Lineage context switches the function of a C. elegans Pax6 homolog in determining a neuronal fate.

The sensory nervous system of C. elegans comprises cells with varied molecular and functional characteristics, and is, therefore, a powerful model for understanding mechanisms that generate neuronal diversity. We report here that VAB-3, a C. elegans homolog of the homeodomain-containing protein Pax6, has opposing functions in regulating expression of a specific chemosensory fate. A homeodomain-only short isoform of VAB-3 is expressed in BAG chemosensory neurons, where it promotes gene expression and cell function. In other cells, a long isoform of VAB-3, comprising a Paired homology domain and a homeodomain, represses expression of ETS-5, a transcription factor required for expression of BAG fate. Repression of ets-5 requires the Eyes Absent homolog EYA-1 and the Six-class homeodomain protein CEH-32. We determined sequences that mediate high-affinity binding of ETS-5, VAB-3 and CEH-32. The ets-5 locus is enriched for ETS-5-binding sites but lacks sequences that bind VAB-3 and CEH-32, suggesting that these factors do not directly repress ets-5 expression. We propose that a promoter-selection system together with lineage-specific expression of accessory factors allows VAB-3/Pax6 to either promote or repress expression of specific cell fates in a context-dependent manner. This article has an associated 'The people behind the papers' interview.

Inducing Controlled Release and Increased Tumor-Targeted Delivery of Chlorambucil via Albumin/Liposome Hybrid Nanoparticles.

Liposomes possess good biocompatibility and excellent tumor-targeting capacity. However, the rapid premature release of lipophilic drugs from the lipid bilayer of liposomes has negative effect on the tumor-targeted drug delivery of liposomes. In this study, a lipophilic antitumor drug-chlorambucil (CHL)-was encapsulated into the aqueous interior of liposomes with the aid of albumin to obtain the CHL-loaded liposomes/albumin hybrid nanoparticles (CHL-Hybrids). The in vitro accumulative release rate of CHL from CHL-Hybrids was less than 50% within 48 h, while the accumulative CHL release was more than 80% for CHL-loaded liposomes (CHL-Lip). After intravenous injection into rats, the half-life (t 1/2ß = 5.68 h) and maximum blood concentration (C max = 4.58 µg/mL) of CHL-Hybrids were respectively 1.1 times and 3.5 times higher than that of CHL-Lip. In addition, CHL-Hybrids had better tumor-targeting capacity for it significantly increased the drug accumulation in B16F10 tumors, which contributed to the significantly control of tumor growth compared with CHL-Lip. Furthermore, CHL-Hybrid-treated B16F10 melanoma-bearing mice displayed the longest median survival time of 30.0 days among all the treated groups. Our results illustrated that the proposed hybrids drug delivery system would be a promising strategy to maintain the controlled release of lipophilic antitumor drugs from liposomes and simultaneously facilitate the tumor-targeted drug delivery.

Renal human organic anion transporter 3 increases the susceptibility of lymphoma cells to bendamustine uptake.

Chronic lymphatic leukemia (CLL) is often associated with nephritic syndrome. Effective treatment of CLL by chlorambucil and bendamustine leads to the restoration of renal function. In this contribution, we sought to elucidate the impact of organic anion transporters (OATs) on the uptake of bendamustine and chlorambucil as a probable reason for the superior efficacy of bendamustine over chlorambucil in the treatment of CLL. We examined the effects of structural analogs of p-aminohippurate (PAH), melphalan, chlorambucil, and bendamustine, on OAT1-mediated [(3)H]PAH uptake and OAT3- and OAT4-mediated [(3)H]estrone sulfate (ES) uptake in stably transfected human embryonic kidney-293 cells. Melphalan had no significant inhibitory effect on any OAT, whereas chlorambucil reduced OAT1-, OAT3-, and OAT4-mediated uptake of PAH or ES down to 14.6%, 16.3%, and 66.0% of control, respectively. Bendamustine inhibited only OAT3-mediated ES uptake, which was reduced down to 14.3% of control cells, suggesting that it interacts exclusively with OAT3. The IC50 value for OAT3 was calculated to be 0.8 µM. Real-time PCR experiments demonstrated a high expression of OAT3 in lymphoma cell lines as well as primary CLL cells. OAT3-mediated accumulation of bendamustine was associated with reduced cell proliferation and an increased rate of apoptosis. We conclude that the high efficacy of bendamustine in treating CLL might be partly contributed to the expression of OAT3 in lymphoma cells and the high affinity of bendamustine for this transporter.

