Paraquat toxicity induced by voltage-dependent anion channel 1 acts as an NADH-dependent oxidoreductase.

Paraquat (PQ), a herbicide used worldwide, causes fatal injury to organs upon high dose ingestion. Treatments for PQ poisoning are unreliable, and numerous deaths have been attributed inappropriate usage of the agent. It is generally speculated that a microsomal drug-metabolizing enzyme system is responsible for PQ toxicity. However, recent studies have demonstrated cytotoxicity via mitochondria, and therefore, the cytotoxic mechanism remains controversial. Here, we demonstrated that mitochondrial NADH-dependent PQ reductase containing a voltage-dependent anion channel 1 (VDAC1) is responsible for PQ cytotoxicity. When mitochondria were incubated with NADH and PQ, superoxide anion (O(2)(*)) was produced, and the mitochondria ruptured. Outer membrane extract oxidized NADH in a PQ dose-dependent manner, and oxidation was suppressed by VDAC inhibitors. Zymographic analysis revealed the presence of VDAC1 protein in the oxidoreductase, and the direct binding of PQ to VDAC1 was demonstrated using biotinylated PQ. VDAC1-overexpressing cells showed increased O(2)(*) production and cytotoxicity, both of which were suppressed in VDAC1 knockdown cells. These results indicated that a VDAC1-containing mitochondrial system is involved in PQ poisoning. These insights into the mechanism of PQ poisoning not only demonstrated novel physiological functions of VDAC protein, but they may facilitate the development of new therapeutic approaches.

Mitochondrial voltage-dependent anion channels (VDACs) as novel pharmacological targets for anti-cancer agents.

Recently, it was demonstrated that some anti-cancer agents used mitochondrial voltage-dependent anion channels (VDAC1-3 isoforms) as their pharmacological target. VDACs are expressed more highly in cancer cells than normal cells; thus the VDAC-dependent cytotoxic agents can have cancer-selectivity. Furanonaphthoquinones (FNQs) induced caspase-dependent apoptosis via the production of NADH-dependent reactive oxygen species (ROS) by VDAC1. The ROS production and the anti-cancer activity of FNQs were increased by VDAC1 overexpression. Meanwhile, erastin induced RAS-RAF-MEK-dependent non-apoptotic cell death via VDAC2. On the other hand, VDACs were needed for transporting ATP to hexokinase (HK), which was highly expressed in cancer cells. We hypothesized that the high glycolysis might induce up-regulation of VDAC. In this review, we propose that VDACs are novel candidates for effective pharmacological targets of anti-cancer drugs.

Bioreductive activation of quinone antitumor drugs by mitochondrial voltage-dependent anion channel 1.

The authors recently demonstrated that the mitochondrial voltage-dependent anion channel 1 (VDAC1) is involved in the sensitivity of cancer cells to furanonaphthoquinone (FNQ). The aim of the present study was to investigate whether mitochondrial VDAC1 reduces quinone antitumor drugs. The VDAC1 purified by immunoprecipitation reduced FNQ in the presence of nicotinamide adenine dinucleotide (NADH) and produced H(2)O(2). Blue native polyacrylamide gel electrophoresis demonstrated that the band that reduced FNQ NADH-dependently mainly included VDAC1. Because H(2)O(2) generation in catalyzing FNQ with NADH caused mitochondrial damage, the cytotoxic activity of FNQ was induced by VDAC1. In the quinone antitumor drugs, menadione (VK3), adriamycin and mitomycin C, mitochondrial VDAC1 bioreductively activated VK3. These results demonstrate that mitochondrial VDAC1 is a pharmacologic target for the treatment of tumor.

Furanonaphthoquinones cause apoptosis of cancer cells by inducing the production of reactive oxygen species by the mitochondrial voltage-dependent anion channel.

The mitochondrial production of reactive oxygen species has been implicated in the anticancer activity of furanonaphthoquinone. However, the mechanism of the activation remains elusive. In the current study, we found that treatment of HeLa cells with 2-methyl-5(or -8)-hydroxy-furanonaphthoquinone (FNQ13) induces mitochondrial swelling, followed by apoptosis. This toxic effect of FNQ13 was reduced by the radical scavengers alpha-tocopherol and trolox. Cytochemical experiments in isolated mitochondria showed that a combination of FNQ13 and NADH induces the production of H(2)O(2) at the exterior mitochondrial membrane surface. This production of H(2)O(2) was reduced by an antibody to the voltage-dependent anion channel (VDAC). Overexpression of the VDAC by transfection with vdac1 cDNA increased the production of H(2)O(2) by HeLa cells, whereas transfection with a small interfering RNA to VDAC reduced FNQ13-induced H(2)O(2) production and cell death due to an almost complete knockdown of VDAC expression. We also found significant correlations between the expression of VDAC and the induction of H(2)O(2) production and cell death by FNQ13 in 11 human cancer cell lines. These results indicate that the anticancer activity of furanonaphthoquinones depends on the production of reactive oxygen species by mitochondrial permeability transition pores including the VDAC.

Growth inhibitory activity of wood of Taxus yunnanensis and its liquid chromatography Fourier-transform mass spectrometry analysis.

The wood of Taxus yunnanensis (Taxaceae) showed growth inhibitory activities against human cancer cell lines, such as cervical HeLa adenocarcinoma. The morphological changes indicated that the cellular growth inhibitions were caused by apoptosis. To determine the active components, T. yunnanensis wood was analyzed by using a liquid chromatography-Fourier-transform MS (LC/FT-MS) technique. As a result, taxane-type diterpenes, such as 10-deacetylcephalomannine and 10-deacetyltaxol, were found to be present in amounts consistent with the growth inhibitory activity.

