All-Trans Retinoic Acid Enhances the Anti-Leukemia Effect of Venetoclax on Acute Myeloid Leukemia Cells

Objective: Acute myeloid leukemia (AML) is a malignant disease of the hematopoietic system in which arrested maturation, excessive proliferation and inhibition of apoptosis have been observed in the process of hematopoietic stem cell differentiation. Some patients, especially elderly patients, do not tolerate cytotoxic chemotherapy, and some patients are resistant to chemotherapeutic drugs, consequently molecular targeting therapy has brought new hope for AML treatment. Venetoclax (ABT-199) is a selective inhibitor of the antiapoptotic protein Bcl-2. Increased expression of Bcl-2 is an important factor in the inhibition of apoptosis in leukemia cells. The use of a single drug is often limited because of resistance, and a better curative effect can be obtained in combination with other drugs. All-trans retinoic acid (all-trans-retinoic acid, ATRA) was first used by Chinese doctor to treat acute promyelocytic leukemia (APL). Many studies have shown that ATRA can inhibit the expression of Bcl-2 in AML cells and promote apoptosis. We aimed to test for the first time whether the combination of ATRA and venetoclax would produce a stronger anti-AML effect.

Methods: The effects of ATRA and ABT-199 on the ABT-199-resistant AML cell lines OCI-AML3 and THP-1 and the sensitive cell lines MV-4-11 and MOLM-13 were observed in vitro. The following methods were used in this study. 1. CCK8 assay was used to detect cell proliferation. 2. Annexin V/FITC and PI double staining were used to detect apoptosis rate by flow cytometry. 3. Flow cytometry was used to detect the expression of CD11b. 4. PI single staining and flow cytometry were used to study the cell cycle. 5. Western blotting was used to detect the expression of Bcl-2, Bax, Mcl-1, PARP, caspase3, GSK-3β and Cyclin-D1 proteins in OCI-AML3 cells.

Results:1. Compared with the control group and the ATRA or ABT-199 single drug group, cell proliferation was significantly inhibited in the combined drug group, the apoptosis rate was significantly increased, and the cell cycle was blocked in G1 phase. 2. The expression of CD11b in the ATRA or ABT-199 group and combination group was significantly higher than that in the control group. 3. In OCI-AML3 cells, compared with the ABT-199 drug group, the expression of Bcl-2, Mcl-1 and Cyclin-D1 decreased, and the expression of Bax, cleaved-PARP, cleaved caspase3 and p-GSK-3β expression increased in the combined treatment group.

Conclusion: All-trans retinoic acid (ATRA) can enhance the anti-leukemia effect of ABT-199 on AML cell lines by inhibiting cell proliferation as seen by the cell cycle arrest of AML cells in G1 phase, promoting apoptosis and inducing differentiation. ATRA can reverse the drug resistance of AML cells to ABT-199 by inhibiting the expression of Bcl-2 and Mcl-1. Our observations may provide new strategies and theoretical support for the clinical treatment of AML.

Key Words:AML, Venetoclax, ATRA, Apoptosis