Thiophene derivative inflicts cytotoxicity via an intrinsic apoptotic pathway on human acute lymphoblastic leukemia cells

Abstract

In an effort to identify novel anti-cancer agents, we employed a well-established High Throughput Screening (HTS) assay to assess the cytotoxic effect of compounds within the ChemBridge DIVERSet Library on a lymphoma cell line. This screen revealed a novel thiophene, F8 (methyl 5-[(dimethylamino)carbonyl]-4-methyl-2-[(3-phenyl-2-propynoyl) amino]-3-thiophenecarboxylate), that displays anti-cancer activity on lymphoma, leukemia, and other cancer cell lines. Thiophenes and thiophene derivatives have emerged as an important class of heterocyclic compounds that have displayed favorable drug characteristics. They have been previously reported to exhibit a broad spectrum of properties and varied uses in the field of medicine. In addition, they have proven to be effective drugs in various disease scenarios. They contain anti-inflammatory, anti-anxiety, anti-psychotic, anti-microbial, anti-fungal, estrogen receptor modulating, anti-mitotic, kinase inhibiting and anti-cancer activities, rendering compounds with a thiophene a subject of significant interest in the scientific community. Compound F8 consistently induced cell death at a low micromolar range on a small panel of cancer cell lines after a 48 h period. Further investigation revealed that F8 induced phosphatidylserine externalization, reactive oxygen species generation, mitochondrial depolarization, kinase inhibition, and induces apoptosis. These findings demonstrate that F8 has promising anti-cancer activity.

Fig 1. Chemical structure of F8 (methyl 5-[(dimethylamino)carbonyl]-4-methyl-2-[(3-phenyl-2-propynoyl)amino]-3-thiophenecarboxylate).

Fig 2. F8’s induction of apoptosis.

F8 compound-induced apoptosis in CEM cells. F8 ability to induce apoptosis was assessed in a phosphatidylserine externalization assay and measured through flow cytometry. Analysis was performed following a 24 h incubation with 24 h CC₅₀ and 2X CC₅₀ (2.89 μM and 5.78 μM). Controls included DMSO as the vehicle, H₂O₂ as the positive, and untreated. Significant phosphatidylserine externalization was evident for the CC₅₀ and 2X CC₅₀, given the p-value p<0.00001 (***).

Fig 3. Depolarization of the mitochondria.

Significant mitochondrial depolarization was induced by compound F8 on CEM cells following a 4 h incubation treated with the CC₅₀ and 2X CC₅₀ (2.89 μM and 5.78 μM). The cells were stained with JC-1 reagent and analyzed by flow cytometry. Statistical analyses were obtained through the two-tailed Students paired t-test. The controls that were included are the same as previously stated.

Fig 4. F8 induced ROS accumulation.

Significant ROS was induced by F8 CC₅₀ and 2X CC₅₀ (2.89 μM and 5.78 μM) in CCRF-CEM cells compared to the vehicle control, DMSO, following an 18 h incubation period. Statistical analyses were acquired using a two-tailed t-test, p = 0.0005 and p = 0.003.

Fig 5. F8 induces caspase activation.

Compound F8 CC₅₀ and 2X CC₅₀ (2.89 μM and 5.78 μM) induced significant caspase 3/7 activation in CEM cells. The cells were incubated for 8 h and stained with NucView 488 caspase-3/7 substrate. Statistical analyses were obtained using the two-tailed Student’s paired t-test compared to the vehicle control (1% DMSO).

Fig 6. F8 induced DNA fragmentation on CEM cells, a hallmark of apoptosis.

F8 cytotoxicity was analyzed via flow cytometer after a 72 h exposure on CEM cells utilizing CC₂₅ and CC₁₀ (1.44 μM and 0.57 μM). NIM-DAPI was used to stain each cell’s DNA amount and quantified in the flow. F8 displays DNA fragmentation evident in the Sub G0-G1 phase. Controls were included, DMSO as the vehicle and H₂O₂ as the positive control, and untreated cells, respectively.

Fig 7. F8 causes hypophosphorylation in JAK/STAT pathway.

Human Phosphorylation Kinase Array C55 was conducted on the CCRF-CEM cell line and treated with 2X CC₅₀ (5.78 μM). This assay was used to determine the phosphorylation status of MAPK and JAK-STAT membranes to gauge phosphorylation. Fold changes were determined using densitometry. Hyperphosphorylation was observed in the MAPK membrane versus the JAK-STAT. Hypophosphorylation was displayed in the JAK 1–2 and STAT 2–5.