Colorectal cancer (CRC) remains a significant burden on global health (Siegel et al., 2023). Millions of people are affected by CRC. Every step of CRC maintenance, including prevention, early detection, therapy, and patient care, faces challenges. Although established chemotherapies such as FOLFOX, FOLFIRI, and FOLFRINOX are effective to some degree, they encounter the emergence of resistance (Tharin et al., 2021). Oxaliplatin (Ox) belongs to platinum-based chemotherapeutics. It has been included in standard regimens for treating CRC (Zheng et al., 2023). Nonetheless, intrinsic and acquired resistance to Ox can develop with the alteration of drug transport, drug metabolism, cell death, DNA repair, and so on (Martinez-Balibrea et al., 2015), and the Ox-resistant cells even secrete growth factors (Bose et al., 2011). While targeted therapies were initially praised for their efficacy, they do not guarantee a complete cure, highlighting the urgent necessity to develop additional molecular targets for treating CRC (Underwood et al., 2024).

The anticancer activity of licochalcone D (LCD), a flavonoid compound originally obtained from the root of Glycyrrhiza inflata (Furusawa et al., 2009), has been demonstrated in various cancers, including skin cancer (Si et al., 2018; Hwang et al., 2023), oral squamous cell carcinoma (Seo et al., 2019), lung cancer (Oh et al., 2020), and breast cancer (Zhang et al., 2024). The anticancer activity of LCD involves the generation of ROS (Si et al., 2018), inhibition of JAK2 kinase (Seo et al., 2019), inhibition of AKT signaling (Hwang et al., 2023), and regulation of other signaling pathways. However, the anticancer activity of LCD against CRC cells has not been reported yet.

Thus, this study pursued the potential of LCD as a therapeutic for treating CRC, especially Ox-resistant CRC cells. Results revealed that LCD effectively suppressed the growth of human CRC cells HCT116 and Ox-resistant HCT116 (HCT116-OxR) cells by inducing apoptosis.

Chemicals and reagents

Licochalcone D (LCD) was synthesized and purified as described previously (Hwang et al., 2023). Culture media RPMI-1640, MEM, and Dulbecco’s modified Eagle’s Medium (DMEM) were procured from Welgene (Gyeongsan, Korea). Dimethyl sulfoxide (DMSO) and N-acetyl-L-cysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS), penicillin streptomycin (p/s), MEM Non-Essential Amino Acids (NEAA), sodium pyruvate (s/p) and MEM Vitamin Solution were purchased from Gibco (Grand Island, NY, USA). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was obtained from BD Biosciences (San Diego, CA, USA). The following primary antibodies for actin (sc-47778), cdc2 (sc-8395), p21 (sc-6246), p27 (sc-56338), caspase 3 (sc-7148), Glucose-regulated protein 78 (GRP78) (sc-1050), C/EBP homologous protein (CHOP) (sc-7351), Death receptor (DR) 4 (sc-8411), DR5 (sc-166624), Bim (sc-374358), Bax (sc-7480), Bcl-2 (sc-7382), Bcl-xL (sc-8392), BID (sc-11423), cytochrome (cyto) c (sc-13156), β-tubulin (sc-166729), COX4 (sc-69359), apoptotic protease activating factor-1 (Apaf-1) (sc-33870), were from Santa Cruz (Santa Cruz Biotechnology, Dallas, TX, USA), and the antibodies for detecting phospho (p) JNK (#9255), pp38 (#9211), JNK (#9251), p38 (#9212), Poly ADP-ribose polymerase (PARP) (#9542), cyclin B1 (#4135), and Mcl-1 (#5453) were purchased from Cell Signaling (Cell Signaling Technology, Danvers, MA, USA).

Cell culture

Colorectal cancer (CRC) HCT116 cells, mouse epidermal JB6 cells, and HaCaT (# PCS-200-011) cells were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). HCT116-OxR [Oxaliplatin-resistant (OxR) CRC cell] were provided by Prof. LM Ellis (The University of Texas MD Anderson Cancer Center, Houston, TX, USA) (Bose et al., 2011). HCT116 and HaCaT cells were maintained in RPMI-1640 and DMEM respectively, supplemented with 10% heat-inactivated FBS and 100 U/mL of p/s. JB6 cells were maintained in MEM supplemented with 5% heat-inactivated FBS, 100 U/mL of p/s, 1% s/p, and 1% MEM NEAA. HCT116-OxR cells were cultured in MEM containing 10% FBS, 100 U/mL of p/s, 1% s/p, 1% MEM NEAA and 1% MEM vitamin solution. In addition, HCT116-OxR cells were maintained in the presence of Ox at 2 μM. All cells were maintained in a humidified 5% CO₂ incubator at 37°C.

