Hispolon induces apoptosis in oral squamous cell carcinoma cells through JNK/HO‐1 pathway activation

Abstract Oral squamous cell carcinoma (OSCC) has a high recurrence rate and poor prognosis. Hispolon, a polyphenolic compound with antiviral, antioxidant, and anticancer activities, is a potential chemotherapy agent. However, few studies have investigated the anti‐cancer mechanism of hispolon in oral cancer. This present study used the cell viability assay, clonogenic assay, fluorescent nuclear staining, and flow cytometry assay to analyse the apoptosis‐inducing effects of hispolon in OSCC cells. After hispolon treatment, the apoptotic initiators, cleaved caspase‐3, −8, and − 9, were upregulated, whereas the cellular inhibitor of apoptosis protein‐1 (cIAP1) was downregulated. Furthermore, a proteome profile analysis using a human apoptosis array revealed the overexpression of heme oxygenase‐1 (HO‐1) by hispolon, which was determined to be involved in caspase‐dependent apoptosis. Moreover, cotreatment with hispolon and mitogen‐activated protein kinase (MAPK) inhibitors revealed that hispolon induces apoptosis in OSCC cells through activation of the c‐Jun N‐terminal kinase (JNK) pathway and not the extracellular signal‐regulated kinase (ERK) or p38 pathway. These findings indicate that hispolon may exert an anticancer effect on oral cancer cells by upregulating HO‐1 and inducing caspase‐dependent apoptosis by activating the JNK pathway.

or combination usage of chemotherapeutic agents, such as carboplatin, 5-fluorouracil (5-FU), cisplatin, and docetaxel, are often managed for oral cancer treatment. [10][11][12][13] These agents are able to induce sequence of cellular response that influence on tumour cell proliferation and survival. The deficient of apoptosis is the basis of tumour development; therefore, to activatee this programmed cell death become important to development of cancer treatment.
The initiation of apoptosis depends on the initiator and effector cysteine-containing asparte-specific proteases (caspases), which activate apoptosis signal and execute proteolysis. 14 Through the death receptor stimulation, the cleaved initiator caspase-8 subsequently provokes two pathways, the effector caspase-3/-7 pathway and mitochondrial pathway via Bid. 15 The cleaved initiator caspase-9 also essential for activating apoptosome complex via mitochondrial pathway. 16 These mechanisms resulted in cellular morphological changes of apoptosis such as membrane blebbing and DNA fragmentation. 17 Hispolon belongs to a polyphenol compound, which was found in several types of mushrooms such as Inonotus hispidus, Phallus linteus, and Phellinus igniarius. 18 To date, hispolon has been widely discovered to have the activities of anti-virus, 18 anti-oxidant, 19 anti-inflammatory, 20 hepato-protective, 21 and anticancer. 22 Huang et al. have shown the apoptotic effect of hispolon by modulating ERK pathway on hepatocellular carcinoma cells. 23 Previously, our study also reported that hispolon suppressed metastasis on cervical cancer cells 24 and nasopharyngeal carcinoma cells. 25 Although the anticancer effect of hispolon has been found in various of tumour types, the mechanism of hispolon still lack on OSCC. In our study, we explored the anti-proliferative effect on OSCC cell lines, including SCC-9 and HSC-3 cells, and analysed the underlying mechanism of hispolon-induced apoptosis. Our study merited to provide the evidence to support the availability of hispolon in chemotherapeutic treatment.

| Cells and cell culture
Human oral squamous carcinoma cell lines SCC-9 and HSC-3 were obtained from American Type Culture Collection (ATCC); human gingival epithelioid cell line Smulow-Glickman (SG) was established by Smulow, J.B. and I. Glickman. 26

| Clonogenic assay
The clonogenic assay has described previously. 27 Briefly, SCC-9 and HSC-3 cells were seeded in 6 cm dish in 80-90% confluent, respectively. Various doses of hispolon (25,50,75, and 100 μM) or vehicle control were treated after 16 h cells adhesion. After 24 h treatment, 500-cells of SCC-9 and HSC-3 cells were seeded in 6-well plates for 10 days cell growth, respectively. Fresh medium was replaced every 2-3 days. Finally, colonies were fixed with methanol and stained with 10% Giemsa staining buffer (Sigma Aldrich). Colony numbers of SCC-9 and HSC-3 cell lines were captured and counted under a microscope.

