Tackling the Cytotoxic Effect of a Marine Polycyclic Quinone-Type Metabolite: Halenaquinone Induces Molt 4 Cells Apoptosis via Oxidative Stress Combined with the Inhibition of HDAC and Topoisomerase Activities

Abstract

A marine polycyclic quinone-type metabolite, halenaquinone (HQ), was found to inhibit the proliferation of Molt 4, K562, MDA-MB-231 and DLD-1 cancer cell lines, with IC50 of 0.48, 0.18, 8.0 and 6.76 μg/mL, respectively. It exhibited the most potent activity against leukemia Molt 4 cells. Accumulating evidence showed that HQ may act as a potent protein kinase inhibitor in cancer therapy. To fully understand the mechanism of HQ, we further explored the precise molecular targets in leukemia Molt 4 cells. We found that the use of HQ increased apoptosis by 26.23%–70.27% and caused disruption of mitochondrial membrane potential (MMP) by 17.15%–53.25% in a dose-dependent manner, as demonstrated by Annexin-V/PI and JC-1 staining assays, respectively. Moreover, our findings indicated that the pretreatment of Molt 4 cells with N-acetyl-l-cysteine (NAC), a reactive oxygen species (ROS) scavenger, diminished MMP disruption and apoptosis induced by HQ, suggesting that ROS overproduction plays a crucial rule in the cytotoxic activity of HQ. The results of a cell-free system assay indicated that HQ could act as an HDAC and topoisomerase catalytic inhibitor through the inhibition of pan-HDAC and topoisomerase IIα expression, respectively. On the protein level, the expression of the anti-apoptotic proteins p-Akt, NFκB, HDAC and Bcl-2, as well as hexokinase II was inhibited by the use of HQ. On the other hand, the expression of the pro-apoptotic protein Bax, PARP cleavage, caspase activation and cytochrome c release were increased after HQ treatment. Taken together, our results suggested that the antileukemic effect of HQ is ROS-mediated mitochondrial apoptosis combined with the inhibitory effect on HDAC and topoisomerase activities.

Antileukemic Scalarane Sesterterpenoids and Meroditerpenoid from Carteriospongia (Phyllospongia) sp., Induce Apoptosis via Dual Inhibitory Effects on Topoisomerase II and Hsp90

“…Compound 1 can damage the integrity of mitochondria, which are the major production sites of the superoxide anion, ozone37. The induction of the intracellular formation of ROS by 1 was determined with a carboxyl derivative of fluorescein, carboxy-H 2 DCFDA dye using flow cytometric analysis38. As shown in Fig.…”

“…These assays were performed as described previously38. MMP disruption, calcium accumulation and ROS generation were detected with JC-1 cationic dye (5 μg/mL), the fluorescent calcium indicator (Fluo 3, 5 mM) and the carboxy derivative of fluorescein (carboxy-H 2 DCFDA, 1.0 mM), respectively.…”

“…The assay was performed as described previously3538. Standard relaxation reaction mixtures (20 μL) containing 50 mM Tris–HCl (pH 8.0), 10 mM MgCl 2 , 200 mM potassium glutamate, 10 mM dithiothreitol, 50 μg/mL bovine serum albumin, 1 mM ATP, 0.3 μg of pHOT1 plasmid DNA, two units of human topoisomerase II (Topogen, Columbus, OH, USA), and the indicated concentrations of etoposide and the compounds were incubated at 37 °C for 30 min.…”

“…Cell lysates were prepared by treating the cells for 30 min in RIPA lysis buffer, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), 1 mM sodium orthovanadate, 100 μg/mL phenylmethylsulfonyl fluoride and 30 μg/mL aprotinin) (all chemicals were from Sigma)38. The lysates were centrifuged at 20,000 ×  g for 30 min, and the protein concentration in the supernatant was determined using a BCA protein assay kit (Pierce).…”

Isoaaptamine Induces t-47D Cells Apoptosis and Autophagy via Oxidative Stress

“…The induction of the intracellular formation of ROS by IAp was determined with a carboxyl derivative of fluorescein, carboxy-H2DCFDA dye using flow cytometric analysis [12]. The levels of ROS at different time intervals following IAp treatment were determined to examine whether the IAp-induced apoptosis and autophagy in T-47D cells involve the overproduction of ROS.…”