Synergetic bitherapy in mice with xenografts of human prostate cancer using a methional mimic (METLICO) and an aldehyde dehydrogenase 3 inhibitor (MATE): systemic intraperitoneal (IP) and targeted intra-tumoral (IT) administration.

An intraperitoneal (IP) monotherapy in nu/nu mice with subcutaneous xenografts of a human prostate epithelial cancer cell line:DU145 was undertaken with an aldehyde dehydrogenase 3 inhibitor MATE, that is a potent apoptogen on (DU145) in culture but not on their human prostate epithelial normal counterparts [13] . Tumour growth was slowed down but treatment had to be done 5days/week. To try to potentiate the action of MATE in vivo, a bitherapy was undertaken based on the synergetic apoptotic effect that had been observed previously in culture on DU145 treated with a methional mimic METLICO and DIMATE, an inhibitor of ALDH1 and ALDH3 [19]. The bitherapy with METLICO/MATE administered IP was as effective as the monotherapy with MATE alone by IP, but at a 2-fold lower dose of MATE and at a dose of METLICO that had no growth-inhibitory effect as a monotherapy . Hence there was definite synergism with bitherapy. To try to increase the efficacy of bitherapy, it was administered by the intra-tumoral (IT) route using the recently developed 20-bars-pressurized microinjection system from CERMA [16, 17]. IT administration of the bitherapy was indeed more effective than that by IP as regards tumour volumes are concerned. Histopathological analysis of IT-treated tumours confirmed that there were many necrotized zones but intact cells were still present. Approaches for treating a wider zone of tumour tissue by IT-bitherapy are discussed.

The effect of a novel irreversible inhibitor of aldehyde dehydrogenases 1 and 3 on tumour cell growth and death.

Aldehyde dehydrogenases (ALDHs) are a family of several isoenzymes expressed in various tissues and in all subcellular fractions. In some tumours, there is an increase of ALDH activity, especially that of class 1 and 3. The increase in the activity of these isoenzymes is correlated with cell growth and drug resistance shown by these cells. It has been observed that hepatoma cells expressing low ALDH3 activity are more susceptible to growth inhibition by low concentration of lipid peroxidation products than hepatoma cells expressing high ALDH3 activity. The products of lipid peroxidation are good substrates for ALDH, but when their intracellular levels are increased in hepatoma cells treated repeatedly with prooxidants, they inhibit ALDH3 and bring about growth inhibition or cell death. As a follow up to the work previously reported on S-methyl 4-amino-4-methylpent-2-ynethioate, a synthetic suicide inhibitor of ALDH1, which induced bcl2 overexpressing cells into apoptosis and exhibited an ED50 of 400 microM, a novel broad spectrum inhibitor of ALDH1 and ALDH3 was synthesised. This new compound (ATEM) is a suicide inhibitor of ALDH1, an irreversible inhibitor of ALDH3 and exhibits an ED50 of 10-25 microM on rat cultured hepatoma cells. Four hours after treatment with 25 microM ATEM, ALDH activity using benzaldehyde or propionaldehyde in hepatoma cells was decreased by 40% and cell number by 15% compared with controls. As cell growth did not resume when the inhibitor was removed from the culture medium, it suggested strongly that ALDHs play a pivotal role in mediating cell death.

Inhibition of growth and induction of apoptosis by androgens of a variant of LNCaP cell line.

Here are described the effects of androgens, and other molecules known to bind to androgen receptors (AR), on MOP cells established from the human prostate cancer cell line LNCaP. MOP cells contained AR: 100000 binding sites/cell, K(D) for 5alpha dihydrotestosterone (DHT) 0.5 nM, size 110 kDa. The AR gene has the same repetition polymorphism in exon 1 and the T876A mutation in exon 8 as LNCaP. The proliferation of MOP cells in culture was repressed by the synthetic androgen 17beta-hydroxy-17-methyl-estra-4,9,11-trien-3-one (R 1881), the antiandrogen cyproterone acetate (CYPA), estradiol (E2), progesterone and the synthetic progestin promegestone: 17,21 dimethyl-19 nor-4,9 pregnandiene-3,20 dione (R 5020). The number of cells recovered after 7 days decreased to approximately 40% of controls. ED(70)s ranged between 50 pM for R 1881 to 50 nM for E2 and CYPA. Treatment with R 1881 decreased [3H]thymidine incorporation into DNA and increased dramatically the doubling time. R 1881, CYPA and E2 blocked the cell cycle between G1 and S phases and they induced apototosis as demonstrated by the increase of blebs on the plasma membrane, nuclear fragmentation, TdT-mediated dUTP nick end-labeling (TUNEL)-positive cells and internucleosomal DNA breaks. In athymic nude mice, testosterone enanthate prevented the growth of MOP tumors and, when tumors did develop, brought about regression. However, the tumors did not regress completely and finally escaped treatment. In conclusion, a variant of the LNCaP cell line has been established. With these cells it was possible to confirm that androgens paradoxically repress the growth of some prostate cancer cells both in culture and in vivo. In addition it is demonstrated in culture but not in vivo, for the first time to the authors' knowledge, that a synthetic androgen is able to induce apoptosis of cells established from human prostate carcinoma.