| Fluorescent nuclear stain assay
SCC-9 and HSC3-cells were seeded and treated different doses (25,50,75, and 100 μM) of hispolon or vehicle control for 24 h. Then, the cells were fixed with 4% paraformaldehyde and stained with 50 mg/mL DAPI staining buffer for 5 min, avoiding light. The fluorescence of cells was analysed by Olympus IX73 inverted fluorescence microscope.

| Cell apoptosis analysis
The SCC-9 and HSC-3 cell lines were seeded in 6-well plates at 80-90% confluent. Cells were collected after being treated with various doses of hispolon (25,50,75, and 100 μM) or vehicle control for 24 h.

| Human apoptosis proteome profiler analysis
A membrane-based antibody Human Apoptosis Array (R&D System) was used to detect 35 apoptosis-related proteins from SCC-9 cells after 24 h of hispolon (100 μM) or vehicle treatment. Image-Pro Plus software has been used to quantify each spot. Spot density is normalized to each reference points, and then normalized to the vehicle control group.

| Western blot assay
The SCC-9 and HSC-3 cell lines were collected after being treated with various doses of hispolon (25,50,75, and 100 μM) or vehicle control for 24 h. For Western blot, cells lysates were collected and separated by 10-15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

| RNA Interference analysis
Human siRNAs for non-specific control and HO-1 were obtained from Ambion Inc. The transfection of siRNA was using Lipofectamine RNAi MAX reagent (Invitrogen) by following a previously described process. 28,29 At 48 h after transfection, cells were used for HO-1 detection by Western blotting assay.

| Statistical analysis
All results were replicated at least three times and reported as mean ± standard deviation (SD). One-way analysis of variance with Tukey's honest significant difference was used to examine the variation between control group and treatment groups in all experiments. The present statistical measurements were performed by Sigmaplot v10.0. (Systat Software Inc.). A p < 0.05 was regarded as statistically significant.

| Hispolon possess cytotoxicity in OSCC cells
The chemical structure of hispolon as shown in Figure 1A. were less than 50 μM in clonogenic assay. The results indicated that hispolon not only has short term cytotoxicity in OSCC cells but also showed anti-tumorigenic effect on long term experiment.

| Hispolon trigger programmed cell death of OSCC cells
The morphological markers of apoptosis, including chromatin condensation and phosphatidylserine (PS) exposure, were then examined using DAPI and annexin V/PI stained, respectively. The condensed of DNA was found to increase after treatment with hispolon in both SCC-9 and HSC-3 cell lines (Figure 2A, arrows). With the combination of PS and annexin V-FITC pointed out early apoptosis and late apoptosis cells that were induced by hispolon treatment for 24 h in OSCC cells ( Figure 2B). These characteristics of apoptosis indicate that hispolon have the ability to induce apoptosis of OSCC cells.

| Hispolon increases HO-1 expression and induces caspase-mediated apoptotic cell death of OSCC cells
To clarify the apoptosis-related proteins that are regulated by hispolon in OSCC cells, we execute a proteome profiler human apoptosis array with the SCC-9 cell line. Compared to vehicle and hispolon at a dose of 100 μM, the protein level of cleaved caspase-3 and heme oxygenase-1 (HO-1) were increased while the cellular inhibitor of apoptosis protein-1 (cIAP-1) was decreased after hispolon treatment ( Figure 3A).
Using caspase-3 detection analysis, activated caspase-3 was found to increase with green fluorescence after treatment with different doses of hispolon (25, 50, 75 and 100 μM) ( Figure 3B). Similar to Figure 3A, the overexpression of HO-1 and suppression of cIAP1 were found in a dose-dependent manner of SCC-9 cells after hispolon treatment.
These results indicate that hispolon can stimulate OSCC cell apoptosis possibly through regulation of HO-1, cIAP1, caspases and PARP.

| HO-1 involves in hispolon-regulated apoptosis as an upstream regulator of OSCC cells
Elevated HO-1 has been reported to be involved in the apoptosis process in OSCC cells. 30,31 To understand the role of HO-1 in hispolon-induced apoptosis, the HO-1 RNA interference system is applied in subsequent experiments. First, we detected the HO-1 RNA interference system and confirmed that the hispolon-induced HO-1 protein level is down-regulated by treating with HO-1 siRNA ( Figure 4A,B). Subsequently, as shown in Figure 4C, hispoloninduced cytotoxicity has been rescued in the HO-1 siRNA treatment group compared to the control siRNA group. Meanwhile, Western blot analysis illustrated that the protein level of cleaved caspase-3, -8 and -9 which activated by hispolon was suppressed after HO-1 siRNA treatment ( Figure 4D,E). The above data reveal that HO-1 is involved in hispolon-regulated apoptosis in SCC-9 cells.

