Isoalantolactone induces the apoptosis of oxaliplatin-resistant human colorectal cancer cells mediated by ROS generation and activation of JNK and p38 MAPK

Abstract

Treating colorectal cancer (CRC) poses challenges due to the lack of specific molecular targets. Although oxaliplatin (Ox) is commonly used to treat CRC, resistance frequently develops, necessitating the discovery of new therapeutics. This study explored the anticancer effects of Isoalantolactone (IAL) on human CRC cells HCT116 and Ox-resistant HCT116 (HCT116-OxR). Apoptosis, ROS generation, cell cycle distribution, mitochondrial membrane potential (MMP), and caspase activation were assessed through flow cytometry. Protein levels were determined by Western blot analysis. IAL reduced cell viability, measured by MTT assay, and inhibited anchorage-independent colony formation in CRC cells in a time- and concentration-dependent manner. The IC₅₀ values for 48 h of incubation were below 10 µM. Annexin V/7-AAD double staining demonstrated that IAL induced apoptosis in HCT116 and HCT116-OxR cells, and Western blot analysis confirmed increased phosphorylation of JNK and p38 mitogen-activated protein kinase (MAPK). The inhibition of these kinases by SP600125 or SB203580 blocked the antiproliferative effects of IAL. Additionally, IAL triggered ROS generation and disrupted mitochondrial membranes, leading to caspase activation. Pretreatment with N-acetylcysteine (NAC) or Z-VAD-FMK inhibited the antiproliferative effects of IAL, highlighting the crucial roles of ROS generation and caspase activation in IAL-induced apoptosis in CRC cells. In summary, IAL exhibited anticancer effects in CRC cells by inducing apoptosis by elevating ROS level and activating JNK and p38 MAPK. These findings warrant further study to evaluate the therapeutic potential of IAL in treating CRC with various resistances.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-98499-7.

Introduction

Colorectal cancer (CRC) ranks as one of the most common cancers globally. The mortality rate for CRC stands at 9.3%, following lung cancer, which holds an 18.7% mortality rate; the International Agency for Research on Cancer (IARC, WHO) reported that in 2022, over nine hundred thousand individuals succumbed to CRC¹. The current treatment modalities for CRC encompass surgical removal, chemotherapy, radiotherapy, and combination therapy, particularly for advanced stages of the disease². Surgical intervention in the early stages of CRC can significantly improve patient survival. However, surgery is not an option for metastatic CRC, necessitating a holistic therapeutic approach. To enhance therapeutic outcomes, the use of platinum-based anticancer drugs has been investigated. Cisplatin, as the inaugural platinum-based anticancer drug, demonstrates potent anticancer efficacy. Nevertheless, its utility is hindered by profound side effects, such as damage to the nervous system and DNA alterations due to its non-specific targeting of DNA. Presently, oxaliplatin (Ox), a third-generation platinum-based anticancer drug, is employed clinically. Ox can be used for cancers that are resistant to platin- or carboplatin-based drugs and presents fewer side effects. However, the therapeutic effectiveness of Ox remains inferior to that of cisplatin, and it still causes serious side effects, including myelotoxicity, vomiting, diarrhea, and peripheral neuropathies, among others³. Despite significant progress in CRC therapies, the adverse effects of chemotherapy continue to pose challenges in treating CRC patients². Consequently, there is a pressing necessity to develop anticancer therapies that are efficacious against drug-resistant cancers while reducing toxicity.

Reactive oxygen species (ROS) modulate various biological processes, including cell homeostasis and apoptosis⁴. While low levels of ROS are involved in cellular proliferation and cell death, the accumulation of excessive ROS can lead to apoptosis. Additionally, ROS can directly activate mitochondrial membrane permeabilization, resulting in the loss of mitochondrial membrane potential (MMP)⁵. The loss of MMP, a committed step in cell death, triggers the release of cytochrome c (cyt c) and apoptosis-inducing factor (AIF) from the mitochondria into the cytoplasm, activating a cascade of apoptosis signaling.

