Rottlerin potentiates camptothecin-induced cytotoxicity in human hormone refractory prostate cancers through increased formation and stabilization of topoisomerase I-DNA cleavage complexes in a PKCδ-independent pathway

Abstract

Combination therapy, which can optimize killing activity to cancers and minimize drug resistance, is a mainstream therapy against hormone-refractory prostate cancers (HRPCs). Rottlerin, a natural polyphenolic component, synergistically increased PC-3 (a HRPC cell line) apoptosis induced by camptothecin (a topoisomerase I inhibitor). Using siRNA technique to knockdown protein kinase C-δ (PKCδ), the data showed that rottlerin-mediated synergistic effect was PKCδ-independent, although rottlerin has been used as a PKCδ inhibitor. Rottlerin potentiated camptothecin-induced DNA fragmentation at S phase and ATM phosphorylation at Ser1981. The effect was correlated to apoptosis (r2 = 0.9). To detect upstream signals, the data showed that camptothecin acted on and stabilized topoisomerase I-DNA complex, leading to the formation of camptothecin-trapped cleavage complexes (TOP1cc). The effect was potentiated by rottlerin. To determine DNA repair capability, the time-related γH2A.X formation was examined after camptothecin removal. Consequently, rottlerin significantly inhibited camptothecin removal-mediated decline of γH2A.X formation at S phase, indicating the impairment of DNA repair activity in the presence of rottlerin. The combinatory treatment of camptothecin and rottlerin induced conformational change and activation of Bax and formation of truncated Bad, suggesting the contribution of mitochondria stress to apoptosis. In summary, the data suggest that rottlerin-mediated camptothecin sensitization is through the augmented stabilization of TOP1cc, leading to an increase of DNA damage stress and, possibly, an impairment of DNA repair capability. Subsequently, mitochondria-involved apoptosis is triggered through Bax activation and truncated Bad formation. The novel discovery may provide an anticancer approach of combinatory use between rottlerin and camptothecin for the treatment of HRPCs.

Protease Nexin I is a feedback regulator of EGF/PKC/MAPK/EGR1 signaling in breast cancer cells metastasis and stemness

“…Cells were seeded into 6-well plates at the density of 2 × 10 5 cells/well, and starved for 24 h with fresh serum-free DMEM when the cells reached 80% confluence. Then cells were stimulated with 100 ng/mL EGF for 24 h or 1 μM PMA for 12 h. For inhibitor studies, cells were pretreated with inhibitors for 4 h before EGF or PMA stimulation, and the concentration of inhibitors were as follows according to the reported lectures: 10 μM EGFR inhibitor AG1478 19 (MedChemExpress, USA),10 μM PKC inhibitor Go6983 20 , 5 μM MEK inhibitor U0126 21 , 20 μM PI3K inhibitor LY294002 22 , 10 μM PLC inhibitor U73122 23 , 10 μM JAK inhibitor Ruxolitinib 24 , 5 μM SCH772984 25 , 10 μM BAPTA-AM 26 , 20 μM Rottlerin 27 (Selleck, USA), 20 μM Mithramycin A 28 (APExBIO, USA). For EGF stimulation in vitro, EGF was added into culture medium at the concentration of 50 ng/ml.…”

Characterization and antitumor activity of camptothecin from endophytic fungus <i>Fusarium solani</i> isolated from <i>Camptotheca acuminate</i>

“…Camptothecin and its derivatives are well-known topoisomerase I inhibitors, which inhibit cell growth and induce apoptotic pathways through decelerating the dissociation of topoisomerase I-DNA to trap topoisomerase I-DNA cleavage complexes 27 . In this study, biological activity of fungal CPT was assayed in vitro against Vero cells.…”

Enhanced Liver Targeting of Camptothecin via Conjugation with Deoxycholic Acid

“…CPT (Figure 1A), a natural monoterpene-quinoline alkaloid first isolated from extracts of the Chinese tree Camptotheca acuminata , exhibits anticancer activity through inhibition of topoisomerase I [1,2,3,4]. As a potent inhibitor of DNA topoisomerase I, CPT shows high activity against a broad spectrum of cancers, including breast, small-cell lung, ovarian, pancreas cancer, particularly hepatic and colon cancer [5,6,7,8]. Unfortunately, the clinical application of CPT has been hindered by its water insolubility, severe toxicity to normal tissues and poor stability of the lactone ring [9,10,11].…”