Phytochemical Profiles and Anticancer Effects of Calophyllum inophyllum L. Extract Relating to Reactive Oxygen Species Modulation on Patient-Derived Cells from Breast and Lung Cancers

Reactive oxygen species (ROS) contribute to cancer growth and metastasis. Using antioxidants to modulate cellular ROS levels is a promisingstrategy for cancer prevention and treatment. Calophyllum inophyllum L., or tamanu, is a medicinal plant renowned for its anti-inflammatory, antioxidant, and anticancer properties in traditional medicine systems. However, the anticancer effects of C. inophyllum extract on cellular ROS remain unexplored. This study represents the first report on such effects and provides the potential mechanisms underlying the anticancer properties of C. inophyllum extract. The branches of C. inophyllum were extracted, and the extract was comprehensively analyzed for phytochemical constituents, antioxidant capacity, total phenolic content, and total flavonoid content. Subsequently, the extract's potential anticancer properties were evaluated using patient-derived cells from breast and lung cancer. The results revealed that the C. inophyllum extract possesses notable antioxidant activity and demonstrated no cytotoxicity within the initial 24 h of treatment. However, after 72 h, it exhibited significant antiproliferative effects. Moreover, the extract exhibited inhibitory properties against migration and invasion at concentrations below the IC50, which corresponded to the expression of related genes. Notably, these effects correlated with the reduction of intracellular ROS levels. Overall, our findings highlight the anticancer potential of C. inophyllum extract, emphasize its ability to modulate cellular ROS levels and target key molecular pathways involved in cancer progression. This study sheds light on the promising therapeutic implications of C. inophyllum extract as a novel agent for cancer treatment, which is safe for normal cells.


Introduction
Reactive oxygen species (ROS) play many crucial roles in cancer progression and metastasis leading to the death of cancerous patients. As we have known for many decades that lung cancer in males and breast cancer in females are the leading causes of mortality worldwide [1]. ROS is accused of cancer death due to the involvement of cancer progression and metastasis including cell growth, proliferation, migration, and invasion by activating upstream-proliferative signaling cascades, growth factor receptors, adhesion molecules, and transcription factors [2]. Nevertheless, disrupting redox homeostasis in cancer cells by rapidly increasing intracellular ROS ultimately results in cell death which is a property of several FDA-approved drugs for cancer treatment including paclitaxel, 5-fuorouracil, doxorubicin, and cisplatin [3]. Tese efective chemotherapies also result in serious side efects on surrounding normal cells by oxidative damage. Increasing the ROS level above the redox balance in cancer cells efectively kills the cells while the ROS level below that alleviates aggressiveness by deceleration of proliferation, metastasis, and cell death induction [4]. Reduction of intracellular ROS by antioxidants on ROS-dependent cancer cells enhanced the potential of success in the treatment of metastatic solid tumors demonstrated in either in vitro or in vivo models with promising results [5,6]. In addition, numerous plantderived phytochemicals, including polyphenols and favonoids, exhibit the potential for antiproliferation and antimetastasis against cancer cells without causing cytotoxicity [5][6][7].
Calophyllum inophyllum L., commonly known as tamanu, is a tropical evergreen tree belonging to the family Calophyllaceae. It distributes across Tailand, especially in the coastal regions. With its medium-sized stature, reaching heights of 20-30 meters, it thrives in diverse landscapes, including forests, mangroves, coastal areas, and tropical regions [8,9]. Tis plant has been recognized as one of the most important medicinal plants in Ayurvedic and Tai traditional medicine with various parts including leaves, fowers, and stem barks with medicinal properties [10]. Te plant extract from various parts is rich in antioxidants identifed as polyphenols, phenolic acids, favonoids, and other antioxidant structuring phytochemicals which exhibit anti-infammation, antimicrobial, and anticancer efects [8,11,12]. Te phytochemicals obtained from the ethanolic leaf extract and oil seed of this plant have demonstrated cytotoxic efects on breast and lung cancer cells, leading to the induction of apoptosis [13,14]. In addition, the extract from this plant was noncytotoxic to many noncancerous cells including keratinocytes, dermal fbroblasts [15], preosteoblasts [16], and conjunctival epithelial cells [17].
Despite its traditional use and documented properties, there is a signifcant knowledge gap in the scientifc literature regarding the specifc efects of C. inophyllum on cancer treatment and its relationship with intracellular ROS. Further research is needed to explore the potential anticancer efects of C. inophyllum extract and its modulation of ROS. With its potent antioxidant, anti-infammatory, and anticancer properties, C. inophyllum presents an intriguing candidate for investigating cellular ROS modulation in cancer cells. Its ability to impact multiple pathways involved in cancer development makes C. inophyllum a promising subject for scientifc inquiry.
Te objectives of this study were to investigate the phytochemical properties of C. inophyllum extract and to elucidate the efects of the C. inophyllum extract on cancer cells derived from the patients in comparison with the cancer cell lines. While previous literature has extensively explored the therapeutic potential of various parts of C. inophyllum, the branches of the tree have received limited attention until now. Te study assessed the biochemical properties of the C. inophyllum branch extract and identifed its phytochemical constituents. It was hypothesized that using cancer cells derived from the patients might provide better outcomes due to the fact that they demonstrate characteristics of cancer tissues in the body which promptly respond to the extract. Te antioxidant activity of C. inophyllum extract was estimated in vitro based on the scavenging activity of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical, as described by Blois [18]. Te stock solution of the extract was diluted in methanol and pipetted into each well of a 96-well plate. Ten, the DPPH solution was added to each well and thoroughly mixed with the plate shaker. Te mixture was further incubated in the dark at room temperature for 30 min. Te optical density values at 517 nm were measured by using a microplate reader (Rayto, China). Te percentage of DPPH free radical scavenging activity was calculated. Te half-maximal 2 Scientifca inhibitory concentration (IC 50 ) was calculated from the plotted equation. Ascorbic acid and quercetin were used as standard antioxidants and positive controls.

