Proteasomal inhibition compromises microvascular integrity via distinct effects on non-immune endothelial cells and
Prajakta Sawant1,2, Hannah Wolf1,2, Aleena Mathew2
1Doctoral Program in Biology (Molecular, Cellular & Developmental Biology), The Graduate Center, The City University of New York, New York, NY, United States.
Abstract:
Chemotherapeutic proteasomal inhibitors Bortezomib (BTZ, reversible inhibitor) and Carfilzomib (CFZ, irreversible inhibitor) are established frontline therapies for multiple myeloma (MM). However, their on-target versus off-target bystander effects in the bloodstream are not clearly known. To elucidate the acute adverse effects of intravenous proteasomal inhibition, we studied some of the key cell types of blood microvasculature: barrier endothelial cells, peripheral blood mononuclear cells (PBMC)s and multiple myeloma (MM1.S) cells. BTZ, but not CFZ, is cytotoxic to microvascular endothelial cells from lung or brain at 100 nM concentration. In human pulmonary microvascular endothelial cell (HPMEC)s, BTZ induces multiple stress pathways: proteotoxic stress, endoplasmic reticulum (ER) stress, and mitochondrial reactive oxygen species (ROS) accumulation. This is associated with activation of apoptotic caspase (CASP)9 (mediator of intrinsic-apoptosis), CASP8 (mediator of extrinsic-apoptosis) and CASP3 (terminal executioner of apoptosis). BTZ-treated HPMECs significantly die and lose vascular barrier function by 24 hours post treatment. These events occur independently of external cues. When we compared 100 nM of BTZ to 250 nM of CFZ (1:2.5 being the ratio of concentration in patients), both compounds elicit comparable cytotoxicity, but elevated ER stress and apoptotic markers are driven by BTZ. Both compounds kill multiple myeloma cells comparably. PBMCs resist drug-mediated changes. Neither inhibitor elicits inflammatory response from HPMECs, MM1.S cells or PBMCs, demonstrating that proteasomal inhibition-mediated death is not inflammatory. As MM is an inflammatory cancer, the natural microvascular environment of patients contains inflammatory cytokines. To model this, we tested the effect of exogenously added tumor necrosis factor alpha (TNF), a key inflammatory cytokine found in MM patients or supernatants from drug-treated MM1.S or PBMCs on HPMECs. Exogenously added TNF or cell-free supernatant from proteasomal inhibitor-treated immune or multiple myeloma cells combined with TNF, further compromises the cellular integrity of HPMECs when compared to drug alone conditions. Physiological levels of TNF enhance stress response in both BTZ- and CFZ-treated cells. This indicates that although proteasomal inhibition is not inflammatory, TNF present in the microvascular environment synergizes with the drugs to compromise endothelial function. Our observations provide an explanation for how microvascular damage potentially underlies tissue injury driven by Bortezomib or Carfilzomib.
Insights
Bortezomib (BTZ) and Carfilzomib (CFZ) are multiple myeloma (MM) drugs. BTZ damages blood vessel cells by inducing stress and apoptosis, while TNF exacerbates this effect, explaining potential tissue injury.
Area of Science:
- Pharmacology
- Cell Biology
- Oncology
Background:
- Bortezomib (BTZ) and Carfilzomib (CFZ) are proteasome inhibitors used for multiple myeloma (MM).
- Their effects on blood vessel cells and potential for tissue injury are not fully understood.
- Understanding these off-target effects is crucial for managing treatment-related adverse events.
Purpose of the Study:
- To investigate the acute adverse effects of intravenous proteasome inhibitors on key blood microvasculature cell types.
- To compare the cytotoxic and stress-inducing effects of BTZ and CFZ on endothelial cells, PBMCs, and MM cells.
- To determine the role of inflammatory cytokines, like TNF, in potentiating drug-induced endothelial cell damage.
Main Methods:
- Treatment of human pulmonary microvascular endothelial cells (HPMECs), peripheral blood mononuclear cells (PBMCs), and multiple myeloma (MM1.S) cells with BTZ and CFZ.
- Assessment of cell viability, apoptosis markers (caspases), and stress pathways (ER stress, ROS).
- Evaluation of endothelial barrier function and the synergistic effects of proteasome inhibitors with tumor necrosis factor alpha (TNF).
Main Results:
- BTZ, but not CFZ, was cytotoxic to HPMECs, inducing proteotoxic, ER, and mitochondrial stress, leading to apoptosis and barrier dysfunction.
- Both BTZ and CFZ exhibited comparable cytotoxicity against MM1.S cells, while PBMCs were resistant.
- TNF significantly exacerbated the cellular damage and stress response in drug-treated HPMECs, indicating a synergistic effect.
Conclusions:
- BTZ induces significant stress and apoptosis in microvascular endothelial cells, compromising vascular barrier function.
- Proteasome inhibitor-mediated cell death is not inherently inflammatory, but inflammatory cytokines like TNF can synergize with these drugs.
- The findings suggest that microvascular damage, potentiated by the inflammatory tumor microenvironment, may underlie tissue injury observed in patients treated with BTZ or CFZ.
