Suppressive Potential of Ethanolic Extracts of Parkia speciosa Hassk. Empty Pods Against Colon Cancer Cell Migration and Invasion

Abstract

Parkia speciosa (P. speciosa), a plant utilized in traditional medicine, has shown promise in various therapeutic applications and contains multiple bioactive components (saponins, alkaloids, flavonoids, polyphenols, and terpenoids). These bioactive compounds have attracted increasing scientific interest due to their ability to modulate key cancer-associated pathways, including the inhibition of cell proliferation and migration and the suppression of oxidative stress and inflammation mechanisms. However, despite P. speciosa’s historically long and wide-ranging usage, a comprehensive investigation of these properties has not been conducted for its pod. This study investigated the effects of P. speciosa empty pod extract (PSET) on human colorectal cancer cells. The extract demonstrated significant dose-dependent inhibition of colorectal cell migration, invasion, and colony formation while exhibiting no cytotoxicity toward normal colon epithelial cells. Western blot analysis confirmed reduced expression of Matrix metalloproteinases 2 (MMP2), Matrix metalloproteinases 9 (MMP9), and N-cadherin, indicating suppression of the epithelial–mesenchymal transition (EMT). These findings demonstrate that the PSET effectively inhibits metastasis in colorectal cancer cells through the EMT pathway, suggesting its potential as a dietary supplement or therapeutic agent for colorectal cancer treatment. Our research provides support for the development of natural, less toxic alternative cancer treatments. Therefore, PSET shows potential for development as a dietary supplement or therapeutic agent for the treatment of colon cancer.

1. Introduction

The global health landscape is currently facing a formidable trajectory regarding oncological mortality. Predictive models suggest that by 2030, cancer will claim the lives of an additional 13 million individuals, representing a staggering 60% escalation over a span of just 15 years [1]. Within cancer’s broad spectrum of malignancies, colorectal cancer (CRC)—pathologies affecting the colon or rectum—has emerged as a public health emergency of international concern due to its remarkably high fatality rates [2]. Consequently, the scientific community has intensified its focus on deciphering the molecular underpinnings of colon cancer pathogenesis with the aim of discovering more effective therapeutic strategies.

The lethality of cancer is largely driven by metastasis, a sophisticated and deadly cellular journey in which epithelial tumor cells liberate themselves from the primary site to colonize distant organs [3]. This “great escape” begins with tumor cells breaching the basal membrane and navigating through the extracellular matrix (ECM) to infiltrate the circulatory system [4]. Once in the bloodstream, these wayward cells may undergo transformation, eventually exiting the vasculature to adapt to foreign microenvironments, shifting their biological machinery from a migratory state to a metastatic one [5]. A pivotal mechanism in this invasion is the degradation of the ECM, orchestrated by proteolytic enzymes such asMMPs and serine proteinases, which are secreted by both tumor and endothelial cells [6]. The breakdown of type IV collagen and other structural proteins by MMPs clears the path for invasion; thus, these enzymes serve as critical biomarkers, and their inhibition represents a strategic bottleneck for halting metastatic spread [7].

In the clinical arena, the arsenal against colon cancer currently comprises surgery, radiation, and targeted therapies, yet chemotherapy remains the cornerstone of treatment. However, this modality is often a double-edged sword. Chemotherapeutic agents are non-selective, decimating healthy cells alongside malignant ones, which precipitates severe systemic toxicity—a particularly dire outcome for patients with compromised vitality. Furthermore, the efficacy of chemotherapy is frequently undermined by resistance, with recurrence rates oscillating between 20% and 80%, thereby compounding the risk of mortality. These profound limitations highlight the urgent imperative to transition towards alternative therapeutic paradigms that offer safety without the debilitating side effects of conventional cytotoxic drugs [8,9].

In this pursuit, modern biomedical research is increasingly revisiting nature’s pharmacy. A growing body of evidence suggests that medicinal herbs are reservoirs of potent bioactive compounds capable of combating specific cancers, including CRC [10]. Plant-derived agents, such as the flavonoids rutin and quercetin, have demonstrated the capacity to modulate complex signaling networks—including the STAT-3, PI3K/Akt, and EGFR pathways. By intervening in these pathways, these natural compounds can arrest cell proliferation, trigger apoptosis (programmed cell death), and crucially, block the EMT that fuels metastasis [11,12]. Beyond their direct antineoplastic activity, these bioactives offer a sustainable, biodegradable, and less toxic platform for future drug development, potentially enhancing the efficacy of standard treatments while mitigating drug resistance [13].

