Synthesis of hydroxy- and amino-substituted benzohydroxamic acids: inhibition of ribonucleotide reductase and antitumor activity.

Benzohydroxamic acids inhibit mammalian ribonucleotide reductase and exhibit antineoplastic activity in L1210 leukemic mice. Five new hydroxy- and amino-substituted benzohydroxamic acids (3,4- and 3,5-OH, 3,4-NH2, 2,3,4- and, 3,4,5-OH) were prepared and tested along with 12 previously reported benzohydroxamic acids (BHA) for enzyme inhibition and antitumor activity. The most potent enzyme inhibitor in this series was 2,3,4-OH-BHA (ID50 = 3.5 microM), which is 140 times more potent than hydroxyurea, but its toxicity limited the antitumor activity to a 30% increase in life span of L1210 bearing mice at 125 (mg/kg)/day ip for 8 days. The most effective antitumor agent in this series was 3,4-OH-BHA which prolonged the life span of L1210 bearing mice 103% at 600 (mg/kg)/day ip for 8 days.     

Biochemical mechanism of acetaminophen (APAP) induced toxicity in melanoma cell lines

In this work, we investigated the biochemical mechanism of acetaminophen (APAP) induced toxicity in SK-MEL-28 melanoma cells using tyrosinase enzyme as a molecular cancer therapeutic target. Our results showed that APAP was metabolized 87% by tyrosinase at 2 h incubation. AA and NADH, quinone reducing agents, were significantly depleted during APAP oxidation by tyrosinase. The IC(50) (48 h) of APAP towards SK-MEL-28, MeWo, SK-MEL-5, B16-F0, and B16-F10 melanoma cells was 100 microM whereas it showed no significant toxicity towards BJ, Saos-2, SW-620, and PC-3 nonmelanoma cells, demonstrating selective toxicity towards melanoma cells. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, enhanced APAP toxicity towards SK-MEL-28 cells. AA and GSH were effective in preventing APAP induced melanoma cell toxicity. Trifluoperazine and cyclosporin A, inhibitors of permeability transition pore in mitochondria, significantly prevented APAP melanoma cell toxicity. APAP caused time and dose-dependent decline in intracellular GSH content in SK-MEL-28, which preceded cell toxicity. APAP led to ROS formation in SK-MEL-28 cells which was exacerbated by dicoumarol and 1-bromoheptane whereas cyslosporin A and trifluoperazine prevented it. Our investigation suggests that APAP is a tyrosinase substrate, and that intracellular GSH depletion, ROS formation and induced mitochondrial toxicity contributed towards APAP's selective toxicity in SK-MEL-28 cells.     

Antitumor effects of biologic reducing agents related to 3,4-dihydroxybenzylamine: dihydroxybenzaldehyde, dihydroxybenzaldoxime, and dihydroxybenzonitrile

This report describes a structure-activity analysis of isomers of three classes of dihydroxybenzene derivatives, including dihydroxybenzaldoxime, dihydroxybenzaldehyde, and dihydroxybenzonitrile. These derivatives were examined for their effect on ribonucleotide reductase activity, macromolecular synthesis, cell growth, and in vivo antitumor activity against the L1210 murine leukemia. One of the compounds studied exhibited significant antitumor activity against the growth of L1210 leukemia cells. A comparison of the various analogues revealed a possible correlation for 3,4-dihydroxybenzaldoxime between its potent inhibitory effect toward ribonucleotide reductase activity (IC50 = 38 microM) and its superior L1210 antitumor activity [percent increased life span (% ILS) = 100].     

