Enhancement of antitumor effects by the combination of cytotoxic agents and cytokines

The characteristics and feasibility of combination therapy of cytotoxic agents and cytokines were discussed from the theoretical as well as experimental basis. The content was divided into two parts depending upon requirements and elements for tumor cells to proliferate. One was concerning the in vitro conditions in which tumor cells were supplied all of them sufficiently. The other was concerning the host conditions in which cells were not necessarily supplied all of them freely and sufficiently. On the basis of characteristic tumor cell and host modifications achieved through receptor binding of cytokines, the combination of cytotoxic agents with cytokines, particularly with interferons, would produced the therapeutic effects that are distinct from those produced by the combination among cytotoxic agents. The clinical and experimental findings were reviewed and summarized along with this proposal.     

Enhancement of antitumor effect by intratumoral administration of bax gene in combination with anticancer drugs in gastric cancer

Proapototic gene is an important target to enhance the chemotherapeutic effect in cancer cells. Based on in vitro study showing that the introduction of bax gene enhanced the sensitivity to anticancer drugs, we examined whether the intratumoral administration of bax gene could enhance the anti-tumor effect in combination with anticancer drugs in gastric cancer. The human gastric cancer cell line, MKN45 was transplanted into nude mice, and the intratumoral administration of bax gene was performed using the bax cDNA plasmid complexed with a cationic lipopolyamine. The enhancement of antitumor effect was examined in the combination with 5-fluorouracil (5-FU) and cisplatin (CDDP). The anticancer drugs were administered intraperitoneally with one third of LD50 four times at 4-day intervals, and the antitumor effect was assessed by the NCI protocol. The expression of bax gene was analyzed by RT-PCR and the apoptotic cell death was assessed by TUNEL method. The intratumoral administration of bax gene alone showed slight anti-tumor effect as compared to that of control tumor injected with vector alone. The antitumor effect of 5-FU and CDDP was significantly enhanced in the combination with intra-tumoral administration of bax gene as compared to that of CDDP and 5-FU alone (p<0.05, Student's t-test). The enhancement of antitumor effect was associated with the constitutive overexpression of bax gene and with the induction of apoptosis in the tumor treated with anticancer drug and bax gene. These results indicate that the combination therapy of intratumoral administration of bax gene complexed with a cationic lipopolyamine and anticancer drugs may provide us a new strategy for cancer chemotherapy to enhance its therapeutic efficacy in gastric cancer as termed with gene-chemotherapy.     

Enhancement of the effects of anticancer agents on B16 melanoma cells by combination with cepharanthine--I. Alkaloids]

The anticancer effect of a podophyllotoxin derivative, etoposide (ETOP), and the vinca alkaloids, vincristine (VCR) and vindesine (VDS), on the colony forming efficiency (CFE) of B16 melanoma cells, and the CFE inhibition by the combination with a biscoclaurine alkaloid, Cepharanthine (Ceph) were studied in vitro. By combination with Ceph, CFE was reduced about 44% for ETOP, 31% for VCR and 30% for VDS compared to CFE without Ceph. Therefore, the effect of each anticancer agent was enhanced by combination with Ceph, especially that of Ceph and ETOP. The combination effect was greater when the low concentration of the agents was used. From the present study, the combination of ETOP and Ceph appears to be the more effective form of treatment.     

Combination of the minor groove-binder U73-975 or the intercalator mitoxantrone with antitumor alkylating agents in MCF-7 or MCF-7/CP cells.