Among these botanical candidates is Parkia speciosa (P. speciosa), colloquially known as the stink bean, a species indigenous to Southeast Asia. Traditionally, the seeds of this plant are celebrated both as a culinary staple and a medicinal remedy for ailments ranging from diabetes to cardiovascular disease [14,15,16,17]. However, its empty pods have been largely ignored in the agricultural value chain, with these pods being discarded as waste despite their potential utility. Advanced chemical profiling liquid chromatography–mass spectrometry (LC-MS/MS) and gas chromatography–mass spectrometry (GC-MS) has revealed that this “waste” is chemically rich, harboring a phytochemical profile analogous to the prized seeds, including compounds like 1,2,3-benzenetriol (pyrogallol), hexadecenoic acid (palmitoleic acid), vitamin E, a-Amyrin, epigallocatechin gallate, quercetin, and piperine [18].

Although P. speciosa has been reported to possess various pharmacological properties, including antioxidant and anti-inflammatory activities, its potential effects on cancer metastasis remain largely unexplored. Specifically, there is a lack of scientific evidence regarding the inhibitory mechanism of PSET on the migration and invasion of human colorectal carcinoma cells. To address this knowledge gap, the present study aimed to investigate the anti-metastatic potential of PSET on HT-29 and HCT-116 colorectal cancer cell lines. We specifically focused on assessing its effect on cell migration, invasion, and the expression of key EMT markers, including MMP-2, MMP-9, and N-cadherin.

2. Results

2.1. Cytotoxicity of the PSET Against Cell Lines

MTT assays were performed to evaluate the effect of the PSET on cell viability. Normal colon cells (CCD-18co) and colon cancer cells (HT-29 and HCT-116) were treated separately with the PSET at various concentrations (25, 50, 100, 200, 400, 600, 800, and 1000 µg/mL) for 24 h. Cells treated with 1% DMSO were used as vehicle controls. CCD-18co cell viability was significantly reduced following treatment with more than 600 µg/mL of the PSET (Figure 1; black line with circles). HCT-116 cells showed a reduced cell viability following treatment with more than 600 µg/mL of the PSET (Figure 1; black line with squares). Similarly, HT-29 cell viability decreased at more than 600 µg/mL of PSET (Figure 1; black dashed line with triangles). Specifically, at concentration of 800 µg/mL, cell viability decreased to 45.38 ± 0.63% and 69.06 ± 2.97% in HT-29 and HCT-116 cells, respectively, compared to the control. Moreover, at the highest concentration of 1000 µg/mL, cell viability decreased to 26.09 ± 2.31% and 39.03 ± 1.33% in HT-29 and HCT-116 cells, respectively, compared to the control. Furthermore, the half-maximal inhibitory concentrations (IC₅₀) of the PSET for HT-29 and HCT-116 cells were 796.42 and 898.14 µg/mL, respectively. Concentrations of PSET (25–400 µg/mL) that demonstrated continued cell viability (95–105%) for all cell lines were selected for further experiments.

2.2. The PSET Inhibits the Migration and Invasion of Colon Cancer Cells

The effects of the PSET on colon cancer cell migration were investigated using wound scratch assays. Treatment with the PSET significantly decreased wound closure in HT-29 and HCT-116 cells starting at 50 µg/mL compared to the untreated group (Figure 2A,C). Higher extract concentrations appeared to induce more extensive wound closure compared to the lowest concentration, which elicited a statistically significant effect. Exposure to PSET at concentration of 50 and 300 µg/mL achieved wound closures of HT-29 cells to 63.92% and 29.38%, respectively, compared to the untreated control (100%). A similar trend was observed in HCT-116 cells, where migration was suppressed by 52.55% and 13.07% at the concentration of 50 and 300 µg/mL. Cell invasion was also assessed using a Transwell assay. A significant decrease in cell invasion was observed in the PSET-treated groups at all tested concentrations compared to the untreated group (Figure 2B,D). The percentage of cell invasion in HT-29 and HCT-116 cells treated with the PSET was reduced in a dose-dependent manner. Exposure to PSET at concentration of 50 and 200 µg/mL significantly decreased the percentage of invasion of HT-29 cells to 37.77% and 8.88%, respectively, compared to the untreated control (100%). A similar trend was observed in HCT-116 cells, where migration was suppressed by 58.25% and 16.37% at the concentration of 50 and 200 µg/mL.

