The aqueous extract contains phenolic compounds, total tannins and total anthocyanin being the most significant quantitatively (Table 1). Regarding the antioxidant capacity, the ORAC (Oxygen Radical Absorbance Capacity) value corresponds to the most active mechanism, followed by the ABTS (2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) value and DPPH (2,2-diphenyl-1 picrylhydrazyl) value (Table 1).
Table 1 Phytochemicals and antioxidant activity of aqueous extract from Andean Berry.
Phenols
|
Tannins 2
|
Anthocyanins3
|
ORAC4
|
ABTS4
|
DPPH4
|
2186 ± 51.92
|
918.22 ± 79,5
|
25219 ± 0.07
|
32927,37 ± 1334,9
|
1102,55 ± 38.3
|
129.25 ± 4.7
|
The results are expressed as the mean ± SD of three independent experiments in triplicates. 1mg Gallic Acid Equivalents /100 g. 2mg Catechin Equivalents/100 g. 3mg Cyanidin-3-Glycoside Equivalents /100 g. 5µmoles Trolox Equivalents/g.
Subsequently, SW480 cell viability decreased with longer exposure to the extract, because the IC50 value obtained was 33.62 ± 1.3 mg/mL and 5.91 ± 1.0 mg/mL at 24 and 48 hours, respectively. The extract produced a detachment of the spheroid cells that increased with exposure time to the extract and concentration. It is also observed that the center of the spheroid is becoming opaquer (darker) with increasing extract concentration and exposure time (Fig. 1).
Figure 2 shows a statistically significant increase in the percentage of spheroid cells in SubG1 (dead or in apoptosis) after treatment for 48 h at different concentrations of the extract (AEM) compared to untreated cells (negative control). Spheroid cells in G1 decreased significantly in all treatments compared to the negative control. In addition, S-phase (proliferative) cells increased significantly in the spheroids after treatment with the extract at different concentrations compared to the untreated cells. The G2 phase (proliferative) cells also decreased significantly after treatment compared to the negative control. Finally, the results obtained with the extract are comparable to the effect observed with 5-Fluorouracile (5-FU).
Figure 3A shows the distribution of cells after double staining with Propidium Iodide (PI) and Annexin-V as follows: necrotic cells (Q1: annexin V-FITC- / IP+), cells in late apoptosis (Q2: annexin V-FITC+/ IP+), cells in early apoptosis (Q3: annexin V-FITC+ / IP-), and viable cells (annexin V-FITC/IP-). The percentage of necrotic cells decreased significantly in spheroids exposed to different concentrations of AEM. In contrast, the rate of apoptotic cells increased significantly in treated spheroids at different AEM concentrations concerning the negative control (Fig. 3b).
Considering the importance of caspases in the process of cell death by apoptosis, the detection or not of active caspases in spheroid cells after treatment was performed. Figure 4 shows that the extract (AEM) induced an increase in the percentage of cells positive for activated caspases. It is noteworthy that a concentration of 7 mg/mL of the extract produced the highest percentage of cells with active caspases, and induced the highest percentage of apoptotic cells (Fig. 4). This result is comparable with the effect of 5-FU induced apoptotic cells, although also a higher percentage of necrotic cells was observed (Fig. 4) when comparing these results with untreated cells.
Since the percentage of cells in the G1 phase of the cell cycle decreased and is associated with senescent cells [12], we verified if the extract induces senescence because this mechanism of death does not occur due to apoptosis. Figure 5 shows that the extract and 5-FU (positive control) compared to untreated cells (negative control) significantly decreased the percentage of senescent cells from spheroids after 24 h of treatment under the same conditions that induce apoptosis.
Monolayer or 2D cell models present several limitations because they do not mimic the in vivo tumor development, in which cell-cell or cell-cell plus extracellular matrix interactions occur; these interactions are related to cell proliferation events, gene expression, and drug response, among others [8, 13-15]. Another disadvantage of 2D cultures is that cells lose their polarity, which has an impact on cell response to apoptosis [16]. By contrast, 3D cultures allow us to obtain results that approximate the results of in vivo assays.
