Apoptotic and Non-Apoptotic Modalities of Thymoquinone-Induced Lymphoma Cell Death: Highlight of the Role of Cytosolic Calcium and Necroptosis

Simple Summary Diffuse large B cell lymphoma (DLBCL) represents the most common type of non-Hodgkin lymphoma with a high curability rate. However, 40% of patients will relapse or exhibit refractory disease, and compromised apoptotic pathways is among the prognosis-worsening factors. Therefore, drugging non-apoptotic modalities might be therapeutically promising. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells. Herein, we show that TQ selectively kills DLBCL cells, either cell lines or primary lymphoma cells bearing resistance features to standard treatment. Investigations show that, although TQ induced apoptotic markers, non-apoptotic death was the major mechanism responsible for TQ-induced cellular demise. We demonstrate critical and selective roles of cytosolic calcium and necroptosis in TQ-induced non-apoptotic cell death. Finally, TQ exhibits an improved selectivity profile over conventional chemotherapy. Collectively, this work provides new insights into the mode of action of TQ and points to the therapeutic relevance of non-apoptotic modalities as a fail-safe mechanism for pro-apoptotic DLBCL therapies. Targeting non-apoptotic modalities might be therapeutically promising in diffuse large B cell lymphoma (DLBCL) patients with compromised apoptotic pathways. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells, but little is known about its effect on non-apoptotic pathways. This work investigates TQ selectivity against DLBCL cell lines and the cell death mechanisms. TQ reduces cell viability and kills cell lines with minimal toxicity on normal hematological cells. Mechanistically, TQ promotes the mitochondrial caspase pathway and increases genotoxicity. However, insensitivity of most cell lines to caspase inhibition by z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) pointed to a critical role of non-apoptotic signaling. In cells dying through non-apoptotic death, TQ increases endoplasmic reticulum (ER) stress markers and substantially increases cytosolic calcium ([Ca2+](c)) through ER calcium depletion and activation of store-operated calcium entry (SOCE). Chelation of [Ca2+](c), but not SOCE inhibitors, reduces TQ-induced non-apoptotic cell death, highlighting the critical role of calcium in a non-apoptotic effect of TQ. Investigations showed that TQ-induced [Ca2+](c) signaling is primarily initiated by necroptosis upstream to SOCE, and inhibition necroptosis by necrostatin-1 alone or with z-VAD-fmk blocks the cell death. Finally, TQ exhibits an improved selectivity profile over standard chemotherapy agents, suggesting a therapeutic relevance of the pro-necroptotic effect of TQ as a fail-safe mechanism for DLBCL therapies targeting apoptosis.

Automated summary

Thymoquinone (TQ) selectively kills DLBCL cells through non-apoptotic cell death mechanisms, with cytosolic calcium and necroptosis playing critical and selective roles. TQ shows improved selectivity over standard chemotherapy, suggesting therapeutic relevance of its pro-necroptotic effect as a fail-safe mechanism for DLBCL therapies targeting apoptosis.