Identification of a small molecule as inducer of ferroptosis and apoptosis through ubiquitination of GPX4 in triple negative breast cancer cells.

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.

Identification of Parthenolide Dimers as Activators of Pyruvate Kinase M2 in Xenografts of Glioblastoma Multiforme in Vivo.

Herein we detail the discovery of a series of parthenolide dimers as activators of PKM2 and evaluation of their anti-GBM activities. The most promising compound 5 showed high potency to activate PKM2 with an AC50 value of 15 nM, inhibited proliferation and metastasis, and induced apoptosis of GBM cells. Compound 5 could promote tetramer formation of PKM2 and reduce nucleus translocation of PKM2 in GBM cells without influence on the expression of total PKM2, thereby inhibiting the STAT3 signal pathway in vitro and in vivo. PKM2 knockdown assay demonstrated that the anti-GBM effect of 5 mainly depended on the expression of PKM2 in vitro and in vivo. Compound 16, a prodrug of 5, markedly suppressed U118 tumor xenograft growth and reduced the weight of tumor. On the basis of these investigations, we propose that 16 might be considered as a promising lead compound for discovery of anti-GBM drugs.

ACT001 modulates the NF-κB/MnSOD/ROS axis by targeting IKKß to inhibit glioblastoma cell growth.

Glioblastomas are high-grade brain tumors with poor prognoses, and new therapeutic approaches for these tumors are critically needed. This study revealed the underlying mechanisms of a new orphan drug, ACT001, that is currently in clinical trials for the treatment of advanced glioblastoma in Australia and China. ACT001 significantly suppressed glioma cell proliferation and induced apoptosis and cell cycle arrest in vitro, as determined by Cell Counting Kit-8 assays and flow cytometry. In addition, U-118 MG cells with high expression of p-IKKß were sensitive to ACT001. Changes in the oxidative stress pathway in U-118 MG cells were detected with the isobaric tags for relative and absolute quantitation (iTRAQ) method. We further verified that ACT001 elevated the levels of reactive oxygen species (ROS) by regulating NF-κB-targeted MnSOD. ACT001 markedly inhibited NF-κB activation by directly binding IKKß and inhibiting its phosphorylation. Overexpression of IKKß markedly attenuated the changes in MnSOD and NOX1, indicating that ACT001 increased the levels of ROS by reducing the protein expression of p-IKKß. Furthermore, ACT001 reduced cyclin B1/CDC2 expression and triggered G2/M phase arrest by increasing ROS production. ACT001 also upregulated the expression of Bax and Bim and induced apoptosis in a ROS-dependent manner. ACT001 effectively suppressed the growth of U-118 MG tumors in BALB/c nude mice and GL-261-luciferase tumors in C57BL/6 J mice. Finally, ACT001 downregulated the expression of p-p65, MnSOD, cyclin B1, CDC2, and Ki67 in U-118 MG tumor tissues. Patients with activated NF-κB signaling should thus be given priority for enrollment in future phase II clinical trials. KEY MESSAGES: ACT001 directly bind to IKKß and inhibited its phosphorylation. The inhibition of p-IKKß induced the generation of ROS. ACT001 promoted the generation of ROS by regulating MnSOD expression to induce G2/M phase arrest.

Dimethylaminomicheliolide (DMAMCL) Suppresses the Proliferation of Glioblastoma Cells via Targeting Pyruvate Kinase 2 (PKM2) and Rewiring Aerobic Glycolysis.