Enzyme-triggered delivery of chlorambucil from conjugates based on the cell-penetrating peptide BP16.

The undecapeptide KKLFKKILKKL-NH2 (BP16) is a non-toxic cell-penetrating peptide (CPP) that is mainly internalized into cancer cells through a clathrin dependent endocytic mechanism and localizes in late endosomes. Moreover, this CPP is able to enhance the cellular uptake of chlorambucil (CLB) improving its cytotoxicity. In this work, we further explored the cell-penetrating properties of BP16 and those of its arginine analogue BP308. We investigated the influence on the cytotoxicity and on the cellular uptake of conjugating CLB at the N- or the C-terminal end of these undecapeptides. The effect of incorporating the cathepsin B-cleavable sequence Gly-Phe-Leu-Gly in CLB-BP16 and CLB-BP308 conjugates was also evaluated. The activity of CLB was significantly improved when conjugated at the N- or the C-terminus of BP16, or at the N-terminus of BP308. While CLB alone was not active (IC50 of 73.7 to >100 µM), the resulting conjugates displayed cytotoxic activity against CAPAN-1, MCF-7, PC-3, 1BR3G and SKMEL-28 cell lines with IC50 values ranging from 8.7 to 25.5 µM. These results were consistent with the internalization properties observed for the corresponding 5(6)-carboxyfluorescein-labeled conjugates. The presence of the tetrapeptide Gly-Phe-Leu-Gly at either the N- or the C-terminus of CLB-BP16 conjugates further increased the efficacy of CLB (IC50 of 3.6 to 16.2 µM), which could be attributed to its selective release in the lysosomal compartment. Enzymatic assays with cathepsin B showed the release of CLB-Gly-OH from these sequences within a short time. Therefore, the combination of BP16 with an enzymatic cleavable sequence can be used as a drug delivery system for the effective uptake and release of drugs in cancer cells.

Enthalpy-driven nuclease-like activity and mechanism of peptide-chlorambucil conjugates.

We report the results of attaching the anticancer drug chlorambucil (CLB) to two high-affinity DNA binding peptides: Met-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyM-10) and Gln-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyQ-10). These CLB-peptide conjugates cleave DNA very effectively and sequence-selectively without the use of chemicals, heat, or UV irradiation. Polyacrylamide gel electrophoresis identifies the sites where CLB-HyM-10 and CLB-HyQ-10 attack a complementary pair of 5'-(32)P-labeled duplexes derived from pBR322 in the absence of piperidine or other chemical additives. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has confirmed the preferential cleavage sites as well as a novel stepwise cleavage mechanism of sequence-selective DNA cleavage. Resembling restriction endonucleases, the CLB-peptide conjugates appear to be capable of producing double strand DNA breaks. Circular dichroism studies show that CLB-HyM-10 and CLB-HyQ-10 induce significant local conformational changes in DNA via the minor groove, possibly with dimeric binding stoichiometry. The energetic basis of DNA binding by these conjugates has been investigated by isothermal titration calorimetry, revealing that the binding of both the peptides and their CLB conjugates is overwhelmingly enthalpy-driven. The maintenance of a conserved negative binding free energy in DNA-conjugate interactions is a crucial feature of the universal enthalpy-entropy compensation phenomenon. The strongly enthalpy-driven binding of CLB-peptide conjugates to preferred loci in DNA furnishes the required proximity effect to generate the observed nuclease-like sequence-selective cleavage.

Chlorambucil and ascorbic acid-mediated anticancer activity and hematological toxicity in Dalton's ascites lymphoma-bearing mice.