2.3. Effect of the PSET on Colony Formation During Colon Cancer Cell Growth

A colony formation assay was used to study the PSET’s effect on cancer cell growth and colony formation. The PSET significantly reduced the number and average size of colonies when compared to the untreated group, starting at 50 µg/mL for HT-29 cells (Figure 3A–C) and 100 µg/mL for HCT-116 cells (Figure 3D–F). Exposure to PSET at concentration of 50 and 200 µg/mL significantly decreased the percentage of colony forming efficiency of HT-29 cells to 28% and 2%, the average size of colonies to 51.33% and 38.17%, respectively, compared to the untreated control (100%). A similar trend was observed in HCT-116 cells, where the percentage of colony forming efficiency was decreased to 10.64% and 0.85%, the average size of colonies to 35.34% and 3.51%, respectively, compared to the untreated control (100%) at the concentration of 100 and 200 µg/mL.

2.4. Effect of PSET Treatment on Colorectal Cancer Cell Proteomes

To elucidate the molecular mechanisms underlying the observed inhibition of migration and invasion, Western blot analysis was performed to assess the expression of key proteins involved in the EMT and extracellular matrix degradation, including MMP2, MMP9 and N-cadherin. In both HT-29 and HCT-116 cells, MMP2, MMP9 and N-cadherin protein levels significantly decreased following PSET treatment at 200 µg/mL (Figure 4). Meanwhile, PSET treatment at 100 µg/mL increased MMP2 and N-cadherin levels.

3. Discussion

In the ethnomedical traditions of Southeast Asia, P. speciosa has long been revered as a botanical treasure. Its seeds, leaves, and roots are ubiquitously utilized both as culinary staples and natural remedies for a spectrum of ailments [14]. Paradoxically, the empty pods of this plant have been historically marginalized, relegated to the status of agricultural refuse with little perceived economic or therapeutic value. This study challenges that paradigm, presenting compelling evidence that these discarded pods are not merely biological debris but are, in fact, reservoirs of potent pharmacological agents.

Our investigation builds upon a foundation of phytochemical profiling, which established that the seeds of P. speciosa are abundant with bioactive secondary metabolites, including saponins, alkaloids, terpenoids, and polyphenols [19]. Crucially, recent analyses of the ethanol-extracted empty pods (the PSET) have unveiled a chemical mirroring of the seeds; the pods are replete with analogous flavonoids and phenolic compounds such as gallic acid, epigallocatechin, theasinensin A, myricitrin, and eucommin A [20]. Among the constellation of identified constituents are robust antioxidants and anti-inflammatory agents such as pyrogallol (1,2,3-benzenetriol), palmitoleic acid, vitamin E, alpha-amyrin, and quercetin [18]. This chemical symmetry between the prized seed and the discarded pod underscores a significant opportunity to reclaim value from waste, transforming a byproduct into a source of health-promoting biomedicines.

To the best of our knowledge, the current research stands as first investigation into the anticancer potential of the PSET specifically against human colorectal cancer. The in vitro assays yielded striking results, delineating a clear anticancer profile. Treatment with the PSET resulted in a marked, dose-dependent suppression of cell viability in both HCT-116 and HT-29 colorectal cancer cell lines. This antiproliferative capacity was further corroborated by colony formation assays, which demonstrated the extract’s ability to severely cripple the reproductive potential of surviving cancer cells. Beyond mere cytotoxicity, the extract exhibited a sophisticated capacity to impede the physical progression of cancer. At concentrations exceeding 100 µg/mL, the PSET effectively paralyzed the metastatic machinery of the tumor cells, significantly inhibiting both migration and invasion. This suggests that the extract acts as a “molecular brake,” potentially preventing circulating tumor cells from adhering to and colonizing distant epithelial surfaces—a critical intervention point in preventing metastasis.