Total Phenolic Content.
Te total phenolic content of C. inophyllum extract was estimated by colorimetric assay, Folin-Ciocalteu's reagent as described elsewhere [19]. In brief, 40 μl of the sample was pipetted into glass tubes, 800 μl 10% Folin-Ciocalteu's reagent was topped up, and the mixture was well mixed and incubated at room temperature for 5 min. Ten, 800 μl 7% sodium carbonate and 360 μl ultrapure water were added to each tube of the mixture followed by thorough mixing. Te reaction mixture was incubated for 2 h in the dark at room temperature. Te OD 760 was measured using a UV-VIS spectrophotometer (Hitachi, Japan). Te OD 760 values of quercetin were plotted to obtain the calibration curve. Estimation of total phenolic content in C. inophyllum extract is exhibited as mg of gallic acid equivalent per gram dry extract (mg GAE/g DW) using the calibration curve of gallic acid.

Total Flavonoid Content.
Total favonoid content of the C. inophyllum extract was assessed by the aluminum chloride colorimetric assay as described previously [20]. 0.2 ml of either C. inophyllum extract or standard was mixed with 4.8 ml of ultrapure water in a test tube. 0.3 ml of 5% NaNO 2 was topped up and mixed well by using a vortex mixer. After that, the mixture was left at room temperature for 5 min prior to the addition of 0.3 ml of 10% AlCl 3 6H 2 O as well as 2 ml of 1 M NaOH solution into each tube. Te ultrapure water was dispensed to reach the fnal volume of 10 ml. Te optical density values at 415 nm were recorded using a UV-VIS spectrophotometer (Hitachi, Japan). Quercetin was used as a standard. Te total favonoid content was expressed as mg of quercetin equivalents per gram of dry extract (mg QE/g DW) using the calibration curve of quercetin.

Gas Chromatography-Mass Spectrophotometry
Analysis. In order to identify certain phytochemical constituents as well as to illustrate the chromatogram fngerprint in the C. inophyllum extract, GC-MS analysis was performed using Agilent Technologies GC-MS 7890A/5975C Series (Agilent, USA) equipped with a capillary column (30 m in length × 0.25 mm in diameter × 0.25 μm flm thickness) (partly modifed from Kadir [21] colorimetric assay was applied. After seeding cells into each well of a 96-well culture plate and incubating for 24 h, the cells were exposed to the complete medium containing various concentrations of C. inophyllum extracts for 24 and 72 h, along with quercetin. Te assessment of cell viability was performed using the sulforhodamine B colorimetric assay, as described by Vichai and Kirtikara [22]. In brief, cells were fxed with 10% trichloroacetic acid and incubated with 0.057% sulforhodamine B solution (Sigma, USA) at room temperature for 30 min. After washing with 1% acetic acid (Sigma, USA), the dye was solubilized with 10 mM tris solution (pH 10.5) (Sigma, USA) with constant agitation for 15 min. Te optical density values at 510 nm were read by a microplate reader (Rayto, China) and the IC 50 of the extract was determined by using PriProbit Program ver. 1.63 [23]. Te criteria for cytotoxicity were adhered to when the IC 50 value was higher than 20 μg/ml [24]. In addition, selectivity index values were calculated by dividing the IC 50 values of the normal cell line by the IC 50 values of the cancer cells. A selectivity index higher than 1.00 indicates specifcity towards cancer cells [25]. Te IMR-90 cell line was used as the normal cell line in this experiment.