Mitogen-activated protein kinases (MAPKs) consist of multiple proteins, including extracellular signal-regulated kinases ERK1 and ERK2, c-Jun N-terminal kinase (JNK), and p38. MAPKs play a crucial role in regulating a variety of cellular processes such as proliferation, differentiation, and apoptosis^6,7. ERK1/2 primarily transmits cell survival signaling, while JNK and p38 MAPK are often activated by stress and they regulate cellular differentiation or cell death⁸. MAPK signaling is involved in ROS-mediated cell death^9–11, suggesting that ROS signaling may activate MAPK signaling.

Recent studies underscore the molecular targeting anticancer activity of various natural products^12,13. Isoalantolactone (IAL), a sesquiterpene lactone, has demonstrated a range of activities, including antioxidant, antibacterial, and anti-inflammatory effects¹⁴. In spite of several pharmacological activities, only toxicity reported so far is cytotoxicity toward cancer cells¹⁵. Additionally, IAL has shown anticancer activity in multiple cancers, such as prostate, pancreas, breast, esophagus, and lung cancers. However, the anticancer activity of IAL in Ox-resistant CRC has not yet been investigated.

Materials and methods

Reagents

Isoalantolactone (IAL) was acquired from Chemfaces (Wuhan, Hubei, China). RPMI-1640, DMEM, and MEM Media were sourced from WelGENE Inc (Gyeongsan, Korea). Tris-glycine-SDS buffer was procured from BioSolution (Seoul, Korea). Fetal bovine serum (FBS), Trypsin, penicillin/streptomycin, MEM non-essential amino acid solution, sodium pyruvate, and MEM vitamin solution were obtained from GIBCO (Invitrogen GmbH, Karlsruhe, Germany). SP600125, SB203580, N-acetyl-cysteine (NAC), N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-FMK), and Basal Medium Eagle (BME) were procured from Sigma-Aldrich (St. Louis, MO, USA). RIPA buffer was purchased from iNtRON (PRO-PREP™, Seongnam, Korea), and 2′,7′-dichlorofluorescin diacetate (DCFH-DA) was from Thermo fisher (Waltham, MA, USA).

Cell lines and culture conditions

HCT116 (human colorectal cancer cells) and HEKa (normal human epidermal keratinocytes) were acquired from the ATCC (Manassas, VA, USA), while HCT116-OxR (oxaliplatin-resistant HCT116 cells) were courtesy of MD Anderson Cancer Center (Houston, TX, USA)¹⁶. HCT116, HCT116-OxR, and HEKa cells were cultured in six-well plates at densities of 5 × 10³, 4 × 10³, and 8 × 10³cells per well, respectively. The colorectal cancer cells and HEKa cells were maintained under the same conditions previously described¹⁷. The cells were treated with IAL (4, 8, and 12 µM) for 48 h. The cells were pre-treated with inhibitors, including SP600125, SB203580, NAC, or Z-VAD-FMK, at the specified concentrations for 3 hours before IAL (12 µM) treatment for 48 h, when required.

Cell viability assay

Following the treatment of cells with IAL, inhibitors, or Ox, cell viability was assessed using the MTT assay. The experiments adhered to methods previously described¹⁰. A microplate reader (Thermo Fisher, Vantaa, Finland) detected absorbance at 570 nm.

Colony formation assay

To evaluate the anchorage-independent growth in response to IAL treatment, a colony formation assay was conducted. The experiments used agar prepared under the same compositional conditions as outlined in prior studies¹⁸. After a 2-week incubation period, colonies larger than 40 μm were counted and analyzed using a microscope (Leica Microsystems, Wetzlar, Germany) and i-Solution™ software (Vancouver, BC, Canada). Colony numbers were quantified and images captured for further analysis.

