M5076 Ovarian Sarcoma Research Articles

Effect of Liposomes with Different Double Arms Polyethyleneglycol on Hepatic Metastasis Model Mice and Evaluation Using a Fluorescent Imaging Device.

We have previously reported the synthesis of a novel polyethyleneglycol (PEG) lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (DDA-PEG). This study aimed to clarify the anti-metastatic effect and localization of DDA-PEG-modified liposomes on a murine hepatic metastasis model. M5076 ovarian sarcoma cells were inoculated for hepatic metastasis model mice. The accumulation of liposomes in the tumor and metastatic sites was detected by fluorescent imaging device. In metastasis study, doxorubicin (DOX) loaded DDA-PEG-modified liposome (DDA-LDOX) was injected. Alexa Fluor 790 NHS Ester loaded DDA-PEG-modified liposomes were used to detect fluorescence intensity at metastatic sites when visualized topically using a fluorescence imaging device. DDA-PEG-modified liposomes accumulated at the sites of hepatic metastasis but not in the normal hepatocytes. Furthermore, the DDA-LDOX inhibited metastasis in this model. The survival time of M5076 ovarian sarcoma bearing mice in DDA-LDOX group was longer than those in control, DOX solution and the other PEG-modified liposomal DOX groups, and the survival ratio in DDALDOX group remained 66.7% until 60 days after treatment. It is expected that the DDA-PEG-modified liposomes will extensively contribute in clinical practice as a superior drug carrier because this liposomes proved to be effective against metastasis.

Abstract 2139: The effects of cyclodipeptide on the transport of doxorubicin and its cytotoxicity on tumor cell

Abstract Purpose: Chemotherapy using antitumor agents plays an important role in clinical cancer therapy. Among the treatments involving antitumor agents, the enhancement of antitumor activity was observed by combined chemotherapy. We have shown previously that some amino acids included theanine, taurine and anserine et al. as food components, increased doxorubicin (DOX) induced antitumor effect in vitro and in vivo. These effects by combined amino acids have depended on the increased DOX concentration in the tumor cells. Furthermore, these suppressions of DOX efflux by theanine and taurine were caused by the inhibitions of glutamate transporter and taurine transporter, respectively. The effect of anserine on DOX influx might connect with dipeptide transporter. Thus, other amino acid and unique peptides may act to DOX transport. In this study, the effects of cyclodipeptide on the transport of DOX and its cytotoxicity on tumor cell were clarified. Methods: Cyclo-leucine-proline (cyclo-Leu-Pro), cyclo-phenylalanine-proline (cyclo-Phe-Pro) and cyclo-glycine-proline (cyclo-Gly-Pro) as cyclodipeptide was used in this study. In transport of DOX, DOX influx or efflux was determined with combined cyclodipeptide in M5076 ovarian sarcoma cells or P388 leukemia cells. DOX concentration in tumor cell was measured using spectrofluorometer (Ex. 470 nm, Em. 580 nm). The cytotoxic study of DOX with combined cyclodipeptide was performed using WST-8. Results and Discussion: Cyclo-Phe-Pro and cyclo-Gly-Pro did not affect DOX influx and efflux in M5076 ovarian sarcoma cells and P388 leukemia cells. By combined cyclo-Leu-Pro, DOX influx into both M5076 ovarian sarcoma cells and P388 leukemia cells changed, compared to that of DOX only group. Into M5076 ovarian sarcoma, cyclo-Leu-Pro had strong effect in increased DOX influx in particular. And cyclo-Leu-Pro showed a tendency to suppress DOX efflux from M5076 ovarian sarcoma. In this studies of influx and efflux, concentration of cyclo-Leu-Pro existed optimum amount for increased DOX concentration in tumor cells. It is expected that cyclo-Leu-Pro induces the increment of DOX concentration in the tumor in vivo. DOX had the cytotoxic effects on M5076 ovarian sarcoma cells or P388 leukemia cells in culture. The combined cyclo-Leu-Pro with DOX increased cytotoxic effect, compared to that in the DOX alone group. In conclusion, cyclo-Leu-Pro has increased effect on DOX influx and inhibited effect on DOX efflux, and DOX induced cytotoxicity. It is expected that the combined cyclo-Leu-Pro will improve cancer chemotherapy by DOX. Moreover, cyclo-Leu-Pro may be help to prevent adverse effect since it will be able to decrease DOX dose. Citation Format: Ikumi Sugiyama, Yasuyuki Sadzuka. The effects of cyclodipeptide on the transport of doxorubicin and its cytotoxicity on tumor cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2139. doi:10.1158/1538-7445.AM2017-2139

