Anti-cancer activities of pure curry feeding in cancer cell-transplanted mouse.

To confirm the cytotoxic effect of instant curry containing combined spices on cancer cells in vivo, cancer was induced by transplanting cancer cells to mice, and the development of cancer upon feeding pure curry were examined. The concentration of lipid peroxide in the groups transplanted with cancer cells which were fed with normal feed was 19.6 nM, and it was increased as the amount of pure curry was increased. The concentration of cytochrome P-450 was decreased in the group transplanted with cancer cells which were fed with pure curry and the group without the transplant which were fed with pure curry when compared with the groups which were fed with normal feed. The activity of cytochrome P-450 was decreased as the concentration of cytochrome P-450 was decreased in the groups transplanted with cancer cells. However, it was increased in the groups without cancer cell transplant when over 2% of pure curry was fed. The amount of glutathione was increased in the groups transplanted with cancer cells when over 2% of pure curry was fed. The activities of glutathione peroxidase and glutathione S-transferase were decreased in the groups transplanted with cancer cells which were fed with over 1% of pure curry, and were restored to the level of the group without cancer cell transplant which were fed with normal feed. The superoxide dismutase activity in the groups transplanted with cancer cells was restored to the level of the group without cancer cell transplant which was fed with normal feed when over 1% of pure curry was fed.

Inhibition of cell proliferation by nobiletin, a dietary phytochemical, associated with apoptosis and characteristic gene expression, but lack of effect on early rat hepatocarcinogenesis in vivo.

Dietary phytochemicals can inhibit the development of certain types of tumors. We here investigated the effects of nobiletin (Nob), garcinol (Gar), auraptene (Aur), beta-cryptoxanthin- and hesperidine-rich pulp (CHRP) and 1,1'-acetoxychavicol acetate (ACA) on hepatocarcinogenesis in a rat medium-term liver bioassay, and also examined their influence on cell proliferation, cell cycle kinetics, apoptosis and cell invasion of rat and human hepatocellular carcinoma (HCC) cells, MH1C1 and HepG2, respectively. While there were no obvious suppressive effects on the development of putative preneoplastic liver lesions, inhibition of hepatocarcinoma cell proliferation was evident in the Nob group. Nob also caused G2/M cell cycle arrest and apoptosis. Microarray analysis identified a set of genes specifically regulated by Nob, and these are likely to be involved in the observed growth suppression of HCC cells. These results suggest that phytochemicals might have chemopreventive potential in late stages of hepatocarcinogenesis.

Hypericum perforatum methanolic extract inhibits growth of human prostatic carcinoma cell line orthotopically implanted in nude mice.

The antiproliferative effect of serotonin-reuptake inhibitors (SSRI) and serotonin antagonists has been demonstrated in prostate tumors. Since Hypericum perforatum components act as serotonin-reuptake inhibitors and exert cytotoxic effects on several human cancer cell lines, in this work we analyzed the effect of a treatment with Hypericum perforatum extract (HPE) on the growth of human prostate cancer cells in vitro and in vivo. This study highlighted a significant reduction of tumor growth and number of metastasis suggesting that this natural compound may be useful in the treatment of prostate cancer.

Effect of LY293111 in combination with gemcitabine in colonic cancer.

New adjuvant therapies are needed for the treatment of stage III colon cancer. The essential fatty acids, linoleic and arachidonic acid enhance tumorigenesis through the cyclooxygenase and lipoxygenase pathways. Leukotriene B4 (LTB4) is a product of 5-lipoxygenase (5-LOX) which has tumor-promoting effects. The LTB4 receptor antagonist, LY293111 inhibited tumor growth and induced apoptosis in vitro. The effectiveness of LY293111, alone and in combination with gemcitabine was investigated in a heterotopic xenograft model in athymic mice using HT29 and LoVo human colonic cancer cells. The combined therapy markedly inhibited tumor growth and could warrant consideration as a new therapeutic option.

Systemic therapy of malignant human melanoma tumors by a common cold-producing enterovirus, coxsackievirus a21.

