Cypripedin Induces Apoptosis and Synergizes with Bortezomib via ER Stress Mediated Ubiquitination of GRP78 in T-Cell Acute Lymphoblastic Leukemia.

BACKGROUND

T-cell acute lymphoblastic leukemia (T-ALL) remains a challenging malignancy with limited targeted therapies. Natural phenanthrene derivatives represent a promising source of antileukemic agents.

OBJECTIVE

We screened a library of natural phenanthrene-type compounds to identify cytotoxic leads in Jurkat T-ALL cells and investigated the mechanisms underlying their activity, including potential synergy with the proteasome inhibitor bortezomib (BTZ).

METHODS

Jurkat cells were treated with thirteen natural compounds at 10 and 20 µM for 48 h; cell viability was assessed by WST-1 cell viability assay. Dose-response curves were generated to calculate IC50 values. Apoptosis was evaluated by Hoechst 33342/PI staining and Annexin V/PI flow cytometry. Synergy with BTZ was analyzed using a fixed-ratio combination index (CI) approach and IC50 shift analysis. ER stress signaling was characterized by Western blotting, quantitative RT-PCR of UPR genes (GRP78, ATF6), and immunoprecipitation of GRP78 followed by ubiquitin immunoblotting.

RESULTS

Among the compounds screened, Cypripedin showed the most potent cytotoxicity with an IC50 of 6.52 µM. It induced a dose-dependent increase in apoptosis. Combination with BTZ yielded a CI < 0.5 and reduced BTZ IC50 from 3.43 to 1.88 ng/mL. Cypripedin activated the unfolded protein response (UPR), modulated key ER stress markers including GRP78, p-PERK, p-eIF2α, p-JNK, and ATF6, downregulated UPR gene transcripts, and promoted GRP78 ubiquitination. Molecular docking predicted strong binding of Cypripedin to the GRP78 ATPase domain (Vina score -7.630 kcal/mol), supporting its mechanism of action.

CONCLUSION

Cypripedin induces apoptosis in Jurkat T-ALL cells, synergizes with BTZ, and modulates ER stress through GRP78 ubiquitination. These findings support its further development as a potential T-ALL therapeutic.