Next Article in Journal
Myrtinols A–F: New Anti-Inflammatory Peltogynoid Flavonoid Derivatives from the Leaves of Australian Indigenous Plant Backhousia myrtifolia
Next Article in Special Issue
Switching from Aromatase Inhibitors to Dual Targeting Flavonoid-Based Compounds for Breast Cancer Treatment
Previous Article in Journal
Trapping of Small Molecules within Single or Double Cyclo[18]carbon Rings
Previous Article in Special Issue
Regiospecific Hydrogenation of Bromochalcone by Unconventional Yeast Strains
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Molecules
Volume 28
Issue 5
10.3390/molecules28052159
molecules-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Antitumor Effect of Chalcone Derivatives against Human Prostate (LNCaP and PC-3), Cervix HPV-Positive (HeLa) and Lymphocyte (Jurkat) Cell Lines and Their Effect on Macrophage Functions

by
Bruno Horta
1,2,3,
Joana Freitas-Silva
4,
Jani Silva
1,5,
Francisca Dias
1,
Ana Luísa Teixeira
1,
Rui Medeiros
1,2,6,7,*,
Honorina Cidade
4,8,
Madalena Pinto
4,8 and
Fátima Cerqueira
1,6,7
1
Molecular Oncology and Viral Pathology GRP—IC, Portuguese Institute of Oncology of Porto (IPO Porto), Rua António Bernardino de Almeida, 4200-072 Porto, Portugal
2
ICBAS—School of Medicine and Biomedical Sciences, Port University, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal
3
CBQF—Centre for Biotechnology and Fine Chemistry, Associated Laboratory, Higher School of Biotechnology, Portuguese Catholic University, Rua Diogo Botelho, 1327, 4169-005 Porto, Portugal
4
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4450-208 Matosinhos, Portugal
5
AquaValor—Centro de Valorização e Transferência de Tecnologia da Água, Rua Júlio Martins, n°1, 5400-342 Chaves, Portugal
6
FP-I3ID, FP-BHS, Universidade Fernando Pessoa, Praça 9 de Abril, 349, 4249-004 Porto, Portugal
7
Faculty of Health Sciences, University Fernando Pessoa, Rua Carlos da Maia, 296, 4200-150 Porto, Portugal
8
Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, Faculty of Pharmacy, University of Porto, Rua Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal
*
Author to whom correspondence should be addressed.
Molecules 2023, 28(5), 2159; https://doi.org/10.3390/molecules28052159
Submission received: 15 January 2023 / Revised: 19 February 2023 / Accepted: 20 February 2023 / Published: 25 February 2023
(This article belongs to the Special Issue Natural Products with α,β-Unsaturated Carbonyl Units and Their Synthetic Derivatives: Recent Advances and Future Perspectives)
Browse Figures
Versions Notes

Abstract

:
Chalcones are synthetic and naturally occurring compounds that have been widely investigated as anticancer agents. In this work, the effect of chalcones 1–18 against the metabolic viability of cervical (HeLa) and prostate (PC-3 and LNCaP) tumor cell lines was tested, to compare the activity against solid and liquid tumor cells. Their effect was also evaluated on the Jurkat cell line. Chalcone 16 showed the highest inhibitory effect on the metabolic viability of the tested tumor cells and was selected for further studies. Recent antitumor therapies include compounds with the ability to influence immune cells on the tumor microenvironment, with immunotherapy being one actual goal in cancer treatment. Therefore, the effect of chalcone 16 on the expression of mTOR, HIF-1α, IL-1β, TNF-α, IL-10, and TGF-β, after THP-1 macrophage stimulation (none, LPS or IL-4), was evaluated. Chalcone 16 significantly increased the expression of mTORC1, IL-1β, TNF-α, and IL-10 of IL-4 stimulated macrophages (that induces an M2 phenotype). HIF-1α and TGF-β were not significantly affected. Chalcone 16 also decreased nitric oxide production by the RAW 264.7 murine macrophage cell line, this effect probably being due to an inhibition of iNOS expression. These results suggest that chalcone 16 may influence macrophage polarization, inducing the pro-tumoral M2 macrophages (IL-4 stimulated) to adopt a profile closer to the antitumor M1 profile.

1. Introduction

Cancer continues to be a leading cause of death worldwide, although cancer treatments have improved over recent decades. Most chemotherapeutic drugs act as antiproliferative agents; however, the tumor surrounding microenvironment has an essential effect on the cancer cells’ capabilities [1]. Therefore, searching for new compounds with broad antitumor activity remains an extraordinary challenge. Macrophages are important cells in the tumor microenvironment, designated as tumor-associated macrophages (TAMs) [1,2]. The amount and phenotype of TAMs can influence tumor initiation, progression, angiogenesis, and metastization, promoting disease prognosis [3,4,5,6]. The M1 phenotype, also mentioned as classically activated, is considered pro-inflammatory, and the M2 phenotype, also referred to as alternatively activated, exerts pro-tumoral effects [2]. M1 macrophages produce reactive oxygen (ROS), nitrogen species (RNS), and pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 [5,7]. M2 macrophages mainly produce IL-10 and TGF-β [5,7]. Nowadays, reprogramming TAMs into an antitumor phenotype is recognized as one of the antitumor immunomodulation strategies developed in the fight against cancer [2,3,4].
Recent studies revealed that the hypoxic microenvironment of tumors promotes macrophage infiltration [8]. As such, macrophages located at hypoxic regions of the tumor express Hypoxia Inducible Factor (HIF-1), and are able to promote angiogenesis [9] and increasing tumor hypoxia. TAMs’ interference with tumor cells’ metabolism also increases aerobic glycolysis, which is one of the mechanisms responsible for tumor resistance to anticancer immunotherapy [10].
Chalcones are a class of flavonoids recognized for their extensive range of biological activities, including antitumor and anti-inflammatory [11,12,13]. The antitumor effect of chalcones is not limited to apoptosis induction in tumor cells, which makes them promising compounds for cancer therapy [14]. In fact, some chalcones were shown to affect the tumor microenvironment through the modulation of immune mediators released by tumor cells and, therefore, have cancer chemoprevention effects. For instance, some methoxy derivatives of 2′-hydroxychalcone significantly reduced ICAM-1 and IL-8 released by SW480 colon tumor cells [14].
Over recent decades, our research group has identified several chalcones with notable growth inhibitory activity in human tumor cell lines [15,16,17,18,19]. Chalcone derivatives 1–18 (Figure 1), in particular, revealed promising antiproliferative activity against A375-C5 (melanoma), MCF-7 (breast adenocarcinoma), and NCI-H460 (non-small cell lung cancer) cell lines, this effect being associated with an antimitotic effect [20]. Aiming to pursue our research on anticancer immunotherapy, namely on their activity on macrophages [21,22,23,24], these chalcones were tested against liquid- and solid tumor-derived cell lines (Jurkat, LNCaP, PC-3, and HeLa). The chalcone that showed the highest antiproliferative effect (16) was selected to explore its effect on some human macrophage functions, as the expression of mTORC1, HIF-1α, cytokine characteristics of an M1 (IL-1β and TNF-α) and M2 (IL-10 and TGF-β) profile. The inhibitory effect of the chalcone on NO production by RAW264.7 murine macrophages was also tested.