MTT cell viability assay

To examine the cytotoxicity of LCD, the MTT cell viability assay was performed on human CRC cells (HCT116 and HCT116-OxR) and non-cancer normal cells (JB6 and HaCaT) after LCD treatment as previously described (Park et al., 2024). Both cells were seeded on 96-well plate at 5,000 cells per well (HCT116) and 4,000 cells per well (HCT116-OxR) and grown overnight at 37°C. Afterward, cells were treated with varying concentrations of LCD (0, 2, 4, and 6 μM) for 24 h and 48 h. To monitor the cell viability, MTT reagent dissolved in PBS was added to each well, and the culture was incubated at 37°C for 45 or 80 min. After incubation, the formazan crystals were dissolved in 100 μL of DMSO and the absorbance at 570 nm was measured with a Multiskan SkyHigh spectrophotometer (Thermo Fisher Scientific, Vantaa, Finland).

Soft agar assay

To evaluate the anchorage-independent growth of CRC cells, soft agar assay was performed as described previously (Lee et al., 2024). Briefly, cells at a density of 8,000 cells in culture media containing agar, BME, 10% FBS, 5 μg/mL gentamicin, 2 mM L-glutamine were treated with LCD (0, 2, 4, 6 μM) or Ox (2 μM, positive control) in a well 6-well plate for a week. The formation of colonies was observed under microscope and the size and number of colonies were analyzed using an IMT i-solution software (IMT i-solution Inc, Vancouver, BC, Canada).

Annexin V/7-aminoactinomycin D (7-AAD) double staining assay

To determine the apoptosis induced by LCD, annexin V/7-AAD double staining assay was performed. First, cells were seeded in 6-well plates and allowed to attach. Then, the cells with ~85% confluency were treated with varying concentrations of LCD for 48 h. The cells were stained with a Muse Annexin V and Dead Cell Kit (Luminex, Austin, TX, USA) according to the manufacturer’s instruction, and the fluorescence was analyzed by Muse™ Cell Analyzer (Merck Millipore, Darmstadt, Germany).

Cell cycle analysis

To examine the cell cycle distribution after treatment with LCD, we used a Muse™ cell cycle kit (Luminex, MCH100106) was used for flow cytometry. Briefly, LCD treated cells were fixed with 70% ethanol by incubating overnight at –20°C. The cells were then washed and resuspended in Muse Cell Cycle Reagent. The DNA content was analyzed by Muse™ Cell Analyzer.

Measurement of Reactive Oxygen Species (ROS)

To determine the effects of LCD on the generation of ROS in CRC cells, Muse™ Oxidative Stress Kit (MCH100111, Luminex) was used. The CRC cells treated with LCD were mixed with Muse™ Oxidative Stress Reagent working solution (37°C, 30 min) following the manufacturer’s manual. The level of fluorescence was detected with a Muse™ Cell Analyzer.

Mitochondrial Membrane Potential (MMP) assay

To examine whether LCD affects the MMP in CRC cells, Muse™ MitoPotential Kit (MCH100110, Luminex) was used. The CRC cells treated with LCD cells were mixed Muse™ MitoPotential working solution and 7-AAD and incubated for 5 min. The MMP was measured using a Muse™ Cell Analyzer.

Western blot analysis

To monitor the level of proteins in the CRC cells treated with LCD, western blot analysis was performed. Cells were lysed with radio-immunoprecipitation assay (RIPA) buffer. Protein mixtures from the whole cell lysates were resolved by SDS-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to PVDF membranes. The PVDF membranes were blocked with 3 or 5% skim milk, then appropriate primary and horseradish peroxidase-conjugated secondary antibodies were used to probe the proteins of interest. The probed proteins were visualized by chemiluminescence using a LAS-Amersham Imager 600 (GE Healthcare, Uppsala, Sweden). The blots were quantified using ImageJ (Schneider et al., 2012).

