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We studied the expression of Nrf2 transcription factor and antioxidant system proteins in drug-resistant murine leukemia strains P388 in vivo, as well as the redox status of cells under conditions of induced oxidative stress. Immunoblotting and real-time PCR showed that the cyclophosphamide-resistant strain P388 (P388/CP) exhibits Nrf2-mediated drug resistance. Cells of the P388/CP strain are characterized by high expression of Nrf2, which leads to a significant increase in the expression of ARE genes and antioxidant system proteins, as well as to the effective maintenance of redox homeostasis under conditions of induced oxidative stress. Taking into account the important role of Nrf2 overexpression in reducing the effectiveness of chemotherapy in patients with different leukemias, the P388/CP strain can be of great interest as a model in the development of new drugs for the treatment of malignant neoplasms., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

Background: High consumption of flavonoids has been associated with a decrease risk of cancer. Alfalfa (Medicago sativa) leaves have been widely used in traditional medicine and is currently used as a dietary supplement because of their high nutrient content. We previously reported the cytotoxic activity of alfalfa leaf extracts against several sensitive and multidrug resistant tumor cell lines., Hypothesis/purpose: We aimed to determine whether medicarpin and millepurpan, two isoflavonoids isolated from alfalfa leaves, may have pro-apoptotic effects against drug-sensitive (P388) and multidrug resistant P388 leukemia cells (P388/DOX)., Study Design/methods: Cells were incubated with medicarpin or millepurpan for the appropriate time. Cell viability was assessed by the MTT assay. DNA fragmentation was analyzed by agarose gel electrophoresis. Cell cycle analysis was realized by flow cytometry technics. Caspases 3 and 9 activities were measured using Promega caspACE assay kits. Proteins and genes expression were visualized respectively by western-blot using specific antibodies and RT-PCR assay., Results: P-glycoprotein-expressing P388/DOX cells did not show resistance to medicarpin (IC50 ≈ 90 µM for P388 and P388/DOX cells) and millepurpan (IC50 = 54 µM and 69 µM for P388 and P388/DOX cells, respectively). Treatment with medicarpin or millepurpan triggered apoptosis in sensitive as well as multidrug resistant P388 cells. These effects were mediated through the mitochondrial pathway by modifying the balance pro/anti-apoptotic proteins. While 3 µM doxorubicin alone could not induce cell death in P388/DOX cells, concomitant treatment with doxorubicin and subtoxic concentration of medicarpin or millepurpan restored the pro-apoptotic cascade. Each compound increased sensitivity of P388/DOX cells to doxorubicin whereas they had no effect in sensitive P388 cells. Vinblastine cytotoxicity was also enhanced in P388/DOX cells (IC50 = 210 nM to 23 and 25 nM with medicarpin and millepurpan, respectively). This improved sensitivity was mediated by an increased uptake of doxorubicin in P388/DOX cells expressing P-gp. P-gp expression was not altered by exposure to medicarpin and millepurpan., Conclusion: These data indicate that medicarpin and millepurpan possess pro-apoptotic properties and potentiate the cytotoxicity of chemotherapy drugs in multidrug resistant P388 leukemia cells by modulating P-gp-mediated efflux of drugs. These flavonoids may be used as chemopreventive agents or as sensitizer to enhance cytotoxicity of chemotherapy drugs in multidrug resistant cancer cells., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

Purpose: Pluronic block copolymers are potent sensitizers of multidrug resistant cancers. SP1049C, a Pluronic-based micellar formulation of doxorubicin (Dox) has completed Phase II clinical trial and demonstrated safety and efficacy in patients with advanced adenocarcinoma of the esophagus and gastroesophageal junction. This study elucidates the ability of SP1049C to deplete cancer stem cells (CSC) and decrease tumorigenicity of cancer cells in vivo., Experimental Design: P388 murine leukemia ascitic tumor was grown in BDF1 mice. The animals were treated with: (a) saline, (b) Pluronics alone, (c) Dox or (d) SP1049C. The ascitic cancer cells were isolated at different passages and examined for 1) in vitro colony formation potential, 2) in vivo tumorigenicity and aggressiveness, 3) development of drug resistance and Wnt signaling activation 4) global DNA methylation profiles, and 5) expression of CSC markers., Results: SP1049C treatment reduced tumor aggressiveness, in vivo tumor formation frequency and in vitro clonogenic potential of the ascitic cells compared to drug, saline and polymer controls. SP1049C also prevented overexpression of BCRP and activation of Wnt-β-catenin signaling observed with Dox alone. Moreover, SP1049C significantly altered the DNA methylation profiles of the cells. Finally, SP1049C decreased CD133(+) P388 cells populations, which displayed CSC-like properties and were more tumorigenic compared to CD133(-) cells., Conclusions: SP1049C therapy effectively suppresses the tumorigenicity and aggressiveness of P388 cells in a mouse model. This may be due to enhanced activity of SP1049C against CSC and/or altered epigenetic regulation restricting appearance of malignant cancer cell phenotype.

The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C-glycosyltransferase (C-GT) that forms the C-C glycosidic bond between the sugar and the SF2575 tetracycline-like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT-B fold wherein the TDP and docked putative aglycon are consistent with the overall C-glycosylation reaction. As one of only a few existing structures for C-glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C-glycosylation., (Copyright © 2013 Wiley Periodicals, Inc.)

The cytotoxin family of cobra venom proteins, also called cardiotoxins, can activate both necrotic and apoptotic cell death pathways in cancer cells. Cytotoxin 1 (CTX1)from Naja atra Cantor venom is a 60 amino acid, 6698 Da protein with as yet untested anticancer efficacy and cell selectivity. We tested the toxicity of CTX1 on a number of cancer cell lines (MCF-7, P388, K562, and H22) and on one normal human cell line (16HBE). The rank order of cytotoxicity was MCF-7 > P388 ≈ K562 >H22 ≈ 16HBE, indicating that the effect of CTX1 on certain cancer cell types was relatively selective.Treatment with CTX1 greatly prolonged the survival of P388 ascites tumors bearing KM mice compared to cyclophosphamide treatment. Cell viability, apoptosis, and lysosomal permeability assays all demonstrated that CTX1 induced dose- and time-dependent cell death, with most cells exhibiting the morphological and biochemical features of late apoptosis and necrosis. Mitochondrial membrane potential was lost in CTX1-treated P388 cells. In addition, CTX1 induced an increase in both lysosomal membrane permeability and cathepsin B protease activity. These analyses reveal that CTX1 possesses significant and selective anticancer activity, likely by inducing programmed cell death through mitochondrial and/or lysosomal pathways.

Apoptosis-associated speck-like protein (ASC) is a bipartite adaptor molecule that participates in inflammation and apoptosis. ASC silencing has been observed in a significant proportion of human cancers. Here, we examined the role of ASC overexpression in the metastasis of the P388D1 murine lymphoma cell line to the liver, lung, spleen and kidney. First, we determined that the P388D1 cells express ASC. Then, ASC overexpression in P388D1 was achieved by transfecting pEGFP-ASC-C2 into the P388D1 cells. Furthermore, after the ASC-overexpressing P388D1 cells were injected into DBA/2 mice through the vena caudalis, their metastasis to the lung and the liver was significantly reduced in the pEGFP-ASC-C2-transfected group. These data indicate that ASC overexpression affects the in vivo metastatic properties of P388D1 cells., (Copyright © 2011 John Wiley & Sons, Ltd.)

