In recent years, scientists worldwide have been conducting research to find a detailed chemical composition of and the anti-proliferating, cytotoxic and proapoptotic properties of propolis, which is confirmed by the results of various experiments and publications in scientific journals. The resistance of neoplastic cells to standard chemotherapy inspires a continuous search for new compounds with cytostatic activity. One assumption of the chemoprevention concept is to prevent the initiation of cancerogenesis or the inhibition of this process at its early stages. This is aimed at exclusion of the development of a tumor capable of invading neighboring tissues and metastasis. Among the chemopreventive substances, there are non-steroid anti-inflammatory medicines, folic acid, vitamins C and A, vitamin E, carotene, cellulose and many more medicines of a natural origin, including propolis and its components, such as the caffeic acid phenethyl ester.
2.2. The Biological Effects of CAPE and Propolis on Morphological Changes of Hs578T and MDA-MB-231 Breast Carcinoma Cells
The results of our in vitro investigation demonstrated that triple-negative MDA-MB-231 and Hs578T human breast carcinoma cells exposed to CAPE and EEP phytochemicals reveal diminished metabolic activity and viability in a dose-dependent and time-dependent manner. Microscopic assessment demonstrated numerous changes in cellular morphology of examined breast carcinoma cells, including a decreased number of affected cells, cell shrinkage and cytoplasmic condensation. These data support the hypothesis that the exposure to some phytochemicals, components of propolis, including derivatives of caffeic acid, may hypothetically reduce the growth of breast cancer cells, compared to the non-cancerous IMR-90 fibroblast control line.
The 72-h incubation of MDA-MB-231 and Hs578T cells with biologically-active substances—propolis and CAPE—resulted in a decreased number of vital cells, which is shown in
Figure 1. Moreover, the cells’ detachment and the changes of their shapes were also observed.
Figure 1.
Morphological and cytological features of triple-negative breast cancer (TNBC) MDA-MB-231 (A–C) and Hs578T (D–F) treated with 0.2% DMSO solution (vehicle control) (A,D), treated with CAPE in a concentration of 80 μM (B,E) and treated with propolis in a concentration of 200.0 μg∙mL−1 (C,F) after 72 h of cell exposure to the investigated phytochemicals. (A) MDA-MB-231 grown as a control with DMSO solvent, showing the characteristic monolayer and suspension of carcinoma cells, with the standard features of cellular atypia: nuclear and cytoplasmic pleomorphism, increased nucleus:cytoplasm ratio, highly irregularly-shaped cells (tadpole, caudate), irregular nuclear shapes and hyperchromasia. (B) MDA-MB-231 with CAPE addition shows a decreased number of breast carcinoma cells and necrotic cells in suspension. (C) MDA-MB-231 with propolis addition, showing a decreased number of breast carcinoma cells, cytoplasmic shrinkage, condensed chromatin and stained fragments of decomposed cancer cells. (D) Hs578T in the DMSO solvent, showing blurred cytoplasmic structure and the characteristic future for cellular atypia. (E) Hs578T with CAPE addition, showing a decreased number of cells without significant nucleus and cytoplasmic changes. (F) Hs578T with propolis addition, showing similar, but slightly less obvious morphological changes as for the MDA-MB-231 line, i.e., a decreased number and condensed chromatin.
Figure 1.
Morphological and cytological features of triple-negative breast cancer (TNBC) MDA-MB-231 (A–C) and Hs578T (D–F) treated with 0.2% DMSO solution (vehicle control) (A,D), treated with CAPE in a concentration of 80 μM (B,E) and treated with propolis in a concentration of 200.0 μg∙mL−1 (C,F) after 72 h of cell exposure to the investigated phytochemicals. (A) MDA-MB-231 grown as a control with DMSO solvent, showing the characteristic monolayer and suspension of carcinoma cells, with the standard features of cellular atypia: nuclear and cytoplasmic pleomorphism, increased nucleus:cytoplasm ratio, highly irregularly-shaped cells (tadpole, caudate), irregular nuclear shapes and hyperchromasia. (B) MDA-MB-231 with CAPE addition shows a decreased number of breast carcinoma cells and necrotic cells in suspension. (C) MDA-MB-231 with propolis addition, showing a decreased number of breast carcinoma cells, cytoplasmic shrinkage, condensed chromatin and stained fragments of decomposed cancer cells. (D) Hs578T in the DMSO solvent, showing blurred cytoplasmic structure and the characteristic future for cellular atypia. (E) Hs578T with CAPE addition, showing a decreased number of cells without significant nucleus and cytoplasmic changes. (F) Hs578T with propolis addition, showing similar, but slightly less obvious morphological changes as for the MDA-MB-231 line, i.e., a decreased number and condensed chromatin.

