Skip to main content
Download PDF

Costunolide inhibits the progression of TPA-induced cell transformation and DMBA/TPA-induced skin carcinogenesis by regulation of AKT-mediated signaling

Cancer Cell International volume 25, Article number: 106 (2025) Cite this article

Abstract

Background

Costunolide (COS), a sesquiterpene lactone extracted from the roots of Saussurea costus, is known to possess anticancer properties in various cancers, including colon, oral, and lung cancers, but its mechanism of action in skin carcinogenesis has not yet been explored. Present study investigates the chemopreventive mechanism of COS on skin inflammation and carcinogenesis both in vitro and in vivo.

Methods

The cytotoxicity of COS was examined on a normal murine epidermal cell line, JB6, by treating with COS using the WST-8 assay. Subsequently, the effect of COS on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cellular transformation was assessed through a soft-agar assay. Furtherly, cell cycle and apoptosis analysis and the expression of related proteins were determined via flow cytometry and Western blotting, respectively. The effects of COS on tumor promotion induced by DMBA/TPA treatment and the underlying molecular mechanisms in mouse skin carcinogenesis were identified through H&E staining and immunohistochemical analysis.

Results

COS significantly inhibited colony growth and number in TPA-induced JB6 cells transformation, arrested the cell cycle at the G2/M phase, increased p21 expression, and decreased cyclin B expression. In addition, COS induced cell apoptosis and increased the related markers expression including cleaved caspase-3 and − 7. COS suppressed the expression of phosphorylated AKT and its downstream signaling proteins and effectively reduced the translocation of phosphorylated NF-κB from the cytosol to the nucleus. Moreover, COS reduced papilloma formation in mouse skin and inhibited hyperplasia and phosphorylated AKT expression in tissues.

Conclusion

These results demonstrate that COS inhibits TPA-induced cellular transformation and skin carcinogenesis both in vitro and in vivo through the AKT signaling pathway. Our findings suggest the potential of COS as a chemopreventive agent for skin carcinogenesis, highlighting its significance for further investigation in cancer prevention and therapy.

Graphical Abstract

Introduction

Skin cancer ranks among the most prevalent cancers globally, with non-melanoma skin cancer (NMSC), including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), being the predominant forms [1]. Over recent decades, an upward trend in skin cancer incidence has been observed [2,3,4], a phenomenon attributed to environmental factors and lifestyle changes. Notably, exposure to ultraviolet radiation (UVR) has been identified as a primary cause, with additional contributions from inflammation, chemical exposures and genetic predispositions [5,6,7,8,9,10]. The management of skin cancer is contingent upon its symptoms and severity, with surgical excision being the standard treatment approach. Other therapeutic strategies encompass curettage and electrosurgery, radiation therapy, cryotherapy, and cytotoxic drug therapy, alongside combinations of chemotherapy, immunotherapy, and radiation therapy [11, 12]. Nonetheless, the prognosis often remains suboptimal, accompanied by potential side effects.

The carcinogenic process of the skin is acknowledged as a multifaceted sequence of events, including initiation, promotion, progression, and metastasis [9, 13], with cellular transformation being a critical early phase where normal cells morph into cancerous entities. 7,12-Dimethylbenz(a)anthracene (DMBA) is used as an initiator in chemically induced skin cancer models, while 12-O-tetradecanoylphorbol-13-acetate (TPA) is widely employed in skin cancer research as a potent tumor promoter that triggers cellular transformation, thereby emulating the inflammatory responses and cell proliferation characteristic of skin cancer. The DMBA/TPA-induced two-stage skin cancer model is a strategic methodology that closely mimics human SCC in experimental paradigms [14, 15].

In this context, there is a surge of interest in natural compounds, especially plant-derived phytochemicals, due to their potential health benefits, including anticancer properties. Sesquiterpene lactones, a class of phytochemicals found in diverse plants, have attracted attention for their anti-inflammatory, antioxidant, and significantly, anticancer effects [16,17,18,19,20]. These compounds disrupt cancer cell growth through various mechanisms such as apoptosis induction, cell cycle regulation, and the inhibition of metastasis and invasion [21,22,23,24]. Costunolide (COS, Fig. 1A), a sesquiterpene lactone extracted from the roots of Saussurea costus, has been reported to exhibit a range of biological effects, including anticancer activity in various cancers like colon, oral, and lung cancers [24,25,26,27,28,29,30,31]. However, its mechanisms of action against skin carcinogenesis have largely remained unexplored.

Fig. 1
figure 1

Evaluation of the structure and cytotoxicity of COS and its inhibition of TPA-induced cell transformation. (A) Structure of COS. (B) Cytotoxicity evaluation of COS was performed by WST-8 assay in JB6 cells, a normal mouse epidermal cell. (C) Colony formation was investigated by soft agar assay after treatment with TPA (10ng/ml) and/or COS. (D) Confirmation of colony formation inhibition effect of COS using Image-Pro Plus 6.0 program. Significantly different at: ###p < 0.001 compared to the control; ***p < 0.001 compared to group treated with TPA

Full size image

This study aims to investigate the anti-proliferation effects of COS on TPA-induced cellular transformation and to assess its anti-cancer efficacy in a DMBA/TPA-induced two-stage skin carcinogenesis mouse model. By elucidating the manner in which COS regulates the process of cellular transformation and the development of skin cancer, as well as the involved molecular mechanisms, suggesting to present novel alternative approaches for the prevention and treatment of skin cancer. Therefore, we provide valuable insights into the dimension of skin cancer prevention and highlight the potential of COS as effective agents.

Materials and methods

Reagents

Eagle’s minimum essential medium (MEM), fetal bovine serum (FBS), penicillin/streptomycin (P/S), MEM non-essential amino acid (MEM NEAA), sodium pyruvate (SP), L-glutamine, and trypsin-EDTA were purchased from Gibco (Grand Island, NY, USA). DMBA, TPA and Basal Medium Eagle (BME) were obtained from Sigma-Aldrich, Inc. (St. Louis, MO, USA). FxCycle™ PI/RNase Staining Solution and FITC-conjugated Annexin V and propidium iodide (PI) kits were obtained from ThermoFisher Scientific (Rockford, Illinois, USA). The antibodies against p21, phosphorylated NF-κB p65 (p-NF-κB, Ser 536), and NF-κB were obtained from Santa Cruz (Santa Cruz Biotechnology, CA, USA). The antibodies against phosphorylated AKT (p-AKT, Ser 473), panAKT, Cyclin B1, Caspase-3 (Cas-3), and Caspase-7 (Cas-7) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against phosphorylated GSK3β (p-GSK3β, Ser 9), GSK3β, phosphorylated mTOR (p-mTOR, Ser 2448), mTOR, and β-actin were purchased from Invitrogen (Beverly, MA, USA). COS was purchased from National Institute of Food and Drug Safety Evaluation (Cheongju-si, Republic of Korea). Stock solution (100 mM) of COS was prepared with DMSO. It was aliquoted and stored at -20 ℃ until use.

Cell culture

Mouse epithelial cell line, JB6 Cl41 (JB6), were purchased from American Type Culture Collection (Manassas, VA, USA). JB6 cells were cultured in MEM supplying 5% FBS, 100 U/mL P/S, 1X MEM NEAA, and 1X SP. The cells were cultured at 37℃ under 5% CO2, and the medium were changed every 2–3 days.