The P2X7 receptor (P2X7R) is a nucleotide receptor expressed predominantly on hemopoietic, bone, and epithelial cells. The P2X7R can be activated by extracellular ATP and induces the influx of calcium, releases cytokines, and participates in cell proliferation and apoptosis. CD44 is an adhesion molecule. The effects of CD44 include cell-cell and cell-matrix adhesion interactions, lymphocyte activation, and cell migration. Many studies have shown that P2X7R and CD44 play important roles in hematological malignancies, but no study exists regarding the relationship between P2X7R and CD44. In the present study, we characterized P388D1 cells for the surface expression of CD44 and analyzed ATP-induced shedding. The data showed that P388D1 cells express CD44. Incubation of P388D1 cells with ATP induced a rapid loss of CD44 from the P388D1 cell surface. In addition, using a receptor inhibitor and P2X7R short hairpin RNA, we showed that the loss of CD44 is mediated via the P2X7R. Finally, we demonstrated that activation of P2X7R by ATP induces CD44 shedding., (Copyright © 2011 John Wiley & Sons, Ltd.)

Purpose: Atrasentan is a highly potent and selective endothelin receptor A (ET(A)) antagonist under development for the treatment of prostate cancer. Only little data exist on its interaction with drug-metabolising enzymes and drug transporters possibly influencing its safety and effectiveness. Our study evaluated whether atrasentan can induce the expression of relevant human drug transporters and cytochrome P450 isozymes (CYPs), whether it retains its efficiency in multidrug resistant cell lines, and whether it inhibits P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP)., Methods: Induction of transporters and enzymes was quantified at the mRNA level by real-time RT-PCR in LS180 cells and for P-gp also at the protein level by Western blot. P-gp inhibition was evaluated by calcein assay in P388/dx and L-MDR1 cells and BCRP inhibition in MDCKII-BCRP cells by pheophorbide A efflux. Substrate characteristics were evaluated by growth inhibition assays in MDCKII cells overexpressing particular ABC-transporters., Results: Atrasentan profoundly induced several CYPs and drug transporters (e.g. 12-fold induction of CYP3A4 at 50 μM). It was a moderate P-gp inhibitor (IC(50) in P388/dx cells = 15.1 ± 1.6 μM) and a weak BCRP inhibitor (IC(50) in MDCKII-BCRP cells = 59.8 ± 11 μM). BCRP or P-gp overexpressing cells were slightly more resistant towards antiproliferative effects of atrasentan., Conclusions: Our data provide a comprehensive analysis of the induction profile of atrasentan and its interaction with P-gp and BCRP. The profound induction effects stress the need for thorough assessment of its interaction potential in vivo.

Fluorescent probes are frequently employed for the detection of different reactive oxygen and nitrogen species formed during the irradiation of photosensitized cells and tissues. Investigators often interpret the results in terms of information provided with the different probes without examining specificity or determinants of fluorogenic reactions. We examine five fluorescent probes in a cell-free system: reduced 2',7'-dichlorofluorescein, dihydroethidine, dihydrorhodamine, 3'-(p aminophenyl) fluorescein (APF), and 4',5'-diaminofluorescein. Of these, only APF demonstrates a high degree of specificity for a single reactive species. There is a substantial influence of peroxidase activity on all fluorogenic interactions. The fluorescence of the photosensitizing agent also must be taken into account in evaluating results.

Cucurbitacin E increases the doxorubicin (DOX) level in M5076 ovarian sarcoma via suppressed DOX efflux in vitro. An increase in DOX induced antitumor activity by cucurbitacin E in vivo has been reported previously. This paper attempts to clarify the mechanism of cucurbitacin E induced increments in the antitumor activity of DOX. MK-571, a multidrug resistance associated protein (MRP) inhibitor, significantly suppressed DOX efflux from M5076 ovarian sarcoma cells. The combination of cucurbitacin E with MK-571 also inhibited DOX efflux, whereas the efficacy was the same in each treatment. Namely, the inhibition of DOX efflux by cucurbitacin E was expected to be related to MRP. In contrast, it appeared that the effect of cucurbitacin E on DOX permeability did not relate to P-gp. The cucurbitacin E co-treatment significantly increased DOX concentration in the tumor within a short time after DOX administration, whereas the same treatment decreased the DOX concentration in normal tissues. The different effects of cucurbitacin E between tumor and normal tissues was speculated to be related to differences in DOX transport system on cell membrane. In DOX therapy, cucurbitacin E co-treatment was expected to increase DOX induced antitumor activity without an increase in adverse reactions due to DOX.

Heparin is known to attenuate tumor metastasis mainly by inhibiting the interaction between L-selectin and its ligand. However, the mechanism of heparin on lymphoma is unclear. This report demonstrates that both L-selectin/h IgG chimeric protein and heparin treatment can significantly inhibit the adhesion of P388D1 cells onto lymphatic sinusoids and marginal sinusoids in vitro, that heparin can attenuate P388D1 cell homing to lymph nodes in vivo at 12 hours, and that heparin significantly reduced P388D1 cells metastasis to lymph nodes 18 days after injection. These results indicate that heparin may act as a ligand for L-selectin on the P388D1 macrophage-like lymphoma cell line to attenuate tumor growth and metastasis.

Oxidative dimerization of ferulic acid methyl ester afforded dihydrobenzofuran derivative and new linear compound identified by X-ray crystallography. The gallate derivatized dihydrobenzofuran analogue was obtained and all compounds were evaluated for potential antiatherogenic, antiplasmodial (best IC(50)=0.8 microM) and cytotoxic activities.

Determination of multidrug resistance (MDR) activity of tumor cells could provide important information for the personalized therapy of cancer patients. The functional calcein assay (MultiDrug Quant Assay, Solvo Biotechnology, Budaörs, Hungary) has been proven to be clinically valuable in hematological malignancies by determining the transporter activity of MDR protein 1 (MDR1, ATP-binding cassette protein [ABC] B1, P-glycoprotein-170) and MDR-related protein 1 (MRP1, ABCC1). In this study, we evaluated if the same functional test was adaptable for the analysis of MDR activity in solid tumors. For this purpose, tissue specimens of human colorectal cancer samples were subjected to limited enzymatic digestion by collagenase to provide a single-cell suspension; dead cells were excluded by 7-aminoactinomycin D staining, and epithelial cancer cells were detected by Cy5-conjugated anti-BerEP4 monoclonal antibody. The transporter functions of MDR1 and MRP1 in viable epithelial cells were assessed by flow cytometry detecting the intracellular accumulation of calcein dye after exposing cells to various MDR inhibitors. Collagenase disintegration preserved the MDR activity and the antigenicity of tumor cells. Thus using the extended calcein assay provided sufficient viable and functionally active tumor cells from surgical biopsies to determine the functional MDR activity. In conclusion, the newly described modified calcein assay may be applicable for evaluating the MDR phenotype in solid tissue specimens from colorectal forceps biopsy to surgical samples.

