western blotting was used to measure the expression of regulated proteins (A, B) the AKT and MAPK pathway, (C-F) the caspase pathway proteins. Quantitative relative density of each protein levels was normalized to beta-actin. Data are presented as mean ± SD (n = 3). *p < 0.05, compared with the control group. #p < 0.05, compared with the cells treated with 7-E (100 nM).

| CON CLUS ION
The study demonstrates the anticancer efficacy of a major pacli-

FU N D I N G I N FO R M ATI O N
This research received no external funding.

CO N FLI C T O F I NTE R E S T
The authors declare no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data availability statementThe data that support the findings of this study are available from the corresponding author upon reasonable request.