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Cellular movement is mainly orchestrated by the actin and microtubule cytoskeleton in which Rho GTPases closely collaborate. We studied whether cytoskeleton-interfering agents at subtoxic and 50% growth-inhibiting concentrations affect motility of five unselected human ovarian cancer cell lines. Cisplatin and doxorubicin as control cytotoxic agents were not effective, the microtubule-targeting agents docetaxel, epothilone B and vinblastine only marginally inhibited cell motility, while the actin-targeting agent cytochalasin D was most potent in hampering both cell migration and invasion. Disturbance of microtubule dynamics by docetaxel did not importantly affect the cellular structures of beta-tubulin and F-actin. In contrast, hindrance of actin dynamics by cytochalasin D resulted in loss of lamellipodial extensions, induced thick layers of F-actin and disorder in cellular organization. In OVCAR-3 cells the activity of Rac1 was only slightly diminished by docetaxel, but clearly reduced by cytochalasin D. In conclusion, targeting the actin cytoskeleton might provide a means to prevent metastasis formation.

Celecoxib, an inhibitor of cyclooxygenase-2 (COX-2), is being investigated for enhancement of chemotherapy efficacy in cancer clinical trials. We determined whether continuous exposure to celecoxib would increase the antiproliferative effects of a 1-h treatment with docetaxel or cisplatin in four human ovarian cancer cell lines. COX-2 protein could not be detected in these cell lines, because of which three COX-2 positive human colon cancer cell lines were included. Multiple drug effect analysis demonstrated additive to borderline antagonistic effects of celecoxib combined with docetaxel. Combination indices with values of 1.4-2.5 in all cancer cell lines indicated antagonism between celecoxib and cisplatin regardless whether celecoxib preceded cisplatin for 3h, was added simultaneously or immediately after cisplatin. Apoptotic features measured in COX-2-negative H134 ovarian cancer cells and COX-2-positive WiDr colon cancer cells, such as the activation of caspase-3 and the number of cells in sub-G0 of the cell cycle, induced by docetaxel were increased in the presence of celecoxib, but were abrogated upon addition of celecoxib to cisplatin. Moreover, the G2/M accumulation in cisplatin-treated cells was less pronounced when celecoxib was present. Drugs did not affect p-Akt. Celecoxib upregulated p-ERK1/2 in H134 cells, but not in WiDr cells. Platinum-DNA adduct formation measured in WiDr cells, however, was reduced when celecoxib was combined with cisplatin. Taken together, our data demonstrate clear antagonistic effects when celecoxib is given concurrently with cisplatin, which is independent of COX-2 expression levels.

Vascular Endothelial Growth Factor (VEGF) and its transcriptional regulator Hypoxia-inducible Factor 1 (HIF-1) play an important role in the process of angiogenesis in many types of cancer, including ovarian cancer. We have examined whether the DNA-damaging drugs cisplatin and doxorubicin and the microtubule inhibitors docetaxel and paclitaxel can affect VEGF expression and HIF-1 activity in three human ovarian cancer cell lines. We demonstrate that cisplatin and doxorubicin abolish hypoxia-induced VEGF mRNA expression in all cell lines, while basal VEGF mRNA expression was also downregulated. Transient transfection with a HIF-1-responsive luciferase construct indicated that cisplatin and doxorubicin inhibited hypoxic activation of HIF-1. Cisplatin repressed HIF-1alpha protein expression in all cell lines. Stimulation of HIF-1alpha protein degradation by cisplatin was observed in the only cell line expressing wild-type p53. Cisplatin also inhibited the synthesis of HIF-1alpha protein for which p53 was dispensable. Interestingly, cisplatin strongly reduced the protein levels of the HIF-1 coactivators p300 and CREB-binding protein (CBP) under hypoxia in all cell lines. Although doxorubicin inhibited hypoxic activation of HIF-1, this drug had no significant effect on the expression levels of HIF-1alpha and hypoxic expression of p300 and CBP was only weakly reduced. Docetaxel and paclitaxel did neither influence VEGF expression nor hypoxia-induced HIF-1 activity. In total, our findings indicate that cisplatin and doxorubicin can repress hypoxic induction of VEGF expression by inhibiting HIF-1 through different mechanisms. This knowledge may be useful for future treatment schedules including agents that target the HIF-1 signalling pathway.