Cell viability assay

To investigate the effect of the COS on the proliferation of JB6 cells, WST-8 assay was conducted. JB6 cells (8 × 103 cells/well) were seeded in a 96-well plate. After 24 h, the cells were then treated with various concentrations of COS. Following incubation for the designated time, 24 h and 48 h, 10 µL of WST-8 (#QM1000, BIOMAX, Guri-si, Republic of Korea) was added to each well. The absorbance was measured at 450 nm using a Multiskan SkyHigh spectrophotometer (Thermo Scientific, Vantaa, Finland).

Soft agar colony formation assay

To evaluate JB6 cell transformation, a cellular anchorage-independent transformation assay was performed according to a previous study [32]. BME, supplemented with 10% FBS, 2 mM L-glutamine, and 25 µg/mL gentamicin from Lonza (Basel, Switzerland), was prepared with the inclusion of 10ng/mL TPA and varying concentrations of COS (5, 10 and 20 µM). This mixture was then combined with 0.6% agar to create a bottom agar solution. Subsequently, 3mL of this bottom agar mixture was carefully dispensed into each well of 6-well plates. 10 ng/mL TPA and various concentrations of COS (5, 10 and 20 µM) were combined with JB6 cells, which were prepared at a density of 8 × 103 cells per well, along with BME medium containing 0.3% agar. This cell mixture was then carefully layered over the bottom agar in the wells. Following this, the plates were placed in an incubator at 37℃ with a 5% CO2 atmosphere and incubated for 14 days. Using a Leica microscope (Leica Microsystems, Wetzlar, Hesse, Germany), colonies were observed, and their numbers were quantified with Image-Pro Plus software version 6.1 (Media Cybernetics, Rockville, MD, USA).

Cell cycle analysis

To assess the effects of the COS on the cell cycle distribution in TPA-induced transformed cells, JB6 cells were plated at a density of 1.8 × 105 cells per well in a 6-well plate and allowed to incubate for 24 h. The cells were serum-starved for 18 h before being treated with TPA (10 ng/mL) and varying concentrations of COS (5, 10 and 20 µM) in serum-free MEM. Subsequently, the cells were incubated for 18 h, respectively. Culture supernatants and cells detached with trypsin treatment were collected, followed by centrifugation to obtain cell pellets, which were then washed once with cold 1X PBS. These cells were fixed in 70% ethanol at -20 ℃ for more than 24 h. After fixation, the cells were stained for 15 min in the dark at room temperature (RT) using FxCycle™ PI/RNase Staining Solution. The analysis was performed using a CytoFLEX flow cytometer, employing CytExpert software version 2.2 (Beckman Coulter, CA, USA).

Apoptosis analysis

To evaluate the effect of COS on apoptosis in TPA-induced transformed JB6 cells, cells were seeded in 6-well plates at a density of 1.8 × 105 cells per well and incubated for 24 h to allow adherence. Afterward, cells were serum-starved in serum-free MEM for 18 h. Following starvation, cells were treated with TPA (10 ng/mL) and COS at various concentrations (5, 10, and 20 µM) in serum-free MEM and incubated under standard culture conditions for 24 h. Both culture supernatant and detaching adherent cells using trypsin were collected. For apoptosis analysis, cells were resuspended in 1X binding buffer provided with the Invitrogen™ Annexin V/PI kit and stained with Annexin V-FITC and PI according to the manufacturer’s instructions. The staining reaction was performed in the dark at room temperature for 15 min. The stained cells were analyzed using a CytoFLEX flow cytometer (Beckman Coulter, CA, USA) with CytExpert software version 2.2.

Western blot analysis

To elucidate the molecular mechanisms underlying the growth inhibitory effects of COS on TPA-induced transformed cells, western blot analysis was conducted. JB6 cells were seeded at a density of 0.8 × 106 cells per 100 mm dish and incubated for 24 h. Subsequently, the cells were serum-starved for 18 h and treated under the following conditions: To analyze AKT signaling-related proteins, the cells were first treated with 5, 10, and 20 µM of COS for 30 min, followed by treatment with TPA (10 ng/mL) for an additional 30 min prior to harvesting. For the analysis of cell cycle-related proteins, the cells were treated with TPA and 5, 10, and 20 µM of COS simultaneously and incubated for 18 h. For apoptosis-related proteins, the cells were treated under the same conditions for 24 h. After harvest cells, proteins were extracted using the PRO-PREP Protein Extraction Solution from iNtRON Biotechnology (Seongnam-si, Republic of Korea), and protein concentrations were determined with the Pierce BCA Protein Assay Kit by ThermoFisher Scientific. The proteins were then separated through SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane. To block non-specific binding, the membrane was treated with 3% skim milk in 1X Tris-buffered saline with Tween-20 (TBS-T) at RT for 1 h, followed by overnight incubation at 4 °C with primary antibodies targeting Cyclin B1 (dilution ratio 1:1,000), p21 (dilution ratio 1:1,000), Cas-3 (dilution ratio 1:1,000), Cas-7 (dilution ratio 1:1,000), p-AKT (dilution ratio 1:2,000), panAKT (dilution ratio 1:5,000), p-GSK3β (dilution ratio 1:1,000), GSK3β (dilution ratio 1:3,000), p-mTOR (dilution ratio 1:1,000), mTOR (dilution ratio 1:1,000), p-NF-κB (dilution ratio 1:1,000), NF-κB (dilution ratio 1:1,000). After three times wash with 1X TBS-T, the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (#31430 or #31460; Invitrogen) at a dilution ratio of 1:2,000 ~ 7,000 for 1 h at RT. Protein expression was detected using enhanced chemiluminescence (ECL) horseradish peroxidase (HRP) substrate (ThermoFisher Scientific) and visualized using a LAS-Amersham Imager 600 (GE Healthcare, Uppsala, Sweden). The blots were quantified using ImageJ software version 1.53 (National Institutes of Health, Bethesda, Maryland, USA).

Animals experiment

For the animal experiments, female mice of the Institute Cancer Research (ICR) strain, all five-weeks-old, were acquired from Samtako (Osan-si, Republic of Korea), and acclimatized for one week. A total of 29 mice were randomly divided into five groups, with each group comprising five to six mice. In the mouse skin tumor model, tumors were induced by a single topical application of the mutagen DMBA followed by repeated topical applications of the inflammation-inducing agent TPA [15, 33, 34]. Five days prior to the experimental procedures, the dorsal skin of each mouse was shaved using an electronic shaver, followed by depilation with hair removal cream. The first group served as the vehicle control group (Vehicle), which received topical treatment of acetone containing 2% DMSO on the shaved dorsal skin twice a week. The second group, designated as the cancer control group (DMBA/TPA), was treated with 200 nmol/200 µL of DMBA at week 0, followed by topical treatment of 5 nmol/200 µL of TPA twice a week from week 1 to week 21. The third group received topical treatment with COS 0.5 µmol/200 µL and 5 nmol/200 µL of TPA twice a week for 21 weeks, following DMBA treatment. The fourth group was treated with COS 1 µmol/200 µL and 5 nmol/200 µL of TPA twice a week for 21 weeks, subsequent to DMBA treatment. The fifth group received only COS 1 µmol/200 µL treatment without DMBA and TPA treatment. COS was topically treated 30 min prior to TPA exposure. The body weight, tumor number and volume of papilloma (at least 2 mm3 in volume) were determined twice weekly during the experimental period. The diameter of the tumors was measured using calipers, and the individual tumor volumes were calculated using the following equation: Volume [mm3] \(\:=\:\frac{4}{3}{\uppi\:}\times\:\frac{\text{l}\text{e}\text{n}\text{g}\text{t}\text{h}}{2}\times\:\frac{\text{w}\text{i}\text{d}\text{t}\text{h}}{2}\times\:\frac{\text{h}\text{e}\text{i}\text{g}\text{h}\text{t}}{2}\). The mice were euthanized by cervical dislocation after the 21-week experimental period, and the back skin was preserved in 10% formalin. All animal experimental procedures were approved by the Dongshin University Animal Ethical Committee under the approval number DSU2021-01-05. All animal procedures were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee (IACUC), ensuring adherence to ethical standards for animal experiment.

