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Article

Prenylated Chalcones as Anticancer Agents Against Castration-Resistant Prostate Cancer

by
Marcos Morales-Reyna
1,
Elisa Elvira Figueroa-Angulo
1,
José Espinoza-Hicks
2,
Alejandro Camacho-Dávila
2,
César López-Camarillo
3,
Laura Isabel Vázquez-Carrillo
1,
Alfonso Salgado-Aguayo
4,
Ángeles Carlos-Reyes
4,
Violeta Deyanira Álvarez-Jiménez
5,
Jonathan Puente-Rivera
1,6 and
María Elizbeth Alvarez-Sánchez
1,*
1
Laboratorio de Patogénesis Celular y Molecular Humana y Veterinaria, Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México, Ciudad de México 03100, Mexico
2
Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, México, Circuito Universitario s/n, Campus Universitario II, Chihuahua 31125, Mexico
3
Posgrado en Ciencias Genómicas, Universidad Autónoma de la Ciudad de México, Ciudad de México 03100, Mexico
4
Laboratorio de Investigación en Enfermedades Reumáticas, Instituto Nacional de Enfermedades Respiratorias, Ciudad de México 14080, Mexico
5
Laboratorio de Biología Molecular y Bioseguridad Nivel 3, Centro Médico Naval-SEMAR, Ciudad de México 04470, Mexico
6
División de Investigación, Hospital Juárez De México, México City 07760, Mexico
*
Author to whom correspondence should be addressed.
Sci. Pharm. 2025, 93(2), 25; https://doi.org/10.3390/scipharm93020025
Submission received: 25 March 2025 / Revised: 25 May 2025 / Accepted: 31 May 2025 / Published: 5 June 2025
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Abstract

Prenylated chalcones have garnered attention as potential anticancer agents due to their ability to modulate multiple cancer-related pathways. In this study, we synthesized and evaluated nine novel prenylated chalcone derivatives for their antiproliferative effects against castration-resistant prostate cancer (CRPC) cell lines, DU145 and PC3. Among these, compounds 6d and 7j demonstrated potent cytotoxic activity, with IC50 values comparable to cisplatin, and exhibited selective toxicity towards cancer cells over non-tumorigenic RWPE-1 cells. Mechanistic investigations revealed that these compounds induce apoptosis via mitochondrial membrane depolarization and increased late apoptotic events. Flow cytometry confirmed activation of both early and late apoptotic pathways. These findings highlight the potential of chalcone derivatives 6d and 7j as promising therapeutic candidates for CRPC treatment and support further development of chalcone-based molecules in precision oncology.

