A synthetic triptycene bisquinone, which blocks nucleoside transport and induces DNA fragmentation, retains its cytotoxic efficacy in daunorubicin-resistant HL-60 cell lines.

In contrast to the parent triptycene (code name TT0), triptycene bisquinone (code name TT2) is cytostatic (IC50: 300 nM) and cytotoxic (IC50: 230 nM) in wild-type (WT), drug-sensitive HL-60 cells (HL-60-S) at day 4. Therefore, the effects of this new quinone antitumor drug were assessed and compared to those of daunorubicin (DAU, daunomycin) in the multidrug-resistant (MDR) HL-60-RV and HL-60-R8 sublines, which respectively overexpress P-glycoprotein (P-gp) or multidrug resistance-associated protein (MRP). In contrast to DAU, which loses its cytostatic [resistance factors (RFs): 22.9-35.7] and cytotoxic (RFs: 23.8-31.3) activities in MDR sublines, TT2 decreases tumor cell proliferation (RFs: 0.9-1.3) and viability (RFs: 0.9-1.5) as effectively in HL-60-S as in HL-60-RV and HL-60-R8 cells at days 2 and 4. Similarly, DAU inhibits the rate of DNA synthesis less effectively in MDR than in parental HL-60 cells (RFs: 8.1-11.9) but TT2 decreases the incorporation of 3[H]-thymidine into DNA to the same degree in HL-60-S, HL-60-RV and HL-60-R8 cells (RFs: 1.2). In contrast to DAU, which is inactive, the advantage of TT2 is its ability to block the cellular transport of purine and pyrimidine nucleosides in WT tumor cells, an effect which persists in both MDR sublines (RFs: 1.0-1.2). Moreover, the concentrations of DAU which induce maximal DNA cleavage in HL-60-S cells at 24 h lose all or most of their DNA-damaging activity in HL-60-RV and HL-60-R8 cells, whereas treatments with 4 microM TT2 produce similar peaks of DNA fragmentation in all WT and MDR cell lines. Since TT2 not only mimics the antitumor effects of DAU but also blocks nucleoside transport and retains its effectiveness in MDR cells that have already developed different mechanisms of resistance to DAU, this new quinone antitumor drug might be valuable to develop new means of polychemotherapy.

Synthetic 1,4-anthracenediones, which block nucleoside transport and induce DNA fragmentation, retain their cytotoxic efficacy in daunorubicin-resistant HL-60 cell lines.

Anthracene-1,4-dione and 6,7-dichloro-1,4-anthracenedione (code names AQ1 and AQ4, respectively) are cytostatic (IC50: 53 and 110 nM, respectively) and cytotoxic (IC50: 100 and 175 nM, respectively) in wild-type drug-sensitive HL-60-S tumor cells at day 4 in vitro. Therefore, the antitumor effects of these drugs were assessed and compared to those of daunorubicin (DAU) in HL-60-RV and HL-60-R8 tumor cells, which are, respectively, P-glycoprotein-positive and -negative multidrug-resistant (MDR) sublines. In contrast to DAU, which loses its cytostatic [resistance factors (RFs): 30.3-31.8] and cytotoxic (RFs: 48.8-58.1) activities in MDR sublines, AQ1 inhibits cell proliferation (RFs: 0.9-1.3) and cell viability (RFs: 1.4-1.6) as effectively in HL-60-RV and HL-60-R8 as in HL-60-S cells. Similarly, DAU decreases the rate of DNA synthesis less effectively in MDR sublines (RFs: 8.0-13.3) but AQ1 inhibits the incorporation of [3H]thymidine into DNA to the same degree in HL-60-S as in HL-60-RV and HL-60-R8 cells (RFs: 0.9-1.1). In contrast to DAU, which is ineffective, the advantage of AQ1 is its ability to block the cellular transport of purine and pyrimidine nucleosides in HL-60-S cells, an effect which persists in the MDR sublines (RFs: 1.1). AQ4, which mimics to a lesser degree all the antitumor effects of AQ1, except the inhibition of adenosine transport, also retains its effectiveness in MDR sublines (RFs: 1.1-3.1). The peaks of DNA cleavage caused by DAU and AQ1 in HL-60-S cells shift to lower concentrations with increasing times of drug exposure but DAU loses most of its ability to induce DNA fragmentation in MDR sublines, whereas the levels of AQ1-induced DNA cleavage at 16 and 24 h are nearly equivalent in HL-60-S, HL-60-RV and HL-60-R8 cells. Because they not only mimic the antitumor effects of DAU in the nM range but also block nucleoside transport and remain effective in tumor cells that have developed different mechanisms of MDR, AQ1 and AQ4 analogs might be valuable to develop new means of polychemotherapy.

Quinone isomers of the WS-5995 antibiotics: synthetic antitumor agents that inhibit macromolecule synthesis, block nucleoside transport, induce DNA fragmentation, and decrease the growth and viability of L1210 leukemic cells more effectively than ellagic acid and genistein in vitro.