Abstract 178: Stromal expression of long Pentraxin-3 impairs tumor growth and metastasis

Abstract Long Pentraxin-3 (PTX3) is a soluble pattern recognition receptor expressed by endothelial and immune cells in inflammatory contexts. We have previously demonstrated that PTX3 binds to different members of the FGF family, thus inhibiting their biological activity. The FGF/FGFR system strongly contributes to cancer progression by inducing tumor growth and neovascularization. To date, recombinant PTX3 protein or PTX3-overexpressing tumor cell lines have been exploited to assess the antitumor effects of this natural FGF trap. Here we generated C57BL/6 transgenic mice expressing human (h)PTX3 under the control of the endothelial specific Tie2/Tek transcription regulatory sequences. These animals were used to investigate the impact of PTX3 overexpression by the host stroma on tumor growth, vascularization and metastasis. Transgenic Tie2-hPTX3 mice were generated by cloning the hPTX3 cDNA into the late-generation, self-inactivating lentiviral vector Tie2p/e to obtain the Tie2-hPTX3 lentiviral transfer vectors that were injected into fertilized oocytes. Expression of the transgene was confirmed by RT-PCR and western blot analyses of different organs from Tie2-hPTX3 mice that showed increased levels of circulating PTX3 (80-180 ng/ml) when compared to wild type (wt) animals (<1.8 ng/ml). Also, histological analysis confirmed the perivascular accumulation of hPTX3 in transgenic animals. To assess the anti-angiogenic activity of endothelium-derived hPTX3, we performed ex vivo aorta ring and in vivo matrigel plug assays. Both assays revealed a significant inhibition of FGF2-driven angiogenesis in Tie2-hPTX3 mice that maintained their responsiveness to VEGF-A, thus confirming the specificity of the effect. Next, different syngeneic FGF-dependent tumor cell lines, including TRAMP-C2 prostate carcinoma, B16-F10 melanoma and Lewis Lung carcinoma cells, were subcutaneously injected in Tie2-hPTX3 mice. Notably, the growth of all tumor grafts was significantly reduced in Tie2-hPTX3 mice when compared to wt animals. Also, histological analysis of TRAMP-C2 tumors grown in Tie2-hPTX3 mice showed a strong perivascular expression of hPTX3 and a significant reduction of FGFR1 phosphorylation. This was paralleled by a significant decrease of tumor vascularity and tumor cell proliferation whereas no difference in tumor growth was observed for TRAMP-C2 grafts expressing a constitutively activated form of FGFR1. Finally, B16-F10 melanoma and M5076 ovarian sarcoma cells showed a dramatic decrease of their capacity to form experimental metastases in the lung and liver, respectively, after intravenous injection in Tie2-hPTX3 mice. PTX3 is a natural FGF ligand trap. Our findings demonstrate for the first time that the production of PTX3 by the host stroma may exert a dramatic impact on tumor growth, vascularization and metastasis with potential exploitation for the therapy of FGF-dependent tumors. Citation Format: Arianna Giacomini, Emanuela Di Salle, Daniela Coltrini, Mirella Belleri, Marco Presta, Roberto Ronca. Stromal expression of long Pentraxin-3 impairs tumor growth and metastasis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 178. doi:10.1158/1538-7445.AM2014-178

Abstract 5408: Liposomal doxorubicin with modified EGCG increased antitumor activity by topical targeting efficiency into tumor