Metabolic bioactivation and toxicity of ethyl 4-hydroxybenzoate in human SK-MEL-28 melanoma cells

The metabolism and toxicity of ethyl 4-hydroxybenzoate (4-HEB) were investigated in vitro using tyrosinase enzyme, a melanoma molecular target, and CYP2E1 induced rat liver microsomes, and in human SK-MEL-28 melanoma cells. The results were compared to 4-hydroxyanisole (4-HA). At 90 min, 4-HEB was metabolized 48% by tyrosinase and 26% by liver microsomes while the extent of 4-HA metabolism was 196% and 88%, respectively. The IC50 (day 2) of 4-HEB and 4-HA towards SK-MEL-28 cells were 75 and 50 microM, respectively. Dicoumarol, a diaphorase inhibitor, and 1-bromoheptane, a GSH depleting agent, increased 4-HEB toxicity towards SK-MEL-28 cells indicating o-quinone formation played an important role in 4-HEB induced cell toxicity. Addition of ascorbic acid and GSH to the media was effective in preventing 4-HEB cell toxicity. Cyclosporin A and trifluoperazine, inhibitors of permeability transition pore in mitochondria, were significantly potent in inhibiting 4-HEB cell toxicity. 4-HEB caused time-dependent decline in intracellular GSH concentration which preceded cell death. 4-HEB also led to reactive oxygen species (ROS) formation in melanoma cells which exacerbated by dicoumarol and 1-bromoheptane whereas cyclosporin A and trifluoperazine prevented it. Our findings suggest that the mechanisms of 4-HEB toxicity in SK-MEL-28 were o-quinone formation, intracellular GSH depletion, ROS formation and mitochondrial toxicity.     

Kinobeon A as a potent tyrosinase inhibitor from cell culture of safflower: in vitro comparisons of kinobeon A with other putative inhibitors

Kinobeon A is produced from cell cultures of the medicinal plant, safflower. Mushroom tyrosinase activity was inhibited in a concentration-dependent manner when treated with kinobeon A using L-tyrosine or L-3,4-dihydroxyphenylalannine (L-DOPA) as substrates. IC50 values were 22 microM (substrate: L-tyrosine) and 27 microM (L-DOPA). Inhibition of human tyrosinase activity also increased with increasing concentrations of kinobeon A using L-DOPA as the substrate, with an IC50 value of 2.5 microM. Kinobeon A was a more potent competitive inhibitor than kojic acid, arbutin or L-ascorbic acid for both mushroom and human tyrosinase as determined from Lineweaver-Burk plots. These results suggested that kinobeon A could be a potent natural tyrosinase inhibitor.     

Cucurbitacins from Trichosanthes kirilowii as the inhibitory components on tyrosinase activity and melanin synthesis of B16/F10 melanoma cells

Cucurbitacins 1 and 2 were isolated from the root of Trichosanthes kirilowii by tyrosinase inhibitory activity-guided fractionation. Spectroscopic analysis revealed that compounds 1 and 2 were cucurbitacin D and 23,24-dihydro-cucurbitacin D, respectively. Compounds 1 and 2 effectively inhibited the activity of tyrosinase (IC(50) = 0.18 microM and 6.7 microM, respectively), and the synthesis of melanin (IC(50) = 0.16 microM and 7.5 microM, respectively) in B16/F10 melanoma cells.     

Inhibitory effects of esculetin on melanin biosynthesis

To investigate the structure-activity relationship of coumarins for the inhibitory activity on mushroom tyrosinase, the 50% inhibitory concentration (IC50 values) of 18 coumarins and four cinnamic acid derivatives were measured. Among these compounds, esculetin had the strongest inhibitory activity (IC50=43 microM) on mushroom tyrosinase. Introduction of a hydroxy group to the C6 and C7 positions of the coumarin ring and no substitution on the lactone ring played an important role in the expression of the strong inhibitory activity of esculetin. We performed further studies to estimate the in vitro inhibitory effects of esculetin on melanogenesis. Esculetin 5 microM significantly suppressed melanin production in murine B16 melanoma cells without affecting cell growth. Furthermore, the number of 3,4-dihydroxyphenylalanine (DOPA)-positive melanocytes in the split-epidermal sheets treated with 0.05% or 0.1% esculetin was significantly lower than that in the control. From these results, it is suggested that esculetin has inhibitory effects on tyrosinase activity in vitro. However, further detailed studies are necessary to understand the inhibitory mechanism of esculetin.     

