Cacalol Acetate as Anticancer Agent: Anti-proliferative, Pro-apoptotic, Cytostatic and Anti-migratory Effects

1. Introduction

According to the World Health Organization (WHO), cervical cancer (CC) is the worldwide fifth most frequently occurring cancer [1] and is one of the leading causes of cancer death among women [2]. Although CC is now relatively uncommon in developed countries as United States, it continues to be the first or second leading cause of cancer-related death among women in many low and middle-income countries, with Latin America and the Caribbean region accounting for almost 12% of the world’s CC deaths [3]; actually, 85% of deaths derived from CC occur in underdeveloped or developing countries, and the death rate is 18 times higher in low-income and middle-income countries compared with wealthier countries [4]. Mexico has the second place in CC only below the African continent [5].

Although CC is a preventable disease, many women present with invasive disease requiring radical surgery (early stage disease) and/or combined radiation therapy and chemotherapy (locally advanced disease) [6], but nevertheless these strategies are not successful interventions, and the incomplete removal of the cancer and the negative effect on normal cells could not be excluded [7]. Fortunately, a large amount of information dealing with clinical aspects of cancer chemotherapy are generated, including the finding and application of natural origin drugs.

According to those background, there are two principal requirements on chemotherapeutic antitumor based research, to achieve an efficient antitumoral agent that satisfy a broad population specter: to find a more efficient natural antitumoral agent without adverse effect and a more profitable source of this kind of agent. In this way, we previously reported that indole-3-carbinol (I3C), a non-carcinogenic agonist compounds of the aryl hydrocarbon receptor (AhR), promotes the activation of AhR and decreases cell proliferation, possibly through UBE2L3 (also known as UBCH7, UbcH7, and E2 Ubiquitin-Conjugating Enzyme L3) mRNA induction, which would result in the ubiquitination of HPV (Human Papilloma Virus) E7 protein [8]. However, it is still unknown whether I3C affects other hallmarks of cancer.

On the other hand, another bioactive molecule is cacalol (C), a phytochemical compound with anti-inflammatory and antioxidant properties [9,10]. It has a strong anti-proliferative effect against breast cancer cells, inducing apoptosis by activating a pro-apoptotic pathway [11]. Furthermore, its derivative compound Cacalol Acetate (CA) (see Scheme 1), has shown anti-inflammatory properties [9] but its antitumoral activity is not well known yet.

Although there is no evidence about the antitumoral properties of cacalol acetate in CC, this compound had shown potential utility as a chemopreventive and chemotherapeutic agent against cancer biological models. Particularly, in this study we investigated the anti-proliferative, pro-apoptotic and anti-migratory properties of CA and I3C in a cervical cancer cell line.

2. Materials and Methods

2.1. Cell Cultures

Cervical cancer cell lines, HeLa (positive to HPV-18) was obtained from the American Type Culture Collection (ATCC Rockville, MD, USA). For all assays, HeLa cells were culture in RPMI-1640 media (GIBCO, Carlsbad, CA. USA) and supplemented with 5% newborn calf serum (NCS, GIBCO, USA), L-glutamine, red phenol, and benzylpenicillin. Cultures were maintained in an incubator (Nauire, US) with a humidified atmosphere at 5% CO₂ and 37 °C. All cell-based assays were performed using culture cells in the exponential growth phase.

2.2. Tested Compounds

CA were solubilized in 100 µl dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA) and 900 µl of RPMI-1640 medium resulting in a final concentration of 1 µg/ µl. I3C (Sigma-Aldrich, St. Louis, MO, USA; purity ≥ 96%) was solubilized in DMSO and using in at final concentration of 150 µM.

