Marsdenia tenacissimae extraction (MTE) inhibits the proliferation and induces the apoptosis of human acute T cell leukemia cells through inactivating PI3K/AKT/mTOR signaling pathway via PTEN enhancement

Abstract

Marsdenia tenacissimae extraction (MTE) as a traditional Chinese herb has long been used to treat some diseases such as tumors in China. However, the potential effectiveness of MTE in leukemia has not yet been fully understood, and the related molecular mechanism is still unknown. In the present study, we aimed to evaluate the effects of MTE on the proliferation and apoptosis of Jurkat cells (T-ALL lines) and lymphocytes from T-ALL (T-cell acute lymphoblastic leukemia) patients. Firstly, CCK8 assays and flow cytometry assays revealed that MTE dose-dependently reduced the proliferation of Jurkat cells by arresting cell cycle at S phase. Secondly, Annexin V-FITC/PI-stained flow cytometry and TUNEL staining assays showed that MTE promoted the apoptosis of Jurkat cells. Mechanistically, MTE enhanced PTEN (phosphatases and tensin homolog) level and inactivated PI3K/AKT/mTOR signaling pathway in Jurkat cells, which mediated the inhibition of cell proliferation by MTE and MTE-induced apoptosis. Finally, MTE significantly inhibited the proliferation and promoted the apoptosis of lymphocytes from T-ALL patients, compared with lymphocytes from healthy peoples. Taken together, these results reveal an unrecognized function of MTE in inhibiting the proliferation and inducing the apoptosis of T-ALL cells, and identify a pathway of PTEN/PI3K/AKT/mTOR for the effects of MTE on leukemia therapy.

Keywords: Marsdenia tenacissimae extraction; PTEN; T-cell acute lymphoblastic leukemia; apoptosis; proliferation.

Figure 1. MTE reduced the viability of Jurkat and Molt-4 cell lines

A. CCK8 assays were performed on Jurkat cells after 24 h of MTE treatment at an ascending concentration range (from 0 to 640 μg/ml) (n=18). Effects on cell viability were presented as a function of μg drug concentration (log scale). Corresponding IC₅₀ value was calculated with the appropriate software (graphpad prism). B. CCK8 assays were performed on Jurkat cells after 24 h, 48 h, 72 h of MTE treatment at 60, 120, 240 μg/ml, respectively (n=18)(^**P < 0.01). C. CCK8 assays were performed on Molt-4 cells after 24 h of MTE treatment at 60, 120, 240 μg/ml (n=18). D. CCK8 assays were performed on Molt-4 cells after 48 h of MTE treatment at 60, 120, 240 μg/ml (n=18). Data were mean ± s.d. ^**P < 0.01, Student's t-test, compared with control. Controls were treated with 0.1% DMSO.

Figure 2. MTE inhibited the proliferation of Jurkat cells by arresting cell cycle at S phase

A-C. Flow cytometric analysis of cell cycle distributions of Jurkat cells treated by control (A), 60 μg/ml MTE (B), 120 μg/ml MTE treatment (C). D. Quantified data of cell cycle distribution of Jurkat cells under various conditions (n=3). Data were mean ± s.d. **P < 0.01, Student's t-test, compared with control. Controls were treated with 0.1% DMSO.

Figure 3. MTE induced the apoptosis of Jurkat cells

A-C. Flow cytometric analysis of Annexin-V-FITC/PI stained Jurkat cells treated with control (0.1% DMSO, A), MTE 60 μg/ml (B), MTE 120 μg/ml (C). Cells are characterized as healthy cells (bottom left quadrant), necrotic (top left quadrant), early apoptotic (bottom right quadrant), and late apoptotic (top right quadrant). D. Quantitative analysis of apoptosis rate of Jurkat cells under various concentrations of MTE as shown in (A-C) (n=3). E. TUNEL assay detected the apoptosis of Jurkat cells treated without or with MTE 60 μg/ml for 24 h. Scale bar, 20 μm. F. Quantitative analysis of the percentages of TUNEL positive cells over total cells in one filed shown in (E) (n=3). G and H. Western blot detected the downstream signaling, cleaved-PARP (c-PARP), Bcl-2, Bax (G) and cleaved-Caspase3 (c-Caspase3) (H) by MTE treatment. I-L. Quantitative analysis of the relative Bcl-2 (I), c-PARP (J), Bax (K) and c-Caspase3 (L) in (G-H) (n=3 per group, normalized to control). Data were mean ± s.d. **P < 0.01, Student's t-test, compared with control. Controls were treated with 0.1% DMSO.