Differential effect of the calmodulin inhibitor trifluoperazine in modulating cellular accumulation, retention and cytotoxicity of doxorubicin in progressively doxorubicin-resistant L1210 mouse leukemia cells. Lack of correlation between cellular doxorubicin levels and expression of resistance

Calmodulin inhibitors are effective in enhancing cytotoxic effects of doxorubicin (DOX) in DOX-resistant cells, possibly by enhancing cellular levels of drug. In the present study, L1210 mouse leukemia cells adapted to grow in vitro, in the presence of 0.025 to 0.25 microgram/ml DOX, and identified as L1210/DOX0.025, L1210/DOX0.05, L1210/DOX0.1, and L1210/DOX0.25 were approximately 5-, 10-, 20-, and 40-fold DOX resistant, respectively, compared to parent-sensitive cells (L1210/S). Using a soft agar colony assay and 3-hr drug exposure, the IC50 concentration of DOX in the progressively DOX-resistant (5- to 40-fold) L1210 cells ranged from 0.25 to 2.0 micrograms/ml and from 0.08 to 0.25 microgram/ml in the absence and presence of a non-cytotoxic concentration of 5 microM trifluoperazine (TFP) respectively. Further, based on the observed in vitro cytotoxic response, the IC50 concentration of DOX in the presence of 5 microM TFP was 2.5-, 4-, 6.7- and 8-fold lower than DOX without 5 microM TFP in the L1210/DOX0.025, L1210/DOX0.05, L1210/DOX0.1, and L1210/DOX0.25 resistant sublines respectively. In contrast, the IC50 of DOX in L1210/S cells was approximately 0.05 microgram/ml with or without 5 microM TFP. Cellular accumulation of DOX was 15-50% lower in the progressively resistant L1210 sublines compared to similarly treated L1210/S cells. However, in the presence of 5 microM TFP, cellular accumulation of DOX in the L1210/DOX0.05 and L1210/DOX0.1 but not L1210/DOX0.25 was comparable to the L1210/S cells. Cellular retention of DOX in the absence or presence of 5 microM TFP was comparable in similarly treated L1210/S, L1210/DOX0.05 and L1210/DOX0.1 cells, and a 2-fold reduction in the retention of DOX in the absence versus the presence of 5 microM TFP was apparent only in L1210/DOX0.25 cells. At the IC50 of DOX in the presence of 5 microM TFP, although cellular accumulation of DOX was concentration dependent over the range of 1-20 microM TFP, enhancement in cytotoxicity of DOX was dose dependent at 1-5 microM TFP but not 5-20 microM TFP. In cells treated for 3 hr at the IC50 concentration of DOX alone or DOX plus 5 microM TFP, cellular accumulation of DOX was 7- to 14-fold and 2.5- to 3.5-fold higher, respectively, in resistant than in sensitive cells. Additionally, following treatment for 3 hr at the IC50 dose of DOX in the absence or presence of 5 microM TFP, drug retention at 3 hr was 4- to 6-fold and 1.5-fold higher, respectively, in the resistant versus sensitive cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Costunolide, a sesquiterpene from the stem bark of Magnolia sieboldii, inhibits the RAS-farnesyl-proteintransferase.

Costunolide, a germacrane sesquiterpene lactone isolated from the stem bark of Magnolia sieboldii demonstrated a significant inhibition upon the farnesylation process of human lamin-B by farnesyl-proteintransferase (FPTase), in a dose dependent manner in vitro (IC50 value was calculated as 20 microM). It was also found to exhibit an inhibition upon the proliferation of cultured human tumor cells, i.e., A549 (non small cell lung), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF498 (central nerve system) and HCT-15 (colon), in vitro.     

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.     