Abstract Purpose: The aim of this study is increase of antitumor activity using novel liposome with (-)-epigallocatechin-3-gallate (EGCG) as one of the green tea components. Liposome has been known as a one of useful drug carriers for delivering drug into targeting site. Moreover, some ligand is able to modify around liposomal membrane by a simple method. On the other hand, it was reported that surface of high grade tumor cell expresses 67kDa laminin receptor (67LR) which intermediates of cytostatic effect of EGCG, and EGCG is able to induce apoptosis selectively. Our strategy was to prepare liposomal doxorubicin with EGCG as carrier ligand for getting antitumor activity by both apoptosis and doxorubicin at tumor site. In this study, we tried to prepare EGCG-modified liposome and evaluated the antitumor activity in vitro. Methods: The liposomal doxorubicin was prepared by L-α-distearoylphosphatidylcholine, cholesterol, L-α-distearoylphosphatidyl-DL-glycerol and doxorubicin (100:100:60:18 μmol). Doxorubicin included EGCG-modified liposome (EL-DOX) was prepared to add EGCG-dipalmitate. Amount of EGCG modification on liposomal membrane was determined using DPPH assay. On P388 leukemia cells and M5076 ovarian sarcoma cells, cytotoxicity was evaluated by WST-8 assay and shown IC50. DOX uptake into tumor cells was evaluated in vitro. The concentration of doxorubicin into tumor cells was determined with a fluorescence spectrophotometer at an excitation wavelength of 500 nm and an emission wavelength of 550 nm. Results and Discussion: Particle sizes of EL-DOX were 147 nm on average, zeta potential was -33.1 mV and encapsulated ratio of doxorubicin was 96.5 %. These data of sizes and zeta potentials were better for intravenous administration of liposomes. EGCG ratio on liposomal membrane (71.0 %) was enough to show its ability. In the study of doxorubicin uptake into tumor cells, EL-DOX group was increased amount of doxorubicin for 30 min. The doxorubicin level of EL-DOX was significantly higher compared with that of unmodified liposomal doxorubicin (p<0.05). At cytotoxicity, EL-DOX was stronger than unmodified liposomal doxorubicin. It was provided that IC50 of EL-DOX was also 125 times higher than EGCG solution which was reported to have been antitumor activity. Furthermore, co-incubation of EGCG solution and liposomal doxorubicin was not shown the enhancement effect both doxorubicin uptake into cells and the cytotoxicity. It was suggested that superior effect were shown in modification of EGCG around liposomal membrane, not mixture of EGCG and liposome. In conclusion, EL-DOX exhibited strong antitumor activity efficiently. Citation Format: Ikumi Sugiyama, Kunihiro Kaihatsu, Nobuo Kato, Yasuyuki Sadzuka. Liposomal doxorubicin with modified EGCG increased antitumor activity by topical targeting efficiency into tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5408. doi:10.1158/1538-7445.AM2014-5408

Abstract 4515: Improvement of tissue distribution by the treatment of novel different double-arms polyethyleneglycol modified liposome encapsulating doxorubicin.

