Anti-inflammatory, antiangiogenic, and apoptosis-inducing activity of DLBS1442, a bioactive fraction of Phaleria macrocarpa, in a RL95-2 cell line as a molecular model of endometriosis

Figures & data

Figure 1 Effect of DLBS1442 on angiogenesis-regulating factors and cell migration.

Notes: DLBS1442 synergistically reduced VEGF (A), HIF-1α (B), and MMP-9 (C) transcriptional levels. RL95-2 cells were treated with DLBS1442 in various concentrations and total RNA was extracted after 24 hours of incubation. Expression levels of VEGF, HIF-1α, and MMP-9 mRNA were analyzed by real-time polymerase chain reaction and normalized to β-actin. Expression of MMP-2 and MMP-9 was also measured by gelatin zymography (D). Treatment with DLBS1442 inhibited migration of RL95-2 cells, suggesting an antiangiogenic effect (E).
Abbreviations: C, control; DMSO, dimethyl sulfoxide; HIF-1α, hypoxia-inducible factor-1α; MMP, matrix metalloproteinase; VEGF, vascular endothelial growth factor.

Figure 2 Expression of ERβ and PR-B in DLBS1442-treated RL95-2 cells.

Notes: To evaluate the effect of DLBS1442 on ERβ and PR-B mRNA, RL95-2 cells were treated with four concentrations of DLBS1442 (25, 50, 75, and 100 μg/mL) for 24 hours. mRNA was prepared from the cells and amplified by specific target genes with a β-actin primer as the internal control. From the real-time polymerase chain reaction data, DLBS1442 was shown to downregulate expression of ERβ (A) and PR-B (C). These results are similar to the conventional polymerase chain reaction result (B) and (D). *P<0.05.
Abbreviations: C, control; ERβ, estrogen receptor beta; PR-B, progesterone receptor B.

Figure 3 DLBS1442 works via eicosanoid pathway.

Notes: COX-2 (A), cPLA2 (B), NFκβ (C), and iNOS (D) expression in DLBS1442-treated RL95-2 cells. To evaluate the effect of DLBS1442 on COX-2, cPLA2, NFκβ, and iNOS mRNA, RL95-2 cells were treated with various concentrations of DLBS1442 for 24 hours. Messenger RNA was prepared from the cells and amplified by specific target genes. The figure shows that DLBS1442-mediated inhibition of COX-2, cPLA2, and iNOS expression is attributed to suppressed NFκB activation at the transcriptional level. *P<0.05.
Abbreviations: COX-2, cyclooxygenase-2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; iNOS, inducible nitric oxide synthase; NFκβ, nuclear factor kappa β; cPLA2, cytosolic phospholipase A2.

Figure 4 DLBS1442 inhibits RL95-2 cell viability and induces cellular apoptosis.

Notes: (A) Cell viability assay conducted in RL95-2 cells in the presence of DLBS1442 for 24 hours. (B) FACS cell cycle analysis in control and treated RL95-2 cells (DLBS1442 25 and 100 μg/mL). (C) Immunoblotting analysis of activated caspase-8 and caspase-9 expression in RL95-2 cells.