GO-Y078 Triggers Program Cell Death in Human Cervical Cancer Cells Via MAPK Activation-Dependent Apoptotic Caspases Signaling

Abstract

Apoptosis is a regulated process of programed cell death that removes damaged ells. GO-Y078, a new curcumin analog, has been studied in the oncology field and shown to exert anti-proliferative and anti-angiogenic effects in multiple tumor types. However, its detailed signaling mechanisms and functional effects in human cervical cancer have not been clarified. Herein, GO-Y078 was employed to examine the anti-cancer mechanism in cervical cancer cells. GO-Y078 reduced the cell viability and elicited chromatin condensation and apoptotic cells of human cervical SiHa and HeLa cancer cells. Active PARP and active caspase-9, -8, and -3 were involved in GO-Y078-stimulated apoptosis. GO-Y078 also elevated phosphorylation of mitogen-activated protein kinase (MAPK) pathway. Co-treatment with GO-Y078 and either the ERK inhibitor U0126 or the p38 inhibitor SB203580 significantly reduced GO-Y078-induced activation of caspase-9, -8, and -3. In conclusion, GO-Y078 is a potential therapeutic candidate that induces apoptotic cell death in cervical cancer cells through phosphorylation-dependent activation of MAPK signaling followed by caspase activation.

Introduction

Cervical cancer remains a significant global health challenge, ranking as the fourth most common cancer among women worldwide. ¹ According to the World Health Organization (WHO), the total incidence of cervical cancer remains substantial and continues to grow slowly, reaching 0.67 million cases in 2021. ² While current prevention strategies, such as HPV vaccination and regular Pap smears, have dramatically reduced incidence in developed regions, many patients are still diagnosed at advanced stages when treatment options are limited. ³

Apoptotic cell death, a homeostatic mechanism that maintains cellular balance, is a common therapeutic target in cancer treatment. It is characterized by morphological changes such as cell shrinkage, nuclear envelope disassembly, chromatin condensation, cytoskeletal collapse, DNA fragmentation, and mitochondrial depolarization.^4,5 The process involves a series of signaling molecules, such as Bcl-2 family members, stress-responsive mitogen-activated protein kinase (MAPK), caspases cascade, etc. that coordinately modulate damage to nuclear DNA of the cell and mediate mitochondrial disruption by the impairment of DNA repair and release of pro-apoptotic proteins from the mitochondria. ⁶ Caspases, a family of cysteine proteases, are synthesized as inactive zymogens (pro-caspases) containing an N-terminal prodomain. Upon proteolytic cleavage, these precursors are converted into active caspases, playing key roles in inflammation and apoptosis. ⁷ Apoptotic caspases have been classified into 2 categories, initiator (caspase-2, -9, -8, and -10) and effector (caspase -3, -7, and -6) caspases, and activation of these caspases can be triggered by death ligand and extrinsic stimulus through extrinsic and intrinsic pathway that leads to DNA fragmentation and mitochondrial dysfunction.

The MAPK, protein Ser/Thr kinases, including ERK, JNK and p38, have been believed to be critically mediating participation of ER-mitochondrial crosstalk, cell cycle, mitosis, and apoptotic phenomena.^8,9 ERK activity can contribute to extrinsic or intrinsic apoptotic pathway induced by antitumor compound (quercetin) in prostate cancer cells ¹⁰ and chemotherapeutic DNA-damaging agent (doxorubicin) in A549 lung cancer cells. ¹¹ MAPK p38 activation has been linked to the stress responses and apoptotic machinery by modulation of BCL-2 family proteins. ¹² JNK promotes activation of transcription factor activator protein 1 and apoptosis-related proteins to enhance apoptotic cell death. ¹³