Efects on Cell Migration.
To assess the inhibitory efects of the C. inophyllum extract on cancer cell migration, a migration assay was employed. After the cells reached 75% confuence and formed a monolayer, the cell layers were scratched with a sterile pipette tip and washed with PBS. Te complete medium containing C. inophyllum extract and reference compound was then added to the scratched cell layer prior to the incubation under the standard culture condition. Gap closure was monitored and photomicrographed after 24 h using an Optika IM-3 inverted microscope equipped with a C-B10+ digital camera (Optika, Italy) in comparison with 0 h of incubation. Te gap distances were measured using ImageJ software (NIH, USA), and the results were expressed as percentages of the gap distance in the treatment group relative to the gap distance in the untreated control at 0 h. control. Te results were quantifed as the percentage of invaded cells, calculated by multiplying the ratio of cell numbers in the treatment group to the cell numbers in the negative control by 100.

Efects on Intracellular Reactive Oxygen Species (ROS)
Modulation. To determine intracellular levels of ROS in the form of hydrogen peroxide (H 2 O 2 ) and superoxide anions (O 2 · − ), 2,7-dichlorodihydrofuorescein diacetate (DCFDA) was applied. After seeding the cells and incubating them in standard culture conditions for 24 h, the cells were exposed to the complete medium containing either the C. inophyllum extract, the reference compound, or 100 μM tert-butyl hydroperoxide (TBHP, a ROS-inducing standard compound) [26]. Intracellular ROS determination was conducted after 24 h of exposure to the targeted substances using the DCFDA/H2DCFDA-Cellular ROS Assay Kit (Abcam, UK). According to the manufacturer's instructions, the medium was discarded, and the cells were rinsed with PBS and with 1X reaction bufer. 1X DCFDA solution was dropped onto the cell surface and incubated at 37°C for 45 min in the dark. Te cells were rinsed with PBS prior to the observation under a fuorescence microscope (Olympus, Japan). Fluorescence intensity was measured by using ImageJ software (NIH, USA) through digital photomicrographs. Te data were represented as the percentages of fuorescence intensity in comparison to the negative control, untreated cells.

Statistical Analysis.
Te data presented in this study are expressed as the mean ± standard deviation (SD) and were derived from fve independent replicates of each treatment. Te signifcant diferences among groups were evaluated by ANOVA followed by the Tukey post hoc test with a confdence interval p < 0.01.

Extraction of Plant Materials.
Te extraction yield was approximately 9.06% viscous mass obtained from 100 g powder branches of C. inophyllum after 72 h of continuous extraction in ethanol.

GC-MS Analysis of C. inophyllum Extract.
Te GC-MS profle of the ethanolic extract of C. inophyllum is shown in Figure 1. Te main constituents identifed in the extract are reported in Table 2. Sixteen phytochemicals were identifed (the quality index >80%). Most components were phenolic (approximately 31% of total identifed compounds). Other components were classifed as furoic acid esters, dihydropyranones, catechols, benzofurans, furans, fatty acids, fatty acid esters, isoquinolines, and xanthones. Te three highest abundant phytochemicals found in the C. inophyllum extract were 5hydroxymethylfurfural (5-HMF), antiarol, and syringol which were classifed as furans, phenols, and phenols, respectively.

Cancer Tissue Collection, Establishment, and Culture of
Patient-Derived Cells. Cancer biopsies were collected from patients (Table 3) and disaggregated to primary cancer cells. Cells were outgrown from the cancer tissue samples and subcultured to obtain PDCs of breast (BC-1, BC-2, and BC-3) and lung (LC-1, LC-2, and LC-3) cancer ( Figure 2). All breast and lung cancer samples were diagnosed by the pathologists as invasive carcinoma and adenocarcinoma, respectively. Phenotypic heterogeneity of the populations was found in all BC and LC cells. Fibroblast-like cells were the dominant phenotype in most of the cells in the population, while mesenchymal-like and epithelial-like cells were in the minority.