1. Introduction

The incidence rate of cancer increased during 2020 around the world. A total of 19,292,789 estimated cases of cancer were registered, with a mortality of 9,958,133. In men, lung and prostate cancer (PCa) present the highest incidence rate, with 1,435,943 and 1,414,259 cases per year, respectively [1]. The PCa progression is a multi-step process that begins with the prostatic intraepithelial neoplasia, followed by a localized growth of carcinogenic cells that degenerate into locally invasive advanced prostate adenocarcinoma. Traditionally, the Gleason score is used to determine the progression status of cancer, where a score of 6 indicates the presence of low-grade cancer, 7 indicates medium-grade cancer, and a score between 8 and 10 is reserved for high-grade cancer with a time to progression to metastatic PCa, characterized by a high-hormonal response [2,3,4]. This type of response is one of the main treatments throughout the progression of the disease, which uses androgen deprivation in combination with radiation therapies, obtaining, in most cases, the desired therapeutic effect [5,6,7]. Although this high-hormonal therapy helps ameliorate disease progression, 10 to 20% of patients will develop castration-resistant PCa (CRPC) within 5 years after diagnosis, with a probability equal to or greater than 84% of metastases at the time of diagnosis of CRPC [8].
CRPC indicates a phase of PCa progression in which the cells do not respond to androgen deprivation therapy, which aims to suppress circulating testosterone levels. Despite the absence of circulating testosterone, the tumor remains functionally dependent on estrogen and estrogen receptors [9]. The CRPC treatment is palliative, and normal disease progression is associated with high mortality [9,10]. Although several drugs have been developed to treat this type of cancer, in many cases, they are usually aggressive and are associated with adverse side effects in patients, and resistance to multiple drugs contributes as another impediment to adequate treatment of this disease [11]. Hence, the development of new molecules with specific anticancer properties is a challenge that has already been addressed by now [12].
Several reports have proposed two mechanisms through which this CRPC can be generated: ligand-dependent and ligand-independent mechanisms [9,13]. The ligand-dependent pathway suggests that castration resistance can be caused by variants of androgen receptors (AR) detected in patients with this condition, and a lack of ligand binding domain, particularly AR-V7, has been identified as a significant mechanism contributing to resistance in CRPC. AR-V7 lacks the ligand-binding domain, rendering it constitutively active and unresponsive to conventional AR-targeted therapies, thereby promoting disease progression [14,15]. These constitutively active variants are capable of carrying out their functions without the presence of androgens, resulting in non-effective therapies [16,17]. The ligand-independent pathway suggests an aberrant activation of ARs due to the overexpression of heat shock proteins, especially HSP90, which is increased in patients with radiation-resistant PCa [13,18]. The anti-cancer potential of natural compounds, such as chalcones, is currently evaluated due to their low-cost synthesis, high availability, and relatively non-toxic characteristics. Moreover, some chalcones are capable of reaching molecular reactions that are key to the genesis and progression of cancer [19,20].
Chalcones are aromatic ketones, part of many important biological compounds, and can usually be produced by condensation reactions of bases or acid catalysis. Even though chalcones are unsaturated ketones, they are easily synthesized with interesting biological activities. The synthesis strategies, general methods, catalysts, and conditions used for the synthesis of chalcone scaffolds are summarized below [21,22]. In brief, the Claisen–Schmidt reaction to chalcone synthesis takes place when two different molecules of aldehyde or aromatic ketone are combined in the presence of a strong base. The ion enolate produced in this reaction attacks the carbonyl group of the other molecule, forming a conjugated double bond, leading to the formation of chalcone [23,24].
Generally, chalcones present a common structure of 1,3-diaryl-2-propen-1-ones, which can be modified to increase their biological activities through the addition of functional groups, such as halogen, hydroxyl, carboxylic, and benzene, among other substituents [19,22]. Chalcones modified with the addition of a prenyl group have shown increased anticancer activity [25]. Some chalcones having this functional group, such as Xanthohumol, Desmethylxanthohumol, and Xanthogalenol exhibit anticancer activities in various cancer cell lines, alongside the reported anticancer effects of chalcones, including inhibition of cell proliferation, induction of apoptosis, and interference with signaling pathways, such as PI3K/AKT, p38/mTOR/STAT3, BRAF, and NF-κB (Figure 1) [26,27,28,29,30,31,32,33,34,35,36,37]. Therefore, we suggested that prenylated chalcone derivatives may exhibit an anticancer effect on CPRC cells.
One of the causes of the multiple anti-cancer activities of the chalcones may be Michael-type acceptors, which can usually form covalent bonds with cysteine sulfhydryl or other thiol groups to obtain the Michael adduct [22]. It has been observed that this type of acceptor can act as an inhibitor of cysteine proteases, which are involved in the development of different diseases ranging from cardiovascular, inflammatory, neurological, respiratory, viral, musculoskeletal, immunological, and of the central nervous system, to cancer [38,39,40].
This study was designed to evaluate the potential anticancer effects of a series of novel prenylated chalcones on PC3 and DU145 prostate cancer cell lines. We initially assessed their impact on cell viability, apoptosis induction, cellular morphology, and mitochondrial membrane potential. The findings provided important insights into the interactions of these chalcones with key therapeutic targets, highlighting their promising properties as potential candidates for pharmacological intervention.

2. Materials and Methods

2.1. Chalcones

Nine chalcones were evaluated for their anticancer properties (Table 1). The chalcone derivatives synthesis was carried out using the Claisen–Schmidt condensation reaction following the procedure described by Hernández-Rivera [41]. The organic synthesis of O-prenylchalcone 7e started from the condensation between vanillin and acetovanillin, which were prenylated by reacting with prenyl chloride in the presence of potassium carbonate to obtain precursors 3a and 3b. Thereafter, the Claisen–Schmidt condensation with substituted benzaldehydes and acetophenones was performed with the prenylated precursor.

2.2. Cell Lines

PC3 (ATCC CRL 1435) and DU145 (ATCC HTB-81) human prostatic cancer cell lines and RWPE-1 epithelial prostate cell line (ATCC CRL-11609), were obtained from the American Type Culture Collection (ATCC 10801, University Boulevard Manassas, Manassas, VA 20110-2209, USA). DU145 and PC3 cell lines were grown in DMEM medium (Gibco™, Waltham, MA, USA) and RPMI 1640 (Gibco™) medium, respectively. The RWPE-1 cell line was grown in K-SFM medium supplemented with Epidermic Growth Factor and Bovine Pituitaries Factor (Gibco™). All mediums were supplemented with 10% Fetal Bovine Serum (Gibco™), and 100 mg/mL of Penicillin-Streptomycin (Sigma™, Kanagawa, Japan). The cultures were maintained in a humidified atmosphere containing 5% CO2 at 37 °C for 24 h.