 Phytochemicals found in plants as chemo-preventive agents have been shown to be effective in reducing the risk of occurrence of cancer.^14-16 Among the bioactive phytochemicals, GO-Y078, a newly synthesized curcumin analog, has a wide range of pharmacological effect for clinical conditions, such as multidrug resistance, ¹⁷ angiogenesis, ¹⁸ and cancer. ¹⁹ GO-Y078 treatment caused an inhibitory effect of the function of MDR-related ABC transporters ABCG2 by suppressing the efflux of the ABCG2 substrates and interacting at the substrate-binding site in K562/BCRP cells. ¹⁷ Treatment of human umbilical venous epithelial cells with GO-Y078 suppressed the cell mobility and growth through regulating of fibronectin expression. ²⁰ GO-Y078 has been demonstrated to possess anticancer properties including antiproliferative phenomenon, G2/M phase arrest of cell cycle, and induction of apoptosis through targeting caspase cascade and upregulating heme oxygenase-1 in oral squamous cell carcinoma. ¹⁹ Recently, GO-Y078 induced sub-G1 arrest and both apoptotic pathways of osteosarcoma U2OS and 143B cells by repressing inhibitor of apoptosis proteins and activating p38 and JNK signaling pathway. ²¹ GO-Y078 exhibits pro-apoptotic properties against oral squamous cell carcinoma and osteosarcoma and antiangiogenic activities of vascular endothelial cells.^19,21 Nevertheless, the anticancer effect of GO-Y078 on human cervical cancer, and the role and mechanism of such effects thoroughly remain unclear. This study investigates the mechanistic action for the pro-apoptotic activities of GO-Y078 and contributes to its potential role in antitumor activity and clinical development of human cervical cancer.

Materials and Methods

Reagents and Antibodies

The chemicals and reagents commonly used in the present study were purchased from Sigma–Aldrich (St. Louis, MO, USA); these include DAPI staining solution, blocking reagent, phosphate-buffered saline (PBS), and sodium dodecyl sulfate (SDS). GO-Y078 was purchased from Tokyo Chemical Industry Co., Ltd., (Cat: H1525, Tokyo, Japan). The specific antibodies information: anti-c-IAP1 (CST, Cat #7065, RRID: AB_10890862), anti-c-IAP2 (CST, Cat #3130, RRID: AB_10693298), anti-XIAP (CST, Cat #2045, RRID: AB_2214866), anti-caspase-8 (CST, Cat #9746, RRID: AB_2275120), anti-caspase-9 (CST, Cat #9502, RRID: AB_2068621), anti-caspase-3 (BD, Cat 610323, RRID: AB_397713), anti-poly (ADP-ribose) polymerase (PARP; CST, Cat #9542, RRID: AB_2160739), anti-cleaved caspase-8 (CST, Cat #9496, RRID: AB_561381), anti-cleaved caspase-9 (CST, Cat #9505, RRID: AB_2290727), anti-cleaved caspase-3 (CST, Cat #9664, RRID: AB_2070042), anti-phospho (p)-ERK (CST, Cat #4370, CST, RRID: AB_2315112), anti-ERK (CST, Cat #9102, RRID: AB_330744), anti-p-JNK (Cat #4668, RRID: AB_823588), anti-JNK (CST, Cat #9258), anti-p-p38 (SC, Cat sc-166182, RRID: AB_2141746), anti-p38 (SC, Cat sc-7972), anti-human β-actin (SC, Cat ab8226, RRID: AB_306371). The U0126 (Cat 662009), JNK-IN-8 (Cat 420150), and SB203580 (Cat 559389) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Cell Culture

HeLa (RRID: CVCL_0030), a human cervical adenocarcinoma, and SiHa (RRID: CVCL_0032), a human cervical squamous cell carcinoma, were obtained from the American Type Culture Collection (Manassas, VA). Both cell lines were authenticated by short tandem repeat (STR) profiling. SiHa and HeLa cells were grown in DMEM containing 2 mM glutamine, 1% streptomycin/penicillin, and 10% FBS.