PURPOSE: The incidence of malignant melanoma continues to increase worldwide; however, treatment of metastatic melanoma remains unsatisfactory, and there is an urgent need for development of effective targeted therapeutics. A potential biological target on the surface of malignant melanoma cells is the up-regulated expression of intercellular adhesion molecule (ICAM)-1 and decay-accelerating factor (DAF), relative to surrounding benign tissue. Coxsackievirus A21 (a common cold virus) targets and destroys susceptible cells via specific viral capsid interactions with surface-expressed virus receptors comprising ICAM-1 and DAF. EXPERIMENTAL DESIGN: The oncolytic capacity of a genetically unmodified wild-type common cold-producing human enterovirus (Coxsackievirus A21, CAV21) was assessed against in vitro cultures and in vivo xenografts of malignant human melanoma cells. RESULTS: In vitro studies established that human melanoma cells endogenously express elevated levels of ICAM-1/DAF and were highly susceptible to rapid viral oncolysis by CAV21 infection, whereas ICAM-1/DAF-expressing peripheral blood lymphocytes were refractile to infection. In vivo studies revealed that the tumor burden of nonobese diabetic severe combined immunodeficient mice bearing multiple s.c. melanoma xenografts was rapidly reduced by oncolysis mediated by a single administration of CAV21. The antitumor activity of CAV21 was characterized by highly efficient systemic spread of progeny CAV21, with oncolysis of tumors also occurring at sites distant to the primary site of viral administration. CONCLUSIONS: Overall, the findings presented herein demonstrate an important proof of principle using administration of replication-competent CAV21 as a potential biological oncolytic agent in the control of human metastatic melanoma.

Prevention of intrahepatic metastasis by curcumin in an orthotopic implantation model.

Curcumin has been shown to have potent anti-metastatic activity, however, its mechanism of action is still unclear. Here, we analyzed the anti-metastatic mechanism using hepatocellular carcinoma, CBO140C12 cells. Daily oral administration of curcumin suppressed intrahepatic metastasis in a dose-dependent manner, whereas the growth of implanted tumors was not affected. We next examined the effect of curcumin on several metastatic properties in vitro. Curcumin inhibited the invasion of tumor cells through Matrigel-coated filters and the production of MMP-9. In addition, curcumin significantly inhibited adhesion and haptotactic migration to fibronectin and laminin without affecting the expression of integrins on the cell surface. Furthermore, the formation of actin stress fibers was affected by treatment with curcumin. These results suggested that curcumin suppressed the intrahepatic metastasis mediated by the inhibiton of several metastatic properties, in which the functional alteration of cytoskeletal organization, at least in part, could play an important role.

Forced expression of cytidine deaminase confers sensitivity to capecitabine.

OBJECTIVE: Cytidine deaminase (CDD) is involved in the metabolism of new pyrimidine analogues, capecitabine (N(4)-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) and gemcitabine (2',2'-difluorodeoxycytidine). The purpose of the present study was to directly examine the role of CDD in tumor cells themselves in mediating the sensitivity to capecitabine compared with gemcitabine. METHODS: The human bladder cancer cell line T24 was transfected with human CDD2 cDNA by the lipofectin method. RESULTS: Transfection of CDD2 cDNA did not change the levels of thymidine phosphorylase, dihydropyrimidine dehydrogenase and thymidylate synthase (TS) but increased the CDD activity significantly (p < 0.01). Forced expression of CDD made T24 sensitive to 5'-deoxy-5-fluorocytidine (5'DFCR) in vitro and capecitabine in vivo, but resistant to gemcitabine both in vitro and in vivo. Tetrahydrouridine, a specific CDD inhibitor, abrogated the changes in the in vitro sensitivity to 5'DFCR and gemcitabine by transfection of CDD2 cDNA. Transfection of CDD2 cDNA resulted in a significant increase in cellular 5-fluorouracil level (p < 0.01) and inhibition of TS activity (p < 0.01) after treatment with 5'DFCR in vitro. CONCLUSIONS: The present study clearly showed direct evidence for the contribution of CDD in tumor cells themselves to the sensitivities to capecitabine and gemcitabine.

Selective hyperthermia using magnetoliposomes to target cervical lymph node metastasis in a rabbit tongue tumor model.