Isolation of cytosolic and mitochondrial fractions

To determine the release of cytochrome c from mitochondria, cytosolic and mitochondrial fractions were isolated. Briefly, cells were homogenized using 0.1% digitonin for 5 min and resuspended in a plasma membrane extraction buffer (Lee et al., 2023). The resuspended cells were centrifuged for 5 min at 13,000 rpm: the supernatant was collected and centrifuged again for 30 min to obtain the cytosolic fraction. The pellet was washed again with the plasma membrane extraction buffer, and the supernatant was used as the mitochondrial fraction after centrifugation for another min at 13,000 rpm.

Multi-caspase assay

To examine the activation of caspase in the CRC cells treated with LCD, Multi-caspase (caspase 1, -3, -4, -5, -6, -7, -8, and -9) activity were measured out as instructed in the Muse™ Multi-Caspase kit (MCH100109, Luminex). Cells treated with LCD were incubated with caspase buffer with 50 µL of Muse™ Multi-Caspase Reagent working solution at 37°C for 30 min. Then, each sample was mixed with 7-AAD working solution. Multi-caspase activity was analyzed by measuring fluorescence with a Muse™ Cell Analyzer.

Statistical analysis

All results are presented as mean ± standard deviation (SD) from three independent experiments. Statistical differences were analyzed using the student’s t-test and one-way or two-way analysis of ANOVA. P-value to differences were considered significant when *p<0.05, **p<0.01, and ***p<0.001 compared to the control group. The level of significance was considered between LCD treatment and inhibitor treatment: ^#p<0.05, ^##p<0.01, and ^###p<0.001.

Effects of LCD on cell proliferation and apoptosis in human CRC cells

We examined the antiproliferative activity of LCD in HCT116 cells by performing MTT cell viability assay. Human colorectal cancer cell lines HCT116 and HCT116-OxR were treated with LCD at different concentrations (0, 2, 4, and 6 μM) for 24 h or 48 h. LCD significantly decreased cell viability in a concentration-dependent manner (Fig. 1A, 1B). IC₅₀ value of LCD after 48 h treatment was 5.09 μM for HCT116 cells and 3.28 μM for HCT116-OxR cells. However, LCD did not show an antiproliferative effect in HaCaT or JB6 cells under the same condition (0, 2, 4, and 6 μM) (Fig. 1C, 1D). Treatment of HaCaT and JB6 cells with Ox (2μM) for 48 h resulted in decreased cell viability (50.23% and 48.38%, respectively). Similarly, treatment of HCT116 with Ox (2 μM) for 48 h decreased cell viability (42.28%), whereas the cell viability of HCT116-OxR did not change significantly (93.50%) after such treatment. Encouraged by the selective antiproliferative activity of LCD against CRC cells, we performed soft agar assays to evaluate effects of LCD and Ox on anchorage-independent cell growth. It was observed that LCD treatment inhibited the formation of colonies both in HCT116 and HCT116-OxR cells in a concentration-dependent manner. Both size and number of colonies decreased as LCD concentration increased (Fig. 1E, 1F). As expected, Ox treatment inhibited the formation of colonies in HCT116 cells, but not in HCT116-OxR cells. These results show that treatment using LCD can inhibit cell viability and colony formation in human colorectal cancer cells. To determine whether the antiproliferative activity of LCD induced apoptosis in CRC cells, we performed annexin V/7-AAD double staining assay. Indeed, it was observed that LCD induced apoptosis in both HCT116 and HCT116-OxR cells in a concentration-dependent manner (Fig. 1G, 1H). After treatment with 6 μM LCD, percentages of HCT116 and HCT116-OxR cells undergoing apoptosis (upper and lower right quadrants in a flow cytometry plot) were 38.00% and 46.57%, respectively.