2.3. The Assessment of Viability of MDA-MB-231 and Hs578T Cells Exposed to CAPE and EEP with the MTT Assay
The cytotoxic activity of various concentrations of propolis and the caffeic acid phenethyl ester on the breast cancer cells is presented in
Figure 2A–D. The viability of MDA-MB-231 and Hs578T cells decreased depending on the concentration and time of exposure to the studied compounds. The cell viability decrease upon exposure to CAPE was stronger for the breast cancer cells of the Hs578T line in the 48th and 72nd hour. In the case of the application of the ethanol extract of the Polish propolis, it could be observed that the cytotoxic activity was stronger against the Hs578T cells than MDA-MB-231 cells, which is presented in
Figure 2C,D. The control assay performed with normal fibroblasts of the IMR-90 line showed a slight effect of the studied compounds on the viability and proliferation of the cells in the analyzed concentrations; however, this effect was not statistically significant. Results are presented in
Figure 3.
Figure 2.
Cytotoxic effects of EEP and CAPE on MDA-MB-231 and Hs578T breast cancer cells. Cells were incubated with 6.25–200.0 μg∙mL−1 EEP or with 2.5–80.0 μM CAPE for 24, 48 and 72 h. The values represent the mean ± SD of three independent experiments performed in quadruplicate (n = 12). (A) Cytotoxic activity of CAPE against MDA-MB-231 cells. (B) Cytotoxic activity of CAPE against Hs578T cells. (C) Cytotoxic activity of EEP against MDA-MB-231 cells. (D) Cytotoxic activity of EEP against Hs578T cells. The percentage of cell death was measured using the MTT cytotoxicity assay. Results are presented as the means of cytotoxicity ± SD. *, ^, # indicate statistically-significant differences compared to the control: * after 24 h of incubation, # after 48 h and ^ after 72 h.
Figure 2.
Cytotoxic effects of EEP and CAPE on MDA-MB-231 and Hs578T breast cancer cells. Cells were incubated with 6.25–200.0 μg∙mL−1 EEP or with 2.5–80.0 μM CAPE for 24, 48 and 72 h. The values represent the mean ± SD of three independent experiments performed in quadruplicate (n = 12). (A) Cytotoxic activity of CAPE against MDA-MB-231 cells. (B) Cytotoxic activity of CAPE against Hs578T cells. (C) Cytotoxic activity of EEP against MDA-MB-231 cells. (D) Cytotoxic activity of EEP against Hs578T cells. The percentage of cell death was measured using the MTT cytotoxicity assay. Results are presented as the means of cytotoxicity ± SD. *, ^, # indicate statistically-significant differences compared to the control: * after 24 h of incubation, # after 48 h and ^ after 72 h.
Figure 3.
Cytotoxic effects of EEP and CAPE on the normal lung fibroblast IMR-90 cell line. Cells were incubated with 6.25–200.0 μg∙mL−1 EEP or with 2.5–80.0 μM CAPE for 24, 48 and 72 h. The values represent the mean ± SD of three independent experiments performed in quadruplicate (n = 12). (A) Cytotoxic activity of CAPE against IMR-90 cells. The percentage of cell death was measured using the MTT cytotoxicity assay. (B) Cytotoxic activity of CAPE against IMR-90 cells. The percentage of cell death was measured using the lactate dehydrogenase (LDH) cytotoxicity assay. (C) Cytotoxic activity of EEP against IMR-90 cells. The percentage of cell death was measured using the MTT cytotoxicity assay. (D) Cytotoxic activity of EEP against IMR-90 cells. The percentage of cell death was measured using the LDH cytotoxicity assay. Results are presented as the means of cytotoxicity ± SD. *, ^, # indicate statistically-significant differences compared to the control: * after 24 h of incubation, # after 48 h and ^ after 72 h.
Figure 3.