2.3. Cell Proliferation Assays

HeLa cells (6 × 10³ cell/well) were cultivated in a 96-well tissue culture plate (Corning, USA) with a 100 µl of RPMI-1640 medium and growth for 24 h at 37 °C and 5% CO₂. Then, CA was added at several concentrations (from 20 µg/ml to 29 µg/ml) and incubated as mentioned above. I3C was added at final concentration of 150 µM. As controls, we added 5 µg/ml of DMSO (vehicle) in cell culture sample and we included one cell culture without treatment (control). In addition, HeLa cells were treated with 1 µM of beta-naphthoflavone (BNF), a agonist synthetic ligand of AhR with anticancer activity against mammary carcinoma cells [13] and cervical cancer cells [14] as a control. After 24 h of incubation with CA, we determine the antiproliferative activity (IC₅₀) by crystal violet staining as previously reported [15,16]. Finally, the cell count was performed spectrophotometrically at 590 nm (Awareness Technology INC, Chromate 4300, USA). Data were analyzed in a dose-response curve to estimate the concentration at which 50% of the cell population decreases (IC₅₀).

2.4. Determination of Apoptosis by Evaluation of Active Caspase-3

HeLa cells (6 × 10³ cell/well) were incubated for 24 h with 102.72 μM of CA and I3C (150 µM). As controls, cells were incubated with 5 µg/ml DMSO (vehicle), and 1 mg/ml colchicine (positive apoptotic cell death control) and beta-naphthoflavone (BNF) (1 µM). For immunodetection of active caspase-3 we followed a reported protocol [16]. Briefly, cells were permeabilized with Triton X-100 (1%) for 20 minutes, after which cells were washed with phosphate buffered saline (PBS) pH 7.3. Then, cells were incubated with rabbit anti-human active caspase-3 polyclonal antibody (Sigma-Aldrich, USA) diluted in PBS (1:1000) for 18 h at 4 °C. Following cells were washed and incubated with goat anti-rabbit IgG secondary antibody coupled to fluorescein-5-isothiocyanate (FITC) diluted 1:1000 in PBS at room temperature for 2 h. Then samples were stained with 4′6-diamidino-2-phenylindole (DAPI) and analyzed under epifluorescence microscopy (Eclipse E600, Nikon, USA) and phase contrast microscopy (Eclipse TS2R-FL, Nikon, Japan). We use a DXMI200F digital camera (Nikon, Melville, NY, USA) for recorded images.

2.5. Determination of Autophagic (p62 and LC3B) Biomarkers by Western Blot Analysis

HeLa cells treated with CA (102.72 μM), I3C (150 µM), DMSO (5 µg/ml, vehicle), BNF (1 µM) and untreated cells (control) were grown for 24 h and were subjected to lysis using RIPA buffer (Santa Cruz Biotechnology, Inc., sc-24948) supplemented with 0.1% protease inhibitors (Complete Protease Inhibitor Cocktail, Roche, catalog number 11697498008). The lysates were then incubated at 4 °C for 30 minutes, followed by centrifugation at 13,000× g at 4 °C for 25 minutes. The soluble protein concentrations in the cell lysates were measured spectrophotometrically at 280 nm using the EPOCH Microplate Spectrophotometer (Bio Tek). Equal amounts of protein from each sample were combined with 5X Laemmli sample buffer (10% SDS, 50% glycerol, 0.02% bromophenol blue, and 0.3125 M Tris HCl, pH 6.8), supplemented with β-mercaptoethanol, and boiled for 10 minutes. Subsequently, 50 µg of protein from each sample were separated on 12% SDS-PAGE gels using a vertical electrophoresis system (Mini Trans-Blot^® Cell, Bio-Rad, catalog number 1703810). The separated proteins were then transferred onto 0.45 µm polyvinylidene difluoride (PVDF) membranes (Thermo Scientific, catalog number 88518) using a Trans-Blot Turbo chamber (Bio-Rad) at 25 V and 1 mA for 30 minutes. The membranes were subsequently blocked with 5% non-fat milk in TBS (pH 7.0) containing 1% Tween-20 for 2 hours at room temperature, followed by incubation with primary antibodies against p62 Rabbit pAb (1:3000 dilution, ABclonal), LC3B Rabbit pAb (1:3000 dilution, ABclonal), and β-actin mouse mAb (1:10,000 dilution, ABclonal, catalog number AC004) overnight at 4 °C on a rocking platform. After washing the membranes five times with TBS (pH 7.0) containing 0.1% Tween-20, membranes were incubated for 1 hour at 25 °C with peroxidase-conjugated secondary antibodies anti-rabbit IgG (1:10,000 dilution, Cell Signaling Biotechnology, catalog number 7074s) or anti-mouse IgG (1:10,000 dilution, Cell Signaling Biotechnology, catalog number 7076s) for β-actin detection. Finally, the membranes were washed with TBS (pH 7.0) containing 0.1% Tween-20 and developed using chemiluminescence with Clarity MaxTM Western ECL (Bio-Rad, catalog number 1705062) following the manufacturer's instructions. Image analysis was performed using the C-DiGit Blot (LI-COR). All experiments were conducted in triplicate. β-actin was consistently detected as a loading control and used for normalizing the densitometry of the target proteins with Image J software. Data normalization was carried out by dividing the densitometry data of the target protein by that of the loading control protein (β-actin).