Activation of macrophages and macrophage cell lines by bacterial LPS elicits a delayed phase of PG biosynthesis that appears to be entirely mediated by cyclooxygenase-2 (COX-2). In previous work, we found that a catalytically active group V secreted phospholipase A(2) (sPLA(2)-V) was required for COX-2 induction, but the nature of the sPLA(2)-V metabolite involved was not defined. In this study, we identify lysophosphatidylcholine (lysoPC) as the sPLA(2)-V downstream mediator involved in COX-2 induction by LPS-stimulated macrophages. Inhibition of sPLA(2)-V by RNA interference or by the cell-permeable compound scalaradial blocked LPS-induced COX-2 expression, and this inhibition was overcome by incubating the cells with a nonhydrolyzable lysoPC analog, but not by arachidonic acid or oleic acid. Moreover, inhibition of sPLA(2)-V by scalaradial also prevented the activation of the transcription factor c-Rel, and such an inhibition was also selectively overcome by the lysoPC analog. Collectively, these results support a model whereby sPLA(2)-V hydrolysis of phospholipids upon LPS stimulation results in lysoPC generation, which in turn regulates COX-2 expression by a mechanism involving the transcriptional activity of c-Rel.

Here we demonstrate the inducible mouse Hsp72 binds markedly to lymphoid neoplastic macrophage-like P388D1 cells. To examine whether mouse CD40 can play a role in signaling exogenously administered HSP70 in a fashion similar to that of human CD40, we established mouse CD40-transfectants of both human 293 cells and murine-pro-B cell line Ba/F3. A small portion of mouse CD40 expressed on 293-derived transfectants was the mature form with a signal-transducible C-terminal domain, whereas a majority of expressed antigen showed the molecular size smaller than we expect. Flow cytometry showed that mouse Hsp72, but neither its deletion variants nor the related Escherichia coli DnaK, bound to the 293-derived transfectants regardless of CD40 expression. CD40 molecules expressed on the transfectants showed the binding of soluble form of CD40L but this binding was not inhibited by excess amount of HSP70. CD40L, but not any HSP70 recombinant proteins, stimulated the production of chemokine RANTES in the transfectants. Furthermore, no RANTES production was induced by HSP70-RCMLA complex in the transfectants, although it binds to 293-derived cells in a CD40-independent manner. No interaction between mouse CD40 and HSP70 recombinant proteins was detected by using the Ba/F3-derived transfectants that express the mature form of mouse CD40. The present results imply that mouse CD40 expressed on the transfectants differs from its human homolog in the binding of exogenously administered HSP70.

Several novel isoquino[4,5-bc]acridine derivatives have been designed and synthesized. Their DNA-binding, anti-tumor and DNA-photo-damaging properties were investigated. A4 exhibited the highest anti-tumor activities against both A 549 (human lung cancer cell) and P388 (murine leukemia cells). All these compounds were found to be more cytotoxic against P388 than against A549. Under 365-nm light irradiation, A3 damaged plasmid DNA pBR322 at <2 microM and cleaved DNA from form I to 100% form II by 50 microM. The mechanism studies revealed that A3 damaged DNA by electron transfer mechanism and singlet oxygen species.

Reactive oxygen species generated by photosensitizers are efficacious remedy for tumor eradication. Eleven cycloimide derivatives of bacteriochlorin p (CIBCs) with different N-substituents at the fused imide ring and various substituents replacing the 3-acetyl group were evaluated as photosensitizers with special emphasis on structure-activity relationships. The studied CIBCs absorb light within a tissue transparency window (780-830 nm) and possess high photostability at prolonged light irradiation. The most active derivatives are 300-fold more phototoxic toward HeLa and A549 cells than the clinically used photosensitizer Photogem due to the substituents that improve intracellular accumulation (distribution ratio of 8-13) and provide efficient photoinduced singlet oxygen generation (quantum yields of 0.54-0.57). The substituents predefine selective CIBC targeting to lipid droplets, Golgi apparatus, and lysosomes or provide mixed lipid droplets and Golgi apparatus localization in cancer cells. Lipid droplets and Golgi apparatus are critically sensitive to photoinduced damage. The average lethal dose of CIBC-generated singlet oxygen per volume unit of cell was estimated to be 0.22 mM. Confocal fluorescence analysis of tissue sections of tumor-bearing mice revealed the features of tissue distribution of selected CIBCs and, in particular, their ability to accumulate in tumor nodules and surrounding connective tissues. Considering the short-range action of singlet oxygen, these properties of CIBCs are prerequisite to efficient antitumor photodynamic therapy.

Purpose: The influence of serum components on the intracellular uptake of an 111In-oligonucleotide (ODN) against mdr1 mRNA was investigated in the murine leukemia cell line, P388/S, and its mdr1-overexpressed P388/R., Methods: 111In-ODNs naked and vectorized with lipids were analyzed for binding with serum components using high-performance liquid chromatography (HPLC). 111In-ODN was incubated in albumin and transferrin solutions. 111In-DTPA and 111In-mononucleotide were incubated in serum. Degradation of naked 111In-ODN was detected in phosphate buffered saline (PBS) and serum containing endonuclease S1. Cellular uptakes of naked and vectorized 111In-ODN in the above cells were examined with and without fetal calf serum (FCS)., Results: Time-dependent binding of naked and vectorized 111In- ODN with serum components was observed throughout 24 hours. Transchelation of 111In to transferrin was not detected. HPLC profiles of 111In-DTPA and 111In-mononucleotide did not change in serum. Degradation of 111In-ODN by S1 was less remarkable in serum than in PBS. Specific accumulation of vectorized 111In-ODN in P388/R cells was achieved in culture with and without 10% FCS., Conclusions: This study verified the intense binding of ODN with serum components, leading to no inhibition on ODN intracellular specific uptake. Binding with serum components protects 111In-ODN from degradation by endonuclease and thus may facilitate ODN transmembrane delivery.

CD1 molecules are MHC class I-like glycoproteins specialized in presenting lipid/glycolipid Ags to T cells. The distinct cell-type specific expression of CD1D1 plays an important role in the development and function of NKT cells, a unique subset of immunoregulatory T cells. However, the mechanisms regulating CD1D1 expression are largely unknown. In this study, we have characterized the upstream region of the CD1D1 gene and identified a minimal promoter region within 200 bp from the translational start site of CD1D1 that exhibits cell-type specific promoter activity. Analysis of this region revealed an Ets binding site critical for CD1D1 promoter activity. Gel shift assays and chromatin immunoprecipitation experiments showed that Elf-1 and PU.1 bind to the CD1D1 promoter. Furthermore, we found that gene disruption of Elf-1 resulted in decreased CD1D1 expression on B cells but not other cell types, whereas conditional activation of PU.1 negatively regulated CD1D1 expression in PU.1-deficient myeloid cells. These findings are the first to demonstrate that Ets proteins are involved in the transcriptional regulation of CD1D1 and that they may function uniquely in different cell types.