In an effort to improve the cytotoxicity of clinically used anticancer alkylating agents, the topoisomerase II inhibitory drugs U73-975 or mitoxantrone were added to cell cultures exposed to CDDP, carboplatin, BCNU, melphalan or thiotepa. In the MCF-7 human breast cancer cell line and in the MCF-7/CP (CDDP resistant) subline, U73-975 and mitoxantrone were both potent cytotoxic agents (IC50 0.002 microM and 0.006 microM for U73-975, respectively and 0.8 microM and 0.1 microM for mitoxantrone, respectively). As evaluated by isobologram analysis, the addition of either U73-975 or mitoxantrone to 1 h exposure to CDDP resulted in greater-than-additive killing in the MCF-7 parent cells. While U73-975 was also greater-than-additive in cytotoxicity with CDDP in the MCF-7/CP line, mitoxantrone and CDDP were only additive in cytotoxicity in these cells. In the case of carboplatin, the addition of U73-975 or mitoxantrone to treatment with the drug resulted in greater-than-additive cell killing in the MCF-7 parental cell line but in the MCF-7/CP cell line these combinations were only additive in cell killing. Addition of U73-975 to treatment with BCNU resulted in only additive cytotoxicity in both cell lines; however, the combination of mitoxantrone with BCNU resulted in greater-than-additive cell killing in both the parental and CDDP resistant cell lines. When either U73-975 or mitoxantrone was added to treatment with melphalan greater-than-additive cytotoxicity resulted in both cell lines except at low melphalan concentrations in the MCF-7/CP cell line. Finally, the addition of either modulator to treatment with thiotepa in the MCF-7 cell line produced variable interactions depending on thiotepa concentration, but in the MCF-7/CP cell line either modulator in combination with thiotepa caused greater-than-additive cell killing. These results indicate that the addition of topoisomerase II inhibitory drugs may substantially increase the cytotoxicity of some alkylating agents. In vivo experiments are necessary, however, to ascertain whether a therapeutic gain is achievable.     

Enhancement of the DNA cross-linking activity of melphalan by misonidazole in vivo.

The technique of alkaline elution has been adapted for the study of drug-induced DNA cross-link formation in vivo. Pretreatment with misonidazole (MISO) enhances the number of cross-links formed in a fibrosarcoma and in the spleen and gut of mice for periods up to 48 h following a single injection of melphalan (MEL). The tumour was sensitized by a greater factor (2.05) than either of the normal tissues (enhancement factor 1.4-1.5). This enhancement did not appear to be related to inhibition of the repair of actual cross-links. Rather, the effect was explicable in terms of one of two alternative models. Firstly, MISO pretreatment could result in a greater amount of binding of MEL to DNA at early times after injection. This may be the result of altered pharmacokinetics of MEL, or of enhanced intracellular uptake of MEL due to MISO pretreatment. Secondly, MISO may exert its affect by inhibition of the repair of cross-links or monoadducts at early times post-injection, which would not be observed in this study. The possible involvement of glutathione depletion in chemosensitization by MISO was investigated by comparison with the effect of diethyl maleate (DEM), a known thiol-depleting reagent. Glutathione depletion, while perhaps being important, could not account for all of the effects observed.     

Enhancement of the effects of anticancer agents on B16 melanoma cells by combination with cepharanthine--II. Antimetabolite, alkylating agent, nitrosourea]

We have studied chemotherapy enhancement by the combination of the biscoclaurine alkaloid, Cepharanthine (Ceph), using the inhibition of colony forming efficiency (CFE) of B16 melanoma cells in vitro. Hydroxyurea, antimetabolite, showed a time-dependent inhibition of CFE, and the combination with Ceph enhanced CFE inhibition by 29%, which is stable and independent of the treatment time. Dacarbazine, a biological alkylating agent, given with Ceph showed a time-dependent inhibition of CFE (43%). MCNU, a nitrosourea, with Ceph showed the greatest inhibition of CFE (67%) among the anticancer agents used. The CFE inhibition was time-dependent and required a higher concentration of MCNU. In this series of studies using CFE inhibition of B16 melanoma cells in vitro, etoposide showed the best effect by the combination with Ceph, which seems to be a candidate for in vivo evaluation.     