2.6. Determination of Cell Migration

To assess cell migration, a wound healing assay was conducted under sterile conditions. In 96-well plates, adhesive tape (0.5 mm width) was carefully positioned, and a mark with a 2-mm division was applied at the center of each well to demarcate the area for subsequent photographic analysis. Following this, HeLa cells were seeded at a density of 3×10⁴ cells per well in 100 µl of RPMI medium supplemented with 10% neonatal serum and incubated for 24 hours at 37 °C with 5% CO₂. After the initial incubation period, 10 µM cytarabine (ara-C) was added and cells were further incubated for 2 hours under the same conditions. The adhesive tape was then carefully removed to create a wound. The samples were gently washed with PBS (pH 7.0), and HeLa cells were treated with CA (102.72 μM), I3C (150 µM), DMSO (5 µg/ml, vehicle), and BNF (1 µM). Negative controls (untreated cells) and cells treated with 105 mM TGF-β were included in each experimental set. Cell migration was observed at various time points (0, 24, 48, and 72 hours) and documented using a Canon camera. Each experiment was conducted in triplicate, with three biological replicates and technical triplicates for each measurement. The thickness of the wound was analyzed using bioinformatics tools (Image J) by assessing images captured at different time points within the designated area and quantifying the migration rate of the cells.

2.7. Determination of Cell Cycle Arrest

Cells (3 × 10⁵) were seeded in a 6-well plate and incubated for 24 hours at 37 °C with 5% CO₂. Subsequently, the cells were divided into untreated and treated groups. Treatment involved the addition of 20 µL of colchicine (1 mg/ml, as a positive control), 102.72 μM of CA, 150 µM of I3C, 5 µg/ml DMSO (vehicle), and 1 µM of BNF. The cells were then incubated for an additional 24 hours at 37 °C with 5% CO₂. Following the incubation period, the cells were trypsinized and collected by centrifugation at 212× g for 3 minutes at room temperature. The collected cells were fixed in ice-cold methanol (500 µL) with PBS (pH 7.0) (500 µL) at 4 °C for 1 hour. After fixation, the cells were washed three times with PBS (pH 7.0) (1 mL each wash) and centrifuged as described above. Next, the cells were treated with 30 µL of RNase A (100 U/ml) and incubated for 30 minutes at 37 °C. After another round of centrifugation, the cells were resuspended in 500 µL of PBS containing 5 µl of propidium iodide (20 µg/ml) and analyzed using the Beckman-Coulter CytoFlex cytometer (Beckman Coulter, USA). The concentration of colchicine chosen for treatment was based on reported values known to arrest the cell cycle at the G2 phase. Each measurement was conducted with three biological replicates and technical duplicates.

2.8. Statistical Analysis

All data were reported in term of mean and standard errors (SE). Statistical analyses were performed by variance differences (ANOVA) using SPSS 10.0.

3. Results

3.1. Cytotoxicity Activity of Phytochemical Compounds on HeLa Cells

First, we determined cellular cytotoxicity by measuring lactate dehydrogenase (LDH), an enzyme that catalyzes the conversion of lactate to pyruvate and is released from cells after membrane dissolution by a toxic stimulus. We did not find cytotoxic activity on HeLa cells from CA or I3C at concentrations of 102.72 μM or 150 μM, respectively, compared to the untreated control or vehicle (Figure 1).