Altered methional homoeostasis is associated with decreased apoptosis in BAF3 bcl2 murine lymphoid cells.

Methional is a potent inducer of apoptosis in an interleukin 3-dependent murine lymphoid cell line BAF3 b0 when it is added to the culture medium. In these cells transfected with the bcl2 gene, BAF3 bcl2, the apoptotic-inducing activity of methional is dramatically reduced. The addition of disulfiram (an inhibitor of aldehyde dehydrogenase) in order to reduce methional oxidation brought about an increase in apoptosis in BAF3 b0 but not in BAF3 bcl2 cells. In contrast, the addition of quercetin (an inhibitor of aldehyde reductase) in an attempt to diminish methional reduction increased apoptosis in both BAF3 b0 and BAF3 bcl2 cells. The extent of DNA fragmentation in BAF3 bcl2 cells approached that in BAF3 b0 cells in the presence of quercetin and exogenous methional, suggesting a defect in methional biosynthesis in BAF3 bcl2 cells. Direct evidence for this was obtained by measuring labelled methional in cells incubated with the sodium, salt of [U-14C]4-methylthio-2-oxobutanoic acid (MTOB), the precursor of methional. The 80% decrease in labelled methional in BAF3 bcl2 compared with BAF3 b0 cells was accompanied by a concomitant rise in the transamination of [14C]MTOB to [14C]methionine in BAF3 bcl2 cells. Inhibition of the transaminase, however, by a synthetic transition-state-type compound, pyridoxal-L-methionine ethyl ester, induced apoptosis in BAF3 b0 but not in BAF3 bcl2 cells, confirming that the defect in BAF3 bcl2 cells was not in the transaminase itself but rather in the oxidative decarboxylation step MTOB --> methional. In addition, no evidence was obtained for the synthesis of [14C]malondialdehyde from [14C]methional in BAF3 bcl2 cells. As these cells show no deficiency in their content of reactive oxygen species compared with that of BAF3 b0 cells, they may possess some other defect in the beta-hydroxylase enzyme system itself.

Methional derived from 4-methylthio-2-oxobutanoate is a cellular mediator of apoptosis in BAF3 lymphoid cells.

4-Methylthio-2-oxobutanoic acid is the direct precursor of methional, which is a potent inducer of apoptosis in a BAF3 murine lymphoid cell line which is interleukin-3 (IL3)-dependent. Cultures treated for 8 h with methional in the presence of IL3 show extensive DNA double-strand breaks on flow cytometric analysis, increases in DNA fragmentation as measured by the amount of non-sedimentable DNA present in the 30,000 g supernatant of cell lysates and the typical laddering pattern of multiples of 180 bp seen upon agarose gel electrophoresis. No such features of apoptosis were found in cells treated with 4-methylthio 2-oxobutanoic acid or propanal, suggesting that the simultaneous presence of the methylthio group on the propanal moiety is essential for apoptosis to take place. Methional is further metabolized in cells by two reactions: oxidation via aldehyde dehydrogenase to (methylthio)propionic acid or beta-hydroxylation to malondialdehyde. The formation of malondialdehyde from methional in vitro by chemical hydroxylation under the conditions of the Fenton reaction provides a mechanism for the beta-hydroxylation which takes place in vivo. During apoptosis induced by IL3 deprivation, the ratio of 2,4-DNPH MDA to 2,4-DNPH methional is 0.94 in cells in IL3- medium compared with 0.54 in cells in IL3+ medium. These results support a role of cellular methional and malondialdehyde in apoptosis.

Inactivation of human cytomegalovirus by sodium periodate oxidation.

Human cytomegalovirus (HCMV), oxidized by sodium periodate (NaIO4), is incapable of giving rise to viral progeny in cell culture. At a NaIO4 concentration as low as 5 mM, there is a loss of at least 6 logs of viral infectivity which occurs very rapidly (less than 5 min). Further, the inactivation is a first-order reaction depending on the periodate concentration. Adsorption to the cell surface, penetration into cells, and penetration of the viral DNA into cell nuclei were found to occur identically in mock oxidized and oxidized HCMV. Since the carbohydrate moiety of viral glycoproteins was the target of periodate attack, these observations strongly suggest that the structural integrity of the sugar residues is not a prerequisite for adsorption and penetration. Nevertheless, no evidence for viral DNA or protein synthesis was detected in cells infected with oxidized virus, and even after 3 weeks in culture, no cytopathic effect was observed.

Contribution of 4-methylthio-2-oxobutanoate and its transaminase to the growth of methionine-dependent cells in culture. Effect of transaminase inhibitors.