Glioblastoma (GBM) is the most prevalent malignant tumor in the central nervous system. Aerobic glycolysis, featured with elevated glucose consumption and lactate production, confers selective advantages on GBM by utilizing nutrients to support rapid cell proliferation and tumor growth. Pyruvate kinase 2 (PKM2), the last rate-limiting enzyme of glycolysis, is known to regulate aerobic glycolysis, and considered as a novel cancer therapeutic target. Herein, we aim to describe the cellular functions and mechanisms of a small molecular compound dimethylaminomicheliolide (DMAMCL), which has been used in clinical trials for recurrent GBM in Australia. Our results demonstrate that DMAMCL is effective on the inhibition of GBM cell proliferation and colony formation. MCL, the active metabolic form of DMAMCL, selectively binding to monomeric PKM2 and promoting its tetramerization, was also found to improve the pyruvate kinase activity of PKM2 in GBM cells. In addition, non-targeting metabolomics analysis reveals multiple metabolites involved in glycolysis, including lactate and glucose-6-phosphate, are decreased with DMAMCL treatment. The inhibitory effects of DMAMCL are observed to decrease in GBM cells upon PKM2 depletion, further confirming the importance of PKM2 in DMAMCL sensitivity. In conclusion, the activation of PKM2 by DMAMCL results in the rewiring aerobic glycolysis, which consequently suppresses the proliferation of GBM cells. Hence, DMAMCL represents a potential PKM2-targeted therapeutic agent against GBM.

Design and synthesis of parthenolide and 5-fluorouracil conjugates as potential anticancer agents against drug resistant hepatocellular carcinoma.

A series of twenty-three parthenolide-5-fluorouracil (5-FU) conjugates ware synthesized and evaluated for their anti-cancer activities against human hepatocellular carcinoma cell line Bel-7402 and 5-fluorouracil resistant human hepatocellular carcinoma cell line Bel-7402/5-FU. The preliminary structure-activity relationships were discussed. The most active compound 15d showed high activity against Bel-7402/5-FU cell line with IC50 value of 2.25 µM, which demonstrated 5.8-fold improvement compared to that of the parent compound parthenolide (IC50 = 12.98 µM). The investigation of preliminary molecular mechanism of 15d revealed that 15d could reverse drug resistance by inhibiting MDR1, ABCC1 and ABCG2 to increase the intracellular drug accumulation and induce apoptosis of Bel-7402/5-FU cells through mitochondria mediated pathway. On the base of these results, compound 15d is deserved to be further investigated as a potential anti-HCC lead compound for ultimate discovery of pathenolide-based anti-cancer drug.

Design and synthesis of parthenolide-SAHA hybrids for intervention of drug-resistant acute myeloid leukemia.

A series of parthenolide-SAHA hybrids were synthesized and evaluated for their anti-AML activities against HL-60 and HL-60/ADR cell lines. The most active compound 26 exhibited high activity against HL-60/ADR cell line with IC50 value of 0.15 µM, which demonstrated 16.8-fold improvement compared to that of the parent compound PTL (IC50 = 2.52 µM). Moreover, it was six times more potent than the reference drug SAHA (IC50 = 0.90 µM) and fifty-one times more potent than ADR (IC50 = 7.72 µM). The preliminary molecular mechanism of 26 indicated that compound 26 could significantly induce apoptosis of HL-60/ADR cells. The effect of compound 26 was mainly through mitochondria pathway. Further investigation revealed that the protein level of HDAC1 and HDAC6 were reduced after the treatment of compound 26 with a dose-dependent manner. Compound 26 could significantly decrease ABCC1 expression, which increased the accumulation of intracellular drug for overcoming the drug resistance. On the base of these results, compound 26 might be considered as a promising candidate for further evaluation as a potential anti-AML drug.

Synthesis and structure-activity relationship studies of parthenolide derivatives as potential anti-triple negative breast cancer agents.

Triple-negative breast cancer (TNBC) is the most aggressive cancers with a high recurrence rate and rapidly acquired drug resistance among various breast cancer subtypes. There is no specific drug for treatment of TNBC. Discovery of therapeutic agents with unique modes of actions is urgently needed. In this study, a series of seventy parthenolide derivatives was designed, synthesized, and evaluated for their anti-TNBC activities. Compound 7d exhibited the most potent activity against different breast cancer cells with IC50 values ranging from 0.20 µM to 0.27 µM, which demonstrated 11.6- to 18.6-fold improvement comparing to that of the parent compound parthenolide with IC50 values of 2.68-4.63 µM. It is worth to note that 7d was more active than the positive control drug ADR. Moreover, compound 7d could induce apoptosis of SUM-159 cells through mitochondria pathway and cause G1 phase arrest of SUM-159 cells. These findings indicate that compound 7d deserves further studies as a lead compound for ultimate discovery of effective anti-TNBC drug.