Western blot analysis

Following IAL treatment, cells were lysed using RIPA buffer with protease inhibitors. Protein concentrations were determined using the BCA assay, and equivalent amounts of protein were separated by SDS-PAGE. Proteins were transferred onto polyvinylidene difluoride membranes, blocked with 3–5% low-fat milk, and incubated with primary antibodies overnight. After washing, membranes were treated with HRP-conjugated secondary antibodies from Thermo Fisher Scientific (#31400, #31430, #31460, Waltham, MA, USA) for 120 min at RT. Protein bands were detected using the ImageQuant™ LAS500 System (GE Healthcare, Uppsala, Sweden) and quantified with ImageJ software (Version 1.4.3.67, NIH, Bethesda, MD, USA). Primary antibodies against β-actin (sc-47778), p21 (sc-6246), p27 (sc-56338), cyclin B1 (sc-166210), cdc2 (sc-8395), Mcl-1 (sc-12756), Bid (sc-11423), Bax (sc-7480), Bad (sc-8044), Bcl-xL (sc-8392), Bcl-2 (sc-7382), cytochrome c (cyt c) (sc-13156), α-tubulin (sc-5546), cytochrome c oxidase subunit 4 (COX4) (sc-69359), Apaf-1 (sc-33870), poly (ADPribose) polymerase (PARP) (sc-8007), caspase-3 (sc-7148), Bim (#2933), phospho (p)-JNK (#9251), JNK (#9252), p-p38 (#9211), and p38 (#9212) were from Santa Cruz (Dallas, TX, USA) and Cell Signaling (Danvers, MA, USA).

Cell cycle analysis

Cell cycle distribution was determined by MACSQuant analyzer (MACSQuant, Miltenyi Biotec, Bergisch Gladbach, Germany) following the same method used in prior studies¹⁷. Flow cytometry assessed the distribution of cells across the sub-G1, G0/G1, S, and G2/M phases.

Reactive oxygen species (ROS) measurement

Following the manufacturer’s guidelines, intracellular ROS levels were assessed using a DCFH-DA (CellROX™ Green Reagent, C10444, Thermo Fisher, Waltham, MA, USA) probe after IAL treatment. A MACSQuant analyzer measured the fluorescence intensity of the cells.

JC-1 assay

To assess mitochondrial membrane potential (MMP), the JC-1 assay was performed using the MitoProbe™ JC-1 Assay Kit (Thermo Fisher). Experiments were conducted according to the manufacturer’s instructions. MMP was assessed using a MACSQuant analyzer, where depolarized mitochondria were indicated by a shift in fluorescence from red to green. Data were analyzed using MACS Quantify software.

Isolation of cell fractions

Cytoplasmic and mitochondrial fractions were isolated from cell lysates as described previously¹⁷. The mitochondrial pellet was washed and resuspended in mitochondrial lysis buffer, while the cytosolic fraction was stored separately for subsequent analysis. Protein concentrations in each fraction were measured using the BCA assay, and fraction purity was verified by Western blot assay. COX4 served as a mitochondrial marker, while α-tubulin was used as a cytoplasmic marker. Primary antibodies for COX4 and α-tubulin detection were both from Santa Cruz.

Cell death assay

Apoptotic cells were quantified using the Annexin V-FITC/7-AAD Apoptosis Detection Kit (MCH100105, Merck Millipore, Darmstadt, Germany). After IAL treatment, cells were harvested, and the proportion of apoptotic cells was determined according to the manufacturer’s instructions. A Muse cell analyzer (Merck Millipore, Burlington, MA, USA) analyzer measured the fluorescence intensity of the cells. Early and late apoptotic cells were evaluated using flow cytometry.

MultiCaspase activity assay

Caspase activity in cells treated with IAL was determined using the Muse MultiCaspase Kit (MCH100109). Experiments were conducted 48 h post IAL treatment following the manufacturer’s instructions. The cells underwent analysis using a Muse Cell Analyzer.