Hematoxylin and Eosin (H&E) staining

Skin samples from the dorsal area of mice were fixed in 10% formalin for more than 48 h, followed by dehydration, clearing and filtration with paraffin wax using an automated tissue processor. The paraffin-embedded skin tissues were sectioned at a thickness of 5 μm and placed on glass slides to dry. After deparaffinization, sections were rehydrated through gradiented alcohols, then stained with hematoxylin (Vector Laboratories, Inc., Newark, CA, USA) and eosin Y (Sigma), and finally mounted with coverslips.

Immunohistochemical (IHC)-Hematoxylin staining

Deparaffinized and rehydrated sections through a series of xylene and gradient percent of ethanols underwent antigen retrieval in heated 10 mM sodium citrate buffer. The sections were then treated with 0.3% peroxidase to reduce endogenous peroxidase activity. Subsequently, sections were incubated in serum blocking solution to reduce non-specific binding. Sections were then incubated with anti-p-AKT (diluted 1:200), anti-cyclooxygenase-2 (COX-2) (Cayman #160106, diluted 1:300) and anti-Ki-67 (Invitrogen #PA5-19462, diluted 1:500) overnight at 4 °C. The sections were then incubated with ImmPRESS® HRP Universal Antibody Polymer Detection Kit (Vector Laboratories) was used as the secondary antibody, followed by development with diaminobenzidine (DAB) peroxidase substrate (Vector Laboratories). Afterwards, the sections were stained with hematoxylin. Stained sections with H&E or IHC were scanned using Pannoramic SCAN II (3DHISTECH, Sysmex, Switzerland), and image magnification was performed using SlideViewer software (version 2.6, 3DHISTECH). The quantitative analysis of p-AKT, COX-2 and Ki-67 expression and epidermal thickness measurements were performed using Image-Pro Plus software (v.6.0, Media Cybernetics, Silver Spring, MD, USA). A minimum of three samples per group were analyzed, and for each sample, measurements were performed at 400× magnification in randomly selected non-overlapping fields. The average value for each group was recorded.

Immunofluorescence (IF) staining

JB6 cells were seeded into a 4-well chamber at a density of 4 × 104 cells, followed by stabilization and starvation before treatment with various concentrations of COS along with TPA (10 ng/mL). Cells were blocked using a BSA solution after fixing with formalin, then incubated overnight at 4°C with the primary antibody p-NF-κB (SantaCruz #136548, diluted 1:200). Subsequently, cells were treated for 1 h at RT with fluorophore-conjugated secondary antibody (Alexa Fluor 488, Invitrogen #A11001, diluted 1:2000). DAPI staining was performed using a mounting medium containing DAPI. Samples were analyzed using a DMi8 CS Promium confocal microscope (Leica Microsystems CMS GmbH, Wetzlar, Germany). Images were acquired using LAS X Office (Leica Microsystems).

Statistical analysis

The results of each experiment were expressed as the mean ± standard deviation (SD) (n = 3). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test using GraphPad Prism (version 6.01 for Windows, California, USA, www.graphpad.com). A p-value less than 0.05 was considered statistically significant.

Results

Cytotoxicity evaluation of COS in JB6 cells

Before exploring the chemopreventive signaling mechanisms of COS, we first assessed its potential cytotoxic effects on JB6 cells, a normal mouse epidermal cell line. The cells were treated with COS at various concentrations (0, 5, 10 and 20 µM) for durations of 0, 24, and 48 h. Cytotoxicity evaluation performed using the WST-8 assay demonstrated that COS exhibited no cytotoxicity at all tested concentrations (Fig. 1B). These results ensure the safety of COS in normal epidermal cells and paves the way for further investigation into its chemopreventive properties.

Inhibitory effects of COS on cell transformation and growth

After confirming the non-toxicity of COS in normal cells, we evaluated the effect of COS on cellular transformation, which is a critical early stage in cancer development. Cell transformation represents the process where normal cells evolve into cancerous cells, with TPA known as a classic tumor promoter that induces this transformation. Through soft-agar assays, we aimed to elucidate whether COS could effectively counter this transformation process. The results indicated that treatment of JB6 cells with TPA led to an increase in colony formation, simulating the transformation within the normal cell population. However, the addition of COS alongside TPA resulted in a concentration-dependent inhibition of both cell transformation and proliferation (Fig. 1C and D). These effects underscore the potential of COS not only as an anticancer agent but also highlights its ability to inhibit the early stages of cancer cell development, offering a promising direction for anticancer therapy.

Effect of COS on cell cycle progression

Following the confirmation that COS inhibits colony formation in TPA-transformed JB6 cells, we aimed to elucidate the cellular mechanisms contributing to the chemopreventive effects of COS, focusing particularly on its effect on cell cycle distribution in transformed JB6 cells. The results of flow cytometry analysis indicated an increase in the S phase distribution following TPA treatment, which shifted to a pronounced G2/M phase arrest with increasing concentrations of COS, suggesting a dose-dependent regulation of cell cycle checkpoints (Fig. 2A and B). This observation was supported by western blot analysis, which indicated a significant increase in p21 levels and a reduction in cyclin B1 levels, key regulators of the G2/M transition, further substantiating the COS-induced cell cycle arrest (Fig. 2C). Quantitative assessments of these protein expressions were graphically represented, providing a clear visual depiction of how COS modulates cell cycle regulatory proteins (Fig. 2D and E).