2. Results

2.1. Effect of Chalcones on the Metabolic Activity of Tumor Cell Lines

All chalcones were able to affect the metabolic activity of solid- and liquid-derived tumors cell lines, including the cervix HPV-positive tumor cell HeLa, androgen-dependent (LNCaP) and -independent (PC-3) prostate cell lines, and the human lymphocyte cell line Jurkat. All the compounds were able to affect the metabolic viability of the four cell lines tested (Table 1). Doxorubicin was used as a positive control, and the values obtained corresponded to those in the literature [25,26].
For the HeLa cell line, compounds revealed low to moderate inhibition of metabolic activity when tested at 5 μM (9.8–46.8%) and 10 μM (20.3–57.8%), while displaying moderate to high inhibition (26.8–85.8%) at 20 μM. Chalcone 12 with a 3,4,5-trimethoxyphenyl B ring demonstrated the highest capacity for decreasing HeLa cell line metabolic viability (85.8%) at 20 μM. Contrarily, the less active compound was chalcone 18, possessing chlorine substituents in the B ring (26.8 % inhibition at 20 μM).
Considering LNCaP, the inhibition of the metabolic activity ranged 16.0–37.9% for compounds tested at 5 μM, 28.4–58.9% for compounds tested at 10 μM, and 37.0–78.4% at 20 μM. The highest inhibitory activity (78.4%) was observed for chalcone 7 with a 3,5-dimethoxyphenyl B ring, while polymethoxylated chalcone 17 showed the lowest inhibition (37.0%) at 20 μM.
Against the PC-3 cell line, the metabolic activity inhibition was also depended on the concentration of the compounds tested: 15.4–47.0% for 5 μM; 27.0–64.7% for 10 μM; and 37.0% to 87.6% for 20 μM. Compounds 3, 10, and 16, all with a methoxylated B ring, were the chalcone derivatives that strongly inhibited the metabolic activity of the PC-3 cell (84.6%, 84.7%, and 87.6%, respectively), while chalcone 15 with methoxy groups on both rings showed the weakest inhibition (37.0%).
The metabolic inhibitory effect of chalcones against the non-adherent Jurkat cell line was 6.8–69.4% for compounds tested at 5 μM, 13.0–97.0% when compounds were tested at 10 μM and ranged from 77.3% to total inhibition for compounds tested at 20 μM.
Doxorubicin was used as positive control, and the values obtained were like those reported in the literature [25,26].
Compound 16 with two methoxy groups in both aromatic rings stands out for strongly reducing the metabolic activity of all the cell lines at 20 μM, not demonstrating any selectivity towards any of the adherent cell lines for all tested concentrations (p > 0.05). Nevertheless, chalcone 16 at 20 μM is more active in Jurkat than in all the adherent cell lines (p < 0.05 for all comparisons).
At 5 μM, chlorinated chalcone 8 resulted in significantly higher inhibition values in LNCaP in comparison to PC-3′s (p < 0.05). Chlorinated chalcone 4 was significantly more cytotoxic against LNCaP, while 18 also possessing two chlorine substituents at the B ring demonstrated its highest activity against PC-3 (p < 0.05), both at 10 μM. PC-3 revealed a higher sensitivity in comparison to LNCaP for some of the derivatives with methoxylated B rings such as 3, 10, and 11 at 20 μM (p < 0.05). Chalcones 2 and 4 strongly inhibited the metabolic activity of the HeLa cell line when compared to LNCaP (p < 0.05), while 4 and 18 had a weaker effect on the cervix cell line when compared to PC-3 (p < 0.05).
In general, when comparing the metabolic inhibitory effect of chalcones possessing the same A ring, those with a 3,5-dimethoxyphenyl B ring showed the most pronounced inhibitory effect, independently of the A ring. Interestingly, most of chalcones with chlorine at B ring displayed a lower activity than those with methoxy groups at the B ring, except for compound 15.
As compound 16 showed to be the most potent inhibitor of the metabolic activity of all tested tumor cell lines, this chalcone was selected for the study of its effect on macrophage activity modulation and its anticancer potential.

2.2. Effect of Chalcone 16 on Macrophage Functions

2.2.1. Effect of Chalcone 16 on THP-1 Macrophage HIF-1α, mTORC1 and Cytokine Expression

As seen in Figure 2A, for unstimulated macrophages, compound 16 treatment significantly increases the expression of mTORC1 (fold-change = 4.0), HIF-1α (fold-change = 4.1), and the pro-inflammatory cytokine IL-1β (fold-change = 5.1), but no effect is observed on the expression of the other cytokines tested. When macrophages were stimulated with IL-4 (Figure 2B), representing M2 macrophages, chalcone 16 treatment increases the expression of mTORC1 (fold-change = 3.5), but HIF-1α expression, an effector of the hypoxia conditions of tumors, is not influenced. M1 characteristic pro-inflammatory cytokines’ (IL-1β and TNF-α, fold change = 4.5 and 5.7, respectively) expression increased, as well as IL-10 (fold-change = 2.4).
For LPS-stimulated macrophages (Figure 2C) representing the M1 phenotype, an increase was observed for all the parameters tested (mTORC1 fold-change = 7.9; HIF-1α fold-change = 3.4; TGF-β1 fold-change = 2.5; IL-1β fold-change = 4.7 and TNF-α fold-change = 4.4), except for IL-10 expression, which was not affected.
Independent of the stimulation, the treatment of macrophages with chalcone 16 significantly increases the expression of mTORC1.