Cytotoxic effects of EEP and CAPE on the normal lung fibroblast IMR-90 cell line. Cells were incubated with 6.25–200.0 μg∙mL−1 EEP or with 2.5–80.0 μM CAPE for 24, 48 and 72 h. The values represent the mean ± SD of three independent experiments performed in quadruplicate (n = 12). (A) Cytotoxic activity of CAPE against IMR-90 cells. The percentage of cell death was measured using the MTT cytotoxicity assay. (B) Cytotoxic activity of CAPE against IMR-90 cells. The percentage of cell death was measured using the lactate dehydrogenase (LDH) cytotoxicity assay. (C) Cytotoxic activity of EEP against IMR-90 cells. The percentage of cell death was measured using the MTT cytotoxicity assay. (D) Cytotoxic activity of EEP against IMR-90 cells. The percentage of cell death was measured using the LDH cytotoxicity assay. Results are presented as the means of cytotoxicity ± SD. *, ^, # indicate statistically-significant differences compared to the control: * after 24 h of incubation, # after 48 h and ^ after 72 h.

Our results based on MTT and LDH assays are coherent with the data obtained by Wu
et al. [
23] which confirm the hypothesis that breast cancer cells’ viability gradually decreases depending on the increasing dose of CAPE. The estimated IC
50 value amounted to 15 μM for MDA-MB-231 and MCF-7 cell lines by Wu
et al. was only slightly higher than the results obtained in our experiment, with 14.08 μM and 8.01 μM for MDA-MB-231 and Hs578T, respectively. The
in vivo study confirms that administration of CAPE for 3–4 weeks decreases the volume of the tumor in mice from 40%–60% in the case of a heterograft of breast cancer cells of the lines MDA-MB-231 and MCF-7. Interestingly, this study showed that a more aggressive phenotype of breast cancer, MDA-MB-231, was more sensitive to CAPE than the MCF-7 cells. Moreover, both models confirmed that low CAPE doses (approximately 10 nmol CAPE/mouse/day) produce better results for inhibiting the volume of the tumor than much higher doses. The same study also showed that CAPE induces the inhibition of the cell cycle in the S-phase and a complete elimination of breast cancer cells in the G
2-/M-phase.
2.4. The Assessment of the Cytotoxic Activity of CAPE and EEP against MDA-MB-231 and Hs578T Cells with the LDH Assay.
In order to confirm the cytotoxic activity of the studied substances, the method based on enzymatic reactions was applied. This assay assesses the activity of lactate dehydrogenase (LDH), which is a cytosolic enzyme that in physiologic conditions is not released to the environment. The obtained results confirmed that the studied compounds show cytotoxic activity, however not as strongly as could have been concluded from the analysis of the results obtained with the MTT assay. The cytotoxic activity results of the ethanol extract of propolis and the caffeic acid phenethyl ester are presented in
Figure 4.
Figure 4.
Effect of CAPE and EEP on the viability of MDA-MB-231 and Hs578T cells. The cytotoxicity was evaluated by the LDH assay after 24, 48 and 72 h of incubation of cells with 6.25–200.0 μg∙mL−1 EEP or with 2.5–80.0 μM CAPE. The values represent the mean ± SD of three independent experiments (n = 12). (A) Cytotoxic activity of CAPE against MDA-MB-231 cells. (B) Cytotoxic activity of CAPE against Hs578T cells. (C) Cytotoxic activity of EEP against MDA-MB-231 cells. (D) Cytotoxic activity of EEP against Hs578T cells. The percentage of cell death was measured using the LDH cytotoxicity assay. Results are presented as the means of cytotoxicity ± SD. *, ^, # indicate statistically-significant differences compared to the control: * after 24 h incubation, # after 48 h and ^ after 72 h.
Figure 4.
Effect of CAPE and EEP on the viability of MDA-MB-231 and Hs578T cells. The cytotoxicity was evaluated by the LDH assay after 24, 48 and 72 h of incubation of cells with 6.25–200.0 μg∙mL−1 EEP or with 2.5–80.0 μM CAPE. The values represent the mean ± SD of three independent experiments (n = 12). (A) Cytotoxic activity of CAPE against MDA-MB-231 cells. (B) Cytotoxic activity of CAPE against Hs578T cells. (C) Cytotoxic activity of EEP against MDA-MB-231 cells. (D) Cytotoxic activity of EEP against Hs578T cells. The percentage of cell death was measured using the LDH cytotoxicity assay. Results are presented as the means of cytotoxicity ± SD. *, ^, # indicate statistically-significant differences compared to the control: * after 24 h incubation, # after 48 h and ^ after 72 h.