Fig. 2
figure 2

COS arrests the cell cycle and inhibits cell growth. (A) Representative plots of flow cytometry analysis of cell cycle. Cells were untreated (-TPA), treated with TPA or TPA coupled with 0, 5, 10, and 20µM COS. (B) Bar graph of cell cycle distribution. The data are presented as means of triplicate samples for each treatment. (C) Effect of COS on cell cycle regulatory proteins on TPA-induced cell transformation were evaluated by Western blot analysis. β-actin levels served as a loading control. (D, E) Quantitation of band intensity from blots by Image-J software. The relative expression of p21 and cyclin B was shown after normalization against actin, respectively. Significantly different at: #p < 0.05 compared to the control; *p < 0.05, **p < 0.01, and ***p < 0.001 compared to group treated with TPA only

Full size image

Effect of COS on apoptosis induction

To investigate the effect of COS on apoptosis in TPA-induced JB6 cells, Annexin V-FITC/PI staining was performed, followed by flow cytometry analysis. The results demonstrated a significant increase in apoptosis in the TPA and COS co-treatment groups compared to the TPA-only control group (Fig. 3A and B). In the untreated group, the proportion of viable cells was 77.4%, and the proportion of total apoptotic cells was 12.7%. In contrast, the TPA-only control group exhibited an increase in viable cells (94.3%) and a decrease in total apoptotic cells (2.7%). However, in the COS treatment groups, there was a dose-dependent increase in both early and total apoptotic cells. Notably, at a concentration of 20 µM COS, the proportion of viable cells decreased to 87.4%, while the proportion of total apoptotic cells increased to 11.3%, showing a significant change compared to the TPA-only control group. These findings were further confirmed through Western blot analysis (Fig. 3C and E). The expression levels of cleaved caspase-3 and caspase-7, key markers of apoptosis, were analyzed. The results showed a dose-dependent increase in the expression of cleaved caspase-3 and caspase-7 in the COS-treated groups.

Fig. 3
figure 3

COS induces apoptosis in TPA-induced JB6 cells. (A) Flow cytometry analysis of apoptosis in JB6 cells treated with TPA (10 ng/mL) and various concentrations of COS (5, 10, and 20 μM). (B) Quantitative analysis of apoptotic cell populations. Data are expressed as the mean ± SD (n=3). Significantly different at: ### p<0.001 compared to the control; ** p<0.01, and *** p<0.001 compared to group treated with TPA only. (C) Western blot analysis of caspase-3 and caspase-7 expression in TPA-treated JB6 cells. β-actin levels served as a loading control. (D) Quantitative analysis of cleaved caspase-3 and caspase-7 protein levels. Results are presented as the mean ± SD.

Full size image

COS-mediated modulation of the AKT pathway and its downstream effectors

Considering the pivotal role of the AKT signaling cascade in coordinating various cellular processes, including cell proliferation and survival, we explored the impact of COS on this pathway in the context of TPA-induced cellular transformations in JB6 cells. A significant increase of phosphorylated-AKT was observed 30 min following TPA treatment, highlighting the activation of this pathway in response to cellular stress (Fig. 4A). To further elucidate the influence of COS on the AKT signaling pathway, we conducted a series of western blot analyses. The analysis revealed that in cells exposed to TPA, there was an increase in the phosphorylation of GSK3β, mTOR, and NF-κB, which was significantly reduced in a dose-dependent manner following COS treatment (Fig. 4). While the total forms of AKT, GSK3β, mTOR, and NF-κB were not much changed. These findings underscore the potential of COS to modulate crucial signaling nodes involved in cell growth and survival. Furthermore, immunofluorescence analysis revealed that COS effectively inhibited the translocation of phosphorylated NFκB (p-NFκB) from the cytosol to the nucleus induced by TPA treatment (Fig. 5). Quantification of p-NFκB fluorescence intensity, normalized against DAPI staining using Image-pro Plus software, showed that in JB6 cells treated with TPA alone, the nuclear translocation of p-NFκB increased approximately 17.8-fold compared to the untreated group. However, when treated with TPA along with varying concentrations of COS (10 and 20 µM), a significant decrease in p-NFκB translocation was observed. Specifically, treatment with 10 µM COS resulted in a 54.3% decrease, and 20 µM COS showed the greatest reduction at approximately 80.1%. The analysis demonstrated that the nuclear levels of p-NFκB decrease in a COS dose-dependent manner, supporting the inhibitory effect of COS on NFκB activation induced by TPA. This evidence highlights the potential of COS in modulating key signaling pathways involved in inflammatory responses, thereby contributing to its anticancer and anti-inflammatory properties.

Fig. 4
figure 4

COS inhibits AKT downstream signaling. JB6 cells were treated with TPA and/or COS (0, 5, 10, and 20 µM) and whole cell lysates were harvested after 30 min. (A) Protein levels of p-AKT, AKT, p-GSK3β, GSK3β, p-mTOR, mTOR, p-NF-κB and NF-κB were assessed by Western blotting. β-actin levels served as a loading control. (B-E) The relative expression of p-AKT, p-GSK3β, p-mTOR, and p-NF-κB was shown after normalization against panAKT, GSK3β, mTOR, and NF-κB, respectively. Significantly different at: #p < 0.05 compared to the control; *p < 0.05, and **p < 0.01 compared to group treated with TPA only

Full size image
Fig. 5
figure 5

COS suppresses p-NF-κB activation in JB6 cells treated with TPA. JB6 cells were treated with various concentrations of TPA and COS (0, 10, and 20 µM), then immunostained against p-NF-κB (shown in green). Nuclei were counterstained with DAPI (shown in blue). (A) Representative images of p-NF-κB, DAPI, and merged visualization to illustrate the subcellular localization of p-NF-κB. (B) Fluorescence intensity of p-NF-κB was quantified using Image-pro Plus software and normalized against DAPI staining to assess the relative changes in p-NF-κB levels. The images were captured at 20x magnification using a confocal microscope. Scale bar: 20 μm

Full size image

Therapeutic effects of COS on skin carcinogenesis mouse model

To transition our observations from in vitro to an in vivo, we utilized a DMBA/TPA-induced skin carcinogenesis mouse model to evaluate the chemopreventive capabilities of COS. The experimental design, outlined in Fig. 6A, was constructed to assess the effect of COS on key markers of skin carcinogenesis, specifically skin hyperplasia and inflammation. Comprehensive assessments including body weight monitoring, papilloma counting, and tumor volume measurement were conducted, with detailed results presented in Fig. 6B, C and D. Notably, we observed a potent protective effect of COS, demonstrated by a significant reduction in papilloma number and volume in COS-treated groups compared to DMBA/TPA group (Fig. 6E). Histological examination through H&E staining affirmed the suppression of epidermal hyperplasia in a concentration-dependent manner with COS treatment (Fig. 7A and B). Concurrently, immunohistochemical staining showed that the expression of p-AKT, COX-2 and Ki-67 was increased in the DMBA/TPA group, while TPA/COS treatment significantly reversed the phenomena. These findings underscore the potential of COS as an effective chemopreventive agent against skin inflammation or skin cancer, as depicted in Fig. 7C, D, E, F, G and H.