2.2.2. Effect of Chalcone 16 on NO Production by RAW264.7 Macrophages

The treatment of RAW 264.7 macrophages with 1.3 and 2.5 µM of compound 16 revealed no cytotoxic effect on RAW 264.7 cells (viability of 95.5 ± 3.2% and 98.2 ± 1.3% for 2.5 and 1.3 μM, respectively) (Table 2). The NO inhibition showed a significant decrease (p < 0.05) when comparing the activity of chalcone 16 when added at different incubation times after LPS stimulation. Dexamethasone, a known inhibitor of iNOS enzyme production, revealed the same profile. These results suggest that the chalcone 16 mechanism of action is similar to dexamethasone, inhibiting iNOS enzyme production.

3. Discussion

In a tumor, along with the cancer cells, immune cells (as macrophages or T-cells) are also present in the tumor microenvironment (TME) [1,27]. The interplay of all the factors of the TME results in the development of the tumor. Macrophages are the majority of immune cells in the TME, and the amount and phenotype of tumor-associated macrophages (TAMs) are determinants for tumor development and progression [2,28]. Briefly, and excluding a variety of subpopulations, macrophages can adopt two opposite phenotypes: M1, or classically activated, with pro-inflammatory activity (producing TNF-α, IL-1β); and M2, or activated, with pro-tumoral characteristics (producing IL-10 and TGF-β) [2,28,29]. TAMs adopt an M2-similar phenotype [2,29]. Docetaxel and tasquinimod are drugs used in cancer chemoimmunotherapy that redirect macrophages to an antitumorigenic M1 profile [1,30,31].
mTORC1 plays an important role in macrophage polarization, mediated by its action at the level of metabolic and inflammatory signaling pathways [32,33]. The effect of mTORC1 on macrophages translates into macrophage polarization to M1, an increase in pro-inflammatory cytokine production by LPS-stimulated macrophages, and suppression of IL-4-induced polarization to M2 [32,33]. In the present study, chalcone 16 treatment induced an increased expression of mTOC1 in either unstimulated, IL-4-stimulated, and LPS-stimulated macrophages.
Usually, the core of the tumor is hypoxic due to anomalous vascularization that implies a deficient oxygen supply [27]. As a response, HIF-1α is activated and acts as a pro-angiogenic factor [28]. HIF influences immune cells, including those associated with tumors [27,28]. Considering TAMS, HIF-1α plays a determining role in the maturation and infiltration of macrophages, as well as in their polarization to an M2 phenotype [27,28]. Chalcone 16 significantly increased the expression of HIF-1α in unstimulated and LPS-stimulated macrophages. However, in IL-4-stimulated macrophages, chalcone 16 did not significantly change HIF-1α expression.
TNF-α and IL1-β are pro-inflammatory cytokines that increase macrophages’ cytotoxicity against tumor cells. They are also characteristic of an M1 macrophage profile [2]. Chalcone 16 significantly increased the expression of IL-1β, independently of macrophage stimulation.
TNF-α expression was only significantly increased for IL-4 and LPS-stimulated macrophages. These results suggest an augmentation in the pro-inflammatory profile of the macrophages, justified by an increase in M1-specific pro-inflammatory cytokines.
With respect to M2-characteristic cytokines, chalcone 16 only exhibited a change in the expression of IL-10 in IL-4-stimulated macrophages. IL-10 is an immunosuppressor cytokine that inhibits the antigen-presenting process and unviable T-cells to recognize and eliminate tumor cells [2,28]. IL-10 levels correlate directly with tumor development [2]. However, chalcone 16 treatment did not cause significant changes in its expression on IL-4 stimulated and unstimulated macrophages when analyzing TGF-β1, another M2-characteristic cytokine that promotes angiogenesis [28]. Only LPS-stimulated macrophages had a significant increase in TGF-β1 expression after chalcone 16 treatment.
NO has dual and controversial effects on cancer, which depend on the type of cancer, concentration, and/or time exposed. In cancers such as prostate, cervical, or melanoma, increased iNOS expression is correlated with a poorer prognosis, while for ovarian and non-small cell lung cancer, increased iNOS expression is considered a positive prognostic marker [34]. M1 macrophages produce high levels of iNOS [1,34]. However, NO production by human macrophages is hard to detect in vitro.
In this study, the RAW 264.7 murine macrophage cell line was used to evaluate the chalcone 16 effect on NO production. The results highlighted the ability of chalcone 16 to inhibit NO production and, by the inhibition profile, a probable inhibitory effect on iNOS expression [23]. These results can reinforce the most likely beneficial effect of compound 16 on macrophage immunomodulation.
Giving special focus to macrophages stimulated with IL-4, which leads to an M2-like macrophage phenotype, treatment with chalcone 16 increased the expression of mTORC1, which induces macrophages polarization to M1, and IL-1β and TNF-α (characteristic of M1). The expression of HIF-1α and TGF-β1, a fulcrum factor for angiogenesis and tumor progression, was not affected by chalcone 16 treatment. However, IL-10 expression increased significantly compared to control cells. Since TAMs are M2-like macrophages, and based on all the results obtained, it was possible to hypothesize that chalcone 16 can influence TAMs, redirecting them to macrophage a phenotype more M1-related. The effect of 16 is also relevant since the M1/M2 ratio in tumors is predictive of disease prognosis [1]. Thus, a higher amount of M2 macrophages is associated with a worse prognosis, while a higher percentage of M1 is associated with a better disease prognosis [1]. Chalcone 16 treatment of IL-4 stimulated macrophages also provoked a significant augmentation in the M1-specific pro-inflammatory cytokines IL1-β and TNF-α. These results suggest a potential chalcone 16 effect on macrophage polarization to an antitumor M1 profile.

4. Materials and Methods

4.1. Reagents

The acquisition of the reagents and media were as follows: Roswell Park Memorial Institute-1640 (RPMI-1640) medium with Ultraglutamine from Lonza (Verviers, Belgium); fetal bovine serum (FBS) from GE Health Care Life Sciences (GE Health Care, UT, USA), 2-mercaptoethanol from VWR International (Leuven, Belgium); Dulbecco’s Modified Eagle Medium/F-12 Nutrient Mixture (Ham) (DMEM/F-12; 1:1) from Gibco (Paisley, UK); phosphate-buffered saline (PBS) from Fisher Reagent (Geel, Belgium); dimethyl sulfoxide (DMSO) and phosphoric acid from Merk (Darmstadt, Germany); dimethylformamide (DMF) from Romil (Cambridge, UK), TripleXtractor and RNA Kit—Blood & Cultured Cells from GRiSP (Porto, Portugal), recombinant human IL-4 from R&D Systems (Minneapolis, USA). High-Capacity RNA-to-cDNA Kit and Master Mix from Applied Biosystems (Foster City, CA, USA) were used. When not specified, the reagents were from Sigma-Aldrich (ST. Louis, MO, USA).