Chlorambucil is an anticancer drug with alkylating and immunosuppressive activities. Considering various reports on the possible antioxidant/protective functions of ascorbic acid (vitamin C), it was aimed at to explore the modulatory effect of ascorbic acid on therapeutic efficacy and toxicity induced by chlorambucil. Dalton's ascites lymphoma tumor serially maintained in Swiss albino mice were used for the present experiments. The result of antitumor activity showed that combination treatment with ascorbic acid and chlorambucil exhibited enhanced antitumor activity with 170% increase in life span (ILS), which is significantly higher as compared to chlorambucil alone (ILS 140%). Analysis of apoptosis in Dalton's lymphoma tumor cells revealed a significantly higher apoptotic index after combination treatment as compared to chlorambucil alone. Blood hemoglobin content, erythrocytes and leukocytes counts were decreased after chlorambucil treatment, however, overall recovery in these hematological values was noted after combination treatment. Chlorambucil treatment also caused morphological abnormalities in red blood cells, majority of which include acanthocytes, burr and microcystis. Combination treatment of mice with ascorbic acid plus chlorambucil showed less histopathological changes in kidney as compared to chlorambucil treatment alone, thus, ascorbic acid is effective in reducing chlorambucil-induced renal toxicity in the hosts. Based on the results, for further development, hopefully into the clinical usage, the administration of ascorbic acid in combination with chlorambucil may be recommended.

Photocontrolled nuclear-targeted drug delivery by single component photoresponsive fluorescent organic nanoparticles of acridin-9-methanol.

We report for the first time an organic nanoparticle based nuclear-targeted photoresponsive drug delivery system (DDS) for regulated anticancer drug release. Acridin-9-methanol fluorescent organic nanoparticles used in this DDS performed three important roles: (i) ″nuclear-targeted nanocarrier″ for drug delivery, (ii) ″phototrigger″ for regulated drug release, and (iii) fluorescent chromophore for cell imaging. In vitro biological studies reveal acridin-9-methanol nanoparticles of ~60 nm size to be very efficient in delivering the anticancer drug chlorambucil into the target nucleus, killing the cancer cells upon irradiation. Such targeted organic nanoparticles with good biocompatibility, cellular uptake property, and efficient photoregulated drug release ability will be of great benefit in the field of targeted intracellular controlled drug release.

[Prolonged release of chlorambucil and etoposide from poly-3-oxybutyrate-based microspheres].

Microspheres were obtained on the basis of poly(3-oxibutyrate) (POB) with the inclusion of the Chlorambucil and Etoposide cytostatic drugs in a polymer matrix, and the morphology, kinetics of drug release from microspheres, and the interaction between microspheres and tumor cells in vitro were studied. Data on the kinetics of drug release suggests that a prolonged release occurs by drug diffusion from the polymer matrix at the initial stage and at the expense of hydrolytic degradation of the polymer at a later stage. A study of the biocompatibility and biological activity of biopolymeric microspheres showed that chlorambucil operates actively and strongly inhibits the growth of cultured cells for a short time (24 h). Etoposide acts weaker (the percentage of cell growth suppression during 48 h does not exceed 50%), but subsequently it has a basis for the creation of new dosage forms with prolonged action of Etoposide and chlorambucil for cancer therapy.

A two-photon responsive naphthyl tagged p-hydroxyphenacyl based drug delivery system: uncaging of anti-cancer drug in the phototherapeutic window with real-time monitoring.

We report a two-photon responsive drug delivery system (DDS), namely, p-hydroxyphenacyl-naphthalene-chlorambucil (pHP-Naph-Cbl), having a two-photon absorption (TPA) cross-section of ≥20 GM in the phototherapeutic window (700 nm). Our DDS exhibited both AIE and ESIPT phenomena, which were utilized for the real-time monitoring of anti-cancer drug release.

Enzyme-responsive sulfatocyclodextrin/prodrug supramolecular assembly for controlled release of anti-cancer drug chlorambucil.

