Proline prodrug of melphalan,prophalan-l,demonstrates high therapeutic index in a murine melanoma model

The therapeutic efficacy of prophalan-l, the l-proline prodrug of melphalan that demonstrated prolidase-dependent bioactivation to melphalan, was examined in vivo in a mouse melanoma model. Prophalan-l exhibited 2- to 2.5-fold higher hydrolytic and cytotoxic activity than prophalan-d, the d-analog, in B16-F10 murine melanoma cells in vitro. Prophalan-l cytotoxicity in B16-F10 cells was lower (GI₅₀ = 221 μM) than that of melphalan (GI₅₀ = 173 μM). The tumor growth profiles in C57BL/6J mice injected with B16-F10 cells and treated with melphalan (5.5 μg/g i.p.) and equimolar concentrations of the prodrugs demonstrated significant difference between the control (buffered saline) and melphalan or prophalan-l but no significant difference between control and prophalan-d or between melphalan and prophalan-l. Prophalan-l was significantly less toxic than melphalan, while no significant difference was observed in toxicity, measured as percent weight loss, between the prodrugs and saline control. Tumor reduction efficacy at high doses (12 μg/g i.p.) was similar for melphalan and prophalan-l; however, fatal toxicity was associated with melphalan while prophalan-l exhibited significantly lower systemic toxicity. An excellent correlation between GI₅₀ and tumor reduction efficacy was observed for the tested drugs (r² = 0.95). Prophalan-l thus demonstrates higher therapeutic index than melphalan in the murine melanoma model.     

Proline Prodrug of Melphalan Targeted to Prolidase,a Prodrug Activating Enzyme Overexpressed in Melanoma

Purpose To determine the bioactivation and uptake of prolidase-targeted proline prodrugs of melphalan in six cancer cell lines with variable prolidase expression and to evaluate prolidase-dependence of prodrug cytotoxicity in the cell lines compared to that of the parent drug, melphalan. Materials and Methods Hydrolysis, cell uptake, and cell proliferation studies of melphalan and the L- and D-proline prodrugs of melphalan, prophalan-L and prophalan-D, respectively, were conducted in the cancer cell lines using established procedures. Results The bioactivation of prophalan-L in the cancer cell lines exhibited high correlation with their prolidase expression levels (r ² = 0.86). There were no significant differences in uptake of melphalan and its prodrugs. The cytotoxicity of prophalan-L (GI₅₀) in cancer cells also showed high correlation with prolidase expression (r ² = 0.88), while prophalan-D was ineffective at comparable concentrations. A prolidase targeting index (ratio of melphalan to prophalan-L cytotoxicity normalized to their uptake) was computed and showed high correlation with prolidase expression (r ² = 0.82). Conclusions The data corroborates the specificity of prophalan-L activation by prolidase as well as prolidase-targeted cytotoxicity of prophalan-L in cancer cell lines. Hence, prophalan-L, a stable prodrug of melphalan, exhibits potential for efficiently targeting melanoma with reduced systemic toxicity.     

Proline prodrug of melphalan, prophalan-L, demonstrates high therapeutic index in a murine melanoma model.

The therapeutic efficacy of prophalan-L, the L-proline prodrug of melphalan that demonstrated prolidase-dependent bioactivation to melphalan, was examined in vivo in a mouse melanoma model. Prophalan-L exhibited 2- to 2.5-fold higher hydrolytic and cytotoxic activity than prophalan-D, the D-analog, in B16-F10 murine melanoma cells in vitro. Prophalan-L cytotoxicity in B16-F10 cells was lower (GI50=221 microM) than that of melphalan (GI50=173 microM). The tumor growth profiles in C57BL/6J mice injected with B16-F10 cells and treated with melphalan (5.5 microg/g i.p.) and equimolar concentrations of the prodrugs demonstrated significant difference between the control (buffered saline) and melphalan or prophalan-L but no significant difference between control and prophalan-D or between melphalan and prophalan-L. Prophalan-L was significantly less toxic than melphalan, while no significant difference was observed in toxicity, measured as percent weight loss, between the prodrugs and saline control. Tumor reduction efficacy at high doses (12 microg/g i.p.) was similar for melphalan and prophalan-L; however, fatal toxicity was associated with melphalan while prophalan-L exhibited significantly lower systemic toxicity. An excellent correlation between GI50 and tumor reduction efficacy was observed for the tested drugs (r2=0.95). Prophalan-L thus demonstrates higher therapeutic index than melphalan in the murine melanoma model.     