Cell Viability Assay

Cells were seeded in 24-well plates and then exposed to GO-Y078 (0, 2.5, 5, 10, 20 and 40 μM) for 24 h. The cytotoxic effects of different concentrations of GO-Y078 were evaluated using MTT-based colorimetric assays, following a previously established protocol. ²² Cell viability was quantified by measuring the absorbance at 563 nm with a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Annexin V-FITC and PI Staining Assay

For apoptotic cells detection assay, GO-Y078-treated cells were collected and defined with FITC Annexin V Apoptosis Detection Kit (Cat: 556547, BD Biosciences, San Diego, CA, USA) via PI uptake and annexin V binding. Afterward, flow cytometry was performed to calculate the rate of GO-Y078-induced apoptosis. ²³

Chromatin Condensation Assay

GO-Y078-treated SiHa and HeLa cells were washed with PBS and fixed in 70% ethanol. After staining with 0.6 μg/mL DAPI, fluorescent-labeled chromatin of cells was detected under a UV-light microscope. ²⁴

Human Apoptosis Array

For GO-Y078 treatment, cells were exposed to GO-Y078 (0 and 10 μM) for 24 h and then solubilized in lysis buffer. Whole-cell lysates were analyzed with R&D Systems human apoptosis array (Cat: ARY009; Minneapolis, MN, USA). Briefly, whole-cell lysates were added to arrays overnight and followed by probing with anti-phosphotyrosine-HRP antibodies. After incubation with antibodies, dot blots of the arrays were photographed and assessed by a Bio-Rad Molecular Imager Gel Doc XR system. ²⁵

Western Blot Analysis

Cells were seeded onto culture dish and then exposed to GO-Y078 (0, 2.5, 5, and 10 μM) for 24 h. Otherwise, cells were pre-treated with MAPK specific inhibitor (U0126, JNK-IN-8 or SB203580) for 1 h and followed by incubated with or without 5 μM GO-Y078 for another 24 h. After treatment, total cell lysates were lysed and collected. Cell lysates of SiHa and HeLa were separated by SDS-polyacrylamide gel electrophoresis. The sample were transferred onto a PVDF. The membranes were incubated with blocking reagent and probed with antibodies. The signal of specific protein was quantified using an ImageQuant LAS 4000 mini (Fuji, Tokyo, Japan). ²⁶

Statistical Analysis

Statistically significant differences were calculated using the Student’s t-test (Sigma-Stat 2.0, CA, USA). P value < .05 was considered statistically significant. The values involve the means ± standard deviation (SD) of at least 3 separate experiments.

Results

Suppression of Viability and Induction of Apoptosis and Chromatin Condensation by GO-Y078 in Cervical Cancer Cells

Results of MTT assay showed that GO-Y078 markedly inhibited the viability of both SiHa (Figure 1A) and HeLa (Figure 1B) cancer cell lines in 2.5 to 40 µM concentrations for 24 h of treatment. The IC₅₀ for SiHa cells was 7.76 µM and for HeLa cells was 11.94 µM. Specifically, treatment with 20 μM GO-Y078 resulted in a greater than 75% reduction in SiHa cell viability, while 40 μM led to approximately a 90% decrease. Therefore, to ensure the feasibility of subsequent experiments, lower concentrations ranging from 0 to 10 μM were selected for further analysis. In order to explore whether GO-Y078 induced apoptosis in cervical cancer cells, alterations of nuclear morphology were assessed using DAPI staining after 24 h treatment. The results revealed that GO-Y078 induced chromatin condensation of nuclear pattern in SiHa and HeLa cells (Figure 1C). Additionally, flow cytometry was used to evaluate whether the SiHa and HeLa cells treated with GO-Y078 underwent increase in apoptotic cell death by annexin V/PI staining. The result showed that GO-Y078 elevated the proportion of second quadrant (red dot; late apoptotic cells) and fourth quadrant (blue dot; early apoptotic cells) in both cell lines (Figure 2A). At the concentration of 10 μM, GO-Y078 increased the percentage of apoptotic cells (including late and early apoptotic stages) to 35.0% and 22.2% in SiHa and HeLa, respectively (Figure 2B).

Figure 1.

