Combination of Curcumin and Silibinin Exhibits Anti-Tumorigenic Activity Against Colon Cancer in a Xenograft Animal Model, Causing Cell Death by Increased ROS, Activation of AMPK, and Inhibition of mTORC1

1. Introduction

Among noncommunicable diseases, cancer is a leading cause of death and one of the most important barriers to increasing life expectancy globally. Colorectal cancer (CRC) ranks third in terms of incidence and second in terms of mortality globally [1,2]. While rates of colon cancer in humans in the 50+ age group are dropping due to effective screening, the age group under 50 is experiencing an alarming increase in colon and rectal cancers in the USA [2]. CRC incidence in 20-34 year-olds is predicted to increase up to 90% and 124% for colon and rectal cancers respectively, and recent estimates further underscore this trend, with approximately 11% of new CRC cases occurring in individuals under the age of 55 [3,4,5]. Even though the reason behind the sharp increase in younger adults is not easily explained, epidemiology studies suggest that a diet lacking in vegetables and fruits but rich in processed foods may be a cause of CRC [5,6].

Development of CRC is multifactorial. Genetic predisposition contributes 5-10% towards CRC onset; the remaining 90-95% of CRCs are due to lifestyle issues such as diet and alcohol or tobacco use [6]. However, some inflammatory bowel disorders (Crohn’s and ulcerative colitis) are also associated with CRC [7,8]. CRC development is a multistep process involving genetic aberrations and epigenetic dysregulation [9]. Epigenetics (specifically involving DNA methylation, histone modification, and noncoding RNA) is implicated in every aspect of tumor formation, from early neoplasia to malignant transformation [9,10,11,12].

Current treatment for CRC is based on removing primary tumors followed by radiotherapy and/or chemotherapy. Unfortunately, both radiation and chemotherapy cause adverse side effects and increased chemoresistance, with an associated increased rate of metastasis [13,14]. Based on results from several clinical studies, recurrence rates rate for colon cancer and for rectal cancer vary [15]. Thus, a preventive approach is needed to avoid CRC and the adverse effects associated with current treatment modalities. CRC prevention should not only focus on preventing CRC in young people but should also concentrate on reducing CRC recurrence in CRC-treated patients. CRC development occurs in those with diets conducive to CRC (low intake of fruits and vegetables, high intake of processed foods), and CRC initiation occurs in those with a genetic predisposition for CRC [16,17,18,19,20].

Some phytochemicals have been shown to protect against cancer. Epidemiological studies show that populations consuming greater amounts of spices and vegetables have a lower incidence of CRC [6,21,22,23,24,25,26,27]. For example, in Asian Indians, the overall rate of colorectal, prostate, and lung cancers in both males and females is the lowest among all populations studied, and this low incidence is attributed to the phytochemical curcumin present in the spice turmeric [26]. Phytochemicals offer alternative approaches to cancer prevention, with reduced host toxicity (due to minimal/no host toxicity) and diminished side effects compared to those of current chemotherapeutic agents [28,29,30,31]. However, when these phytochemicals have been studied in vitro and translated into mice and human studies, the pharmacologically effective dose is several-fold higher than that indicated by epidemiological studies [32]. For example, in clinical trials, curcumin has shown promising anticancer effects and is well tolerated at a dose of 12 g/day [32,33,34]. But the normal dietary intake of turmeric in the Asian population is about 2-2.5 g/day, which corresponds to 60-100 mg/day of curcumin [35,36]. In addition, the low bioavailability of curcumin has raised questions about how epidemiological studies and several research publications can demonstrate medicinal effects in animal and human studies against diseases such as cancer, Alzheimer’s, and arthritis [32,37,38,39,40,41,42,43,44]. Despite the low bioavailability of curcumin and its relatively low amount of regular dietary intake [35,37,38], if there is a beneficial protective effect against cancer, as suggested by epidemiological studies, it could be because curcumin is always consumed in combination with other substances. We hypothesized that curcumin, together with other phytochemicals present in a regular diet, acts synergistically in cancer prevention, thereby explaining the results observed in epidemiological studies [22,23,24,26,39].