Statistical analysis

All experiments were conducted in triplicate, and the data are presented as mean ± SD. Statistical analyses were carried out using one-way and two-way ANOVA, followed by Tukey’s post hoc test with Synergy Software KaleidaGraph 4.5 (Reading, PA, USA). Statistical significance was denoted as *p < 0.05, **p < 0.01, and ***p < 0.001 when compared to the untreated group, and ^#p < 0.05, ^##p < 0.01, and ^###p < 0.001 when compared to the IAL-treated group.

Results

IAL suppresses the growth of CRC cells

To assess the cytotoxicity of IAL in human CRC cells, we conducted the MTT cell viability assay on HCT116 and HCT116-OxR cells treated with IAL (4, 8, and 12 µM) for 24–48 h. We observed that IAL reduced the cell viability of both HCT116 and HCT116-OxR cells in a time- and dose-dependent manner. The IC₅₀ values after 48 h of incubation were 8.51 µM for HCT116 and 8.84 µM for HCT116-OxR respectively (Fig. 1A and B). To determine if IAL’s cytotoxicity was cancer-specific, we assessed the cell viability of HEKa cells treated with IAL, noting no effect on HEKa cell viability (Fig. 1C). Conversely, the cell viability of HCT116 and HEKa cells treated with Ox at 2 µM decreased respectively to 66.00% and 65.48% at 24 h, and 63.94% at 48 h for HEKa cells, and 34.01% for HCT116 cells. The cell viability of HCT116-OxR cells was 94.72% at 24 h and 93.17% at 48 h in the presence of 2 µM Ox. We also examined IAL’s effects on the anchorage-independent colony growth of CRC cells and observed a concentration-dependent decline in both number and size of colonies in HCT116 and HCT116-OxR cells, as illustrated in Fig. 1D, E, and F. Expectedly, Ox inhibited colony growth in HCT116 cells, but not in HCT116-OxR cells. Collectively, these findings demonstrate that IAL possesses antiproliferative activity in CRC cells.

Fig. 1.

Inhibition of growth in CRC cells by IAL. (A-C) Cell viability was measured in CRC cells (HCT116 and HCT116-OxR) and HEKa treated for 24 (filled) or 48 h (empty) with IAL (0, 4, 8, and 12 µM), and Ox (2 µM) using the MTT cell viability assay. Data are presented as the mean ± SD (n = 3). IC₅₀ values for 48 h incubation. (D) Micrograph of a soft agar assay to determine the anchorage-independent colony growth in CRC cells over 10 days. (E, F) Size and number of colonies. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to vehicle only.

IAL induces apoptosis by activating JNK and p38 MAPK in CRC cells

To determine whether the antiproliferative activity of IAL is linked to apoptosis induction, we conducted flow cytometry analysis using annexin V/7-AAD double staining. The proportion of HCT116 cells undergoing apoptosis increased from 2.60 ± 0.20% to 13.66 ± 0.53%, 26.92 ± 0.44%, and 39.33 ± 0.45% with increasing IAL concentrations (4, 8, and 12 µM). In HCT116-OxR cells, the corresponding proportions rose from 4.71 ± 0.57% to 6.23 ± 0.53%, 21.64 ± 0.81%, and 44.03 ± 2.37% respectively (Fig. 2A and B). Noticing that IAL induced apoptosis, we evaluated its effect on JNK and p38 MAPK signaling pathways through western blot analysis. Indeed, increased phosphorylation levels of JNK and p38 MAPK correlated with higher IAL concentrations, while overall protein levels remained stable (Fig. 2C and D). To verify if kinase activation via phosphorylation mediated IAL-induced apoptosis, we assessed the antiproliferative activity of IAL following a 3-hour pretreatment with SP600125 (a JNK inhibitor) or SB203580 (a p38 inhibitor). Figure 2E and F show that kinase inhibitor pretreatment prevented IAL’s antiproliferative effect by more than 30%. These results indicate that IAL triggers apoptosis by activating JNK and p38 MAPK in CRC cells.

Fig. 2.

