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. 2023 Jan 16;27(3):446–455. doi: 10.1111/jcmm.17671

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

Chung‐Yuan Lee 1,2, Yi‐Hsuan Hsiao 3,4,5, Pei‐Ni Chen 6,7, Heng‐Hsiung Wu 8, Chih‐Yun Lu 6, Shun‐Fa Yang 6,7,✉, Po‐Hui Wang 6,9,✉
PMCID: PMC9889609  PMID: 36645157

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.

Keywords: apoptosis, cervical cancer, CLEFMA, ERK, p38

1. INTRODUCTION

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.

2. MATERIALS AND METHODS

2.1. 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% CO2 humidified atmosphere. 25

2.2. Cell viability

Cell viability was assessed using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐25‐diphenyltetrazolium bromide (MTT; Sigma‐Aldrich) assay. The HeLa cells (7 × 104 cells/well) and SiHa cells (7 × 104 cells/well) were seeded in a 24‐well plate with 100 μl of the culture medium. The medium was removed after 24 h of culturing, and 100 μl of a medium containing 0.5 mg/ml MTT was applied. 26

2.3. Flow cytometry for apoptosis assay

Approximately, 6.5 × 105 HeLa cells and 7.0 × 105 SiHa cells were cultured in one 6‐cm dish in DMEM and treated with CLEFMA for 24 h at one of the following concentrations: 0, 5, 10, 20, or 40 μM. 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.

2.4. 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.

2.5. 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 × 105 HeLa and 7 × 105 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 × 105 HeLa and 7.0 × 105 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.

2.6. 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.

3. RESULTS

3.1. Effects of CLEFMA on HeLa and SiHa cervical cancer cell viability

The MTT assay revealed that the cell viability of the HeLa cervical cancer cells treated with 2.5, 5, 10, 20, and 40 μM CLEFMA for 24 h was 96%, 91%, 83%, 61%, and 33%, respectively, of that of the control (no CLEFMA) cells. The cell viability of the SiHa cancer cells treated with 2.5, 5, 10, 20 and 40 μM CLEFMA for 24 h was 99%, 96%, 85%, 73%, and 37%, respectively, of that of the control (no CLEFMA) cells. The viability of both the HeLa and SiHa cells treated with 5, 10, 20, or 40 μM CLEFMA was statistically different from that of their respective control (Figure 1A,B).

FIGURE 1.

FIGURE 1

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The effects of CLEFMA on cell viabilities of the HeLa and SiHa uterine cervical cancer cell lines using MTT assay. (A) HeLa cells and (B) SiHa cells were treated with different concentrations of CLEFMA (0, 2.5, 5, 10, 20, and 40 μM). Triplet experiments were done. The data were expressed 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).

3.2. 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).

FIGURE 2.

FIGURE 2

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Effects of CLEFMA on the apoptosis of HeLa and SiHa cervical cancer cells. (A) HeLa and SiHa were treated with 0, 5, 10, 20, and 40 μM of CLEFMA and subsequently subjected to flow cytometry after fluorescein isothiocyanate (FITC)‐labelled annexin V and propidium iodide (PI). Both FITC annexin V and PI negative indicate cells that were regarded as visible; FITC annexin V positive and PI negative indicate cells that were in in early apoptosis; both FITC annexin V positive and PI positive indicate cells that were in late apoptosis. The quantitative analysis of early and late apoptosis for (B) HeLa and (C) SiHa cancer cells was summarized as cell apoptosis. Triplet experiments were done. The data were expressed 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).

3.3. CLEFMA increases cleaved caspase‐3 expression in HeLa cervical cancer cells

To clarify which apoptosis‐related proteins' expression was increased by CLEFMA in the HeLa cervical cancer cells, a human apoptosis array was employed. The results revealed a significant increase in cleaved caspase‐3 as well as decreases in heat shock protein 27 and cellular inhibitor of apoptosis protein 1 in the HeLa cells treated with 20 μM CLEFMA for 24 h (Figure 3A,B).

FIGURE 3.