2.3. Cell Viability by MTT Assay and Selectivity Index

To determine the cytotoxic effect of the chalcones, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT, Sigma-Aldrich, Saint Louis, MO, USA) bromide assay was carried out. Next, 1 × 104 cells/well of DU145, PC3, and RWPE-1 cell lines were cultured in 96-well plates for 24 h at 37 °C, until reaching 80–90% confluence. After that, the cells were exposed to 25, 50, 75 or 100 µM of each chalcone for 0, 24 and 48 h. The medium without chalcones was used as a control. At the end of these incubation times, 5 mg/mL of MTT was added to each well and incubated for 3 h in the dark at 37 °C. Subsequently, the MTT solution was removed, and the formazan crystals were dissolved in isopropanol (Sigma-Aldrich). The plates were read in a Biotek Elx800 Universal Plate Reader (BioTek Instruments, Inc., Winooski, VT, USA) at 570 nm. Cell viability was expressed as a relative percentage of untreated control cells. IC50 (half-maximal inhibitory concentration) values and their 95% confidence intervals were obtained by nonlinear regression of three independent experiments performed in triplicate.
The selectivity index was calculated with the following equation: Selectivity Index (SI) = IC50 non-cancer line/IC50 cancer line. A selectivity index equal to or greater than 1 indicates higher selectivity towards the cancer cell line [30]. The best treatment time was selected, as well as the IC50 of the chalcones with the strongest activity to perform the subsequent evaluations.

2.4. Apoptosis Assay

The apoptotic effect of chalcones was carried out by Annexin V-FITC/PI staining (Abcam/Thermo Fisher, Waltham, MA, USA). First, 8 × 105 cells were grown in 6-well plates for 24 h until reaching 80% confluence and incubated with 100 µM of 6d or 7j chalcones or 2 µg/mL of cisplatin (Sigma Aldrich, St. Louis, MO, USA) for 48 h at 37 °C. After incubation, the cells were harvested and washed twice with 100 µL of PBS pH 7, and incubated in the dark at RT for 15 min with 5 μL of Annexin V-FITC diluted in 500 μL of binding buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 2.5 mM CaCl2, PBS pH 7.4). Then, 5 μL of propidium iodide (PI) at 50 μg/mL was added, and all the samples were processed on a flow cytometer (FACS Canto II BD, Stockholm, Sweden) and quantified with the FlowJo software version 10.8.1 [42]. This assay was performed in biological duplicates by technical triplicate.

2.5. Mitochondrial Membrane Potential Analysis

First, 8 × 105 cells were grown in 6-well plates for 24 h until reaching 80–90% confluence, and were then treated with the selected chalcones and the MTT viability assay. The medium culture of each cellular line was used as a negative control. The specific medium with 50 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) (Merck, Darmstadt, Germany) was taken as a positive control. After the treatment time, cells were collected in 15 mL falcon tubes, and 2 µL of the MitoOrange 200X solution (Abcam) was added and incubated for 15 min at 37 °C, 5% CO2. Subsequently, cells were centrifuged at 1000 rpm for 4 min (Biofuge primo R) and resuspended in 1 mL of PBS pH 7. Fluorescence intensity was monitored using a flow cytometer BD FACSCanto™ II (BD Biosciences, Stockholm, Sweden) in the FL 2 channel (Ex/Em = 540/590 nm), creating a cell gate of interest excluding debris. The percentage of cells with membrane potential collapse was determined using the FlowJo software version 10.8.1 [42].

2.6. Confocal Microscopy

First, 8 × 105 cell lines were grown in a 6-well plate until they reached around 80–90% confluence and incubated for 48 h with the selected chalcones. The cells were washed three times with PBS and fixed with 4% paraformaldehyde (PFA) (Merck) for 10–15 min at room temperature (RT). Then, the cells were washed in PBS pH 7 to remove any remaining PFA residues and permeabilized with 0.1% Triton X-100 (Thermo Fisher) for 5 min at RT. Cells were washed three times with PBS pH 7 to remove any remaining Triton X-100. Subsequently, the treated cell was incubated with 5 µL Phalloidin Rhodamine (Thermo Fisher) in 200 µL of PBS pH 7 for 30 min at RT to label the actin filaments. Cells were washed with PBS pH 7 twice to remove any traces of rhodamine phalloidin and incubated with DAPI (Thermo Fisher) for 10 min at RT to label the nucleus. Finally, the cells were mounted on a slide using a VECTASHIELD H1000 (Vector Laboratories Inc., Newark, CA, USA). The cells were observed with an Olympus FV1000 confocal microscope (Olympus, Tokyo, Japan).

2.7. Statistical Analysis

All experiments were carried out three times, and the statistical analyses were performed with Student’s t-test or one-way analysis of variance; p < 0.05 is estimated to be statistically significant. * p < 0.05, ** p < 0.01, *** p < 0.001.