Synthesis of Cucurbitacin B Derivatives as Potential Anti-Hepatocellular Carcinoma Agents.

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.

Synthesis and biological evaluation of dithiocarbamate esters of parthenolide as potential anti-acute myelogenous leukaemia agents.

A series of dithiocarbamate esters of parthenolide (PTL) was designed, synthesised, and evaluated for their anti- acute myelogenous leukaemia (AML) activities. The most promising compound 7l showed greatly improved potency against AML progenitor cell line KG1a with IC50 value of 0.7 µM, and the efficacy was 8.7-folds comparing to that of PTL (IC50 = 6.1 µM). Compound 7l induced apoptosis of total primary human AML cells and leukaemia stem cell (LSCs) of primary AML cells while sparing normal cells. Furthermore, 7l suppressed the colony formation of primary human leukaemia cells. Moreover, compound 12, the salt form of 7l, prolonged the lifespan of mice in two patient-derived xenograft models and had no observable toxicity. The preliminary molecular mechanism study revealed that 7l-mediated apoptosis is associated with mitogen-activated protein kinase signal pathway. On the basis of these investigations, we propose that 12 might be a promising drug candidate for ultimate discovery of anti-LSCs drug.

Copper(I) oxide nanoparticles catalyzed click chemistry based synthesis of melampomagnolide B-triazole conjugates and their anti-cancer activities.

A series of thirty one melampomagnolide B-triazole conjugates was synthesized via Copper(I) oxide nanoparticles catalyzed click chemistry. These conjugates were evaluated for their anti-cancer activities against a panel of five human cancer cell lines. The most active compound 6e showed high activity against HCT116 cell line with IC50 value of 0.43 µM, which demonstrated 11.5-fold improvement compared to that of the parent compound melampomagnolide B (IC50 = 4.93 µM). Compound 6e showed significant efficacy of inducing apoptosis, inhibiting proliferation and migration of HCT116 cells. The preliminary molecular mechanism of 6e was also investigated. On the base of these results, compound 6e might be considered as a promising candidate for further evaluation as a potential anti-cancer drug.

Synthesis and anti-acute myeloid leukemia activity of C-14 modified parthenolide derivatives.

Parthenolide (PTL) selectively ablates leukemia stem cells (LSCs). A series of PTL derivatives with modifications on C-14 of PTL was synthesized, and most of the derivatives showed high activities against HL-60 and KG1a. The most potent compound 6j exhibited IC50 values of 0.4 µM and 1.1 µM against KG1a and HL-60, respectively, which were 8.7 and 3.8 folds more potent than those of PTL, respectively. Moreover, compound 6j showed relatively low toxicity to normal cells (IC50 = 12.3 µM) comparing with its high anti-AML activity. The selectivity indexes for AML cells KG1a and HL-60 were 30.8 and 11.2, respectively. Preliminary study revealed that compound 6j could induce apoptosis of KG1a cells.

Syntheses and Biological Evaluation of Costunolide, Parthenolide, and Their Fluorinated Analogues.

Inspired by the biosynthesis of sesquiterpene lactones (SLs), herein we report the asymmetric total synthesis of the germacrane ring (24). The synthetic strategy features a selective aldol reaction between ß,γ-unsaturated chiral sulfonylamide 15a and aldehyde 13, as well as the intramolecular α-alkylation of sulfone 21 to construct a 10-membered carbocylic ring. The key intermediate 24 can be used to prepare the natural products costunolide and parthenolide (PTL), which are the key precursors for transformation into other SLs. Furthermore, the described synthetic sequences are amenable to the total synthesis of SL analogues, such as trifluoromethylated analogues 32 and 45. Analogues 32 and 45 maintained high activities against a series of cancer cell lines compared to their parent PTL and costunolide, respectively. In addition, 32 showed enhanced tolerance to acidic media compared with PTL. To our surprise, PTL and 32 showed comparable half-lives in rat plasma and in the presence of human liver microsomes.