In vitro and in vivo activity of antifungal agents in combination with fleroxacin, a new quinolone

The in vitro and in vivo interaction of fleroxacin with amphotericin B (Amph B), flucytosine (5-FC) and azoles against Candida albicans strains was tested. In vitro the interaction between fleroxacin and various antifungals was not dependent on the incubation time. Fleroxacin neither enhances nor antagonizes the in vitro activity of Amph B at high concentration (50-100 micrograms/ml). Fleroxacin has a synergistic effect with ketoconazole (KETO), but this is not observed with itraconazole (ITRA) or fluconazole (FLU). In no instance antagonism was observed. The activity of 5-FC was antagonized by fleroxacin being generally reduced by 2-4 dilution steps. In murine candidosis the efficacies of all antifungal drugs were not influenced by addition of 100 mg/kg fleroxacin. Therefore, the effects seen in in vitro tests are most probably not relevant for the clinical use of a combination of fleroxacin with antifungal drugs.     

Phase I dose-escalation study of brostallicin, a minor groove binder, in combination with cisplatin in patients with advanced solid tumors

Purpose

Brostallicin is a DNA minor groove binder which shows enhanced antitumor activity in cells which are resistant to several anticancer agents due to their high glutathione S-transferase (GST)/glutathione content. Phase I and II clinical trials of single-agent brostallicin have shown that myelotoxicity is the dose-limiting toxicity (DLT), while hints of antitumor activity were mainly observed in soft tissue sarcoma. Preclinical studies showing a more than additive antitumor effect of the cisplatin–brostallicin combination paved the way to clinical combination studies. In particular, we set up the first clinical combination study of brostallicin and cisplatin in patients with advanced solid tumors. This study was to be followed by a phase II study in patients with recurrent squamous cell carcinoma of the head and neck (SCCHN).

Methods

Escalating doses of brostallicin were administered in combination with a fixed dose of cisplatin (75 mg/m²) in patients with recurrent or metastatic advanced solid tumors who had previously received a cumulative dose of cisplatin not higher than 475 mg/m². The recommended dose of brostallicin was expanded in order to have a better estimate of antitumor activity and to better define the safety profile of the combination.

Results

Twenty-one patients were treated. Two DLTs (grade 3 fatigue and febrile neutropenia) were observed at dose level 3 (brostallicin 9 mg/m²). Dose level 2 (brostallicin 7 mg/m² and cisplatin 75 mg/m²) was recommended for future phase II studies. Main toxicity was hematologic; in fact, only 1 patient out of 21 did not develop neutropenia and only 2 patients did not have thrombocytopenia. Grade 3–4 neutropenia was observed in 90.5% of patients, grade 3–4 thrombocytopenia in 38.1%, grade 3–4 anemia in 23.8%. The cycle 1 nadir (ANC < 500 × 10⁹/L) for neutrophils was Day 14 (median; range 11–17) with recovery to an ANC of >1,500 3.5 days after nadir (median; range 2–4) at dose level 3. The cycle 1 nadir (median of 51,000 × 10⁹/L) for platelets occurred on Day 13 (median; range 10–15) with recovery to a platelet count of >100,000 4 days after nadir (median; range 2–8). No objective responses were observed, but seven patients had a long lasting (>18 weeks) stable disease.

Conclusions

Further studies of the combination of brostallicin and cisplatin are warranted.     

Potent antitumor activity of MS-247, a novel DNA minor groove binder, evaluated by an in vitro and in vivo human cancer cell line panel.