Activation of JNK and p38 MAPK signaling in IAL-induced apoptosis. CRC cells HCT116 and HCT116-OxR were treated with IAL (0, 4, 8, and 12 µM) for 48 h and analyzed by flow cytometry with annexin V/7-AAD double staining or western blot analysis. (A) Representative plot from flow cytometry analysis. (B) Proportion of cells undergoing apoptosis. (C) Western blot analysis for determining levels of p-JNK, JNK, p-p38, and p38. β-actin was used as the loading control. (D) The ratio of phosphoprotein/total protein signal for JNK and p38. (E, F) Cell viability was measured using the MTT assay for CRC cells treated for 48 h with IAL, SP600125, SB203580, or Ox as indicated. Data are from three replicates. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to the control group. ###p < 0.001 compared with the IAL alone-treated group.

IAL stimulates the generation of ROS to induce apoptosis in CRC cells

While excessive ROS generation can lead to oxidative stress and apoptosis, a moderate level is essential for cellular homeostasis and signaling⁴. To determine if IAL affects ROS generation in CRC cells, we assessed cellular ROS levels using flow cytometry and a Muse Oxidative™ Stress Kit (Fig. 3A and B). The data indicated that ROS levels in HCT116 cells increased from 1.92 to 3.23%, 22.91%, and 45.93% following IAL treatment at 4, 8, and 12 µM. Similarly, in HCT116-OxR cells, ROS levels rose from 6.32 to 8.82%, 14.63%, and 23.43% under the same conditions. To investigate if ROS induction mediates IAL-induced apoptosis, we pretreated CRC cells with NAC, an ROS scavenger, at 4 mM. Following this pretreatment, cell viability, which had decreased to 29.74% and 29.36% in HCT116 and HCT 116-OxR cells respectively at 12 µM IAL, recovered to 79.55% and 80.22%. The viability of CRC cells treated with NAC alone was 96.77% in HCT116 cells and 96.57% in HCT116-OxR cells, indicating minimal toxicity at this concentration (Fig. 3C). Furthermore, NAC pretreatment effectively reversed the phosphorylation of JNK and p38 MAPK, and the activation of caspase-3 and PARP cleavage (Fig. 3D). These findings suggest that ROS generation, and the subsequent activation of JNK, p38 MAPK, and caspase-3, are critical mediators of IAL-induced apoptosis in CRC cells.

Fig. 3.

ROS generation induced by IAL. CRC cells were treated with IAL, NAC, and Ox for 48 h. (A) The cells were subjected to flow cytometry analysis using a DCFH-DA probe. (B) Proportion of cells with induced ROS. (C) Cell viability was assessed with the MTT assay after 48 h treatment with IAL, NAC, or Ox as indicated. (D) Western blot analysis was performed to determine levels of p-JNK, p-p38, caspase-3, and PARP. β-actin served as the loading control. Data are presented as mean ± standard deviation (n = 3). **p < 0.01 and ***p < 0.001 compared with the control group. ###p < 0.001 compared with the IAL alone-treated group.

IAL induces cell cycle arrest at the G2/M transition in CRC cells

We analyzed the cell cycle distribution in CRC cells treated with IAL (0, 4, 8, and 12 µM) for 48 h using flow cytometry with PI staining (Fig. 4A). The percentage of HCT116 cells in the sub-G1 phase increased from 0.63 to 2.28%, 7.89%, and 16.33% after treatment with IAL at 4, 8, and 12 µM. Similarly, in HCT116-OxR cells, these percentages increased from 1.21 to 4.28%, 3.14%, and 10.01% (Fig. 4B). In addition, IAL treatment resulted in an increase in the cell population in the G2/M phase and a decrease in cells in the G1 and S phases in a concentration-dependent manner (Fig. 4C and D). This shift in cell cycle distribution was accompanied by changes in protein levels involved in cell cycle regulation. Western blot analysis revealed that the levels of p21 and p27 increased while those of cyclin B1 and cdc2 decreased following IAL treatment (Fig. 4E). Collectively, these results demonstrate that IAL induces cell cycle arrest at the G2/M phase, contributing to its antiproliferative effects in CRC cells.

