In Vitro Investigation of the Cytotoxic Activity of Emodin 35 Derivative on Multiple Myeloma Cell Lines

Abstract

Background: Bortezomib is used for treating multiple myeloma (MM); however, it has considerable adverse effects. Emodin has been reported to exhibit inhibitory effects on MM cell lines. We investigated the efficacy of emodin 35 (E35), an emodin derivative, using U266 and MM1s cell lines in treating MM and the efficacy of combining bortezomib and E35.

Methods: MTT assays were used to observe the effects of E35 on MM cell growth. The effects on cellular apoptosis were then observed using Annexin V/propidium iodide (PI) staining assay. The expression of apoptosis-related genes, including the caspase family, was examined. The efficacy of combining bortezomib and E35 was investigated by examining the expression of the Akt/mTOR/4EBP1 signaling pathway-related proteins.

Results: We report that E35 inhibited the growth of U266 and MM1s cells by inducing cellular apoptosis. Moreover, E35 downregulated the expression of apoptosis-related genes and suppressed the phosphorylation of Akt/mTOR/4EBP1 signaling pathway-related genes, thus exhibiting synergistic effects with bortezomib. All observed effects were dose-dependent.

Conclusion: The results showed that E35 exhibited cytotoxic effects in MM cell lines in protein levels. Thus, E35, particularly in combination with bortezomib, may be considered as a promising treatment for MM; however, this requires further investigation in vivo.

Figure 1

Effects of emodin derivative (E35) on myeloma cell lines. (a) Chemical structure of E35. (b) Inhibitory effects of E35 on U266 and MM1s cell growth after 48 h incubation. (c) Growth curve of U266 cells in different E35 levels. (d) Growth curve of MM1s cells in different E35 levels. Data are presented as means ± SD; OD = Optical Density.

Figure 2

Results of the Annexin V/PI staining assay. (a) Representative images of the Annexin V/PI staining assay of E35 effects on U266 cells. (b) Representative images of the Annexin V/PI staining assay of E35 effects on MM1s cells. (c) Quantitative analysis of the Annexin V/PI staining assay of the apoptotic rate in the U266 and MM1s cell lines induced by E35. Data are presented as means ± SD. ∗∗ means p < 0.01 vs. control group.

Figure 3

Quantitative analysis of mRNA expression of C-Myc (a), Bcl-2 (b), Mcl-1 (c), and Pim2 (d) in U266 and MM1s cell lines treated by E35. Data are presented as means ± SD. ∗ means p < 0.05 vs. control group.

Figure 4

Relative expression of apoptosis-related proteins and caspase family induced by E35. (a) Representative images of western blot. (b) Quantitative results of relative expression of apoptosis-related proteins and caspase family in U266 cells induced by E35 treatment. (c) Quantitative results of relative expression of apoptosis-related proteins and caspase family in MM1s cells induced by E35 treatment. Data are presented as means ± SD. ∗ means p < 0.05; ∗∗ means p < 0.01 vs. control group.

Figure 5

Relative expression of the Akt/mTOR/4EBP1 signaling pathway-related proteins induced by E35. (a) Representative images of western blot. (b) Quantitative results of relative expression of Akt/mTOR/4EBP1 signaling pathway-related proteins in U266 cells induced by E35 treatment. (c) Quantitative results of relative expression of Akt/mTOR/4EBP1 signaling pathway-related proteins in MM1s cells induced by E35 treatment. Data are presented as means ± SD. ∗ means p < 0.05; ∗∗ means p < 0.01 vs. control group.

Figure 6

Relative expression of C-Myc, Mcl-1, NF-κB, 4EBP1, p-4EBP1, EIF4E, and p-EIF4E proteins affected by administration of E35 and bortezomib in U266 cells. (a) Representative images of western blot. (b) Quantitative results of relative expression of C-Myc, Mcl-1, NF-κB, 4EBP1, p-4EBP1, EIF4E, and p-EIF4E proteins. Data are presented as means ± SD. ∗ means p < 0.05; ∗∗ means p < 0.01 vs. control group.