Antibiotic WS-5995A (code name J4) and two of its synthetic analogs, o-quinone J1 and model p-quinone J7, which show some structural similarity with both ellagic acid (EA) and genistein (GEN), were compared for their antileukemic activity in L1210 cells in vitro. Overall, J4 is more cytostatic and cytotoxic than J1 and J7, suggesting that methyl and methoxy substitutions, a p-quinone moiety, and a hydrogen bonding phenolic group may enhance the antitumor potential of these naphthoquinone lactones, which are all more potent than EA and GEN. For instance, the lead compound J4 inhibits tumor cell proliferation and viability at day 4 (IC(50): 0.24--0.65 microM) more effectively than EA (IC(50): 5--6 microM) and GEN (IC(50): 7 microM). Since J4 does not increase but rather decreases the mitotic index of L1210 cells at 24 h, it is not an antitubulin drug but might arrest early stages of cell cycle progression like EA and GEN. A 1.5- to 3-h pretreatment with J4 is sufficient to inhibit the rates of DNA, RNA and protein syntheses (IC(50): 2.0--2.5 microM) determined over 30- to 60-min periods of pulse-labeling in L1210 cells in vitro, whereas EA (IC(50): 20-130 microM) and GEN (IC(50): 40--115 microM) are less effective against macromolecule synthesis. In contrast to 156 microM EA, which is inactive, a 15-min pretreatment with 10--25 microM J4 has the advantage of also inhibiting the cellular transport of both purine and pyrimidine nucleosides over a 30 s period in vitro, an effect which can be mimicked by 156 microM GEN. Hence, the WS-5995 analogs and GEN may prevent the incorporation of [(3)H]adenosine and [(3)H]thymidine into DNA because they rapidly block the uptake of these nucleosides by the tumor cells. After 24 h, the concentration-dependent induction of DNA cleavage by J4 peaks at 10 microM and declines at 25 microM, whereas EA and GEN are ineffective at 10 microM but maximally stimulate DNA cleavage at 62.5 microM. Like EA and GEN, the mechanism by which J4 induces DNA fragmentation is inhibited by actinomycin D, cycloheximide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone and ZnSO(4), suggesting that J4 triggers apoptosis by caspase and endonuclease activation. Because they are more potent than EA and GEN, and affect both nucleoside transport and DNA cleavage, the WS-5995 antitumor antibiotics might be valuable in polychemotherapy to potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

Antileukemic activity of synthetic daunomycinone derivatives bearing modifications in the glycosidic moiety.

The antileukemic activities of the daunomycinone glycosides synthesized in our laboratories (compounds 4 and 7, code names S12 and S13, respectively) were characterized in L1210 cells in vitro. S13 inhibits tumor cell proliferation and viability at day 4 (IC50: 150-200 nM) more effectively than S12 (IC50: 250-450 nM), suggesting that the 4'-trifluoracetamido substitution of the glycosidic moiety of these 3'-halo daunonycinone derivatives has greater antitumor potential than the 4'-azido substitution. Since S12 and S13 do not increase but rather decrease the mitotic index of L1210 cells at 24 hours, they are not antitubulin drugs but might arrest the early stages of cell cycle progression. Pretreatments for 1.5-3 hours with S12 and S13 are sufficient to partially inhibit the rates of DNA and RNA syntheses (IC50: 4-10 microM) determined over 30- to 60-minute periods of pulse-labeling in L 1210 cells in vitro, but these daunomycinone glycosides alter neither the cellular transport of purine and pyrimidine nucleosides nor the rate of protein synthesis. After 24 hours, the concentration-dependent induction of DNA cleavage by S13 reaches a plateau at 10 microM but the weaker S12 requires 48 hours to maximally stimulate DNA cleavage like S13. The mechanism by which S13 induces DNA fragmentation is inhibited by actinomycin D, cycloheximide, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, benzyloxycarbonyl-Ile-Glu-Thr-Asp-fluoromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone and ZnSO4, suggesting that S13 triggers apoptosis by caspase and endonuclease activation. Since microM concentrations of S12 and S13 are cytostatic and cytotoxic, but do not sufficiently inhibit RNA and protein syntheses to block their own ability to sustain the active process of apoptosis and DNA fragmentation, such 3'-halo daunomycinone glycosides might be valuable to develop new means of polychemotherapy.

1,4-Anthraquinone: an anticancer drug that blocks nucleoside transport, inhibits macromolecule synthesis, induces DNA fragmentation, and decreases the growth and viability of L1210 leukemic cells in the same nanomolar range as daunorubicin in vitro.