In our study, the aqueous extract from Andean Berry (V. meridionale) presented a higher content of total phenols such as anthocyanins compared to other studies employing the same extract (2546 mg gallic acid and 150.7 mg cyanidin-3-glucoside/100 g) [16]. A similar situation was presented for the antioxidant activity analyzed by three methods based on different antioxidant mechanisms that could be attributed to their phenolic content. these findings agree with the results of Maldonado-Celis et al, 2014 [8], who reported an ORAC value of an aqueous extract from V. meridionale berry corresponding to 41775.2 µmol Trolox/100 g, a higher value than the one obtained in our analysis. Factors that may affect the variation of the antioxidant activity of the aqueous extract from Andean berries among different studies include the method of extraction, fruit maturity, and storage conditions of the fruit and/or extract [17].
The aqueous extract decreased the growth of SW480 (2D) cells at lower concentrations compared to SW480 spheroids. This could be due to differences in metabolism that make them sensitive to test compounds, and to the cell proliferation rate of monolayer culture that is higher compared to cells in 3D cultures. This suggests a higher efficacy of test compounds or extract; thus, data collected from 3D cell cultures concerning resistance to antitumor agents are generally more accurate than in 2D cultures [18, 19].
Regarding the cell cycle, it was found that spheroids with three days of growth treated with aqueous extract from Andean Berry at different concentrations for 24h, showed an accumulation of cells in sub G1. This result differs from the findings reported by Arango-Varela et al. [20], where SW480 cells in 2D culture exposed for 24 h with V. meridionale berry juice 18 mg/mL (30% v/v) presented a cell cycle arrest in the S and G2/M phases (SW480). This difference in cell distribution could be attributed to the fact that SW480 cell culture in monolayer was not synchronized, and the cell population was segregated in different phases of the cycle. Therefore, exposition to bioactive berry compounds such as ellagic acid, gallic acid, and methyl gallate present in Vaccinium meridionale might activate p53 and thus regulate the cell cycle [21, 22]. Moreover, SW480 spheroid cells have a more complex environment than monolayer culture with the same cell line, and bioactive compounds may interact differently; furthermore, spheroid cells do not have the same exposure to oxygen and nutrients, which may favor the increased percentage of dead or dying hypodiploid cells [23].
To know if apoptosis is involved in the increase of SubG1 cells in the spheroids treated with the extract mentioned in this study, the cells were analyzed by double staining with Annexin-V/PI, as well as the activity of caspases. The aqueous extract from Andean Berry induced apoptosis and activation of caspases, although the strategy available for this research did not enable the identification of a differential activity of each enzyme. These results were comparable with 5-FU. The effect of the extract observed here on spheroids coincides with that described by Agudelo et al. in 2017 [6] and Arango-area et al., [20] in SW480 cells treated with Andean Berry juice that inhibited the proliferation of these cells by apoptotic mechanisms through perturbation of the intracellular oxidative state, activation of caspase-3 and p53.
The 5-FU was employed in this study as a positive control because is a cytotoxic agent, an antimetabolite that interferes with RNA and DNA synthesis by inhibiting the enzyme thymidylate synthase [24]. In cancer cells, 5-FU unbalances the autoregulatory system that controls and limits cell division leading to cell death. Thus, 5-FU is used as a chemotherapeutic agent in the treatment of various types of cancer, including colorectal [24]. SW480 cells have been reported to be resistant to 5-FU, which cytotoxic, antiproliferative, and apoptotic effect has been evaluated in 2D culture at 48 h incubation times at concentrations of 0.84 to 89 mg/mL [25], 2.0 µg/mL [26], 1 to 200 µM for 24 h to 72 h [27], 25 mg/mL for 48 h [28]. In our study, it was found that 5µM 5-FU induced apoptosis in spheroids obtained from SW480 treated for 24 h; however, the mechanisms of action involved in this result were not analyzed. Further studies are required to do so and to determine whether this effect can be potentiated by combining 5-FU with plant extracts such as the one evaluated here.
Finally, our data suggest that extract and 5FU behaved as senolytic agents under the same conditions that induced apoptosis. Senescent cells are resistant to apoptosis [29]; however, it is necessary to analyze the status of anti-apoptotic proteins in the spheroids before and after treatments —as well as the selectivity of the extract concerning non-malignant colon cells— to know their selectivity and safety, since senescent cells also play an important role in tissue damage repair [30].