The effect of hyperthermia on cervical lymph node metastasis of VX7 tongue cancer in female Japanese white rabbits was investigated. Magnetoliposomes (MLs) with a neutral surface charge and a size of 94.1 nm were used as heating mediators. MLs were injected into the tongue 20 days after tumor transplantation, and we examined whether they reached the metastatic deep cervical lymph node. The highest magnetite concentration 24 h after ML injection was detected in the lymph node, followed by tongue, spleen, blood, and liver. Rabbits were separated into three groups: group I as the control; group II with ML injection alone; and group III with ML injection and hyperthermia 24 h after ML injection, generated by applying an alternating magnetic field (118 kHz, 384 Oe) to the neck region. The hyperthermic effect was evaluated in terms of the percentage of necrosis in proportion to the metastatic tumor and the apoptotic index (AI), defined as the ratio of TUNEL-positive cells. The temperature of lymph nodes in group III reached over 44 degrees C. The mean area of necrosis in group III was 58.0%, which was significantly higher than that in group I (19.6%) or group II (20.4%). The AI in group III was 22.9%, significantly higher than in group I (1.67%) or II (1.42%). The difference between group I and II was not statistically significant. Group III tumor sites around MLs showed necrosis or apoptosis-positive cells induced by hyperthermia. These results indicate that MLs injected into the tongue can target cervical lymph node metastases and accumulate there at concentrations sufficient to generate therapeutically effective temperatures.

n-3 polyunsaturated fatty acids decrease mucosal/epidermal reactions and enhance antitumour effect of ionising radiation with inhibition of tumour angiogenesis.

The purpose of this study was to evaluate the effect of n-3 polyunsaturated fatty acids (n-3 PUFAs) on normal tissue (lip mucosa) and tumour growth when combined with ionising radiation. The oral region (snout) of C57 black mice was irradiated with 16.5 Gy and n-3 PUFAs (100 microl) were injected intravenously for 2 weeks. After exposure to irradiation, the degree and duration of the acute reactions decreased significantly when mice were treated with n-3 PUFAs as compared to the control group. Interestingly, the range of the reactions in the n-3 PUFAs-treated group compared favourably to the group receiving amifostine (27.5 mg/kg i.v.). the effect of n-3 PUFAs was further evaluated in HEP-2 human carcinoma xenograft transplanted in nude mice. An inhibition of tumour growth was observed when mice were treated with n-3 PUFAs alone and this effect was maximal when combined with irradiation. Similar results were obtained using eicosapentaenoic acid. The effect of n-3 PUFAs was associated with inhibition of angiogenesis and tumour proliferation, and significantly decreased expression of cyclooxygenase-2. In conclusion, n-3 PUFAs administration decrease mucosal response, while moderately enhancing the antitumour effect of irradiation. The magnitude of the differential effect suggests that n-3 PUFAs need to be further investigated in the clinic.

SU11248 inhibits KIT and platelet-derived growth factor receptor beta in preclinical models of human small cell lung cancer.

The purpose of this study was to evaluate the activity of the indolinone kinase inhibitor SU11248 against the receptor tyrosine kinase KIT in vitro and in vivo, examine the role of KIT in small cell lung cancer (SCLC), and anticipate clinical utility of SU11248 in SCLC. SU11248 is an oral, multitargeted tyrosine kinase inhibitor with direct antitumor and antiangiogenic activity through targeting platelet-derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor, KIT, and FLT3 receptors. Treatment of the KIT-expressing SCLC-derived NCI-H526 cell line in vitro with SU11248 resulted in dose-dependent inhibition of stem cell factor-stimulated KIT phosphotyrosine levels and proliferation. The biological significance of KIT inhibition was evaluated in vivo by treating mice bearing s.c. NCI-H526 tumors with SU11248 or another structurally unrelated KIT inhibitor, STI571 (Gleevec), which is also known to inhibit Bcr-Abl and PDGFRbeta. SU11248 treatment resulted in significant tumor growth inhibition, whereas inhibition from STI571 treatment was less dramatic. Both compounds reduced phospho-KIT levels in NCI-H526 tumors, with a greater reduction by SU11248, correlating with efficacy. Likewise, phospho-PDGFRbeta levels contributed by tumor stroma and with known involvement in angiogenesis were strongly inhibited by SU11248 and less so by STI571. Because platinum-based chemotherapy is part of the standard of care for SCLC, SU11248 was combined with cisplatin, and significant tumor growth delay was measured compared with either agent alone. These results expand the profile of SU11248 as a KIT signaling inhibitor and suggest that SU11248 may have clinical potential in the treatment of SCLC via direct antitumor activity mediated via KIT as well as tumor angiogenesis via vascular endothelial growth factor receptor FLK1/KDR and PDGFRbeta.

Usefulness of combined treatment with mild temperature hyperthermia and/or tirapazamine in the treatment of solid tumors: its independence of p53 status.