Abstract [Purpose] Polyethyleneglycol (PEG) modification on liposomal membrane increases therapeutic index by improvement of targeting based on prolonged blood stream circulation. In contrast, some accumulation of drug after liposome treatment has also been observed in normal tissues, it is necessary to improve liposomal composition on the decrease of adverse reactions. Particularly, doxorubicin (DOX) encapsulated liposome has some adverse reactions as cardiac toxicity or hand-foot syndrome,etc. To contribute superior liposome formulation, different double arms PEG in a molecule(DDA-PEG) was synthesized and was clarified effectiveness in our previous reports. In this study, we examined physical properties of novel synthesized DDA-PEG, and the tissue distribution of the DOX encapsulated liposome with modification of these DDA-PEGs. [Methods] Two DDA-PEGs with (PEG1000 and PEG500) or (PEG2000 and PEG500) were synthesized. Critical micelle concentration (cmc) in 10 mM Tris-HCl buffer (pH7.4) and 10 mM lactate buffer (pH4.0) was measured by the method of florescent probe using the 8-anilinonaphthalene-1-sulfonate (ANS). Octanol-buffer partitioning value was determined. DOX encapsulated liposomes with the modification of each DDA-PEGs were prepared by DSPC/cholesterol/DSPG-Na/DOX/PEG-lipid =100:100:60:18:7.5 μmol according to the method of Bangham. M5076 ovarian sarcoma cells were transplanted into the backs of BDF1 mice, and each liposome was injected intravenously at a dose of 2.5 mg DOX/kg on 15, 18 and 21 day after tumor inoculation. The mice were sacrificed after 2 day from last administration, and the tumor was removed and weighted. Moreover, DOX concentrations in each tissue were determined. [Results and discussion] The cmc of all PEG-lipids were same level. In PEG(2000, 500)-modified liposome, DOX concentrations in tumors were equal to that in PEG2000-modified liposome. On the other hand, DOX concentration in the heart after PEG(2000, 500) modified liposome and PEG(1000,500)-modified liposome treatments were one second and one sixth compared with DOX solution group, respectively. According to these results, it was expected that novel DDA-PEG-modified liposome was able to reduce the cardiac toxicity which is adverse reaction by DOX. The DOX concentration in the liver was showed a low level by PEG (1000, 500) modified liposome. It was suggested that novel DDA- PEG-modified liposome had the effect of avoiding the uptake into the liver. In conclusion, it was suggested that PEG(1000,500) modified liposome could decrease the DOX level in heart, and had ability of accumulation into tumor and avoidance from reticuloendothelial cells as same as PEG2000-modified liposome as known liposome. Citation Format: Mai Yagi, Ikumi Sugiyama, Yasuyuki Sadzuka. Improvement of tissue distribution by the treatment of novel different double-arms polyethyleneglycol modified liposome encapsulating doxorubicin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4515. doi:10.1158/1538-7445.AM2013-4515

Enhanced antitumor activity of different double arms polyethyleneglycol-modified liposomal doxorubicin

The effect of polyethyleneglycol (PEG)-modified liposome as a drug carrier has been demonstrated clinically. We designed and synthesized a novel PEG-lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (different double arms PEG, DDA-PEG) which had two PEG chains of 500 and 2000 in one molecule to develop more useful PEG-modified liposome. DDA-PEG-modified liposomal doxorubicin (DDA-LDOX(7.5)) had the biggest fixed aqueous layer thickness (FALT) compared with other PEG-lipid-modified liposomes even if the added amount was a few. It was thought that FALT was the indication of blood circulation time. In DOX uptake in tumor cells, DDA-LDOX(7.5) group increased the DOX concentration in tumor cells because it had contact ability with tumor cells. Hence, DDA-LDOX(7.5) which has long circulation time in the bloodstream and contact ability with tumor cells, also had a strong antitumor effect on mice bearing M5076 ovarian sarcoma cells which were DOX low sensitive cells according to the expression of multidrug resistance protein. Furthermore, this liposome maintained a high DOX concentration in a tumor for a long time. These results indicated that the useful antitumor effect of DDA-LDOX(7.5) against M5076 ovarian sarcoma cells is a promising DDS carrier for therapies against drug resistant tumors.

Abstract 1961: Evaluated mechanism of dual targeting using liposomal doxorubicin-modified novel PEG by imaging analysis