Supramolecular drug delivery systems are becoming an increasingly important part in controlled drug release. In this work, we report a novel enzyme-responsive supramolecular assembly directly constructed using biocompatible sulfato-ß-cyclodextrin (SCD) and an anti-cancer prodrug, i.e. choline modified anti-cancer drug chlorambucil (QA-Cbl). The supramolecular assembly acts as an effective drug delivery system via the controlled drug loading and enzyme-responsive drug release, because the butyrylcholinesterase (BChE) can cleave the ester bond of QA-Cbl prodrug, resulting in the release of anti-cancer drug chlorambucil (Cbl). Compared to other sophisticated drug delivery systems, the present system provides a feasible and functional approach for achievement of controlled drug release.

Synthesis, in vivo antileukemic evaluation and comparative study of novel 5alpha-7-keto steroidal esters of chlorambucil and its active metabolite.

Recent structure-antileukemic activity studies showed that the steroidal part of complex molecules containing DNA alkylators does not play only the role of the "biological carrier". New such compounds designed to possess an allylic 7-ketone showed enhanced antileukemic potency compared with derivatives with a simple steroidal skeleton. In order to investigate whether the enhancement of the antileukemic potency is attributed to the introduction of the 7-ketone or to the Delta5-7-keto conjugated steroidal system we decided to reduce the Delta5 double bond. The 5alpha-7-keto-steroidal skeletons synthesized were tethered to chlorambucil and phenyl acetic acid's nitrogen mustard and studied against leukemia P338 in vivo. The reduction of the double bond had a negative impact on the antileukemic potency since the comparative study of the novel derivatives showed that a series of very potent Delta 5-7-keto-steroidal esters were converted by this modification to compounds with marginally accepted activity.

Construction of drug-drug conjugate supramolecular nanocarriers based on water-soluble pillar[6]arene for combination chemotherapy.

The synergistic effect of two anticancer drugs can significantly overcome the multidrug resistance of tumor cells and improve the drug bioavailability. Herein, two different anticancer drugs, camptothecin and chlorambucil, are successfully connected together by a disulfide linkage to get a novel drug-drug conjugated prodrug (G). Using water-soluble pillar[6]arene (WP6) as a host molecule, a supramolecular host-guest complex WP6⊃G is formed, which can further self-assemble into supramolecular vesicles in aqueous solution. In the specific microenvironment of cancer cells, the disulfide linkage is destroyed and the two anticancer drugs can be released efficiently to achieve a better synergistic effect than a single anticancer drug. Notably, these prodrug nanocarriers can not only effectively kill the cancer cells but also obviously reduce the undesirable side effects on normal cells.

Reactions of {4-[bis(2-chloroethyl)amino]phenyl}acetic acid (phenylacetic acid mustard) with 2'-deoxyribonucleosides.

Phenylacetic acid mustard (PAM; 2), a major metabolite of the anticancer agent chlorambucil (CLB; 1), was allowed to react with 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), 2'-deoxy-5-methylcytidine (dMeC), and thymidine (T) at physiological pH (cacodylic acid, 50% base). The reactions were followed by HPLC and analyzed by HPLC/MS and/or (1)H-NMR techniques. Although the predominant reaction observed was hydrolysis of PAM, 2 also reacted with various heteroatoms of the nucleosides to give a series of products: compounds 5-31. PAM (2) was found to be hydrolytically slightly more stable than CLB (1). The principal reaction sites of 2 with dA, dG, and with all pyrimidine nucleosides were N(1), N(7), and N(3), resp. Also, several other adducts were detected and characterized. There was no significant difference in the reactivity of 1 and 2 with dG, dA or T, but the N(3) dC-PAM adduct was deaminated easier than the corresponding CLB derivative. The role of PAM-2'-deoxyribonucleoside adducts on the cytotoxic and mutagenic properties of CLB (1) is discussed.

[Rat microsomal glutathione S-transferase 1 alters cytotoxic effects of chlorambucil on PC-3, K562, HepG2 and P388D1 cell lines].