The synthetic furanonaphthoquinone induces growth arrest, apoptosis and differentiation in a variety of leukaemias and multiple myeloma cells.

2-methyl-naphtho[2,3-b]furan-4,9-dione (FNQ3), a synthetic analogue of the quinone kigelinone, has demonstrated a real potential for use in the treatment of a variety of solid tumours. Unlike other quinones, such as mitomycin-C and adriamycin, the cytotoxicity of FNQ3 is often 10- to 14-fold more potent towards the tumour cells than their normal counterparts. We report, for the first time, that the drug had activity against a broad spectrum of leukaemias and multiple myeloma cells. It decreased the growth of acute myeloid leukaemia (AML) and multiple myeloma cell lines in a dose-dependent fashion (50% inhibitory concentration approximately 1.25 microg/ml against most of the leukaemia cell lines). This dose apparently initiated mitochondrial collapse as measured by depolarisation of the mitochondrial membrane. FNQ3 potentiated the differentiation of HL-60 myeloid cells in the presence of either 1alpha, 25(OH)(2) dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] or all-trans-retinoic acid (ATRA). FNQ3 inhibited the proliferation of primary AML cells while inducing apoptosis. Eleven of 14 (79%) AML marrow samples had a prominent decrease in their clonogenic growth when cultured in the presence of the drug. In summary, this drug has growth inhibitory, apoptotic and differentiative effects against myeloid leukaemias and multiple myeloma cells. FNQ3 may represent a new therapeutic approach to these malignancies.

Mitochondrial damage prior to apoptosis in furanonaphthoquinone treated lung cancer cells.

The mechanisms of the antitumor reactions of 2-methylnaphtho[2,3-b]furan-4,9-dione (FNQ3) to human lung adenocarcinoma A549 cells were investigated. A549 cells that received 1.25 microg/ml FNQ3 (IC(50) at 0.35 microg/ml) developed intensive mitochondrial H(2)O(2) production at 1 h. Selective structural mitochondrial swelling, alteration of mitochondrial membrane potential, and cytochrome c and caspase-9 release from the mitochondria occurred 18-24 h later. alpha-Tocopherol inhibited the alteration of both mitochondrial permeability and the leakage of procaspase-9. The caspase-9 was then activated in the cytosol. The expression of Bcl-2 oncoprotein was suppressed by FNQ3, and resulted in apoptosis. The higher dose of 5 microg/ml induced necrosis via severe mitochondrial breakage. These results showed that FNQ3 targets the mitochondria of A549 cells to produce a reactive oxygen species resulting in apoptosis and necrosis.

Alpha-tocopherol protects cultured human cells from the acute lethal cytotoxicity of dioxin.

The possible protection of cultured human cells from acute dioxin injury by antioxidants was investigated. The most potent dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), caused vacuolization of the smooth endoplasmic reticulum and Golgi apparatus in cultured human conjunctival epithelial cells and cervical cancer cells. Subsequent nuclear damage included a deep irregular indentation resulting in cell death. A dosage of 30-40 ng/mL TCDD induced maximal intracellular production of H2O2 at 30 minutes and led to severe cell death (0-31% survival) at two hours. A dose of 1.7 mM alpha-tocopherol or 1 mM L-dehydroascorbic acid significantly protected human cells against acute TCDD injuries (78-97% survivals), but vitamin C did not provide this protection. These results indicate that accidental exposure to fatal doses of TCDD causes cytoplasmic free radical production within the smooth endoplasmic reticular systems, resulting in severe cytotoxicity, and that vitamin E and dehydroascorbic acid can protect against TCDD-induced cell damage.

Paraquat detoxicative system in the mouse liver postmitochondrial fraction.

We examined the paraquat detoxicative system in mouse livers. The survival rate of mice receiving 50 mg/kg paraquat was 41% at 7 days and significantly rose to 88, 64, 69% with pretreatment with phenytoin, phenobarbital, and rifampicin, respectively. Phenytoin induced activity in NADPH-cytochrome P450 reductase, CYP3A, CYP2B, and CYP2C that was 3 to 4 times higher than that of the controls. Phenobarbital induced CYP2B and rifampicin induced CYP3A, respectively, in addition to NADPH-cytochrome P450 reductase. 3-Methylcholanthrene did not induce these enzymes and did not alter the survival rate. All the mice pretreated with CoCl(2) (a CYP synthesis inhibitor) or SKF 525-A (a CYP inhibitor) were dead after 5 days, and troleandomycin (a CYP3A-specific inhibitor) also reduced the survival rate. When cell homogenates were incubated with paraquat and NADPH, paraquat decreased and its metabolic intermediate paraquat-monopyridone was formed. Troleandomycin inhibited the decrease in paraquat and increased the monopyridone. After making a subfraction of the homogenate, monopyridone was produced in the postmicrosomal 105,000g supernatant, but not in the microsomes. The pretreatment of mice with phenytoin decreased the monopyridone in the postmitochondrial fraction, but did not affect the supernatant. These results indicated that paraquat was first metabolized in the postmicrosomal supernatant into monopyridone, and that may have been subsequently hydroxylated by the microsomes. Repeated intravenous injections of alpha-tocopherol to paraquat-loaded mice significantly reduced the paraquat mortality and when these mice were pretreated with rifampicin, 100% of them survived. These studies demonstrate that postmitochondrial fractions play an important role in paraquat detoxication metabolism, and that the combination of CYP induction and alpha-tocopherol administration is highly useful for the survival of paraquat-exposed mice.