Poly(gamma-benzyl L-glutamate) (PBLG)/poly(ethylene glycol) (PEG) diblock copolymer endcapped with galactose moiety (abbreviated as GEG) was synthesized and characterized for study of liver-specific targeting. From dynamic light scattering measurement, particle sizes of copolymeric nanoparticles were decreased with an increase of PEG in the copolymer. The morphology of GEG-3 nanoparticles observed by transmission electron micrograph was observed as almost spherical shapes and ranged about 50-300 nm. From the structural characterization using 1H nuclear magnetic resonance, both characteristic peaks of PBLG and PEG were visible in CDCl3 but the characteristic peaks of PBLG were invisible in D2O, indicating that GEG block copolymers are found to the core-shell type nanoparticles in water with PBLG innercore and PEG outershell, exposing that galactose moiety of GEG block copolymers are outerwards oriented on the nanoparticle surfaces. By galactose-specific aggregation test of particles using beta-galactose specific lectin, and flow cytometry measurement, specific interaction between asialoglycoprotein receptors (ASGPR) of HepG2, human hepatoma cell line, and galactose moieties of the GEG nanoparticles was confirmed. From cell cytotoxicity test, HepG2 cells with ASGPR are more sensitive to paclitaxel (TX)-loaded nanoparticles than free TX whereas, P388 cells, murine leukemia cell line, and SK-Hep 01, human hepatoma cell line, without ASGPR is less sensitive to TX-loaded nanoparticles than free TX, suggesting that specific interaction between HepG2 cells and galactose moiety of the nanoparticles occurred.

Background: SN-38, 7-ethyl-10-hydroxycamptothecin, is a biologically active metabolite of irinotecan. Its poor solubility restricted its development as an anticancer agent. We have developed an easy-to-use liposome-entrapped SN-38 (LE-SN38) and evaluated its toxicology, pharmacokinetics and antitumor efficacy profile., Materials and Methods: Toxicity and pharmacokinetics studies were conducted in CD2F1 mice and beagle dogs. Therapeutic efficacy studies were performed in murine leukemia (P388 and P388/ADR) and in a human pancreatic (Capan-1) tumor models., Results: Multiple dose administration (i.v. x 5) of LE-SN38 indicated a maximum tolerated dose (MTD) of 5.0 and 7.5 mg/kg/day for male and female mice, respectively. The MTD of LE-SN38 in dogs was 1.2 mg/kg. The elimination half-life (t1/2) of SN-38 in mouse plasma was 6.38 h with volume of distribution (VdSS) 2.55 L/kg. In dogs, t1/2 and VdSS were 1.38-6.42 h and 1.69-5.01 L/kg; respectively. P388 tumor-bearing mice dosed with LE-SN38 at 5.5 mg/kg (i.v. x 5) showed 100% survival. LE-SN38 at 4 or 8 mg/kg (i. v. x 5) inhibited 65% and 98% tumor growth, respectively, in a human pancreatic tumor model., Conclusion: LE-SN38 showed a favorable pharmacokinetics profile and can be administered safely at therapeutically effective doses.

Several types of lymphoid and myeloid tumor cells are known to be relatively resistant to radiation-induced apoptosis compared to normal lymphocytes. The intracellular generation of reactive oxygen species was measured in irradiated spleen cells from C57BL/6 and BALB/c mice and murine tumor cells (EL-4 and P388) by flow cytometry using dichlorodihydrofluoresceindiacetate and dihydrorhodamine 123 as fluorescent probes. The amount of reactive oxygen species generated per cell was low in the tumor cells compared to spleen cells exposed to 1 to 10 Gy of gamma radiation. This could be due to the higher total antioxidant levels in tumor cells compared to normal cells. Further, the changes in mitochondrial membrane potential and cytoplasmic Ca2+ content were appreciable in lymphocytes even at a dose of 1 Gy. In EL-4 cells, no such changes were observed at any of the doses used. About 65% of spleen cells underwent apoptosis 24 h after 1 Gy irradiation. However, under the same conditions, EL-4 and P388 cells failed to undergo apoptosis, but they accumulated in G2/M phase. Thus the intrinsic radioresistance of tumor cells may be due to a decreased generation of reactive oxygen species after irradiation and down-regulation of the subsequent events leading to apoptosis.

The cytotoxicity of two nitroheterocyclic compounds (NHCD), Nitracrine, 1-nitro-9(3'3'-dimethylaminopropylamino) acridine (Polfa, Poland) and Quinifuryl, 2-(5'-nitro-2'-furanyl) ethenyl-4-[N-[4-(N,N-diethylamino)-1'-methylbutyl] carbamoyl] quinoline (Dr. N. M. Sukhova, Institute of Organic Synthesis, Riga, Latvian Republic), towards two lines of leukaemic cells and a line of non-transformed cells, was determined under normoxia conditions. Although both drugs showed significant cytotoxicity to all cell lines (LC(50) for 24h, < or = 2 microM) with that of Nitracrine exceeding Quinifuryl, their toxicity towards murine leukaemia P388 was substantially higher, compared to murine fibroblasts NIH3T3. In addition, the rate of cell death was also two- to three-fold higher in case of P388 cells versus NIH3T3. Interestingly, human erythroleukaemia K562 cells were shown to uptake the drugs 10 min after their addition to the tissue culture medium, while the LC(50) values were reached after a substantial delay of 3h. This delay might be due to the intracellular transformation of drugs required for cell killing.

TZT-1027 (Soblidotin), an antimicrotubule agent, has been demonstrated to show potent antitumor effects, though the relationships among antitumor effect, cytotoxicity and anti-vascular effect of TZT-1027 have not been studied. We established in vivo human lung vascular-rich tumor models using a vascular endothelial growth factor-secreting tumor (SBC-3/VEGF). SBC-3/VEGF tumors exhibited a high degree of angiogenesis in comparison with the mock transfectant (SBC-3/Neo) tumors in a dorsal skinfold chamber model and grew much faster and larger than SBC-3/Neo tumors in the tumor growth study. The antitumor activity of antimicrotubule agents, including TZT-1027, was evaluated in both early- and advanced-stage SBC-3/Neo and SBC-3/VEGF tumor models to elucidate the relationship between the antitumor activity and anti-vascular effect of these agents. TZT-1027 exhibited potent antitumor activity against both early- and advanced-stage SBC-3/Neo and SBC-3/VEGF tumors, whereas combretastatin A4 phosphate did not. Vincristine and docetaxel exhibited potent antitumor activity against early-stage SBC-3/Neo and SBC-3/VEGF tumors, and advanced-stage SBC-3/Neo tumors, but did not exhibit activity against advanced-stage SBC-3/VEGF tumors. The difference in antitumor activity between these agents could be ascribed to differences in direct cytotoxicity and anti-vascular effect. Furthermore, a prominent accumulation of erythrocytes in the tumor vasculature, followed by leakage and scattering of these erythrocytes from the tumor vasculature, was observed after TZT-1027 administration to mice bearing advanced-stage SBC-3/VEGF tumors. These findings strongly suggest that TZT-1027 has a potent anti-vascular effect, in addition to direct cytotoxicity.