Human head and neck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector as a control (SAS/neo) were inoculated subcutaneously into both hind legs of Balb/cA nude mice. Mice bearing the tumors received 5-bromo-2'-deoxyuridine (BrdU) continuously to label all proliferating (P) cells in the tumors. The mice then received tirapazamine (TPZ) with or without mild temperature hyperthermia (40 degrees C, 60 min) (MTH), gamma-ray irradiation with or without MTH and/or TPZ, cisplatin (CDDP) with or without MTH and/or TPZ, or paclitaxel (TXL) with or without MTH and/or TPZ. After each treatment, the tumors were excised, minced and trypsinized. The tumor cell suspensions thus obtained were incubated with a cytokinesis blocker (cytochalasin-B), and the micronucleus (MN) frequency in cells without BrdU labeling (i.e., quiescent (Q) cells) was determined by using immunofluorescence staining for BrdU. Meanwhile, 6 h after gamma-ray irradiation or 24 h after other cytotoxic treatments, tumor cell suspensions obtained in the same manner were used for determining the frequency of apoptosis in Q cells. The MN frequency and apoptosis frequency in total (P+Q) tumor cells were determined from the tumors that were not pretreated with BrdU. On the whole, gamma-ray irradiation and CDDP injection induced a higher frequency of apoptosis and lower frequency of MN in SAS/neo cells than SAS/mp53 cells. There were no apparent differences in the induced frequency of apoptosis and MN between SAS/neo and SAS/mp53 cells after TPZ or TXL treatment. MTH sensitized cells to TPZ-inducing cytotoxicity more markedly in SAS/mp53 and Q cells than in SAS/neo cells and total cells, respectively. In gamma-ray irradiation and CDDP treatment, the enhancement in combination with MTH and/or TPZ was more remarkable in SAS/mp53 cells and Q cells than in SAS/neo and total tumor cells, respectively. Also in the case of TXL treatment, the combination with MTH and/or TPZ induced a slightly greater enhancement effect in SAS/mp53 cells and Q cells. In view of the difficulty in controlling mutated p53 status tumors and intratumor Q cells, combination treatment with MTH and/or TPZ as a cooperative modality in cancer therapy is considered to have potential for controlling solid tumors as a whole.

Optimizing the dosing schedule of TNP-470 [O-(chloroacetyl-carbamoyl) fumagillol] enhances its antitumor and antiangiogenic efficacies.

Many drugs vary in potency and/or toxicity according to the time of day when they are administered. In this study, we investigated whether antitumor efficacy of angiogenesis inhibitor, TNP-470 [O-(chloroacetyl-carbamoyl) fumagillol], could be improved by optimizing the dosing schedule. Tumor-bearing mice were housed under standardized light/dark cycle conditions (lights on at 7:00 AM, off at 7:00 PM) with food and water ad libitum. The antitumor effect of TNP-470 (30 mg/kg s.c.) was more potent in mice injected with the drug at the early light phase than it was when administered at the early dark phase. The diurnal change in the antitumor effect of TNP-470 was parallel to that in its antiangiogenic activity. The variation in the effects of TNP-470 was closely related to the diurnal variations in its inhibitory action on methionine aminopeptidase activity in tumor masses. There was a significant dosing time-dependent change in the concentration of TNP-470 in plasma. The higher concentration of TNP-470 in plasma was observed when its antitumor and antiangiogenic activities were increased. These results suggest that therapeutic efficacy of TNP-470 can be enhanced by choosing the most appropriate time of day to administer the drug.

Immuno-gene therapy with adenoviruses expressing fms-like tyrosine kinase 3 ligand and CD40 ligand for mouse hepatoma cells in vivo.

Introduction of genes encoding immuno-stimulatory cytokines into cancer cells is known to enhance antitumor immunity. CD40 ligand (CD40L, CD154) and fms-like tyrosine kinase 3 ligand (Flt3L) are recently identified cytokines, which have been demonstrated to stimulate antitumor immunity in several cancer models. However little is known about antitumor activity of Ftl3L and CD40L against hepatocellular carcinoma (HCC). In the present study, we constructed replication-defective adenoviruses expressing Flt3L and CD40L and examined their therapeutic efficacy on mouse HCC, MH134 cells. Subcutaneous injection of MH134 cells genetically engineered to express Flt3L and/or CD40L developed tumors in all the syngeneic immunocompetent mice, but tumor growth was significantly delayed as compared to control mice. Partial inhibition of this antitumor effect in athymic nude mice suggests that both innate and adaptive immunity appear to play a role. It was shown by immunodepletion of NK cells with anti-asialo-GM1 antibody that the effector cells involved in innate immunity are NK cells. In a therapeutic setting, however, injection of adenovirus expressing Flt3L or CD40L into pre-established MH134 tumors exhibited no efficacy. These data demonstrate that Flt3L and CD40L induce significant, but only weak, antitumor immunity against MH134 cells presumably through both innate and adaptive immunity. Our results suggest that immuno-gene therapy with Flt3L and CD40L may need adjuvant modalities to achieve strong immune response.