Fig. 6
figure 6

COS effectively inhibits the growth of mouse skin carcinoma. (A) A schematic diagram of the DMBA/TPA carcinogenesis. 5 weeks old ICR female mice were treated with COS, followed by treatments with DMBA/TPA for 21-week. (B) Body weight change in mice. (C) Average number of papillomas per mouse. (D) Mean tumor volume per mouse. The data are shown as the mean ± SEM values; n = 5–8. Significantly different at: *p < 0.05, and **p < 0.01 compared to the control. (E) The images of the dorsal skin of representative mice from the Vehicle, DMBA/TPA, low dose COS, high dose COS, only COS groups

Full size image
Fig. 7
figure 7

COS inhibits AKT downstream signaling and reduces skin hyperplasia and expression of p-AKT, COX-2, and Ki-67. (A) Representative images of epidermal proliferation and hyperplasia in the DMBA/TPA-induced mice with different concentrations of COS treatments analyzed by H&E staining. (B) The effects of COS on the histology of mouse skin carcinogenesis induced by DMBA/TPA were evaluated by quantifying epidermal thickness following H&E staining. (C, E, G) Immunohistochemical analysis was used to confirm p-AKT, COX-2, and Ki-67 levels in the skin tissue samples obtained from mice following different treatments. (D, F, H) Quantitative analysis of p-AKT, COX-2, and Ki-67 levels after treatment. All measurements were made using the Image-Pro plus 6.0 program. Significantly different at: ###p < 0.001 compared to the control; **p < 0.01, and ***p < 0.001 compared to group treated with DMBA/TPA only

Full size image

Discussion

This study investigated the effects of COS on skin cancer prevention both in vitro and in vivo. There is growing interest in exploring the therapeutic potential of natural compounds extracted from plants, especially phytochemicals known for their diverse pharmacological properties, including anticancer effects. Our study focused on COS, a naturally occurring sesquiterpene lactone extracted from the plant Saussurea costus. TPA is a potent tumor promoter, which is widely used in the study of tumor promotion mechanisms. PKC, stimulated by TPA, regulates cell proliferation, growth, survival, and inflammatory responses through pathways such as MAPK/ERK, PI3K/AKT, AP-1, and NF-κB [35,36,37,38,39,40,41,42,43]. The action mechanism of COS against cell transformation induced by TPA and skin carcinogenesis induced by DMBA/TPA remains largely unexplored. Cell transformation, the process in which normal cells are transformed into malignant ones, is a critical initial phase in the progression of cancer, playing a crucial role in cancer development and metastasis [15, 44, 45]. Inhibiting cell transformation can thus be a vital strategy in blocking cancer development at an early stage. This study aimed to provide important insights into the anticancer mechanisms of COS in cell transformation and skin carcinogenesis induced by TPA treatment. As a result, it was confirmed that COS demonstrated the ability to inhibit TPA-induced cell transformation and skin carcinogenesis both in vitro and in vivo by inducing cell cycle arrest and blocking the downstream signaling pathways associated with the AKT pathway.

Since COS does not adversely affect normal epidermal cells, it has become a potential candidate for a cancer prevention strategy (Fig. 1B). COS significantly inhibited colony formation and proliferation in TPA-treated JB6 cells (Fig. 1C and D), and flow cytometry analysis confirmed that COS induces cell cycle arrest at the G2/M phase. Cyclin B1 are critical proteins regulating the G2/M transition of the cell cycle and are overexpressed in various malignant tumors, promoting proliferation. Therefore, inhibiting the expression of cyclin B1 at this stage can effectively block cell division and growth, making them potential targets for cancer therapy [46,47,48]. Additionally, p21 interacts with various cell signaling pathways to regulate the cell cycle at both the G1 and G2/M phases. Specifically, it inhibits CyclinB1-CDK1 activity, thereby inducing cell cycle arrest at the G2/M phase, preventing mitotic entry, and facilitating DNA repair [49, 50]. COS upregulates p21 while simultaneously downregulating cyclin B, thereby arresting the cell cycle at the G2/M phase (Fig. 2).

Flow cytometry analysis demonstrated that COS treatment significantly increased the proportions of early and total apoptotic cells in a dose-dependent manner. This indicates that COS effectively disrupts the survival pathways activated by TPA, thereby reactivating apoptotic mechanisms. Consistently, Western blot analysis revealed an upregulation of cleaved caspase-3 and caspase-7, key executioners of apoptosis. The activation of these caspases suggests that COS induces apoptosis via the intrinsic apoptotic pathway. This study highlights the potential of COS to restore apoptotic pathways in TPA-induced JB6 cells, effectively eliminating transformed cells.

Furthermore, we confirmed through western blotting that COS inhibited the expression of AKT and its downstream signaling proteins. TPA plays a vital role in activating various downstream signaling proteins in the PI3K/AKT pathway, which is involved in cellular processes such as growth, survival, and metabolism [39, 51,52,53,54]. This pathway is abnormally activated in many types of cancer, and its effective inhibition can be a vital strategy for suppressing tumor growth and metastasis. As shown in Fig. 4, COS inhibited AKT phosphorylation and decreased the expression of downstream signaling proteins including GSK3β, mTOR, and NF-κB. The ability of COS to inhibit this pathway and block essential downstream processes for cancer cell growth and survival is a significant finding. Moreover, fluorescence microscopy analysis confirmed that nuclear translocation of p-NF-κB decreased upon COS treatment (Fig. 5), showing that inhibition of the AKT pathway plays an important role in TPA-induced cell transformation. Interestingly, while immunofluorescence staining showed significant nuclear translocation of p-NF-κB upon TPA treatment and its substantial reduction with COS treatment, Western blot results revealed minimal changes in the total protein levels of p-NF-κB following TPA treatment. Additionally, in COS-treated groups, the expression of p-NF-κB was further reduced to levels lower than those observed in the untreated group. This discrepancy can likely be attributed to the different aspects measured by these two methods. Immunofluorescence staining is highly sensitive to activation states and subcellular localization changes, such as nuclear translocation, while Western blot measures the total protein levels across the entire cell sample. The observed reduction in nuclear translocation of p-NF-κB in COS-treated groups, as shown by immunofluorescence staining, indicates that COS effectively inhibits the activation and nuclear entry of p-NF-κB. This aligns with the decrease in total p-NF-κB protein levels observed in the Western blot analysis, confirming that the inhibition of p-NF-κB nuclear translocation by COS is dose-dependent.

Additionally, to demonstrate the effectiveness of COS in ameliorating skin hyperplasia and inflammation in a mouse model of skin cancer, H&E staining and IHC analyses were conducted. H&E staining is widely used to evaluate changes in epidermal thickness, which can serve as an indicator of tumor growth and inflammatory responses. To verify the effects of COS, we compared the epidermal thickness of skin tissues between the TPA group and the COS-treated group to evaluate the anticancer and anti-inflammatory effects of COS. COS not only effectively reduced papilloma formation in TPA-induced mouse skin (Fig. 6) but also significantly decreased epidermal thickness (Fig. 7A and B). Moreover, the decreased expression of p-AKT in the skin tissues of mice treated with TPA/COS compared to the DMBA/TPA group further suggests that COS regulates the AKT signaling pathway in vivo (Fig. 7C and D). The fact that COS can regulate the AKT signaling pathway, a crucial pathway for the survival and growth of skin cancer cells, reaffirms the importance of targeting the AKT signaling pathway in cancer therapy. Meanwhile, COX-2 is a key mediator of inflammation, and its overexpression promotes the production of inflammatory cytokines and shapes the tumor microenvironment, supporting the growth, survival, and metastasis of cancer cells [55,56,57,58]. Thus, assessing changes in COX-2 expression is important for understanding the anti-inflammatory and anticancer mechanisms of COS. Additionally, Ki-67 is a protein used as a marker to evaluate cell proliferation, expressed in all stages of the cell cycle including G1, S, G2, and mitosis. Its expression is associated with the proliferation and growth of various tumor cells [59,60,61,62]. Through IHC analysis, we observed that the expression of tumor progression markers such as COX-2 and Ki-67, which increased in the skin tissue of TPA-treated mice, decreased in a concentration-dependent manner in the TPA/COS group (Fig. 7E and H). The reduction in COX-2 and Ki-67 expression suggests a potential mechanism by which COS may suppress inflammatory responses and the development and progression of skin cancer.