We synthesized a novel anticancer agent MS-247 (2-[[N-[1-methyl-2-[5-[N-[4-[N,N-bis(2-chloroethyl) amino] phenyl]] carbamoyl]-1H-benzimidazol-2-yl] pyrrol-4-yl] carbamoyl] ethyldimethylsulfonium di-p-toluenesulfonate) that has a netropsin-like moiety and an alkylating residue in the structure. We evaluated antitumor activity of MS-247 using a human cancer cell line panel coupled with a drug sensitivity database and subsequently using human cancer xenografts. The average MS-247 concentration required for 50% growth inhibition against a panel of 39 cell lines was 0.71 microM. The COMPARE analysis revealed that the differential growth inhibition pattern of MS-247 significantly correlated with those of camptothecin analogues and anthracyclins, indicating that MS-247 and the two drug groups might have similar modes of action. MS-247 exhibited remarkable antitumor activity against various xenografts. A single i.v. injection of MS-247 significantly inhibited the growth of all 17 xenografts tested, which included lung, colon, stomach, breast, and ovarian cancers. In many cases, MS-247 was more efficacious than cisplatin, Adriamycin, 5-fluorouracil, cyclophosphamide, VP-16, and vincristine and was almost comparable with paclitaxel and CPT-11; these are the most clinically promising drugs at present. MS-247 was noticeably more effective than paclitaxel (in HCT-15) and CPT-11 (in A549, HBC-4, and SK-OV-3). The toxicity of MS-247, indicated by body weight loss, was reversible within 10 days after administration. The MS-247 mode of action showed DNA binding activity at the site where Hoechst 33342 bound, inhibited topoisomerases I and II (as expected by the COMPARE analysis) blocked the cell cycle at the G2-M phase, and induced apoptosis. These results indicate that MS-247 is a promising new anticancer drug candidate to be developed further toward clinical trials.     

Augmentation of antitumor efficacy by the combination of recombinant tumor necrosis factor and chemotherapeutic agents in vivo

We evaluated the in vivo antitumor effects of the combination of recombinant human tumor necrosis factor (rhTNF) and three chemotherapeutic agents in an established murine tumor model. C57BL/6 mice bearing a subdermal weakly immunogenic 3-methylcholanthrene-induced sarcoma (MCA-106) received one i.v. dose of cyclophosphamide (Cy) (100 mg/kg), doxorubicin (5 mg/kg), or 5-fluorouracil (75 mg/kg) on either Day 8, 10, or 12. All animals received one i.v. dose of rhTNF (4 or 6 micrograms/mouse) on Day 10. The most effective time for administration of the chemotherapeutic agent was determined to be 48 h following rhTNF administration of all agents tested. The combined results of four separate experiments evaluating tumor size on Day 28 following tumor inoculation revealed that the groups treated with 4 or 6 micrograms of rhTNF and Cy (on Day 12) had tumor size reductions of 70 and 94%, respectively, compared to untreated controls (P2 less than 0.005). Mice treated with Cy alone, or with 4 or 6 micrograms of rhTNF alone had tumor size reductions of 30, 35, and 41%, respectively, compared to untreated controls (P2 less than 0.02). Analysis of cure rates demonstrates that the combination of Cy with 4 or 6 micrograms tumor necrosis factor cured 35 and 48% of the animals, respectively (P2 less than 0.01), compared to 10, 0, and 14% of mice treated with single agent Cy, 4 micrograms rhTNF, or 6 micrograms rhTNF, respectively. The timing of Cy and TNF administration was critical since administration of Cy prior to or concurrent with rhTNF was not effective in reducing tumor area or increasing cure rates over those achieved with either agent alone. Mice treated with doxorubicin alone had an increase in tumor size of 139 +/- 29% over untreated controls (P2 less than 0.05) on Day 28 following tumor inoculation and none were cured. In contrast, mice treated with doxorubicin plus 4 or 6 micrograms rhTNF exhibited early reductions in tumor size such that on Day 28 the average tumor areas were decreased by 66 +/- 34% (P2 less than 0.05) and 73 +/- 1% (P2 less than 0.02) of untreated controls with cure rates of 29% and 43% (P2 less than 0.02), respectively. However, the combination of 6 micrograms rhTNF plus doxorubicin led to substantial lethal toxicity with only 29% of mice surviving treatment. 5-Fluorouracil alone resulted in an increase in tumor area of 164% (P2 less than 0.05) over that of untreated controls on Day 28 following tumor inoculation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Potential antagonism of tubulin-binding anticancer agents in combination therapies.