The aim of this study was to investigate the in vivo ability of O/W cationic emulsions to deliver oligonucleotides (ON) in leukemic P388/ADR cells in ascite, after intraperitoneal (IP) administration in mice. Cationic emulsions were prepared by microfluidization as previously described by Teixeira et al. [Pharm. Res 16 (1999) 30]. The formulations consisted mainly of medium chain triglycerides, phosphatidylcholine (PC), poloxamer, and either a monocationic lipid stearylamine (PC/SA-emulsion) or a polycationic lipid RPRC(18) (PC/RPRC(18)-emulsion). A model ON (33P-pdT(16)) was associated with cationic emulsions by single addition at the end of the manufacturing process. Seven days after P388/ADR inoculation IP to mice, ON free or associated with PC/SA or PC/RPRC(18) emulsions was injected IP at a dose of 0.5 mg/kg. At different interval times, ascite including cells, blood and the main organs were collected and the radioactivity counted by liquid scintillation. The overall results showed significantly high amounts of ON in the leukemic cell pellet, 24 h after administration of ON associated to either PC/SA (AUC(0-24 h)=13634, %injected dose/min) or PC/RPRC(18) (AUC(0-24 h)=22592, % injected dose/min), contrary to the free ON solution (AUC(0-24 h)=3095, %injected dose/min), which displayed only reduced capture by cancer cells. In conclusion, complexation of ON with cationic emulsions had a beneficial effect in increasing tumor cells uptake in vivo (up to sevenfold for PC/RPRC(18)-emulsion) after IP administration. This could open interesting prospects for the treatment of ovarian cancers.

alpha-Hederin, a pentacyclic triterpene saponin isolated from the seeds of Nigella sativa, was recently reported to have potent in vivo antitumor activity against LL/2 (Lewis Lung carcinoma) in BDF1 mice. In this study we observed that alpha-hederin caused a dose- and time-dependent increase in apoptosis of murine leukemia P388 cells. In order to evaluate the possible mechanisms for apoptosis, the effects of alpha-hederin on intracellular thiol concentration, including reduced glutathione (GSH), and protein thiols, and the effects of pretreatment with N-acetlycysteine (NAC), a precursor of intracellular GSH synthesis, or buthionine sulfoxime (BSO), a specific inhibitor of intracellular GSH synthesis, on alpha-hederin-induced apoptosis were investigated. It was found that alpha-hederin rapidly depleted intracellular GSH and protein thiols prior to the occurrence of apoptosis. NAC significantly alleviated alpha-hederin-induced apoptosis, while BSO augmented alpha-hederin-induced apoptosis significantly. The depletion of cellular thiols observed after alpha-hederin treatment caused disruption of mitochondrial membrane potential (deltapsi(m)) and subsequently increased the production of reactive oxygen species (ROS) in P388 cells at an early time point. Bongkrekic acid (BA), a ligand of the mitochondrial adenine nucleotide translocator, and cyclosporin (CsA) attenuated the alpha-hederin-induced loss of deltapsi(m), and ROS production. Thus, oxidative stress after alpha-hederin treatment is an important event in alpha-hederin-induced apoptosis. As observed in this study, permeability transition of mitochondrial membrane occurs after depletion of GSH and precedes a state of reactive oxygen species (ROS) generation. Further, we observed that alpha-hederin caused the release of cytochrome c from the mitochondria to cytosol, leading to caspase-3 activation. Our findings thus demonstrate that changes in intracellular thiols and redox status leading to perturbance of mitochondrial functions are important components in the mechanism of alpha-hederin-induced cell death.

1. The present study found that, compared with mouse heart and liver, P388 ascitic tumour had significantly lower superoxide dismutase (SOD) activity and that compared with the mouse liver, the heart had significantly lower SOD and catalase activities, as well as a lower glutathione content. 2. At 7.5 mg/kg, doxorubicin (DOX), a superoxide radical inducer, induced significant lipid peroxidation only in the tumour, whereas 15.0 mg/kg DOX induced lipid peroxidation in both the tumour and heart, but not in the liver. 3. Overall, the results of the present study suggest that the differential anti-oxidant activities in P388 ascitic tumour, heart and liver in mice may explain their differential responses and, hence, susceptibility to DOX-induced lipid peroxidation.

We studied the effect of oxidative stress induced by hyperoxia, hydrogen peroxide, or menadione on mouse leukemia P388 cells at early (4 days) and late (7 days) stages of tumor growth. Oxidative stress proved to inhibit cell division and to induce apoptosis. Seven-day leukemia cells feature lower proliferative potential and higher sensitivity to oxidative stress and platidiam.

This study was undertaken to compare the effect of glucose injection on the pharmacokinetic behavior of a soluble dye in normal and tumoral tissues. The measurements were done using a noninvasive fluorescent spectroscopy in situ and in real time. The experiments were performed on three groups of animals with calcein as a soluble pH-insensitive fluorescent dye combined or not with glucose. Glucose solution was injected 5 or 30 min before calcein. Fluorescence emission intensity was recorded on normal and tumor tissues with an optical multichannel analyzer. Calcein concentration was also measured in blood using repetitive blood sampling. In the control group (without glucose injection), calcein is rapidly cleared from the blood, with a slow tissue clearance. Fluorescence of normal tissue was higher than fluorescence measured in tumor tissue. When glucose is injected 5 min before calcein, there was a rapid increase of tissue fluorescence followed by a plateau remaining during the whole experiment. No difference between tumor and normal tissue fluorescence intensity was observed. When glucose was injected 30 min before calcein, the plateau phase was reduced to 50 min in normal tissue. Tumor tissue fluorescence displays no distinct plateau phase. These results clearly showed the effect of glucose injection in situ and in real time, by a noninvasive method, on the pharmacokinetic of a soluble dye in a tumor tissue compared to a normal tissue. Differences between blood compartment and tissues kinetic profiles were also clearly demonstrated.

It was reported that xanthine derivatives (caffeine and 1-methyl-3-propyl-7-butylxanthine) enhanced the antitumor activity of doxorubicin (DOX) with increasing DOX concentrations in tumors in vivo in our previous papers. In addition, these actions were found to be related to the inhibitory activity toward DOX efflux from tumor cells in vitro. In this study, we searched for novel biochemical modulators of DOX among 3-n-propylxanthines with functional groups at the 1- or 7-position by using an assay system for their inhibitory effect on DOX efflux from P388 leukemia and DOX resistant P388 leukemia (P388/DOX) cells. 1-Substituted xanthines facilitated the DOX efflux from P388 cells. In contrast, among 7-substituted xanthines, XT-141 and XT-139 significantly inhibited the DOX efflux from P388 cells. In addition, XT-141 inhibited the DOX efflux from P388/DOX cells, and P-glycoprotein (P-gp) inhibitor facilitated DOX influx and inhibited DOX efflux from P388/DOX cells in a dose-dependent manner. These results indicated that the resistance of P388/DOX might depend on the over-expression of P-gp, and that XT-141 inhibited DOX efflux through its interaction with P-gp. We suspect that XT-141 is a useful biochemical modulator of DOX in DOX-resistant tumors with over-expression of P-gp in addition in DOX-sensitive tumors.

