P2Et is a C. spinosa-derived fraction mainly composed of hydrolyzable tannins, gallotannins, and minor concentrations of pentagalloylglucose (PGG), as previously stated . Our study gives evidence of the fraction’s cytotoxic effects on 4T1 cells, inducing apoptosis by the mitochondrial pathway, a path selective for cells with high proliferative rates when compared with normal mononuclear cells or human fibroblasts. Apoptosis is a type of programmed cell death that can be activated by two pathways: extrinsic and intrinsic. The latter involves mitochondrial translocation of pro-apoptotic members of the Bcl-2 family, such as Bax, causing MMP loss followed by the release of several apoptosis mediators, including cytochrome c, into the cytosol , which binds Apaf-1 and, in association with other proteins, the apoptosome is formed. This in turn recruits and activates pro-caspase 9, leading to the activation of effector caspases such as caspase 3. Finally, DNA endonucleases are activated [30, 31]. This cell death pathway involving caspase activation produces typical morphological changes such as chromatin condensation, nucleosomal DNA fragmentation, nuclear membrane disruption, PS externalization, and formation of apoptotic bodies . We herein demonstrated that P2Et fraction activity induces apoptosis with MMP loss, PS externalization, caspase 3 activation, and DNA fragmentation.
The activities of isolated compounds that are also present in P2Et fraction have been previously studied. Gallotannins or gallic acid derivatives, specifically alkyl gallates and gallamides, have proven cytotoxic effects on L1210 leukemia cells in a dose-dependent manner . Eugenia jambos L. (Myrtaceae) plant extract exerts cytotoxic activity on HL60 leukemia cells. This extract contains hydrolyzable tannins (1-O-galloyl castalagin, casuarinin, and gallotannins) and induces apoptosis and DNA fragmentation . Similarly, we previously reported that P2Et fraction has a cytotoxic effect on leukemia cells (K562), inducing apoptosis . The compound’s broad activity range can also be seen in Terminalia chebula fruit extract, rich in gallotannins capable of inducing apoptosis in the murine and human breast cancer cells MCF-7 and S115, respectively, among other activities .
PGG is a compound also present in P2Et fraction, and it is capable of inducing G1-phase arrest , suppressing the phosphorylation and protein level of estrogen receptor alpha by promoting degradation in the lysosome and negatively modulating the ErbB/PI3K/Akt pathway in MCF-7 cells . This causes the cells to become refractory to estrogenic stimulation. In our previous studies, we also observed a G1-phase arrest in MCF-7 cells , but not in 4T1 cells, suggesting a cell line-selective mechanism. We also showed that the activities of gallotannins and PGG are regulated by the same cell diversity.
Caesalpinia spinosa fruits are well known to be good tannins sources, yet evidence of their cytotoxic or antitumor activity is quite limited. Gali-Muhtasib’s group reported that C. spinosa hydrolyzable tannins such as TA lower skin cancer biochemical marker levels, delaying tumor development in mice [13, 14]. Similarly, epigallocatechin 3-gallate (EGG), a gallic acid condensed tannin present in green tea, is capable of inducing apoptosis through the mitochondrial pathway in murine breast cancer cells (4T1) . In general, such compounds exhibit low toxicity to normal cells, as observed herein and previously demonstrated for 1-O-galloyl castalagin, casuarinin, and gallotannins from Eugenia jambos, which are slightly cytotoxic to human lymphocytes .
Our study evaluated the fraction’s ability to modulate tumor cell cytokine production. We found that both P2Et fraction and doxorubicin induced early IL-6 secretion from ER(+) 4T1 cells and in both treatments there was apoptosis induction, inferring that IL-6 favors their latter removal . No significant changes in the levels of the other tested cytokines were found in tumor cell secretion. To control tumor growth in vivo, is necessary to induce direct death of tumor cells and regulate microenvironmental and tissue factors involved in primary tumor proliferation and migration. IL-6 is a mediator with dual activity in tumor growth and metastatic processes. Intrinsic production of IL-6 in the ER(+) breast cancer cell lines MCF-7, T47D, ZR-75-1, and SK-BR promotes cell death in response to drugs, inducing morphological changes, and apoptosis with DNA fragmentation [37–39]. On the contrary, in the ER(-) breast cancer cell lines HS578T and MDA-MB-231, IL-6 is related to a drug-resistant phenotype with increased expression of MDR-1 . In our system, IL-6 production by 4T1 cells favored apoptotic cell death.
On the other hand, when overexpressed in organs such as the liver, brain, or lung, IL-6 can promote metastasis by attracting and promoting tumor cells . In patients with breast cancer, IL-6 is associated with a poor prognosis because the levels increase in advanced stages and are associated with a higher number of metastases . We observed a decrease in circulating IL-6 that may have been correlated with the reduction in tumor volume and metastasis; however, it also may have been correlated with negative regulation of the inflammatory microenvironment, favoring the activation of mechanisms implied in tumor elimination.
Additional results suggest that P2Et fraction could modulate different mediators. When 4T1 cells are orthotopically transplanted to BALB/c mice, a leukemoid reaction is induced. This reaction is characterized by leukocytosis of >50,000 cells/μL with predominance of immature granulocytes Gr-1dim and splenomegaly due to massive granulocyte infiltration into the red pulp . P2Et fraction-treated animals showed a reduced number of peripheral blood leukocytes at day 30. The leukemoid reaction has been associated with a poor prognosis in animal cancer models and human cancers . The decrease in response by P2Et fraction therapy could improve the course of the disease.
The in vivo P2Et fraction activity showed that the fraction favors not only a decrease in tumor diameter and weight, but also a significant decrease in spleen and liver metastases. 4T1 tumor cells are highly aggressive and metastatic in BALB/c mice, causing death mostly by metastasis and not by the primary tumor [16, 43]. Polyphenols, particularly EGG, reportedly have a role in metastasis control in 4T1 models  and that their activity is linked to a decrease in proliferating cell nuclear antigen, resulting in a decreased cell proliferation rate.
Tumor progression into a malignant phenotype is favored by different signals generated by microenvironmental factors such as proinflammatory cytokines and adhesion molecules as well as conditions that support the tumor microenvironment by interacting with specific tumor cell receptors , promoting breast cancer progression . Thus, high IL-6 serum levels are associated with lower survival, and at later stages, IL-6 can contribute to tumor growth progression . We found that IL-6 serum levels in mice treated with P2Et fraction were significantly lower than those in untreated mice. Similarly, MCP-1 levels were reduced, although not significantly. The latter findings of the decrease in leukemoid reaction and direct activity over tumor cells allow us to conclude that P2Et fraction, besides reducing primary tumor diameter and weight, lessens the migration to distant organs by mechanisms involving the tumor microenvironment as shown by the decreased serum IL6. With regards to data demonstrating that treatment of tumor-bearing mice with P2Et fraction results in significant reduction in serum IL-6, we hypothesize that locally produced IL-6 may improve apoptosis mechanisms and are consumed immediately by tumor cells during its death process thus acting in an autocrine fashion as previously discussed. We concur that tumor reduction after P2Et treatment cannot simply explain IL-6 serum reduction and hypothesize that some other immune-regulatory mechanisms are mediated by the P2Et fraction cannot be excluded, including the inhibition of suppressor macrophages.
Overall, P2Et fraction decreases associated risk factors and primary tumor size, supporting its potential use as an adjuvant in ER(+) breast cancer treatment. Moreover, it is important to note that antitumor activity of the fraction is observed in immunocompetent mice; consequently, tumor microenvironment modulation together with antitumor immune response activation allows for control of distant metastases. We are currently evaluating this hypothesis in our laboratory.