Chlorambucil-induced high mutation rate and suicidal gene downregulation in a base excision repair-deficient Escherichia coli strain

Chlorambucil (CLB; N,N-bis(2-chloroethyl)-p-aminophenylbutyric acid) is a bifunctional alkylating agent widely used as an anticancer drug and also as an immunosuppressant. Its chemical structure and clinical experience indicate that CLB is mutagenic and carcinogenic. We have investigated the ability of CLB to induce mutations and gene expression changes in the wild-type (WT) Escherichia coli strain AB1157 and in the base excision repair-deficient (alkA1, tag-1) E. coli strain MV1932 using a rifampicin (rif) forward mutation system and a cDNA array method. The results showed that CLB is a potent mutagen in MV1932 cells compared with the E. coli WT strain AB1157, emphasizing the role of 3-methyladenine DNA glycosylases I and II in protecting the cells from CLB-induced DNA damage and subsequent mutations. Global gene expression profiling revealed that nine genes in WT E. coli and 100 genes in MV1932, of a total of 4290 genes, responded at least 2.5-fold to CLB. Interestingly, all of these MV1932 genes were downregulated, while 22% were upregulated in WT cells. The downregulated genes in MV1932 represented most (19/23) functional categories, and unexpectedly, many of them code for proteins responsible for genomic integrity. These include: (i) RecF (SOS-response, adaptive mutation), (ii) RecC (resistance to cross-linking agents), (iii) HepA (DNA repair, a possible substitute of RecBCD), (iv) Ssb (DNA recombination repair, controls RecBCD), and (v) SbcC (genetic recombination). Our results strongly suggest that in addition to the DNA damage itself, the downregulation of central protecting genes is responsible for the decreased cell survival (demonstrated in a previous work) and the increased mutation rate (this work) of DNA repair-deficient cells, when exposed to CLB.     

Immunoreactive-beta-endorphin, -adrenocorticotropin and -calcitonin in extracts of anaplastic or differentiated (rat) medullary thyroid carcinoma.

Fifteen analogs of luliberin (2, LRH) were synthesized by the solid phase method and examined for their ability to block ovulation in the rat. Two analogs, [Ac-DAla¹,DPhe²,DTrp^3,6]-LRH and [Ac-DPhe¹,DPhe²,DTrp^3,6]-LRH, each blocked ovulation at a single injection dose of 250 μg administered at noon on the day of proestrus; three peptides, [Ac-DPro¹,DPhe²,DTrp^3,6]-LRH, [Ac-DThi¹,DPhe²,DTrp^3,6]-LRH and [Ac-DTrp¹,DPhe²,DTrp^3,6]-LRH, were effective at doses of 500 μg each; and four others, [Ac-DTrp¹,DPhe²,DTrp³,DTrp(Nps)⁶]-LRH, [Chlorambucil-DPhe¹, DPhe², DTrp^3,6]-LRH, [1,DThi²,DTrp^3,6]-LRH and [(2-DLys⁶]-LRH, gave partial inhibition at doses tested.     

Rational design,synthesis and preliminary antitumor activity evaluation of a chlorambucil derivative with potent DNA/HDAC dual-targeting inhibitory activity

Histone deacetylases (HDACs) play a pivotal role not only in gene expression but also in DNA repair. Herein, we report the successful design, synthesis and evaluation of a chlorambucil derivative named vorambucil with a hydroxamic acid tail as a DNA/HDAC dual-targeting inhibitor. Vorambucil obtained both potent DNA and HDACs inhibitory activities. Molecular docking results supported the initial pharmacophoric hypothesis and rationalized the potent inhibitory activity of vorambucil against HDAC1, HDAC2 and HDAC6. Vorambucil showed potent antiproliferative activity against all the test four cancer cell lines with IC₅₀ values of as low as 3.2–6.2 μM and exhibited 5.0–18.3-fold enhanced antiproliferative activity than chlorambucil. Vorambucil also significantly inhibits colony formation of A375 cancer cells. Further investigation showed that vorambucil remarkably induced apoptosis and arrested the cell cycle of A375 cells at G2/M phase. Vorambucil could be a promising candidate and a useful tool to elucidate the role of those DNA/HDAC dual-targeting inhibitors for cancer therapy.     