ZD6126 is a vascular targeting agent, developed for the treatment of solid tumors. In vivo, ZD6126 is rapidly converted into the tubulin-binding agent N-acetylcolchinol. We have previously reported that in vitro N-acetylcolchinol disrupts microtubules and induces rapid changes in endothelial cell morphology, which in a tumor would lead to a rapid loss of tumor vessel integrity and subsequent extensive tumor necrosis. The aim of this study was to investigate the effect of cytotoxic antineoplastic drugs-cisplatin, doxorubicin, vincristine, paclitaxel, and docetaxel-on endothelial cell response to N-acetylcolchinol. We found that cisplatin and doxorubicin did not interfere with the ability of N-acetylcolchinol to cause morphologic changes in human umbilical vein endothelial cells, whereas vincristine showed additive effects. In contrast, the microtubule-stabilizing agents paclitaxel (1-10 micromol/L) and docetaxel (0.1-1 micromol/L) prevented the morphologic changes induced by N-acetylcolchinol in human umbilical vein endothelial cells. The effect was observed when cells were exposed to paclitaxel and N-acetylcolchinol together or when paclitaxel was given shortly before N-acetylcolchinol. Paclitaxel and N-acetylcolchinol interacted at the level of microtubule organization, as shown in immunofluorescence analysis of the cytoskeleton. The protective effect was reversible because 4 hours after paclitaxel wash out, cells recovered the sensitivity to N-acetylcolchinol. In vivo, pretreatment of mice with paclitaxel inhibited the vascular targeting activity of ZD6126 on newly formed vessels in the Matrigel plug assay and ZD6126-induced necrosis in tumors. These findings indicate that paclitaxel, depending on the timing and schedule of administration, can affect the vascular targeting activity of ZD6126, which may have an effect on the optimal scheduling of therapies based on the combined use of microtubule-stabilizing and microtubule-destabilizing agents.     

The effects of ICRF-154 in combination with other anticancer agents in vitro.

We studied the effects of ICRF-154 in combination with 11 anticancer agents on four human leukaemia cell lines. Cells were incubated for 3 days in the presence of two drugs (ICRF-154 and one other), and cell growth inhibition was determined by MTT assay. Effects of drug combinations at the ID50 level were analysed using the isobologram method (Steel). In the lymphoblastic leukaemia cell lines, MOLT-3, HSB, and B-ALL, supra-additive effects were observed for ICRF-154 in combination with amsacrine, bleomycin, doxorubicin, and etoposide. Additive effects were observed for its combinations with cisplatin, CPT-11, cytosine arabinoside, 5-fluorouracil, mitomycin C, and vincristine. Sub-additive to protective effects were observed in combination with methotrexate. In an erythroleukaemia cell line, K-562, no drug showed supra-additive effects with ICRF-154, while sub-additive to protective effects were observed for ICRF-154 in combination with cisplatin and methotrexate. The other drugs showed additive effects with ICRF-154. These results indicate that the combined effects of ICRF-154 with other agents vary, depending on the cell line. Against lymphoid malignancies, ICRF-154 would be advantageous when administered simultaneously with many anticancer agents. Of such agents, amsacrine, bleomycin, doxorubicin, and etoposide are the most suitable, while methotrexate is least suitable for such combined treatment.     

Enhancement of the antitumor activity of adriamycin by Tween 80

This paper describes the effect of Tween 80 on the antitumor activity and on the distribution of adriamycin in mice. The dilution of adriamycin in a 10% water solution of Tween 80 produced a significant increase of the antitumor activity in mice against ascites tumors (L 1210 leukemia), disseminated leukemias (transplanted leukemias originally induced by Gross leukemai virus and Moloney leukemia virus), and solid tumors (Sarcoma 180, MS-2 sarcoma). In all these experiments the drug was administered i.v., according to different schedules. Higher antitumor activity at the optimal dose and an increase of activity at lower doses were observed in different experimental systems. Toxicity was also slightly enhanced. Tissue distribution was studied in normal mice and in tumor-bearing mice (Gross leukemia and MS-2 sarcoma). In animals give i.v. adriamycin diluted in 10% Tween 80 there was a higher drug concentration in spleen, lung and kidney than there was in mice given the drug in a water solution. In all the other organs examined (heart, liver, small intestine) and in the MS-2 tumor tissue, no significant increase was observed. In L1210 leukemia-bearing mice, i.p. treatment with adriamycin diluted in 10% Tween 80 resulted in a significantly higher toxicity than that which resulted from treatment with adriamycin in a wa ter solution; no increase of antitumor activity was observed.     

