Background: TNBC is the most aggressive breast cancer with higher recurrence and mortality rate than other types of breast cancer. There is an urgent need for identification of therapeutic agents with unique mode of action for overcoming current challenges in TNBC treatment. Methods: Different inhibitors were used to study the cell death manner of DMOCPTL. RNA silencing was used to evaluate the functions of GPX4 in ferroptosis and apoptosis of TNBC cells and functions of EGR1 in apoptosis. Immunohistochemical assay of tissue microarray were used for investigating correlation of GPX4 and EGR1 with TNBC. Computer-aided docking and small molecule probe were used for study the binding of DMOCPTL with GPX4. Results: DMOCPTL, a derivative of natural product parthenolide, exhibited about 15-fold improvement comparing to that of the parent compound PTL for TNBC cells. The cell death manner assay showed that the anti-TNBC effect of DMOCPTL mainly by inducing ferroptosis and apoptosis through ubiquitination of GPX4. The probe of DMOCPTL assay indicated that DMOCPTL induced GPX4 ubiquitination by directly binding to GPX4 protein. To the best of our knowledge, this is the first report of inducing ferroptosis through ubiquitination of GPX4. Moreover, the mechanism of GPX4 regulation of apoptosis is still obscure. Here, we firstly reveal that GPX4 regulated mitochondria-mediated apoptosis through regulation of EGR1 in TNBC cells. Compound 13, the prodrug of DMOCPTL, effectively inhibited the growth of breast tumor and prolonged the lifespan of mice in vivo, and no obvious toxicity was observed. Conclusions: These findings firstly revealed novel manner to induce ferroptosis through ubiquitination of GPX4 and provided mechanism for GPX4 inducing mitochondria-mediated apoptosis through up-regulation of EGR1 in TNBC cells. Moreover, compound 13 deserves further studies as a lead compound with novel mode of action for ultimate discovery of effective anti-TNBC drug.

Cucurbitacin B shows potent activity against tumor cells, but its high toxicity limits its application in the clinic. A series of cucurbitacin B derivatives was synthesized and evaluated for their anti-hepatocellular carcinoma (HCC) activities against the HepG-2 cell line. These compounds were also tested for their toxicity against the L-O2 normal cell line. The compound with the most potential, 10b, exhibited potent activity against the HepG-2 cell line with an IC₅₀ value of 0.63 μM. Moreover, compound 10b showed the highest TI value (4.71), which is a 14.7-fold improvement compared to its parent compound cucurbitacin B. A preliminary molecular mechanism study of 10b indicated that 10b could inhibit P-STAT3 to induce the activation of mitochondrial apoptotic pathways. An in vivo acute toxicity study indicated that the compound 10b has preferable safety and tolerability compared with cucurbitacin B. These findings indicate that compound 10b might be considered as a lead compound for exploring effective anti-HCC drugs.