Synthesis and cytotoxic properties of new fluorodeoxyglucose-coupled chlorambucil derivatives

Frequently used in the treatment of malignant cells, alkylating agents, like most anticancer substances, produce adverse side effects caused by the toxicity of the agents toward normal tissues and lose efficiency through poor distribution to target sites. Our approach to developing more selective drugs with low systemic toxicity is based on the premise that the body distribution and cell uptake of a drug can be altered by attaching a neoplastic cell-specific uptake enhancer, such as 2-fluoro-2-deoxyglucose (FDG), the radiotracer most frequently used in PET for tumor imaging. Two properties of deoxyglucose, namely preferential accumulation in neoplastic cells and inhibition of glycolysis, underpin this targeting approach. Here, we report the synthesis of 19 new chlorambucil glycoconjugates in which the alkylating drug is attached to the C-1 position of FDG, directly or via different linkages. This set of compounds was evaluated for in vitro cytotoxicity against different human normal and tumor cell lines. There was a significant improvement in the in vitro cytotoxicity of peracetylated glucoconjugates compared with the free substance. Four compounds were finally selected for further in vivo studies owing to their lack of oxidative stress-inducing properties.     

Reactions of 4‐[Bis(2‐chloroethyl)amino]benzenebutanoic Acid (Chlorambucil) with DNA

4‐[Bis(2‐chloroethyl)amino]benzenebutanoic acid (=chlorambucil, 1 ; 2.5 mM ) was allowed to react with single‐ and double‐stranded calf thymus DNA at physiological pH (cacodylic acid, 50% base) at 37°. The DNA–chlorambucil adducts were identified by analyzing the DNA hydrolysates by NMR, UV, HPLC, LC/ESI‐MS/MS techniques as well as by spiking with authentic materials. ssDNA was more reactive than dsDNA, and the order of reactivity in ssDNA was Ade‐N1>Gua‐N7>Cyt‐N3>Ade‐N3. The most reactive site in dsDNA was Ade‐N3. The Gua‐N7 and Ade‐N3 adducts were hydrolytically labile. Ade‐N7 adduct could not be identified in the hydrolysates of ssDNA or dsDNA. The adduct Gua‐N7,N7, which consists of two units of Gua bound together with a unit derived from chlorambucil, is a cross‐linking adduct, and it was detected in the hydrolysates of ssDNA and dsDNA. Also several other adducts were detected which could be characterized by spiking with previously isolated authentic adducts or tentatively by MS. The role of chlorambucil–DNA adducts on the cytotoxicity and mutagenity of 1 is also discussed.     

Synthesis and anticancer activity of open-resorcinarene conjugates

The first example of conjugation of open-resorcinarenes with chlorambucil, ibuprofen, naproxen and indomethacin are presented. The cytotoxic properties of the obtained conjugates were tested against the cancer cell lines U-251, PC-3, K-562, HCT-15, MCF-7 and SKLU-1. It was found that the conjugate with chlorambucil, naproxen or indomethacin (having 8 moieties) was toxic towards cancer cell lines U-251 and K-562, with no activity against non-cancerous COS-7 cells. The conjugates with naproxen and indomethacin showed high selectivity towards U-251 tumor 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.     

Ascaridia galli: effects of immunosuppressive drugs or bursectomy in chickens

Immunosuppressive drugs significantly increasing numbers of A. galli and incidences of infection were: cortisone, cortisol, 9-α-fluorohydrocortisone, 2-methyl-9-α-fluorohydrocortisone, prednisone, prednisolone, 6-mercaptopurine, 2, 6-diaminopurine, 6-thioguanine, 5-bromodeoxyridine, 5-fluorouracil, methotrexate, chlorambucil, and actinomycin D. These drugs and/or worm burdens significantly suppressed weight gains of hosts, and neither altered the male:female ratio of worms nor their growth. The following drugs neither altered worm burdens nor increased incidences of infection: corticosterone, 2-azaadenine, 8-azaguanine, azathioprine, 6-azauridine, busulfan, thio-TEPA, triethylenemelamine, vincristine, acriflavine, reserpine, and l-phenylalanine.Worm burdens and incidences of infection were increased significantly in chickens surgically bursectomized when 3 or 14 but not 35 days old. Chicks bursectomized in ovo with testerosterone propionate on Day 5 or 14 of incubation and infected on Day 14 after hatching developed significantly increased worm burdens and incidences of infection.Applying the Kolmogorov-Smirnov test for goodness of fit to data on increased worm burdens showed that the immunosuppressive drugs or bursectomy had a normalization effect on the statistical distribution.