Fig. 4.

Induction of cell cycle arrest at the G2/M phase by IAL. CRC cells HCT116 and HCT116-OxR were treated with IAL (0, 4, 8, and 12 µM) for 48 h. (A) Flow cytometry analysis was performed using PI staining. (B) Proportion of cells in the Sub-G1 phase. (C, D) Cell cycle distribution of HCT116 and HCT116-OxR cells. Data are presented as mean ± SD from three replicates across three independent experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to vehicle only. (E) Western blot analysis was conducted to determine levels of p21, p27, cyclin B1, and cdc2. β-actin served as the loading control.

IAL dysregulates MMP in CRC cells

Maintaining MMP is essential for preserving mitochondrial function and cellular physiology¹⁹. We investigated whether IAL-induced apoptosis associates with mitochondrial dysfunction using flow cytometry and JC-1 staining (Fig. 5A and B). We noted an increase in the green fluorescence of JC-1, indicative of mitochondrial damage, in CRC cells treated with IAL. Conversely, the red fluorescence of JC-1, which indicates healthy and functional mitochondria, decreased. The proportion of cells exhibiting mitochondrial dysfunction rose from 2.17 to 3.53%, 24.39%, and 40.71% at IAL concentrations of 4, 8, and 12 µM in HCT116 cells. Similarly, in HCT116-OxR cells, this proportion escalated from 2.51 to 4.27%, 14.71%, and 23.98%. These findings imply that IAL triggers mitochondrial membrane depolarization, leading to apoptosis in CRC cells. Adding to the mitochondrial membrane depolarization, we observed a shift in the balance of Bcl-2 family proteins via western blotting (Fig. 5C). Levels of Bim, Bad, and Bax increased, while those of Mcl-1, BID, Bcl-xL, and Bcl-2 decreased. Furthermore, we detected the release of cyt c from mitochondria into the cytoplasm, as evidenced by western blot analysis of cytoplasmic and mitochondrial fractions. The release of cyt c was followed by the activation of Apaf-1, caspase-3, and cleavage of PARP. Collectively, these results suggest that IAL disrupts MMP to induce apoptosis in CRC cells.

Fig. 5.

Dysregulation of mitochondrial membrane by IAL. CRC cells HCT116 and HCT116-OxR were treated with IAL (0, 4, 8, and 12 µM) for 48 h. (A) Flow cytometry analysis was conducted using JC-1 staining. (B) Proportion of cells with JC-1 green fluorescence. Data are shown as mean ± SD from three independent experiments. ***p < 0.001 compared to vehicle only. (C) Western blot analysis was performed to assess proteins regulating mitochondrial membrane permeability (Bim, Mcl-1, Bad, Bax, BID, Bcl-xL, and Bcl-2), the release of cyt c into the cytoplasm, and levels of apoptosis-related proteins Apaf-1, caspase-3, and PARP. β-actin was used as the control for proteins from cell lysates, α-tubulin for cytoplasmic fraction, and COX4 for mitochondrial fraction.

IAL activates caspases to induce apoptosis in CRC cells

To explore caspase activation in CRC cells treated with IAL, we conducted flow cytometry using a Muse™ Multi-Caspase Kit. The proportion of cells with activated caspases increased following IAL treatment (4, 8, and 12 µM, Fig. 6A and B). In HCT116 cells, the values ascended from a baseline of 3.05  15.80%, 27.20%, and 39.07%, while those in HCT116-OxR cells rose from 2.54 to 4.30%, 37.08%, and 53.78%, respectively. We hypothesized that caspase activation plays a role in IAL-induced apoptosis. Therefore, we assessed the viability of CRC cells treated with IAL, with and without pre-treatment with Z-VAD-FMK, a pan-caspase inhibitor. The viability of HCT116 and HCT116-OxR cells treated with 12 µM IAL was 33.52% and 42.57%, respectively. In contrast, viability increased to 78.71% and 93.91% when the cells were pretreated with Z-VAD-FMK (Fig. 6C). These findings support the hypothesis that caspase activation mediates IAL-induced apoptosis in CRC cells.