Extracts of the dorid nudibranch Asteronotus cespitosus from two geographically separate regions of Australia and from the Philippines were compared using thin-layer, high-performance liquid and gas chromatography and 1H NMR analysis. Halogenated metabolites were detected in all mollusk specimens. The major component detected in digestive tissue of specimens from the Great Barrier Reef in northeastern Australia was 4,6-dibromo-2-(2'.4'-dibromophenoxy)phenol (1), with minor amounts of 3,5-dibromo-2-(3',5'-dibromo-2'-methoxyphenoxy)phenol (2). In a specimen collected from northwestern Australia, only 3,5-dibromo-2-(3',5'-dibromo-2'-methoxyphenoxy)phenol was found. The specimen from the Philippines contained 2,3,4,5-tetrabromo-6-(2'-bromophenoxy)phenol (3) together with a novel chlorinated pyrrolidone (4). In addition, the sesquiterpenes dehydroherbadysidolide (5) and spirodysin (6) were detected in the digestive organs and mantle tissue of the nudibranchs from the Great Barrier Reef and from the Philippines, whereas these chemicals were not found in the specimen from northwestern Australia. All of the chemicals (1-3,5, and 6) have previously been isolated from the sponge Dysidea herbacea, as have chlorinated metabolites related to 4. This is the first time the characteristic halogenated metabolites that typify Dysidea herbacea have been reported from a carnivorous mollusk, which implies a dietary origin as opposed to de novo synthesis.

The sialic acid binding lectin from bullfrog oocytes (cSBL) is known to have anti-tumor activity. In a previous report, to elucidate the relationship between the net charge and anti-tumor activity of cSBL, we examined the effect of chemical modifications of cSBL with a water-soluble carbodiimide in the presence of various nucleophiles. The results suggested that the anti-tumor activity and internalization into tumor cells increased with an increase in the net charge of cSBL. However, in the chemically modified cSBL, a modification site was observed on average in two of the carboxyl groups of cSBL. To confirm these previous results and to determine which modified carboxyl group contributes to the increase in anti-tumor activity, we prepared mutants with substitutions of Asn/Gln and Arg at three acidic amino acid residues of cSBL and studied their anti-tumor activity and internalization efficiency. The results showed the enhancing effect of charge on anti-tumor activity and internalization, and suggested that the replacement of D24 and E88 of cSBL with arginine is more effective than that of E97. The double mutant D24RE88R showed comparable anti-tumor activity to the ethylenediamine-modified cSBL reported previously. The mutant was well-characterized as a pure cSBL derivative suitable for studying the mechanism of the anti-tumor action of cSBL.

When ginsenoside R(c) was anaerobically incubated with human fecal microflora, all specimens metabolized ginsenoside R(c) to compound K and protopanaxadiol. The main metabolite was compound K. Among the bacteria isolated from human fecal microflora, most bacteria, such as Bacteroides sp., Eubacterium sp., and Bifidobacterium sp. potently transformed ginsenoside R(c) to compound K. Bifidobacterium K-103 and Eubacterium A-44 transformed it to compound K via ginsenoside R(d) and Bacteroides HJ-15 and Bifidobacterium K-506 metabolized to compound K via ginsenoside Mb, which was isolated as a new metabolite (M.W. 940[+Na]). Among ginsenoside R(c) and its metabolites, compound K exhibited the most potent antiallergic activity on the IgE-induced RBL cell line as well as potent cytotoxic activity against tumor cell lines.

Although the ability of chemosensitizers to modulate P-glycoprotein (PGP)-based multidrug resistance (MDR) has been extensively studied, relatively little is known about the cellular pharmacology of the PGP inhibitors themselves in MDR cells. The studies described here have correlated the in vitro accumulation and retention properties of verapamil (VRP) in murine P388 (sensitive) and P388/ADR (MDR) cells with doxorubicin (DOX) uptake and cytotoxicity modulation characteristics in order to better understand VRP-tumor cell interactions that give rise to MDR modulation. VRP is rapidly taken up by DOX-sensitive and -resistant P388 cells where greater than 50% maximal VRP uptake occurs within 10 min of initial exposure at 37 degrees C. Whereas chemosensitization and DOX uptake in P388/ADR cells increase with increasing VRP concentration until a plateau is achieved at approximately 5 microM VRP, cellular modulator levels increase proportionally with increasing VPR concentrations beyond 20 microM. Subsequent to removal of noncell-associated modulator, VRP levels in both sensitive and resistant cells rapidly fall below 10% of those obtained at uptake equilibrium. However, a residual amount of VRP remains associated with the cells for extended time periods after the cells are washed. Pulse exposures of P388/ADR cells to high concentrations of VRP (50-100 microM) are capable of providing extended cell-associated VRP levels comparable to those obtained with continuous exposure at biologically active VRP concentrations (1-3 microM) and this leads to chemosensitization. These results are consistent with the existence of high- and low-affinity intracellular VRP pools in P388 MDR cells, both of which can contribute to the reversal of drug resistance. It is suggested that these properties should be taken into consideration during the design and evaluation of preclinical in vivo MDR models where pulsed exposure to high concentrations of resistance modulators often occurs. Special attention must be given to whether such high concentration pulses are desirable and/or achievable in relevant clinical settings.

MR1 is a major histocompatibility complex (MHC) class I-related gene conserved among mammals, and its predicted amino acid sequence is relatively closer to the classical MHC class I molecules among several divergent class I molecules. However, as its molecular nature and function have not yet been clarified, we set out in this study to establish transfected P388 murine cell lines that stably produce a large number of MR1 proteins and conducted analyses to investigate the molecular nature of MR1. Immunoprecipitation and Western blot analyses with specific antisera revealed that the MR1 protein can associate with beta(2)-microglobulin, suggesting its molecular form of a typical class I heterodimer composed of a heavy and a light chain (beta(2)-microglobulin), like the classical MHC class I molecules.

Specific phosphorylation of serine- and arginine-rich pre-mRNA splicing factors (SR proteins) is one of the key determinants regulating splicing events. Several kinases involved in SR protein phosphorylation have been identified and characterized, among which human DNA topoisomerase I is known to have DNA-relaxing activity. In this study, we have investigated the mechanism of splicing inhibition by a glycosylated indolocarbazole derivative (NB-506), a potent inhibitor of both kinase and relaxing activities of topoisomerase I. NB-506 completely inhibits the capacity of topoisomerase I to phosphorylate, in vitro, the human splicing factor 2/alternative splicing factor (SF2/ASF). This inhibition is specific, because NB-506 does not demonstrate activity against other kinases known to phosphorylate SF2/ASF such as SR protein kinase 1 and cdc2 kinase. Importantly, HeLa nuclear extracts competent in splicing but not splicing-deficient cytoplasmic S100 extracts treated with the drug fail to phosphorylate SF2/ASF and to support splicing of pre-mRNA substrates containing SF2/ASF-target sequences. Native gel analysis of splicing complexes revealed that the drug affects the formation of the spliceosome, a dynamic ribonucleoprotein structure where splicing takes place. In the presence of the drug, neither pre-spliceosome nor spliceosome is formed, demonstrating that splicing inhibition occurs at early steps of spliceosome assembly. Splicing inhibition can be relieved by adding phosphorylated SF2/ASF, showing that extracts treated with NB-506 lack a phosphorylating activity required for splicing. Moreover, NB-506 has a cytotoxic effect on murine P388 leukemia cells but not on P388CPT5 camptothecin-resistant cells that carry two point mutations in conserved regions of topoisomerase I gene (Gly361Val and Asp709Tyr). After drug treatment, P388 cells accumulated hypophosphorylated forms of SR proteins and polyadenylated RNA in the nucleus. In contrast, neither SR protein phosphorylation nor polyadenylated mRNA distribution was affected in P388 CPT5-treated cells. Consistently, NB506 treatment altered the mRNA levels and/or splicing pattern of several tested genes (Bcl-X, CD 44, SC35, and Sty) in P388 cells but not in P388 CPT5 cells. The study shows for the first time that indolocarbazole drugs targeting topoisomerase I can affect gene expression by modulating pre-mRNA splicing through inhibition of SR proteins phosphorylation.