Our previous study showed that a combination of curcumin and the whole milk thistle phytochemical silymarin (CS) elicited synergistically enhanced anticancer activity in vitro [40], supporting our postulate. In our study using a xenograft mouse model, only the CS-treated group showed a significant reduction in tumor size compared with single compound phytochemical treatment or control groups. To understand the mechanism of the anti-tumor effect of combination compounds, we used an in vitro model, which was the DLD-1 CRC cell line [40]. Phytochemical combinations such as CS may exert anticancer effects by disrupting redox homeostasis and metabolic signaling in tumor cells. Phytochemicals can disrupt this balance by inhibiting ROS-scavenging enzymes, thereby pushing ROS levels beyond the cytotoxic threshold and inducing cell death [41,42]. Our preliminary findings indicate that CS treatment, compared to single compound treatment and vehicle control, increases intracellular ROS, thereby activating AMPK and suppressing mTORC1 signaling—two key pathways that regulate cellular growth and survival. This coordinated increase in oxidative stress and inhibition of anabolic signaling promotes apoptosis and autophagy. Because cancer cells already maintain elevated basal ROS levels [41], the additional oxidative burden imposed by CS may overwhelm their antioxidant capacity, pushing them beyond their survival threshold and inducing cell death.

2. Materials and Methods

2.1. Cell Line and Propagation

Human colon cancer DLD-1 cells were obtained from the American Type Culture Collection (Manassas, VA) and maintained at 37 °C in a humidified incubator with 5% CO₂. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, penicillin/streptomycin, glutamine, sodium pyruvate, and HEPES buffer (all from Hyclone, Logan, UT). Cultures were propagated under standard conditions until reaching confluence, at which point the cells were detached using 0.25% trypsin and neutralized with enriched DMEM. Viable cells were then counted using a hemocytometer and prepared for tumor induction.

2.2. Tumor Formation in Nude Mice

Six-week-old athymic nude mice (Charles River Laboratories) were housed in a pathogen-free facility and handled in accordance with Institutional Animal Care and Use Committee (IACUC) guidelines. Athymic nude mice are immunocompromised and readily accept human tumor cells, allowing for consistent tumor development. DLD-1 cells were centrifuged, washed with DPBS, and resuspended at 5 × 10⁶ cells per 100 μL of serum-free medium. This suspension was mixed with 100 μL of Matrigel (8 mg/mL) and kept on ice until injection. Seven-week-old mice were injected subcutaneously with 5 × 10⁶ DLD-1 cells in a total volume of 200 μL (cell suspension mixed with Matrigel) into the right and left flanks. The day of injection was designated as day 0. By day 5, tumors were visible with an average volume of approximately 50 mm³. On day 5, mice were randomly assigned to four groups (10 mice per group; five males and five females). Phytochemicals or vehicle were administered daily for 30 days. Treatments consisted of phytochemicals suspended in a carboxymethylcellulose (CMC) solution (0.5% w/v in 0.025% Tween-20 in water) or vehicle alone (CMC solution). Dosing was performed by oral gavage on days 5 through 34. Treatment groups were: (1) vehicle control (CMC only); (2) curcumin (C:200 mg/kg); (3) silibinin B (S:200 mg/kg); and (4) curcumin + silibinin B (CS:200 mg/kg each). All mice were gavaged using a 22-gauge ball-tipped feeding needle, and the gavage volume did not exceed 10 mL/kg.

Mice were monitored daily, weighed weekly, and tumors were measured twice weekly using calipers. Tumor volume was calculated using the formula: ½ × (length × width²). On day 34, animals were euthanized according to IACUC guidelines, and tumors were excised and weighed.

2.3. Immunohistochemistry

During apoptosis, inactive caspase-3 is proteolytically cleaved to generate its active form, and the presence of cleaved caspase-3 serves as a reliable marker of apoptotic cells. Tumor sections were therefore stained with an antibody against cleaved caspase-3 (Cell Signaling Technology, Cat. No. 9664), and images were captured at 20× magnification. Quantification was performed using QuPath software [43]. Each tumor section was defined as a separate region of interest, and a three-tier thresholding strategy was applied to classify staining intensity as background (0), low (1+), or high (2+) based on intensity peaks. Cells were segmented from hematoxylin-stained images, and the proportion of high-intensity (2+) cells was used to determine the percentage of cleaved caspase-3–positive cells.