Fig. 6.

Activation of caspases induced by IAL. CRC cells HCT116 and HCT116-OxR were treated with IAL (0, 4, 8, and 12 µM) for 48 h. (A) Flow cytometry analysis was performed using a Muse MultiCaspase Kit. (B) Proportion of cells with activated caspases. (C) Cell viability was assessed by the MTT assay after 48 h treatment with IAL, Z-VAD-FMK, and Ox as specified. Data are presented as the mean of three replicates. *p < 0.05, **p < 0.01, and ***p < 0.001 compared to vehicle only. ###p < 0.001 compared with the IAL alone-treated group.

Discussion

Cancer treatment has made significant progress over time. However, cancer continues to pose a major challenge to public health, the economy, and society²⁰. Certain characteristics of cancer, observed in the cell cycle and signaling pathways involving PI3 kinase, Akt, Ras, and MAP kinases, are critical to understanding. A deeper comprehension of these aspects in cancer biology could significantly enhance the discovery of effective treatments. Recently, there has been a surge in research focusing on anticancer therapeutics that target specific protein molecules, underscoring the critical need to identify and discover additional molecular targets. Natural products show considerable promise in developing new therapeutics, either by uncovering previously unrecognized molecular targets or by providing novel chemical entities^13,21. An increasing number of studies focus on utilizing natural products for cancer treatment.

Ox exerts its anticancer activity primarily by forming adducts between two adjacent guanine nucleotides or between guanine and adenine, which inhibits both DNA replication and transcription. Ox also disrupts ion channel regulation, damaging neurons and the central nervous system, leading to hyperactivity, oxidative stress, and neurodegeneration. These side effects are significant limitations of Ox-based cancer therapy and can result in treatment failure. Additionally, resistance to Ox-based therapy presents a further challenge²².

In this study, we aimed to assess the anticancer activity of IAL in CRC cells and to elucidate the underlying mechanisms. IAL, a sesquiterpene lactone compound originally isolated from Inula helenium L, has been traditionally used in the treatment of bronchitis and tuberculosis²³. The anticancer efficacy of IAL has been demonstrated in various cancers including prostate²⁴, pancreas^23,25, breast²⁶, esophagus²⁷, and lung²⁸. Our data indicate that IAL exhibited cytotoxic effects on human CRC HCT116 cells. This supports a previous study²⁹, and reveals that IAL reduced cell viability of HCT116 cells in both a time- and concentration-dependent manner (Fig. 1). Conversely, IAL did not exhibit significant cytotoxic effects on HEKa cells, suggesting that its cytotoxic effects may be cancer-specific. Since IAL has long been used in traditional Chinese medicine for multiple purposes³⁰, any off-target activity would not be a serious concern.The antiproliferative effects of IAL can be attributed to its ability to induce apoptosis, as demonstrated by annexin V/7-AAD double staining analysis. IAL induced apoptosis in CRC cells in a concentration-dependent manner (Fig. 2A and B). JNK and p38 MAPK are activated by various stimuli, such as inflammation, exposure to ultraviolet light, DNA damage, and oxidative stress^6,11. Our findings indicate that both JNK and p38 MAPK were activated by IAL, promoting apoptosis (Fig. 2C and D), and that kinase inhibitors SP600125 and SB203580 could partially mitigate the cytotoxicity induced by IAL (Fig. 2E and F). These results confirm that JNK and p38 MAPK are key mediators in IAL-induced apoptosis.