In vivo antitumor activity of choline kinase inhibitors: a novel target for anticancer drug discovery.

Transformation by some oncogenes is associated with increased activity of choline kinase (ChoK), resulting in elevated constitutive levels of phosphorylcholine, a proposed second messenger required for DNA synthesis induced by growth factors. Here we describe the characterization of ChoK inhibitors with antiproliferative properties against human tumor-derived cell lines. The new molecules were tolerated in mice at doses that showed in vivo antitumor activity against human tumor xenografts derived from HT-29 and A431 cell lines implanted s.c. in nude mice. This first generation of inhibitors provides in vivo evidence that blockade of phosphorylcholine production is a valid strategy for the development of new anticancer agents, opening a new avenue for the development of antitumor drugs with a novel mechanism of action.     

Enhancement of in vivo antitumor activity of a novel antimitotic 1-phenylpropenone derivative, AM-132, by tumor necrosis factor-alpha or interleukin-6.

TK5048 and its derivatives, AM-132, AM-138, and AM-97, are recently developed antimitotic (AM) compounds. These 1-phenylpropenone derivatives induce cell cycle arrest at the G2 / M phase of the cell cycle. TK5048 inhibited tubulin polymerization in human lung cancer PC-14 cells in a concentration-dependent manner. In a polymerization assay using bovine brain tubulin, AM-132 and AM-138 were quite strong, AM-97 was moderately strong, and TK5048 was a relatively weak inhibitor of tubulin polymerization. A murine leukemia cell line resistant to a sulfonamide antimitotic agent, E7010, which binds to colchicine-binding sites on tubulin, was cross-resistant to the in vitro growth-inhibitory effect of AM compounds. Inhibition of tubulin polymerization is therefore one of the mechanisms of action of these AM compounds against tumor cells. To profile the antitumor effect of AM compounds, the in vivo antitumor effect of AM-132 was evaluated against cytokine-secreting Lewis lung carcinoma (LLC). Tumor-bearing mice were treated with intravenous AM-132 using three different treatment schedules. LLC tumors expressing tumor necrosis factor-alpha (TNF-alpha), granulocyte macrophage colony-stimulating factor (GM-CSF), or interleukin (IL)-6 were very sensitive to AM-132. In particular, LLC tumors expressing IL-6 were markedly reduced by AM-132 treatment, and showed coloring of the tumor surface and unusual hemorrhagic necrosis. These results suggest a combined effect of AM-132 and cytokines on the blood supply to tumors.     