2.4. Treatment of DLD-1 Cells for ROS, ADP/ATP, and Western Blot Analysis

Curcumin and silibinin B were prepared as 100 mM stock solutions in dimethyl sulfoxide (DMSO) and stored at −20 °C. DMSO alone (0.01%) served as the vehicle control. Cells were treated with 12.5 µM curcumin (C), 12.5 µM silibinin B (S), or a combination of 12.5 µM curcumin plus 12.5 µM silibinin B (CS) for 48 hours. Following treatment, cells were analyzed for reactive oxygen species and the ADP/ATP ratio. Cell lysates were quantified for protein using a BCA assay (Pierce Biotechnology, Rockford, IL) and subsequently used for SDS-PAGE and Western blot analysis.

2.4a. Measurement of Reactive Oxygen Species

Intracellular ROS levels were quantified using the oxidation-sensitive probe 2′,7′-dichlorofluorescin diacetate (DCFDA). Cells were treated with vehicle control or 12.5 µM of each compound for 48 hours. After treatment, the growth medium was removed and replaced with DCFDA diluted in Hank’s balanced salt solution, followed by incubation at 37 °C for 30 minutes. Cells were then visualized using a fluorescence microscope, and fluorescence intensity was measured using a microplate reader (Cytation 3, BioTek, Vermont, USA) at λ_excitation = 485 ± 20 nm and λ_emission = 528 ± 20 nm. Relative ROS levels in DLD-1 cells were calculated as fold change compared with untreated controls. Data were presented as mean ± SEM from at least three independent experiments (**p ≤ 0.01).

2.4b. Analysis of ATP/ADP Ratio

The ATP/ADP ratio was measured using the ApoSENSOR™ ADP/ATP Ratio Bioluminescent Assay Kit (BioVision). First, 100 µL of reaction mix was added to a 96-well plate, and background luminescence was recorded (Data A). Cells were treated with phytochemical compounds for 48 hours, after which the medium was removed, and cells were incubated with 50 µL of nucleotide-releasing buffer for 5 minutes. The resulting lysate was transferred to the wells containing the reaction mix, and luminescence was measured after 2 minutes (Data B). Samples were then read again to determine ADP-dependent signal (Data C). Subsequently, 1 µL of ADP-converting enzyme was added to each well, and luminescence was recorded (Data D). The ATP/ADP ratio was calculated using the formula:

ATP/ADP ratio = Data B-Data A

Data D- Data C

The ADP/ATP ratio was normalized to protein concentration.

2.4c. Analysis by Western Blot

DLD-1 cells treated with vehicle control (DMSO), curcumin (C, 12.5 µM), silibinin B (S, 12.5 µM), or the curcumin plus silibinin B combination (CS, 12.5 µM each) were lysed and analyzed by Western blot. Cells were lysed in buffer containing 50 mM Tris, 100 mM NaCl, protease inhibitors, and 1 mM phenylmethylsulfonyl fluoride. Lysates were centrifuged at 10,000 × g for 15 min, and the resulting supernatant was collected. Protein concentration was determined using the BCA assay (Pierce Biotechnology, Rockford, IL). Equal amounts of protein (20 µg per sample) were separated by SDS–PAGE and electrophoretically transferred onto nitrocellulose membranes (Pierce Biotechnology, Rockford, IL). Membranes were blocked for 1 h at room temperature in TBST blocking buffer (20 mM Tris, pH 7.6, 100 mM NaCl, 0.1% Tween-20, 5% nonfat dry milk) with gentle agitation. After blocking, membranes were washed and incubated overnight at 4 °C with primary antibodies against AMPK, phospho-AMPK, phospho-4E-BP1, phospho-p70S6, LC3I, LC3-II, Noxa, cleaved caspase 3, and GAPDH (Cell Signaling Technology, Danvers, MA) diluted in TBST blocking buffer. Following primary antibody incubation, membranes were washed three times with TBST and incubated with horseradish peroxidase–conjugated secondary antibodies for 1 h at room temperature. Immunoreactive bands were visualized using a chemiluminescent detection system (Pierce Biotechnology, Rockford, IL). GAPDH was used as the loading control.