4.2. Chalcone Derivatives 1–18

Chalcone derivatives 1–18 were synthesized and characterized by the Laboratory of Organic and Pharmaceutical Chemistry, Department of Chemical Sciences, Faculty of Pharmacy/CIIMAR research group as previously described [20]. The powered compound was dissolved in dimethyl sulfoxide (Acros Organics) and stored at −20 °C and diluted, before each assay, at the desired concentration, in the appropriate culture media.

4.3. Cell Lines and Cell Culture

PC-3 cell lines were obtained from the European Collection of Cell Cultures (ECCAC) and LNCaP from the American Type Cell Culture (ATCC). The other cell lines were kindly provided by Maria José Oliveira (THP-1 and HeLa), Institute for Investigation and Innovation in Health (i3S), Portugal; Henrique Almeida (Jurkat), i3S, Portugal; Maria São José Nascimento (RAW 264.7), Faculty of Pharmacy, University of Porto, Portugal. The complete culture medium for tumor cell lines was composed of RPMI-1640 supplemented with 10% of FBS and 1 μg/mL of gentamicin. In the THP-1 cell line, we supplemented all the culture media described with 2-mercaptoethanol (0.05 mM). RAW 264.7 cell line was cultured in DMEM/F-12 supplemented with FBS and gentamicin. All cell lines were incubated in a humidified atmosphere containing 5% CO2, at 37 °C.

4.4. Cytotoxic Assay (MTT) for Adherent Tumor Cell Lines

The MTT colorimetric assay was conducted based on the original procedure proposed by Mosmann [35], with modifications [24]. Cell lines (HeLa, PC-3, or LNCaP) were seeded at a concentration of 1.5 × 104 cells/well (96-flat-bottom well culture plate) and incubated for cell adherence (24 h) [36]. After removing the supernatants, the cells were treated with chalcone derivatives, at the desired concentrations, for 48 h [36]. Additionally, included non-treated and doxorubicin-treated cells in the assays as controls [26].
Once the incubation period was over, we washed the cells, and MTT (0.2 mg/mL) was placed in contact with the cells for 4 h at 37 °C in a CO2 incubator [21]. The MTT formazan product was solubilized with DMSO while shaking for 10 min, and absorbance was measured 545/630 nm (STAT FAX 3200) [24]. The formula used to calculate the cytotoxicity was as follows:
cellular metabolic viability inhibition (% of control) = 100 − (abs sample/abs control × 100).

4.5. Cytotoxic Assay (MTT) for Non-Adherent Tumor Cell Lines

Jurkat cells (50 μL; 1.5 × 104 cells/well) were placed in 96-well plates, and after a 24 h incubation [36], the desired test concentrations of the compounds (50 μL) were added. Several controls were included in the experiments consisting of untreated cells, doxorubicin-treated cells, compound blank, and medium blank. A new 48 h period of incubation was carried out [36], after which an MTT solution 0.2 mg/mL per well was added [21] and left for 4 h in a CO2 incubator at 37 °C. After MTT reduction by viable cells, 50 μL of an SDS solution (20% SDS in DMF/H2O (1:1)) was added to the wells to dissolve formazan [21]. After reading absorbance, as already stated, cytotoxicity was calculated as follows:
Inhibition of metabolic viability (% of control) = 100 − [(abs sample-abs blank)/(abs control − abs negative control) × 100]

4.6. THP-1 Macrophage-Phenotype Differentiation

The human leukemic monocyte cell line THP-1 (1 × 106 cell/mL) was differentiated into macrophages by phorbol 12-myristate 13-acetate (PMA, 0.1 μg/mL) treatment for 72 h [37]. After washing and an additional incubation in the culture medium for 24 h [37,38], an M0 phenotype was obtained. The effect of chalcone 16 on the various macrophage phenotypes was achieved by their treatment for 24 h [39], in three different stimulation conditions: unstimulated, LPS-stimulated (1 μg/mL) [39], and IL-4 stimulated (20 ng/mL) [37]. LPS or IL-4 stimulation was performed simultaneously to chalcone 16 treatment. The control used were non-treated macrophages.

4.7. Assay for Quantification of mRNA Expression

For mRNA cytokines expression quantification, THP-1 derived macrophages (M0) were obtained as stated in Section 4.6. The influence of chalcone 16 with cytokine mRNA expression was studied exposing the macrophages to different concentrations of compound 16 for 6 h [37] in three different conditions described above: un-stimulated, LPS-stimulated and IL-4 stimulated. Non-treated macrophages, exposed or not to LPS or IL-4 stimulation, were also used. After incubation, the media were removed and cells were washed. TripleXtractor reagent (GRISP) was used for mRNA isolation, stored at −80 °C until use. The RNA fraction was separated, samples were purified (GRS Total RNA Kit—Blood & Cultured Cells commercial kit) and RNA concentration and purity were assessed using a NanoDrop Lite spectrophotometer (Thermo Scientific®, Waltham, MA, USA). For cDNA synthesis, mRNA samples were used (High-Capacity RNA-to-cDNA Kit; Thermo Fisher Scientific). Reactions were carried out in the StepOneTM Plus PCR Real-Time PCR instrument, with 1x Master Mix, 1x probes (TaqMan® Gene Expression assays mTOR, Hs00234508_m1; HIF1α, Hs00153153_m1; TGF-β1, Hs00998133_m1; IL-1β, Hs01555410_m1; IL-10, Hs00961622_m1, and TNF-α, Hs02621508_s1; Applied Biosystems, Foster City, CA, USA). The housekeeping gene used was B2M (TaqMan® Hs99999907_m1; Applied Biosystems, Foster City, CA, USA), which served as an endogenous control to normalize the results. The analysis of the results was conducted utilizing the StepOneTM Software v2.2 (Applied Biosystems, Foster City, CA, USA) with the same baseline and threshold set for each plate, to generate quantification cycle values (Cts) for all the mRNA targets in each sample.

