Salvia officinalis L. exerts oncostatic effects in rodent and in vitro models of breast carcinoma

Abstract

Introduction: Based on extensive data from oncology research, the use of phytochemicals or plant-based nutraceuticals is considered an innovative tool for cancer management. This research aimed to analyze the oncostatic properties of Salvia officinalis L. [Lamiaceae; Salviae officinalis herba] using animal and in vitro models of breast carcinoma (BC). Methods: The effects of dietary administered S. officinalis in two concentrations (0.1%/SAL 0.1/and 1%/SAL 1/) were assessed in both syngeneic 4T1 mouse and chemically induced rat models of BC. The histopathological and molecular evaluations of rodent carcinoma specimens were performed after the autopsy. Besides, numerous in vitro analyses using two human cancer cell lines were performed. Results and Conclusion: The dominant metabolites found in S. officinalis propylene glycol extract (SPGE) were representatives of phenolics, specifically rosmarinic, protocatechuic, and salicylic acids. Furthermore, the occurrence of triterpenoids ursolic and oleanolic acid was proved in SPGE. In a mouse model, a non-significant tumor volume decrease after S. officinalis treatment was associated with a significant reduction in the mitotic activity index of 4T1 tumors by 37.5% (SAL 0.1) and 31.5% (SAL 1) vs. controls (set as a blank group with not applied salvia in the diet). In addition, salvia at higher doses significantly decreased necrosis/whole tumor area ratio by 46% when compared to control tumor samples. In a rat chemoprevention study, S. officinalis at a higher dose significantly lengthened the latency of tumors by 8.5 days and significantly improved the high/low-grade carcinomas ratio vs. controls in both doses. Analyses of the mechanisms of anticancer activities of S. officinalis included well-validated prognostic, predictive, and diagnostic biomarkers that are applied in both oncology practice and preclinical investigation. Our assessment in vivo revealed numerous significant changes after a comparison of treated vs. untreated cancer cells. In this regard, we found an overexpression in caspase-3, an increased Bax/Bcl-2 ratio, and a decrease in MDA, ALDH1, and EpCam expression. In addition, salvia reduced TGF-β serum levels in rats (decrease in IL-6 and TNF-α levels were with borderline significance). Evaluation of epigenetic modifications in rat cancer specimens in vivo revealed a decline in the lysine methylations of H3K4m3 and an increase in lysine acetylation in H4K16ac levels in treated groups. Salvia decreased the relative levels of oncogenic miR21 and tumor-suppressive miR145 (miR210, miR22, miR34a, and miR155 were not significantly altered). The methylation of ATM and PTEN promoters was decreased after S. officinalis treatment (PITX2, RASSF1, and TIMP3 promoters were not altered). Analyzing plasma metabolomics profile in tumor-bearing rats, we found reduced levels of ketoacids derived from BCAAs after salvia treatment. In vitro analyses revealed significant anti-cancer effects of SPGE extract in MCF-7 and MDA-MB-231 cell lines (cytotoxicity, caspase-3/-7, Bcl-2, Annexin V/PI, cell cycle, BrdU, and mitochondrial membrane potential). Our study demonstrates the significant chemopreventive and treatment effects of salvia haulm using animal or in vitro BC models.

Keywords: Salvia officinalis L.; apoptosis; breast carcinoma; cancer stem cells; cell proliferation; epigenetics; human carcinoma cell lines; inflammatory cytokines.

FIGURE 1

The LC-DAD-analysis of the propylene glycol extract of S. officinalis. (A) The UV signal showing the presence of phenolics, (B) a total ion current chromatogram (fast polarity switching mode).

FIGURE 2

Allograft 4T1 model in mice. (A) The volume of 4T1 mammary adenocarcinomas in mouse allograft model after S.officinalis treatment. (B) The mitotic activity index after the treatment with S. officinalis extract in 4T1 tumors in Balb/c mice. The mitotic figures are highlighted in circles; H&E staining; magnification ×400. CONT–control group, SAL 0.1 – a group with dietary applied salvia at a concentration of 1 g/kg in the diet, SAL 1 – a group with dietary applied salvia at a concentration of 10 g/kg in the diet. Data are shown as mean ± SEM.

FIGURE 3

Immunohistochemical analyses of rat carcinoma cells in vivo after S. officinalis treatment. (A) Immunoexpression of cleaved caspase-3 (cytoplasmic), Bax, Bcl-2, Ki67, VEGFA, VEGFR-2, and MDA in rat tumor samples. (B) Immunoexpression of cancer stem cell markers in rat tumor samples. (C) Immunoexpression of H3K4m3, H3K9m3, H4K16ac, and H4K20m3 markers in rat tumor samples.