Figure 1. LCD suppresses proliferation of human colorectal cancer cells. (A-D) MTT assay of cell viability of human colorectal cancer (CRC) cell lines (HCT116, HCT116-OxR), human adult primary epidermal keratinocytes (HaCaT) cell lines, and mouse epidermal cells JB6 were treated with LCD at various concentrations (0, 2, 4, 6 μM) for 24 or 48 h. (E) Microscopic images of soft agar colonies of CRC cell lines induced by LCD (0, 1, 2, 4 μM) treatment. These results were microscopic data. Colony size was analyzed after 7 days of culture. (F) Graph showing quantitation of colony number data from Fig. 1E. (G) Cells were treated with various concentrations (0, 2, 4, 6 μM) of LCD for 48 h. Apoptosis expressed as dot plots was measured using Annexin V/7-aminoactinomycin D (7-ADD) staining. Non-apoptotic cells (Annexin-V negative/7-AAD negative) are shown on the bottom left side. Early apoptotic cells (Annexin-V positive/7-AAD negative) are shown on the bottom right side. Late apoptotic cells (Annexin-V positive/7-AAD positive) are shown on the top right side and necrotic cells (Annexin-V negative/7-AAD positive) on the top left side. (H) A graph showing quantitation of early and late apoptotic cells induced by LCD. Data are expressed as mean ± SD (n=3). *p<0.05, **p<0.01, and ***p<0.001 compared to the control group.

Effects of LCD on cell cycle regulation in human CRC cells

As the antiproliferative activity of LCD was observed in human CRC cells, we asked whether LCD disturb the cell cycle distribution. Therefore, the effects of LCD treatment on cell cycles of colorectal cancer cells were examined. The percentage of sub-G1 population was increased in response to treatment with LCD (Fig. 2A, 2B). Treatment of cells with LCD at 2, 4, and 6 μM increased the sub-G1 population from 4.47% to 15.87%, 23.00%, and 31.10% in HCT116 cells and from 4.80% to 26.13%, 36.73%, and 50.63% in HCT116-OxR cells, respectively. Levels of proteins involved in cell cycle regulation, such as p21, p27, cyclin B1, and cdc2, were determined by western blot analysis. As shown in Fig. 2C, LCD decreased protein levels of cyclin B1 and cdc2 but increased protein levels of p21 and p27 in a dose-dependent manner. These results imply that LCD can induce cell cycle arrest to exert an antiproliferative effect in CRC cells HCT116 and HCT116-OxR.

Figure 2. LCD induces cell cycle arrest and increases sub-G1 accumulation in CRC. HCT116 and HCT116-OxR cells were treated with LCD (0, 2, 4 and 6 μM) for 48 h. (A) Cell cycle was analyzed with a Muse™ Cell Analyzer. (B) Sub-G1 population of HCT116 and HCT116-OxR cells in Fig. 2A. Figures in the bar graph represent the mean ± SD of triplicate measurements from three separate experiments. **p<0.01 and ***p<0.001 compared to an untreated group. (C) Expression levels of cell cycle regulation proteins in HCT116 and HCT116-OxR cells were decided by western blot. Actin was used as a control. Protein levels of p21, p27, cyclin B1, and cdc2 were analyzed using ImageJ software.

Effects of LCD on phosphorylation of JNK/p38 MAPK in human CRC cells

Upon observing the dysregulation of cell cycle induced by LCD, we examined if JNK and p38 MAPK signaling pathways were involved in the apoptosis induced by LCD. The MAPK signaling cascades are critical in mediating cell cycle, death, and life (Kong et al., 2000). The levels of proteins JNK and p38 MAPK as well as their phosphorylated forms were examined by western blot analysis. LCD increased levels of phosphorylated proteins of both JNK and p38 MAPK in a dose-dependent manner (Fig. 3A-3C), indicating that phosphorylation of these two proteins was involved in the LCD-induced apoptosis. To see whether JNK/p38 MAPK signaling pathways were required for LCD-induced apoptosis in human CRC cells, we examined cell viability of human CRC cells pretreated with an inhibitor SP600125 or SB203580 before LCD treatment. As shown in Fig. 3D, pretreatment with SP600125 (4 μM), a JNK inhibitor, prevented the antiproliferative effect of LCD effectively. Similarly, pretreatment with SB203580 (8 μM), a p38 MAPK inhibitor, reverted the cytotoxicity of LCD efficiently (Fig. 3E). These results suggested that phosphorylation of JNK and p38 MAPK mediated LCD-induced apoptosis in human CRC cells.

