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

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.


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 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 tumorassociated 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].

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]. Results are expressed as mean ± SEM (n ≥ 3). TI-total inhibition. Doxorubicin was used as a positive control.
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. 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).
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.
Independent of the stimulation, the treatment of macrophages with chalcone 16 significantly increases the expression of mTORC1.  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 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 foldchange = 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.

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. Results are expressed as mean ± SEM; n ≥ 3. Dexamethasone was used as a positive control. * p < 0.05.

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 LPSstimulated 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 M2characteristic 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.

Reagents
The acquisition of the reagents and media were as follows:

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.

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 × 10 4 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 CO 2 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).

Cytotoxic Assay (MTT) for Non-Adherent Tumor Cell Lines
Jurkat cells (50 µL; 1.5 × 10 4 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, doxorubicintreated 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 CO 2 incubator at 37 • C. After MTT reduction by viable cells, 50 µL of an SDS solution (20% SDS in DMF/H 2 O (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]

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.

Nitric Oxide Production Assay
RAW 264.7 cells were placed in a 96-well culture plate (1 × 10 6 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]

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.

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are not available from authors.