1,4-Anthraquinone (AQ) was synthesized and shown to prevent L1210 leukemic cells from synthesizing macromolecules and growing in vitro. In contrast, its dihydroxy-9,10anthraquinone precursor, quinizarin, was inactive. The antitumor activity of AQ was compared to that of daunorubicin (DAU), which is structurally different from AQ but also contains a quinone moiety. AQ is equipotent to DAU against L1210 tumor cell proliferation (IC50: 25 nM at day 2 and 9 nM at day 4) and viability (IC50: 100 nM at day 2 and 25 nM at day 4), suggesting that its cytostatic and cytotoxic activities are a combination of drug concentration and duration of drug exposure. Since AQ does not increase but rather decreases the mitotic index of L1210 cells at 24 h, it is not an antitubulin drug but might arrest early stages of cell cycle progression. Like DAU, a 1.5-3 h pretreatment with AQ is sufficient to inhibit the rates of DNA, RNA and protein syntheses (IC50: 2 microM) determined over 30-60 min periods of pulse-labeling in L1210 cells in vitro. In contrast to DAU, which is inactive, a 15 min pretreatment with AQ has the advantage of also inhibiting the cellular transport of both purine and pyrimidine nucleosides (IC50: 2.5 microM) over a 30 s period in vitro. Hence, AQ may prevent the incorporation [3H]thymidine into DNA because it rapidly blocks the uptake of these nucleosides by the tumor cells. After 24 h, AQ induces as much DNA cleavage as camptothecin and DAU, two anticancer drugs producing DNA strand breaks and known to, respectively, inhibit topoisomerase I and II activities. However, the concentration-dependent induction of DNA cleavage by AQ, which peaks at 1.6-4 microM and disappears at 10-25 microM, resembles that of DAU. The mechanism by which AQ induces DNA cleavage is inhibited by actinomycin D, cycloheximide and aurintricarboxylic acid, suggesting that AQ activates endonucleases and triggers apoptosis. The abilities of AQ to block nucleoside transport, inhibit DNA synthesis and induce DNA fragmentation are irreversible upon drug removal, suggesting that this compound may rapidly interact with various molecular targets in cell membranes and nuclei to disrupt the functions of nucleoside transporters and nucleic acids, and trigger long-lasting antitumor effects which persist after cessation of drug treatment. Because of its potency and dual effects on nucleoside transport and DNA cleavage, the use of bifunctional AQ with antileukemic activity in the nM range in vitro might provide a considerable advantage in polychemotherapy to potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

Triptycenes: a novel synthetic class of bifunctional anticancer drugs that inhibit nucleoside transport, induce DNA cleavage and decrease the viability of leukemic cells in the nanomolar range in vitro.

In contrast to their inactive parent compound triptycene (code name TT0), several triptycene (TT) analogs (code names TT1 to TT13), most of them new compounds, were synthesized and shown to prevent L1210 leukemic cells from synthesizing macromolecules and growing in vitro. The most potent rigid tetracyclic quinones synthesized so far are TT2 and its C2-brominated derivative, TT13. The antitumor activity of TT2 has been compared to that of daunomycin (DAU), a clinically valuable anthracycline antibiotic which is structurally different from TT2 but also contains a quinone moiety. TT2 inhibits the proliferation (IC50: 300 nM at day 2 and 150 nM at day 4) and viability (IC50: 250 nM at day 2 and 100 nM at day 4) of L1210 cells to the same maximal degree as DAU, suggesting that the cytostatic and cytotoxic activities of TT2 are a combination of drug concentration and duration of drug exposure. Since TT2 does not increase the mitotic index of L1210 cells at 24 h like vincristine, it is unlikely to be an antimitotic drug that disrupts microtubule dynamics. Like DAU, a 1.5-3 h pretreatment with TT2 is sufficient to inhibit the rates of DNA, RNA and protein syntheses determined over 30-60 min periods of pulse-labeling in L1210 cells in vitro (IC50: 6 microM). In contrast to DAU, which is inactive, a 15 min pretreatment with TT2 has the advantage of also inhibiting the cellular transport of nucleosides occuring over a 30 s period in vitro (IC50: 6 microM), suggesting that TT2 prevents the incorporation of [3H]thymidine into DNA because it rapidly blocks the uptake of [3H]thymidine by the tumor cells. After 24 h, TT2 induces as much DNA cleavage as camptothecin and DAU, two anti-cancer drugs producing DNA strand breaks and known to respectively inhibit DNA topoisomerase I and II activities. Interestingly, the abilities of TT2 to block nucleoside transport, inhibit DNA synthesis and induce DNA fragmentation are irreversible upon drug removal, suggesting that this compound may rapidly interact with various molecular targets in cell membranes and nuclei to disrupt the functions of nucleoside transporters and nucleic acids, and trigger long-lasting antitumor effects which persist after cessation of drug treatment. Because inhibition of nucleoside transport is highly unusual among DNA-damaging drugs, the use of bifunctional TTs with antileukemic activity in the nM range in vitro might provide a considerable advantage in polychemotherapy to potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

6,7,8,9-Tetrahydro-3-methyl-1H-pyrano-[4,3-b]quinolin-1-one.