4.8. Nitric Oxide Production Assay

RAW 264.7 cells were placed in a 96-well culture plate (1 × 106 cell/mL; 200 µL) and incubated for 2 h to allow the cells’ adhesion [22]. The culture media was then discarded, and an LPS (1.5 µg/mL) and chalcone solution were added at equal volume and added together (0 h). Chalcone treatment was also started 6 or 14 h after stimulation with LPS to evaluate the effect of compound 16 on iNOS expression/activity. Cells were incubated for a total of 24 h after stimulation, and after that period, 100 µL of the culture media were placed into a new 96 flat-bottom well plate, and 100 µL of Griess Reagent (1:1 solution of 1% w/v sulphanilamide solution in phosphoric acid (5% v/v) and naphtylethylenediamide (0.1%) in deionized water) were added to every well. The reaction occurred for 10 min protected from light and at room temperature [22]. The optical density was measured (545/630 nm; STAT FAX 3200), and the effect on nitrite production was calculated as follows:
Inhibition of NO production (% of control) = 100 − [(abs sample-abs blank)/(abs control − abs negative control) × 100]

4.9. Statistical Analysis

IBM SPSS Statistics 26.0 for Windows was used. The results were displayed as mean ± SEM. To obtain meaningful results, media ± 2SD was used to perform the statistical evaluation of the effect of chalcone derivatives against the metabolic viability of tumor cell lines. The normality of the distribution of the results was confirmed by the Shapiro–Wilk test of normality, and the homogeneity of variance assumption was checked using Levene’s test. One-way Anova was conducted for the experimental cytotoxicity analysis with Bonferroni’s correction post hoc test. The differences in cytokine mRNA expression were analyzed using the Student’s t-test. Statistical significance was considered for p < 0.05.

Author Contributions

Conceptualization: M.P., F.C., H.C. and R.M.; experimental: F.C., B.H., R.M., J.S., F.D., A.L.T. and J.F.-S.; chemistry: H.C. and M.P.; statistics: R.M., B.H., A.L.T. and J.S.; funding acquisition, M.P.; writing, reviewing and editing: all authors. All authors approved the final version of the work. All authors have read and agreed to the published version of the manuscript.

Funding

This work was sponsored by the national funds of FCT/MCTES—Foundation for Science and Technology I.P. from the Ministry of Science, Technology, and Higher Education (PIDDAC) and the European Regional Development Fund (ERDF) by the COMPETE—Programa Operacional Factores de Competitividade (POFC) under the Strategic Funding UID/Multi/04546/2019 and UIDB/04423/2020, UIDP/04423/2020 (Group of Natural Products and Medicinal Chemistry-CIIMAR) and project PTDC/MAR-BIO/4694/2014 (reference POCI-01-0145-FEDER-016790; Project 3599—Promover a Produção Científica e Desenvolvimento Tecnológico e a Constituição de Redes Temáticas (3599-PPCDT)) under the program PT2020 and the Research Center of the Portuguese Oncology Institute of Porto (project no. PI86-CI-IPOP-66-2019). FD has a junior researcher contract funded by UIDP/00776/2020-4B.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are not available from authors.

