CLEFMA induces intrinsic and extrinsic apoptotic pathways through ERK1/2 and p38 signalling in uterine cervical cancer cells

Abstract Although concurrent chemoradiotherapy is the cornerstone of treatment for locally advanced or recurrent uterine cervical cancer, treatment fails at a high rate. Therefore, the development of novel targeting agents is critical. This study investigated the action of CLEFMA, a potent, synthetic curcumin derivative, on cervical cancer cells and its mechanism of action. We found that CLEFMA negatively regulated the viability of cervical cancer cells, involving induction of cell apoptosis. Cleaved caspase‐3, cleaved poly(adenosine diphosphate‐ribose) polymerase, cleaved caspase‐8, and cleaved caspase‐9 expression were increased by treatment with CLEFMA. After U0126 (ERK1/2 inhibitor) and SB203580 (p38 inhibitor) were applied as cotreatment with CLEFMA, the expression of cleaved caspase‐8, ‐9, and ‐3 was reduced significantly. In conclusion, CLEFMA activates both extrinsic and intrinsic apoptotic pathways through ERK1/2 and p38 signal transduction in cervical cancer cells.


| INTRODUC TI ON
Uterine cervical cancer is the fourth most common cancer diagnosis in women and the most lethal malignancy worldwide, and approximately 580,000 patients received diagnoses in 2018. [1][2][3][4] The annual global incidence of cervical cancer is approximately 14.0 per 100,000 women. 5 However, current treatments are still lacking; platinum-based chemotherapy in combination with radiation and concurrent chemoradiation are the preferred treatments for locally advanced or recurrent cervical cancer, but local relapse is often accompanied by distant failure. 6,7 Therefore, novel agents targeting particular intracellular signalling routes relating to the unique properties of cervical cancer cells are urgently needed.
CLEFMA is a synthetic curcumin analogue (curcuminoid) that was developed as an anticancer agent. 8,9 Curcumin, a bright yellow, natural polyphenolic compound, is extracted from the rhizomes of the Curcuma longa plant. [10][11][12] It exhibits several pharmacological characteristics, such as anti-inflammatory, antioxidant, wound-healing, and antidiabetic properties. 13,14 Moreover, curcumin may protect against a number of cancers by targeting various biological pathways involved in apoptosis, cell cycle arrest, and activity against several protein kinases. [15][16][17][18][19] However, the poor water solubility, rapid metabolism, short half-life, chemical instability, and poor intestinal absorption of curcumin may lead to low plasma levels and thus low bioavailability. 20 To overcome curcumin's low bioavailability, several approaches, such as adjuvant application and the use of its structural derivatives (e.g. CLEFMA), have been proposed. 21 CLEFMA possesses superior bioavailability and solubility to those of curcumin; it also exerts antiproliferative effects on cancer cells 8,22 and is a potential active anticancer compound. 23,24 Moreover, CLEFMA was demonstrated to have the potential to induce cell death in lung adenocarcinoma cells. 9 To date, no study has investigated the effect of CLEFMA on cervical cancer cells. Therefore, the present research investigated whether and how CLEFMA induces cell death in cervical cancer cells as well as the underlying mechanisms.

| Uterine cervical cancer cell culture
HeLa and SiHa cervical cancer cell lines were obtained from the ATCC. Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 ng/ml streptomycin, and 100 ng/ml penicillin was used to culture the cells. For culturing, the HeLa and SiHa cells were maintained at 37°C in an incubator with a 5% CO 2 humidified atmosphere. 25
Thereafter, the HeLa and SiHa cells, along with the floating nonviable cells, were harvested through trypsinization. FITC Annexin V Apoptosis Detection Kit I was used to perform an apoptosis assay in accordance with the manufacturer's protocols (BD Biosciences). 27 Phospholipid phosphatidylserine molecules were translocated from the inner face of the cell membrane to the outer surface of the cell immediately after apoptosis was initiated. Annexin V, a fluorescent conjugated protein exhibiting a high affinity for the phospholipid phosphatidylserine, was applied as a stain to detect apoptosis at an early stage, and propidium iodide (PI) was applied as a stain to detect DNA fragmentation at a later stage. Subsequently, the percentage of cells undergoing apoptosis was determined through flow cytometry. 28 Quantitative analysis was used to detect early (annexin V positive and PI negative) and late apoptosis (annexin V positive and PI positive) in the HeLa and SiHa cancer cells.

| Human apoptosis array
To delineate the underlying mechanisms of induced apoptosis, a Human Apoptosis Array Kit (R&D Systems) was employed to detect protein lysates from cervical cancer cells treated with a vehicle or 20 μM CLEFMA for 24 h, in accordance with the manufacturer's protocols. The kit was used to detect 35 human apoptosis-related proteins simultaneously. 29 Proteins were captured on the nitrocellulose membrane, identified with biotinylated detection antibodies, and visualized using chemiluminescent detection reagents.

| Protein extraction, detection, and Western blotting
To understand the underlying molecular mechanism, the initiator and effector caspases and signalling pathways were examined using Western blot analysis. We seeded 6.5 × 10 5 HeLa and 7 × 10 5 SiHa cervical cancer cells in 6-cm plates; the cells were cultured for 24 h and subsequently treated with different concentrations (0, 5, 10, 20, or 40 μM) of CLEFMA for 24 h; the total cervical cancer cell lysates were subsequently prepared as previously described. 30 Western blotting was performed using primary antibodies against caspase-3, -8, and -9; cleaved caspase-3, -8, and -9; poly(adenosine diphosphate-ribose) polymerase (PARP); and cleaved PARP. By using the specific antibodies binding the unphosphorylated and phosphorylated forms of the three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinases (JNK)1/2, and p38, or their inhibitors (U0126, JNK-IN-8 and SB203580, respectively), the total cell lysates were obtained from the 6.5 × 10 5 HeLa and 7.0 × 10 5 SiHa cells that were cultured for 24 h and then treated or not treated with the three MAPK inhibitors for 2 h and finally treated with various concentrations of CLEFMA. As previously described, 31 the Western blots were incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse immunoglobulin G for 1 h, and the intensity of each band was measured through densitometry.