The condensation reaction of 4-amino-6-methyl-2-pyrone with 1-cyclohexenecarboxaldehyde and a catalytic amount of (S)-(+)-10-camphorsulfonic acid in toluene at 358 K gave a 1:2.5 ratio of the title compound, (1) (C13H13NO2), and 7,8,9,10-tetrahydro-1H-pyrano[4,3-c]isoquinoline-1-one, (2). The formation of (2) presumably proceeds through an intermediate imine. Both (1) and (2) show inhibitory activities against acetylcholinesterase and human aldose reductase. Of the three linear-fused rings of (1), both ring A and ring B are planar and the angle between these planes is 0.46 (13) degrees. While the two C atoms of cyclohexane ring C attached to its common atoms with ring B are in the plane of the latter, as expected, the remaining two C atoms of ring C are out of this plane, by 0.342 (4) and -0.402 (3) A, respectively.

Tricyclic pyrone analogs: a new synthetic class of bifunctional anticancer drugs that inhibit nucleoside transport, microtubule assembly, the viability of leukemic cells in vitro and the growth of solid tumors in vivo.

Tricyclic pyrones (TPs) may represent a novel synthetic class of microtubule (MT) de-stabilizing anticancer drugs previously shown by us to inhibit macromolecule synthesis, tubulin polymerization, and the proliferation of leukemic and mammary tumor cells in vitro. A linear skeleton with a N-containing aromatic ring attached at C3 of the top A-ring, a central pyran B-ring and a six-membered bottom C-ring with no alkylation at C7 are required for the antitumor activities of the lead compounds, a 3-pyridyl benzopyran (code name H10) and its somewhat weaker 2-pyridyl regioisomer (code name H19). Increasing concentrations of H10 do not alter the binding of [3H]vinblastine and [3H]GTP to tubulin but mimic the ability of unlabeled colchicine (CLC) to reduce the amount of [3H]CLC bound to tubulin, suggesting that TPs may interact with the CLC binding site to inhibit tubulin polymerization. Exogenous Mg2+ cations absolutely required for the binding of GTP to tubulin and MT assembly cannot overcome the antitubulin action of H10. H10 reduces the viability of L1210 cells in vitro (IC50: 0.5 microM) but its antitumor activity may be related to its ability to inhibit tubulin polymerization and rapidly increase the mitotic index rather than to induce DNA cleavage and apoptosis. The anticancer potential of TPs in vivo is demonstrated by the fact that i.p. injections of the water-soluble H10-HCl decrease the growth of solid tumors in mice inoculated s.c. with Lewis lung carcinoma. A critical finding is that the antimitotic H10 is a bifunctional anticancer drug, which also blocks the cellular transport of nucleosides (IC50: 6 microM) to inhibit DNA synthesis. Since few CLC site-binding antimitotic agents are active in solid tumor models in vivo, the ability of these new MT destabilizing TPs to totally block nucleoside transport might be valuable in polychemotherapy to arrest tumor cells at several phases of their cycle, potentiate the action of antimetabolites and sensitize multidrug-resistant tumor cells.

Microtubule-disrupting effects of gallium chloride in vitro.

Gallium chloride (GaCl3), an antitumor agent with antagonistic action on iron, magnesium and calcium, was tested for its ability to alter the polymerization of purified tubulin (2.2 mg/ml) in a cell-free system in vitro. GaCl3 (250 microM) does not mimic the effect of 10 microM paclitaxel and, therefore, is not a microtubule (MT)-stabilizing agent that can promote tubulin polymerization in the absence of glycerol and block MT disassembly. In contrast, GaCl3 mimics the effect of 1 microM vincristine (VCR) and inhibits glycerol-induced tubulin polymerization in a concentration-dependent manner (IC50: 125 microM), indicating that GaCl3 is a MT de-stabilizing agent that prevents MT assembly. However, 150 microM GaCl3 must be used to match or surpass the inhibitions of tubulin polymerization caused by 0.25 microM of known MT de-stabilizing agents, such as colchicine (CLC), nocodazole, podophyllotoxin, tubulozole-C and VCR. The inhibitory effect of 250 microM GaCl3 persists in the presence of up to 9 mM MgCl2, suggesting that the exogenous Mg2+ cations absolutely required for the binding of GTP to tubulin and MT assembly cannot overcome the antitubulin action of Ga3+ ions of a higher valence. The binding of [3H]vinblastine (VBL) to tubulin (0.5 mg/ml) is inhibited by unlabeled VBL but enhanced by concentrations of GaCl3 > 200 microM. However, increasing concentrations of GaCl3 mimic the ability of cold CLC to reduce the amount of [3H]CLC bound to tubulin, suggesting that GaCl3 may interact with the CLC binding site to inhibit tubulin polymerization. The binding of [3H]GTP to tubulin is decreased by unlabeled GTP but markedly enhanced by GaCl3, especially when concentrations of this metal salt of 32 microM or higher are added to the reaction mixture before rather than after the radiolabeled nucleotide. These data suggest that changes in protein conformation following GaCl3 binding might increase the interactions of tubulin with nucleotides and Vinca alkaloids. After a 24 h delay, the viability of GaCl3-treated L1210 leukemic cells is reduced in a concentration-dependent manner at days 2 (IC50: 175 microM), 3 (IC50: 35 microM) and 4 (IC50: 16 microM). Since GaCl3 (100-625 microM) increases the percentage of mitotic cells at 2-4 days, it might arrest tumor cell progression in M phase, but its antimitotic activity is much weaker than that of 0.25 microM VCR. Because the concentrations of GaCl3 that inhibit tubulin polymerization also increase the mitotic index and decrease the viability of L1210 cells in vitro, the antitubulin and antimitotic effects of GaCl3 might contribute, at least in part, to its antitumor activity.