Figure 3. LCD induces phosphorylation of JNK/p38 MAPK in human CRC cells. HCT116 and HCT116-OxR cells were treated with LCD (0, 2, 4 and 6 μM) for 48 h. (A) Cell lysates were collected and protein levels of phosphorylated (p)JNK, JNK, pp38, and p38 were analyzed by western blot assay. Actin was used as the internal control. (B, C) Quantitation of the intensity of pJNK and pp38 western blot bands. (D, E) Cells were pre-treated with 4 μM SP600125 (JNK inhibitor) or 8 μM SB203580 (p38 inhibitor) for 3 h and then treated with or without 6 μM LCD for 48 h. Cell viability was analyzed using the MTT assay. Data are presented as mean ± SD of three independent experiments in triplicate. *p<0.05, **p<0.01, and ***p<0.001 compared to the control group; ^###p<0.001 compared to the LCD-treated group.

Effects of LCD on MMP and caspase cleavage in human CRC cells

We then measured the MMP using a MitoPotential Kit after HCT116 and HCT116-OxR cells were treated with LCD. We observed that depolarization of the mitochondrial membrane was induced in the CRC cells when they were treated with LCD (Fig. 4A, 4B). The proportion of cells with depolarized mitochondrial membrane increased from 2.05% to 9.46%, 18.83%, and 41.97% after treatment with LCD at 2, 4, and 6 μM in HCT116 cells and from 1.45% to 5.75%, 16.83%, and 43.73% in HCT116-OxR cells, respectively. In addition, dysregulation of the mitochondrial membrane was associated with an increase in endoplasmic reticulum stress, as evidenced by elevated protein levels of GRP78, CHOP, DR4, and DR5 (Fig. 4C). Levels of proteins involved in mitochondrial apoptosis were monitored too (Fig. 4D). Levels of Bax and Bim as proapoptotic proteins were increased, whereas levels of antiapoptotic proteins Bcl-2, Bcl-xL, and Mcl-1 were decreased by LCD treatment in a concentration-dependent manner. Activation of BID by cleavage, release of cytochrome c into cytoplasm, formation of Apaf-1, and cleavage of caspase 3 and c-PARP were also observed. Overall, these results indicate that LCD could exert its cytotoxicity through mitochondrial membrane dysregulation, resulting in the initiation of apoptosis.

Figure 4. LCD induces depolarization of MMP. HCT116 and HCT116-OxR cells were treated with LCD (0, 2, 4 and 6 μM) for 48 h. (A) Flow cytometry analysis with the Muse™ MitoPotential kit. (B) Total depolarized cells in Fig. 4A. Data are presented as mean ± SD of three independent experiments in triplicate. *p<0.05 and ***p<0.001 compared to the control group. (C) Western blot analysis of levels of proteins involved in endoplasmic reticulum stress (GRP78, CHOP, DR4, and DR5) and Bcl-2 family proteins (Bim, BID, Bax, Bcl-2, Bcl-xL, and Mcl-1). Actin was used as a loading control. (D) Western blot analysis to monitor levels of Cyto c in the cytosol and mitochondria, Apaf-1, caspase 3, and c-PARP. β-tubulin was used as a loading control for the cytosol fraction and COX4 was used as a loading control for the mitochondrial fraction.

Effects of LCD on caspase activation in human CRC cells

The multi-caspase activity in CRC cells treated with LCD was measured with a Muse™ Multi-Caspase kit. Results revealed that the proportion of cells with multi-caspase activity increased by LCD treatment (2, 4, and 6 μM) from the base level 6.16% to 9.28%, 24.26%, and 40.64% in HCT116 cells. In HCT116-OxR cells after treatment with LCD at 2, 4, and 6 μM, corresponding values increased from 6.26% to 14.28%, 26.50%, and 35.06%, respectively (Fig. 5A, 5B). To see if activation of caspase was involved in the antiproliferative activity of LCD, we compared viabilities of CRC cells with LCD-only treatment and cells with LCD and pretreatment of Z-VAD-FMK (4 μM), a pan-caspase inhibitor. Indeed, pretreatment with the pan-caspase inhibitor significantly prevented the cytotoxicity induced by LCD (Fig. 5C). These results suggest that LCD can exert its cytotoxicity by activating caspases.

Figure 5. LCD induces activation of multiple caspases. HCT116 and HCT116-OxR cells were treated with LCD (0, 2, 4 and 6 μM) for 48 h. (A) Flow cytometry analysis with a Muse™ Multi-Caspase kit. (B) Total caspase+ cells in Fig. 5A. (C) CRC cells were pre-treated with Z-VAD-FMK (4 μM) for 3 h and then treated with or without 6 μM LCD for 48 h. Cell viability was determined by MTT cell viability assay. Data are presented as mean ± SD of three independent experiments in triplicate. ***p<0.001 compared to the control group; ^###p<0.001 compared to the LCD-treated group.