Figure 4. MTE inactivated PI3K/AKT/mTOR signaling pathway though enhancing PTEN

A, D. Western blot detected the downstream signaling, PTEN, p-mTOR, mTOR (A), and p-AKT, AKT (D) in Jurkat cells by MTE treatment at the indicated concentration for 24 h. B, C, E. Quantitative analysis of the relative PTEN (B), p-mTOR/mTOR (C) and p-AKT/AKT (E), as shown in (A, D) (n=3 per group, normalized to control). F. Double immunocytochemistry of PTEN (green) and FAS (red) in Jurkat cells treated with MTE at the indicated concentrations for 24 h. Scale bar, 10 μm. G. Quantitative analysis of the relative fluorescent PTEN level as shown in (F) (n=20 cells per group, normalized to control). Data were mean ± s.d. *P < 0.05 and **P < 0.01, Student's t-test, compared with control. Controls were treated with 0.1% DMSO.

Figure 5. PTEN inhibitor BPV blocked MTE's effects in Jurkat cells, whereas PI3K inhibitor wortmanin enhanced MTE's effects in Jurkat cells

A. Western blot detected the downstream signaling, PTEN, p-mTOR, mTOR, p-AKT, AKT and Bax in Jurkat cells treated with control, MTE (60 μg/ml) and/or BPV (1 μM) or wortmanin (50 nM). GAPDH as an internal control. B-D. Quantitative analysis of the relative PTEN (B), Bax (C), p-AKT/AKT (D) as shown in (A) (n=3 per group, normalized to control). Data were mean ± s.d. ^**P < 0.01, Student's t-test, compared with control. ^##P < 0.01, Student's t-test, compared with MTE treatment. Controls were treated with 0.1% DMSO.

Figure 6. PTEN inhibitor BPV blocked MTE's cell cycle arresting effects, whereas PI3K inhibitor wortmanin enhanced MTE's cell cycle arresting effects in Jurkat cells

A-D. Representative photographs of cell cycle distributions analyzed by flow cytometer assay. Jurkat cells were treated with control media (A) or MTE (B, 60 μg/ml) or MTE plus BPV (C, 1 μM) or MTE plus wortmanin (D, 50 nM) for 24 h. E. Quantified data of cell cycle distribution as shown in (A-D) (n=3). Data were mean ± s.d. ^**P < 0.01, Student's t-test, compared with control. ^#P < 0.01 Student's t-test, compared with MTE treatment. Controls were treated with 0.1% DMSO.

Figure 7. PTEN inhibitor BPV blocked MTE's apoptosis induction effects, whereas PI3K inhibitor wortmanin enhanced MTE's apoptosis induction effects in Jurkat cells

A-F. Flow cytometric analysis of Annexin-V-FITC/PI stained Jurkat cells treated with control media (A) or MTE (B) or BPV (C) or wortmanin (D) or MTE plus BPV (E) or MTE plus wortmanin (F). Cells are characterized as healthy cells (bottom left quadrant), necrotic (top left quadrant), early apoptotic (bottom right quadrant), and late apoptotic (top right quadrant). G. Quantified data of apoptosis rate of Jurkat cells were shown as in (A-F). Data were mean ± s.d. H. Working model of signaling pathways induced by MTE and the effects of MTE in T-ALL cells. ^**P < 0.01, Student's t-test, compared with control. ^#P < 0.01 Student's t-test, compared with MTE treatment. Controls were treated with 0.1% DMSO.

Figure 8. MTE reduced the viability and induced apoptosis of primary lymphocytes from T-ALL patients

A and B. CCK8 assays were performed on lymphocytes from healthy people (A) (n=6) or T-ALL patients (B) (n=6) treated by MTE for 24 h. C and D. Flow cytometric analysis of Annexin-V-FITC/PI stained lymphocytes from healthy people treated by control (C) or MTE (D) for 24 h. E. Quantitative analysis of apoptosis rate of lymphocytes from healthy people as shown in (C-D) (n=6 each group). F and G. Flow cytometric analysis of Annexin-V-FITC/PI stained lymphocytes from T-ALL patients treated by control (F) or MTE (G) for 24 h. H. Quantitative analysis of apoptosis rate of lymphocytes from T-ALL patients as shown in (F-G) (n=6 each group). Data were mean ± s.d. ^**P < 0.01, Student's t-test, compared with control. Controls were treated with 0.1% DMSO.