These results suggest that the anti-inflammatory and anticancer effects of COS are mediated through G2/M phase arrest in the cell cycle, inhibition of COX-2 and Ki-67 expression, and suppression of the AKT signaling pathway. COS shows potential as a chemopreventive agent by preventing TPA-induced cell transformation and skin carcinogenesis. Future research should aim to further clarify the molecular mechanisms of COS and evaluate its efficacy in various carcinogenesis models.

Conclusion

Our findings demonstrate the potential of COS, a natural plant-derived compound, with significant anti-skin cancer properties. By revealing its effects on cell transformation, proliferation, and key signaling pathways, our study not only expands our understanding of the pharmacological profile of COS, but also establishes its position as a promising candidate for future development as a skin cancer treatment. The implications of our research extend beyond immediate results, suggesting that integrating natural compounds like COS into the cancer treatment paradigm could offer more holistic and less invasive treatment options. Considering the complexity of cancer onset and the multifaceted role of signaling pathways in tumor biology, our study calls for in-depth exploration of the synergistic effects of COS with other therapeutic modalities, aiming to enhance the efficacy of skin cancer treatment and minimize resistance.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

COS:

Costunolide

DMBA:

7,12-Dimethylbenz(a)anthracene

TPA:

12-O-tetradecanoylphorbol-13-acetate

MEM:

Eagle’s minimum essential medium

FBS:

Fetal bovine serum

BME:

Basal Medium Eagle

References

  1. Eisemann N, Waldmann A, Geller AC, Weinstock MA, Volkmer B, Greinert R, Breitbart EW, Katalinic A. Non-melanoma skin cancer incidence and impact of skin cancer screening on incidence. J Invest Dermatology. 2014;134(1):43–50.

    Article  CAS  Google Scholar 

  2. Rogers HW, Weinstock MA, Feldman SR, Coldiron BM. Incidence estimate of nonmelanoma skin cancer (keratinocyte carcinomas) in the US population, 2012. JAMA Dermatology. 2015;151(10):1081–6.

    Article  PubMed  Google Scholar 

  3. Aggarwal P, Knabel P, Fleischer AB Jr. United States burden of melanoma and non-melanoma skin cancer from 1990 to 2019. J Am Acad Dermatol. 2021;85(2):388–95.

    Article  PubMed  Google Scholar 

  4. Siegel RL, Giaquinto AN, Jemal A. Cancer statistics, 2024. Cancer J Clin 2024;74(1).

  5. Emri G, Paragh G, Tósaki Á, Janka E, Kollár S, Hegedűs C, Gellén E, Horkay I, Koncz G, Remenyik É. Ultraviolet radiation-mediated development of cutaneous melanoma: an update. J Photochem Photobiol B. 2018;185:169–75.

    Article  CAS  PubMed  Google Scholar 

  6. Parker ER. The influence of climate change on skin cancer incidence–a review of the evidence. Int J Women’s Dermatology. 2021;7(1):17–27.

    Article  Google Scholar 

  7. Narayanan DL, Saladi RN, Fox JL. Ultraviolet radiation and skin cancer. Int J Dermatol. 2010;49(9):978–86.

    Article  PubMed  Google Scholar 

  8. Gordon R. Skin cancer: an overview of epidemiology and risk factors. In: Seminars in oncology nursing: Elsevier. 2013;160–9.

  9. Drexler SK, Bonsignore L, Masin M, Tardivel A, Jackstadt R, Hermeking H, Schneider P, Gross O, Tschopp J, Yazdi AS. Tissue-specific opposing functions of the inflammasome adaptor ASC in the regulation of epithelial skin carcinogenesis. Proceedings of the National Academy of Sciences. 2012;109(45):18384–18389.

  10. Radiation. Ultraviolet (UV) radiation and skin cancer [https://www.who.int/news-room/questions-and-answers/item/radiation-ultraviolet-(uv)-radiation-and-skin-cancer]

  11. Samarasinghe V, Madan V. Nonmelanoma skin cancer. J Cutan Aesthetic Surg. 2012;5(1):3–10.

    Article  Google Scholar 

  12. Didona D, Paolino G, Bottoni U, Cantisani C. Non melanoma skin cancer pathogenesis overview. Biomedicines. 2018;6(1):6.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000;100(1):57–70.

  14. Goldstein J, Roth E, Roberts N, Zwick R, Lin S, Fletcher S, Tadeu A, Wu C, Beck A, Zeiss C. Loss of endogenous Nfatc1 reduces the rate of DMBA/TPA-induced skin tumorigenesis. Mol Biol Cell. 2015;26(20):3606–14.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Kong Y-H, Xu S-P. Salidroside prevents skin carcinogenesis induced by DMBA/TPA in a mouse model through suppression of inflammation and promotion of apoptosis. Oncol Rep. 2018;39(6):2513–26.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. Ivanescu B, Miron A, Corciova A. Sesquiterpene lactones from Artemisia genus: biological activities and methods of analysis. J Anal Methods Chem. 2015;2015(1):247685.

    PubMed  PubMed Central  Google Scholar 

  17. Kawasaki BT, Hurt EM, Kalathur M, Duhagon MA, Milner JA, Kim YS, Farrar WL. Effects of the sesquiterpene lactone parthenolide on prostate tumor-initiating cells: an integrated molecular profiling approach. Prostate. 2009;69(8):827–37.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Sokovic M, Ciric A, Glamoclija J, Skaltsa H. Biological activities of sesquiterpene lactones isolated from the genus Centaurea L.(Asteraceae). Curr Pharm Design. 2017;23(19):2767–86.

    Article  CAS  Google Scholar 

  19. Yan S, Ke C, Feng Z, Tang C, Ye Y. The first phytochemical investigation of Artemisia divaricate: sesquiterpenes and their anti-inflammatory activity. Molecules. 2023;28(10):4254.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Anibogwu R, Jesus KD, Pradhan S, Leuven SV, Sharma K. Sesquiterpene lactones and flavonoid from the leaves of basin big sagebrush (Artemisia tridentata subsp. tridentata): Isolation, Characterization and Biological Activities. Molecules. 2024;29(4):802.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kim NY, Sethi G, Um J-Y, Ahn KS. Euphorbiasteroid induces apoptosis as well as autophagy through modulating SHP-1/STAT3 pathway in hepatocellular carcinoma cells. Int J Mol Sci. 2023;24(18):13713.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Cao F, Chu C, Qin J-J, Guan X. Research progress on antitumor mechanisms and molecular targets of Inula sesquiterpene lactones. Chin Med. 2023;18(1):164.