3. Results

3.1. 6d and 7j Chalcones Reduce Viability in DU145 and PC3 Cells

DU145 and PC3 cell lines were selected for this study based on previous reports that validate them as a CRPC model [43,44,45]. The viability of these cell lines treated with different concentrations (25, 50, 75 and 100 µM) of the nine prenylated chalcones incubated at 0, 24 and 48 h was analyzed by MTT assay (Figure 2). At time zero, both cellular lines showed 100% viability; however, after 48 h, the DU145 cell line viability decreased to 48.94% with the 6d and 28.29% with the 7j at the same chalcone concentration (Figure 2A). The PC3 cells reduced their viability to 46.6% with 6d and 26.56% with 7j at a 100 µM chalcone concentration (Figure 2B). These two chalcones were used for subsequent assays at a 100 µM concentration and 48 h incubation.
To determine the SI degree of the 6d and 7j chalcones, we calculated the IC50 values for each chalcone at 48 h (Table 1). The 6d IC50 of chalcone on DU145 and PC3 cells were 88.73 µM (+/−1.086) and 54.96 µM (+/−0.56), respectively, both with 95% confidence intervals (CI). The IC50 values of 7j on DU145 and PC3 cells were 61.71 (+/−0.79) µM and 57.22 (+/−0.85) µM, respectively, with the same CI (Table 2).
These data showed that 6d and 7j chalcones have the best value of IC50 on DU145 and PC3 cells (Figure 3A). Then, we calculated the SI for 6d and 7j, and the data showed that both chalcones had a value ≥ 1 (Figure 3B).

3.2. Chalcones 6d and 7j Induce Apoptosis on Prostate Cancer Cell Lines

To determine whether the reduced cellular viability resulting from treatment with 6d and 7j was caused by the induction of apoptosis, an Annexin V/PI assay was performed. This assay leverages the principle that, during early apoptosis, phosphatidylserine translocates from the inner to the outer leaflet of the plasma membrane, allowing it to be detected by Annexin V. Necrotic cells, characterized by compromised membrane integrity, allow propidium iodide (PI) to access and stain DNA. This differential staining allows for the distinction between apoptotic and necrotic cells. As a control, the CRPC cells were treated with 2 µM/mL of cisplatin, an apoptotic inductor frequently used in PCa treatment. The treatment for 48 h with 6d, 7j, or cisplatin on DU145 and PC3 cells, showed an increase of apoptotic population in comparison with the untreated control (Figure 4A). The treatment with 7j showed 3.67%, 27.95% and 32.9% of the population in early apoptosis (Figure 4D), late apoptosis (Figure 4E), and necrosis, respectively (Figure 4F). The treatment with 6d showed 1.72%, 13.45%, and 49.7% population in early apoptosis (Figure 4D), late apoptosis (Figure 4E), and necrosis (Figure 4F), respectively, on DU145 cells. In the case of PC3 cells, 7j showed 18.9%, 44.15%, 17.5% of the population in early apoptosis, late apoptosis, and necrosis, respectively, and 6d showed 11.45%, 40.75%, and 22% of the population in early apoptosis, late apoptosis, and necrosis, respectively (Figure 4D–F). These results indicate that apoptosis plays a key role in the death machinery in DU145 and PC3 cells treated with both chalcones.

3.3. DU145 and PC3 Cell Lines Change Its Membrane Potential Under 7j and 6d Treatment

A key early event in apoptosis is the disruption of mitochondrial function. Changes in mitochondrial membrane potential (MMP) are associated with membrane permeabilization and the release of cytochrome c into the cytosol, which subsequently activates downstream events in the apoptotic cascade. To assess mitochondrial membrane integrity and changes in MMP following treatment with the chalcones 6d and 7j, a Mito Orange staining assay was performed. After 48 h of treatment, both chalcones induced a significant impairment of mitochondrial function compared to untreated cells. The DU145 and PC3 cells treated with the 6d at an IC50 concentration for 48 h show a membrane potential of 49.6% and 50.2% in both cell lines, whereas the same cells treated with the 7j showed 48% and 47.5% of membrane potential, respectively (Figure 5, Table 3).

3.4. Chalcones 7j and 6d Induce Changes in the Morphology of DU145 and PC3 Cell Lines

To investigate the effects of 7j and 6d chalcones on cell morphology, we examined the actin cytoskeleton and nuclear morphology using confocal microscopy. Our results revealed a distinct pattern of actin filaments in untreated PC3 and DU145 cells compared to chalcone-treated cells. Specifically, in untreated cells, these filaments extended from the perinuclear actin layer to the cortical region of the cell (Figure 6A,B). In both cell lines treated with the chalcones 7j and 6d had a loss of the actin filaments the organization, this was probably due to depolymerization of the filaments and microtubules when the formation of the apoptotic microtubule network (ARM) occurs, similarly, the conformation of DAPI-stained nuclei is altered in both cell lines, with chromatin fragmentation that may be associated with apoptotic processes. These results show that the morphology changes of both PCa cell lines are due to the interaction with chalcones 7j and 6d.

