Ilomastat

Colorectal cancer (CRC) progression is often driven by hypoxia-induced signaling that promotes metastasis through epithelial-mesenchymal transition and invadopodia formation. PX-12, an irreversible thioredoxin-1 (Trx-1) inhibitor, has shown anticancer potential, but its anti-invasive effects under hypoxic conditions remain unclear. This study investigated the cytotoxic, anti-migratory, anti-invasive, and anti-invadopodia activities of PX-12 in hypoxia-induced CRC cells. Human HCT116 cells were exposed to dimethyloxalylglycine (DMOG) to induce hypoxia. Cytotoxicity was assessed using MTT assays, while migration and invasion were evaluated through wound-healing and Transwell assays. Invadopodia formation was quantified using Oregon Green-labeled gelatin degradation assays. Protein expression of HIF-1α and Trx-1 was analyzed via Jess Simple Western system. PX-12 significantly reduced HCT116 cell viability in a dose-dependent manner, with lower cytotoxicity under DMOG-induced hypoxia. PX-12 markedly suppressed cell migration and invasion, particularly under hypoxia-like conditions. Gelatin degradation analysis revealed a significant reduction in invadopodia formation with PX-12 treatment, paralleling effects of the MMP inhibitor GM6001. Although DMOG markedly upregulated HIF-1α expression, PX-12 did not alter Trx-1 protein levels, suggesting that its inhibitory effects may occurs via enzymatic blockade rather than changes in protein expression. PX-12 exerts potent anti-invasive effects by inhibiting migration, invasion, and invadopodia formation in hypoxia-mimicking CRC cells. These findings highlight PX-12 as therapeutic agent targeting hypoxia-driven migratory and invasive behaviors in CRC.

Doxorubicin (Dox) is a potent first-line chemotherapeutic and widely administered against different types of cancer, but is associated with a myriad of side effects, including cancer/chemotherapy-associated thrombosis and drug-induced thrombocytopenia. Although we and others have reported Dox-induced platelet activation, the binding partner of Dox on platelets has not been previously explored. Here, we found human and mouse platelet aggregation triggered via C-type lectin-like receptor-2 (CLEC-2) was obstructed by Dox, but aggregation induced by classical agonists like ADP, collagen/collagen-related peptide, or thrombin receptor-activating peptide 6, was unaffected. By isothermal titration calorimetry, we detected a high binding affinity between Dox and recombinant CLEC-2 at 4.2 ± 2.4 nM. Interestingly, we found significant GPIb⍺ shedding from human and mouse platelet surfaces following Dox treatment. Consistently, GPIb⍺ shedding was recapitulated following anti-CLEC-2 monoclonal antibody treatment. Using Piceatannol to selectively inhibit CLEC-2 intracellular signaling or the pan-matrix metalloproteinases inhibitor GM6001 rescued glycoprotein Ibα (GPIbα) from both Dox and CLEC-2 mAb-induced shedding. Using GI254023X or GW280264X to specifically inhibit ADAM10 or ADAMs10/17, respectively, revealed inhibition of ADAM10/17, but not ADAM10 exclusively, prohibited GPIbα shedding. Collectively, this implicates the classical sheddase of GPIbα, ADAM17. Thus, we pinpointed CLEC-2 as a binding partner for Dox on platelets and a novel pathway of ADAM17-mediated GPIb⍺ shedding via CLEC-2. These data not only provide insights into a mechanism of Dox-induced platelet activation, thrombosis, and drug-induced thrombocytopenia, but also reveal putative precision therapeutic approaches for Dox-treated patients and nominate CLEC-2 inhibition as a means to regulate thrombotic disease and/or bleeding disorders.