Inhibitory effects of GO-Y078 on cell viability in cervical cancer cells. Cell viability of (A) SiHa. (B) HeLa cells through MTT assay. The data represented the mean ± standard deviation (SD) of 3 independent experiments. Docetaxel (DXL; 10 μM), a known inducer of apoptosis, was used as the positive control. (C) The condensed nuclei morphology was examined by DAPI staining under a UV-light microscope in SiHa and HeLa cervical cancer cells. The quantitative data were presented from triplicates independent experiments.

Figure 2.

Induction of apoptotic patterns of GO-Y078 in cervical cancer cells. (A) Cervical cancer cells were treated with various concentrations of GO-Y078, and the number of apoptosis were assessed by annexin V/PI double staining. Quantification results of apoptotic cells of (B) SiHa and HeLa cells are presented as the bar graph.

GO-Y078 Exerts a Promotive Effect on Cleaved Caspase-3 in Cervical Cancer Cells

We examined whether GO-Y078 influences the expression of apoptotic related proteins of cervical cancer cells considering that the GO-Y078 significantly elevates the apoptotic cell death of cervical cancer cells. A human apoptosis array was used to screen for the protein expression of 35 different apoptotic proteins following 24 h treatment of SiHa cells with 0 and 10 μM GO-Y078. The levels of cleaved caspase-3 were increased, while the expression of CIAP-1, CIAP-2, and XIAP decreased under GO-Y078 treatment compared to the 0 µM control group in SiHa cell lines (Figure 3A and B). Moreover, Western blot analysis confirmed that GO-Y078 significantly decreased the expression levels of CIAP-1, CIAP-2, and XIAP in both SiHa and HeLa cells (Figure 3C). Densitometric analysis showed that these decreases were statistically significant compared to the untreated control (P < .01), confirming the ability of GO-Y078 to effectively downregulate IAP family proteins involved in apoptosis process (Figure 3D).

Figure 3.

Promotive effect of GO-Y078 on cleaved caspase-3. (A and B) Cell lysates from SiHa cells were run on a human apoptosis array (R&D systems) to screen the differences among 35 apoptotic proteins expression. (C and D) SiHa and Hela cells were exposed to GO-Y078 at 2.5, 5, or 10 μM for 24 h, then lysed for protein extraction and the subsequent immunodetection of pro-apoptotic proteins. β-actin was used as internal control. The quantitative data were presented from triplicates independent experiments.

GO-Y078 Triggers Caspase-Mediated Apoptotic Cell Death in Cervical Cancer Cells

The western blot showed that the protein levels of PARP, pro-caspase-8, -9, and -3 were remarkably declined in SiHa cells (Figure 4A); while the activation of PARP, caspase-8, -9, and -3 were observed and increased in SiHa cells after GO-Y078 treatment (Figure 4B). Quantification of band intensities revealed that, at the highest concentration of GO-Y078, the protein levels of pro-caspase-8 decreased by approximately 52%, pro-caspase-9 by 92%, pro-caspase-3 by 83%, and PARP levels by 48% compared to untreated controls. Similar phenomena were observed in HeLa cells after 24 hours of GO-Y078 treatment. GO-Y078 significantly reduced the protein levels of PARP, pro-caspase-8, -9, and -3 (Figure 4C P < .05), while the expression levels of cleaved PARP and cleaved caspase-8, -9, and -3 were markedly increased in a dose-dependent manner following treatment with 0 to 10 µM GO-Y078, as shown by Western blot analysis (Figure 4D; P < .05).

Figure 4.

The effects of GO-Y078 on protein levels of caspase cascade and PARP. SiHa cells were treated with GO-Y078 (0, 2.5, 5 and 10 μM), and Western blot analysis were carried out to detect (A) PARP, pro-caspase-9, -8, and -3. (B) The expression of cleaved caspase-8, -9, -3, and cleaved PARP of SiHa cells. The protein levels of (C) PARP, pro-caspase-8, -9, -3, (D) cleaved caspase-8, -9, -3, and cleaved PARP were performed in HeLa cells. The data represented the mean ± standard deviation (SD) of 3 independent experiments. The quantification data is ratio to β-actin.