    Article  PubMed  PubMed Central  Google Scholar 

  23. Cheng M-Y, Chuang Y-T, Chang H-W, Lin Z-Y, Chen C-Y, Cheng Y-B. Chemical constituents from soft coral clavularia spp. Demonstrate antiproliferative effects on oral cancer cells. Mar Drugs. 2023;21(10):529.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Shams A, Ahmed A, Khan A, Khawaja S, Rehman NU, Qazi AS, Khan A, Bawazeer S, Ali SA, Al-Harrasi A. Naturally isolated sesquiterpene lactone and hydroxyanthraquinone induce apoptosis in oral squamous cell carcinoma cell line. Cancers. 2023;15(2):557.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Alamoudi AJ, Badr-Eldin SM, Ahmed OA, Fahmy UA, Elbehairi SEI, Alfaifi MY, Asfour HZ, Mohamed GA, Ibrahim SR, Abdel-Naim AB. Optimized bilosome-based nanoparticles enhance cytotoxic and pro-apoptotic activity of Costunolide in LS174T colon cancer cells. Biomed Pharmacother. 2023;168:115757.

    Article  CAS  PubMed  Google Scholar 

  26. Kumar R, Bhardwaj P, Soni M, Singh R, Choudhary S, Virmani N, Asrani R, Patial V, Sharma D, Gupta V. Modulation of mammary tumour progression using murine model by ethanol root extract of saussurea costus (falc.) Lipsch. J Ethnopharmacol. 2024;319:117302.

    Article  CAS  PubMed  Google Scholar 

  27. Xing W, Wen C, Wang D, Shao H, Liu C, He C, Olatunji OJ. Cardiorenal protective effect of Costunolide against doxorubicin-induced toxicity in rats by modulating oxidative stress, inflammation and apoptosis. Molecules. 2022;27(7):2122.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. El-Far AH, Godugu K, Salaheldin TA, Darwish NH, Saddiq AA, Mousa SA. Nanonutraceuticals: Anti-cancer activity and improved safety of chemotherapy by Costunolide and its nanoformulation against colon and breast cancer. Biomedicines. 2021;9(8):990.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Huang H, Park S, Zhang H, Park S, Kwon W, Kim E, Zhang X, Jang S, Yoon D, Choi S-K. Targeting AKT with Costunolide suppresses the growth of colorectal cancer cells and induces apoptosis in vitro and in vivo. J Experimental Clin Cancer Res. 2021;40:1–18.

    Article  Google Scholar 

  30. Lee SH, Cho Y-C, Lim JS. Costunolide, a sesquiterpene lactone, suppresses skin cancer via induction of apoptosis and blockage of cell proliferation. Int J Mol Sci. 2021;22(4):2075.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Fu D, Wu D, Cheng W, Gao J, Zhang Z, Ge J, Zhou W, Xu Z. Costunolide induces autophagy and apoptosis by activating ROS/MAPK signaling pathways in renal cell carcinoma. Front Oncol. 2020;10:582273.

    Article  PubMed  PubMed Central  Google Scholar 

  32. Hwang S-Y, Wi K, Yoon G, Lee C-J, Lee S-I, Jung J-g, Jeong H-W, Kim J-S, Choi C-H, Na C-S. Licochalcone D inhibits skin epidermal cells transformation through the regulation of AKT signaling pathways. Biomolecules Ther. 2023;31(6):682.

    Article  CAS  Google Scholar 

  33. Kemp CJ. Multistep skin cancer in mice as a model to study the evolution of cancer cells. In: Seminars in cancer biology: Elsevier. 2005:460–73.

  34. Sachs N, Secades P, Van Hulst L, Song J-Y, Sonnenberg A. Reduced susceptibility to two-stage skin carcinogenesis in mice with epidermis-specific deletion of CD151. J Invest Dermatology. 2014;134(1):221–8.

    Article  CAS  Google Scholar 

  35. Li J, Ma C, Huang Y, Luo J, Huang C. Differential requirement of EGF receptor and its tyrosine kinase for AP-1 transactivation induced by EGF and TPA. Oncogene. 2003;22(2):211–9.

    Article  CAS  PubMed  Google Scholar 

  36. Gupta A, Galoforo S, Berns C, Martinez A, Corry P, Guan KL, Lee Y. Elevated levels of ERK2 in human breast carcinoma MCF-7 cells transfected with protein kinase Cα. Cell Prolif. 1996;29(12):655–63.

    Article  CAS  PubMed  Google Scholar 

  37. Park S-H, Kim J-H, Lee D-H, Kang J-W, Song H-H, Oh S-R, Yoon D-Y. Luteolin 8-C-β-fucopyranoside inhibits invasion and suppresses TPA-induced MMP-9 and IL-8 via ERK/AP-1 and ERK/NF-κB signaling in MCF-7 breast cancer cells. Biochimie. 2013;95(11):2082–90.

    Article  CAS  PubMed  Google Scholar 

  38. Noh E-M, Park Y-J, Kim J-M, Kim M-S, Kim H-R, Song H-K, Hong O-Y, So H-S, Yang S-H, Kim J-S. Fisetin regulates TPA-induced breast cell invasion by suppressing matrix metalloproteinase-9 activation via the PKC/ROS/MAPK pathways. Eur J Pharmacol. 2015;764:79–86.

    Article  CAS  PubMed  Google Scholar 

  39. Lee KS, Nam GS, Baek J, Kim S, Nam KS. Inhibition of TPA–induced metastatic potential by Morin hydrate in MCF–7 human breast cancer cells via the Akt/GSK–3β/c–Fos signaling pathway. Int J Oncol. 2020;56(2):630–40.

    CAS  PubMed  Google Scholar 

  40. Newton AC. Regulation of protein kinase C. Curr Opin Cell Biol. 1997;9(2):161–7.

    Article  CAS  PubMed  Google Scholar 

  41. Rundhaug JE, Fischer SM. Molecular mechanisms of mouse skin tumor promotion. Cancers. 2010;2(2):436–82.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Verma AK, Wheeler DL, Aziz MH, Manoharan H. Protein kinase Cε and development of squamous cell carcinoma, the nonmelanoma human skin cancer. Mol Carcinogenesis: Published Cooperation Univ Tex MD Anderson Cancer Cent. 2006;45(6):381–8.

    Article  CAS  Google Scholar 

  43. Lin C-W, Hou W-C, Shen S-C, Juan S-H, Ko C-H, Wang L-M, Chen Y-C. Quercetin Inhibition of tumor invasion via suppressing PKCδ/ERK/AP-1-dependent matrix metalloproteinase-9 activation in breast carcinoma cells. Carcinogenesis. 2008;29(9):1807–15.

    Article  CAS  PubMed  Google Scholar 

  44. Chatterjee N, Alfaro-Moreno E. In vitro cell transformation assays: a valuable approach for carcinogenic potentiality assessment of nanomaterials. Int J Mol Sci. 2023;24(9):8219.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Colacci A, Corvi R, Ohmori K, Paparella M, Serra S, Da Rocha Carrico I, Vasseur P, Jacobs MN. The cell transformation assay: A historical assessment of current knowledge of applications in an integrated approach to testing and assessment for non-genotoxic carcinogens. Int J Mol Sci. 2023;24(6):5659.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hwang A, Maity A, McKenna WG, Muschel RJ. Cell cycle-dependent regulation of the Cyclin B1 promoter. J Biol Chem. 1995;270(47):28419–24.