References

  1. Yan, S.; Wan, G. Tumor-associated macrophages in immunotherapy. FEBS J. 2021, 288, 6174–6186. [Google Scholar] [CrossRef] [PubMed]
  2. Zhou, J.; Tang, Z.; Gao, S.; Li, C.; Feng, Y.; Zhou, X. Tumor-Associated Macrophages: Recent Insights and Therapies. Front. Oncol. 2020, 10, 188. [Google Scholar] [CrossRef] [PubMed]
  3. Horta, B.; Pereira, T.; Medeiros, R.; Cerqueira, F. Cervical Cancer Outcome and Tumor-Associated Macrophages. Res. Evidence. Immuno. 2022, 2, 460–468. [Google Scholar] [CrossRef]
  4. De Nola, R.; Loizzi, V.; Cicinelli, E.; Cormio, G. Dynamic crosstalk within the tumor microenvironment of uterine cervical carcinoma: Baseline network, iatrogenic alterations, and translational implications. Crit. Rev. Oncol. Hematol. 2021, 162, 103343. [Google Scholar] [CrossRef]
  5. Sousa, S.; Brion, R.; Lintunen, M.; Kronqvist, P.; Sandholm, J.; Mönkkönen, J.; Kellokumpu-Lehtinen, P.L.; Lauttia, S.; Tynninen, O.; Joensuu, H.; et al. Human breast cancer cells educate macrophages toward the M2 activation status. Breast Cancer Res. BCR 2015, 17, 101. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  6. Lin, Y.; Xu, J.; Lan, H. Tumor-associated macrophages in tumor metastasis: Biological roles and clinical therapeutic applications. J. Hematol. Oncol. 2019, 12, 76. [Google Scholar] [CrossRef]
  7. Huang, Q.; Liang, X.; Ren, T.; Huang, Y.; Zhang, H.; Yu, Y.; Chen, C.; Wang, W.; Niu, J.; Lou, J.; et al. The role of tumor-associated macrophages in osteosarcoma progression—Therapeutic implications. Cell. Oncol. 2021, 44, 525–539. [Google Scholar] [CrossRef] [PubMed]
  8. Chen, X.J.; Deng, Y.R.; Wang, Z.C.; Wei, W.F.; Zhou, C.F.; Zhang, Y.M.; Yan, R.M.; Liang, L.J.; Zhong, M.; Liang, L.; et al. Hypoxia-induced ZEB1 promotes cervical cancer progression via CCL8-dependent tumour-associated macrophage recruitment. Cell Death Dis. 2019, 10, 508. [Google Scholar] [CrossRef] [Green Version]
  9. Werno, C.; Menrad, H.; Weigert, A.; Dehne, N.; Goerdt, S.; Schledzewski, K.; Kzhyshkowska, J.; Brüne, B. Knockout of HIF-1α in tumor-associated macrophages enhances M2 polarization and attenuates their pro-angiogenic responses. Carcinogenesis 2010, 31, 1863–1872. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  10. Jeong, H.; Kim, S.; Hong, B.J.; Lee, C.J.; Kim, Y.E.; Bok, S.; Oh, J.M.; Gwak, S.H.; Yoo, M.Y.; Lee, M.S.; et al. Tumor-Associated Macrophages Enhance Tumor Hypoxia and Aerobic Glycolysis. Cancer Res. 2019, 79, 795–806. [Google Scholar] [CrossRef] [Green Version]
  11. Fang, W.-Y.; Ravindar, L.; Rakesh, K.; Manukumar, H.; Shantharam, C.; Alharbi, N.S.; Qin, H.-L. Synthetic approaches and pharmaceutical applications of chloro-containing molecules for drug discovery: A critical review. Eur. J. Med. Chem. 2019, 173, 117–153. [Google Scholar] [CrossRef]
  12. Zhao, C.; Rakesh, K.; Ravidar, L.; Fang, W.-Y.; Qin, H.-L. Pharmaceutical and medicinal significance of sulfur (SVI)-containing motifs for drug discovery: A critical review. Eur. J. Med. Chem. 2018, 162, 679–734. [Google Scholar] [CrossRef]
  13. Zhuang, C.; Zhang, W.; Sheng, C.; Zhang, W.; Xing, C.; Miao, Z. Chalcone: A privileged structure in medicinal chemistry. Chem. Rev. 2017, 117, 7762–7810. [Google Scholar] [CrossRef] [PubMed]
  14. Bronikowska, J.; Kłósek, M.; Janeczko, T.; Kostrzewa-Susłow, E.; Czuba, Z.P. The modulating effect of methoxy-derivatives of 2’-hydroxychalcones on the release of IL-8, MIF, VCAM-1 and ICAM-1 by colon cancer cells. Biomed. Pharmacother. 2022, 145, 112428. [Google Scholar] [CrossRef] [PubMed]
  15. Fonseca, J.; Marques, S.; Silva, P.; Brandão, P.; Cidade, H.; Pinto, M.; Bousbaa, H. Prenylated chalcone 2 acts as an antimitotic agent and enhances the chemosensitivity of tumor cells to paclitaxel. Molecules 2016, 21, 982. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Leão, M.; Soares, J.; Gomes, S.; Raimundo, L.; Ramos, H.; Bessa, C.; Queiroz, G.; Domingos, S.; Pinto, M.; Inga, A.; et al. Enhanced cytotoxicity of prenylated chalcone against tumour cells via disruption of the p53–MDM2 interaction. Life Sci. 2015, 142, 60–65. [Google Scholar] [CrossRef] [PubMed]
  17. Masawang, K.; Pedro, M.; Cidade, H.; Reis, R.M.; Neves, M.P.; Corrêa, A.G.; Sudprasert, W.; Bousbaa, H.; Pinto, M.M. Evaluation of 2′,4′-dihydroxy-3,4,5-trimethoxychalcone as antimitotic agent that induces mitotic catastrophe in MCF-7 breast cancer cells. Toxicol. Lett. 2014, 229, 393–401. [Google Scholar] [CrossRef] [PubMed]
  18. Neves, M.P.; Cravo, S.; Lima, R.T.; Vasconcelos, M.H.; Nascimento, M.S.J.; Silva, A.S.; Pinto, M.; Cidade, H.; Corrêa, A.G. Solid-phase synthesis of 2’-hydroxychalcones. Effects on cell growth inhibition, cell cycle and apoptosis of human tumor cell lines. Bioorganic Med. Chem. 2012, 20, 25–33. [Google Scholar] [CrossRef]
  19. Neves, M.P.; Lima, R.T.; Choosang, K.; Pakkong, P.; Nascimento, M.S.J.; Vasconcelos, M.H.; Pinto, M.; Silva, A.M.S.; Cidade, H. Synthesis of a natural chalcone and its prenyl analogues—Evaluation of tumor cell growth inhibitory activity and effects on cell cycle and apoptosis. Chem. Biodivers 2012, 9, 1133–1143. [Google Scholar] [CrossRef]
  20. Pinto, P.; Machado, C.M.; Moreira, J.; Almeida, J.D.; Silva, P.M.; Henriques, A.C.; Soares, J.X.; Salvador, J.A.; Afonso, C.; Pinto, M.; et al. Chalcone derivatives targeting mitosis: Synthesis, evaluation of antitumor activity and lipophilicity. Eur. J. Med. Chem. 2019, 184, 111752. [Google Scholar] [CrossRef]
  21. Pedro, M.; Cerqueira, F.; Sousa, M.E.; MSJ; Pinto, M. Xanthones as inhibitors of growth of human cancer cell lines and Their effects on the proliferation of human lymphocytes In Vitro. Bioorg. Med. Chem. 2002, 10, 3725–3730. [Google Scholar] [CrossRef] [PubMed]
  22. Teixeira, M.; Cerqueira, F.; Barbosa, C.; Nascimento, M.S.J.; Pinto, M.M.M. Improvement of the inhibitory effect of xanthones on NO production by encapsulation in PLGA nanocapsules. J. Drug Target. 2005, 13, 129–135. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  23. Silva, V.; Cerqueira, F.; Nazareth, N.; Medeiros, R.; Sarmento, A.; Sousa, E.; Pinto, M. 1,2-Dihydroxyxanthone: Effect on A375-C5 Melanoma Cell Growth Associated with Interference with THP-1 Human Macrophage Activity. Pharmaceuticals 2019, 12, 85. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Medeiros, R.; Horta, B.; Freitas-Silva, J.; Silva, J.; Dias, F.; Sousa, E.; Pinto, M.; Cerqueira, F. Effect of 1-Carbaldehyde-3,4-dimethoxyxanthone on Prostate and HPV-18 Positive Cervical Cancer Cell Lines and on Human THP-1 Macrophages. Molecules 2021, 26, 3721. [Google Scholar] [CrossRef]
  25. Orzechowska, E.J.; Girstun, A.; Staron, K.; Trzcinska-Danielewicz, J. Synergy of BID with doxorubicin in the killing of cancer cells. Oncol. Rep. 2015, 33, 2143–2150. [Google Scholar] [CrossRef]
  26. Henslee, E.A.; Torcal Serrano, R.M.; Labeed, F.H.; Jabr, R.I.; Fry, C.H.; Hughes, M.P.; Hoettges, K.F. Accurate quantification of apoptosis progression and toxicity using a dielectrophoretic approach. Analyst 2016, 141, 6408–6415. [Google Scholar] [CrossRef] [Green Version]
  27. Petrova, V.; Annicchiarico-Petruzzelli, M.; Melino, G.; Amelio, I. The hypoxic tumour microenvironment. Oncogenesis 2018, 7, 10. [Google Scholar] [CrossRef] [Green Version]