The sialic acid binding lectin from bullfrog Rana catesbeiana oocyte (cSBL) is known to have anti-tumor activity. In order to investigate the relationship between the net charge of cSBL and its anti-tumor effect, cSBL was modified with a water-soluble carbodiimide (EDC) in the presence of three kinds of nucleophiles, taurine, glycine methylester and ethylenediamine. cSBL having four carboxyl groups was partially modified (ca. 2 residues). The anti-tumor activity of modified cSBLs was in the order of ethylenediamine-modified cSBL > glycine methylester-modified cSBL > taurine modified cSBL > or = native cSBL. The results suggested that anti-tumor activity seems to increase with the increase in positive net charge, possibly enhancing the interaction of cSBL with sialoglycoprotein on the surface of tumor cells. The ribonuclease activity of ethylenediamine-modified cSBL decreased with the progress of the reaction, but the number of internalized molecules in the tumor cell increased. Thus, for antitumor activity, a higher incorporation of cSBL with reasonable RNase activity seems to be more important than total RNase activity.

Acquired drug resistance is a major factor in the failure of doxorubicin-based cancer chemotherapy. We determined the ability of cinchonine to reverse doxorubicin drug resistance in a doxorubicin-resistant leukaemia cell line (mouse P388/DOX). A non-cytotoxic concentration of cinchonine (10 microM) increased the sensitivity to doxorubicin of multidrug-resistant P388/DOX cells and significantly enhanced the doxorubicin-induced apoptosis and DNA fragmentation in resistant cells, but had no effect in parent cells. Time-course studies demonstrated that DNA fragmentation was present 24 h after incubation with doxorubicin and cinchonine, indicating that DNA degradation was a preceding event. In cultured cells, cinchonine increased the intracellular accumulation of doxorubicin in the resistant cells in a dose-dependent manner. Using flow cytometry to measure the inhibition of the P-glycoprotein (P-gp) dependent efflux of rhodamine 123, cinchonine was found to be considerably more effective than quinine. The results with cinchonine suggest that there may be quinine derivatives with a similar capacity to inhibit drug transport by P-gp. Additionally, the G2/M phase cell population in resistant cells is increased by doxorubicin/cinchonine treatment. Exposure of resistant cells to 1 microM doxorubicin and 10 microM cinchonine resulted in the expression of Fas (APO-1/CD95) in cells after 6 h. These studies demonstrate that the cell killing effects of doxorubicin and cinchonine in resistant cells

2-Etheny1-2,3-dihydrophthalazine-1,4-diones were successfully synthesized and proved to be effective cytotoxic agents against the growth of suspended murine and human leukemias and lymphomas. Selected compounds were also active in human HeLa uterine carcinoma, suspended effusion breast MCF-7 and glioma HS683 screens. These agents suppressed P388 lymphocytic leukemia DNA synthesis after 60 min at 100 microM. Their target appeared to be the de novo synthesis pathway with significant inhibition of the activities of both regulatory enzymes of the pathway, i.e. PRPP-amide transferase and IMP dehydrogenase resulting in a reduction in the d[NTP] pool levels for DNA incorporation. The compounds did not affect de novo pyrimidine synthesis and its regulatory enzymes. Very minor reduction by the agents was noted for the nucleoside kinases and the DNA and RNA polymerase activities within 60 min. DNA was not a target of the agents in that there was no alkylation of the nucleotide bases, intercalation between base pairs or cross-linking of the DNA strands; however, the agents did cause P388 DNA strand scission after 24 h at 100 microM.

Aragusterol A (YTA0040), isolated from the Okinawan marine sponge of the genus Xestospongia, is a potent anti-tumor marine steroid that possesses a unique structural component. This compound showed broad-spectrum anti-proliferative activity against a panel of 14 human cancer cell lines (IC(50) = 0.01-1.6 microM). P-glycoprotein-mediated, multidrug-resistant cells showed cross-resistance to YTA0040 cells, whereas cisplatin-resistant non-small-cell lung-cancer (NSCLC) sublines showed a collateral sensitivity to YTA0040. In transplantable murine tumor models, YTA0040 displayed a broad spectrum and high degree of anti-tumor activity when administered i.p. or p.o. (life span T/C = 135-234%). In P388 murine leukemia cells, YTA0040 caused dose- and time-dependent suppression of nucleic acid and protein synthesis, with protein synthesis being more potently and rapidly inhibited than nucleic acid synthesis. Flow-cytometric analysis revealed that YTA0040 blocked the entry of human NSCLC-derived A549 cells into S phase, leading to arrest in the G(1) phase of the cell cycle. Western blot analysis demonstrated that YTA0040 caused a dose-dependent decrease in the levels of expression of hyperphosphorylated pRb and cyclin A in A549 cells. The level of p53 protein expression was decreased by YTA0040 treatment. A higher concentration of YTA0040 down-regulated the levels of expression of CDK2, CDK4, cyclin D1 and cyclin E. These findings indicated that YTA0040 arrested human NSCLC cells in late G(1) phase of the cell cycle through inhibition of pRb phosphorylation. Inhibition of pRb phosphorylation by YTA0040 resulted from down-regulation of levels of expression of the CDKs and cyclins involved in the G(1)/S transition and not from induction of p53 and/or the CDK inhibitor p21., (Copyright 2000 Wiley-Liss, Inc.)

P-glycoprotein (P-gp) and multidrug resistance related protein (MRP) overexpression is often responsible of the development of multidrug resistance in cancer therapy. These proteins are also expressed in normal tissues, where their physiological role is related to the extrusion of endogenous toxins or to secretory function in liver and kidney. The LLC-PK1 cell line is derived from normal pig proximal renal tubule and physiologically expresses low levels of P-gp and MRP. A resistant cell line (LLC-PK1/ADR) has been established in our laboratory by chronic exposure to increasing doses of doxorubicin. Cytofluorimetric analysis of P-gp and MRP expression performed by C219 and MRPm6 immunofluorescence detection showed that these cells overexpress P-gp but not MRP. The uptake of doxorubicin and rhodamine 123 has been quantified in LLC-PK1 and LLC-PK1/ADR cells and compared with data obtained using other tumor cell lines commonly used as reference for studying P-gp or MRP overexpression. P388 sensitive cells and its resistant counterpart P388/ADR cells, which overexpress P-gp and PANC-1 cells, which express high levels of MRP were used. A lower fluorescence intensity was evident with both doxorubicin and rhodamine 123 in LLC-PK1/ADR as well as in P388/ADR cells, that overexpresses P-gp, in comparison with the parental lines. The uptake was increased by a pretreatment with verapamil. Verapamil was completely ineffective on PANC-1 cells, confirming a selective effect of this inhibitor on P-gp. Propidium iodide staining, performed after doxorubicin treatment, confirmed a higher cytotoxicity of the antineoplastic drug in the LLC-PK1 cells compared with the resistant counterpart.