Synergistic antitumor activity of capecitabine in combination with irinotecan

5-Fluorouracil (5-FU) and capecitabine alone and in combination with irinotecan/oxaliplatin are clinically active in the treatment of colorectal and other solid tumors. Studies of the antitumor activity and toxicity of capecitabine or irinotecan alone and in combination with each other, were compared with 5-FU and raltitrexed in human tumor xenografts of colorectal and squamous cell carcinoma of the head and neck using clinically relevant schedules. Antitumor activity and toxicity were evaluated in nude mice bearing human colon carcinomas of HCT-8 and HT-29 and in head and neck squamous cell carcinomas of A253 and FaDu xenografts using the maximum tolerable dose of single-agent capecitabine, 5-FU, or raltitrexed, or each of the drugs in combination with irinotecan. Mice were treated with capecitabine and irinotecan alone or in combination using 2 different schedules: (1) capecitabine orally once a day for 7 days and a single dose of irinotecan (50 mg/kg intravenously [I.V.]), with each drug alone or in combination, and (2) capecitabine orally 5 days a week for 3 weeks and irinotecan 50 mg/kg (I.V. injection) once a week for 3 weeks, with each drug alone or in combination. For comparative purposes, the antitumor activity of single-agent capecitabine, 5-FU, or raltitrexed, or each drug in combination with irinotecan was carried out at its maximum tolerated dose (MTD) using a 3-week schedule. Results indicated that HT-29 and A253 xenografts were de novo resistant (no cure) to capecitabine and irinotecan alone at the MTD, whereas HCT-8 and FaDu xenografts were relatively more sensitive, yielding 10%-20% cures. The combination of irinotecan/capecitabine was much more active than either drug alone against all 4 tumor models. The cure rates were increased from 0 to 20% in A253 and HT-29 xenografts and from 10%-20% to 80%-100% in HCT-8 and FaDu tumor xenografts, respectively. Irinotecan/capecitabine had clear advantage over irinotecan/5-FU and irinotecan/raltitrexed in efficacy and selectivity in that they were more active and less toxic. The extent of synergy with irinotecan/capecitabine appears to be tumor-dependent and independent of the status of p53 expression. The potential impact of the preclinical results on clinical practice for the use of these drugs in combination needs clinical validation.     

A novel synthetic DNA minor groove binder, MS-247: antitumor activity and cytotoxic mechanism

Purpose: MS-247 is a novel synthetic compound possessing a DNA-binding moiety and a DNA-alkylating residue, chlorambucil. In this study, we evaluated the antitumor activity of MS-247 against murine tumor cell lines and its effects on DNA molecules in both cell-free and cellular systems. Methods: The in vitro cytotoxic activity of MS-247 was evaluated against four murine tumor cell lines, P388, L1210, Colon26 and B16, and its in vivo antitumor activity was also tested in comparison with Adriamycin (ADM), cisplatin (CDDP) and paclitaxel. The ability of MS-247 to associate with the DNA minor groove was assessed by measuring quenching of Hoechst 33342 fluorescence. DNA-DNA interstrand crosslinks (ICL) were detected by an alkaline elution assay for cellular DNA and a band-shift assay using the plasmid pBR322. The effects of MS-247 on macromolecule synthesis (DNA, RNA and proteins) were examined by measuring incorporation of the radiolabeled precursors. Results: MS-247 exhibited in vitro cytotoxicity with IC₅₀ values ranging 11 to 500 nM, and MS-247 given i.v. showed strong in vivo antitumor activity against i.p.-implanted L1210 leukemia cells and s.c.-implanted Colon26 carcinoma cells, and moderate activity against i.p.-implanted P388 leukemia cells but no apparent activity against s.c.-implanted B16 melanoma cells. MS-247 reversibly displaced Hoechst 33342 bound to DNA within a few minutes, and irreversibly formed ICL within 1–6 h in both the cell-free system and the cellular system. These results suggest that an association of MS-247 with the DNA minor groove occurred more quickly than ICL formation. The inhibition of DNA synthesis was more prominent than the inhibition of RNA and protein synthesis in L1210 cells exposed to MS-247, and a 6-h incubation with MS-247, which formed apparent ICL in the cellular system, strongly inhibited DNA synthesis. This result suggests that impairment of DNA replication preceded the inhibition of RNA and protein synthesis and that ICL formation greatly contributed to the inhibition of macromolecule synthesis. Conclusion: The results of this study suggest that MS-247 exerts its cytotoxic effect through impairment of DNA function by getting into the minor groove of DNA and subsequently forming ICL. MS-247 has potent antitumor activity with a different spectrum from the activity of clinically proven antitumor agents such as paclitaxel, ADM and CDDP against several murine tumor cell lines. This result suggests that MS-247 may be useful for the treatment of human cancers. Received: 6 July 1999 / Accepted: 14 February 2000     