4-(6-Hydroxy-2-naphthyl)-1,3-bezendiol: a potent, new tyrosinase inhibitor

Tyrosinase is a key enzyme for melanin biosynthesis and known to be sensitive to ultraviolet light in the presence of oxygen. Therefore, finding effective tyrosinase inhibitors, either from synthetic or natural sources, can be beneficial in the treatment of melanin-related disorders. We synthesized 4-(6-hydroxy-2-naphthyl)-1,3-bezendiol (HNB), a new family of hydroxyl substituted phenyl naphthalenes, as the isosteres of oxyresveratrol. This study investigated inhibitory effects of HNB on tyrosinase activity. HNB inhibited mushroom tyrosinase with an IC(50) value of 0.07 microM, which is more potent than the anti-tyrosinase activity of kojic acid (IC(50)=38.24), a well-known tyrosinase inhibitor. The kinetic analysis of tyrosinase inhibition revealed that HNB is a competitive inhibitor (K(i) 4.78 x 10(-9) M at 0.125 microM and K(i) 6.21 x 10(-9) M at 0.25 microM). We further found that HNB also inhibited melanin production in B16F10 melanoma cells (B16 cells). In addition to tyrosinase inhibiting activity, melanin biosynthesis was inhibited by HNB in the B16F10 cells. These data strongly suggest that HNB can suppress the production of melanin via the modulation of tyrosinase activity.     

Piperlonguminine from Piper longum with inhibitory effects on alpha-melanocyte-stimulating hormone-induced melanogenesis in melanoma B16 cells

Skin hyperpigmentations such as melasma, freckles and senile lentigines can be subjectively treated by depigmenting agents. In our ongoing study to find melanogenesis inhibitors from natural sources, Piper longum L (fruits, Piperaceae) was discovered to have an inhibitory effect on alpha-melanocyte-stimulating hormone (alpha-MSH)-induced melanogenesis in melanoma B16 cells. Piperlonguminine has been identified as the melanogenesis inhibitor from P. longum by activity-guided extraction and isolation. The compound showed dose-dependent inhibitory effects with 85.1 +/- 4.9% inhibition at 25 microM, 62.1 +/- 6.1% at 12.5 microM, 36.4 +/- 4.6% at 6.3 microM and 18.4 +/- 5.1% at 3.1 microM on alpha-MSH-induced melanogenesis, showing an IC50 value of 9.6 microM. As a positive control, kojic acid exhibited an IC50 value of 44.6 microM on the melanogenesis. As to the mode of action, piperlonguminine showed an inhibitory effect on alpha-MSH-induced tyrosinase synthesis, documented by Western immunoblot analysis. However, piperlonguminine did not show an inhibitory effect on tyrosinase activity or a direct depigmenting effect of melanin.     

4,4'-Dihydroxybiphenyl as a new potent tyrosinase inhibitor

The color of mammalian skin is determined by many factors, for which visible ones are the degree and distribution of melanin pigmentation. Because tyrosinase, (polyphenol oxidase) is the key enzyme for melanin biosynthesis, the use of various tyrosinase inhibitors is a common practice for whitening purpose in cosmetics. In the present study, the inhibition of tyrosinase by 4,4'-dihydroxybiphenyl (44'-BP) was investigated. In addition to tyrosinase inhibiting activity, melanin biosynthesis was assessed in B16F10 melanoma cells (B16 cells). The results showed that 44'-BP exhibits a strong anti-tyrosinase activity with IC50=1.91 microM. The kinetic analysis of tyrosinase inhibition revealed that 44'-BP acts a competitive inhibitor (Ki=4.0 x 10(-4) M at 2.5 microM and Ki =21 x 10(-5) M at 5 microM). Furthermore, data on melanin biosynthesis indicated that the amount of melanin was clearly suppressed by 44'-BP.     