3.2. Anti-proliferative Activity of CA and I3C in HeLa Cells

The antiproliferative effect of CA and I3C was determined for HeLa cells. Antiproliferative activity was measured by a decrease in cell growth 24 h after compound administration. Both compounds had the ability to impair HeLa cell proliferation (Figure 2). A concentration of 150 µM of I3C inhibited cell growth by 70%. In contrast, CA (102.72 μM) inhibited cell growth by 40%. Taken together, these results suggest that CA has greater antiproliferative activity for HeLa cells than I3C.

3.3. Caspase-3-Induced-Apoptosis in HeLa Cells by CA and I3C

The apoptotic pathway, and its initiation may rely on the liberation of cytochrome c and caspase-9 activation, resulting in caspase-3 cleavage [17]. To determine the correlation between Cas-3-induced-apoptosis by CA or I3C, we treated HeLa cells with those compounds (Figure 3). We observed that CA induces the expression of Cas-3, suggesting that CA induces apoptosis via Cas-3. Moreover, after I3C and BNF treatments, the expression of Cas-3 was not increased, suggesting that Cas-3-induced-apoptosis was not triggered by I3C or BNF. The positive control (colchicine) induces Cas-3 expression, thereby activating the Cas-3-induced-apoptosis.

3.4. Determination of Autophagy Induced by CA and I3C Treatments in HeLa Cells

Autophagy biomarkers such as LC3B and p62 were detected by immunoblotting (Figure 4a) and the expression levels of LC3B and p62 were normalized using the expression of β-actin as a loading control (Figure 4b). In all treatments, the expression levels of LC3B or p62 were not statistically significantly different. Neither CA, I3C, nor BFN induced changes in the expression of LC3B or p62, suggesting that these compounds did not induce autophagy.

3.5. Effect of CA and I3C On Cell Cycle Arrest of HeLa cells

To explore the impact of CA and I3C on cell cycle progression, we examined the distribution of HeLa cells treated with both compounds (Figure 5a). Approximately 28% of HeLa cells treated with CA remained in G2 phase. In contrast, the 18% of cells treated with I3C remained in this phase. Therefore, CA has a stronger effect on arresting the cell cycle of HeLa cells in phase 2 than I3C. Additionally, HeLa cells treated with I3C predominantly remained in the S phase (48%), while 58% of cells treated with BNF remained in the G1 phase. Approximately, 95% of cells treated with colchicine (positive control) remained in the G2 phase (Figure 5b).

3.6. Effect of CA and I3C Over Cell Migration of HeLa Cells

Wound assays were conducted to assess whether CA or I3C could influence cell migration in HeLa cells (Figure 6a,b). At 72 hours, cell migration of HeLa cells was significantly enhanced following TGF-β treatment (positive control) compared to untreated cells [negative control, C(-)] and vehicle. Conversely, the presence of CA significantly reduced cell migration by 58% in HeLa cells at 72 h (p>0.05) compared to the positive control. On the other hand, in HeLa cells, relative migration decreased by 40% after 72 hours of treatment with 150 µM I3C compared to the positive control. Additionally, BNF reduced HeLa cell migration by 75%, while the negative control and vehicle reduced cell migration by approximately 30% compared to the positive control These results suggested that CA (102.72 μM) reduced HeLa cell migration 1.45-fold more than I3C did. Therefore, the CA has a greater effect in reducing cell migration of HeLa cells compared to I3C.

4. Discussion

Sesquiterpenes present in several plant species induce apoptosis in cancer cells through different mechanisms [18]. It had been reported that C has shown anti-cancer properties inducing apoptosis in several cancer cells, particularly in breast cancer cells.

Our data showed that CA had no cytotoxic effects on HeLa cells and that this compound exhibited antiproliferative activity in a dose-dependent manner. The IC₅₀ value of cacalol acetate for HeLa cells was 102.72 μM, whereas for I3C it was 150 μM, suggesting the strong potential of CA as anticancer agent through cell growth inhibition. In breast cell cancer (MCF7 and MDA-MD231) and in a xenograft mouse model, cacalol inhibits cell growth without toxic effects and significantly suppress tumor growth when administered intraperitoneally or orally, suggesting its potential as a preventive and therapeutic agent against cancer [11]. Cacalol and its derivative, CA, have antioxidant activity due to their ability to act as potent free radical scavengers, contributing to their anti-cancer properties by reducing oxidative stress in cancer cells [12]. Moreover, this phytochemical compound inhibits the lipid peroxidation induced by free radicals [10].