The glucuronide prodrug of doxorubicin, DOX-GA3, can be selectively activated in tumors by extracellular human beta-glucuronidase, resulting in a better therapeutic index than doxorubicin. DOX-GA3, however, is rapidly excreted by the kidney. We hypothesized that slow release of DOX-GA3 from its methylester, DOX-mGA3, by esterase activity in blood would result in improved circulation half-life (t(1/2)) of DOX-GA3. DOX-mGA3 was synthesized more efficiently with an overall yield of 60% as compared to 37% in the case of DOX-GA3. We showed that DOX-mGA3 was enzymatically converted to DOX-GA3 with a t(1/2) of approximately 0.5 min in mouse plasma to 2.5 h in human plasma, which was in agreement with differences in esterase activity between species. DOX-mGA3, similar to DOX-GA3, was at least 37-fold less potent than the parent drug doxorubicin in growth inhibition of four different human malignant cell lines in vitro. Incubation of OVCAR-3 cells with DOX-mGA3 in combination with an excess of human beta-glucuronidase (0.05 U mL(-1)) resulted in a similar growth inhibition to that of doxorubicin. Intravenous administration of DOX-mGA3 in FMa-bearing mice resulted in an area under the concentration versus time curve (AUC) of DOX-GA3 in tumor and most normal tissues that was 2.5- to 3-fold higher than after the same dose of DOX-GA3 itself. In tumor tissue, this was accompanied by a 2.7-fold increase in the AUC of doxorubicin from DOX-mGA3 than from DOX-GA3. In conclusion, an advantage of DOX-mGA3 over DOX-GA3 is that this prodrug can be produced with a higher yield. Another important advantage is the improved pharmacokinetics of the lipophilic DOX-mGA3 as compared to that of the hydrophilic DOX-GA3. This effect may even be more pronounced in man, because of the lower plasma esterase activity than measured in mice.

Disodium 2,2'-dithio-bis-ethane sulfonate (BNP7787) is under investigation as a potential new chemoprotector against cisplatin-induced nephrotoxicity. The selective protection of BNP7787 appears to arise from the preferential uptake of the drug in the kidneys, where BNP7787 would undergo intracellular conversion into mesna (2-mercapto ethane sulfonate), which in turn can prevent cisplatin induced toxicities. In the present study, we have investigated whether the reduction of BNP7787 into the reactive compound mesna is restricted to the kidney or whether it can also occur in other organs, cells and physiological compartments, including the cytosolic fraction of the renal cortex, plasma, red blood cells (RBCs), liver and small intestine from rats and several tumors (OVCAR-3, MRI-H-207 and WARD). We also determined whether the endogenous thiols glutathione (GSH) and cysteine and the enzyme systems glutaredoxin and thioredoxin, which are all present in the kidney, can be involved in the BNP7787 reduction. UV detection and micro-HPLC with dual electrochemical detection were used to analyze the various incubation mixtures. Our observations are that, in contrast to plasma, a very large reductive conversion of BNP7787 to mesna was measured in RBC lysate. Intact RBCs, however, did not take up BNP7787. Although BNP7787 could be reduced in cytosol of liver and several tumors, this reduction will not be relevant in vivo, since these tissues do not take up large amounts of BNP7787. Kidney cortex cytosol was, similar to the small intestine cytosol, able to substantially reduce BNP7787 to mesna. The ability to reduce BNP7787 in the presence of the endogenous thiols GSH and cysteine, the glutaredoxin system as well as the thioredoxin system, could at least in part explain the high BNP7787 reductive activity of the kidney cortex cytosol. In conclusion, the high reduction of BNP7787 into mesna in the kidney as well as our earlier observation that the distribution of BNP7787 and mesna was mainly restricted to rat kidney are strong arguments in favor of selective protection of the kidney by BNP7787.

