Salidroside enhances the anti-cancerous effect of imatinib on human acute monocytic leukemia via the induction of autophagy-related apoptosis through AMPK activation

Abstract

As the typical tyrosine kinase inhibitor, imatinib has been the first-line antineoplastic agent for both chronic myeloid leukemia and acute lymphoblastic leukemia. However, a large number of patients are still resistant to the benefits of imatinib, and they have a dissatisfactory prognosis. Salidroside, a compound that is extracted from natural plants, has been reported to have an excellent anticancer effect and few side effects. In the present study, we have developed a new combination therapy strategy of salidroside and imatinib for combating the growth of acute lymphoblastic leukemia. As demonstrated by the anti-proliferation assay, salidroside exhibited excellent cytotoxicity against myeloid leukemia cells. Moreover, cells treated by the combination therapy of salidroside and imatinib displayed a clear lower growth rate than cells only treated by imatinib, indicating that salidroside has a positive effect on enhancing the cytotoxicity of imatinib against leukemia cells. Subsequently, the underlying mechanisms were investigated. The results revealed that autophagy marker proteins in leukemia cells, including LC3, p62, and Beclin1, displayed a significant expression change after treating them with salidroside plus imatinib, with the levels of LC3 and Beclin1 dramatically increasing while the expression of p62 was significantly decreased. Moreover, an obvious down-regulation of p-PI3K, p-AKT and p-mTOR expression levels in leukemia cells after treatment with salidroside plus imatinib suggested that the PI3K/mTOR pathway plays an important role in the process of cell apoptosis induced by salidroside or imatinib. Further studies showed that pre-incubating the cells with an autophagy inhibitor dramatically inhibited the ability of imatinib to induce autophagy, but did not inhibit the ability of salidroside. The underlying causes were subsequently explored and the results showed that silencing AMPKα1, the most important regulator of autophagy, dramatically attenuates the ability of salidroside to induce cell apoptosis. These results together indicated that salidroside enhances the cytotoxicity of imatinib on acute monocytic leukemia via the induction of autophagy-related apoptosis through AMPK activation. The unique advantages of combination therapy were further confirmed by in vivo experiments, with the tumor-bearing cells treated with salidroside plus imatinib achieving the best anti-tumor effect.

Fig. 1. Cell viabilities of THP-1 and U937 cells after receiving therapy with salidroside, imatinib, or the combination therapy strategy. (A) Salidroside exists in Rhodiola rosea, and the molecular structure is shown. THP-1 and U937 cells were treated with different concentrations of imatinib (B) and salidroside (C) for 48 h, and the cell viability was detected by a CCK-8 assay. For further determination of the ratio of salidroside and imatinib in the combination therapy strategy, both THP-1 and U937 cells were treated by various combination therapy strategies: 2 mM salidroside + 10 μM imatinib (D), 2 mM salidroside + 20 μM imatinib (E), and 2 mM salidroside + 30 μM imatinib (F).

Fig. 2. Salidroside combined with imatinib inhibits proliferation and promotes apoptosis in AML cells. THP-1 and U937 cells were treated with PBS (control), 2 mM salidroside, 20 μM imatinib, and 2 mM salidroside + 20 μM imatinib, respectively. (A) CCK-8 assays were performed to measure the proliferation abilities of THP-1 and U937 cells (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group). (B and C) Cell apoptosis was examined by Hoechst staining, and the cell apoptosis rate was quantitatively analyzed (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group). (D) Bcl-2 and Bax expressions were examined by Western blot assay, and the protein quantification was analyzed according to the results of an RT-qPCR assay (E). GAPDH was used as the internal reference (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group).

Fig. 3. Salidroside combined with imatinib induces autophagy in AML in vitro. THP-1 and U937 cells were treated with PBS (control), 2 mM salidroside, 20 μM imatinib, and 2 mM salidroside + 20 μM imatinib, respectively. (A) The protein expression levels of LC3, P62 and Beclin 1 were analyzed by Western blot assay in THP-1 and U937 cells, and the expression levels were quantitatively analyzed by RT-qPCR experiments (B) (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group). (C) The protein expression levels of AMPKα1, t-PI3K, p-PI3K, t-AKT, p-AKT, t-mTOR, and p-mTOR were analyzed by Western blot assay in the THP-1 and U937 cells, and the expression levels were quantitatively analyzed by RT-qPCR experiments (D) (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group).

Fig. 4. 3-MA relieves the induction of salidroside in combination with imatinib on AML autophagy in vitro. THP-1 cells were treated with PBS (control), 2 mM salidroside, 2 mM salidroside + 20 μM imatinib, 3-MA + 20 μM imatinib, and 3-MA + 20 μM imatinib + 2 mM salidroside, respectively. (A) LC3, P62 and Beclin 1 expressions were assessed by Western blot assay in THP-1 and U937 cells, and the expression levels were quantitatively analyzed by RT-qPCR experiments (B) (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group, & vs. 3-MA + imatinib group). (C and D) LC3 expression was detected by an immunofluorescence (IF) assay (*P < 0.05 vs. control group; ^#P < 0.05 vs. imatinib group, and vs. 3-MA + imatinib group).

Fig. 5. Silencing of AMPKα1 attenuates the induction of salidroside in combination with imatinib on AML autophagy in vitro. THP-1 cells were transfected with a control (si-Ctrl) and with AMPKα1 siRNAs (si-AMPKα1), respectively. AMPKα1 expression was evaluated by Western blot assay (A), and the relative expression level was recorded by RT-qPCR experiments (B) (***P < 0.001). THP-1 cells were treated with PBS (control), 2 mM salidroside, 20 μM imatinib, and 2 mM salidroside + 20 μM imatinib, respectively. After treatment, the cells were transfected with a control (si-Ctrl) and with AMPKα1 siRNAs (si-AMPKα1), respectively. AMPKα1 expression was assessed by Western blot assay (C), and the relative intensity of LC3II/LC3I was recorded by RT-qPCR experiments (D) (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group). (E) Cell proliferation was measured by the CCK-8 assay in THP-1 cells treated as in B (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group). (F) Cell apoptosis was detected by Hoechst staining in THP-1 cells treated as in B (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group).

Fig. 6. Salidroside in combination with imatinib promotes the anti-tumor effect of AML in vivo. (A) Photographs of tumor and (B) tumor growth curves after treatment with various strategies. (C) The heart, liver, lung and kidney sections of the treated mice were used in an HE-staining assay. The magnification was 400× for heart, 400× for liver, 400× for lung and 200× for kidney. (D) The tumor weight, (E and F) the number of apoptotic cells (TUNEL assay), and (G and H) Ki67 expression (IHC assay) were measured in wild type mice and in AMPKα1 knockout mice (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group). (I) LC3, P62 and Beclin 1 expression levels were analyzed by Western blot assay and RT-qPCR experiments (J) in WT mice and in AMPKα1 knockout mice, and the levels were also quantitatively analyzed (*P < 0.05 vs. control group; ^#P < 0.05 vs. salidroside group, and vs. 3-MA + imatinib group).