4. Discussion

The analysis of the antiproliferative activity of the chalcone derivatives reveals a clear structure–activity relationship (SAR), particularly within the 6x and 7x series. Chalcones 6d and 7j, which showed the strongest cytotoxic effects, share a prenylated aromatic ring and specific halogen substitutions. For instance, 6d (with a fluorine in meta position) displayed superior activity compared to its analog 6c, where the fluorine is located in the ortho position. Similarly, 7j (with a chlorine in the ortho position) was more active than 7k, which contains chlorine in the meta position. These results suggest that both the position of the halogen substituent and the aromatic ring arrangement play a crucial role in enhancing the interaction with molecular targets, possibly by influencing the electronic distribution and steric accessibility of the α,β-unsaturated carbonyl system [46,47,48]. This observation supports previous reports indicating that fine variations in chalcone substitution patterns can significantly affect their anticancer potency and selectivity.
Actin filaments form stress fibers that support the cell and play a significant role in mechanotransduction, which is a characteristic of cells that grow in rigid strata. Both are proteins that pump the drugs supplied to the outside of the cell, causing them to be ineffective [36,49,50,51]. The in vitro antiproliferative activity of prenylated chalcones was evaluated against two PCa cell lines (DU 145 and PC3) using the MTT assay. The IC50 values of the two best compounds obtained after 48 h of treatment are reported in Table 1. This type of antiproliferative activity of chalcones has already been reported in other studies, where it is indicated that the presence of the prenyl group improves the selectivity towards cancer cells [52]. In this study, the best antiproliferative effect was presented by the chalcones 7j and 6d, since these compounds had different activities than their analogs 6c and 7k, suggesting that the arrangement of the functional group causes an important change in the manifestation of their biological activities. This was observed in other studies, where the use of structural variants of chalcones, in which the position of the functional groups or the prenyl group is changed, is determinant for the antiproliferative effect to be weaker or stronger [52,53,54].
The observed differences in sensitivity between DU145 and PC-3 PCa cells may be attributed to the different expression profiles of the estrogen receptors ER-a and ER-b. PC-3 PCa cells express low levels of ER-a mRNA and high levels of ER-b [55,56]. Recently, many new molecules that target apoptosis have entered various stages of clinical trials, targeting various molecules involved in this process, most of which are IAP antagonists and molecules that target the Bcl-2.1 family of proteins [57,58,59,60].
Our results confirmed that both compounds induce apoptosis in the initial stages by externalizing phosphatidylserines in the two cell lines analyzed, so the search for chalcones as new alternatives for cancer treatment may be promising. It has been shown that the mitochondrial membrane potential (Δψm), which reflects the functional state of the mitochondria, is highly related to cancer malignancy. Studies have shown that in cancer cells, it is increased compared to those that do not have it [61,62]. A collapse of Δψm was observed after treatment with the chalcones 7j and 6d, as expected. These results are consistent with what has been reported in other studies, where, in the same way, a reduction in PMM is reported after treatment. In treatment with chalcones [63,64], these results are consistent with the decrease in viability presented after 48 h of treatment with both chalcones.
To determine the effect of the chalcones 7j and 6d on cell morphology, we evaluated their actin cytoskeleton and nucleus. We observed morphological changes as a result of the interaction with chalcones. This type of morphological alteration has already been reported in other studies where chalcones are supplied as cancer treatments [39,65]. Among the changes reported are damages at the core level, adoption of a rounded shape and shrinkage of the same [66]. Both chalcones altered microtubule organization in PC3 and DU145 when compared to control cells, which possessed a well-organized microtubule network. These data suggest that both chalcones may affect microtubule organization in PC3 and DU145 cells, contributing to their induction of apoptosis. Chalcones are a central part of the synthesis of flavonoids, since their structure is a stepping stone for their formation [67,68]. This allows them to exhibit a broad spectrum of biological activities, probably due to their small structure and Michael acceptor characteristics, making them tolerant to different biological molecules, allowing for their rapid or reactive binding with certain molecules [20]. Although chalcones are molecules that may be great candidates to be used for the generation of new drugs, their broad spectrum of bioactivity may indicate a potentially promiscuous targeting profile, which may represent a challenge for clinical development [20,69].
However, the use of chalcones as therapeutic agents in various types of cancer has shown promising results [19,70,71,72], in addition to indicating that chalcones are more active against cancerous disorders, against breast cancer cell lines, and also against lung cancer. From this point of view, it is important to continue research on the improvement of this type of molecule for its medical application. In general, chalcones are easy to synthesize to generate compounds with a wide variety of structural diversity, which makes them attractive as building blocks for the synthesis of targeted agents. In an area where molecularly targeted therapy and personalized medicine, both natural and synthetic chalcones can be particularly useful tools to study the basic mechanisms of cancer treatment and prevention and to develop new agents for targeted cancer therapies.