| Hispolon activates JNK/HO-1 signalling cascade in caspase-mediated apoptotic cell death of OSCC cells
Mitogen-activated protein kinase (MAPK) pathways are signalling cascades that connect the signal from the extracellular to the nucleus and regulate biological processes such as cell proliferation, cell differentiation and cell death. 32 To investigate the role of the MAPK pathway in hispolon-regulated apoptosis, we examined the activated form of MAPK proteins, phosphor-extracellular signal-regulated kinase (p-ERK)1/2, p-Jun kinase (JNK)1/2, and p-p38 using Western blot analysis. As shown in Figure 5A,B, p-ERK1/2, p-JNK1/2 and, p-p38 protein expression was significantly up-regulated after hispolon treatment in a dose-dependent manner. Subsequently, we added MAPK inhibitors, U0126 (ERK inhibitor), JNK-in-8 (JNK inhibitor) and SB203580 (p38 inhibitor) in the experiment to further elucidate the pathway that directly involved in hispolon regulation. Compared with hispolon alone, cleaved caspase-3, −8, −9 and HO-1 were suppressed in a group that cotreated with hispolon and JNK in 8 ( Figure 5C,D). However, the cleaved caspase-3, -8, -9 and HO-1 remained unchanged in hispolon combined with U0126 or SB203580. These results indicate that hispolon-regulated apoptosis via the JNK1/2 pathway in SCC-9 cells. Taken together, our results confirmed the underlying mechanism of hispolon in OSCC cells that hispolon-induced apoptotic cell death is regulated by cleaved caspase-3, -8 and -9, and HO-1 activation through the JNK signalling pathway ( Figure 6).

| DISCUSS ION
Hispolon, a bioactive constituent of traditionally used medicinal mushrooms, has been reported against 13 different types of cancer to date. 22 Due to the broad range of medicinal properties, we aimed to investigate the chemotherapeutic effect of hispolon on OSCC cells. In present the study, we demonstrated that hispolon lead to high cytotoxicity of the OCSS cell lines SCC-9 and HSC-3, but not the human gingival epithelial cell line SG. The mechanism of hispolon-mediated apoptosis is associated with the activation of cleaved caspase-3, -8 -9, and PARP. Moreover, our results showed that hispolon induces OSCC cell apoptosis through the elevation of HO-1 mediated by the JNK pathway. These data supported that the hispolon is a potential agent for OSCC treatment.
Apoptosis is beneficial to cancer therapy, which can be recognized with its characteristic morphological changes, including chromatin condensation, nuclear fragmentation, membrane blebbing and cell shrinkage. 33 Previous studies had reported that chromatin condensation and annexin V positive cells were increased   (HO-1, cIAP1, pro-and cleaved caspases-3, -8 and -9, and PARP) in the apoptosis pathways after 24 h hispolon treatment (25-100 μM) or vehicle treatment were confirmed by Western blots and quantified by Image J. Normalisation of protein levels is performed using β-actin to accurately quantify Western blots. Data from three independent experiments, each in triplicate, are represented as mean ± SD. *p < 0.05, compared to the vehicle control group. after hispolon treatment in nasopharyngeal carcinoma cells 34 and hepatocellular carcinoma cells, 23 which were consistent with our results. As a crucial role that participated in the initiation of apoptosis, caspases can be active by the death receptor pathway and the mitochondrial pathway (also known as extrinsic pathway and intrinsic pathway, respectively). 35 The combination of Fas or tumour necrosis factor (TNF) -related apoptosis-inducing ligand (TRAIL) receptors with Fas ligand or TRAIL caused Fas-associated death domain (FADD) and caspase-8 recruitment and triggers caspase-3 activation. 36 On the other hand, the release of cytochrome c from mitochondria initiated apoptosome complex formation (including cytochrome c, Apaf-1 and caspase-9), which also activated caspase-3 and caused downstream processes of apoptosis. 37 In the present study, caspase-9, -8 and -3 were activated after hispolon treatment in both SCC-9 and HSC-3 cells. These findings confirm those of earlier studies, such as hispolon caused cleaved caspase-3, -8 and -9 increasing in acute myeloid leukaemia 38 and gastric cancer cells. 39 The inhibitor of apoptosis proteins (IAPs) family members are represented a group of endogenic negative modulator of caspases, which are frequently overexpressed in tumour cells and promoted chemo-resistance and tumour survival. 40 Among the IAPs family, cIAP1 has been shown to suppress the pro-caspase-3 activation by interacting with apoptosome complex. 41 Furthermore, Zhang's study indicated that cIAP1/2 functionally suppressed caspase-8dependent cell death in vivo. 42 Similar to the above findings, our present data demonstrated that the down-regulation of cIAP1 and up-regulation of caspase-3, -8 and -9 were found in hispoloninduced cell apoptosis.
Heme oxygenase (HO) is a metabolic enzyme that generates carbon monoxide (CO), biliverdin, and iron in the rate limitation step of heme degradation. 43 Two main HO isoforms, HO-1 (inducible isozyme) and HO-2 (constitutively express isozyme), exert cellular HO activity with different biochemical properties. 44 . Normalisation of protein levels is performed using β-Actin to accurately quantify Western blots. Data from three independent experiments, each in triplicate, are represented as mean ± SD. *p < 0.05, compared to the vehicle control group; #p < 0.05, compared to the siCtrl-transfected group.

