Purpose: Many anticancer drugs induce apoptosis in malignant cells, and resistance to apoptosis could lead to suboptimal or no therapeutic benefit. Two cytoplasmic proteins, B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and Bcl-2, act as a promoter and an inhibitor of apoptosis, respectively. Both Bax and Bcl-2 as well as their ratio have been regarded as prognostic markers in various cancers. However, conflicting results have been reported. A clear understanding of apoptosis has also become crucial due to reports about anti-Bcl-2 chemotherapy. We explored the relationship of Bax and Bcl-2 gene expression and their ratio with the therapeutic response in acute myeloid leukemia (AML) patients.
Patients and methods: Bone marrow and/or blood samples from 90 AML patients treated with cytarabine and daunorubicin were included. Expression of Bax and Bcl-2 was determined through real-time polymerase chain reaction by using ΔΔCt method of relative expression.
Results: Bax and Bcl-2 expression among marrow and blood samples correlated with each other (rs=0.5, p<0.01). Although bone marrow expression of Bax and Bcl-2 tended to remain higher among responders (median 1.01 and 0.29, respectively) as compared to non-responders (median 0.66 and 0.24, respectively), the difference failed to reach statistical significance (U=784.5 and 733; p=0.68 and 0.28, respectively). Conversely, Bax/Bcl-2 ratio was higher among poor responders (median 3.07 vs 1.78), though again failed to reach statistical significance (U=698.5, p=0.07).
Conclusion: Expression of Bax and Bcl-2 does not differ significantly among AML patients treated with cytarabine and daunorubicin in terms of remission, relapse, resistance, overall survival, and disease-free survival, thus questioning the utility of emerging anti-Bcl-2 therapy.
Keywords: anthracyclines, cytarabine, Bcl-2, Bax Bcl-2 ratio, anti Bcl-2 therapy, BH3 mimetic inhibitors
Acute myeloid leukemia (AML) is frequently a fatal malignancy in adults.1,2 Chemotherapy is the primary treatment of AML, which commonly employs cytarabine and anthracyclines such as daunorubicin.3 It is typically administered in two phases: an induction phase followed by a consolidation phase. After induction chemotherapy is given, the patient’s bone marrow and blood are analyzed for complete remission (CR) between days 21 and 28. Unfortunately, a substantial number of patients do not respond to chemotherapy. In addition, AML patients who respond to chemotherapy often relapse later.3–6 Relapse is defined as >5% blast cells in bone marrow, or reappearance of blast cells in blood, or development of blasts from any sites other than bone marrow after CR is achieved. Relapse occurs usually within first 3 years from the end of the chemotherapy,3 especially in young patients.5 Different relapse rates have been reported, ranging between 21% and 39%, from various parts of the world.7,8 Thus, resistance to chemotherapy is a common observation and major obstacle in treating AML patients. Even though patients may respond to anticancer drugs, their overall survival (OS) remains low.5,9
Anticancer drugs eradicate cancer cells either by disrupting cellular pathways vital for cell survival or by activating programmed cell death (apoptosis). Apoptosis is the final executer of many anticancer drugs. This cell suicide can be achieved by extrinsic or intrinsic pathways. The extrinsic pathway is mainly involved in controlling the cell turnover as well as eliminating mutant cells, while the intrinsic pathway is involved in antineoplastic drug action.10,11 DNA strand breaks accumulate after chemotherapy which trigger the intrinsic pathway of apoptosis in cancer cells.11 In apoptosis, B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) protein activates the cascade of reactions by releasing cytochrome c from the mitochondria that helps in successive activation of caspases and ultimately leads to cell death. Bcl-2 is believed to prevent Bax from releasing cytochrome c, thus restricting downstream activation of apoptotic machinery. This will result in cell survival but at the same time Bcl-2 is also involved in retiring proliferating cells back to G0 phase of the cell cycle.12 Both Bax and Bcl-2 are cytoplasmic proteins.13 Release of cytochrome c from mitochondrial matrix is considered to work as an inevitable call for cell death.14 DNA fragmentation activates Bax and inhibits Bcl-2 through p53.10,11
Dysregulation of apoptosis in cancer cells is considered to be one of the mechanisms of multidrug resistance.10 Many studies have investigated Bax and Bcl-2 in AML as well as other cancers and have yielded conflicting results. It has been proposed that high Bax and/or low Bcl-2 as well as high Bax/Bcl-2 ratio favors apoptosis and hence may lead to a favorable outcome,15–19 whereas others have reported contradictory observations.18,20,21 Thus, despite ongoing research, our understanding of this process and its impact on therapeutic outcome is still inadequate. This is further complicated by the current focus to develop anti-Bcl-2 drugs and their anticipated role in cancer therapy.22,23 Hence, we designed this study to explore the relationship of chemotherapeutic response to Bax and Bcl-2 gene expression in newly diagnosed AML patients using their bone marrow as well as peripheral blood samples and investigate their role as biomarkers of chemotherapy outcome.