Adaphostin and other anticancer drugs quench the fluorescence of mitochondrial potential probes

Fluorescent dyes are widely used to monitor changes in mitochondrial transmembrane potential (DeltaPsim). When MitoTracker Red CMXRos, tetramethylrhodamine methyl ester (TMRM), and 3,3'dihexyloxacarbocyanine iodide (DiOC6(3)) were utilized to examine the effects of the experimental anticancer drug adaphostin on intact cells or isolated mitochondria, decreased fluorescence was observed. In contrast, measurement of tetraphenylphosphonium uptake by the mitochondria using an ion-selective microelectrode failed to show any effect of adaphostin on DeltaPsim. Instead, further experiments demonstrated that adaphostin quenches the fluorescence of the mitochondrial dyes. Structure-activity analysis revealed that the adamantyl and p-aminobenzoic acid moieties of adaphostin are critical for this quenching. Anticancer drugs containing comparable structural motifs, including mitoxantrone, aminoflavone, and amsacrine, also quenched the mitochondrial probes. These results indicate the need for caution when mitochondrial dyes are utilized to examine the effects of xenobiotics on DeltaPsim and suggest that some previously reported direct effects of anticancer drugs on mitochondria might need re-evaluation.     

A direct interaction between NQO1 and a chemotherapeutic dimeric naphthoquinone

Background

Multimeric naphthoquinones are redox-active compounds that exhibit antineoplastic, antiprotozoal, and antiviral activities. Due to their multimodal effect on perturbation of cellular oxidative state, these compounds hold great potential as therapeutic agents against highly proliferative neoplastic cells. In our previous work, we developed a series of novel dimeric naphthoquinones and showed that they were selectively cytotoxic to human acute myeloid leukemia (AML), breast and prostate cancer cell lines. We subsequently identified the oxidoreductase NAD(P)H dehydrogenase, quinone 1 (NQO1) as the major target of dimeric naphthoquinones and proposed a mechanism of action that entailed induction of a futile redox cycling.

Results

Here, for the first time, we describe a direct physical interaction between the bromohydroxy dimeric naphthoquinone E6a and NQO1. Moreover, our studies reveal an extensive binding interface between E6a and the isoalloxazine ring of the flavin adenine dinucleotide (FAD) cofactor of NQO1 in addition to interactions with protein side chains in the active site. We also present biochemical evidence that dimeric naphthoquinones affect the redox state of the FAD cofactor of NQO1. Comparison of the mode of binding of E6a with those of other chemotherapeutics reveals unique characteristics of the interaction that can be leveraged in future drug optimization efforts.

Conclusion

The first structure of a dimeric naphthoquinone-NQO1 complex was reported, which can be used for design and synthesis of more potent next generation dimeric naphthoquinones to target NQO1 with higher affinity and specificity.

     

Histological and immunohistochemical effects of Curcuma longa on activation of rat hepatic stellate cells after cadmium induced hepatotoxicity

The liver is a target for toxic chemicals such as cadmium (Cd). When the liver is damaged, hepatic stellate cells (HSC) are activated and transformed into myofibroblast-like cells, which are responsible for liver fibrosis. Curcuma longa has been reported to exert a hepato-protective effect under various pathological conditions. We investigated the effects of C. longa administration on HSC activation in response to Cd induced hepatotoxicity. Forty adult male albino rats were divided into: group 1 (control), group 2 (Cd treated), group 3 (C. longa treated) and group 4 (Cd and C. longa treated). After 6 weeks, liver specimens were prepared for light and electron microscopy examination of histological changes and immunohistochemical localization of alpha smooth muscle actin (αSMA) as a specific marker for activated HSC. Activated HSC with a positive αSMA immune reaction were not detected in groups 1 and 3. Large numbers of activated HSC with αSMA immune reactions were observed in group 2 in addition to Cd induced hepatotoxic changes including excess collagen deposition in thickened portal triads, interlobular septa with hepatic lobulation, inflammatory cell infiltration, a significant increase in Kupffer cells and degenerated hepatocytes. In group 4, we observed a significant decrease in HSC that expressed αSMA with amelioration of the hepatotoxic changes. C. longa administration decreased HSC activation and ameliorated hepatotoxic changes caused by Cd in adult rats.     

Inhibition of interferon secretion by vinblastine

The plant alkaloids vinblastine and colchicine are known to arrest cells in mitosis by virtue of their binding to spindle protein. These drugs are also capable of binding to microtubule protein and causing these structures to disaggregate into nonfunctional subunits (1, 2). Microtubular structures are thought to be involved in the secretory process of a number of proteins including insulin (7), collagen (4), and thyroid hormone (12). In this report we present our findings on the effects of these two drugs on the synthesis and secretion of interferon in a high producing human foreskin fibroblast strain (FS-4) (11).     

Homology model of the CDK1/cyclin B complex

We describe a refined homology model of a CDK1/cyclin B complex that was previously used for the structure-based optimization of the Paullone class of inhibitors. The preliminary model was formed from the homologous regions of the deposited CDK2/cyclin A crystal structure. Further refinement of the CDK1/cyclin B complex was accomplished using molecular mechanics and hydropathic analysis with a protocol of constraints and local geometry searches. For the most part, our CKD1/cyclin B homology model is very similar to the high resolution CDK2/cyclin A crystal structure regarding secondary and tertiary features. However, minor discrepancies between the two kinase structures suggest the possibility that ligand design may be specifically tuned for either CDK1 or CDK2. Our examination of the CDK1/cyclin B model includes a comparison with the CDK2/cyclin A crystal structure in the PSTAIRE interface region, connecting portions to the ATP binding domain, as well as the ATP binding site itself.     

N-(2-hydroxypropyl)methacrylamide copolymer-6-(3-aminopropyl)-ellipticine conjugates. Synthesis, in vitro, and preliminary in vivo evaluation

Ellipticine derivatives have potential as anticancer drugs. Their clinical use has been limited, however, by poor solubility and host toxicity. As N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anticancer conjugates are showing promise in early clinical trials, a series of novel HPMA copolymer conjugates have been prepared containing the 6-(3-aminopropyl)-ellipticine derivative (APE, NSC176328). Drug was linked to the polymer via GFLG or GG peptide side chains. To optimize biological behavior, HPMA copolymer-GFLG-APE conjugates with different drug loading (total APE: 2.3-7% w/w; free APE: <0.1% w/w) were synthesized. Conjugation of APE to HPMA copolymers considerably increased its aqueous solubility (>10-fold). HPMA copolymer-GG-APE did not liberate drug in the presence of isolated lysosomal enzymes (tritosomes), but HPMA copolymer-GFLG-APE released APE to a maximum of 60% after 5 h. The rate of drug release was influenced by drug loading; lower loading led to greater release. Whereas free APE (35 microg/mL) caused significant hemolysis (50% after 1 h), HPMA copolymer-APE conjugates were not hemolytic up to 300 microg/mL (APE-equiv). As would be expected from its cellular pharmacokinetics, HPMA copolymer-GFLG-APE was >75 times less cytotoxic than free drug (IC(50) approximately 0.4 microg/mL) against B16F10 melanoma in vitro. However, in vivo when tested in mice bearing s.c. B16F10 melanoma, HPMA copolymer-GFLG-APE (1-10 mg/kg single dose, APE-equiv) given i.p. was somewhat more active (highest T/C value of 143%) than free APE (1 mg/kg) (T/C =127%). HPMA copolymer-APE conjugates warrant further evaluation as potential anticancer agents.