| Statistical analysis
Statistical analysis of the data was performed using one-way analysis of variance with a post hoc Scheffe test for comparisons of more than two groups. Each experiment was conducted in triplicate. A two-sided p value of <0.05 was considered statistically different.

| Effects of CLEFMA on HeLa and SiHa cervical cancer cell viability
The MTT assay revealed that the cell viability of the HeLa cervi-

| Flow cytometric analysis of CLEFMA-induced HeLa and SiHa cervical cancer cell apoptosis
To clarify the mechanism underlying the CLEFMA-induced reduction of HeLa and SiHa cancer cell viability, cell apoptosis was analysed using flow cytometry with fluorescein isothiocyanate-labelled annexin V and PI after treatment with 0, 5, 10, 20, or 40 μM CLEFMA in these cancer cells for 24 h (Figure 2A). Results indicated that the percentage of apoptotic cells exhibited a statistically significant increase in a concentration-dependent manner in both HeLa and SiHa cells ( Figure 2B,C).

| CLEFMA increases cleaved caspase-3 expression in HeLa cervical cancer cells
To clarify which apoptosis-related proteins' expression was in-

| DISCUSS ION
With advancements in cancer therapy, platinum-based chemotherapy with irradiation has become the preferred treatment for locally advanced or recurrent cervical cancer. However, treatment still fails a relatively high rate. 7  regulating the JNK and p38 pathways. 34 However, no research has examined the anticancer effect of curcumin on cervical cancer cells exerted through the inhibition of cell proliferation and the promotion of apoptosis through the ERK, JNK, and p38 signalling pathways. 35 Despite its efficacy and safety, curcumin has limited bioavailability because it is poorly absorbed and rapidly metabolized. 36 Accordingly, we chose a structural analogue, CLEFMA-which possesses superior bioavailability and solubility to those of curcumin-as the target for our research. 9 Apoptotic mechanisms are triggered by extrinsic apoptotic signals, which mainly respond to extracellular stimuli and are associated with death receptors, and by intrinsic apoptotic processes, which are activated by modulators within the cell itself and relate to the mitochondria. The induction and execution of apoptosis signal transduction require the caspase cascade. 37,38 In this study, a human apoptosis array kit was employed to determine which apoptosisrelated proteins were affected by CLEFMA in cervical cancer cells and revealed significantly increased expression of cleaved caspase-3.
In addition to cleaved caspase-3 and cleaved PARP, whose expression was significantly increased in HeLa and SiHa cancer cells, the upstream initiators of the extrinsic cleaved caspase-8 and intrinsic cleaved caspase-9 pathways were both involved, as determined through Western blotting. This result implies that CLEFMA exerts anticancer effects through extrinsic and intrinsic apoptosis-related pathways in cervical cancer cells.
CLEFMA was demonstrated to activate both extrinsic and intrinsic apoptotic pathways through the action of MAPKs, including JNK and p38 signalling, in human osteosarcoma cells. 27 As an adjuvant of anticancer agents, curcumin has been found to induce apoptosis in human breast cancer, melanoma, and oral cancer through MAPK pathways. [39][40][41][42] GO-Y078, a synthesized curcumin analogue, can induce cell apoptosis through a variety of mechanisms and has been shown to exert anticancer effects through increased phosphorylation of ERK and JNK in osteosarcoma cells. 43,44 Although no study has delineated the signalling pathways by which CLEFMA affects cervical cancer cells, we F I G U R E 3 Effect of CLEFMA treatment on the HeLa cervical cancer cells by the human apoptosis array. (A) Expressions of cleaved caspase-3, heat shock protein 27 (HSP27) and cellular inhibitor of apoptosis protein 1 (cIAP-1) of total cell lysates from HeLa cells cultured for 24 h and then treated with 20 μM CLEFMA for 24 h by human apoptosis array, as compared to controls (no CLEFMA treatment). (B) The quantitative analysis of apoptosis associated proteins. cade. CLEFMA's effect on cleaved caspase-3, -8, and -9 was significantly inhibited by cotreatment with CLEFMA and the ERK or p38 inhibitor. Therefore, these findings suggest that CLEFMA activates both extrinsic and intrinsic apoptotic pathways through ERK and p38 signalling, but not through the JNK signalling, in cervical cancer cells.
In conclusion, CLEFMA triggers ERK and p38 signal transduction. CLEFMA activates both the extrinsic apoptotic pathway to F I G U R E 5 Effects of CLEFMA on the phosphorylation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinases (JNK) and p38 in HeLa and SiHa cervical cancer cells. Expressions of phosphorylation of ERK, JNK, and p38 using total cell lysates from (A) HeLa and (B) SiHa cells after treatment with 0, 5, 10, 20, and 40 μM of CLEFMA for 6 h by Western blotting. The quantitative analysis for the relative expression levels of phosphorylated ERK, JNK, and p38 were indicated as these phosphorylated proteins divided by their total counterpart proteins. Triplet experiments were done. Data are shown as mean ± SD. Significant differences were defined by one-way ANOVA with post hoc analysis. *p < 0.05, as compared to controls (no CLEFMA treatment).

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. Data are shown as mean ± SD. *p < 0.05, as compared to controls (no drug treatment); #p < 0.05, as compared to 40 μM CLEFMA.