Effects of LCD on ROS generation and caspase activation in human CRC cells

We examined if there was an increase in the generation of ROS in CRC cells treated with LCD by flow cytometry. In HCT116 cells treated with 2, 4, and 6 μM LCD, intracellular ROS levels increased from the base level of 9.24% to 41.07%, 52.03%, and 61.20% (Fig. 6A, 6B). Likewise, the ROS level in HCT116-OxR cells treated with LCD at 2, 4, and 6 μM increased from 9.58% to 23.31%, 49.58%, and 63.31% by to see whether the generation of ROS was involved in the antiproliferative effect of LCD, we pretreated CRC cells with NAC, a scavenger of ROS, before LCD treatment. As shown in Fig. 6C, pretreatment of NAC somehow rescued CRC cells treated with LCD. The viability of HCT116 cells, initially decreased to 39.22% by LCD treatment, recovered up to 79.97% with NAC pretreatment. Similarly, the viability of HCT116-OxR treated with LCD recovered from 25.85% to 77.78% with NAC pretreatment. Phosphorylation of JNK and p38 also decreased with NAC pretreatment, implying that the induction of ROS generation was involved in the phosphorylation of these proteins (Fig. 6D). Moreover, western blot analysis showed that the decrease of whole-length caspase 3 could be prevented by NAC pretreatment. Indeed, the multi-caspase assay revealed that the percentage of CRC cells with active caspases increased significantly. The proportion of caspase-positive cells was 4.57% in HCT116 and 3.61% in HCT116-OxR cells without LCD treatment. These ratios increased to 45.26% and 41.90% after treatment with LCD at 6 μM. These values dropped to 11.09% and 10.49%, respectively, when CRC cells were pretreated with NAC (Fig. 6E, 6F). Taken together, these results suggest that the antiproliferative activity of LCD in HCT116 cells is mediated by ROS generation.

Figure 6. LCD induces ROS generation and caspase activation. HCT116 and HCT116-OxR cells were treated with LCD (0, 2, 4 and 6 μM) for 48 h. (A) Flow cytometry analysis with Muse™ Oxidative Stress Kit. (B) ROS+ population of CRC cells in Fig. 4A. (C-F) CRC cells were pre-treated with NAC (4 mM) for 3 h and then treated with or without 6 μM LCD for 48 h. (C) Cell viability assessed by MTT cell viability assay. (D) Western blot analysis of pJNK, pp38, caspase 3. Actin was used as a loading control. (E) Flow cytometry analysis with the Muse™ Multi-Caspase kit. (F) Total caspase+ cells in Fig. 6E. Data are presented as mean ± SD of three independent experiments in triplicate. *p<0.05 and ***p<0.001 compared to the control group; ^###p<0.001 compared to the LCD-treated group.

When treating CRC patients, tumor-related characteristics determines the direction of primary care. Chemotherapy after surgery is very common (Marmol et al., 2017). The drawback of the conventional chemotherapy is the cytotoxicity damaging not only cancer cells, but also non-cancerous normal cells altogether (Oun et al., 2018) and the emergence of resistance in cancer cells (Bose et al., 2011). To ensure an effective cancer treatment, anticancer drugs need to show cancer selectivity with minimal toxicity toward non-cancerous cells. In reality, major treatments for cancer have several side effects, both physiologically and immunologically (Schirrmacher, 2019). Ox has been used as a first-line chemotherapeutic for treating CRC. It is also being used to treat several other cancers with reasonable efficacy (Graham et al., 2004; Meyerhardt and Mayer, 2005; Comella et al., 2009). However, resistance to Ox-based chemotherapy is not negligible, hampering the effort to treat the disease (Martinez-Balibrea et al., 2015). Various mechanisms exist in the resistance development, including reduced cellular uptake, impairment of DNA adducts formation, alterations in DNA repair genes, and so on (Virag et al., 2013). To overcome Ox resistance, we turned to LCD, which demonstrated anticancer activities to several cancers. This is the first report to show the anticancer activity of LCD in CRC cells.