FIGURE 4

Representative pictures of caspase-3, Bax, Bcl-2, Ki67, VEGFA, VEGFR-2, MDA, CD24, CD44, ALDH1A1, EpCam, H3K4m3, H3K9m3, H4K20m3, and H4K16ac expressions gained from rat BC samples. For analysis, polyclonal caspase-3 antibody (Bioss, Woburn, USA), polyclonal Bax and Bcl-2 antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA), monoclonal Ki67 antibody (Dako, Glostrup, Denmark), monoclonal VEGFA and VEGFR-2 antibodies (Santa Cruz Biotechnology, Paso Robles, CA, USA), polyclonal CD24 antibody (GeneTex, Irvine, CA, USA), polyclonal CD44 antibody (Boster, Pleasanton, CA, USA), polyclonal ALDH1A1 antibody (ThermoFisher, Rockford, IL, USA), polyclonal MDA, EpCAM, H3K4m, H3K9m3, and H4K20m3 antibodies (Abcam, Cambridge, MA, USA), and monoclonal H4K16ac antibody (Abcam, Cambridge, MA, USA) were applied. The final microscope magnification of ×400 was used.

FIGURE 5

Relative miRNA expression of miR21, miR155, miR210, miR22, miR34a, and miR145 in rat BC specimens. MiR-191-5p was selected as the internal control miRNA to normalize the cDNA levels of the samples. Data are shown as mean ± SEM. A significant difference, *p < 0.05, ***p < 0.001 vs. CONT, ⁺ p < 0.05 vs. SAL 0.1.

FIGURE 6

Promoter methylation status of ATM, PITX2, RASSF1A, PTEN, and TIMP3 tumor-suppressor genes in rat BC specimens. The level of methylation was designated using all evaluated CpG isles of the above-mentioned promoters. The brackets indicate the number of evaluated isles. Data are shown as mean ± SEM. A significant difference, **p < 0.01, ***p < 0.001 vs. CONT group and ⁺⁺ p < 0.01, ⁺⁺⁺ p < 0.001 vs. SAL 0.1 group.

FIGURE 7

Serum levels of cytokines in rat chemoprevention study. Data are shown as mean ± SEM. TGF-β, transforming growth factor-beta; IL-6, Interleukin 6; IL-10, Interleukin 10; TNF-a, tumor necrosis factor-alpha. A significant difference, *p < 0.05, **p < 0.01 vs. CONT group.

FIGURE 8

Relative concentrations of plasma metabolites in rats with mammary carcinomas. Data are shown as mean ± STD, normalized to the mean of controls set to 1. A significant difference, *p < 0.05, **p < 0.01, ***p < 0.001 vs. CONT.

FIGURE 9

Relative survival of BC cells after salvia extract treatment. (A) Salvia propylene glycol extract (10–1,000 μg/mL) analyzed by resazurin metabolic assay, (B) propylene glycol dilutant analyzed by resazurin metabolic assay, (C) salvia extract (10–1,000 μg/mL) analyzed by BrdU assay. Data are shown as mean ± SD using three independent studies. Significant difference, *p < 0.05, **p < 0.01, ***p < 0.001 vs. CONT (untreated).

FIGURE 10

Sample diagrams of cell cycle distribution in BC cell lines after salvia extract treatment. (A) MCF-7 lines, (B) MDA-MB-231 lines; SPGE was applied at 160 or 130 μg/mL, resp.

FIGURE 11

Sample diagrams of apoptotic cell diversification. (A) MCF-7 line, (B) MDA-MB-231 line; salvia extract was applied at 160 and 130 μg/mL, resp.

FIGURE 12

Effect of salvia extract on apoptosis parameters in BC cell lines analyzed by flow cytometry. (A) caspase-7 activation (MCF-7), (B) caspase-3 activation (MDA-MB-231), (C, D) PARP cleavage (MCF-7 and MDA-MB-231 cells). Salvia extract was applied at 160 and 130 μg/m resp. Data are based on three independent studies. Significant difference, **p < 0.01, ***p < 0.001 vs. CONT.

FIGURE 13

Mitochondrial membrane potential (MMP) in BC cell lines after salvia extract treatment. (A) MCF-7 cell line, (B) MDA-MB-231 cell line; salvia extract was applied at 160 and 130 μg/mL, resp. Data are based on three independent studies. Significant difference, **p < 0.01, ***p < 0.001 vs. CONT.

FIGURE 14

Flow cytometric evaluation of mitochondria-associated Bcl-2 protein in BC cell lines after salvia extract treatment. (A) Total Bcl-2, (B) phosphorylated Bcl-2; salvia extract was applied at 160 and 130 μg/mL, resp. Data are based on three independent studies. Significant difference, *p < 0.05, **p < 0.01, ***p < 0.001 vs. CONT.