Alterations in focal adhesion kinase activity and associated proteins during malignant conversion of mouse keratinocytes.

Focal adhesion kinase (pp125FAK) has well-established functions in the attachment and growth of cells in culture and has been implicated as a marker of malignant progression in human tumors. To evaluate its role in the metastatic conversion of mouse skin tumors, pp125FAK activity and protein expression were examined in normal and transformed keratinocyte cell lines. Malignant mouse keratinocyte lines exhibited a reproducible increase in the specific activity of pp125FAK compared with that of nontransformed control cells. An increase in pp125FAK activity was not observed in papilloma-derived keratinocytes, indicating that this response correlated with malignant progression of cells and not cell transformation per se. Immune complex kinase assays and metabolic labeling with [32P]orthophosphate also revealed the specific loss of pp125FAK-associated proteins in the metastatic keratinocytes. Furthermore, immunocytochemical examination revealed an altered distribution of pp125FAK in the cells with malignant potential compared with normal and papilloma-inducing keratinocytes. The cells with malignant potential also exhibited reduced levels of paxillin and integrin beta1 as well as altered distribution of paxillin, reinforcing the notion that specific changes in the composition of focal adhesions contribute to the malignant conversion of mouse keratinocytes.

Inhibitory effects of plant tannins on ultraviolet light-induced epidermal DNA synthesis in hairless mice.

Naturally occurring hydrolyzable (HT) and condensed (CT) tannins and their monomeric units were tested for their ability to inhibit the stimulation of DNA synthesis by UVB radiation. Hairless mice were irradiated with either single (200 mJ/cm2) or multiple (150 mJ/cm2) doses of UVB applied at 24 h intervals and epidermal DNA synthesis was measured at different times after the last of these treatments. The peak of DNA synthesis that is observed 48-56 h after a single UVB irradiation shifts to an earlier time of 16-24 h after multiple UVB treatments. Interestingly, the early inhibitory period of DNA synthesis observed 8 h after a single UVB treatment is not detected following multiple UVB treatments. Rather, DNA synthesis is stimulated six-fold 24 h after multiple UVB treatment, a response that is higher than the peak occurring 48-56 h after a single UVB irradiation. The disappearance of the early period of inhibition when the peak of DNA synthesis shifts to an earlier time may be linked to reactive oxygen species brought to the epidermis by infiltrating leukocytes, which, in turn, act as second messengers to stimulate growth signals in cells. Topical applications of HT or CT remarkably inhibit the DNA responses to single and multiple UVB treatments, an effect that is dependent on the dose and time of administration. Indeed, the peak stimulation of DNA synthesis is maximally inhibited when 17 mg of Tarapod tannic acid (TA), an HT, are applied topically 20 min before a single UVB treatment. The polymeric tannins inhibited DNA synthesis to a greater degree than equal doses of their monomeric units, gallic acid and catechin. These results suggest that various oligomeric HT and CT may be useful against tumor-promoting responses associated with the exposure of skin to physical carcinogens.

Antitumor activity of novel octalactin A analogs in murine leukemia cells in vitro.

Octalactin A and B (code names K1 and K2) are eight-membered-ring lactones from a marine bacterium. K1 is reportedly cytotoxic. Since access to this natural product is severely limited, the entire synthesis of K1 has been achieved in K. Buszek's laboratory, and several of its structural and stereochemical analogs (code names K3-K9) have been tested for their ability to prevent murine L1210 leukemic cells from synthesizing macromolecules and growing in vitro. At 50 microM, K1 is inactive and the eight-membered lactone K4, an oxocene, is the only compound found to inhibit tumor cell growth by about 90% in the L1210 system. The long-term inhibition of L1210 cell growth by K4 is concentration dependent (IC50 around 10 microM) and not reversible following drug removal. The delayed and weaker cytotoxic effects of K4 suggest that the inhibition of tumor cell proliferation observed 1-4 days after K4 treatment is not solely caused by drug cytotoxicity. When compared to a spectrum of representative anticancer drugs, higher concentrations of K4 must be used to maximally inhibit tumor cell growth. In contrast to its antiproliferative activity, 50 microM K4 fails to alter the rates of DNA, RNA and protein synthesis in L1210 cells. This discrepancy between the ability of K4 to inhibit macromolecule synthesis and leukemic cell growth suggests that other molecular targets are involved in the antitumor action of this drug. At 50 microM, K4 inhibits the polymerization of purified tubulin by about 45%, and therefore may be a novel microtubule de-stabilizing drug weaker than vincristine. Even though other mechanisms may be involved in its antitumor action, the ability of K4 to partially disrupt microtubule dynamics indirectly suggests that this synthetic oxocene may be a cell cycle-specific anticancer drug that blocks mammalian cells in M-phase.