Predictions from a preclinical model: studies of aromatase inhibitors and antiestrogens.

We have developed a tumor model for studying the effects of aromatase inhibitors and antiestrogens in vivo. The model simulates postmenopausal breast cancer patients with estrogen-dependent tumors. This model utilizes human estrogen-dependent breast cancer cells transfected with the aromatase gene (MCF-7(CA)), which are inoculated in Matrigel s.c. into ovariectomized nude mice. The estrogen receptor-positive cells produce sufficient estrogen by aromatization to stimulate their proliferation and the formation of tumors in the mice. Using several different strategies, we have compared the aromatase inhibitors (letrozole and anastrozole) with antiestrogens (tamoxifen and fulvestrant). Aromatase inhibitors were more effective than tamoxifen and were more effective alone than when combined with antiestrogens. In addition, letrozole had a longer duration of effect than tamoxifen. The possibility of delaying the development of resistance to antiestrogens and aromatase inhibitors was investigated by alternating letrozole with tamoxifen every 4 weeks. However, although alternating treatment proved more effective than tamoxifen alone (tumors doubled in 16 weeks), tumors doubled in volume at about 18 weeks when treatment began with tamoxifen. When treatment started with letrozole and alternated with tamoxifen, tumors doubled in volume in about 22 weeks. However, tumors of mice treated with letrozole alone did not double in size until 35 weeks. These results demonstrate that this aromatase inhibitor is more effective and has a longer duration of response as a single agent than tamoxifen or in combination with tamoxifen.

Induction of tumor necrosis factor production and antitumor effect by cabbage extract.

The effect of cabbage extract on the production of tumor necrosis factor and its implication in the antitumor effect were examined in vitro and in vivo. Cabbage extract stimulated the production of tumor necrosis factor by rat spleen cells and showed cytotoxic activity in a rat ascites hepatoma cell line (AH109A) when hepatoma cells were cultured with cabbage-stimulated spleen cells. When the extract was adminstered orally to AH109A-bearing rats in combination with lipopolysaccharide injection, the hepatoma weights were reduced to one-half of the vehicle control. The cytotoxic activity of tumor-infiltrating macrophages was induced by simultaneous treatment with cabbage extract and lipopolysaccharide. These results indicate that cabbage extract contains macrophage-stimulating component(s) and can implement the antitumor effect by stimulating the cytotoxicity of tumor-infiltrating macrophages.

Antitumour effect of abrin on transplanted tumours in mice.

Abrin, a galactose specific lectin was purified using sepharose 4B affinity column from seeds of Abrus precatorius. It exhibited antitumour activity in mice when used at a sublethal dose of 7.5 micrograms/kg every alternate day for 10 days. Both intralesional and intraperiloneal (i.p.) administration of abrin was effective in reducing solid tumour mass development induced by Dalton's Lymphoma Ascites (DLA) and Ehrlich's Ascites Carcinoma (EAC) cells. DLA cell line was more sensitive to abrin than EAC. Abrin when injected i.p. increased the life span of ascites tumour bearing mice. Abrin when used simultaneously with tumour cells brought about maximum antitumour effect. On developed tumour masses, abrin administration brought about significant reduction in tumour volume, especially in DLA induced tumours. Prophylactic administration of abrin was found ineffective.

CHS 828 inhibits neuroblastoma growth in mice alone and in combination with antiangiogenic drugs.

CHS 828 is a new chemotherapeutic drug, a pyridyl cyanoguanidine. CHS 828 has low toxicity and lacks known patterns of multidrug resistance. Here we report that oral, daily treatment with CHS 828 reduced the growth of SH-SY5Y human neuroblastoma tumors in male NMRI nu/nu mice by 82% without apparent toxicity. CHS 828 induced complete tumor regression for at least 5 weeks in four of nine animals (44%). Combination therapy with CHS 828 and the antiangiogenic drugs TNP-470 or SU5416 decreased neuroblastoma growth by a further 10 and 3%, respectively. Combination therapy induced tumor regression at d 4 with CHS plus TNP and d 6 with CHS plus SU5416, compared with d 14 with CHS 828 alone (p < 0.05), and complete tumor regression was seen in nine of 19 animals (47%). Combination treatment of CHS 828 and TNP-470 decreased the total viable tumor volume by 71% compared with treatment with CHS 828 alone. Our findings support CHS 828 as a promising new drug in treatment of childhood cancers. Furthermore, they imply efficiency of daily administration of nontoxic doses of chemotherapy, and a possible additive effect when chemotherapy is combined with angiogenesis inhibitors.