  28. Fu, L.Q.; Du, W.L.; Cai, M.H.; Yao, J.Y.; Zhao, Y.Y.; Mou, X.Z. The roles of tumor-associated macrophages in tumor angiogenesis and metastasis. Cell Immunol. 2020, 353, 104119. [Google Scholar] [CrossRef]
  29. Liu, Y.; Li, L.; Li, Y.; Zhao, X. Research Progress on Tumor-Associated Macrophages and Inflammation in Cervical Cancer. Biomed. Res. Int. 2020, 2020, 6842963. [Google Scholar] [CrossRef]
  30. Zhao, Y.; Zheng, Y.; Zhu, Y.; Li, H.; Zhu, H.; Liu, T. Docetaxel-loaded M1 macrophage-derived exosomes for a safe and efficient chemoimmunotherapy of breast cancer. J. Nanobiotechnology 2022, 20, 359. [Google Scholar] [CrossRef]
  31. Williamson, S.C.; Hartley, A.E.; Heer, R. A review of tasquinimod in the treatment of advanced prostate cancer. Drug Des. Devel. Ther. 2013, 7, 167–174. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  32. Vergadi, E.; Ieronymaki, E.; Lyroni, K.; Vaporidi, K.; Tsatsanis, C. Akt Signaling Pathway in Macrophage Activation and M1/M2 Polarization. J. Immunol. 2017, 198, 1006–1014. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Byles, V.; Covarrubias, A.J.; Ben-Sahra, I.; Lamming, D.W.; Sabatini, D.M.; Manning, B.D.; Horng, T. The TSC-mTOR pathway regulates macrophage polarization. Nat. Commun. 2013, 4, 2834. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  34. Kashfi, K.; Kannikal, J.; Nath, N. Macrophage Reprogramming and Cancer Therapeutics: Role of iNOS-Derived NO. Cells 2021, 10, 3194. [Google Scholar] [CrossRef] [PubMed]
  35. Mosmann, T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J. Immunol. Methods 1983, 65, 55–63. [Google Scholar] [CrossRef]
  36. Gabrani, R.; Jain, R.; Sharma, A.; Sarethy, I.P.; Dang, S.; Gupta, S. Antiproliferative Effect of Solanum nigrum on Human Leukemic Cell Lines. Indian J. Pharm. Sci. 2012, 74, 451–453. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  37. Chanput, W.; Mes, J.J.; Wichers, H.J. THP-1 cell line: An in vitro cell model for immune modulation approach. Int. Immunopharmacol. 2014, 23, 37–45. [Google Scholar] [CrossRef]
  38. Chanput, W.; Reitsma, M.; Kleinjans, L.; Mes, J.J.; Savelkoul, H.F.J.; Wichers, H.J. β-Glucans are involved in immune-modulation of THP-1 macrophages. Mol. Nutr. Food Res. 2012, 56, 822–833. [Google Scholar] [CrossRef]
  39. He, X.; Shu, J.; Xu, L.; Lu, C.; Lu, A. Inhibitory Effect of Astragalus Polysaccharides on Lipopolysaccharide-Induced TNF- and IL-1β Production in THP-1 Cells. Molecules 2012, 17, 3155–3164. [Google Scholar] [CrossRef]
Molecules 28 02159 g001 550
Figure 1. Small library of chalcone derivatives 1–18 investigated in this study.
Figure 1. Small library of chalcone derivatives 1–18 investigated in this study.
Molecules 28 02159 g001
Molecules 28 02159 g002 550
Figure 2. Effect of chalcone 16 on THP-1 macrophage mRNA expression of mTORC1, HIF-1α and cytokines. (A) Unstimulated macrophages; (B) IL-4-stimulated macrophages; (C) LPS-stimulated macrophages. (−∆Cq) of mTOR, HIF-1α, IL-1β, TNF-α, TGF-β1, and IL-10 by THP-1 macrophages. Values represent mean ± SEM (n = 3). * p < 0.05, ** p < 0.001.
Figure 2. Effect of chalcone 16 on THP-1 macrophage mRNA expression of mTORC1, HIF-1α and cytokines. (A) Unstimulated macrophages; (B) IL-4-stimulated macrophages; (C) LPS-stimulated macrophages. (−∆Cq) of mTOR, HIF-1α, IL-1β, TNF-α, TGF-β1, and IL-10 by THP-1 macrophages. Values represent mean ± SEM (n = 3). * p < 0.05, ** p < 0.001.
Molecules 28 02159 g002
Table
Table 1. Effect of chalcones 1–18 on the metabolic viability of tumor cell lines expressed as percentage of cellular inhibition (%) compared to control.
Table 1. Effect of chalcones 1–18 on the metabolic viability of tumor cell lines expressed as percentage of cellular inhibition (%) compared to control.
CompoundConcentration (µM)Inhibition of Metabolic Viability (% of Control)
HeLaLNCaPPC-3Jurkat
1539.1 ± 0.116.0 ± 5.423.5 ± 7.258.6 ± 13.6
1033.5 ± 5.633.5 ± 6.933.6 ± 10.790.8 ± 4.2
2043.6 ± 5.739.1 ± 1.147.6 ± 10.5T.I.
2524.0 ± 12.524.3 ± 1.931.2 ± 4.36.8 ± 3.9
1022.4 ± 7.539.2 ± 5.341.9 ± 4.020.8 ± 1.6
2029.5 ± 4.245.3 ± 4.145.4 ± 8.781.8 ± 7.3
3529.4 ± 10.423.1 ± 8.533.4 ± 5.855.3 ± 15.8
1020.3 ± 5.740.9 ± 5.932.0 ± 5.087.8 ± 5.6
2061.1 ± 10.174.4 ± 0.884.6 ± 2.7T.I.
4521.9 ± 3.830.1 ± 4.933.3 ± 4.729.1 ± 11.5
1029.3 ± 9.740.5 ± 2.727.0 ± 2.866.1 ± 10.9
2026.8 ± 4.956.3 ± 2.452.3 ± 3.991.6 ± 5.9
5531.3 ± 4.731.2 ± 2.128.4 ± 9.260.2 ± 9.5
1033.7 ± 3.449.1 ± 2.450.5 ± 4.057.1 ± 2.4
2045.8 ± 8.658.5 ± 3.763.6 ± 3.788.9 ± 4.0
6525.0 ± 7.730.4 ± 8.526.6 ± 5.435.6 ± 1.1
1052.0 ± 7.837.0 ± 8.332.2 ± 3.130.7 ± 7.6
2074.8 ± 7.563.4 ± 7.178.7 ± 2.379.2 ± 3.7
7525.7 ± 4.023.6 ± 0.819.6 ± 5.147.0 ± 8.0
1045.8 ± 5.934.4 ± 5.038.5 ± 5.789.0 ± 7.4
2060.2 ± 14.578.4 ± 2.662.2 ± 7.5T.I.
8511.4 ± 3.524.4 ± 1.518.3 ± 0.628.0 ± 8.2
1035.7 ± 7.128.4 ± 8.233.6 ± 9.145.7 ± 8.4
2051.6 ± 6.047.8 ± 5.145.5 ± 6.391.6 ± 6.9
9526.4 ± 7.123.5 ± 4.832.0 ± 1.032.2 ± 9.8
1034.4 ± 3.036.9 ± 2.133.4 ± 10.770.6 ± 13.2
2055.8 ± 7.151.5 ± 7.556.3 ± 11.2T.I.
10514.0 ± 5.529.4 ± 6.047.0 ± 5.545.1 ± 10.0
1056.4 ± 8.641.4 ± 1.433.9 ± 11.693.6 ± 6.4
2070.2 ± 7.960.0 ± 5.984.7 ± 2.9T.I.
11529.2 ± 8.928.6 ± 5.018.4 ± 1.918.1 ± 2.2
1031.9 ± 8.931.5 ± 5.030.0 ± 2.240.5 ± 2.5
2051.5 ± 3.349.9 ± 2.160.4 ± 3.499.0 ± 1.6
12535.8 ± 4.427.4 ± 6.015.4 ± 3.810.8 ± 2.0
1042.6 ± 8.339.2 ± 4.830.0 ± 2.747.4 ± 11.7
2085.8 ± 3.769.1 ± 7.980.9 ± 5.1T.I.
13546.8 ± 3.737.9 ± 12.222.1 ± 6.648.9 ± 9.2
1040.5 ± 13.558.9 ± 11.244.5 ± 2.577.7 ± 10.9
2068.0 ± 10.675.1 ± 6.273.1 ± 2.8T.I.
14529.7 ± 11.618.6 ± 2.216.1 ± 1.157.0 ± 11.1
1063.7 ± 8.643.4 ± 9.137.9 ± 11.897.0 ± 6.7
2074.5 ± 9.169.5 ± 5.357.4 ± 15.4T.I.
15533.3 ± 6.224.3 ± 6.417.2 ± 4.414.6 ± 6.5
1024.5 ± 11.244.0 ± 7.137.7 ± 2.413.0 ± 2.3
2033.6 ± 2.439.8 ± 10.137.0 ± 2.377.3 ± 6.3
16537.3 ± 12.830.6 ± 4.440.8 ± 4.869.4 ± 13.7
1057.8 ± 8.952.4 ± 6.964.7 ± 1.881.6 ± 15.1
2078.3 ± 3.076.9 ± 4.687.6 ± 3.7T.I.
17519.5 ± 6.821.3 ± 6.719.6 ± 3.032.1 ± 10.2
1030.3 ± 5.229.3 ± 3.028.0 ± 1.444.2 ± 8.6
2047.0 ± 8.837.0 ± 3.938.9 ± 3.6T.I.
1859.8 ± 3.223.0 ± 9.328.7 ± 1.534.6 ± 11.3
1021.5 ± 14.833.0 ± 1.243.1 ± 2.984.1 ± 13.2
2026.8 ± 3.945.1 ± 15.359.9 ± 7.2T.I.
Doxorubicin582.8 ± 2.562.5 ± 1.873.2 ± 2.8111.4 ± 2.8
Results are expressed as mean ± SEM (n ≥ 3). TI—total inhibition. Doxorubicin was used as a positive control.
Table
Table 2. Effect of chalcone 16 on nitric oxide production by RAW 264.7, expressed as a percentage of inhibition of NO production.
Table 2. Effect of chalcone 16 on nitric oxide production by RAW 264.7, expressed as a percentage of inhibition of NO production.
CompoundConcentration (µM)Time
0 h6 h14 h
Chalcone 161.352.4 ± 10.119.0 ± 0.7 *11.0 ± 1.1 *
2.566.4 ± 3.624.8 ± 4.0 *12.3 ± 2.0 *
Dexamethasone558.1 ± 7.831.6 ± 1.8 *5.9 ± 3.2 *
Results are expressed as mean ± SEM; n ≥ 3. Dexamethasone was used as a positive control. * p < 0.05.
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Horta, B.; Freitas-Silva, J.; Silva, J.; Dias, F.; Teixeira, A.L.; Medeiros, R.; Cidade, H.; Pinto, M.; Cerqueira, F. Antitumor Effect of Chalcone Derivatives against Human Prostate (LNCaP and PC-3), Cervix HPV-Positive (HeLa) and Lymphocyte (Jurkat) Cell Lines and Their Effect on Macrophage Functions. Molecules 2023, 28, 2159. https://doi.org/10.3390/molecules28052159