Human cytosolic beta-glycosidase is a small monomeric enzyme that is active under physiological conditions, which might be ideal for enzyme-prodrug therapy. We have previously reported the synthesis of a galactoside (DNR-GlA3) and a glucoside (DNR-GsA3) prodrug of daunorubicin. In the present study, we established that cellular uptake of DNR-GlA3 and DNR-GsA3 was low in contrast to that of daunorubicin. Recombinant human beta-glycosidase converted both prodrugs to daunorubicin as shown by liquid chromatography. The kinetics of the conversion of DNR-GlA3 and DNR-GsA3 by human beta-glycosidase, however, was unfavorable as the K(m) values were, respectively, 3- and 6-fold higher than those of another mammalian beta-glycosidase of bovine origin. The V(max) values were, respectively, 3.3 and 8.5nmol/hr/mg as compared to 158.3 and 147.8nmol/hr/mg of the bovine enzyme. Treatment of OVCAR-3 cells with human beta-glycosidase (0.5U/mL) and 0.5 microM DNR-GlA3 or DNR-GsA3 resulted in, respectively, 86 and 81% cell growth inhibition, while the prodrugs alone inhibited growth to only 19 and 1%. Treatment of cells with the bovine enzyme and the prodrugs inhibited cell growth more efficiently. We conclude that the endogenous intracellular beta-glycosidase is not available for extracellular prodrug activation. Thus, the incorporation of the enzyme in enzyme-prodrug therapy might be an elegant approach to achieve tumor-specific prodrug conversion. The efficiency of glycoside prodrug conversion might be improved by design of a prodrug that is more readily activated by human beta-glycosidase or by evolution of the enzyme into a mutant form that displays high activity towards these prodrugs.

DX-8951f or exatecan mesylate ((1S,9S)-1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10-13(9H,15H)-dione methanesulfonate dihydrate), is a new water-soluble derivative of camptothecin. We determined the activity of DX-8951f in experimental human colon cancer and ovarian cancer, being tumor types sensitive to camptothecins. With the use of the MTT assay, DX-8951f was more potent than SN-38 in four out of five human colon cancer cell lines and three out of four human ovarian cancer cell lines (P<0.05). DX-8951f was considerably more potent than topotecan in all cell lines tested (P<0.05). Prolonged exposure to DX-8951f resulted in a greater increase in inhibition of cell proliferation as compared to that obtained with SN-38 or topotecan (P<0.05). Overexpression of Pgp, MRP1, and LRP did not affect the in vitro activity of DX-8951f. DX-8951f administered daily x 5 or weekly x 2 resulted in growth inhibition <50% in two human colon cancer xenografts grown s.c. in nude mice. In three human ovarian cancer xenografts, however, >50% growth inhibition was observed at both schedules. In the OVCAR-3 human ovarian cancer model, DX-8951f showed considerably greater activity than topotecan (P<0.01). DX-8951f combined with cisplatin or paclitaxel did not indicate the presence of a pharmacological interaction. In OVCAR-3 xenografts the combination was clearly more effective than DX-8951f alone, as the number of complete remissions increased substantially. In conclusion, this study shows that DX-8951f is highly potent in vitro and highly effective in experimental human ovarian cancer in vivo. Prolonged exposure to DX-8951f in vitro greatly increased the antiproliferative effects, which may be a rationale for testing a continuous infusion schedule in the clinic. Addition of cisplatin or paclitaxel improved the in vivo antitumor effects of DX-8951f.

There is increasing interest in the exploitation of molecular addresses for the targeting of tumor imaging or therapeutic agents. A recent study demonstrated anticancer activity in human xenografts of doxorubicin (DOX)-peptide conjugates targeted to the tumor vascular endothelium, among them DOX coupled to the cyclic pentapeptide CNGRC [Science 279 (1998) 377]. In order to learn more about the mechanism of action of this type of DOX-peptide conjugates, we have studied the interaction of DOX-CNGRC with primary human umbilical cord vein endothelial cells (HUVEC) and tumor cells under defined in vitro conditions. We used a DOX conjugate, in which the cyclic CNGRC peptide, for which an in vivo endothelial address has recently been identified as aminopeptidase N (APN)/CD13, has been coupled via a hydrolysable spacer to the C-14 anthracycline-side chain. First we determined that the t(1/2) of DOX-CNGRC conjugate in human blood was 442 min (at 37 degrees ) allowing sufficient time for endothelial targeting when administered i.v. When cultured cells were exposed for 30 min to DOX-CNGRC a more cytoplasmic localization of fluorescent drug was seen when compared to DOX exposure and intracellular DOX-CNGRC was identified after extraction from the cells. This revealed differences in the cellular uptake process of the conjugate compared to DOX. The antiproliferative effect of DOX-CNGRC was determined by 30 min exposure in medium with a high protein content in order to mimick the in vivo targeting situation. In this medium, the IC(50) was 1.1 microM for highly CD13 expressing HT-1080, 1.45 microM for CD13 negative SK-UT-1 sarcoma cells and 6.5 microM for CD13 positive HUVEC. The IC(50) of DOX for these cells were 1.0, 2.0 and 7.3 microM, respectively. Although DOX-CNGRC inhibited the peptidase activity of CD13 up to 50%, our data do not favor an important role for the enzyme inhibition in the cytotoxic effect of the conjugate. The antitumor activity was tested in nude mice bearing human ovarian cancer xenografts (OVCAR-3). A weekly i.v. administration (3mg/kg DOX-equivalent, 3x) showed a minor (40%) growth delay, which does not indicate efficacy better than that expected for free DOX. In conclusion, this study indicates that the antiproliferative and anti-angiogenic effects of DOX-CNGRC as reported before, are likely caused by the cytostatic effects of intracellularly released parent drug DOX, independent of CD13 expression/activity. More research is needed to identify the optimal specific chemical configuration of DOX-peptide conjugates for in vivo targeting and receptor-mediated cellular uptake.

