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

Abstract

DLBS1442 is a bioactive fraction extracted from the fruit of the native Indonesian plant, Phaleria macrocarpa (Scheff.) Boerl (Thymelaceae). This bioactive fraction is a potential treatment for dysmenorrhea and endometriosis. The present study investigated the pharmacological action of DLBS1442 in endometrial cells. The effect of various doses of DLBS1442 (0-200 μg/mL) over 24 hours was studied using the human endometrial RL95-2 cell line to observe its effect on angiogenesis, cell migration, estrogen and progesterone receptor levels, the eicosanoid pathway, cell viability, and apoptosis. The impact of DLBS1442 on nuclear factor kappa B (NFκB) and the eicosanoid pathway was also studied through its marker gene expression using a quantitative real-time polymerase chain reaction method. DLBS1442 showed an ability to inhibit angiogenesis and cell migration in a dose-dependent manner. At a dose of 100 μg/mL, DLBS1442 increased the cell population in sub-G1 phase from 7% to 34%. DLBS1442 also significantly downregulated the estrogen receptor level and upregulated the progesterone receptor level. Further, it inhibited the eicosanoid signaling pathway by reducing the NFκB transcription level and subsequent reduction of inducible nitric oxide synthase. A dose-dependent decrease in viability and increased apoptosis in RL95-2 cells were also evident after exposure to DLBS1442, where the IC50 was obtained at around 100 μg/mL. In conclusion, DLBS1442 is a potential agent for alleviating symptoms of endometriosis via its antiangiogenic, anti-inflammatory, and proapoptotic activity.

Keywords: anti-inflammatory; eicosanoid pathway; estrogen receptor; progesterone receptor.

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.