4-Methoxydalbergione is a potent inhibitor of human astroglioma U87 cells in vitro and in vivo

Abstract

Astroglioma is the most common primary tumor in the central nervous system without effective treatment strategies. Temozolomide (TMZ) is a chemotherapeutic drug to treat astroglioma but exhibits low potency and has side effects. Therefore, there is an urgent need to develop new compounds to treat astroglioma. Dalbergia sissoo Roxb was the source of Dalbergia odorifera in traditional Chinese medicine (TCM) and has been clinically used as an anti-tumor medicine. 4-Methoxydalbergione (4MOD) is purified from Dalbergia sissoo Roxb., and shows an inhibitory effect on osteosarcoma, but its effects on astroglioma have not been reported. Here, we evaluate its anti-astroglioma effects on both in vitro and in vivo models. In cultured astroglioma U87 cells, 4MOD inhibited cell proliferation and induced cell apoptosis in a time- and concentration-dependent manner. Compared with TMZ, 4MOD exhibited a tenfold greater potency of anti-astroglioma effects. 4MOD effectively stalled the cell cycle in G2 phase. Transcriptome sequencing (RNA-seq) showed that 4MOD upregulated 158 genes and downregulated 204 genes that are mainly enriched in cell membrane, cell division, cell cycle, p53, TNF, and MAPK signaling pathways, which may underlie its anti-tumor mechanisms. In a nude mouse xenograft model transplanted with U87 cells, 10 mg/kg 4MOD slowed down tumor growth rate, while at 30 mg/kg dose, it reduced tumor size. Collectively, this study demonstrates that 4MOD is a potent native compound that remarkably inhibits U87 astroglioma growth in both in vitro and in vivo models.

Keywords: 4‐methoxydalbergione; Dalbergia sissoo Roxb; RNA-seq; apoptosis; cell cycle; human astroglioma U87 cell line.

Fig. 1. Effects of 4MOD and TMZ on cell growth of U87 astroglioma cells.

a Morphological and cell density changes of U87 cells observed after 48 h treatment with different concentrations of 4MOD (top) and TMZ (bottom). b Effects of 4MOD on U87 cell proliferation using a CCK-8 assay. Bar graph shows that 4MOD inhibited U87 cell proliferation in a concentration-dependent manner between 2.5 and 25.0 µM, but did not show a treatment time-dependent manner between 48 and 72 h. c Effect of TMZ on U87 cell proliferation. TMZ inhibited U87 cell proliferation, like 4MOD, but with lower potency. d IC₅₀ after 4MOD and TMZ treatment in U87 cells at different treatment time. Data are presented as the mean ± SEM, n = 5, ***P < 0.001, ****P < 0.0001 vs. control group for (b) and (c), and in (d), **P < 0.01 and ***P < 0.001 present the comparisons between 4MOD and TMZ.

Fig. 2. Effect of 4MOD and TMZ on apoptosis and the cell cycle of U87 astroglioma cells.

a Cells treated with different concentrations of 4MOD and TMZ for 48 h. Cell apoptosis was detected by flow cytometry. The lower left represents normal cells, the lower right represents early apoptotic cells, the upper right represents late apoptotic cells, and the upper left represents dead or late apoptotic cells. b, c Analysis of the altered rates of early apoptosis, late apoptosis, and total apoptosis after 4MOD and TMZ treatment. d Cells treated with different concentrations of 4MOD and TMZ for 48 h, stained with propidium iodide (PI), and subjected to flow cytometry analysis to determine their distribution in each phase of the cell cycle. Different colors from left to right represent G0/G1, S, and G2/M phase cells, respectively. e, f Altered cell cycle distribution of U87 cells after 48 h treatment with different 4MOD and TMZ concentrations. Results are expressed as means ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the control group. g Comparison of apoptosis rates between 4MOD and TMZ, ***P < 0.001, 4MOD vs. TMZ. h Comparison of cell cycle between 4MOD and TMZ, **P < 0.01, ***P < 0.001, 4MOD vs. TMZ.

Fig. 3. GO/KEGG pathway enrichment analysis of DEGs.

a Volcano plots analyzing differential expressions of mRNA in U87 cells. Green dots represent downregulated genes, and red dots represent upregulated genes. b Histogram showing the differentially expressed mRNA in U87 cells, 158 genes were upregulated, and 204 genes were downregulated. c Differentially expressed mRNA on the top 20 pathways identified by KEGG analysis. Bubble size represents the number of DEGs enriched in a signaling pathway; the bubble color represents the q value. d GO enrichment analysis of the significantly upregulated and downregulated genes. Red columns are the numbers of up-regulated DEGs related to this function, and green columns are the numbers of down-regulated DEGs; DEGs were chosen by a fold change ≥ 2 and q < 0.01.

Fig. 4. Validation of DEGs by qRT-PCR.

a Protein–protein interaction network of the differentially expressed genes after 4MOD treatment. The color of the circle represents the action type of the corresponding interaction relationship and is a qualitative attribute; the size of the circle represents quantitative attributes; the line represents the functional correlation of the prediction, and the thickness represents the degree of confidence of the interaction. b Relative expression of DEGs as verified by qRT-PCR. The results indicated that the relative expressions of ATF4, SESN2, HSPB1, and IL-1A in the 4MOD group were higher than control group. c The expression of Bcl-2, IL-6, CCNB1, CCNA2, CCNB2, CDC20, MCM7, PLK1, BUB1B, BUB1, CDK1, MAPSK6, and RRM2 in the 4MOD group were lower than control group. Results are expressed as the means ± SEM, n = 3, **P < 0.01; ***P < 0.001 vs. the control group.

Fig. 5. 4MOD inhibites U87 tumor growth in BALB/c-nu mice.

a Ultrasound images (2D images) of the cross-section view of tumors collected using a small animal ultrasonic 3D imaging platform. The blue lines represent measurements of the tumor boundary at intervals of 1.0 mm on this section. b 3D reconstruction of tumors and the tumor volume determined using 3D fitting. Tumor volume was also calculated by this procedure. c Drug administration time and tumor volume change curves. d Tumor tissue sizes after 4MOD treatment. e Tumor weight plot. Results are expressed as the means ± SEM, n = 6, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs. the control group. f Expression of CCNB1, CCNB2, CDK1, p53, BCL-2, and BAX in tumors of the 4MOD groups and control group. Results are expressed as the means ± SEM, n = 3, *P < 0.05; **P < 0.01; ***P < 0.001, ****P < 0.0001 vs. the control group.