As a result of the conducted experiment assessing the cytotoxic activity of the ethanol extract of propolis and the caffeic acid phenethyl ester towards the triple-negative breast cancer cells, the IC
50 values were determined for the EEP and CAPE compounds depending on the time of cell exposure (
Table 1). The IC
50 values estimated by the MTT cytotoxicity assay are generally lower than with the lactate dehydrogenase release test. However, the MTT assay determines the mitochondrial activity of cells, which may be interrupted due to the activity of certain chemical compounds on the cells, whereas the LDH assay clearly confirms cell death and is a more precise marker of cell necrosis. It can be observed (
Table 2) that the IC
50 estimations in both assays (MTT and LDH) are higher for MDA-MB-231 cells exposed to CAPE, and a higher sensitivity to the studied compounds was revealed by the breast cancer cells of the Hs578T line (lower IC
50 values). The IC
50 after 72 h is almost twice as high for the MDA-MB-231 cells exposed to CAPE than for Hs578T breast cancer cells.
Table 2.
Comparison of IC50 values for MDA-MB-231 and Hs578T cells obtained from the LDH leakage assay and MTT assay following exposure to CAPE and EEP for 24, 48 and 72 h.
Table 2.
Comparison of IC50 values for MDA-MB-231 and Hs578T cells obtained from the LDH leakage assay and MTT assay following exposure to CAPE and EEP for 24, 48 and 72 h.
Breast Cancer Cell Line (TNBC) | CAPE Exposure, EEP Exposure (h) | MTT Assay IC50: CAPE μM (μg∙mL−1) | MTT Assay IC50: EEP μg∙mL−1 | LDH Assay IC50: CAPE μM (μg∙mL−1) | LDH Assay IC50: EEP (μg∙mL−1) |
---|
MDA-MB-231 | 24 | 21.05 (5.99) | 232.31 | 22.93 (6.52) | 731.68 |
48 | 13.78 (3.92) | 63.38 | 18.64 (5.30) | 170.97 |
72 | 11.69 (3.32) | 40.40 | 14.08 (4.00) | 48.35 |
Hs578T | 24 | 16.38 (4.66) | 2538.51 | 32.80 (9.33) | >3000.00 |
48 | 6.60 (1.88) | 38.64 | 11.53 (3.28) | 45.07 |
72 | 4.82 (1.37) | 31.03 | 8.01 (2.28) | 33.68 |
The
in vitro study performed by Omene
et al. [
3] with the use of selected breast cancer cells confirms the substantial cytotoxic effect of CAPE towards the MDA-MB-231, MCF-7 and SK-BR-3 cell lines. The IC
50 values ranged from 15 μM for MCF-7 up to 35 μM for the MDA-MB-231 line. These data are double those gathered in our study (14.08 μM, MDA-MB-231), which can be related to a different technique used for the cytotoxicity assessment of the investigated phytochemicals. Omene
et al. also showed that CAPE causes accumulation of the acetylated histone protein by which they suggest that it reveals the properties inhibiting the inhibitor of deacetylation of histones. This mechanism may be partly responsible for the antitumor activity of the caffeic acid phenethyl ester and strengthen its role as a potential antitumor drug. It was also shown that in the lines of breast cancer with a positive expression of the estrogen receptor (ER+), the application of CAPE itself or CAPE found in propolis leads to the decrease in the estrogen and progesterone receptors’ number, which suggests that the decrease of these genes’ expression causes the anti-hormonal effect. Moreover, this study shows that the application of CAPE in the triple-negative breast cancer is possible accompanying hormonal therapy. It was also shown that CAPE inhibits the expression of the mdr-1 gene responsible for the resistance of neoplastic cells to the applied chemotherapeutics.
A similar situation to the caffeic acid phenethyl ester can be observed in the case of the ethanol extract of propolis, which is presented in
Figure 4 and
Table 2. In this case, the IC
50 values were also higher for MDA-MB-231 cells; however, these discrepancies were considerably smaller than for CAPE. The exceptions were only the values of IC
50 obtained in the 24-h incubation of cells with EEP and CAPE. Values for IC
50 obtained in the LDH test were lower for MDA-MB-231 cells than Hs578T. These finding and the analysis of the IC
50 values for the investigated substances may also suggest that caffeic acid derivative CAPE has a higher biological activity towards breast carcinoma cells compared to ethanol extract of propolis with a complex chemical composition. Possibly, the “attenuation phenomenon” can be responsible for a less effective action of propolis as a mixture of variable ingredients.
The quantitative composition of propolis is heterogeneous; however, it always contains chemical compounds, such as polyphenols, terpenoids, steroids and amino acids. Propolis samples obtained from various plants may therefore differ in terms of chemical composition. The Polish propolis is mainly classified as the poplar-type propolis, and its dominating components are flavonoids and phenolic compounds, which constitute between 35% and 50%.
There is more and more proof that the polyphenol compounds found, e.g., in propolis may serve as a supplement to standard chemotherapy and radiotherapy.