Paclitaxel is able to cause cell death through the induction of apoptosis. Cell death characteristics for docetaxel have not yet been described in detail. We investigated four unselected human ovarian cancer cell lines for the sensitivity to a 1hr exposure to docetaxel and calculated the concentrations inhibiting 50% (IC(50)) and 90% (IC(90)) of cell growth. Of the cell lines A2780, H134, IGROV-1 (all wild-type p53) and OVCAR-3 (mutant, mt p53) A2780 was most sensitive and OVCAR-3 least sensitive. Equitoxic drug concentrations representing IC(90) values (25-510nM) were applied for 1hr to measure cell cycle distribution, DNA degradation, and to count apoptotic cell bodies and cells with multifragmented nuclei at various time-points after drug exposure. H134, IGROV-1 and OVCAR-3 showed a continued mitotic block up to at least 72hr and prolonged presence of cells with multifragmented nuclei. High percentages of apoptosis were calculated at 48hr and at later time-points. In contrast, A2780 cells accumulated in the S-phase of the cell cycle and apoptosis was hardly present. The changes in the expression levels of p53, p21/WAF1, Bax and Bcl-2, were not predictive for docetaxel-induced apoptosis. Caspase-3 activation occurred only in cells with accumulation in the G2/M phase starting as early as 8hr in OVCAR-3. Prolonged Bcl-2 phosphorylation was evident in OVCAR-3, visible at 24hr in H134 and IGROV-1, while this phenomenon did not occur in A2780. The mitogen-activated protein kinase pathway (JNKs/SAPKs or c-Jun N-terminal kinases/stress-activated protein kinases, JNK1/2; extracellular response kinase, ERK1/2; p38) did not seem to be directly involved in Bcl-2 phosphorylation or apoptosis. We conclude that docetaxel is able to activate caspase-3, induce Bcl-2 phosphorylation and apoptosis in cells that show a prolonged G2/M arrest, but cells may also die by a caspase-3-independent cell death mechanism.

N-[4-daunorubicin-N-carbonyl (oxymethyl)phenyl] O-beta-glucuronyl carbamate (DNR-GA3) is a glucuronide prodrug of daunorubicin (DNR) which induced a better tumor growth delay than DNR when studied at equitoxic doses in three human ovarian cancer xenografts. These results suggested that the prodrug DNR-GA3 was selectively activated by human beta-glucuronidase present in tumor tissue. We determined the pharmacokinetics and distribution of DNR-GA3 in nude mice bearing human ovarian cancer xenografts (OVCAR-3, FMa, A2780, and MRI-H-207). Administration of DNR at 10 mg/kg i.v. (maximum tolerated dose) to OVCAR-3-bearing mice resulted in a peak plasma concentration of the drug of 12.18 microM (t = 1 min). DNR-GA3 at 100 mg/kg i.v. (approximately 50% of the maximum tolerated dose [MTD]) resulted in a peak plasma concentration of DNR that was 28-fold lower than that after DNR itself; in normal tissues, prodrug injection resulted in 5- to 23-fold lower DNR concentrations. DNR showed a relatively poor uptake into OVCAR-3 tumors with a peak concentration of 2.05 nmol x g(-1) after injection. In the same xenograft, DNR-GA3 resulted in a significantly higher DNR peak concentration of 3.45 nmol x g(-1) (P < 0.05). The higher area under the curve of DNR in tumor tissue after DNR-GA3 than after DNR itself would be the result of prodrug activation by beta-glucuronidase. In this respect, a considerably higher beta-glucuronidase activity was found in tumor tissue when compared to plasma. The specific activation of DNR-GA3 by beta-glucuronidase at the tumor site relative to normal organs leads to a more tumor-selective therapy, resulting in greater efficacy without increased toxicity.