1. The combined effects of the macrolide antibiotics erythromycin, josamycin, clarithromycin and YM17K (3,4'-dideoxy mycaminosyl tylonolide hydrochloride) on in vitro intracellular accumulation of vinblastine or cyclosporine (Cs)A and on the in vivo antitumour activity of vinblastine were investigated using mouse leukaemia P388 cells (P388/S) and anticancer drug-resistant (P388/ADR) cells. These effects were compared with those of a calcium antagonist (verapamil) or immunosuppressants (FK506 and CsA). 2. All tested macrolide antibiotics increased the accumulation of both vinblastine and CsA in P388/ADR cells in a dose-dependent manner, but their potency was lower than that of verapamil, CsA or FK506. 3. When vinblastine (200 microg/kg) was administered intraperitoneally with each of the macrolide antibiotics (10 or 100 mg/kg) or with verapamil (25 mg/kg) once a day for 10 days in P388/ADR-bearing mice, combined effects of vinblastine with the macrolide antibiotics (erythromycin, clarithromycin and YM17K) or verapamil were observed. 4. The present study suggests that macrolide antibiotics may overcome anticancer drug resistance by inhibiting the binding of vinblastine or CsA to P-glycoprotein in P388/ADR cells. 5. We believe that these results are encouraging for combination chemotherapy to overcome P-glycoprotein-dependent anticancer drug-resistant tumours in clinical practice.

The effects of 1-methyl-3-propyl-7-butylxanthine (MPBX), a xanthine derivative, on idarubicin (IDA)-induced antitumor activity against P388 leukemia cells (P388) and bone marrow suppression were examined. In P388 tumor-bearing mice, the combination of MPBX with IDA increased the antitumor activity of IDA. The IDA concentration in the tumors in the MPBX combination group increased by 2.0-fold compared to the level in the IDA-alone group. On the other hand, as regards IDA-induced bone marrow suppression, the combination of MPBX with IDA reduced the decrease in the bone marrow cell number by 30% compared to that in the IDA-alone group. In addition, the IDA concentration in the bone marrow cells was decreased by the combination of MPBX with IDA. An in vitro experiment showed that MPBX facilitated IDA influx and suppressed IDA efflux in P388 cells. In conclusion, the combination of MPBX with IDA increased the antitumor activity and decreased the bone marrow suppression. Therefore, we expect that the combination of MPBX with IDA will be useful for leukemia chemotherapy.

A new cytotoxic cembrenolide diterpene, lobocrassolide (1), and a known cytotoxic cembrenolide, lobohedleolide (2), were isolated from the Formosan soft coral Lobophytum crassum. The structure of compound 1 was determined by 1D and 2D spectral analysis.

A new cytotoxic lobane diterpene, ineleganene (1), was isolated from the Formosan soft coral Sinularia inelegans. The structure of compound 1 was determined by 1D and 2D spectral analysis.

6-2-(Dimethylamino)ethylămino-3-hydroxy-7H-indeno2, 1-cquinolin-7-one dihydrochloride (TAS-103) is a novel anticancer agent that was developed to target both topoisomerase (Topo) I and Topo II. To elucidate its mechanism of action, we have established and characterized TAS-103-resistant cells, derived from mouse leukemia (P388), human colon cancer (DLD-1), and human lung adenocarcinoma (A549) cell lines, by exposure to stepwisely increasing concentrations of TAS-103 in the culture medium. P388 / TAS cells showed only cross-resistance to VP-16 and adriamycin (ADR). The Topo II activity in these cells was decreased to below one-fourth of that in the parental cells, while the Topo I activity remained unchanged. DLD / TAS cells appeared to be cross-resistant to VP-16, ADR, camptothecin (CPT), SN-38 and vincristine (VCR). The enzymatic activities of both Topo I and Topo II in these cells were decreased to one-fourth of that observed in the parental cells. Furthermore, the decreased activities were accompanied by lower expression at the mRNA and protein levels. A549 / TAS cells acquired cross-resistance to VP-16, ADR and VCR, though the Topo activities were virtually unchanged. In this cell line, the intracellular accumulation of TAS-103 was significantly decreased and the expression of multidrug resistance associated protein (MRP) was elevated when compared with the parental cells. The results indicate that the affected activities of Topo I and / or Topo II, and in some instances decreased accumulation of TAS-103, are associated with the development of resistance to TAS-103, although the main mechanism of resistance to TAS-103 varied among cell lines.

Considering of novel biochemical modulation by some foods and beverages, we have performed screening for green tea components that have enhancing effects on doxorubicin (DOX) induced antitumor activity. Components, such as caffeine, theanine, (-)-epigallocatechin gallate (EGCG) and flavonoids have inhibitory effects on the DOX efflux from Ehrlich ascites carcinoma cells. Thus, it is suggested that EGCG and flavonoids may enhance DOX induced antitumor activity and increase the DOX concentrations in tumors through the inhibition of DOX efflux. It is expected that these components in green tea exhibit low toxicity and that there are few side effects of drinking green tea in combination with an antitumor agent. We think that the intake of a favorite beverage favors a positive mental attitude of a patient and increases the efficacy of the chemotherapeutic index, and that this efficacy is useful for improving the quality of life on cancer chemotherapy. In DOX resistant P388 leukemia cell bearing mice theanine increased the DOX induced efficacy through an increase in the DOX concentrations in the tumors. Theanine attacked the same transport process for DOX in both types of cells, elevated the DOX concentration and increased the DOX induced antitumor activity.

Leukemia cells (HL-60 and P388) treated with the topoisomerase I inhibitor camptothecin (CPT) undergo rapid apoptosis as judged from internucleosomal degradation of genomic DNA, morphological changes and flow cytometry analysis. The intracellular free calcium concentration is not affected by the treatment with a high dose of CPT. In contrast, fluorescence measurements of cells loaded with the pH indicator BCECF-AM indicate that the intracellular pH decreases significantly. Incubation of the leukemia cells with a high drug concentration for 5 h or with lower drug concentrations for 15 h results in a pronounced intracellular acidification. Measurements with the whole cell population show a decrease of 0.3-0.4 pH units. The extent of the acidic shift is proportional to the drug concentration and the period of incubation. No such effects were observed with P388CPT5 cells resistant to CPT. The results support the hypothesis that apoptosis induced in leukemia cells by CPT is associated with decreased intracellular pH. Modification of intracellular pH by topoisomerase inhibitors is viewed as an essential event responsible for the induction and/or propagation of apoptosis. The role of CPT-induced cellular acidification in the mechanism of action of the drug is discussed.