Initial results with the MTT cell viability assay demonstrated that LCD was selectively cytotoxic to both HCT116 and HCT116-OxR cells (Fig. 1A, 1B), whereas it did not affect the cell viability of non-cancerous cells HaCaT or JB6 (Fig. 1C, 1D). LCD effectively inhibited the formation of colony in both CRC cells HCT116 and HCT116-OxR (Fig. 1E, 1F) and induced apoptosis significantly (Fig. 1G, 1H). This result expands the antitumor activity of LCD to CRC cells in addition to breast, skin, and lung cancer, and supports the cancer selectivity: our previous study with xenograft node mice model showed the antitumor effect of LCD without exerting toxicity at 20 mg/kg (Seo et al., 2019). The IC50 values for LCD are slightly lower in HCT116-OxR cells compared to HCT116 cells. Although the exact reason remains unclear, it is suspected that HCT116-OxR cells may have a diminished ability to respond to stress, as they were continuously exposed to Ox in the culture media.

LCD-induced apoptosis was involved in the regulation of cell cycle progression (Fig. 2A, 2B). Results showed increased levels of CDK inhibitors p21 and p27 with decreased levels of cyclin B1 and cdc2 (Fig. 2C), indicating fine regulation of CDK complexes (Malumbres and Barbacid, 2009). Signaling pathways involving p38 and JNK MAPK are very important in determining the fate of cancer cells (Sui et al., 2014). Phosphorylation levels of these proteins were increased by LCD treatment (Fig. 3A-3C). Activation of JNK and p38 signaling pathways by LCD has recently been reported in a breast cancer cell model too (Zhang et al., 2024). In addition, the generation of ROS plays various roles in survival and death of CRC cells (Sreevalsan and Safe, 2013; Chun and Joo, 2022). Inducing heightened production of ROS is a strategy that can lead to the death of cancer cells (Moloney and Cotter, 2018). Previously, it has been shown that LCD can induce ROS generation in various cancer cells (Si et al., 2018; Seo et al., 2019; Oh et al., 2020). Our data clearly showed again the generation of ROS induced by LCD treatment (Fig. 6A, 6B). To establish the signaling mediated by ROS generation, we pretreated CRC cells with NAC before LCD treatment. As shown in Fig. 6C, NAC pretreatment effectively prevented the LCD-induced apoptosis (Fig. 6C). Moreover, the phosphorylation of both JNK and p38 and the activation of caspase 3 were prevented by NAC pretreatment, implying that the ROS generation precedes the activation of both JNK and p38 (Fig. 6D-6F).

The generation of ROS is closely related to mitochondria (Redza-Dutordoir and Averill-Bates, 2016). Our experimental data showed that LCD dysregulated mitochondria function (Fig. 4A, 4B) and induced a shift in the balance of pro- and anti-apoptotic Bcl-2 family proteins (Ola et al., 2011), resulting in the activation of caspase (Fig. 4C, 4D). The activation of multiple caspases in both CRC cells (Fig. 5A, 5B) and the prevention of cell death induced by LCD treatment with Z-VAD-FMK pretreatment (Fig. 5C) imply that LCD can induce apoptosis by activating caspase.

In conclusion, this study showed that LCD exerted antiproliferative activities in both Ox-sensitive and Ox-resistant HCT116 cells. The cytotoxicity of LCD is involved with the cell cycle regulation, JNK and p38 MAPK signaling, dysregulating mitochondrial membrane, and activating multiple caspases, which is triggered by ROS generation (Fig. 7). Further studies are needed to enhance our understanding of antitumor property of LCD.

Figure 7. Schematic representation of antitumor activity of LCD in CRC cells.

This work was supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (2022R1A5A8033794, RS-2024-00336900).

The authors have no conflicts of interest relevant to this study to disclose.

Seung-On Lee: Data curation, Formal analysis, Methodology, Validation, and Investigation. Sang Hoon Joo: Conceptualization, Investigation and Writing–original draft. Seung-Sik Cho: Conceptualization, Methodology, Validation, and Resources. Goo Yoon: Resources Methodology, Validation, and Investigation. Yung Hyun Choi; Conceptualization, Methodology, Investigation, Software, and Resources. Jin Woo Park: Conceptualization, Investigation, and Resources. Kwon-Yeon Weon: Conceptualization and Project administration. Jung-Hyun Shim: Project administration, Supervision, and Funding acquisition. 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.