Tricyclic pyrone analogs: a new class of microtubule-disrupting anticancer drugs effective against murine leukemia cells in vitro.

Novel 1H,7H-5a,6,8,9-tetrahydro-1-oxopyrano [4,3-b][1]benzopyrans were synthesized in Hua's laboratory (code names H5, H10, H14 and H15) and tested for their ability to prevent L1210 leukemic cells from synthesizing macromolecules and growing in vitro. The aryl groups of these tricyclic pyrone (TP) analogs are either 3, 4-dimethoxyphenyl in H5 and H15 or 3-pyridyl in H10 and H14. Since 50 M H5 and H10 both inhibit DNA synthesis and tumor cell growth by 79-100%, concentrations 25 M were used in this study to assess the structure-activity relationships for this class of compounds. At 10-25 M, H5 and H14 are more potent inhibitors of DNA, RNA and protein synthesis than H10. In contrast, at 5-25 M, H10 is much more effective than H5 and H14 at inhibiting the growth of L1210 cells over a 4-day period. Interestingly, H15 inhibits DNA synthesis as much as H10 but fails to alter tumor cell growth. This discrepancy between the ability of TPs to inhibit macromolecule synthesis and leukemic cell growth suggests that other molecular targets may be involved in the antitumor action of these drugs. Their short-term inhibition of nucleic acid synthesis is reversible following drug removal but their long-term inhibition of tumor cell growth is not. Moreover, 25 M H5 and H10 are not cytotoxic at 2 days but equally decrease cell viability at 4 days, suggesting that the potent and irreversible inhibition of cell proliferation observed 1-4 days after H10 treatment is not solely caused by drug cytotoxicity. The effectiveness of H10 as inhibitor of L1210 cell growth is comparable to that of a spectrum of representative anticancer drugs. A critical finding is that 5 M H10 blocks the polymerization of purified tubulin by 90% and, therefore, may be a novel microtubule de-stabilizing drug. Indeed, H10 inhibits tubulin polymerization and L1210 cell growth as much as 5 M of vincristine (VCR). In contrast, 5 M H5 alters neither tubulin polymerization nor tumor cell growth. The ability of H10 to disrupt microtubule dynamics indirectly suggests that TPs may be novel cell cycle-specific anticancer drugs useful for arresting mammalian cells in mitosis.

Antitumor activity of tricyclic pyrone analogs, a new synthetic class of microtubule de-stabilizing agents, in the murine EMT-6 mammary tumor cell line in vitro.

Novel tricyclic pyrone (TP) analogs synthesized in Hua's laboratory (code names H10, H14 and H16) were tested against a spectrum of known antimitotic drugs for their ability to disrupt microtubule (MT) dynamics, alter the mitotic index, and prevent murine EMT-6 mammary sarcoma cells from synthesizing DNA and proliferating in vitro. At 2-10 microM, H10 inhibits DNA synthesis, tubulin polymerization and tumor cell growth to a greater degree than H14, whereas H16 has no effect. A linear skeleton with a pyridyl ring at C-3 of the A-ring, a pyran B-ring and no alkylation at C-7 of the C-ring is required for the antitumor activity of these TPs. Since H10 mimics the effect of vincristine (VCR), but not that of paclitaxel, on tubulin polymerization, TPs may represent a novel synthetic class of MT de-stabilizing anticancer drugs. H10 is less potent than VCR against tubulin polymerization (IC50: 1.5 microM versus 0.15 microM) and tumor cell proliferation (IC50: 1.5 microM versus 5 nM) but inhibits DNA synthesis (IC50: 10 microM) more effectively than all other MT-disrupting agents tested, except tubulozole-C. Although TPs disrupt DNA synthesis and might affect several phases of the cell cycle, the ability of H10 to increase the percentage of mitotic cells indicates that these novel compounds may be cell cycle-specific anticancer drugs useful for arresting mammalian cells in M-phase.

Antitumor activity of novel tricyclic pyrone analogs in murine leukemia cells in vitro.