A pivotal discovery in previous studies was the identification of pyrogallol (1,2,3-benzenetriol), rutin, and quercetin as predominant phytochemicals within the pod extract [18]. We hypothesize that these flavonoids serve as a primary driver of the observed antineoplastic effects. Pyrogallol is a well-documented cytostatic agent, known to exert efficacy against a variety of colorectal cancer lineages (SW620, SW480, HCT-116) [21,22]. Its mechanism of action is multifaceted: it dismantles the oncogenic RAS/PI3K/AKT/mTOR signaling axis, arrests the cell cycle, induces autophagy, and triggers apoptotic cell death [21,23,24]. Furthermore, it has been shown to reverse drug resistance and inflict DNA damage specifically upon malignant cells [23]. While the presence of rutin and quercetin offers a strong mechanistic rationale for our findings, future fractionation studies will be essential to isolate and quantify its specific contribution relative to other components.

Quercetin has been extensively documented as a potent chemotherapeutic agent against colorectal cancer [25]. Its mechanism of action is multifaceted, directly targeting the molecular machinery of metastasis [26]. Specifically, quercetin has been shown to inhibit the expression of MMP2 and MMP9, the enzymes that tumor cells use to degrade the ECM. By suppressing these enzymes, quercetin effectively prevents the physical invasion of cancer cells into surrounding tissues [27]. Furthermore, quercetin modulates critical signaling pathways such as PI3K/Akt/mTOR and Wnt/β-catenin, which are essential for cell survival and proliferation [28]. It also arrests the cell cycle in the G2/M phase and induces apoptosis via the mitochondrial pathway, evidenced by the upregulation of pro-apoptotic proteins such as Bax and caspase-3 and the downregulation of the anti-apoptotic protein Bcl-2 [29].

Rutin, while structurally similar to quercetin, exhibits a distinct yet complementary therapeutic profile [30]. It has demonstrated a unique capacity to sensitize colon cancer cells (e.g., HT-29) to radiation and chemotherapy by impairing cell adhesion and mitigating migration [31]. Rutin’s anticancer activity is also mediated through the induction of apoptosis, triggering both extrinsic (death receptor) and intrinsic (mitochondrial) pathways. Crucially, rutin functions as a powerful antioxidant; however, in the context of cancer cells, it can paradoxically increase reactive oxygen species (ROS) generation to toxic levels, leading to DNA damage and cell death specifically in malignant tissues [32]. Additionally, rutin significantly diminishes the expression of vascular endothelial growth factor (VEGF), thereby inhibiting angiogenesis—the formation of new blood vessels that tumors rely on for growth [33].

The simultaneous presence of both rutin and quercetin in the PSET suggests a potential synergistic effect. While quercetin exhibits superior cytotoxicity and acts as a robust chemosensitizer for drugs like doxorubicin, rutin enhances the efficacy of other agents like 5-fluorouracil. Together, they attack the tumor on multiple fronts: quercetin locks the cells in a non-invasive state by degrading MMPs and arresting the cell cycle, while rutin impairs angiogenesis and induces oxidative stress-mediated apoptosis. This “double-hit” strategy may explain the potent antiproliferative and anti-metastatic activities observed in our PSET-treated cells, offering a compelling rationale for the extract’s efficacy beyond what might be expected from single-compound treatments.

To validate these phenotypic observations at the molecular level, we determined the expression of key proteins governing the EMT—the developmental program hijacked by cancers to facilitate invasion [34]. Western blot analysis revealed a profound downregulation of MMP2 and MMP9 in PSET-treated cells. These enzymes are the “molecular scissors” that tumor cells use to shear through the ECM, a prerequisite for invasion and metastasis. The suppression of these proteins suggests that the PSET operates by locking cells in a non-invasive state. In parallel with the suppression of these proteolytic enzymes, our proteomic analysis highlighted a critical modulation of N-cadherin, a hallmark cell adhesion molecule often upregulated during the “cadherin switch” of the EMT [35]. High levels of N-cadherin typically signal a transition to a motile, invasive phenotype, allowing cancer cells to detach from the primary tumor mass and migrate with “mesenchymal” freedom [36,37]. The observed downregulation of N-cadherin in PSET-treated cells (specifically at 200 µg/mL) provides robust molecular evidence that the extract does not simply kill cancer cells but actively reverses their invasive programming. By reducing N-cadherin expression, the PSET likely stabilizes cell–cell adhesion and reinforces the epithelial integrity of the tumor cells, effectively halting the mesenchymal drift. However, an intriguing biphasic effect was observed at the lower dose of 100 µg/mL, where the expression of MMP2 and N-cadherin transiently increased. This phenomenon can be attributed to a stress-induced compensatory mechanism, or a hormetic response, commonly observed when cancer cells are exposed to sub-lethal concentrations of phytochemicals (well below the IC₅₀ values of ~800 µg/mL) [38]. In an attempt to survive and overcome the initial inhibitory stress, the cells may transiently upregulate specific EMT markers.