Enhancement of antitumor activity of docetaxel by celecoxib in lung tumors

Our study investigates the effect of a highly selective cyclooxygenase-2 (COX-2) inhibitor, celecoxib, on the cytotoxicity of docetaxel in nude mice bearing A549 tumor xenografts and elucidates the molecular mechanisms of the antitumor effect of this combination. Female nu/nu mice, xenografted with s.c. A549 tumors were treated with either celecoxib (150 mg/kg/day), docetaxel (10 mg/kg) or a combination of both. The tumor tissues were quantified for the induction of apoptosis, intratumor levels/expressions of prostaglandin E2 (PGE2), 15 deoxy prostaglandin J2 (15-d PGJ2), microsomal prostaglandin E synthase (mPGES) and cytoplasmic phospholipase A2 (cPLA2). The combination of celecoxib with docetaxel significantly inhibited the tumor growth (p < 0.03) as compared to celecoxib or docetaxel alone, decreased the levels of PGE2 by 10-fold and increased the 15-d PGJ2 levels by 4-fold as compared to control. The combination also enhanced the peroxisome proliferator-activated receptor (PPAR)-gamma expression, decreased the expression of cPLA2, mPGES and vascular endothelial growth factor (VEGF), but had no effect on the expression of COX-1 or COX-2 in tumor tissues. TUNEL staining of the tumor tissues showed a marked increase in the apoptosis in the combination group as compared to the celecoxib- or docetaxel-treated groups and this was associated with an increase in the intratumor p53 expression. In conclusion, the combination of celecoxib with docetaxel produces a greater antitumor effect in s.c. A549 tumors as compared to celecoxib or docetaxel alone and this effect is associated with concomitant alterations in the intratumor levels of PGE2 and 15-d PGJ2.     

SJG-136 (NSC 694501), a novel rationally designed DNA minor groove interstrand cross-linking agent with potent and broad spectrum antitumor activity: part 1: cellular pharmacology, in vitro and initial in vivo antitumor activity

SJG-136 (NSC 694501) is a rationally designed pyrrolobenzodiazepine dimer that binds in the minor groove of DNA. It spans 6 bp with a preference for binding to purine-GATC-pyrimidine sequences. The agent has potent activity in the National Cancer Institute (NCI) anticancer drug screen with 50% net growth inhibition conferred by 0.14 to 320 nmol/L (7.4 nmol/L mean). Sensitive cell lines exhibit total growth inhibition and 50% lethality after treatment with as little as 0.83 and 7.1 nmol/L SJG-136, respectively. COMPARE and molecular target analysis of SJG-136 data versus that of >60,000 compounds tested in the NCI 60 cell line screen shows that, although the agent has similarity to other DNA binding agents, the pattern of activity for SJG-136 does not fit within the clusters of any known agents, suggesting that SJG-136 possesses a distinct mechanism of action. Testing in the NCI standard hollow fiber assay produced prominent growth inhibition in 20 of 24 i.p. and 7 of 24 s.c. test combinations with 5 of 12 cell lines exhibiting cell kill. In addition, SJG-136 produced antitumor activity in mice bearing CH1 and CH1cisR xenografts, a cisplatin-resistant human ovarian tumor model, and also in mice bearing LS174T xenografts, a human colon tumor model. SJG-136 produces DNA interstrand cross-links between two N-2 guanine positions on opposite strands and separated by 2 bp. In human tumor cell lines, the cross-links form rapidly and persist compared with those produced by conventional cross-linking agents such as nitrogen mustards. In mice bearing the LS174T human colon xenograft, DNA interstrand cross-links can be detected in tumor cells using a modification of the single cell gel electrophoresis (comet) assay after administration of a therapeutic dose. Cross-links in the tumor increase with dose and are clearly detectable at 1 hour after i.v. administration. The level of cross-linking persists over a 24-hour period in this tumor in contrast to cross-links produced by conventional cross-linking agents observed over the same time period.