In addition to JNK and p38 MAPK signaling, ROS generation’s induction was crucial in IAL-induced apoptosis. The increase in ROS levels in cancer cells triggers oxidative stress, DNA damage, and cell cycle arrest^4,5. As illustrated in Fig. 3A and B, IAL treatment induced ROS generation. Pretreatment with NAC substantially restored cell viability (Fig. 3C), underscoring the pivotal role of ROS generation. Furthermore, we assessed JNK and p38 MAPK levels to determine whether ROS generation mediated the activation of these kinases, given that ROS generation could trigger several biochemical events, including JNK and p38 MAPK signaling activation^4,31. Indeed, kinase activation was reduced by NAC pretreatment (Fig. 3D), suggesting that ROS generation’s induction preceded JNK and p38 MAPK signaling activation.

The regulation of the cell cycle is mediated by several factors including cyclins and cyclin- dependent kinases (CDKs). Cyclin B1 expression precedes the G2/M phase transition, and cyclin B1, forming a complex with CDK1, induces mitosis³². The Cip/Kip family CDK inhibitors, p21^Cip, p27 ^Kip, and p57 ^Kip, share a conserved N-terminal domain that interacts with CDK proteins. By binding to the cyclin-CDK complex, these proteins inhibit the catalytic activity of CDK, consequently inducing cell cycle arrest³³. Upon examining cyclins and CDKs, it was noted that IAL decreased cyclin B1 and cdc2 levels while increasing those of p21 and p27. It seems that the upregulation of p27 caused G2/M arrest³⁴ (Fig. 4).

The loss of mitochondrial membrane potential (MMP) is a characteristic of cell apoptosis³⁵. Disruption of mitochondrial outer membrane permeability serves as a committed step in apoptosis, initiating the caspase cascade. Bcl-2 family proteins, through a complex interaction network on the outer mitochondrial membrane, are key regulators of apoptosis³⁶. We used flow cytometry with a JC-1 probe to monitor the MMP (Fig. 5A and B) and observed that IAL induced depolarization of the mitochondrial membrane. Additionally, we observed shifts in the balance of Bcl-2 family proteins, the release of cyt c, and the activation of Apaf-1 and caspase-3, along with cleavage of PARP (Fig. 5C). Using the Muse MultiCaspase Kit, the activation of multiple caspases in IAL-induced apoptosis in CRC cells was confirmed by flow cytometry (Fig. 6A and B). Moreover, pretreatment with Z-VAD-FMK indicated that IAL-induced apoptosis in these cells is mediated by caspase activation (Fig. 6C).

In summary, IAL induces apoptosis in CRC cells HCT116 and HCT116-OxR through the generation of ROS and activation of JNK and p38 MAPK, leading to MMP dysregulation, cyt c release, and caspase activation (Fig. 7). The antiproliferative effect of IAL is effective against Ox-resistant CRC, suggesting potential benefits in treating Ox-resistant CRC. While the study was limited to HCT116 cells, further investigation will validate the therapeutic potential of IAL in various CRC cells with EGFR overexpression/activation or kRAS mutation. Also, conducting in vivo studies using a xenograft model will further clarify the anticancer activities of IAL.

Fig. 7.

Schematic representation of apoptosis induced by IAL in HCT116 and HCT116-OxR CRC cells. IAL induces the generation of ROS, resulting in the phosphorylation of JNK and p38 MAPKs and cell cycle arrest. The upregulation of JNK and p38 leads to the release of cyt c from mitochondria and caspase activation, ultimately resulting in the apoptosis of CRC cells.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Made substantial contributions to conception and design of the study and performed data analysis, made figures and wrote manuscript, and interpretation: S- O Lee, S H Joo. Performed data acquisition, as well as provided administrative, technical, and material support: N Y Lee, S- S Cho, G Yoon, K- T Kim, Y H Choi, J W Park. Technical, and material support, performed project administration: J- S Choi. Performed project administration, supervision, and funding acquisition: J- H Shim. All the data were generated in-house. All authors agree to be accountable for all aspects of the work and to ensure their integrity and accuracy.

Data availability

The datasets are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.