Evaluating the effects of various ethanolic medicinal plant extracts on metastatic breast cancer proliferation, invasion, and expression of a novel potential drug target; CD82 metastatic suppressor protein, and on in vivo angiogenesis using the ex ovo yolk sac membrane (YSM) assay.

PURPOSE: Breast cancer metastasis relies on cellular invasion and angiogenesis facilitated by the downregulation of metastatic suppressor proteins like Cluster of Differentiation 82 (CD82). Currently, no medicines target multiple systems to prevent metastatic progression through CD82 upregulation. This study screened for plant extracts displaying effects on cell proliferation, invasion, and CD82 expression in breast cancer cells, and in vivo angiogenesis, and further correlated between the biological activities and effect on CD82 expression. METHODS: Seventeen ethanolic plant extracts were screened for their effect on cell proliferation (against MDA-MB-231 and MCF-7 breast cancer and Hek293 kidney cells), cell invasion and effect on CD82 expression in metastatic MDA-MB-231 cells. Selected extracts were further evaluated for in vivo anti-angiogenesis. RESULTS: Extracts displayed varying antiproliferative activity against the different cell lines, and those that showed selectivity indexes (SI) > 0.5 against MDA-MB-231 were selected for anti-invasion evaluation. Buddleja saligna Willd. (BS), Combretum apiculatum Sond. (CA), Foeniculum vulgare, Greyia radlkoferi, Gunnera perpensa and Persicaria senegalensis (Meisn.) Soják (PS) displayed 50% inhibitory concentration (IC50) values of 44.46 ± 3.46, 74.00 ± 4.48, 180.43 ± 4.51, 96.97 ± 2.29, 55.29 ± 9.88 and 243.60 ± 2.69 µg/mL, respectively against MDA-MB-231, and compared to Hek293 showed SI of 0.9, 0.7, 1.4, 1.1, 2.2 and 0.5. Significant invasion inhibition was observed at both 20 and 40 µg/mL for BS (94.10 ± 0.74 and 96.73 ± 0.95%) and CA (87.42 ± 6.54 and 98.24 ± 0.63%), whereas GR (14.91 ± 1.62 and 41 ± 1.78%) and PS (36.58 ± 0.54 and 51.51 ± 0.83%), only showed significant inhibition at 40 µg/mL, and FV (< 5% inhibition) and GP (10 ± 1.03 and 22 ± 1.31%) did not show significant inhibition at both concentrations. Due to the significant anti-invasive activity of BS, CA and PS at 40 µg/mL, these extracts were further evaluated for their potential to stimulate CD82. BS showed significant (p < 0.05) reduction in CD82 at 20 and 40 µg/mL (13.2 ± 2.2% and 20.3 ± 1.5% decrease, respectively), whereas both CA and PS at 20 µg/mL increased (p < 0.05) CD82 expression (16.4 ± 0.8% and 5.4 ± 0.6% increase, respectively), and at 40 µg/mL significantly reduced CD82 expression (23.4 ± 3.1% and 11.2 ± 2.9% decrease, respectively). Using the yolk sac membrane assay, BS (59.52 ± 4.12 and 56.72 ± 3.13% newly formed vessels) and CA (83.33 ± 3.17 and 74.00 ± 2.12%) at both 20 and 40 µg/egg showed significant (p < 0.001) angiogenesis inhibition, with BS showing statistical similar activity to the positive control, combretastatin A4 (10 nmol/egg), whereas PS only displayed significant (p < 0.001) angiogenesis stimulation at 40 µg/egg (120.81 ± 3.34% newly formed vessels). CONCLUSION: BS exhibits antiproliferative, anti-invasive, and anti-angiogenic activity despite inhibiting CD82, suggesting an alternative mode of action. CA at 20 µg/mL shows moderate anti-invasive and anti-angiogenic potential by stimulating CD82, while at 40 µg/mL it still displays these properties but inhibits CD82, suggesting an additional mode of action. PS, with the least antiproliferative activity, stimulates CD82 and inhibits angiogenesis at 20 µg/mL but inhibits CD82 and increases angiogenesis at 40 µg/mL, indicating CD82 targeting as a major mode of action. Future studies should explore breast cancer xenograft models to assess the extracts' impact on CD82 expression and angiogenesis in the tumor microenvironment, along with isolating bioactive compounds from the extracts.

Evaluation of Wound Healing and Antibacterial Potential of Greyia radlkoferi Szyszyl. Ethanolic Leaf Extract.

Angiogenesis is an essential mechanism in both physiological and pathological functions, such as wound healing and cancer metastasis. Several growth factors mediate angiogenesis, including vascular endothelial growth factor (VEGF) and platelet derived growth factor (PDGF). This study evaluated the potential wound healing activity of Greyia radlkoferi Szyszyl (GR) and its effect on growth factors regulating angiogenesis. The ethanolic leaf extract of GR was evaluated for antibacterial activity against wound associated bacteria; Staphylococcus aureus and Pseudomonas aeruginosa. It exhibited antibacterial activity against two strains of S. aureus (ATCC 25293 and ATCC 6538) displaying a minimum inhibitory concentration (MIC) at 250 and 500 µg/ml, respectively. The antioxidant activity of the extract was investigated for nitric oxide (NO) scavenging activity and showed a fifty percent inhibitory concentration (IC50) of 1266.5 ± 243.95 µg/ml. The extract was further investigated to determine its effect on the proliferation and modulation of growth factors secreted by human keratinocytes (HaCaT). Its effect on wound closure was evaluated using the scratch assay, where non-toxic concentrations were tested, as determined by the antiproliferative assay against HaCat cells (IC50 > 400 µg/ml). Results showed that the extract significantly inhibited wound closure, with a percentage closure of 60.15 ± 1.41% (p < 0.05) and 49.52 ± 1.43% (p < 0.01) at a concentration of 50 and 100 µg/ml, respectively, when compared to the 0.25% Dimethyl sulfoxide vehicle control (65.86 ± 1.12%). Quantification of secreted growth factors from cell-free supernatant, collected from the scratch assay, revealed that the extract significantly decreased the concentration of platelet-derived growth factor (PDGF-AA) at both 50 (p < 0.05) and 100 µg/ml (p < 0.001) (443.08 ± 77.36 and 178.98 ± 36.60 pg/ml) when compared to the 0.25% DMSO vehicle control (538.33 ± 12.64 pg/ml). Therefore, whilst the extract showed antibacterial activity against wound associated bacteria, it did not induce wound healing but rather showed a significant inhibition of wound closure, which was confirmed by the inhibition of PDGF-AA, a major growth factor involved in angiogenesis. Therefore, the GR extract, should be considered for further investigation of anti-angiogenic and anti-metastatic properties against cancer cells.