The growth in culture of methionine-dependent transformed cells of human, rat and mouse origin was arrested in the absence of L-methionine (Met) but took place in the presence of 4-methylthio-2-oxobutanoic acid (MTOB), the keto acid of Met. From 24 hr after seeding, cells grew in 0.1 mM MTOB medium at a rate comparable to that in 0.1 mM Met medium. Using [35S]MTOB, it was found that the Met synthesized was used in normal MRC-5 cells and in transformed HeLa cells to the same extent for protein, adenosylmethionine and adenosylhomocysteine syntheses. However, when the free Met content was examined, it was found to be 3-fold greater in HeLa than in MRC-5 cells. To examine the importance of this free Met for the growth of transformed cells, the transaminase responsible for converting MTOB to Met was chosen as a target enzyme for the synthesis of compounds with potential inhibitory activity. Since this is a multisubstrate enzyme, reduced Schiff bases were prepared containing both pyridoxal or other aromatic groups, as one constituent, and L-Met or other amino-acids in the free acid or ester or amide form, as the other constituent. Only esters containing the pyridoxal moiety and Met or certain of its structural analogues exhibited good selective growth inhibitory activity in that there was little (20%) or no effect on the growth of normal MRC-5 and derm cells, respectively, while that of transformed HeLa, HEp-2 and L1210 cells was strongly inhibited (80%). This inhibition was accompanied by a concomitant decrease in the activity of the MTOB transaminase in both HeLa and MRC-5 cells treated with 3c the most potent inhibitor. However, using [35S]MTOB it was found that MTOB itself accumulated 48% in HeLa but only 12% in MRC-5 cells treated with 3c. On the contrary [35S]Met formed from [35S]MTOB increased 3.7-fold in MRC-5 inhibitor-treated cells showing 20% growth inhibition whereas it decreased 38% in HeLa-treated cells showing 80% growth inhibition. This decrease in cellular Met in HeLa is not responsible for growth arrest. Indeed the growth of HeLa cells could not be restored by adding a 10-fold excess of Met. Since MTOB can alleviate Met-dependence, the intracellular homeostasis of this metabolite may play a hitherto unsuspected role in controlling cell growth.

Transglutaminase activity and N epsilon (gamma glutamyl) lysine isopeptide levels during cell growth: an enzymic and immunological study.

A monoclonal antibody (MAb) 81D1c2, which recognizes the N epsilon (gamma glutamyl) lysine isopeptide produced by the action of transglutaminase activity was prepared. Its reactivity towards the homologous isopeptide was about 3-fold greater than that with either N alpha (alpha glutamyl) lysine (a naturally occurring heterologous dipeptide) or N alpha (gamma glutamyl) lysine, another heterologous peptide not described so far in naturally occurring proteins. When used in an immunohistochemical study on cells in culture derived from human carcinoma of the larynx (HEp2) and from chicken embryo cells (CEC), both fixed in acetone, this MAb detected N epsilon (gamma glutamyl) lysine residues in the nucleus. The amount of N epsilon (gamma glutamyl) lysine isopeptides follows closely transglutaminase activity during the lag phase of growth of both CEC and HEp2 cells. However, during exponential growth, the 2 parameters decrease concomitantly in HEp2 cells, whereas in CEC, transglutaminase activity increases but isopeptide bond levels drop. Compared with other reported methods for measuring isopeptides, this immunohistological approach permits the localization and at least the semi-quantitative determination of N epsilon (gamma glutamyl) lysine in cells in situ.

Synthesis of acetylenic spirobutenolide derivatives and evaluation of their growth inhibitory effect on cells in culture.

Acetylenic spirobutenolide amides and esters and their Mannich bases were synthesized to evaluate their growth inhibitory effect. The biological tests have used both normal and transformed cells and they have shown the selectivity of the prepared compounds. The ester derivatives presented the best selectivity comparable to that of daunorubicin.

The effect of a novel inhibitor of aldehyde dehydrogenase on viral replication.

The effect of AMPAL (4-amino 4-methyl 2-pentyne 1-al), an inhibitor of aldehyde dehydrogenase, on adenovirus type 5 (Ad5) replication was studied. AMPAL at 2 x 10(-4) M clearly reduced the cytopathic effect on HeLa cells but had no effect on cell growth at this concentration. Viral adsorption, penetration and protein synthesis were not affected by adding AMPAL at 2 hr post infection. When viral DNA synthesized in the presence of AMPAL was investigated, no significant inhibition was observed on either synthesis or the physicochemical properties of the neosynthesized DNA. However, there was a 4-fold increase in the amount of condensed DNA. In addition, AMPAL inhibited intracellular viral production (40%) and brought about concomitant inhibition of virus release (70%) into the medium. The absence of a quantitative relationship between the inhibition of viral DNA synthesis on one hand and that of viral production on the other may imply that the antiviral effect of AMPAL is indirectly mediated by the action of the malondialdehyde which has accumulated on some, as not yet identified, membrane constituent.