New tricyclic pyrone derivatives were synthesized and tested for their ability to prevent L1210 leukemic cells from synthesizing DNA and growing in vitro. At 50 microM, a pyripyropene analog has no effect, whereas four pentahydro-3-aryl-1-oxopyrano[4,3-b][1]benzopyrans all inhibit DNA synthesis by 79-91% and tumor cell growth by 93-100%. These inhibitory effects are concentration dependent with IC50 around 8.5 microM for DNA synthesis at 2 hours and 1.1 microM for tumor cell growth at 4 days. The aryl groups of these antitumor agents are either 3,4-dimethoxyphenyl or 3-pyridyl. Introduction of a methyl group at C5a and a formyloxy or hydroxy group at C6 does not alter the antitumor effects of the 3,4-dimethoxyphenyl benzopyrans but reduces those of the 3-pyridyl benzopyrans, which, at 50 microM, inhibit DNA synthesis by only 32-49% and fail to alter tumor cell growth. The 4-hydroxy-6-(3-pyridyl)-2-pyrone has no effect and the tricyclic pyrones lacking aryl groups have very little inhibitory effects on DNA synthesis, suggesting that a greater conjugation is required for the antitumor activity. These molecules have never been reported and might be valuable to develop a new class of anticancer drugs.

Inhibition of ultraviolet-B radiation induced ornithine decarboxylase activity and edema formation by hydrolyzable and condensed tannins in mouse skin in vivo.

Naturally occurring hydrolyzable (HT) and condensed (CT) tannins and their monomeric units were tested for their ability to inhibit the induction of epidermal ODC activity and the formation of skin edema by UVB, two responses that are linked to the hyperplastic and inflammatory components of skin tumor promotion by this agent. Hairless mice were irradiated with either single (200 mJ/cm2/sec) or multiple (150 mJ/cm2/sec) doses of UVB and epidermal ODC activity was assayed at different times following irradiation. The peak of ODC induction which is observed 30-40 hours after a single UVB irradiation increases by 2.5 fold and shifts to a much earlier time of 5 hours after two UVB treatments repeated at 24-hour intervals. Topical applications of the various plant tannins, before or after irradiation, were found to inhibit, in a dose-dependent manner, epidermal ODC activity induced by single and multiple UVB treatments. Furthermore, the various HT and CT samples resulted in significant protection against UVB radiation-caused cutaneous edema. In general, the polymeric tannins inhibited ODC induction and edema to a greater degree than equal doses of their monomeric units, gallic acid and catechin. These results, in conjunction with our prior publications, suggest that various HTs and CTs may be useful against the hyperplastic and inflammatory responses associated with the exposure of skin to the tumor-promoting effects of both physical and chemical environmental carcinogens.

Characterization of the antitumor-promoting activity of camptothecin in SENCAR mouse skin.

(+)-Camptothecin (CPT), a topoisomerase I inhibitor specifically toxic toward S phase cells, was tested topically for its ability to inhibit skin tumor initiation by 7,12-dimethylbenz[a]anthracene (DMBA) and complete tumor promotion by 12-0-tetradecanoylphorbol-13-acetate (TPA) in SENCAR mice. Even though CPT does not prevent the covalent binding of a subcarcinogenic dose of DMBA to DNA, it enhances early inhibition of DNA synthesis caused by this initiator and may decrease the essential role of DNA replication in tumor initiation. Indeed, CPT (400 nmol) applied 4 h before or 1 h after DMBA inhibits the yield, but not the incidence, of skin tumors initiated by this compound. Moreover, because it inhibits TPA-stimulated DNA synthesis at 16 h when applied 12 h after the tumor promoter, CPT partially decreases tumor initiation when DMBA is applied 16 h after a TPA pretreatment. CPT (400 nmol) applied 1 h before or 4, 12, 24 or 48 h after each promotion treatment with TPA remarkably inhibits the incidence and yield of skin tumors promoted by this agent. CPT delays and inhibits promotion of skin tumors the most when applied 12-24 h after each TPA treatment, at times when it can block the stimulation of DNA synthesis that follows the period of early inhibition caused by TPA. The ability of post-treatments with 25, 100 and 400 nmol CPT to inhibit skin tumor promotion is dose dependent. In the TPA (stage I)-mezerein (stage 2) protocol CPT (400 nmol) post-treatment inhibits both the first and second stages of tumor promotion, related to its ability to decrease the DNA and ornithine decarboxylase responses required for stages 1 and 2 respectively. The classic model of multistage skin carcinogenesis, therefore, may be valuable to determine if novel CPT analogs are more effective than their parent compound at inhibiting tumor initiation, promotion and progression.

Camptothecin post-treatments inhibit the biochemical events linked to the tumor-promoting component of carcinogenesis in mouse epidermis in vivo.