Induction of growth inhibition and G1 arrest in human cancer cell lines by relatively low-toxic diamantane derivatives

We describe the discovery of a novel series of antitumor diamantane derivatives which induces G1 arrest in Colo 205 cells. Eight diamantane derivatives were screened for their activity in vitro against 60 human cancer cell lines in the National Cancer Institute (NCI)'s anticancer drug screen. The relationships between structure and in vitro antitumor activity are discussed. The structure-activity relationship (SAR) study of diamantane derivatives clarified that the conformation of 1,6-bis(4-(4-aminophenoxy)-phenyl)diamantane (1,6-DPDONH2) was essential for significant antitumor activity. Very strong growth inhibition of 1,6-DPDONH2 (NSC-706829) was observed against one colon cancer line (Colo 205), four melanoma lines (MALME-3M, M14, SK-MEL-5 and UACC-257) and two breast cancer lines (MDA-MB-435 and MDA-N) with GI50 <1.0 microM, i.e. below 0.01, 0.23, 0.48, 0.5, 0.32, 0.26 and 0.28 microM, respectively. 1,6-DPDONH2 also exhibited particular selectivity against one colon cancer line (Colo 205), four melanoma lines (MALME-3M, M14, SK-MEL-5 and UACC-257) and two breast cancer lines (MAD-MB-435 and MDA-N) with GI50 < or=0.5 microM. In the same cancer subpanel, the selectivity of 1,6-DPDONH2 between these seven most sensitive lines and the least sensitive line ranged from 40- to 100-fold. With the exception of melanoma lines, 1,6-bis(4-(4-amino-3-hydroxyphenoxy)-phenyl)diamantane (1,6-DPD/OH/NH2) (NSC-706831) possessed stronger activity than 1,6-DPDONH2 against almost all tested cancer lines. Very strong growth inhibition of 1,6-DPD/OH/NH2 was observed against one leukemia line (HL-60(TB)), one NSCLC line (HOP-92), one ovarian cancer line (OVCAR-8) and one breast cancer line (T-47D) with GI50 <1.0 microM, i.e. 0.50, 0.85, 0.62 and 0.75 microM, respectively.     

Depigmentation of melanocytes by isopanduratin A and 4-hydroxypanduratin A isolated from Kaempferia pandurata ROXB

This study was carried out to investigate the in vitro effects of isopanduratin A and 4-hydroxypanduratin A isolated from Kaempferia pandurata ROXB. on melanin biosynthesis and tyrosinase activity. Two chalcone compounds, isopanduratin A and 4-hydroxypanduratin A, were isolated from the ethyl acetate fraction of ethanol extract as the active principles. Compared with phenylthiourea (IC(50)=34.3 microM) as a positive control, the depigmentation IC(50) values for isopanduratin A and 4-hydroxypanduratin A were 10.6 microM and 23.2 microM, respectively. The compounds also significantly inhibited the activity of tyrosinase, the enzyme that converts DOPA (3,4-dihydroxyphenylalanine) to dopachrome in the biosynthetic process of melanin. The IC(50) values of isopanduratin A and 4-hydroxypanduratin A for tyrosinase were 10.5 microM and >30 microM, respectively, while that of phenylthiourea was 47.6 microM. The tyrosinase protein level was also significantly decreased by isopanduratin A and 4-hydroxypanduratin A. The results indicate that isopanduratin A and 4-hydroxypanduratin A isolated from K. pandurata ROXB. are promising compounds that could be useful for treating hyperpigmentation as skin-whitening agents.     

Synthesis, biological evaluation, and quantitative structure-activity relationship analysis of new Schiff bases of hydroxysemicarbazide as potential antitumor agents.