Inhibition of melanoma growth and metastasis by combination with (-)-epigallocatechin-3-gallate and dacarbazine in mice.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, was shown to have cancer chemopreventive activity. In this study, we examined the antimetastatic effects of EGCG or the combination of EGCG and dacarbazine on B16-F3m melanoma cells in vitro and in vivo. First, the antimetastatic potentials of five green tea catechins were examined by soft agar colony formation assay, and the results show that EGCG was more effective than the other catechins in inhibiting soft agar colony formation. Second, EGCG dose-dependently inhibited B16-F3m cell migration and invasion by in vitro Transwell assay. Third, EGCG significantly inhibited the spread of B16-F3m cells on fibronectin, laminin, collagen, and Matrigel in a dose-dependent manner. In addition, EGCG significantly inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) and the activity of matrix metalloproteinase-9 (MMP-9). In animal experiments, EGCG alone reduced lung metastases in mice bearing B16-F3m melanomas. However, a combination of EGCG and dacarbazine was more effective than EGCG alone in reducing the number of pulmonary metastases and primary tumor growths, and increased the survival rate of melanoma-bearing mice. These results demonstrate that combination treatment with EGCG and dacarbazine strongly inhibits melanoma growth and metastasis, and the action mechanisms of EGCG are associated with the inhibition of cell spreading, cell-extracellular matrix and cell-cell interactions, MMP-9 and FAK activities.

Osteosarcoma cell apoptosis induced by selenium.

An essential nutrient selenium has been reported to be a potential cancer preventive and inhibitory agent, although no exact mechanism has yet been proposed. Since little is known about the anti-proliferative effect of selenium on osteosarcoma, this issue was addressed in the present study in vitro using three osteosarcoma cell lines, and in vivo using an osteosarcoma transplantable to nude mice. Selenium inhibited the tumor growth in vitro and morphological changes indicative of apoptosis were demonstrated. Osteosarcomas in nude mice were inhibited in growth by selenium with no cytotoxic change in normal tissues. The findings suggested that selenium may offer a novel therapeutic modality for osteosarcoma.

N-(2-hydroxypropyl)methacrylamide copolymer-6-(3-aminopropyl)-ellipticine conjugates. Synthesis, in vitro, and preliminary in vivo evaluation.

Ellipticine derivatives have potential as anticancer drugs. Their clinical use has been limited, however, by poor solubility and host toxicity. As N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anticancer conjugates are showing promise in early clinical trials, a series of novel HPMA copolymer conjugates have been prepared containing the 6-(3-aminopropyl)-ellipticine derivative (APE, NSC176328). Drug was linked to the polymer via GFLG or GG peptide side chains. To optimize biological behavior, HPMA copolymer-GFLG-APE conjugates with different drug loading (total APE: 2.3-7% w/w; free APE: <0.1% w/w) were synthesized. Conjugation of APE to HPMA copolymers considerably increased its aqueous solubility (>10-fold). HPMA copolymer-GG-APE did not liberate drug in the presence of isolated lysosomal enzymes (tritosomes), but HPMA copolymer-GFLG-APE released APE to a maximum of 60% after 5 h. The rate of drug release was influenced by drug loading; lower loading led to greater release. Whereas free APE (35 microg/mL) caused significant hemolysis (50% after 1 h), HPMA copolymer-APE conjugates were not hemolytic up to 300 microg/mL (APE-equiv). As would be expected from its cellular pharmacokinetics, HPMA copolymer-GFLG-APE was >75 times less cytotoxic than free drug (IC(50) approximately 0.4 microg/mL) against B16F10 melanoma in vitro. However, in vivo when tested in mice bearing s.c. B16F10 melanoma, HPMA copolymer-GFLG-APE (1-10 mg/kg single dose, APE-equiv) given i.p. was somewhat more active (highest T/C value of 143%) than free APE (1 mg/kg) (T/C =127%). HPMA copolymer-APE conjugates warrant further evaluation as potential anticancer agents.