2.5. Statistical Analysis

Results were presented as the mean ± standard error of the mean (SEM). All data were analyzed using a one-way ANOVA followed by Dunnett’s multiple comparisons post hoc test. A p-value of less than 0.05 (p < 0.05) was considered statistically significant.

3. Results

3.1. Combination of Phytochemical Treatment Significantly Reduced Tumor Burden

In our previous cell culture studies, we used curcumin and silymarin. Silymarin is a mixture of phytochemicals, including silychristin, silydianin, isosilybin, and silibinin B (silybin), the latter being the major flavonolignan and comprising approximately 40–60% of the total extract [44]. Subsequently, we used pure silibinin B for the current in vivo and in vitro studies, as it represents the primary bioactive component of silymarin.

In our previous studies, we treated colon cancer cell lines (DLD-1, HCT116, LoVo) with a single phytochemical (curcumin or silymarin) or with a combination of curcumin and silymarin to assess anticancer activity [40]. Curcumin inhibited colon cancer cell proliferation in a concentration-dependent manner, whereas silymarin showed significant inhibition only at the highest concentrations [40]. We observed synergistic effects when colon cancer cells were treated with curcumin and silymarin in combination [40]. The combination treatment inhibited colon cancer cell proliferation and increased apoptosis compared with single compound-treated cells [40].

In the present study, we employed immunocompromised athymic nude mice to evaluate the antitumor activity of curcumin and silibinin B (major flavonolignan of silymarin), administered individually or in combination. The human colon cancer cell line DLD-1 was injected subcutaneously into the right and left flanks of mice. On Day 5, tumors were visible (average 50 mm³) and palpable. Animals were randomized to receive either vehicle (control), curcumin alone (C), silibinin B alone (S), or the combination of curcumin and silibinin B (CS). Treatments were administered orally by gavage for 34 consecutive days (Figure 1).

As shown in Figure 2A, only the CS combination group showed a statistically significant reduction in tumor volume observed at each time point (days 8 to 34; *=p<0.05, **=p<0.001). C and S showed a significant reduction only at early time points (days 10-16; p<0.05; Figure 2A). Animals were sacrificed on day 34, and the tumors were excised and weighed. Only the CS group showed a statistically significant reduction in tumor weight (p<0.05) compared with the control C alone or S alone groups (Figure 2B). There was no statistically significant difference in mouse body weight among the groups, indicating no adverse effects from phytochemical administration or cancer cell implantation (Figure 2C). Based on these results, the combination compound showed a statistically significant reduction in tumor volume compared with animals treated with a single compound.

3.2. CS Combination Treatment Increased Apoptotic Cell Death in the Tumor

In a previous in vitro study, we observed that cells treated with the combination (CS) exhibited pronounced cell rounding and membrane blebbing, features characteristic of apoptosis [40]. Measurement of caspase-3/7 activity, a standard indicator of apoptotic cell death, showed that combination-treated cells displayed significantly higher caspase activity than control or single compound-treated cells, indicating that CS induced apoptosis [40]. To extend these findings in vivo, we analyzed cleaved caspase-3, an apoptotic marker, by immunohistochemistry of mouse tumor sections (Figure 3). As shown in Figure 3, tumors from combination-treated animals exhibited significantly greater numbers of apoptotic cells, as evidenced by cleaved caspase-3 staining. These results suggest that apoptosis-mediated cell death is one mechanism by which the combination treatment reduces tumor burden.

3.3. Treatment of DLD-1 Cells with the Curcumin–Silibinin B (CS) Combination Significantly Increased Reactive Oxygen Species (ROS) Levels

Combination treatment produced significantly higher ROS levels in vitro than either compound alone or the vehicle control. DLD-1 colorectal cancer cells were treated with vehicle control (DMSO), 12.5 µM C, 12.5 µM S, or a combination of 12.5 µM CS, and intracellular ROS levels were measured. We observed that cells treated with the combination exhibited markedly increased ROS compared with vehicle-treated or single-compound-treated cells. Figure 4A shows the intensity of green fluorescence, which reflects ROS accumulation in the cells; combination-treated cells display visibly stronger fluorescence than the other groups. The quantification of relative ROS levels is shown in Figure 4B demonstrates that the CS combination significantly increased ROS in DLD-1 colorectal cancer cells.