Antibody-directed enzyme prodrug therapy (ADEPT) aims at the specific activation of a prodrug by an enzyme-immunoconjugate localized in tumor tissue. The use of an enzyme of human origin is preferable in ADEPT because it might not be immunogenic when administered to patients. In the case of human beta-glucuronidase, prodrugs should be designed that are rapidly and completely activated at a neutral pH. Four new daunorubicin glucuronides were synthesized by coupling a glucuronide group to daunorubicin via an aliphatic (GA1 and GB1) or an aromatic (GA3, GB6) carbamate spacer, to be released by electron shift (A-type) or by ring closure (B-type). These prodrugs were characterized in vitro for their usefulness in ADEPT and were compared with the previously described prodrugs epirubicin-glucuronide and doxorubicin-nitrophenyl-glucuronide. The four new prodrugs were stable in serum, hydrophilic when compared to the lipophilic daunorubicin, and at least 20-fold less toxic than the parent compound. The hydrolysis rate at clinically relevant enzyme and prodrug concentrations (1 microgram/mL human beta-glucuronidase, 100 microM prodrug) at pH 6.8 were similar for GA3 (T1/2 160 min) and higher for GB6 (T1/2 40 min) when compared to that of doxorubicin-nitrophenyl-glucuronide (T1/2 170 min). Epirubicin-glucuronide, GA1, and GB1 showed a low hydrolysis rate (T1/2 > 400 min). GA1 and GA3, but not GB1 or GB6, were activated to the parent compound. Complete activation was confirmed in OVCAR-3 cells pretreated with a specific antibody-human beta-glucuronidase conjugate, where GA3 had similar antiproliferative effects to those of daunorubicin.

2',2'-Difluorodeoxycytidine (Gemcitabine, dFdC) is a relatively new deoxycytidine antimetabolite, with established activity against ovarian cancer and non-small-cell lung cancer. dFdC is assumed to exert its antitumour effect mainly by incorporation of the triphosphate dFdCTP into DNA. We determined the sensitivity to dFdC of six cell lines derived from solid tumours; two ovarian carcinoma (A2780 and OVCAR-3), two colon carcinoma (WiDr and C26-10) and two squamous cell carcinoma cell lines (UM-SCC-14C and UM-SCC-22B). In vitro sensitivity to dFdC was strongly time dependent. Under all conditions A2780 was the most sensitive cell line with an IC50 (the concentration of dFdC causing 50% growth inhibition) of 31 and 0.6 nM at 1 and 48 hr exposure, respectively. WiDr and C26-10 cells were relatively insensitive, with IC50s of 468 and 1133 nM, respectively, at 1 hr exposure, but of 11 and 6 nM at 48 hr exposure. Accumulation of the triphosphate dFdCTP was also time dependent. After 4 hr exposure to 10 microM dFdC, A2780, WiDr and C26-10 cells accumulated 223, 136 and 267 pmol/10(6) cells, respectively; after 24 hr exposure they accumulated 1045, 619 and 617 pmol/10(6) cells, respectively. A2780 cells retained the high dFdCTP concentration longer than 24 hr. For comparison purposes we also studied dFdCTP kinetics in the corresponding solid tumours, showing the same sensitivity pattern as the cell lines. In general, sensitivity to dFdC in vitro related with dFdCTP accumulation and retention, but in vivo this relation was less clear. Unexpectedly, remarkable in vitro and in vivo changes were observed in the ribonucleotide pools. The most predominant in vitro cell line dependent changes were a decrease in CTP concentrations, accompanied by an increase in UTP and GTP concentrations. In vivo CTP, UTP and GTP pools increased in all tumours. In conclusion, in this study we demonstrate that dFdCTP is accumulated and retained in solid tumours and cell lines. dFdCTP is not only important as a DNA precursor, but also appears to interfere with normal ribonucleotide metabolism.