5. Conclusions

Our results demonstrate that novel chalcones 6d and 7j exhibit strong and selective antiproliferative activity against CRPC cell lines DU145 and PC3, with minimal effects on non-cancerous cells. These chalcones promote apoptosis through mitochondrial dysfunction and membrane depolarization, suggesting a mechanism involving the intrinsic apoptotic pathway. The structural features of these derivatives, particularly halogen positioning and prenylation, appear to play a key role in their bioactivity, as highlighted in our SAR analysis. This study contributes valuable insights into the design of effective chalcone-based chemotherapeutics and proposes 6d and 7j as lead compounds for further preclinical development against CRPC (Figure 7).

Author Contributions

Conceptualization: M.M.-R., C.L.-C. and M.E.A.-S. Methodology: E.E.F.-A. and L.I.V.-C. Software: M.M.-R. and V.D.Á.-J. Validation: A.S.-A., Á.C.-R. and M.E.A.-S. Formal analysis: M.M.-R., C.L.-C., M.E.A.-S., A.S.-A. and J.P.-R. Investigation: M.M.-R., E.E.F.-A., L.I.V.-C., J.E.-H., A.C.-D. and J.P.-R. Resources: C.L.-C. and M.E.A.-S., Data curation: M.M.-R. and V.D.Á.-J. Writing—original draft preparation:. M.M.-R. and M.E.A.-S. Writing—review and editing: M.M.-R., C.L.-C., J.E.-H., A.C.-D., M.E.A.-S., A.S.-A., J.P.-R. and E.E.F.-A., Visualization: E.E.F.-A., L.I.V.-C. and J.P.-R. Supervision: M.E.A.-S. and C.L.-C. Project administration: M.E.A.-S. and C.L.-C. Funding acquisition: M.E.A.-S. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by UACM and the project CCyT-2023-IMP-03. The funding body had no role in the design of the study, data collection, analysis, interpretation, or writing of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

No new data were created.

Acknowledgments

The authors would like to acknowledge the financial support provided by the Universidad Autónoma de la Ciudad de México and the funding received through the project CCyT-2023-IMP-03.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
PCaProstate Cancer
CRPCcastration-resistant prostate cancer
6c(E)-3-(2-fluorophenyl)-1-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
6e(E)-3-(4-fluorophenyl)-1-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
7e(E)-1-(3-fluorophenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
7a(E)-3-(3-Methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-1-(2-nitrophenyl)prop-2-en-1-one
7j(E)-1-(2-chlorophenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
7l(E)-1-(4-chlorophenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
7d(E)-1-(2-fluorophenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
7k(E)-1-(3-chlorophenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
6d(E)-3-(3-fluorophenyl)-1-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one