OBJECTIVE: To explore the possible association between activation of rat microsomal glutathione S-transferase 1 (mGST1) and chlorambucil toxicity on selected cancer cell lines. METHODS: Hepatic microsomes were prepared from male Sprague-Dawley rats and washed to remove cytosolic contamination. mGST1 was purified and its activity was measured. PC-3, K562, HepG2 and P388D1 cell lines were exposed to chlorambucil (CHB) alone or to CHB with mGST1 at concentrations of 0 ~ 100 micromol/L for 8, 24, 48, 72 h. Cytotoxic effects of CHB were determined by cell growth inhibition (MTT assay), mitochondrial transmembrane potential (DeltaPsim), and fluorescence morphological examination (AO/EB staining). RESULTS: The decreased cytotoxic effects of CHB on the cell lines altered by mGST1 were demonstrated in concentration- and time-dependant manners. The CHB-induced apoptosis on PC-3 and K562 cell lines altered by mGST1 was confirmed using DeltaPsim examination, JC-1 or AO/EB staining. CONCLUSION: mGST1 can reduce the cytotoxic effects of CHB in selected cancer cell lines.

Novel 99mTc-Radiolabeled Anionic Linear Globular PEG-Based Dendrimer-Chlorambucil: Non-Invasive Method for In-Vivo Biodistribution.

Chlorambucil (CBL) is an alkylating agent, which widely use in the treatment of various types of tumors. The main purpose of this study is to evaluate the in-vivo biodistribution of CBL conjugated to the anionic dendrimer, which has a great ability to labeled with 99mTc through binding to carboxylate terminate groups. Whole body scans were used to analyze the percentage of the injected dose in different times. Radiochemical purity (RCP) and in-vivo biodistribution were also calculated with the SPECT/CT instrument. Our study proposes a new method for RCP determination and shows that this carrier is a promising agent to complex with the 99mTc and biological assessment.

Comparison of antitumor activities of 2-chlorodeoxyadenosine and 9-beta-arabinosyl-2-fluoroadenine in chronic lymphocytic leukemia and marrow cells in vitro.

The in vitro antitumor activities of the nucleoside analogs, 2-chlorodeoxyadenosine (CdA) and 9-beta-arabinosyl-2-fluoroadenine monophosphate (Flu), and the alkylating agent, chlorambucil (CLB), were compared in leukemic cells from 28 patients with chronic lymphocytic leukemia (CLL). On a molar basis, the median sensitivities of the cells to these agents were CLB > CdA > Flu. CLL cells from 90% of the patients had similar relative orders of sensitivities to CdA and Flu, while cells from 10% of the patients showed differential sensitivities to these agents. There was no relationship between the sensitivities of the cells to the nucleoside analogs and sensitivity to CLB. CdA and CLB produced similar toxicities to human marrow progenitor cells in vitro, while Flu was less toxic to these cells. An 18 h exposure to CdA produced significantly greater cell kill of both CLL and marrow progenitor cells than an equivalent 2 h treatment; however, the difference in cytotoxicity was greater for the tumor cells resulting in a higher therapeutic index with the 18 h treatment. The intracellular accumulation of drug varied 5-fold for CdA, with the major metabolite being CdAMP, and 15-fold for Flu, with the major metabolite being F-ara-ATP. However, the accumulation of CdA, Flu or their metabolites did not predict for drug sensitivity. These studies suggest that CdA and Flu cross-resistance cannot be assumed in all CLL patients. The therapeutic effectiveness of CdA may be enhanced by use of a prolonged, low-dose drug regimen.

DNA minor groove alkylating agents.

Recent work on a number of different classes of anticancer agents that alkylate DNA in the minor groove is reviewed. There has been much work with nitrogen mustards, where attachment of the mustard unit to carrier molecules can change the normal patterns of both regio- and sequence-selectivity, from reaction primarily at most guanine N7 sites in the major groove to a few adenine N3 sites at the 3'-end of poly(A/T) sequences in the minor groove. Carrier molecules discussed for mustards are intercalators, polypyrroles, polyimidazoles, bis(benzimidazoles), polybenzamides and anilinoquinolinium salts. In contrast, similar targeting of pyrrolizidine alkylators by a variety of carriers has little effect of their patterns of alkylation (at the 2-amino group of guanine). Recent work on the pyrrolobenzodiazepine and cyclopropaindolone classes of natural product minor groove binders is also reviewed.