    Article  CAS  PubMed  Google Scholar 

  47. Wang Z, Fan M, Candas D, Zhang T-Q, Qin L, Eldridge A, Wachsmann-Hogiu S, Ahmed KM, Chromy BA, Nantajit D. Cyclin B1/Cdk1 coordinates mitochondrial respiration for cell-cycle G2/M progression. Dev Cell. 2014;29(2):217–32.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Lim S, Kaldis P. Cdks, cyclins and CKIs: roles beyond cell cycle regulation. Development. 2013;140(15):3079–93.

    Article  CAS  PubMed  Google Scholar 

  49. Rahmani F, Zandigohar M, Safavi P, Behzadi M, Ghorbani Z, Payazdan M, Ferns G, Hassanian SM, Avan A. The interplay between noncoding RNAs and p21 signaling in Gastrointestinal cancer: from tumorigenesis to metastasis. Curr Pharm Design. 2023;29(10):766–76.

    Article  CAS  Google Scholar 

  50. Gartel AL, Tyner AL. The role of the cyclin-dependent kinase inhibitor p21 in apoptosis. Mol Cancer Ther. 2002;1(8):639–49.

    CAS  PubMed  Google Scholar 

  51. Hwang S-Y, Chae J-I, Kwak A-W, Lee M-H, Shim J-H. Alternative options for skin cancer therapy via regulation of AKT and related signaling pathways. Int J Mol Sci. 2020;21(18):6869.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Lu J, Rho O, Wilker E, Beltran L, DiGiovanni J. Activation of epidermal Akt by diverse mouse skin tumor promoters. Mol Cancer Res. 2007;5(12):1342–52.

    Article  CAS  PubMed  Google Scholar 

  53. Wang F, Ma H, Liu Z, Huang W, Xu X, Zhang X. α-Mangostin inhibits DMBA/TPA-induced skin cancer through inhibiting inflammation and promoting autophagy and apoptosis by regulating PI3K/Akt/mTOR signaling pathway in mice. Biomed Pharmacother. 2017;92:672–80.

    Article  CAS  PubMed  Google Scholar 

  54. Hung C-Y, Lee C-H, Chiou H-L, Lin C-L, Chen P-N, Lin M-T, Hsieh Y-H, Chou M-C. Praeruptorin-B inhibits 12-O-tetradecanoylphorbol-13-acetate-induced cell invasion by targeting AKT/NF-¥êB via matrix metalloproteinase-2/-9 expression in human cervical cancer cells. Cell Physiol Biochem. 2019;52(6):1255–66.

    Article  PubMed  Google Scholar 

  55. Sobolewski C, Cerella C, Dicato M, Ghibelli L, Diederich M. The role of cyclooxygenase-2 in cell proliferation and cell death in human malignancies. Int J Cell Biology. 2010;2010(1):215158.

    Google Scholar 

  56. Gandhi J, Khera L, Gaur N, Paul C, Kaul R. Role of modulator of inflammation cyclooxygenase-2 in gammaherpesvirus mediated tumorigenesis. Front Microbiol. 2017;8:538.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Ling F, Baldus S, Khochfar J, Xi H, Neiss S, Brabender J, Metzger R, Drebber U, Dienes H, Bollschweiler E. Association of COX-2 expression with corresponding active and chronic inflammatory reactions in Barrett’s metaplasia and progression to cancer. Histopathology. 2007;50(2):203–9.

    Article  CAS  PubMed  Google Scholar 

  58. Rizzo MT. Cyclooxygenase-2 in oncogenesis. Clin Chim Acta. 2011;412(9–10):671–87.

    Article  CAS  PubMed  Google Scholar 

  59. Scholzen T, Gerdes J. The Ki-67 protein: from the known and the unknown. J Cell Physiol. 2000;182(3):311–22.

    Article  CAS  PubMed  Google Scholar 

  60. Liu Q, Peng Z, Shen L, Shen L. Prognostic and clinicopathological value of Ki-67 in melanoma: a meta-analysis. Front Oncol. 2021;11:737760.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Menon SS, Guruvayoorappan C, Sakthivel KM, Rasmi RR. Ki-67 protein as a tumour proliferation marker. Clin Chim Acta. 2019;491:39–45.

    Article  CAS  PubMed  Google Scholar 

  62. Sobecki M, Mrouj K, Colinge J, Gerbe F, Jay P, Krasinska L, Dulic V, Fisher D. Cell-cycle regulation accounts for variability in Ki-67 expression levels. Cancer Res. 2017;77(10):2722–34.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

None.

Funding

This work was supported by a Korea Innovation Foundation (INNIPOLIS) grant funded by the Korean government (Ministry of Science and ICT) through a science and technology project that opens the future of the region, grant number: 2021-DD-UP-0380, the National Research Foundation of Korea (NRF), funded by the Ministry of Science and ICT (No. 2022R1A5A2029546).

Author information

Author notes

  1. Kwanhwan Wi and Sun-Young Hwang contributed equally to this work.

Authors and Affiliations

  1. College of Korean Medicine, Dongshin University, Naju-si, Jeonnam, 58245, Republic of Korea

    Kwanhwan Wi, Sun-Young Hwang, Young-Gwon Kim, Soong-In Lee & Mee-Hyun Lee

  2. Biopharmaceutical Research Center, Ochang Institute of Biological and Environmental Science, Korea Basic Science Institute (KBSI), Cheongju, 28119, Republic of Korea

    Cheol-Jung Lee

  3. Digital Omics Research Center, Ochang Institute of Biological and Environmental Science, Korea Basic Science Institute (KBSI), Cheongju, 28119, Republic of Korea

    Geul Bang

  4. Daehan Cell Pharm INC, Guri, 11923, Republic of Korea

    Je-Ho Lee

Authors
  1. Kwanhwan Wi
    View author publications

    You can also search for this author inPubMed Google Scholar

  2. Sun-Young Hwang
    View author publications

    You can also search for this author inPubMed Google Scholar

  3. Young-Gwon Kim
    View author publications

    You can also search for this author inPubMed Google Scholar

  4. Soong-In Lee
    View author publications

    You can also search for this author inPubMed Google Scholar

  5. Cheol-Jung Lee
    View author publications

    You can also search for this author inPubMed Google Scholar

  6. Geul Bang
    View author publications

    You can also search for this author inPubMed Google Scholar

  7. Je-Ho Lee
    View author publications

    You can also search for this author inPubMed Google Scholar

  8. Mee-Hyun Lee
    View author publications

    You can also search for this author inPubMed Google Scholar

Contributions

SYH, KHW and MHL were involved in study concept and design, acquisition of data, analysis and interpretation of data, and drafting of the manuscript. SYH, KHW, YGK and MHL performed experiments. SIL, CJL, GB, and JHL supported the data analysis and materials. SYH, KHW and MHL wrote the manuscript. MHL supervised the study. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Mee-Hyun Lee.

Ethics declarations

Ethics approval and consent to participate

All animal experiments were approved by the Animal Ethics Committee of Dongshin University (DSU2021-01-05).

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wi, K., Hwang, SY., Kim, YG. et al. Costunolide inhibits the progression of TPA-induced cell transformation and DMBA/TPA-induced skin carcinogenesis by regulation of AKT-mediated signaling. Cancer Cell Int 25, 106 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-025-03742-w

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12935-025-03742-w

Keywords

Cancer Cell International

ISSN: 1475-2867

Contact us