Andrographolide's Dual Induction of Apoptosis and Ferroptosis Linked to Caspase-3 and FACL4 Regulation in T-Cell Acute Lymphoblastic Leukemia Cells
Authors: Doi Hiroki , Akiyama Hidehiko , Shiogama Kazuya , Fujii Sumie , Matsuura Hideaki , Miura Yasuo 
Published: December 2024

Andrographolide (Andro) was found to have a cell-killing effect on T-cell acute lymphoblastic leukemia cells (T-ALL) in a drug screening. Although, Andro has been shown to inhibit cell proliferation in solid tumors derived from gastric and liver cancers, there have been few reports of its effects in hematologic tumor cell lines. Past studies consistently showed that Andro suppressed cell viability and proliferation although the underlying mechanisms varied between different cell types. This study investigates Andro's potential to induce ferroptosis, an iron-dependent form of cell death, in T-ALL.

Jurkat cells, a T-ALL cell line, were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin at 37℃ with 5% CO2. Cells were treated with Andro, cytarabine (Ara-C), vincristine (VCR), or Erastin. In some experiments, an antioxidant, N-acetyl-L-cysteine (NAC), was added one hour prior to these treatments. Cell viability was measured using an MTT assay. Apoptosis was assessed morphologically using Wright-Giemsa staining and quantified through Annexin V staining with a Muse Cell Analyzer. The production of reactive oxygen species (ROS) was measured using a Muse Oxidative Stress Kit. Mitochondrial membrane potential was evaluated to detect depolarization. Western blotting was used to analyze the expression level of cytochrome c, with detection carried out using a LuminoGraph I Imaging system. Cell cycle analysis was performed to determine the percentages of cells in each phase of the cell cycle, using a Muse Cell Cycle Kit. The iron content within the cells was measured using the LABOSPECT 008 analyzer. The expression of cleaved caspase-3 and the ferroptosis-inducing marker fatty acid CoA ligase 4 (FACL4) was analyzed by immunohistochemistry. Combination index was evaluated by Chou-Talalay method using CompuSyn software (ComboSyn Inc, Paramus, New Jersey) to assess the combined effect of Andro and Ara-C or VCR.

In the MTT assay conducted 24 hours after the addition of Andro (1-100 µM), a concentration-dependent inhibition of cell proliferation was observed, with an IC50 of 7.4 µM. The percentage of Annexin V positive cells was 56.5% after 24 hours of treatment with Andro at a concentration of 50 μM, which decreased to 22.3% following the addition of NAC. Intracellular ROS positivity and mitochondrial membrane potential reduction were 52.8% and 80.0%, respectively, after treatment of Andro at 50 μM. These values decreased to 7.0% and 24.5%, respectively, following the addition of NAC. However, no reduction in the positivity rate was observed when NAC was added to the treatments with Ara-C and VCR. Immunohistochemistry analysis of the expression levels of cleaved caspase-3 showed an increase in expression after treatment with Andro at 50 μM. Cell cycle analysis revealed that untreated cells had 48.5% in the G0/G1 phase, 21.4% in the S phase, and 30.1% in the G2/M phase. Ara-C treatment increased the G0/G1 phase to 70.2%, and VCR treatment led to an increase in the G2/M phase to 65.2%. Ara-C and VCR notably affected cell cycle phases, but Andro did not significantly alter the distribution compared to untreated cells. The Fe2+ content in Jurkat cells was was 4.4 times higher than that in normal lymphocytes, prompting the examination of ferroptosis, an iron-dependent cell death process. The expression of FACL4, a key regulator of ferroptosis, increased following treatment with the addition of Andro at 50 μM, as well as with Erastin at 5 μM, a known ferroptosis-inducing agent, compared to untreated cells. Additionally, transmission electron microscopy revealed mitochondrial contraction, a chacteristic feature of ferroptosis, in cells treated with Andro (50 μM) and Erastin (5 μM), compared to untreated cells. The combination index of Andro with Ara-C or VCR was synergistic effect (CI &lt; 1).

In summary, Andro significantly inhibited Jurkat cell proliferation in a concentration-dependent manner, causing cell shrinkage and nuclear fragmentation. Andro induced apoptosis via the intrinsic pathway, increasing ROS, decreasing mitochondrial membrane potential, and releasing cytochrome c. Additionally, Andro induced ferroptosis, characterized by increased iron content and FACL4. Andro's dual induction of apoptosis and ferroptosis suggests its potential as a therapeutic agent for T-ALL, either alone or in combination with existing anti-cancer drugs like Ara-C or VCR.