Gallotannin-rich Caesalpinia spinosa fraction decreases the primary tumor and factors associated with poor prognosis in a murine breast cancer model

Abstract

Background: Several treatment alternatives are available for primary breast cancer, although those for metastatic disease or inflammation associated with tumor progression are ineffective. Therefore, there is a great need for new therapeutic alternatives capable of generating an immune response against residual tumor cells, thus contributing to eradication of micrometastases and cancer stem cells. The use of complex natural products is an excellent therapeutic alternative widely used by Chinese, Hindu, Egyptian, and ancestral Latin-American Indian populations.

Methods: The present study evaluated cytotoxic, antitumor, and tumor progression activities of a gallotannin-rich fraction derived from Caesalpinia spinosa (P2Et). The parameters evaluated in vitro were mitochondrial membrane depolarization, phosphatidylserine externalization, caspase 3 activation, DNA fragmentation, and clonogenic activity. The parameters evaluated in vivo were tumor growth, leukocyte number, metastatic cell number, and cytokine production by flow cytometry.

Results: The in vitro results showed that the P2Et fraction induced apoptosis with mitochondrial membrane potential loss, phosphatidylserine externalization, caspase 3 activation, DNA fragmentation, and decreased clonogenic capacity of 4T1 cells. In vivo, the P2Et fraction induced primary tumor reduction in terms of diameter and weight in BALB/c mice transplanted with 4T1 cells and decreased numbers of metastatic cells, mainly in the spleen. Furthermore, decreases in the number of peripheral blood leukocytes (leukemoid reaction) and interleukin 6 (IL-6) serum levels were found, which are events associated with a poor prognosis. The P2Et fraction exerts its activity on the primary tumor, reduces cell migration to distant organs, and decreases IL-6 serum levels, implying tumor microenvironment mechanisms.

Conclusions: Overall, the P2Et fraction lessens risk factors associated with tumor progression and diminishes primary tumor size, showing good potential for use as an adjuvant in breast cancer ER(+) treatment.

Figure 1

MMP loss, PS externalization, and activation of caspase 3 in 4T1 cells induced by P2Et fraction. (a) 4T1 cells treated with P2Et fraction for 6, 12, 24, or 48 h, stained with JC-1, and analyzed by flow cytometry. Ethanol (negative control) and valinomycin (positive control) (***p < 0.001). (b) PS externalization in 4T1 cells by flow cytometry after treatment for 24 h and 48 h with P2Et fraction, doxorubicin (positive control), or ethanol (negative control). Top panel is a representative dot plot showing the various treatments; bottom is a representative histogram showing percent (%) PS/PI cells after 24 and 48 h of treatment. (c) Caspase 3 activity by enzyme-linked immunosorbent assay (ELISA) after cell treatment with P2Et fraction or doxorubicin (positive control) for 48 h (***p <0.001).

Figure 2

P2Et fraction decreases 4T1 cell nuclear segmentation and clonogenic activity. (a) 4T1 cells treated with P2Et fraction for 48 h and stained with DAPI. White arrows show apoptotic cells or fragmented nuclei observed under a fluorescence microscope (40×). (b) 4T1 cells treated with P2Et fraction, ethanol (negative control), and doxorubicin (positive control). Data represent the mean number of colonies ± SEM of three independent experiments (***p < 0.001) versus control.

Figure 3

IL-6 and MCP-1 production by 4T1 cells after treatment with P2Et fraction in vitro. 4T1 cells were treated with P2Et fraction and doxorubicin for 6, 12, 24, or 48 h, and IL-6 (a, b) or MCP-1 (c, d) Secretion was determined by flow cytometry (***p < 0.001) versus control.

Figure 4

Tumor growth inhibition and decrease in leukemoid reaction in BALB/C mice by P2Et fraction. BALB/c mice implanted SC with 1 × 10⁴ 4T1 cells for 5 days and randomly divided into three groups. Group 1 was treated with PBS (vehicle), group 2 was treated with 18.7 mg/kg of P2Et fraction, and group 3 was treated with 9.3 mg/kg of P2Et fraction. Leukocyte count was determined weekly using a hemocytometer (ABX Micros 60). After 31 days, all animals were sacrificed. (a) Tumor volume. The graph represents the mean of each group with 7–8 animals per group. (b) Tumor weight. The graph represents the total animals by each group. For these experiments we used 7–8 animals per group and each experiment was performed independently two times. (c) Leukocyte count. The graph represents the total number animals used per group. For these experiments we used 7–8 animals per group and each experiment was performed independently two times.

Figure 5

Reduction of spleen metastasis and decreased IL-6 serum levels in BALB/c mice treated with P2Et fraction. After treatment, primary tumors and lung, liver, and spleen tissues were dissected and fixed in 10% formaldehyde, embedded in paraffin, and stained with H&E. Metastatic infiltrations were evaluated in each tissue and counted using a microscope with a micrometric grid (magnification power, 10×). (a) Tissue Images. White arrows show metastatic tumor infiltration, white dotted arrows show alveolar septum, black arrows show adipocytes, and black dotted arrows show necrotic areas. (b) Graphic shows tissue cell number in each (***p < 0.001) versus control. The graphs represents mean of each group with 7–8 animals by group and each experiment was performed independently two times. (c) After treatment, mice serum was collected by cardiac puncture, and cytokine estimation was performed using a CBA mouse inflammation kit. (c) IL-6 secretion. (d) MCP-1 secretion (***p < 0.001) versus control. The graphs represents mean of each group with 7–8 animals by group and each experiment was performed independently two times.

Figure 6

Tumor growth and lung metastases formation is decreased after treatment with P2Et fraction. BALB/c mice were randomly assigned in control or treatment groups (7 mice per group). One week prior to tumor cell inoculation (day 0), the treatment group started P2Et fraction consumption administered in water. 4T1 cells (1 × 10⁴) were injected into the right mammary fat pad SC on day 7. After 33 days, all animals were sacrificed. (a) Tumor volume. The graph represents the mean of each group with 7 animals per group. (b) Lung metastatic number. White tumor nodules against a blue lung background were counted by two independent observers. The graph represents the total animals by each group. For these experiments we used 5 animals per group.