Crucially, this molecular upregulation at 100 µg/mL does not translate into a “pro-oncogenic” phenotype. Our functional phenotypic data specifically the wound healing and Transwell invasion assays clearly demonstrated that cell migration and invasion were already significantly suppressed at concentrations as low as 50 µg/mL and 100 µg/mL. This discrepancy between protein expression and cellular behavior suggests that while the cells attempt to produce more MMP2 and N-cadherin, PSET likely inhibits the actual enzymatic activity of the secreted MMPs or simultaneously disrupts other critical signaling nodes required for motility. Ultimately, at the higher dose of 200 µg/mL, the robust multi-target efficacy of PSET completely overwhelms the cells’ compensatory resistance, resulting in the profound suppression of these metastatic proteins and securely locking the cells in a non-invasive state.

The downregulation of N-cadherin, MMP2, and MMP9 observed in this study aligns with the typical inhibition of the EMT process often reported in flavonoid-rich plant extracts [39,40,41,42]. However, EMT is a complex regulatory network involving multiple other markers. For instance, while PSET significantly reduced mesenchymal markers like N-cadherin, other studies on similar polyphenolic compounds have demonstrated a concomitant increase in E-cadherin—a primary epithelial marker—thereby reversing the EMT phenotype [43,44,45]. Furthermore, the modulation of Vimentin and the nuclear translocation of β-catenin are also hallmarks of EMT progression in cancer [46,47,48,49]. Although these specific markers were not assessed in the current investigation, the suppression of N-cadherin and MMPs strongly suggests that PSET interferes with the mesenchymal properties of HT-29 and HCT-116 cells. Future studies incorporating a broader panel of markers, including E-cadherin and Vimentin, would further elucidate the comprehensive regulatory mechanism of PSET on the EMT landscape.

It is noteworthy that a significant discrepancy exists between the concentrations required for cytotoxicity (IC50 of ~800–900 µg/mL) and those required for functional inhibition of metastasis (50–200 µg/mL). This phenomenon indicates that PSET’s anti-metastatic effects and its cytotoxic effects are uncoupled and governed by distinct molecular thresholds. While cytotoxicity generally requires higher concentrations of the crude extract to induce irreversible intracellular damage and apoptosis, the metastatic machinery is profoundly more sensitive to the extract’s bioactive constituents. Phytochemicals present in PSET, such as quercetin and rutin, can potently modulate highly sensitive signaling networks—such as the EMT pathway—downregulating the expression of MMP2, MMP9, and N-cadherin at sub-lethal, non-toxic doses. Pharmacologically, this discrepancy represents a highly desirable therapeutic profile. It underscores that PSET functions specifically as a targeted “molecular brake” on cancer dissemination, rather than functioning merely as a non-selective cellular poison. By exerting robust anti-migratory and anti-invasive effects at doses that maintain 95–105% cell viability, PSET presents a promising avenue for the development of safer adjuvant therapies designed to prevent metastasis without exacerbating the severe systemic toxicity commonly associated with conventional high-dose chemotherapeutics.

While the present study demonstrates the potent in vitro anti-metastatic properties of PSET, several limitations must be acknowledged. First, the transient upregulation of certain EMT markers at the 100 µg/mL dose suggests a complex, stress-induced compensatory cellular response that warrants further mechanistic investigation, although our functional assays confirmed significant phenotypic inhibition at this dose. Second, this preliminary investigation did not include a standard chemotherapeutic agent (such as 5-Fluorouracil) as a positive control. Future studies incorporating such controls will be essential to accurately benchmark the relative pharmacological potency of PSET. Finally, while these in vitro results are highly promising, they do not account for the complexities of systemic metabolism, pharmacokinetics, and tumor microenvironment interactions. Therefore, future in vivo studies using appropriate animal models are strictly necessary. These in vivo evaluations will be critical to confirm the extract’s bioavailability, establish a comprehensive safety profile, and validate its efficacy against colorectal cancer metastasis in a complex biological system before any clinical applications can be considered.