F I G U R E 5
The JNK1/2 signalling pathway is involved in hispolon-mediated HO-1 overexpression and cell apoptosis. (A) The protein expression levels of ERK1/2, p38, and JNK1/2 were detected by Western blot analysis with hispolon treatment (25-100 μM) for 6 h in SCC-9 cells. (B) Normalisation of phospho-MAPKs levels is performed using total MAPKs level to accurately quantify western blots. (C) SCC-9 cells were pretreated with U0126, JNK-in-8 or SB203580 for 1 h followed by vehicle or hispolon treatment (100 μM) treatment for an additional 24 h. The expression levels of cleaved caspase-3, -8 and -9 protein and HO-1 were detected by a Western blot analysis. Quantitative results of protein levels are shown in (D). Normalisation of protein levels is performed using β-actin to accurately quantify Western blots. Data from three independent experiments, each in triplicate, are represented as mean ± SD. *p < 0.05, compared to the vehicle control group; # p < 0.05, compared to the hispolon-treated group. modulating cellular autophagy, apoptosis, ferroptosis or pyroptosis programs. 46 Interestingly, previous reports showed that HO-1 may play a dual role in apoptosis among different types of cancer. The elevation of HO-1 is supposed to promote tumour cell proliferation and survival, such as in gastrointestinal tumours, 47 non-small cell lung cancer, 48 and prostate cancer. 49 Nevertheless, up-regulation of HO-1 is also found in drug-mediated tumour apoptosis, especially in head and neck squamous cell carcinoma. Our previous studies have indicated that curcumin analogues increased HO-1 expression in OSCC apoptosis through MAPK pathways, 28,29 which agrees with our present study that HO-1 is overexpressed in hispolon-induced apoptosis.
Among apoptosis, the MAPKs signalling pathway can be an activator or inhibitor, depending on different stimulus and cell types.
Huang et al. have reported that hispolon-induced caspase-mediated apoptosis through inhibition of ERK in hepatocellular carcinoma. 23 Additionally, hispolon has been found to promote the activation of ERK1/2, JNK1/2, and p38 in cells induced by apoptosis of nasopharyngeal carcinoma. 34 Similar results were obtained in the present study that p-ERK1/2, p-JNK1/2, and p-p38 increased after hispolon treatment. However, the combination of hispolon and MAPK inhibitor confirmed that the JNK pathway is the main pathway which involved in hispolon-regulated apoptosis in OSCC cells.
It is worth noting that the apoptotic effect of hispolon has been recorded to result in loss of mitochondrial membrane potential and caspase-3 activation in the KB cell line. 50

This study was supported by Chung Shan Medical University
Hospital, Taiwan (CSH-2022-D-003).

CO N FLI C T O F I NTE R E S T S TATE M E NT
The authors declare that there is no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of the present study are available from the corresponding author upon request.