Abstract
Background: Multiple myeloma (MM) is a malignant neoplasm of plasma cells. Clinically, the proteasome inhibitor bortezomib (BTZ) in combination with other agents remains the mainstay of MM treatment. However, the rising incidence of drug resistance in MM patients underscores the urgent need for novel therapeutic strategies. Triptonide (TN), a small-molecule monomer extracted from the traditional Chinese herb Tripterygium wilfordii Hook, has recently shown potential as an anticancer agent. In this study, we identified the synergistic effect of BTZ combined with triptonide on MM cells and investigated the underlying mechanisms.
Materials and Methods: A high-throughput screening of a 198-compound ubiquitination library was conducted using the cell counting kit-8 (CCK-8) assay. The combination indices (CIs) were calculated by using CompuSyn software. The value of CI below 1 indicates synergy. Cell proliferation was examined by CCK-8, EdU assay, and soft-agar clonogenicity assay. Cell apoptosis and viability were evaluated by flow cytometry and calcein-AM/PI staining, respectively. DNA damage of MM cells was examined by western blotting and comet assay. The synergistic effects between TN and BTZ were determined in MM cell lines (RPMI-8226 and OCI-My5), primary MM cells (n = 10), and MM mouse models. RNA sequencing was carried out to investigate the underlying mechanisms of synergy between TN and BTZ.
Results: To identify novel anti-MM agents, we screened 198 compounds from a ubiquitination compound library in OCI-My5 and RPMI 8226 cell lines. Among those compounds, eleven compounds showed over 90% inhibition and were selected as anti-MM candidates. Testing the synergy between BTZ and these compounds revealed that the combination of BTZ and TN had a strong synergistic effect on MM cells. Subsequently, CCK-8 assay, soft agar colony formation assay, flow cytometry, and calcein-AM/PI staining were performed on MM cells treated with TN alone or in combination with BTZ. As a result, TN effectively inhibited MM cell proliferation and induced apoptosis. Notably, combining TN with BTZ significantly enhanced the inhibition of proliferation, induction of apoptosis, and reduction of cell viability compared to single-agent treatments. The synergy between TN and BTZ was further confirmed in primary MM cells and xenograft MM mouse models.
To explore the synergistic mechanisms, RNA sequencing was conducted to analyze TN-induced gene expression changes in MM cells. Enrichment analysis of differentially expressed genes showed that TN mainly affected DNA damage, cell cycle, and apoptosis-related pathways. Western blotting further confirmed that TN significantly increased γ-H2AX expression, a marker of DNA double-strand breaks. The DNA comet assay also showed that TN markedly elongated comet tail length in MM cells, indicating substantial DNA damage. Notably, combining TN with BTZ caused greater DNA damage than either agent alone. These results indicate that DNA damage may be a key mechanism underlying the synergistic effect of TN and BTZ in MM cells.
Data from our group and others indicated that BTZ induces DNA damage in MM cells primarily by increasing reactive oxygen species (ROS) levels. In contrast, TN did not induce DNA damage through ROS elevation or telomere disruption; instead, it exerted this effect through the inhibition of DNA repair pathways, including non-homologous end joining (NHEJ) and homologous recombination (HR). Our findings further showed that TN suppressed these DNA repair pathways by downregulating thyroid hormone receptor-interacting protein 13 (TRIP13), which is involved in BTZ resistance and DNA repair. TN effectively overcame TRIP13 overexpression-mediated BTZ resistance, while TRIP13 silencing partially attenuated TN-induced DNA damage and cell death and diminished the synergistic effect of TN and BTZ in MM cells. These findings demonstrated that TN and BTZ synergistically augment DNA damage burden through distinct mechanisms, thereby exerting significant anti-MM synergy.
Conclusions: Our findings indicate that TN exerts a significant inhibitory effect on MM cells. Combining TN and BTZ synergistically inhibits MM by enhancing DNA damage through blocking TRIP13-mediated DNA repair. This study provides a novel combination strategy for the clinical management of MM.
Disclosures: No relevant conflicts of interest to declare.