Thirty Schiff bases of hydroxysemicarbazide (Ar-CH=NNHCONHOH) have been synthesized and tested against L1210 murine leukemia cells. The IC(50) values were found to be in a range from 2.7 x 10(-6) to 9.4 x 10(-4) M. A total of 17 out of the 30 compounds had higher inhibitory activities than hydroxyurea (an anticancer drug currently used for the treatment of melanoma, leukemia, and ovarian cancer) against L1210 cells. Six compounds with IC(50) values in micromolar range were 11- to 30-fold more potent than hydroxyurea (IC(50) = 8.2 x 10(-5) M). The partition coefficient (log P) and ionization constants (pK(a)) of a model compound [1-(3-trifluoromethylbenzylidene)-4-hydroxysemicarbazide, 1] were measured by the shake-flask method, and the measured log P was used to derive Hansch-Fujita pi constant of -CH=NNHCONHOH. On the basis of the newly derived pi and those of other moieties, the partition coefficients (SlogP) of the other 29 compounds were calculated by the summation of pi values. Quantitative structure-activity relationship (QSAR) analysis showed that, besides the essential pharmacophore (-NHCONHOH), hydrophobicity (SlogP), molecular size/polarizability (calculated molar refractivity), and the presence of an oxygen-containing group at the ortho position (I) were important determinants for the antitumor activities. In conclusion, the results obtained in this study show that several Schiff bases of hydroxysemicarbazide are potent inhibitors of tumor cells and warrant further investigation as cancer chemotherapeutic agents.     

Inhibitory effects of kurarinol, kuraridinol, and trifolirhizin from Sophora flavescens on tyrosinase and melanin synthesis

Previously, it was reported that some prenylated flavonoids contained in the dichloromethane fraction of the ethanolic extract of Sophora flavescens, such as kuraridin, sophoraflavanone G, kurarinone, and kushenol F, are tyrosinase inhibitors; however, based on the level of these inhibitors in the extract, its inhibitory effect on tyrosinase activity was higher than expected. This has led us to further investigate other possible constituents that may contribute to the extract's strong inhibitory activity. The results of this study indicate that kurarinol (1), kuraridinol (2), and trifolirhizin (3), from the ethyl acetate fraction of Sophora extract, can inhibit tyrosinase activity. Compared with kojic acid (16.22+/-1.71 microM), compounds 1-3 possessed potent tyrosinase inhibitory activity with IC(50) values of 8.60+/-0.51, 0.88+/-0.06, and 506.77+/-4.94 microM, respectively. These three compounds were further tested for their inhibitory effects on melanogenesis. In cultured B16 melanoma cells, 1-3 markedly inhibited (>50%) melanin synthesis at 50 microM. This is the first study indicating that 1-3 exert varying degrees of inhibition on tyrosinase-dependent melanin biosynthesis, and therefore, are candidates as skin-whitening agents.     

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.     

A new macromolecular antitumor antibiotic, C-1027. III. Antitumor activity

C-1027, a new macromolecular antitumor antibiotic produced by Streptomyces globisporus C-1027, showed extremely potent cytotoxicity toward cultured cancer cells. Compared in terms of IC50 values, antibiotic C-1027 showed much more potent cytotoxicity than doxorubicin, mitomycin C and neocarzinostatin. Spermatogonial assay, a prescreen for anticancer drugs, was highly sensitive for detection of C-1027. At tolerable doses, C-1027 exhibited marked inhibition on a panel of transplantable tumors in mice, which included leukemia L1210, P388, ascites hepatoma H22, sarcoma 180 and melanoma Harding-Passey.     

Molecular basis of the antitumor activities of 2-crotonyloxymethyl-2-cycloalkenones

The antitumor activity of 2-crotonyloxymethyl-2-cyclohexenone (COMC-6) is not the result of the GSH conjugate (GSMC-6) formed inside tumor cells, as the diethyl ester prodrug form of GSMC-6 displays little antitumor activity with B16 melanotic melanoma in vitro (IC(50) > 460 microM) versus COMC-6 (IC(50) 0.041 microM) and its five- and seven-membered ring homologues. Antitumor activity probably results from a reactive intermediate that forms during conjugation of the COMCs with intracellular GSH.