Selective chemotherapeutic strategies using catalytic antibodies: a common pro-moiety for antibody-directed abzyme prodrug therapy.

Prodrug activation by catalytic antibodies (abzymes) conjugated with anti-tumor antibodies, called antibody-directed abzyme prodrug therapy (ADAPT), has been proposed as a strategy for site-specific drug delivery systems for anti-tumor drugs. The delivery of abzymes is achieved by making a bi-specific antibody with a monovalent catalytic antibody and a monovalent binding antibody. To achieve ADAPT, we focused on specific requirements for prodrugs and catalytic antibodies, the stability of the prodrugs against natural enzymes, and the applicability of abzymes for a wide range of prodrugs. Attention was paid to the design of a pro-moiety rather than a parent drug. As a common pro-moiety, we chose vitamin B(6), because the bulky vitamin B(6) esters are relatively stable against hydrolytic enzymes in serum. We have generated catalytic antibodies by immunization of a vitamin B(6) phosphonate transition state analog. The elicited antibodies were found to hydrolyze several anti-cancer and anti-inflammatory prodrugs with the vitamin B(6) pro-moiety. Finally, we evaluated antibody-catalyzed prodrug activation by examining the growth inhibition of human cervical cancer (HeLa) cells with the vitamin B(6) ester of butyric acid. These results suggest that the pro-moiety of vitamin B(6) ester is stable enough to resist natural enzymes in serum and is removed by the tailored catalytic antibodies. The combination of catalytic antibodies and prodrugs masked with vitamin B(6) would allow hydrophobic and highly toxic drugs to be used.

Studies of chlorambucil-DNA adducts.

Chlorambucil (CLB) is a bifunctional nitrogen mustard whose therapeutic and major side-effects are thought to be caused by binding to DNA. HPLC analysis of hydrolyzed DNA from L1210 cells incubated with [14C]CLB generated two peaks of radioactivity, indicating the formation of two or more major adducts. Since DNA incubated with [14C]CLB in a cell-free system gave rise to the same profile, experiments were conducted with DNA from cells exposed to radiolabeled DNA precursors, which was then reacted with CLB. DNA containing [8-14C]guanine gave rise to one peak of radioactivity, while DNA containing [2,8-3H]adenine gave rise to two peaks. These peaks corresponded to the peaks seen in the experiment with intact L1210 cells treated with [14C]CLB. Experiments with DNA containing [5-3H]cytosine indicated that no cytosyl adducts were formed. No adducts were seen in hydrolysates prepared from labeled DNA incubated with drug solvent alone. These data indicate that the majority of adducts induced by CLB are guanyl adducts, but a substantial quantity of adenyl adducts has also been identified.

Uptake and decomposition of chlorambucil by L5178Y lymphoblasts in vitro.

The uptake of [14C]chlorambucil by L5178Y lymphoblasts was studied using thin-layer chromatography to identify the various radioactive components that enter or leave cells. Theoretical calculations predicted that entry of chlorambucil into cells by simple diffusion would be rapid and essentially complete in 45 sec or less. Uptake of intact chlorambucil was rapid, reaching a cell/medium ratio of approximately 1.5 in less than 15 sec at both 37 degrees and 4 degrees, consistent with a simple diffusion mechanism. In cells treated with [14C]chlorambucil for 60 min, the intracellular level of intact drug decreased with time, and this decay was attributed to hydrolysis and alkylation. The level of intact drug in the medium decreased at a similar rate resulting in a nearly constant cell/medium distribution ratio. Intact chlorambucil in the cells was found to be entirely ethanol- and trichloroacetic acid-soluble. Efflux of intact chlorambucil was very rapid and temperature-insensitive. These findings suggest that chlorambucil efflux, as well as influx, is by a simple diffusion mechanism. A derivative of chlorambucil was found in ethanol solutions of the drug. This derivative, which may be the ethyl ester of the drug, is highly concentrated in cells and may interfere with pharmacological investigations of chlorambucil.