20(S)-Camptothecin (CPT), a topoisomerase I inhibitor specifically toxic toward S-phase cells, was tested topically for its ability to inhibit the biochemical markers of skin tumor promotion. CPT has no or very little inhibitory effect on the covalent binding of an initiating dose of 7,12-dimethylbenz-[a]anthracene (DMBA) to DNA at 24 hr, but CPT post-treatments remarkably inhibit stimulations of DNA synthesis caused by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) at 16 hr and a carcinogenic dose of DMBA at 7 days. CPT is a much more potent inhibitor if it is applied 10-14 hr after TPA or 4-6 days after DMBA, when DNA synthesis starts being stimulated after the periods of early inhibition caused by TPA and DMBA. When applied 12 hr after the tumor promoter, the ability of 3-3,000 nmol of CPT to inhibit TPA-stimulated DNA synthesis at 16 hr is dose-dependent. A single dose of 500 nmol of CPT inhibits the entire time course for the stimulation of DNA synthesis observed 16-64 hr after TPA. CPT also reduces the various DNA responses to chronic TPA treatments and structurally different non-TPA-type tumor promoters. CPT may indirectly decrease the ornithine decarboxylase-inducing activity of multiple TPA treatments because it can inhibit the stimulation of RNA synthesis by this compound. However, CPT fails to alter TPA-stimulated hydroperoxide production in relation to its inability to inhibit TPA-stimulated protein synthesis. On an equal dose basis, topotecan and 10-hydroxycamptothecin are more and less effective than CPT, respectively, whereas 10,11-methylenedioxycamptothecin is much more potent than its parent compound at inhibiting the DNA response to TPA. A single dose of 400 nmol of CPT has no effect on tumor initiation when applied 4 hr before or 1 hr after a single subcarcinogenic dose of DMBA. In contrast, 400 nmol of CPT chronically applied 1 hr before or 24 hr after each treatment with TPA remarkably inhibits the complete tumor-promoting activity of this agent. CPT post-treatments also inhibit the respective activities of TPA and mezerein in the 1st and 2nd stages of skin tumor promotion.

Ability of okadaic acid and other protein phosphatase inhibitors to mimic the stimulatory effects of 12-O-tetradecanoylphorbol-13-acetate on hydroperoxide production in mouse epidermis in vivo.

The non-12-O-tetadecanoylphorbol-13-acetate (TPA)-type tumor promoters, okadaic acid (OA) and calyculin-A (CAL-A), which neither interact with the phorbol ester receptor nor directly activate protein kinase C, mimic the stimulatory effects of and thapsigargin on hydroperoxide (HPx) production in mouse epidermis in vivo. The time course and dose dependency for the stimulation of HPx production by O and TPA are similar. HPx production is maximally stimulated 16 h after two applications of 2 nmol of OA at a 48-h interval. However CAL-A is a stimulator of HPx production about 4 times more potent than OA or TPA. Combinations of TPA and OA or CAL-A have subadditive effects on HPx production. The discrepancies between the abilities of various serine/threonine protein phosphatase (PP) inhibitors to stimulate HPx production suggest that PP inhibition alone is not sufficient for this response. Cycloheximide, Ca2+ antagonists, oxypurinol, diphenyliodonium, nordihydroguaiaretic acid, bromophenacyl bromide, antiinflammatory agents, and antihistamines block or decrease OA-stimulated HPx production. Although most of these inhibitors may have more than one action, their effects suggest that protein synthesis, Ca2+, xanthine oxidase and NADPH oxidase activities, the lipoxygenase pathway of arachidonic acid metabolism, and vascular permeability may be involved in the inflammatory and HPx responses that occur after tumor promoter treatment. The increased HPx-producing activity of the epidermis, therefore, may be a common event resulting from the inflammatory and tumor-promoting actions of diverse TPA- and non-TPA-type agents.

Ability of m-chloroperoxybenzoic acid to induce the ornithine decarboxylase marker of skin tumor promotion and inhibition of this response by gallotannins, oligomeric proanthocyanidins, and their monomeric units in mouse epidermis in vivo.

m-Chloroperoxybenzoic acid (CPBA) was tested for its ability to induce the ornithine decarboxylase (ODC) marker of skin tumor promotion. In contrast to benzoyl peroxide, dicumyl peroxide, and 2-butanol peroxide, 5 mg of CPBA applied twice at a 72-h interval induce ODC activity at least as much as 3 micrograms of 12-O-tetradecanoylphorbol-13-acetate (TPA). ODC induction peaks 36 h after a single CPBA treatment but is maximal 5 h after two applications of CPBA at a 48-h interval. The ODC-inducing activity of CPBA is dose dependent and sustained after chronic treatment. In contrast to TPA, two CPBA treatments at 12-24 h intervals produce no refractory state against ODC induction. The mechanism of ODC induction by CPBA is iron dependent. Various hydrolyzable tannins, condensed tannins (CTs) and their monomeric units remarkably inhibit the ODC response to multiple CPBA treatments. At 12 mg, gallic acid, Aleppo gall tannic acid (TA), catechin, and loblolly pine bark CT inhibit the most CPBA-induced ODC activity. Aleppo gall TA is even effective when applied several hours before CPBA. The tumor-promoting activity of CPBA and its inhibition by plant tannins remain to be evaluated.