Activation of MAPK Signaling Cascades by GO-Y078 in HeLa and SiHa Cells

The MAPK cascade has recently been reported to mediate cell growth and apoptosis through directly or indirectly inciting the activation of caspases -9, -8, and -3 pathways.^23,27 Figure 5 illustrates the effect of GO-Y078 treatment on MAPK signaling pathway activation in HeLa and SiHa cervical cancer cells. Specifically, cells were treated with increasing concentrations (0-10 μM) of GO-Y078, and the phosphorylation levels of ERK, JNK, and p38 were analyzed by Western blot. The results showed that GO-Y078 treatment significantly increased the phosphorylation of ERK (Figure 5A), JNK (Figure 5B), and p38 (Figure 5C) in a dose-dependent manner in both HeLa and SiHa cells. In HeLa cells treated with 10 μM GO-Y078, p-ERK, p-JNK, and p-p38 levels increased by approximately 12.6, 23.4, and 17.9-fold, respectively, compared to untreated controls. These data indicate robust activation of the MAPK pathway in response to GO-Y078.

Figure 5.

Active effects of GO-Y078 on protein levels of MAPK pathway. After treatment with GO-Y078, Western blot was employed to evaluate the protein levels of (A) p-ERK, total-ERK. (B) p-JNK, total-JNK. (C) p-p38, and total-p-38 in in HeLa and SiHa cervical cancer cells. The data represented the mean ± standard deviation (SD) of 3 independent experiments.

GO-Y078-Induced Caspase-Mediated Apoptotic Cell Death in HeLa Cells is Dependent on ERK and p38 Activation

To further investigate the role of MAPK signaling in GO-Y078-induced apoptosis, HeLa cells were pretreated with individual MAPK pathway inhibitors: the ERK inhibitor U0126 (10 μM), the JNK inhibitor JNK-IN-8 (1 μM), or the p38 inhibitor SB203580 (10 μM), followed by treatment with or without 5 μM GO-Y078. Western blot analysis revealed that pretreatment with U0126 or SB203580 significantly reduced the GO-Y078-induced increase in cleaved caspase-3, caspase-8, and caspase-9 protein levels (Figure 6A). Specifically, pretreatment with the p38 inhibitor SB203580 (10 μM) prior to GO-Y078 exposure reduced the levels of cleaved caspase-3 by 79%, cleaved caspase-8 by 50%, and cleaved caspase-9 by 38% compared to cells treated with GO-Y078 alone (Figure 6B). This indicates that activation of the ERK and p38 pathways is required for GO-Y078-induced caspase activation and apoptosis in HeLa cells. In contrast, pretreatment with the JNK inhibitor JNK-IN-8 did not significantly alter cleaved caspase levels, suggesting that JNK activation is not critically involved in GO-Y078-mediated apoptosis under these conditions. Together, these results demonstrate that GO-Y078 induces apoptosis through ERK- and p38-dependent caspase activation in HeLa cells.

Figure 6.

Caspase-regulated the apoptosis by GO-Y078 is dependent on phosphorylation of ERK and p38. (A) MAPK specific inhibitor (U0126, JNK-IN-8 or SB203580) were added in HeLa and for 1 hour and then reacted with the presence or absence of GO-Y078 for 24 hours. Cells were subjected to Western blot to evaluate the cleaved caspase-8, -9, and -3. Quantitative results of protein levels are shown in (B). Normalization of protein levels is performed using β-actin to accurately quantify Western blots. The data represented the mean ± standard deviation (SD) of 3 independent experiments.