Proline analogue of melphalan as a prodrug susceptible to the action of prolidase in breast cancer MDA-MB 231 cells

Proline analogue of melphalan (Mel-pro) was synthesized as a prodrug susceptible to the action of ubiquitously distributed, cytosolic imidodipeptidase-prolidase [E.C.3.4.13.9]. Conjugation of melphalan (Mel) with proline (Pro) through imido-bond resulted in formation of a good substrate for prolidase. Cytosolic location of prolidase in neoplastic cell suggests that proline analogue of melphalan (Mel-pro) may serve as a prolidase convertible prodrug. We have compared several aspects of pharmacologic actions of Mel and Mel-pro in estrogen-independent breast cancer MDA-MB 231 cells. It has been found that Mel-pro is more effectively transported into the MDA-MB 231 cells, evokes higher cytotoxicity, similar inhibitory effect on DNA synthesis, lower inhibitory effect on collagen biosynthesis and reduces IGF-I receptor and MAPkinase expression in MDA-MB 231 cells, compared to Mel. The results suggest that targeting of prolidase as a Mel-pro-converting enzyme may serve as a potential strategy in pharmacotherapy of breast cancer.     

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.     

Cytotoxicity and effect on collagen biosynthesis of proline analogue of melphalan as a prolidase-convertible prodrug in cultured human skin fibroblasts.

Proline analogue of melphalan (MEL-PRO) was synthesised as a prodrug susceptible to the action of ubiquitously distributed, cytosolic imidodipeptidase--prolidase [E.C.3.4.13.9]. Conjugation of melphalan (MEL) with proline (PRO) through an imido-bond resulted in formation of a good substrate for prolidase. The susceptibility of MEL-PRO to the action of prolidase was found to be similar, compared to glycyl-proline--the most abundant, endogenous substrate for prolidase and about 6-fold higher compared to its substrate--glycyl-hydroxyproline. We have compared the transport of MEL and its prodrug through cell membrane, their antimitotic activity, cytotoxicity and effect on collagen biosynthesis in cultured, normal human skin fibroblasts. The prodrug was found to be more effectively transported into the cells than the free drug. Moreover, a lower cytotoxicity, antimitotic activity and inhibitory effect on collagen biosynthesis of the prodrug, compared to the free drug were observed after 24 h of incubation. MEL and MEL-PRO at concentrations of 12 microM led to the decrease in cell viability in confluent human skin fibroblasts by about 40 and 20%, respectively, during 24 h of incubation. IC50 of MEL for DNA synthesis (measured by thymidine incorporation assay) was found at about 7 microM, while MEL-PRO used at this concentration produced about 35% reduction in thymidine incorporation. Similarly, MEL and MEL-PRO used at 7 microM concentrations inhibited collagen biosynthesis in fibroblasts cultured for 24 h to about 30 and 80% of control values, respectively. However, when the cells were cultured with the drugs for 72 h, similar effects of both drugs on DNA and collagen biosynthesis were observed. The data suggest that MEL-PRO may serve as a prolidase-convertible prodrug that evokes lower cytotoxicity, antimitotic activity, and lower inhibitory effect on collagen biosynthesis in fibroblast cultures, compared to the free drug.     

Novel amidine analogue of melphalan as a specific multifunctional inhibitor of growth and metabolism of human breast cancer cells

A novel amidine analogue of melphalan (AB4) was compared to its parent drug, melphalan in respect to cytotoxicity, DNA and collagen biosynthesis in MDA-MB-231 and MCF-7 human breast cancer cells. It was found that AB4 was more active inhibitor of DNA and collagen synthesis as well more cytotoxic agent than melphalan. The topoisomerase I/II inhibition assay indicated that AB4 is a potent catalytic inhibitor of topoisomerase II. Data from the ethidium displacement assay showed that AB4 intercalated into the minor-groove at AT sequences of DNA. The greater potency of AB4 to suppress collagen synthesis was found to be accompanied by a stronger inhibition of prolidase activity and expression compared to melphalan. The phenomenon was related to the inhibition of beta(1)-integrin and IGF-I receptor mediated signaling caused by AB4. The expression of beta(1)-integrin receptor, as well as Sos-1 and phosphorylated MAPK, ERK(1) and ERK(2) but not FAK, Shc, and Grb-2 was significantly decreased in cells incubated for 24h with 20 microM AB4 compared to the control, not treated cells, whereas in the same conditions melphalan did not evoke any changes in expression of all these signaling proteins, as shown by Western immunoblot analysis. These results indicate the amidine analogue of melphalan, AB4 represent multifunctional inhibitor of breast cancer cells growth and metabolism.     

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.     