Apoptosis is one of the most potent defenses against cancer, because this process eliminates potentially deleterious, mutated cells. Many dietary cancer-preventive compounds, including propolis and its active derivatives, induce apoptosis in cancer cells. The mechanism of evoking apoptosis by propolis seems to be independent of the type of the studied neoplastic cell; however, it is directly dependent on the concentration of the applied extract for the purpose of the study. Some studies suggest that propolis induces apoptosis by releasing cytochrome c from mitochondria to cytosol by means of the caspases’ cascade and with the pro-apoptotic proteins.
The studies of Watabe
et al. [
36] depict the mechanism of CAPE activity as dependent on the nuclear transcription factor NF-κB. These factors play a significant role in the regulation of death and survival of cells. It is known that the caffeic acid phenethyl ester strongly inhibits NF-κB activation. The study also confirms that CAPE is responsible for the activation of apoptosis in neoplastic cells of various types, but does not lead to the activation of this pathway in the case of the normal fibroblast cells WI-38. It may be caused by the fact that the neoplastic cells with a high base of NF-κB activity are more sensitive to CAPE than normal cells. The study also analyzes the pathways of apoptosis activation in the neoplastic cells. It confirms the activation of apoptosis both in the receptor pathway dependent on the Fas receptor and the mitochondrial pathway with the release of cytochrome c. Additionally, this mechanism may be responsible for the difference in the results regarding the assessment of the cytotoxic activity in the MTT and LDH assays.
Therefore, the results of the experiment conducted by Lee
et al. [
54] are of special interest, since they defined the effect of the caffeic acid phenethyl ester on the activity of proteins p53 and p38 in the C6 glioma cells. This study confirmed that propolis has cytotoxic activity. The scientists proved that CAPE leads to the release of cytochrome c from mitochondrion to cytosol and the activation of caspase-3. What is most important, the expression of the p53 protein, Bax and Bak increased only after 3 h of incubation with CAPE, simultaneously causing a decrease of the expression of the anti-apoptotic protein Bcl-2 upon a 36-h incubation. Similar results were also obtained by Jin
et al. [
55], who carried out an experiment on the line of human myeloid leukemia U937. They proved, similarly to Lee
et al., that the caffeic acid phenethyl ester (propolis component) has cytotoxic properties dependent on the concentration and exposure time of the substance on the neoplastic cells. With DAPI staining, they observed in the fluorescent microscope some changes characteristic for apoptosis in the cellular nuclei. They did not confirm the expression of the Fas protein on the surface of the studied cells; however, they observed the release of cytochrome c to cytosol, inhibition of the expression of the anti-apoptotic protein Bcl-2 and an increase of the pro-apoptotic protein Bax. Our results confirm the observations of Jin
et al., since, similarly to the Korean team, we revealed that the caffeic acid phenethyl ester and the ethanol extract of the Polish propolis show cytotoxic activity dependent on the concentration of the studied compound and the time of cell exposure to these compounds.
Szliszka
et al. [
56] in their experiment showed that artepillin C found in the Brazilian propolis causes an increase in neoplastic cells’ sensitivity to the TRAIL protein. This protein is a strong stimulator of apoptosis in neoplastic cells and an important factor responsible for the elimination of developing tumors. However, a number of cells undergoing oncogenesis is resistant to the apoptotic death with the participation of the TRAIL protein. Therefore, Szliszka
et al. decided to sensitize the neoplastic cells to the TRAIL protein by adding substances of a natural origin, such as flavonoids and phenolic acids. In order to confirm the advanced thesis, they used the cytotoxic assays (MTT, LDH), fluorescent staining (to define apoptosis), flow cytometry (to assess the death receptors) and the immune-enzymatic tests. It was proven that the application of the propolis component with the TRAIL protein led to an increase of the number of dying cells though apoptosis by an increase of caspase-3 and -8 activity and inhibition of the nuclear factor NF-κB.
It should be noted that the obtained differences in the MTT and LDH assays suggest the changes at the level of mitochondrial metabolism in the studied compounds. The possible mechanism of the anticancer property of propolis and one of its active derivate, CAPE, seems to be the augmentation of apoptosis phenomenon in human breast cancer cells. The detected changes of carcinoma cells’ morphology following EEP exposure may suggest and indicate the carcinoma cells’ death due to the apoptosis pathway. Therefore, the authors’ next research aim shall be the determination of a hypothetical way for the breast cancer cells’ metabolic death, after exposure to these highly-active phytochemicals inhibiting malignant cells’ metabolism.