4. Materials and Methods

4.1. National Product of Parkia speciosa Extraction

The empty pods sample (lot: PS-P-2023-001) was used in this study [18]. The comprehensive phytochemical profiling of this specific batch, including the identification and quantification of pyrogallol, rutin, and quercetin via LC-MS/MS and GC-MS, has been previously completely characterized and reported by Samrit et al. [18]. The empty pods were extracted using absolute ethanol (pod/ethanol ratio, 500 g:3 L) for 14 days. The extract was then concentrated using a rotary evaporator. The yield of the extract was 11.26 %. The resulting crude extract was dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich, Saint Louis, MO, USA) to prepare a 1 mg/mL stock solution. In all experimental assays, the final concentration of DMSO was kept below at 1% (v/v) in the culture medium. A vehicle control group containing the same concentration of DMSO was also included to confirm that the observed effects were not due to the solvent.

4.2. Cell Cultures

The cell lines were purchased from the American Type Culture Collection (ATCC). The human colon cell line CCD-18Co (ATCC No. CRL-1459^TM; lot no. 70024131) was used at passage 11. The human colon carcinoma cell line HCT-116 (ATCC No. CCL-247^TM; lot no. 70019042) was used at passage 6 after receipt. The human colon adenocarcinoma cell line HT-29 (ATCC No. HTB-38^TM; lot no. 70035209) was used at passage 129. Briefly, CCD-18Co was grown in DMEM supplemented with 1 g/L of D-glucose and L-glutamine, 110 mg/L of sodium pyruvate, 10 U/mL of penicillin G, 10 µg/mL of streptomycin, and 10% fetal bovine serum. HCT-116 and HT-29 cells were grown in McCoy’s 5A medium with L-glutamine but without sodium bicarbonate and supplemented with penicillin G (10 U/mL), streptomycin (10 µg/mL), and 10% fetal bovine serum. All cell lines were maintained at 37 °C in a humidified incubator with a 5% CO₂ atmosphere and monitored daily. The culture medium was changed every 2–3 days. Cells at the logarithmic stage were harvested before performing each experiment. For all experiments, passage numbers of CCD-18Co (passages 14 to 17), HT-29 (passages 133 to 136) and HCT-116 (passaged ten to thirteen times after receipt) were used to ensure phenotypic stability.

4.3. MTT Assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma-Aldrich, Saint Louis, MO, USA) was used to evaluate cell viability. The cells were seeded into 96-well plates at 1 × 10⁴ cells per well, containing 100 µL of the respective culture medium, and allowed to grow for 24 h at 37 °C. After this time, the cells were treated with various concentrations of the extract (25–1000 µg/mL) or 1% DMSO as a negative control for 24 h. After treatment, the medium was replaced with 100 μL of fresh medium containing 0.5 mg/mL MTT (stock solution 5 mg/mL) to each well and incubated for 3 h at 37 °C. Finally, the MTT was removed, and 100 µL/well of DMSO was added. Absorbance was determined using a microplate spectrophotometer (VersaMax, Marshall Scientific, Hampton, VA, USA) at wavelengths of 570 and 690 nm. The percentage of viable cells was calculated after normalization with the negative control, which was considered 100% cell viability and calculated using the following equation:

$$\mathrm{\%} \mathrm{C}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=\left({\displaystyle \frac{{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\left(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)}}{{\mathrm{A}\mathrm{b}\mathrm{s}\mathrm{o}\mathrm{r}\mathrm{b}\mathrm{a}\mathrm{n}\mathrm{c}\mathrm{e}}_{\left(\mathrm{n}\mathrm{e}\mathrm{g}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}}}\right)\times 100$$

(1)

4.4. Cell Migration by Wound Scratch Assay

The cell migratory ability of HCT-116 and HT-29 cells was determined using a wound scratch assay. Cells were seeded into 6-well plates at 1 × 10⁶ cells per well, containing 2 mL of the respective culture medium, and were allowed to grow for 24 h at 37 °C. After this time, the monolayer cells were scratched using sterile pipette tips and washed twice with phosphate-buffered saline (PBS). Fresh medium was added, and the cells were treated for 24 h with various concentrations of the extract. Five concentrations were selected based on the percentage of cell viability achieved in the previous step, ranging from 105% to 95%, with untreated cells serving as the negative control. The analysis involved removing the plate from the incubator and placing it under an inverted microscope to take a snapshot and check for wound closure. Experiments were carried out in triplicate, and 4–5 fields of view of each wound were recorded. ImageJ software (version 6.1) was used to measure the scratched area. The cell migratory ability during wound healing was assessed using the following formula:

$$\mathrm{\%} \mathrm{W}\mathrm{o}\mathrm{u}\mathrm{n}\mathrm{d} \mathrm{C}\mathrm{l}\mathrm{o}\mathrm{s}\mathrm{u}\mathrm{r}\mathrm{e}=\left({\displaystyle \frac{{\mathrm{A}}_{\mathrm{t}=0\mathrm{h}}-{\mathrm{A}}_{\mathrm{t}=∆\mathrm{h}}}{{\mathrm{A}}_{\mathrm{t}=0\mathrm{h}}}}\right)\times 100$$

(2)

4.5. Cell Invasion by Transwell Assay

In this assay, a Corning Matrigel Basement Membrane Matrix (Corning 356234, Corning, NY, USA) was used. The Matrigel matrix was diluted in serum-free medium to a final concentration of 20 µg in the center of each Transwell^® insert (8 µm PET membrane, Corning 3464, Corning, NY, USA). The plate was incubated at 37 °C for 1 h to allow the Matrigel matrix to form a gel. After that, HCT-116 and HT-29 cells were seeded at 250 µL into the upper chamber of each Transwell insert. The final cell density was 1 × 10⁴ cells per well using the treatment extract in serum-free medium (without fetal bovine serum). At the same time, 800 µL of medium (supplemented with 10% fetal bovine serum) was used as a chemoattractant and added to the lower chambers followed by 24 h of culture in a humidified incubator at 37 °C with 5% CO₂. The Transwell inserts were washed twice with PBS. The cells inside the Transwell inserts were gently removed using moistened cotton swabs. After this, the cells on the lower surface of the membrane were fixed with the inserts using absolute methanol for 5 min and then stained with 0.1% crystal violet for 3 min. The Transwell inserts were washed twice with PBS to remove unbound crystal violet. Invaded cells that migrated through the pores to the lower surface of the filter were observed and counted under a microscope. Values were calculated by averaging the total number of cells from three filters. The cell invasion ability was assessed using the following formula:

$$\% \mathrm{I}\mathrm{n}\mathrm{v}\mathrm{a}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}=\left({\displaystyle \frac{{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{i}\mathrm{n}\mathrm{v}\mathrm{a}\mathrm{d}\mathrm{e}}_{\left(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)}}{{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{i}\mathrm{n}\mathrm{v}\mathrm{a}\mathrm{d}\mathrm{e}}_{\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}}}\right)\times 100$$

(3)

4.6. Colony Formation

A colony formation assay was performed to evaluate cell proliferation. HCT-116 and HT-29 cells were seeded at a density of 1 × 10³ cells per well into 6-well plates containing 2 mL of the respective culture medium and allowed to grow for 24 h at 37 °C. After this time, the medium was discarded, and the cells were treated for 24 h with various concentrations of the extract. After incubation at 37 °C for 10 days, when macroscopic cell colonies appeared on the bottom of the plate, the cells were washed twice with PBS and fixed with absolute methanol for 5 min. Then, the cell colonies were stained with 0.1% crystal violet for 3 min. The cells were washed twice with PBS to remove unbound crystal violet, and the number of cell colonies was visually measured using a microscope (clusters containing around 50 cells were counted as a single colony). The analysis of colony formation efficiency was defined by the number of cell colonies and the size of the colonies. ImageJ software (version 6.1) was used to determine the colony size using the following formula:

$$\% \mathrm{C}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y} \mathrm{f}\mathrm{o}\mathrm{r}\mathrm{m}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{r}\mathrm{a}\mathrm{t}\mathrm{e}=\left({\displaystyle \frac{{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y}}_{\left(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)}}{{\mathrm{N}\mathrm{u}\mathrm{m}\mathrm{b}\mathrm{e}\mathrm{r} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{y}}_{\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}}}\right)\times 100$$