AMA Style

Horta B, Freitas-Silva J, Silva J, Dias F, Teixeira AL, Medeiros R, Cidade H, Pinto M, Cerqueira F. Antitumor Effect of Chalcone Derivatives against Human Prostate (LNCaP and PC-3), Cervix HPV-Positive (HeLa) and Lymphocyte (Jurkat) Cell Lines and Their Effect on Macrophage Functions. Molecules. 2023; 28(5):2159. https://doi.org/10.3390/molecules28052159

Chicago/Turabian Style

Horta, Bruno, Joana Freitas-Silva, Jani Silva, Francisca Dias, Ana Luísa Teixeira, Rui Medeiros, Honorina Cidade, Madalena Pinto, and Fátima Cerqueira. 2023. "Antitumor Effect of Chalcone Derivatives against Human Prostate (LNCaP and PC-3), Cervix HPV-Positive (HeLa) and Lymphocyte (Jurkat) Cell Lines and Their Effect on Macrophage Functions" Molecules 28, no. 5: 2159. https://doi.org/10.3390/molecules28052159

APA Style

Horta, B., Freitas-Silva, J., Silva, J., Dias, F., Teixeira, A. L., Medeiros, R., Cidade, H., Pinto, M., & Cerqueira, F. (2023). Antitumor Effect of Chalcone Derivatives against Human Prostate (LNCaP and PC-3), Cervix HPV-Positive (HeLa) and Lymphocyte (Jurkat) Cell Lines and Their Effect on Macrophage Functions. Molecules, 28(5), 2159. https://doi.org/10.3390/molecules28052159

Article Metrics

Back to TopTop