3.4. Curcumin -Silibinin B Treatment Elevates ADP/ATP Ratio and p-AMPK Levels in DLD-1 Cells

An increase in ROS leads to mitochondrial impairment, which elevates the ADP/ATP ratio; therefore, we measured the ADP/ATP ratio under our treatment conditions. Cells were treated with vehicle control (DMSO), 12.5 µM curcumin (C), 12.5 µM silibinin B (S), or a combination of 12.5 µM curcumin plus 12.5 µM silibinin B (CS), and the ADP/ATP ratio was quantified. As shown in Figure 4C, only curcumin-treated and CS-treated cells exhibited an increased ADP/ATP ratio. Notably, the combination treatment (CS) induced a significantly higher ADP/ATP ratio compared with vehicle control and each compound alone.

Mitochondrial impairment caused by ROS is expected to increase the ADP/ATP ratio first, followed by an increase in the AMP/ATP ratio. This progressive energy depletion activates AMPK, leading to phosphorylation of AMPK (p-AMPK). Therefore, we assessed p-AMPK levels in treated cells to evaluate its activation and the downstream inhibitory effects on the mTORC1 pathway. As shown in Figure 4D, only the curcumin–silibinin B combination (CS) induced AMPK phosphorylation, whereas no phosphorylated AMPK was detected in vehicle- or individually-treated DLD-1 cells.

3.5. Treatment of DLD-1 Cells with the Curcumin–Silibinin B (CS) Combination Leads to Inhibition of the mTORC1 Pathway

To examine the effect of CS on signaling pathways, DLD-1 cells were treated with vehicle control, C (12.5uM), S (12.5uM), or CS (12.5uM of each) for 48 hrs. Then, the cells were lysed and analyzed by Western blot for the expression of phosphorylated 4E-BP1 and phosphorylated p70S6 kinase. The downstream proteins involved in the mTORC1 pathway, 4E-BP1 and p70S6 kinase, exhibited significantly decreased phosphorylation in CS-treated cells compared to C or S treatments alone, indicating mTORC1 was downregulated (Figure 5A,B). Phospho-4E-BP1 levels were significantly reduced in curcumin-treated and CS-treated cells, with the CS combination producing a greater inhibition than curcumin alone (Figure 5E). Inhibition of the mTORC1 pathway increases autophagy and apoptosis, leading to cell death [45,46,47,48]. During autophagy, vesicles or autophagosomes form around damaged organelles and proteins and fuse with lysosomes to degrade contents and recycle them. A hallmark of autophagy is the lipidation and conversion of LC3-I to LC3-II [49]. As shown in Figure 5C, increased LC3-II was evident in CS compared to C. But we observed that LC3-I and LC3-II bands were undetectable in vehicle- and S-treated cells. A potential explanation is that DLD-1 colorectal cancer cells exhibit low basal autophagy under nutrient-rich culture conditions, keeping autophagosome machinery below the threshold of detection during rapid proliferation [50,51]. To determine whether mTORC1 inhibition combined with increased reactive oxygen species (ROS) levels induces apoptosis in DLD-1 cells, we evaluated the expression of the pro-apoptotic markers Noxa and cleaved caspase-3. ROS can independently promote apoptosis through Noxa upregulation and caspase-3 activation, while mTORC1 inhibition can indirectly induce apoptosis by reducing protein synthesis and sensitizing cells to apoptotic signaling [52,53]. Both proteins were upregulated in C- and CS-treated cells compared to both the vehicle control and S-treated cells (Figure 5D). Based on these results, combination compounds exhibit anticancer activity by inhibiting mTORC1, which in turn can lead to apoptotic and autophagy-induced cell death.