FIGURE 3

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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.

3.4. CLEFMA can trigger the activation of caspase cascade in HeLa and SiHa cervical cancer cells

To elucidate the effect of CLEFMA on the caspase cascade of the apoptotic signalling pathway, the effector caspase‐3; its upstream initiators, caspase‐8 and caspase‐9; and their respective cleaved forms were detected in HeLa and SiHa cervical cancer cells through Western blotting. After treatment with 0, 5, 10, 20, or 40 μM CLEFMA for 24 h, the expression of the apoptosis‐associated markers procaspase‐8, ‐9, and ‐3 and PARP was inversely proportional to the concentrations of CLEFMA in the HeLa and SiHa cells (Figure 4A,C). By contrast, higher expression of the cleaved forms of caspase‐8, ‐9, and ‐3 and PARP was significantly associated with higher concentrations of CLEFMA in the HeLa and SiHa cells, constituting a directly proportional relationship (Figure 4B,D).

FIGURE 4.

FIGURE 4

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Effects of CLEFMA on the activation of caspase cascade in HeLa and SiHa cervical cancer cells. Less expressions of proform caspases, pro‐caspases‐8, ‐9, and ‐3, as well as poly adenosine diphosphate‐ribose polymerase (PARP) correspond to the higher concentrations of CLEFMA using total cell lysates from (A) HeLa cells and (C) SiHa cells after treatment with 0, 5, 10, 20, and 40 μM of CLEFMA for 24 h by Western blotting. β‐Actin as internal control. The quantitative analysis reveals an inverse proportion relationship. *p < 0.05, as compared to controls (no CLEFMA treatment). Higher expressions of cleaved caspases, caspases‐8, 9, and 3, as well as cleaved PARP correspond to the higher concentrations of CLEFMA using total cell lysates from (B) HeLa cells and (D) SiHa cells by Western blotting. β‐Actin as internal control. Triplet experiments were done. The data were expressed as mean ± SD. The quantitative analysis reveals a direct proportion relationship. *p < 0.05, as compared to controls (no CLEFMA treatment).

3.5. CLEFMA activates extrinsic and intrinsic apoptotic pathways, possibly through MAPK pathways in HeLa and SiHa cervical cancer cells

Western blot analysis was performed to determine the underlying molecular mechanisms through which MAPK‐related proteins influence the expression of caspase‐8, ‐9, and ‐3 and PARP and thus induce apoptosis in HeLa and SiHa cells. Our experiments revealed that CLEFMA increased the phosphorylation of ERK1/2, JNK1/2, and p38 significantly in a concentration‐dependent manner in HeLa and SiHa cells (Figure 5A,B). These findings suggest that the ERK1/2, JNK1/2, and p38 pathways are involved upstream in the regulation of CLEFMA‐mediated apoptotic signalling through the extrinsic caspase‐8 pathway and intrinsic caspase‐9 pathway and in their downstream effect exerted through caspase‐3 in HeLa and SiHa cervical cancer cells.

FIGURE 5.

FIGURE 5

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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).

To determine which MAPK proteins (i.e. ERK1/2, JNK1/2, or p38) trigger the activation of the caspase cascade, inhibitors of ERK1/2 (U0126, 10 μM), JNK1/2 (JNK‐IN‐8, 1 μM), and p38 (SB203580, 10 μM) were used to assess the proteins' influence on the expression of caspase‐8, ‐9, and ‐3 in combination with 40 μM CLEFMA treatment in HeLa cells (Figure 6A). After the application of the ERK1/2 (U0126) and p38 (SB203580) inhibitors and CLEFMA, the expression of cleaved caspase‐8, ‐9, and ‐3 was reduced significantly, as determined through Western blotting (Figure 6B–D, respectively). However, the JNK inhibitor (JNK‐IN‐8) did not significantly reduce the expression of cleaved caspase‐8, ‐9, or ‐3 after treatment with 40 μM CLEFMA. These results indicate that CLEFMA activates the extrinsic caspase‐8, intrinsic caspase‐9, and caspase‐3 apoptotic pathways mainly through ERK1/2‐ and p38‐mediated pathways.