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Figure 1. Representative chalcones with reported anticancer activity and their mechanisms of action. Chemical structures of four natural chalcone derivatives—Isoliquiritigenin, Licochalcone A, Xanthoangelol, and Butein—are shown alongside a summary of their reported selective anticancer effects in different cancer models, including colorectal, melanoma, hepatocellular carcinoma, neuroblastoma, breast, and PCa (↓) indicates low expression.
Figure 1. Representative chalcones with reported anticancer activity and their mechanisms of action. Chemical structures of four natural chalcone derivatives—Isoliquiritigenin, Licochalcone A, Xanthoangelol, and Butein—are shown alongside a summary of their reported selective anticancer effects in different cancer models, including colorectal, melanoma, hepatocellular carcinoma, neuroblastoma, breast, and PCa (↓) indicates low expression.
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Figure 2. Cell viability determination of DU145 and PC3 CRPC cells treated with different chalcone concentrations. (A) DU145 and (B) PC3 cells were treated with nine chalcones at different concentrations of 25, 50, 75, and 100 µM for 48 h. * p < 0.05, statistically significant differences between treatments and control are shown.
Figure 2. Cell viability determination of DU145 and PC3 CRPC cells treated with different chalcone concentrations. (A) DU145 and (B) PC3 cells were treated with nine chalcones at different concentrations of 25, 50, 75, and 100 µM for 48 h. * p < 0.05, statistically significant differences between treatments and control are shown.
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Figure 3. Selectivity index (SI) of chalcones 6d and 7j on CRPC cell lines. (A) Bar graph representing the selectivity index (SI) values of chalcones 6d and 7j for DU145 and PC3 cells after 48 h of treatment. The SI was calculated as the ratio of IC50 in RWPE-1 (non-cancerous prostate epithelial cells) to IC50 in cancer cell lines. (B) Numeric representation of the SI values shown in panel (A). An SI ≥ 1 indicates preferential cytotoxicity against PCa cells over non-cancerous cells. Values represent the mean ± SD of three independent experiments.
Figure 3. Selectivity index (SI) of chalcones 6d and 7j on CRPC cell lines. (A) Bar graph representing the selectivity index (SI) values of chalcones 6d and 7j for DU145 and PC3 cells after 48 h of treatment. The SI was calculated as the ratio of IC50 in RWPE-1 (non-cancerous prostate epithelial cells) to IC50 in cancer cell lines. (B) Numeric representation of the SI values shown in panel (A). An SI ≥ 1 indicates preferential cytotoxicity against PCa cells over non-cancerous cells. Values represent the mean ± SD of three independent experiments.
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Figure 4. Apoptotic effect of chalcones 6d, 7j on CRPC cells. (A) DU145 cell line and (B) PC3 cells treated with 100 µM of 6d, 7j, or 2 µg/mL of cisplatin, analyzed by Annexin V/PI flow cytometry assay after 48 h of treatment. A representative image of three independent experiments is shown and compared with untreated control or cisplatin-treated cells. Graphic representations of a total alive population (C), the population in early apoptosis (E), the population in late apoptosis (F), and the necrotic population (D) are shown. (C) The bar graph shows the standard deviation represented by the error bars. *** p < 0.001, significant differences.
Figure 4. Apoptotic effect of chalcones 6d, 7j on CRPC cells. (A) DU145 cell line and (B) PC3 cells treated with 100 µM of 6d, 7j, or 2 µg/mL of cisplatin, analyzed by Annexin V/PI flow cytometry assay after 48 h of treatment. A representative image of three independent experiments is shown and compared with untreated control or cisplatin-treated cells. Graphic representations of a total alive population (C), the population in early apoptosis (E), the population in late apoptosis (F), and the necrotic population (D) are shown. (C) The bar graph shows the standard deviation represented by the error bars. *** p < 0.001, significant differences.
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Figure 5. The membrane potential of DU145 and PC3 treated with 7j and 6d. On the Y-axis the membrane potential is shown; on the X-axis, the treatments applied are shown. CCCP: Carbonyl Cyanide 3-Chlorophenylhydrazone. The standard deviation is represented by the error bars of three independent experiments. ΨPMM: Potential Mitochondrial Membrane. *** p < 0.001, statistically significant differences between treatments and control are shown.
Figure 5. The membrane potential of DU145 and PC3 treated with 7j and 6d. On the Y-axis the membrane potential is shown; on the X-axis, the treatments applied are shown. CCCP: Carbonyl Cyanide 3-Chlorophenylhydrazone. The standard deviation is represented by the error bars of three independent experiments. ΨPMM: Potential Mitochondrial Membrane. *** p < 0.001, statistically significant differences between treatments and control are shown.
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Figure 6. Morphology changes induced by chalcone treatment on PC3 and DU145 cell lines. Confocal microscopic images of the effect of chalcones 7j or 6d on (A) DU145 and (B) PC3 cell lines. Bright-field (first panel), Phalloidin- FITC (second panel), DAPI stained (third panel) and merged channel (fourth panel) are shown. The orange arrow shows the actin filaments.
Figure 6. Morphology changes induced by chalcone treatment on PC3 and DU145 cell lines. Confocal microscopic images of the effect of chalcones 7j or 6d on (A) DU145 and (B) PC3 cell lines. Bright-field (first panel), Phalloidin- FITC (second panel), DAPI stained (third panel) and merged channel (fourth panel) are shown. The orange arrow shows the actin filaments.
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Figure 7. Proposed mechanism of action of prenylated chalcones in castration-resistant prostate cancer (CRPC) cells. Figure illustrates how prenylated chalcones (6d and 7j) exert cytotoxic effects on CRPC cells inducing mitochondrial membrane potential disruption and actin filament destabilization, which can ultimately lead to apoptotic cell death in PCa cells. PCa. Up-red arrow (↓) indicates decrease function. (→) indicates effect on mitochondria, actin filaments and apoptosis.
Figure 7. Proposed mechanism of action of prenylated chalcones in castration-resistant prostate cancer (CRPC) cells. Figure illustrates how prenylated chalcones (6d and 7j) exert cytotoxic effects on CRPC cells inducing mitochondrial membrane potential disruption and actin filament destabilization, which can ultimately lead to apoptotic cell death in PCa cells. PCa. Up-red arrow (↓) indicates decrease function. (→) indicates effect on mitochondria, actin filaments and apoptosis.
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Table 1. Structural and analytical data of prenylated chalcone derivatives evaluated for anticancer activity.
Table 1. Structural and analytical data of prenylated chalcone derivatives evaluated for anticancer activity.
StructureCodeMWSMILEIUPAC Name
Scipharm 93 00025 i001(6c)340.39C/C(C)=C\COC1=CC=C(C(/C=C/C2=C(F)C=CC=C2)=O)C=C1OC(E)-3-(2-fluorophenyl)-1-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
Scipharm 93 00025 i002(6e)340.39C/C(C)=C\COC1=CC=C(C(/C=C/C2=CC=C(F)C=C2)=O)C=C1OC(E)-3-(4-fluorophenyl)-1-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
Scipharm 93 00025 i003(7e)340.39C/C(C)=C\COC1=CC=C(/C=C/C(C2=CC(F)=CC=C2)=O)C=C1OC(E)-1-(3-fluorophenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
Scipharm 93 00025 i004(7a)367.40C/C(C)=C\COC1=CC=C(/C=C/C(C2=C([N+]([O-])=O)C=CC=C2)=O)C=C1OC(E)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)-1-(2-nitrophenyl)prop-2-en-1-one
Scipharm 93 00025 i005(7j)356.84C/C(C)=C\COC1=CC=C(/C=C/C(C2=C(Cl)C=CC=C2)=O)C=C1OC(E)-1-(2-chlorophenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
Scipharm 93 00025 i006(7l)356.84C/C(C)=C\COC1=CC=C(/C=C/C(C2=CC=C(Cl)C=C2)=O)C=C1OC(E)-1-(4-chlorophenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
Scipharm 93 00025 i007(7d)340.39C/C(C)=C\COC1=CC=C(/C=C/C(C2=C(F)C=CC=C2)=O)C=C1OC(E)-1-(2-fluorophenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
Scipharm 93 00025 i008(7k)356.84C/C(C)=C\COC1=CC=C(/C=C/C(C2=CC(Cl)=CC=C2)=O)C=C1OC(E)-1-(3-chlorophenyl)-3-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
Scipharm 93 00025 i009(6d)340.39C/C(C)=C\COC1=CC=C(C(/C=C/C2=CC(F)=CC=C2)=O)C=C1OC(E)-3-(3-fluorophenyl)-1-(3-methoxy-4-((3-methylbut-2-en-1-yl)oxy)phenyl)prop-2-en-1-one
Table 2. The IC50 value of 6d and 7j at 48 h.
Table 2. The IC50 value of 6d and 7j at 48 h.
ChalconeStructureIC50 DU145 (μM)IC50 PC3 (μM)IC50 RWPE-1 (μM)
6dScipharm 93 00025 i01088.73 (+/−1.086)54.969 (+/−0.56)62.34 (+/−0.47)
7jScipharm 93 00025 i01161.71 (+/−0.79)57.22 (+/−0.85)1 139.5 (+/−0.7)
Table 3. Membrane potential of DU 145 and PC3 cell lines.
Table 3. Membrane potential of DU 145 and PC3 cell lines.
TreatmentControl7j6dCCCP
Cell line
DU 14599%48%49.6%68.5%
PC399.8%47.549.6%50.2%
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Morales-Reyna, M.; Figueroa-Angulo, E.E.; Espinoza-Hicks, J.; Camacho-Dávila, A.; López-Camarillo, C.; Vázquez-Carrillo, L.I.; Salgado-Aguayo, A.; Carlos-Reyes, Á.; Álvarez-Jiménez, V.D.; Puente-Rivera, J.; et al. Prenylated Chalcones as Anticancer Agents Against Castration-Resistant Prostate Cancer. Sci. Pharm. 2025, 93, 25. https://doi.org/10.3390/scipharm93020025