HeLa cells treated with CA showed increased levels in the expression of caspase-3. In contrast, when cells were treated with I3C, the expression of this apoptotic biomarker was not observed, suggesting that CA induces apoptotic cell death via Cas-3, whereas I3C does not. These results are consistent with our previous report, in which we measured apoptosis by cytometry in I3C-treated HeLa cells and did not observe increased levels of apoptosis [8]. Our evidence suggested that the induction of apoptosis by CA is independent of Ahr. However, the molecular mechanism of CA is still unknown. Previous reports suggest that cacalol has an anti-breast cancer effect by inhibiting Fatty Acid Synthase (FAS) at transcriptional and post-transcriptional levels and modulating the Akt-SREBP (sterol regulatory element-binding protein) pathways. In this way, cacalol blocks P13K/Akt signaling resulting in a inhibition of SREBP1 which is the main transcriptional regulator of FAS [11]. Therefore, cacalol induces apoptosis in breast cancer cells by modulating the Akt-SREBP-FAS signaling pathway, leading to the activation of pro-apoptotic proteins DAPK2 and caspase 3 [11]. Furthermore, cacalol inhibits the FAS gene, essential in fatty acid biosynthesis and energy homeostasis, causing apoptosis through its antioxidant activity [19]. Intrinsic apoptosis is a cell death centered in the mitochondrion [20]. Activation of Bax and Bak (BCL-2 family members) resulted in mitochondrial outer membrane permeabilization (MOMP) and the releasing of proapoptotic proteins and cytochrome c from the inter-membrane mitochondrial space into the cytosol where cytochrome c bind to Apaf-1 forming apoptosome and activating caspase-9 which cleave and activate caspase-3 and -7 [21,22,23,24,25]. Therefore, CA induced apoptotic cell death though promoting that more cervical cancer cells expressed caspase-3. The biomarker of late stage of apoptosis (caspase-3) was not significantly up-regulated in HeLa cells treated with CA treatment. Previous reports indicated that the treatment of tumor cells with cacalol promote the expression of DAPK2 and caspase-3 [11]. Our results agree, showing that CA induces the expression of caspase-3, suggesting that the acetylation of cacalol might enhance the apoptotic induction mechanism of this compound. Although some evidence suggests that cacalol induces apoptosis through the mechanisms described above, the acetylated molecule (CA) might trigger apoptosis through a different pathway, which we are elucidating.

Furthermore, cacalol has synergistic activity that enhances the apoptotic effect of chemotherapeutic drugs such as taxol and cyclophosphamide, helping to overcome chemoresistance [11]. It is still unclear, if CA might have synergistic activity.

In addition, neither CA nor I3C induces autophagic cell death. CA arrest the cell cycle at G2 phase, while I3C does arrest cell cycle at this phase, albeit to a lesser extent. CA could inhibit cell migration and has a relatively low cytotoxicity, as it does not affect HeLa cell morphology as does BNF (1uM). It is worth mentioning that CA does not cause a morphological cytotoxic effect associated with its ability to inhibit cell migration (Figure 7).

5. Conclusions

CA had lower cytotoxic effect on HeLa cells than I3C. CA and I3C inhibited cell growth, but CA is more effective to inhibit cell proliferation than I3C. I3C did not affect the expression of Cas-3, suggesting that this compound did not induce apoptosis via Cas-3. In contrast, CA upregulated the expression of Cas-3, suggesting that CA induces apoptosis via Cas-3.CA and I3C did not induce the expression of p62 and LC3B levels, suggesting that these phytochemical compounds not induce autophagic cell death. CA arrests cell cycle and inhibits cell migration of HeLa cells at a higher rate than I3C. Although CA and I3C are promising anticancer agents for the treatment of cervical cancer, the low cytotoxicity of CA compared to I3C and the fact that CA induces apoptosis vía Cas-3, cell cycle arrest and inhibits cell proliferation and migration in a higher rate than I3C, make CA a more promising agent than I3C.