(4)

$$\% \mathrm{A}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{s}\mathrm{i}\mathrm{z}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{e}\mathrm{s}=\left({\displaystyle \frac{{\mathrm{A}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{s}\mathrm{i}\mathrm{z}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{e}\mathrm{s}}_{\left(\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\right)}}{{\mathrm{A}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{a}\mathrm{g}\mathrm{e} \mathrm{s}\mathrm{i}\mathrm{z}\mathrm{e} \mathrm{o}\mathrm{f} \mathrm{c}\mathrm{o}\mathrm{l}\mathrm{o}\mathrm{n}\mathrm{i}\mathrm{e}\mathrm{s}}_{\left(\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l}\right)}}}\right)\times 100$$

(5)

4.7. Western Blot Analysis

After treatment with the pod extract, the colon cancer cells were collected and placed in a prepared cold lysis buffer (8 M urea, 0.8 M NH₄HCO₃, and pH 8) containing protease inhibitors. Then, the cells were homogenized before a BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA, #23227) was used to determine their concentration. The cell lysates (30 µg) were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to a nitrocellulose membrane (0.2 µm, Bio-Rad Laboratories, Inc., Hercules, CA, USA). After blocking with 4% skim milk in PBS buffer for 1 h at room temperature, the membrane was incubated with the primary antibody overnight at 4 °C. The primary antibodies used were rabbit polyclonal anti-human MMP2 (#4022), MMP9 (#3852), N-cadherin (#4061), and GAPDH (#2118) (dilution 1:500; Cell Signaling Technology, Danvers, MA, USA). Then, the membranes were washed with 0.1 M PBS and 0.05% Tween 20 (PBST) three times. The membrane was incubated with the goat anti-rabbit IgG (H + L) secondary antibody–HRP conjugate for 2 h at room temperature and washed three times with PBST. Finally, the membrane was imaged using a chemiluminescence imaging instrument (ChemiDoc™ Imaging System #12003153, Bio-Rad Laboratories, Inc., Hercules, CA, USA) and quantified using Quantity One software (Bio-Rad Laboratories, Inc., USA).

4.8. Statistical Analysis

The experimental data are presented as the means ± standard deviations (SDs) and were analyzed using GraphPad Prism 7 software (version 7.04). Comparisons between the extracts and controls were conducted using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. The IC₅₀ for cell viability was calculated from dose–response data using a non-linear regression model with four-parameter logistic (4PL) curve fitting. Differences were considered statistically significant at * p < 0.05 for cell viability, cell migration, cell invasion, colony formation, and Western blotting.

5. Conclusions

In this study, we investigated the effects of an extract of PSET (Lot: PS-P-2023-001) on colon cancer HCT-116 and HT-29 cells, providing evidence that the extract possesses anticancer properties and elucidating the cellular and molecular components that are impacted. Phytochemical screening from a previous study of the empty pod extract demonstrated that it contains polyphenols, phytosterols, triterpenes, oxaloacetic acid, and unsaturated fatty acids. The most abundant chemical was 1,2,3-benzenetriol, followed by epigallocatechin gallate, hyperin and quercetin. The combined results of in vitro cell assays and protein analyses support the bioactivity of the extract’s compounds in the suppression of colon cancer cell migration and invasion and the inhibition of colony formation in colon cancer through the EMT pathway. At sub-lethal concentrations, PSET significantly suppressed cell migration, invasion, and colony formation. While the high dose (200 µg/mL) effectively downregulated key EMT-related proteins (MMP2, MMP9, and N-cadherin), a transient compensatory molecular response was observed at a lower dose (100 µg/mL), highlighting the complex dose-dependent mechanism of the extract. In addition, the results of cellular experiments demonstrate its promising anticancer effects; however, several limitations must be acknowledged. As an initial in vitro screening, this study did not incorporate a standard chemotherapeutic drug (e.g., 5-Fluorouracil) as a positive control, which limits the comparative evaluation of the extract’s relative potency. Further validation through in vivo studies and specific tests to confirm its efficacy, safety, and mechanisms of action under more complex physiological conditions is required. While PSET demonstrates a degree of inhibitory effect on colorectal cancer cell lines, its high IC₅₀ values suggest that it may be more suitable as a functional food ingredient or a source for identifying bioactive lead compounds, rather than a potent therapeutic agent. Further bioassay-guided fractionation is required to isolate compounds with improved potency. This study provides support for the anticancer activity of extracts of empty P. speciosa pods, which could assist farmers by presenting new economic opportunities from their crops and thereby increase the commercial value of P. speciosa in Thailand.