Ki-67 Immunocytochemistry for Cancer Cell
Characterization. Immunocytochemistry of Ki-67 was applied to BC and LC cells in comparison to A549 and MDA-MB-231. Te positive cells expressed brown nuclei (Figure 3(a)) were randomly counted and calculated to the percentage of positive cells (Figure 3(b)). Tere   (Table 5). Te acceptable SI value was >1.00 indicating the specifcity of the compounds to cancer cells. Te results revealed that both C. inophyllum extract and quercetin exhibited specifcity towards most cancer cell types, not all cell types were afected by these substances.

Evaluation of C. inophyllum Extract on Cancer
Based on the obtained results, subsequent experiments were conducted using C. inophyllum extract at concentrations of 100 and 200 μg/ml, as well as quercetin at concentrations of 10 and 20 μg/ml. Tese concentrations were selected based on their demonstrated antioxidant capacity and nontoxicity to cells, as evidenced by the cell survival rate of 80% after 24 h of treatment.

Efects on Cell Migration.
After treating the cells with C. inophyllum extract and quercetin in the migration assay, the gap distances were measured to assess their migratory ability. Te results showed that both the C. inophyllum extract and quercetin treatments signifcantly closed the gap compared to the control group at 0 and 24 h (Figure 4(a)). In the photomicrographs, the white-dotted lines are used as guidelines to measure the gap distances between cell margins. Te highest percentage of gap distance indicates the largest space observed between cell margins at 0 h (Figure 4(b)). After 24 h, the control group (Ctrl) displayed a signifcant decrease in the percentage of gap distance for all cells, suggesting cell migration and gap closure. However, the efects of C. inophyllum extract and quercetin treatments on gap distances varied depending on the cell types. At 200 μg/ml of C. inophyllum extract exhibited signifcant  Figure 1: Te GC-MS chromatogram of the C. inophyllum extract.  inhibition of cell migration in most cancer cell types, except for MDA-MB-231. However, the efects of the extract at 100 μg/ml were less pronounced, whilst quercetin at a concentration of 20 μg/ml demonstrated signifcant inhibition of cell migration in most cancer cell types. However, at a concentration of 10 μg/ml, quercetin showed less inhibitory ability, particularly in BC-1 and LC-1 cells. When comparing the efects of C. inophyllum extract to quercetin, the percentage of gap distance in the quercetin-treated cells indicated a greater potential for inhibiting cancer cell migration compared to the C. inophyllum extract across all cancer cell types.

Efects on Cell Invasion.
Te most efective concentrations of C. inophyllum extract and quercetin were found to be 200 and 20 μg/ml, respectively. Using these concentrations, the ability of the compounds to inhibit cancer cell invasion was tested using a Transwell insert with the Matrigel

Efects on Intracellular ROS Modulation.
After exposing the cells to 200 μg/ml of C. inophyllum extract or 20 μg/ml of quercetin for 24 h, the intracellular ROS levels were visualized in green using fuorescence microscopy ( Figure 6(a)). tert-Butyl hydroperoxide (TBHP), a compound known to induce ROS production, was used as a positive control and successfully increased ROS levels in all cancer cell types. Te treatments with C. inophyllum extract and quercetin signifcantly reduced intracellular ROS levels compared to the untreated controls in all cancer cell types ( Figure 6(b)). Notably, in many cases, the C. inophyllum extract exhibited a greater ability to decrease ROS levels compared to quercetin, except in MCF-7, BC-1, BC-2, and LC-3 cells where both C. inophyllum extract and quercetin treatments showed similar potency.

