Curcumin analog HO‐3867 triggers apoptotic pathways through activating JNK1/2 signalling in human oral squamous cell carcinoma cells

Abstract Human oral squamous cell carcinoma (OSCC) is the common head and neck malignancy in the world. While surgery, radiotherapy and chemotherapy are emerging as the standard treatment for OSCC patients, the outcome is limited to the recurrence and side effects. Therefore, patients with OSCC require alternative strategies for treatment. In this study, we aimed to explore the therapeutic effect and the mode of action of the novel curcumin analog, HO‐3867, against human OSCC cells. We analysed the cytotoxicity of HO‐3867 using MTT assay. In vitro mechanic studies were performed to determine whether MAPK pathway is involved in HO‐3867 induced cell apoptosis. As the results, we found HO‐3867 suppressed OSCC cells growth effectively. The flow cytometry data indicate that HO‐3867 induce the sub‐G1 phase. Moreover, we found that HO‐3867 induced cell apoptosis by triggering formation of activated caspase 3, caspase 8, caspase 9 and PARP. After dissecting MAPK pathway, we found HO‐3867 induced cell apoptosis via the c‐Jun N‐terminal kinase (JNK)1/2 pathway. Our results suggest that HO‐3867 is an effective anticancer agent as its induction of cell apoptosis through JNK1/2 pathway in human oral cancer cells.


| INTRODUC TI ON
Oral squamous cell carcinoma (OSCC) is the most common oral cavity cancer with over 90% of cases. 1 Betel nut chewing, smoking and drinking are the most common risk factors for oral cancer in Taiwan. [2][3][4] OSCC often develops regional and distant lymph node. 5,6 Although surgery, radiotherapy and chemotherapy have applied for the treatments of OSCC, the prognosis of OSCC is still poor due to the recurrence and resistance to treatments. 7,8 According to its high incidence and mortality, development of new and effective treatments for OSCC is an urgent and unmet goal.
Programmed cell death (apoptosis) is a process of eliminating cells to maintain cell population and the normal growth of an organism during development, ageing and DNA damage. 9,10 On the contrary, defects in apoptosis result in neoplastic cells survival as dysregulated cell proliferation, increased cell motility and tumour progression. 11 Apoptosis is triggered through intrinsic, extrinsic and endoplasmic reticulum (ER) pathways. 12,13 Caspases drive apoptosis through activation of caspases initiator 8, 9 and 10 (initiators), caspases 3 and 7 (executioners), and caspases 1, 4 and 5 (inflammatorys). 10,14 The inhibitors of apoptosis proteins are known as the inhibitor of apoptosis protein (IAP) family, including cellular inhibitors of apoptosis 1, 2 (cIAP-1 and cIAP-2), X-linked inhibitor of apoptosis (XIAP) and survivin prohibit death receptor-mediated apoptosis through binding caspases. [15][16][17][18][19] The mitogen-activated protein kinase (MAPKs) (ERKs, JNKs and p38 signalling) also mediate progression of apoptosis; however, the role of MAPKs is relied on status of activated MAPKs, cell types, stimuli or cell stress. 20 Among them, activation of apoptotic via JNKs is through transcriptionally upregulating pro-apoptotic genes or phosphorylating mitochondrial pro-and anti-apoptotic proteins. 21 Curcumin analog HO-3867 is an antioxidant and antiproliferative compound as it is also known as an antitumour agent through blocking the Janus kinase/signal transducer and activator of transcription (JAK/STAT3) pathway and downregulation focal adhesion kinase and fatty acid synthase in human breast cancer, ovarian cancer and human pancreatic cancer cells alone or in combined with cisplatin or doxorubicin. [22][23][24][25][26][27][28][29][30][31] In addition, HO-3867 is also found to activate phosphatase and tensin homolog (PTEN) in human smooth muscle cells and in lung and heart tissues. 32,33 A more recent study found that HO-3867 transcriptionally converts mutant p53 protein to active wild-type p53 in cancer cells. 34 Nevertheless, HO-3867 is revealed to rescue suppression of placenta-specific protein 1 (PLAC1) level in ovarian cancer cells. 35 As OSCC has over 40% mutant rate of TP53 8,36 and mutant TP53 leads cancer progression, 37 we aimed to explore whether HO-3867 is capable of suppressing OSCC cell growth. We analysed its therapeutic effect on OSCC and to discover the inside mechanisms involved in HO-3867 induced apoptosis and attempted to define its underlying mechanisms.