Prolidase-activated prodrug for cancer chemotherapy cytotoxic activity of proline analogue of chlorambucil in breast cancer MCF-7 cells

Although prolidase [EC 3.4.13.9] is found in normal cells, substantially increased levels are found in some neoplastic tissues. Because prolidase possesses the ability to hydrolyse imido bonds of various low molecular weight compounds coupled to L-proline, we hypothesized that coupling of L-proline through an imido bond to anticancer drugs might create prodrugs which would be locally activated by tumour-associated prolidase and consequently would be less toxic to normal cells that evoke lower prolidase activity. To test this concept we have synthesized a conjugate of chlorambucil-proline (CH-pro) as a possible prodrug. Treatment of this prodrug with prolidase generated the L-proline and the free drug, demonstrating its substrate susceptibility to prolidase. We have compared several aspects of biological actions of chlorambucil (CH) and its prodrug in breast cancer MCF-7 cells. IC50 values for chlorambucil and for CH-pro in DNA synthesis were found to be 54 and 16 microM, respectively. CH-pro also exhibited a lesser ability to inhibit collagen biosynthesis in breast cancer MCF-7 cells compared to the free drug. The IC50 values for chlorambucil and for CH-pro in collagen biosynthesis were found to be about 32 and 80 microM, respectively. This suggests that the targeting of prolidase may serve as a potential strategy for converting antineoplastic prodrugs.     

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.     

Synthesis of some new quinoxalines and 1,2,4-triazolo[4,3-a]-quinoxalines for evaluation of in vitro antitumor and antimicrobial activities

Two novel series of quinoxalines derived from 3-phenylquinoxalin-2(1H)-one and 2-hydrazino-3-phenylquinoxaline, namely 1-substituted-3-phenylquinoxaline-2(1H)-ones, 2a-c, 3a-d, and 4; 2-(3-oxo-3,3a,4,5,6,7-hexahydroindazol-2-yl)-3-phenylquinoxaline 6; N- cyclopentylidene or benzylidene-N'-(3-phenylquinoxaline-2-yl)hydrazines, 7 and 18; 1-substituted-4-phenyl-1,2,4-triazolo[4,3-a]quinoxalines, 9, 10, 12, 13, 14, and 16 have been synthesized in order to evaluate their antitumor and antimicrobial activities. Preliminary screening at NCI showed that compounds 2b, 2c, 3b, 3c, and 9 exhibited a moderate to strong growth inhibition activity on various tumor panel cell lines between 10(-6) to 10(-5) molar concentrations. Compound 3b was the most active with a broad spectrum of activity. Compound 3c showed selectivity towards CNS-cancer SF-639, leukemia CCRF-CEM, and melanoma SK-MEL-5 (GI(50) = 4.03, 6.46, and 4.17 microM, respectively). On the other hand, the in vitro microbiological data revealed that the prepared compounds showed mild antimicrobial activity.     

Decreased cytotoxicity and increased antimitotic activity of a proline analogue of chlorambucil as a prodrug susceptible to the action of fibroblast's prolidase

We synthesized an proline analogue of chlorambucil (CH-pro) as a prodrug susceptible to the action of ubiquitously distributed, cytosolic imidopeptidase--prolidase [E.C.3.4.13.9]. A conjugation of chlorambucil (CH) with proline through an imido-bond resulted in the formation of a good substrate for prolidase. We have compared several aspects of biological actions of CH and its prodrug in cultured normal human skin fibroblasts. The prodrug was found to be more effectively transported into the cells than the free drug. Moreover, in opposition to CH, CH-pro had no inhibitory effect on fibroblast's prolidase activity against the endogenous substrate, glycyl-L-proline. Lower cytotoxicity and a higher antimitotic activity of the prodrug, compared to the free drug, was observed. CH and CH-pro at concentrations of 25 microM led to a 30% and 10%, decrease in cell viability in confluent human skin fibroblasts. IC50 values of CH and CH-pro for DNA synthesis was found to be 30 microM and 7 microM, suggesting higher antimitotic potency of the pro-drug compared to the free drug. CH-pro also evoked lower ability to inhibit collagen biosynthesis in cultured fibroblasts than the free drug. IC50 values of CH and CH-pro for collagen biosynthesis were found at about 15 microM and 30 microM, respectively. Targeting of prolidase as a prodrug-converting enzyme may serve as a novel strategy in pharmacotherapy of various diseases, leading to the increase in therapeutic efficacy and reduction in untoward side effects of antineoplastic agents.     