Efects on Gene Expression.
Te results demonstrated that treatment with 200 μg/ml of C. inophyllum extract and 20 μg/ml of quercetin signifcantly decreased the expression of genes involved in cancer cell migration, invasion, and ROS modulation, with the exception of E-cadherin, which showed signifcant activation (Figure 7). Te expression of migration genes including E-cadherin and Twist-1, determined that E-cadherin was signifcantly activated in all cells treated, except LC-2. In contrast, the expression of Twist-1 was signifcantly reduced, except in NCI-H1299. In addition, NCI-H1299 responded to either C. inophyllum extract or quercetin by signifcantly increasing the expression of Twist-1. Expressions of MMP-2 and MMP-9 as invasion-involved genes declined upon either C. inophyllum extract or quercetin treatments observed in all cancer cell types. However, a statistically insignifcant reduction of those genes could be observed in certain cancer cell types including NCI-H1299, LC-1, LC-3, and BC-2. Te ROSresponsive genes, NRF2 and HIF-1α responded to the C. inophyllum extract and quercetin in a similar manner. Te reductions of NRF2 and HIF-1α were observed in all cell types, except LC-1 and BC-1. Te C. inophyllumextractexposed LC-1 enhanced the expression of NRF2 and HIF-1α.
In BC-1, the expression of HIF-1α from the treatments of C. inophyllum extract and quercetin was relatively higher than the controls; however, signifcant diferences were found in the quercetin treatment.

Discussion
In this study, we investigated the efects of an ethanolic extract derived from branches of C. inophyllum on cancer     cell viability, migration, and invasion. For the frst time, we examined the extract's ability to alleviate intracellular ROS levels, an important factor in cancer progression. Our study was driven by the assumption that antioxidant extracts can efectively reduce free radicals in cells and potentially impede cancer progression. We optimized the extraction conditions of C. inophyllum extract by using temperatures of 40-50°C and ethanol concentrations of 70-100% to preserve antioxidant properties and maximize bioactive compound yields [31]. Te extract exhibited strong antioxidant activity (ranking from 100 to 150 μg/ml) [32], demonstrated by its efective free radical scavenging capabilities (Table 1). GC-MS analysis confrmed the presence of phenolic compounds, favonoids, and other bioactives, including the newly discovered 5-HMF, contributing to the extract's antioxidant activity ( Table 2). Tese compounds, characterized by benzene rings with hydroxy or methoxy groups, possess direct and indirect antioxidant properties [33]. Previous studies consistently support the antioxidant capacity of C. inophyllum, including its stem bark and wood [34], highlighting its broad range of bioactivities and antioxidant potential [10]. Tus, C. inophyllum represents a valuable natural resource abundant in antioxidants and bioactive compounds. Tese compounds hold great promise for diverse therapeutic applications, including the development of anticancer therapies.
Te PDCs ofer distinct advantages over immortalized cell lines, as they faithfully preserve the cellular assembly, tissue architecture, microenvironment, and cancer niches found in the body. Tis unique characteristic enables PDCs to exhibit more accurate responses to cytotoxic compounds and enhanced detection of cancer markers, surpassing traditional cell lines [35]. A notable biomarker associated with cancer cell proliferation and metastasis is Ki-67, which is highly expressed in malignant breast and lung cancer cells but is minimally detected in normal proliferating cells [36]. In this study, the PDCs derived from breast or lung cancer demonstrated high Ki-67 expression (>50%) (Figure 3), a signifcant feature resembling that of cancer cells [37]. Tis further underscores the resemblance of these PDCs to malignant breast and lung cancer cells, emphasizing their utility as a valuable model for studying cancer biology.
Cytotoxicity and antiproliferation efects of C. inophyllum extracts were evaluated using both PDCs and standard cancer cell lines. A noncancerous cell line was included as a reference to assess the selective index. Quercetin, a natural favonoid known for its high antioxidant activity, protective efects against oxidative damage, and anticarcinogenic properties, was used as a reference substance [38]. Our fndings demonstrate that C. inophyllum extract and quercetin exhibited antiproliferative efects according to the NCI criteria [24] and consistent with previous studies [15][16][17], while showing no cytotoxicity (Table 4). Te selectivity index indicated cancer specifcity (>1.0) for most cells, with PDCs showing higher sensitivity to quercetin (Table 5). While the C. inophyllum extract lacked specifcity for PDCs, it exhibited timedependent antiproliferative efects. Te presence of bioactive compounds, including 5-HMF and xanthones, likely contributed to the observed anticancer properties, either directly or indirectly [11,12,39]. Importantly, the extract demonstrated selectivity for cancer cells, making it a promising candidate for further anticancer drug development.
Te antioxidant properties and cancer cell specifcity of the C. inophyllum extract were found to be associated with its potential for inhibiting migration and invasion (Figures 4  and 5), which correlated with a reduction in intracellular ROS levels ( Figure 6). Tis was further supported by the upregulation of E-cadherin and downregulation of MMP-9, MMP-2, Twist-1, NRF2, and HIF-1α (Figure 7). Te extract and quercetin, known for their antioxidant capabilities, likely alleviated intracellular ROS, leading to the      downregulation of NRF2 and HIF-1α. Consequently, the expressions of MMP-2 and MMP-9 were partially restricted, inhibiting epithelial-mesenchymal transition (EMT) [40]. Previous studies have shown that reducing intracellular ROS can disrupt metastasis in various types of cancer by decreasing MMP-2 and MMP-9 expression [41,42]. Similar efects have been observed with other antioxidants such as silibinin and quercetin derivatives [43,44]. In addition, the downregulation of NRF2 and HIF-1α, which are associated with cancer cell growth and propagation, has been reported in response to antioxidants [45]. Te antioxidant phytochemicals in C. inophyllum extract, including 5-HMF, 2methoxy-4-vinylphenol, cinnamic derivatives, xanthones, and isoquinolines, likely contribute to the anticancer efects by directly reducing intracellular ROS and inhibiting cell migration and invasion [11,46]. However, our fndings showed diferential expression patterns of migration and invasion-related genes in LC-3 and NCI-H1299 cell lines treated with C. inophyllum extract and quercetin. Both treatments resulted in the downregulation of E-cadherin and Twist-1 (Figure 7), suggesting their potential role in inhibiting metastasis. Notably, in highly metastatic lung cancer cells, the association between EMT and MMPs with metastasis appears to be mediated through integrin-and proteaseindependent mechanisms [47,48]. Furthermore, 70% of cellular deformation under hypoxia was dependent on Twist-1 and MMPs but not EMT process for their migration [49].
In addition, the exposure to C. inophyllum extract and quercetin led to the reduction of NRF2 and HIF-1α in most cells, with BC-1 and LC-1 cells showing distinct responses (Figure 7). In BC-1, high expression of HIF-1α led to increased ROS levels, a common characteristic of canceractivated HIF-1α without upstream NRF2 interference. On the other hand, LC-1 cells exhibited increases in both NRF2 and HIF-1α expressions. NRF2 upregulation in response to rising free radicals and ROS serves to maintain redox homeostasis and rescue cells from death, while HIF-1α induction is associated with cancer cell survival mechanisms under fuctuating intracellular ROS levels [50]. Hence, the observed inhibition of migration and invasion in BC-1 and LC-1 cells may not solely be attributed to intracellular ROS levels but likely involves other mechanisms or signaling molecules [51,52].
Te involvement of ROS in various cellular signaling transduction processes, including cancer cell growth, EMT, and metastasis, highlights its signifcance in cancer biology. However, it is important to acknowledge that cellular responses to ROS are highly diverse and depend on specifc cell types. Further purifcation and investigation of phytochemicals, along with comprehensive cellular biology studies, are needed to unravel the intricate mechanisms underlying cancer cell migration, invasion, and ROS levels. Tese endeavors hold the potential to provide crucial insights into the complex interplay between ROS and cancer, thereby paving the way for the development of innovative therapeutic strategies targeting ROS-mediated processes in cancer.