4. Discussion

Tumor growth and progression require increased cell survival, unchecked cell proliferation, activation of anti-apoptotic pathways, induction of angiogenesis, deregulation of oncogenes, repression of tumor suppressor genes, and modulation of signaling pathways [54]. Therefore, targeting a single event may not be sufficient to prevent CRC. Anticancer agents that target multiple tumorigenic events may be more effective in inhibiting CRC tumor growth and progression. Rodent models have been extensively used for preclinical cancer drug development [55]. Animal models provide valuable insights into the physiological interactions between tumor cells and their environment in vivo. In this study, we used a xenograft model in athymic nude mice, which are immunocompromised and accept human tumor cells, thereby allowing tumor development. Human xenograft tumor growth depends on the interplay between the human tumor cells and murine stromal cells, such as endothelial cells, leukocytes, and fibroblasts recruited to generate a pro-tumor microenvironment [58]. Athymic nude mice used in our xenograft model have a T cell deficiency, allowing for the growth of the xenografted tumor cells. Even though the immunodeficiency is severe in this model, both the humoral and innate adaptive immune systems remain intact. Despite the immunologically deficient environment, the model supports the formation of three-dimensional tumors and incorporates a tumor microenvironment (TME) that provides extracellular matrix, tumor–stroma interactions, and vascularization, thereby partially recapitulating human tumor biology [56]. Our results suggest that combination compound treatment significantly reduced tumor formation, and this reduction was due to increased apoptosis.

Our long-term goal is to elucidate the mechanism of CS action in CRC using in vitro and in vivo models, thereby reducing CRC incidence in high-risk groups. To understand how some of the crucial pathways are affected by CS compounds that may explain the anticancer activity, we explored the phytochemical effects on anticancer activity in vitro. We treated DLD-1 CRC cells with single or combination compounds and evaluated the effects on ROS, AMPK activation, and mTOR1 pathway status. Compared to normal cells, cancer cells have elevated ROS, because of increased metabolic activity [41,42]. They maintain high energy levels by increased glycolysis followed by lactic acid fermentation in the presence of abundant oxygen. In other words, they use aerobic glycolysis [48,57]. Any compound that increases ROS in cancer cells will reach a high ROS threshold, causing cell death [41]. Normal cells cannot undergo cell death from these compounds because basal ROS levels are low, so they cannot reach a higher ROS threshold that would trigger apoptosis. Phytochemicals such as C bind to ROS-scavenging enzymes in cancer cells, thereby increasing ROS to much higher levels [41]. The increased ROS kill leukemic cells but not normal cells [41]. Several dietary phytochemicals that increase ROS have been shown to selectively target cancer cells for killing [58,59]. The apoptosis we observed in vivo is likely due to increased ROS, among other mechanisms. To verify the increase in ROS, we treated DLD-1 cells with vehicle, C, S, or CS and found that only CS-treated cells exhibited significantly increased ROS (section 3.3). Elevated ROS can independently trigger apoptosis by inducing oxidative damage to DNA, lipids, and proteins [60]. Compared with normal cells, cancer cells maintain intrinsically higher ROS levels due to increased glycolysis, mitochondrial dysfunction, and oncogenic signaling; however, they rely heavily on antioxidant systems to survive this oxidative stress [61]. Phytochemicals can disrupt this balance by inhibiting ROS-scavenging enzymes, thereby pushing ROS levels beyond the cytotoxic threshold and inducing cell death, as observed in mouse tumors (Figure 3) [62,63].

ROS levels can impair mitochondrial function, leading to an increased ADP/ATP ratio. To assess this under our treatment conditions, we quantified the ADP/ATP ratio in cells treated with vehicle control (DMSO), 12.5 µM curcumin (C), 12.5 µM silibinin B (S), or the combination of curcumin plus silibinin (CS). As shown in Figure 4C, only curcumin- and CS-treated cells exhibited an increased ADP/ATP ratio. Notably, the combination treatment induced a significantly greater increase compared with vehicle control and each compound alone.