PTD4-apoptin protein and dacarbazine show a synergistic antitumor effect on B16-F1 melanoma in vitro and in vivo

PTD4-apoptin protein enters cells and harbors tumor-selective cell death activity. Dacarbazine is the mainstay of treatment for malignant melanoma. In this study, we investigated the cytotoxic effect of PTD4-apoptin protein and/or dacarbazine in mouse B16-F1 and human A875 and SK-MEL-5 melanoma cells in vitro and by means of a mouse B16-F1 melanoma model in vivo. PTD4-apoptin protein inhibits the growth of B16-F1, A875 and SK-MEL-5 melanoma cells in a dose-dependent manner, but not in normal human cell lines WI-38 and L-02. PTD4-apoptin combined with dacarbazine revealed a synergistic cytotoxic effect (coefficient of drug interaction < 1) in all three different tumor cell lines. In vivo, PTD4-apoptin protein and dacarbazine alone effectively inhibited the growth of B16-F1 melanoma in C57BL/6 mice. Strikingly, combined PTD4-apoptin/dacarbazine treatment significantly increased the antitumor effect in comparison to the single treatments. As important, a combined PTD4-apoptin/dacarbazine treatment with a 50% reduction of dacarbazine revealed similar antitumor activities, without detectable hematologic side effects. A combined PTD4-apoptin/dacarbazine treatment represents a promising novel efficient and safe anticancer strategy.     

Induction of G(1) cell cycle arrest and p27(KIP1) increase by panaxydol isolated from Panax ginseng

Polyacetylenic compounds of Panax ginseng roots have been shown to inhibit growth of several human malignant tumor cell lines. Panaxydol is known to be one of the cytotoxic polyacetylenic compounds of P. ginseng. In this study, we first showed that panaxydol decreased markedly the proliferation, and to a lesser extent, the number of cells in a human melanoma cell line, SK-MEL-1. Next, the effect of panaxydol on cell cycle progression and its mechanism of action were investigated. Cell cycle analysis revealed that panaxydol inhibited cell cycle progression of a human malignant melanoma cell line, SK-MEL-1, at G(1)-S transition. At the same time, panaxydol increased the protein expression of p27(KIP1) as early as 1 hr after treatment. Cyclin-dependent kinase 2 (Cdk2) activity was decreased in a dose-dependent manner after 24 hr of panaxydol treatment. Protein levels of p21(WAF1), p16(INK4a), p53, pRb (retinoblastoma protein), and E2F-1 were not changed. It was also found that cycloheximide reversed the growth inhibition induced by panaxydol and partially abrogated the increase in p27(KIP1) expression. These results indicate that panaxydol induces G(1) cell cycle arrest by decreasing Cdk2 activity and up-regulating p27(KIP1) protein expression.     

Two cytotoxic sesquiterpene lactones from the leaves of Xanthium strumarium and their in vitro inhibitory activity on farnesyltransferase

Two xanthanolide sesquiterpene lactones, 8- epi-xanthatin (1) and 8- epi-xanthatin epoxide (2), isolated from the leaves of Xanthium strumarium (Compositae), demonstrated a significant inhibition on the proliferation of cultured human tumor cells, i. e., A549 (non-small cell lung), SK-OV-3 (ovary), SK-MEL-2 (melanoma), XF498 (central nervous system) and HCT-15 (colon) in vitro. They were also found to inhibit the farnesylation process of human lamin-B by farnesyltransferase (FTase), in a dose-dependent manner in vitro (IC 50 value was calculated as 64 and 58 microM, respectively). Due to the relatively high concentrations of 1 and 2 required to obtain an FTase inhibition as compared with those necessary for a cytotoxic effect on tumor cells, it remains unclear whether a relationship between these two activities exists.     