AMA Style

Morales-Reyna M, Figueroa-Angulo EE, Espinoza-Hicks J, Camacho-Dávila A, López-Camarillo C, Vázquez-Carrillo LI, Salgado-Aguayo A, Carlos-Reyes Á, Álvarez-Jiménez VD, Puente-Rivera J, et al. Prenylated Chalcones as Anticancer Agents Against Castration-Resistant Prostate Cancer. Scientia Pharmaceutica. 2025; 93(2):25. https://doi.org/10.3390/scipharm93020025

Chicago/Turabian Style

Morales-Reyna, Marcos, Elisa Elvira Figueroa-Angulo, José Espinoza-Hicks, Alejandro Camacho-Dávila, César López-Camarillo, Laura Isabel Vázquez-Carrillo, Alfonso Salgado-Aguayo, Ángeles Carlos-Reyes, Violeta Deyanira Álvarez-Jiménez, Jonathan Puente-Rivera, and et al. 2025. "Prenylated Chalcones as Anticancer Agents Against Castration-Resistant Prostate Cancer" Scientia Pharmaceutica 93, no. 2: 25. https://doi.org/10.3390/scipharm93020025

APA Style

Morales-Reyna, M., Figueroa-Angulo, E. E., Espinoza-Hicks, J., Camacho-Dávila, A., López-Camarillo, C., Vázquez-Carrillo, L. I., Salgado-Aguayo, A., Carlos-Reyes, Á., Álvarez-Jiménez, V. D., Puente-Rivera, J., & Alvarez-Sánchez, M. E. (2025). Prenylated Chalcones as Anticancer Agents Against Castration-Resistant Prostate Cancer. Scientia Pharmaceutica, 93(2), 25. https://doi.org/10.3390/scipharm93020025

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