FIGURE 6.

FIGURE 6

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The influences of inhibitors of mitogen‐activated protein kinases (MAPKs) on the expression of activation of cleaved caspases‐8, ‐9, and ‐3 on treatment of CLEFMA in HeLa cervical cancer cells. (A) The expressions of cleaved caspases‐8, ‐9, and ‐3 were assessed based on total cell lysates from HeLa cells after inhibitors of extracellular signal‐regulated kinase (ERK; U0126, 0 or 10 μM), c‐Jun N‐terminal kinases (JNK; JNK‐IN‐8, 0 or 1 μM) or p38 (SB203580, 0 or 10 μM) were given for 2 h and then CLEFMA (0 or 40 μM) for 24 h by Western blotting in HeLa cells. β‐Actin for internal control. Expressions of (B) cleaved caspase‐8 (C) cleaved caspase‐9 and (D) cleaved caspase‐3 in quantitative analysis. Triplet experiments were done. 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.

4. DISCUSSION

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 Therefore, new modalities, drugs, or agents for the treatment of cervical cancer require development. In addition, the mechanisms or pathways underlying their effects should be delineated. Recently, curcumin has demonstrated potential anticancer effects.

Curcumin has been demonstrated to induce apoptosis in human leukaemia cells, possibly through both intrinsic and extrinsic pathways, which are triggered by MAPK pathways. 32 , 33 Moreover, curcumin exhibits antitumor effects on retinoblastoma cells by 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 apoptosis‐related 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 could infer that CLEFMA, a potent curcumin analogue, activates extrinsic and intrinsic apoptotic pathways, possibly through the phosphorylation of ERK, JNK, or p38, in HeLa and SiHa cervical cancer cells. In this study, the expression of phosphorylated ERK, JNK, and p38 progressively increased with the concentration of CLEFMA in HeLa and SiHa cervical cancer cells. To determine the underlying mechanism by which CLEFMA increases the expression of cleaved caspase‐3, ‐8, and ‐9, ERK, JNK, and p38 pathway inhibitors were applied to determine whether they could inhibit the CLEFMA‐induced activation of the caspase cascade. 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 increase caspase‐8 expression and the intrinsic apoptotic pathway to increase caspase‐9 expression and subsequently increases the expression of cleaved caspase‐3 and cleaved PARP in cervical cancer cells. By this mechanism of apoptosis induction, CLEFMA reduces the viability in cervical cancer cells.

AUTHOR CONTRIBUTIONS

Chung‐Yuan Lee: Conceptualization (equal); writing – original draft (equal); writing – review and editing (equal). Yi‐Hsuan Hsiao: Writing – original draft (equal). Pei‐Ni Chen: Methodology (equal). Heng‐Hsiung Wu: Methodology (equal). Chih‐Yun Lu: Methodology (equal). Shun‐Fa Yang: Conceptualization (equal); writing – original draft (equal); writing – review and editing (equal). Po‐Hui Wang: Conceptualization (equal); writing – original draft (equal); writing – review and editing (equal).

FUNDING INFORMATION

This study was supported by research grants from the Chung Shan Medical University Hospital, Taiwan (CSH‐2022‐D‐002). This research was also funded by China Medical University, Taiwan (CMU‐108‐MF‐10).

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

Lee C‐Y, Hsiao Y‐H, Chen P‐N, et al. CLEFMA induces intrinsic and extrinsic apoptotic pathways through ERK1/2 and p38 signalling in uterine cervical cancer cells. J Cell Mol Med. 2023;27:446‐455. doi: 10.1111/jcmm.17671

Contributor Information

Shun‐Fa Yang, Email: ysf@csmu.edu.tw.

Po‐Hui Wang, Email: wang082160@gmail.com.

DATA AVAILABILITY STATEMENT

The data used to support the findings of the present study are available from the corresponding author upon request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data used to support the findings of the present study are available from the corresponding author upon request.

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