The mechanism for anthracycline-induced inhibition of collagen biosynthesis

One of the recognized side effects accompanying anti-neoplastic anthracyclines administration is poor wound healing resulting from impairment of collagen biosynthesis. However, the precise mechanism of anthracyclines-induced inhibition of collagen synthesis has not been established. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for anthracyclines-induced inhibition of synthesis of this protein. Prolidase [EC 3.4.13.9] cleaves imidodipeptides containing C-terminal proline, providing large amount of proline for collagen synthesis. Therefore, we compared the effect of daunorubicin and doxorubicin on prolidase activity and collagen biosynthesis in confluent cultured human skin fibroblasts. We have found that daunorubicin and doxorubicin coordinately induced the inhibition of prolidase activity (IC(50)=0.3 and 10 microM, respectively) and collagen biosynthesis (IC(50)=1 and 15 microM, respectively) in cultured human skin fibroblasts. The inhibitory effect of daunorubicin or doxorubicin on prolidase activity and collagen biosynthesis was not due to anti-proliferative activity of these drugs as shown by cell viability tetrazoline test. The decrease in prolidase activity due to the treatment of confluent cells with the anthracyclines was not accompanied by any difference in the amount of enzyme protein recovered from these cells as shown by Western immunoblot analysis. It may be suggested that the inhibition is a post-translational event. Since prolidase is metalloprotease, requiring manganese for catalytic activity, and anthracyclines are known as chelators of divalent cations, we considered that the chelating ability of anthracyclines might be an underlying mechanism for the anthracyclines-induced inhibition of prolidase activity. In order to determine the ability of daunorubicin or doxorubicin to form complexes with manganese (II), potentiometric method was employed based on the measurement of protonation constant by pH-metric titrated assay. We have found that both anthracyclines form stable complexes with manganese (II). The composition of the daunorubicin-Mn(II) complex was calculated as 3:1 while that of doxorubicin-Mn(II) complex was 2:1. The constant stability value for the investigated complexes were calculated as beta(av)=(1.74+/-0.01)x10(23) for daunorubicin, and beta(av)=(1.99+/-0.025)x10(11) for doxorubicin. The higher ability of daunorubicin vs. doxorubicin to chelate manganese and inhibit prolidase activity may explain the potential mechanism for its greater potency to inhibit collagen biosynthesis.     

Fatty acid conjugates of 2'-deoxy-5-fluorouridine as prodrugs for the selective delivery of 5-fluorouracil to tumor cells.

We have prepared a novel class of prodrugs by coupling 2'-deoxy-5-fluorouridine (5dFU) to oleic (18:1) and docosahexaenoic (22:6) acids, respectively. The cytotoxic activity of the drug and its conjugates (5dFU-18:1 and 5dFU-22:6) has been assayed in vitro upon HT-29, a colon carcinoma cell line of human origin. After short term (2-hr) treatments with the drugs, both fatty acid conjugates of 5dFU showed cytotoxic activity in a dose-dependent way, while 5dFU alone was devoid of toxic effects within the whole range of concentrations (10-200 microM) tested. Following long term (24- or 48-hr) incubations only a fraction of the HT-29 cell population was sensitive to 5dFU, the rest of the population being resistant even at the highest concentration tested (200 microM). In contrast, 5dFU-oleic acid and, particularly, 5dFU-docosahexaenoic acids appeared toxic for the whole population of HT-29 cells under the same experimental conditions. The considerable gain in cell toxicity and, to a lesser extent, in selectivity resulted from the conjugation since the toxic effect of the drug alone was not modified when equimolar mixtures of 5dFU and fatty acids were assayed. These results confirm a previous study on the cytotoxicity of fatty acid derivatives of chlorambucil toward malignant lymphoblastoid cells and reinforce the potential use of fatty acid conjugates as efficient anti-tumor prodrugs.     

Induction of alkylator (melphalan) resistance in HL60 cells is accompanied by increased levels of topoisomerase II expression and function.

Human leukemic HL60 cells were selected for resistance to alkylating agents by stepwise exposure to increasing concentrations of L-phenylalanine mustard (melphalan). The resulting resistant cell line (R-HL60) was 4-fold resistant (melphalan IC50 value, 27.84 +/- 4.2 microM) to melphalan compared with parental HL60 cells (melphalan IC50 value, 6.9 +/- 1.78 microM). Nuclear extracts from R-HL60 cells possess a approximately 4-fold increase in DNA topoisomerase II activity compared with parental HL60 cells. As determined using Western blot analysis, the level of topoisomerase IIalpha protein expressed in R-HL60 cells was approximately 3-fold that of parental HL60 cells. However, there were no differences observed in the level of topoisomerase IIbeta protein, in the topoisomerase I activity, or in the level of topoisomerase I protein expression comparing the two cell lines. R-HL60 cells were 5-fold more sensitive than parental HL60 cells to the cytotoxic effect of the topoisomerase II inhibitor doxorubicin. The sensitivity to the cytotoxic effects of the topoisomerase I inhibitor camptothecin did not differ in R-HL60 and parental HL60 cell lines. Preincubation with doxorubicin significantly increased melphalan-induced interstrand DNA cross-link formation and cytotoxicity in R-HL60 cells compared with the parental HL60 cells. The affinity of topoisomerase II for UV-irradiated cross-linked HL60 DNA was increased by approximately 2.5-fold compared with that of HL60 native DNA. The affinity of topoisomerase II for both UV-irradiated (cross-linked) and native DNA was significantly decreased after doxorubicin pretreatment. Elevated topoisomerase II activity and the increased affinity of topoisomerase II for cross-linked DNA in melphalan-resistant cells seems to contribute to alkylator resistance by changing DNA topology, thereby facilitating DNA repair.