Discussion

In cervical cancer, the lack of cell death and uncontrolled cell growth are associated with impaired apoptosis, which has been observed to result from genetic mutations in apoptosis-related proteins. ²⁸ Herein, GO-Y078 induced chromatin condensation of nuclear pattern and increased the percentage of apoptosis in HeLa and SiHa cells. The activation of caspase-3, -9, and -8 were observed in HeLa and SiHa cells after GO-Y078 treatment. Taken together, these results suggest that GO-Y078 induces apoptosis through dual targeting of both the intrinsic (mitochondrial) and extrinsic (death receptor-mediated) pathways in cervical cancer cells. Numerous studies have reported that activation of the caspase-3/-9 cascade can lead to a feedback amplification of mitochondrial dysfunction by inhibiting the anti-apoptotic functions of Bcl-xL and Bcl-2. ²⁹ Therefore, GO-Y078 may enhance therapeutic efficacy against cervical cancer by stimulating mitochondria-mediated apoptotic signaling. To confirm this mechanism, future studies should investigate the expression levels of Bcl-2 family proteins and assess changes in mitochondrial membrane potential.

During apoptosis, another characteristic event is inactive cleaved-PARP. Caspases, in particular caspase-7 and 3, not only trigger mitochondrial disruption but can also inhibit PARP-mediated DNA repair through cleaving the 116 kDa form of PARP to generate a proteolytic cleavage of PARP. ³⁰ PARP is a nuclear chromatin-associated enzyme involved in transcriptional regulation, DNA stability, and DNA repair. In the presence of DNA damage, PARP binds to the DNA and trigger the recruitment of the DNA-repairing enzyme, such as DNA polymerase β and DNA ligase III, to sites of damaged DNA. In this study, the expression levels of cleaved PARP noticeably elevated after treating cells with GO-Y078, thus preventing DNA repair of HeLa and SiHa cells.

We next explored GO-Y078-mediated upstream signal of caspase in modulating apoptosis. The MAPK signaling plays the role of an early step prior to the caspase activation and mitochondrial damage during apoptosis.^23,31,32 Earlier reports have indicated that MAPK plays a more important role in differentiation, mitosis, survival, motility, proliferation, and gene expression through relaying extracellular stimuli to intracellular responses. ³³ Accumulating evidence indicates that aberrant activation and high-expression of JNK signaling pathway of tumor cells may lead to activation of DNA damage-inducible genes and apoptotic cell death through phosphorylation of transcription factor c-Jun. ⁵ Curcumin has an impact on active MAPK p38 to exert ability of pro-apoptosis and properties anti-cancer and anti-inflammation. ⁹ Phosphorylation of ERK promotes apoptosis through modulation of c-IAP1 expression in cancer cells. ³⁴ In the present study, the stimulation of active caspase-9, -8, -3 by GO-Y078 was reversed by the ERK inhibitor and p38 inhibitor, suggesting that the phosphorylation of ERK and p38 MAPK is necessary for the anti-cancer ability and apoptotic effect of GO-Y078. Earlier reports have indicated that induction of HO-1 expression is closely linked to the MAPK pathway. The effect of HO-1 of GO-Y078-treated SiHa and HeLa cells will be examined in the future.

This study has several limitations. All experiments were performed in vitro using cervical cancer cell lines without animal or clinical validation, so the in vivo efficacy and safety of GO-Y078 remain unclear. In addition, the study did not evaluate the combinational effects of GO-Y078 with existing chemotherapeutic or targeted agents currently used in cervical cancer treatment, which could provide insights into potential synergistic or antagonistic interactions. Finally, the lack of normal cervical epithelial cell controls limits the assessment of GO-Y078’s cancer specificity effects. Further in vivo and combination studies are needed to confirm its therapeutic potential.

Overall, these results suggest that phosphorylation of the ERK and p38 MAPK pathways may lead to increased cleaved-PARP levels and activation of caspase-dependent apoptosis induced by GO-Y078 in cervical cancer cells (Figure 7). Therefore, GO-Y078 represents a potential therapeutic candidate with pro-apoptotic activity against cervical cancer. Future studies should focus on evaluating the in vivo efficacy and safety of GO-Y078 using animal models. In addition, combining GO-Y078 with current cervical cancer therapies could help determine possible synergistic effects. Further exploration of other signaling pathways will also be important to clarify the specificity and mechanism of GO-Y078 in cervical cancer treatment.

Figure 7.

Schematic diagram model of the molecular mechanism on anticancer effects of GO-Y078 of cervical cancer cells.