Mitochondrial dysfunction driven by ROS is expected to elevate the ADP/ATP ratio first, followed by an increase in the AMP/ATP ratio. This progressive decline in cellular energy activates AMPK, resulting in phosphorylation of AMPK (p-AMPK). Therefore, we evaluated p-AMPK levels in treated cells to determine the extent of AMPK activation and its downstream inhibitory effects on the mTORC1 pathway. We found that in CS-treated cells, there is an increase in p-AMPK, which is an active form and functions as an energy stress sensor. Active p-AMPK shuts down the major anabolic pathway mTORC1. Therefore, we next assessed the status of the mTORC1 pathway in phytochemical-treated cells.

mTORC1 is a central regulator of cellular growth that integrates nutritional and environmental cues, including energy availability and redox status [64,65,66]. When activated, mTORC1 promotes the transcriptional and translational programs required for cell growth, while simultaneously suppressing autophagy and apoptosis [47,67]. Therefore, sustained activation of mTORC1 inhibits programmed cell death and enhances cell proliferation, contributing to tumorigenesis [68]. A key initial event in autophagy is the formation of an autophagosome mediated by ULK1-complex and the first step is initiated by ULK1 kinase activity [47,69]. To inhibit autophagy, activated mTORC1 inactivates ULK1 [47,70]. mTORC1 regulates multiple components of the protein-synthesis machinery, including translation initiation and elongation factors as well as ribosome biogenesis [71]. Upon activation, mTORC1 phosphorylates two major downstream effectors, 4E-BP1 and p70S6K. 4E-BP1 normally functions as a translational repressor by binding to the initiation factor eIF4E; phosphorylation of 4E-BP1 causes its dissociation from eIF4E, thereby permitting translation initiation [71,72]. Phosphorylation of p70S6K1 activates a set of downstream targets that collectively enhance mRNA translation and promote protein synthesis. Therefore, reduced phosphorylation of 4E-BP1 and p70S6K indicates inhibition of the mTORC1 pathway. We found that in CS-treated cells, the phosphorylated forms of 4E-BP1 and p70S6K were markedly reduced (Figure 5A,B). Compared with control-treated cells, the CS combination significantly suppressed phospho-4E-BP1 expression (Figure 5E).

If the mTORC1 pathway is inhibited, autophagy is expected to increase. During autophagy, double-membrane vesicles (autophagosomes) form around damaged organelles and proteins, which subsequently fuse with lysosomes to degrade and recycle their contents. A hallmark of autophagy is the lipidation and conversion of LC3-I to LC3-II [49]. We observed an increase in LC3-II levels, with the strongest induction in CS-treated cells, moderate induction in C-treated cells, and no LC3-II detected in vehicle-treated or silibinin-treated cells. The reason there is no detection of LC3-I in vehicle- and S-treated cells may be that some cell lines show very low basal autophagy in nutrient-rich growth conditions that cannot be detected [50,51]. We observed both in the in vivo experiment and our earlier study that curcumin and silymarin increased apoptosis [40]. Our findings support the hypothesis that simultaneous mTORC1 inhibition and elevated ROS levels drive DLD-1 cells toward programmed cell death. This is evidenced by the marked upregulation of the pro-apoptotic markers Noxa and cleaved caspase-3 in C- and CS-treated cells compared with the control and S-treated groups (Figure 5D). These results indicate that ROS independently promotes apoptosis through Noxa induction and caspase-3 activation, whereas mTORC1 inhibition indirectly facilitates this process. By limiting de novo protein synthesis, mTORC1 suppression sensitizes malignant cells to downstream apoptotic signaling, thereby amplifying the overall cytotoxic response.

In the present study, we demonstrated that the CS combination inhibits the mTORC1 pathway, significantly increases ROS production, and elevates markers of autophagy and apoptosis in DLD-1 cells. Based on these findings, we postulate that CS engages multiple cellular mechanisms, including pathways governing cell survival and proliferation, and elicits anticancer activity (Figure 6). Future studies will focus on characterizing additional signaling cascades affected by CS, such as the Wnt pathway in vitro. Furthermore, we aim to utilize a xenograft mouse model to investigate how CS modulates the tumor microenvironment (TME), particularly regarding its role in fostering pro-tumorigenic cytokines, growth factors, and chronic inflammation [73].