| Cell culture and HO-3867 treatment
Being purchased from the American Type Culture Collection (Manassas, VA, USA) and the Japanese Collection of Research Bioresources (Osaka, Japan), the human OSCC SCC-9 and HSC-3 cells were supplemented with 10% FBS, 5 mL glutamine and 1% penicillin/streptomycin, and cultured in DMEM. HO-3867 was dissolved initially in 100% DMSO to achieve a 100 mM stock solution of HO-3867, and appropriate amounts of stock solution were subsequently added into the culture medium to achieve the indicated concentrations.

| Protein extraction and western blot analysis
To investigate the molecular mechanism further, the initiator and effector caspases and signalling pathways were detected using Western blot analysis. As described previously, 7.0 x 10 5 /dish SCC-9 and HSC-3 cells were cultured in 6 cm plates for 16 h and treated with different concentrations (0, 2.5, 5, 10 and 20 µM) of HO-3867 for 24 h, and the total cell lysates of SCC-9 and HSC-3 cells were prepared. 42,43 Blots were then incubated with a horseradish peroxidase goat anti-rabbit or anti-mouse.

| Statistical analysis
The SigmaStat 2.0 software package (Jandel Scientific, San Rafael, CA, USA) was applied for statistical analyses. Differences between untreated and HO-3867-treated groups were calculated by Student's t-test, and a p value of <0.05 was considered statistically significant. Each experiment was done in triplicate at least (n ≥ 3) were performed.

| Cytotoxicity of HO-3867 in human oral squamous cell carcinoma SCC-9 and HSC-3 cells
Curcumin and its analogs have been shown their anticancer effects, including suppression of oral squamous cell carcinoma ( Table 1). The main goal of this study is to examine whether the novel curcumin analog HO-3867 exhibits antitumour activity ( Figure 1A). We first performed cytotoxicity assay in human oral cancer SCC-9 and HSC-3 cells using MTT assay. We found HO-3867 effectively suppressed SCC-9 and HSC-3 cells growth at the dose region from 10 to 20 µM ( Figure 1B). Moreover, cell proliferation was assessed by using the CCK-8 method in SCC-9 and HSC-3 cells. As shown in Figure 1C

| HO-3867 induces apoptosis and sub-G1 fraction arrest of SCC-9 and HSC-3 cells
Given that HO-3867 potently suppressed cell viability in SCC-9 and HSC-3 cells, we assumed that HO-3867 may affect with cell cycle progression. To examine this hypothesis, we tested the cell cycle progression of oral cancer SCC-9 and HSC-3 cells by

| Analysis of activating extrinsic and intrinsic apoptotic processes by HO-3867 in SCC-9 and HSC-3 cells
The next question is how apoptosis was activated in HO-3867treated cells. Since curcumin can increase apoptotic levels through multiple signalling, such as TNF and caspase 8, 44 we hypothesized HO-3867 may affect signalling associated with apoptosis or cell survival. To test this hypothesis, we first examined human apoptosis array (ARY009, R&D systems) in SCC-9 cells. The human apoptosis array contains 35 proteins that associated with apoptotic process as our previous reports. 45 We identified a serial change in the protein amounts ( Figure 4A) We found that cleaved caspase-3 was increased by approximately 2.3-fold in HO-3867-treated SCC-9 cells compared to the vehicle control ( Figure 4B). Nevertheless, we also found that XIAP, cIAP and Survivin were reduced by 60% upon HO-3867 ( Figure 4B). These results indicate that HO-3867 induces apoptotic pathway through activating cleaved caspase-3 in human oral cancer cells.
The clarify the capability of HO-3867 triggering apoptotic pathway through activating cleaved caspase proteins, we measured both total and cleaved forms of apoptotic proteins, including caspase

| HO-3867 activates extrinsic and intrinsic apoptotic processes via JNK1/2 pathways in SCC-9 and HSC-3 cells
Mitogen-activated protein kinase pathway is known to mediate the apoptotic pathway. 20 To determine whether the treatment of HO-3867 could activate MAPK signalling in human oral cancer cells, we

CO N FLI C T O F I NTE R E S T
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.