Conclusions
Te ethanolic extract of C. inophyllum demonstrated selective cytotoxicity, antiproliferation, and migration/invasion inhibition which was related to intracellular ROS reduction. Te potent anticancer efects might be due to the availability of total phenolic content and total favonoid content and their strong antioxidant properties. Notably, the prominent phytochemical component responsible for these antioxidant efects is 5-HMF. When tested on both PDCs and standard commercial cell lines of breast and lung cancers, the C. inophyllum extract exhibited specifc antiproliferative efects that selectively targeted cancer cells. Te extract's antioxidant properties efciently attenuated intracellular ROS levels, consequently impeding the migration and invasion processes. Tis observation was further substantiated by gene expression analysis, revealing a signifcant increase in E-cadherin expression alongside notable decreases in Twist-1, MMP-2, MMP-9, NRF2, and HIF-1α. Terefore, the fndings of this study suggest that the C. inophyllum extract could have the potential to be an alternative therapeutic agent for cancer treatment. Nevertheless, further research and development are still required to fully understand the mechanism of cancer therapy.

Data Availability
Te generated or analyzed data used to support the fndings of this study are included within the article.

Ethical Approval
Te study and all processes were conducted in accordance with the Declaration of Helsinki, and the exemption was approved by the Institutional Research Ethics Committee of the Faculty of Medicine, Chiang Mai University, for studies involving humans (EXEMPTION 8600/2021).