Apigenin and Luteolin Attenuate the Breaching of MDA-MB231 Breast Cancer Spheroids Through the Lymph Endothelial Barrier in Vitro

Abstract

Flavonoids, present in fruits, vegetables and traditional medicinal plants, show anticancer effects in experimental systems and are reportedly non-toxic. This is a favorable property for long term strategies for the attenuation of lymph node metastasis, which may effectively improve the prognostic states in breast cancer. Hence, we studied two flavonoids, apigenin and luteolin exhibiting strong bio-activity in various test systems in cancer research and are readily available on the market. This study has further advanced the mechanistic understanding of breast cancer intravasation through the lymphatic barrier. Apigenin and luteolin were tested in a three-dimensional (3-D) assay consisting of MDA-MB231 breast cancer spheroids and immortalized lymph endothelial cell (LEC) monolayers. The 3-D model faithfully resembles the intravasation of breast cancer emboli through the lymphatic vasculature. Western blot analysis, intracellular Ca2+ determination, EROD assay and siRNA transfection revealed insights into mechanisms of intravasation as well as the anti-intravasative outcome of flavonoid action. Both flavonoids suppressed pro-intravasative trigger factors in MDA-MB231 breast cancer cells, specifically MMP1 expression and CYP1A1 activity. A pro-intravasative contribution of FAK expression in LECs was established as FAK supported the retraction of the LEC monolayer upon contact with cancer cells thereby enabling them to cross the endothelial barrier. As mechanistic basis, MMP1 caused the phosphorylation (activation) of FAK at Tyr397 in LECs. Apigenin and luteolin prevented MMP1-induced FAK activation, but not constitutive FAK phosphorylation. Luteolin, unlike apigenin, inhibited MMP1-induced Ca2+ release. Free intracellular Ca2+ is a central signal amplifier triggering LEC retraction through activation of the mobility protein MLC2, thereby enhancing intravasation. FAK activity and Ca2+ levels did not correlate. This implicates that the pro-intravasative contribution of FAK and of Ca2+ release in LECs was independent of each other and explains the better anti-intravasative effects of luteolin in vitro. In specific formulations, flavonoid concentrations causing significant anti-intravasative effects, can certainly be achieved in vivo. As the therapeutic strategy has to be based on permanent flavonoid treatment both the beneficial and adverse effects have to be investigated in future studies.

Structural Insight into the In Vitro Anti-Intravasative Properties of Flavonoids

“…MMP2 and MMP9 contribute to the intravasation in the MCF-7/LEC model [13] and this can explain the anti-intravasative properties of chrysin and luteolin. This is also in agreement with the observation that 20 µM luteolin inhibited the matrix metallo-protease 1 (MMP1) in MDA-MB231 cells [16]. MMP1, MMP2, and MMP9 are triggering the retraction of LECs [37] and the formation of CCIDs.…”

“…In comparison to 14, the most active compound, and its 4 -O-methylderivative 15, with only one-tenth of its activity, the 6-methoxyderivative nepetin (16) resulted in an IC 50 value of 79 µM.…”

“…Thus, luteolin, chrysin, and apigenin stabilize the endothelial barrier function by inhibiting NF-κB in LECs despite the de-stabilizing effects of 12(S)-HETE. Another mechanism of luteolin and apigenin is the inhibition of FAK phosphorylation at Tyr397 and thereby attachment/detachment oscillations at the matrix, which is necessary for LEC migration and CCID formation [16].…”

Apigenin induces apoptosis and counteracts cisplatin‑induced chemoresistance via Mcl‑1 in ovarian cancer cells

“…Apigenin is a part of the average daily diet (7)(8)(9). It has been shown that apigenin can significantly suppress malignant cell growth in cultivated cells and in vivo malignant models (10)(11)(12)(13). Apigenin can also inhibit malignant invasion and metastasis, while downregulating downstream mitogen-activated protein kinases and oncogenes (14).…”

Whole Transcriptomic Analysis of Apigenin on TNFα Immuno-activated MDA-MB-231 Breast Cancer Cells

“…Apigenin is a natural compound which has the capacity to antagonize several mitigating events in human tumor development and tumor immune evasion. These include the capability to reduce inflammation, inhibit casein kinase 2 (CK2), matrix metalloproteinases 9 and 1, protein kinase C (18), cytochrome P450 1A1 (19) topoisomerase I (20) tumor growth factors (21), CCL2-induced release by TNFαactivated breast cancer (22) and blockade of drug resistanceassociated extrusion pumps (23,24). In the current study, we expand on our previous work by evaluating the effects of apigenin on TNFα-treated MDA-MB-231 cells (22) by analyzing the whole transcriptome.…”