Abstract [Purpose] Liposome is one of the useful drug carriers and it may be applying to a lot of diseases from now on. One of the most reasons which liposomes were using as medicine was improvement of circulation time in bloodstream by modifying polyethyleneglycol (PEG)-lipid. We synthesized novel PEG-lipid with two different PEG lengths of 2000 and 500 (different double arms-PEG; DDA-PEG) for developing more superior passive targeting liposome. In our previous study, doxorubicin (DOX) containing liposome which was modified DDA-PEG (DDA-PEG modified liposomal DOX; DDA-LDOX) had dual effect for primary and hepatic metastasis cancer. In this study, we observed tissue distribution at primary tumor using the near infrared fluorescence and analyzed hepatic metastasis by histological observation for explain this dual effect. Moreover, this effect for pulmonary metastasis was also evaluated. [Methods] Liposome (DSPC/cholesterol/DSPG-Na/DDA-PEG = 100:100:60:7.5 μmol) was prepared according to the method of Bangham. For observing tissue distribution of liposome at primary tumor model, liposome was contained indocyanine green (ICG) as fluorescence probe. This liposome was injected in tail vein and sequentially observed using fluorescent imaging device (Clairvivo OPT, SHIMADZU). In histological observation at hepatic metastasis murine model, M5076 ovarian sarcoma cells were inoculated in tail vein of mice. These mice were injected liposome at a dose of 2.5 mg/kg (i.v.) as DOX on 6, 9 and 12 d after tumor inoculation. At 14 d, this liver was removed and examined by microscope after HE stain. At assessment of pulmonary metastasis, B16F10 melanoma cells were implanted in tail vein and treated similarly to liver metastasis.[Results and discussion] In fluorescent imaging of primary tumor, DDA-PEG modified liposome assembled into tumor at 48 h after injection. This analysis supported our previous finding that DDA-LDOX significantly decreased tumor size. Furthermore, it was proved that DDA-PEG modified liposome or encapsulated drug remained in tumor site for a long time. In histological examination for hepatic metastasis, the tumor area in the liver was 5% by DDA-LDOX injection (normal group: 90%). More lymphocytes around tumor thrombus were positive for CD45. It was suggested that macrophage or lymphocytes, especially T cell, were concerned in this therapy. Pulmonary metastasis was depressed by DDA-LDOX compared with control or DOX containing PEG unmodified liposome group. In DDA-LDOX group, DOX concentration in lung was 2.6 times higher than that in PEG unmodified liposome group. It is known that metastasis is the most severe factor in cancer chemotherapy. Antitumor agent containing DDA-PEG modified liposome may have not only superior effect for primary tumor but also hepatic or pulmonary metastasis. Hence, it was suggested that DDA-PEG modified liposome was useful drug carrier for passive targeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1961. doi:1538-7445.AM2012-1961

Enhancement of doxorubicin-induced antitumor activity and reduction of adverse reactions by cucurbitacin I

Using the concept of biochemical modulation, we have previously shown that cucurbitacin E enhances doxorubicin (DOX)-induced antitumor activity. Unlike cucurbitacin E, another derivative called cucurbitacin I has antioxidative ability. However, it is feared that the antioxidative ability of cucurbitacin I will improve DOX-induced adverse reactions whereas reduce its antitumor activity by cucurbitacin I induced suppression on DOX generated reactive oxygen species. In the present study, compared to DOX treatment alone, a combination of cucurbitacin I and DOX produced a significant increase in cytotoxicity (M5076 ovarian sarcoma cell) in vitro and decreased tumor size and weight with a significant increase in DOX concentration in the tumor, using M5076 ovarian sarcoma bearing mice in vivo. Furthermore, the combination significantly suppressed the DOX-induced increase in lipid peroxide (LPO) level in the heart, which is considered to be an index of DOX-induced cardiotoxicity. Thus, it was surmised that cucurbitacin I was responsible for reducing heart damage. It was speculated that the radical trapping effect of cucurbitacin I on free radical lipids directly suppressed the increase in LPO level and DOX-induced cardiotoxicity. Although cucurbitacin I did not have a significant effect on the DOX influx system, it suppressed DOX efflux and increased DOX concentration in tumor cells. In M5076 ovarian sarcoma cells, multidrug resistance-associated protein inhibitors significantly inhibited DOX efflux, similar to cucurbitacin I. Furthermore, cucurbitacin I significantly decreased glutathione (GSH) levels in tumors, similar to DL-buthionine sufoximine (an inhibitor of GSH synthesis). Thus, it is speculated that cucurbitacin I acted via both routes.Based on these results, we conclude that cucurbitacin I increases DOX-induced antitumor activity with enhanced cytotoxicity and elevates DOX levels, thereby inhibiting DOX efflux from tumor cells.

Abstract 1443: Dual effect of novel polyethyleneglycol modified liposome on antitumor activity and liver metastasis

Abstract [Purpose] We synthesized polyethyleneglycol-lipid with different double arms (DDA-PEG) as the novel modification material on liposomal membrane, and reported that doxorubicin (DOX) contained DDA-PEG modified liposome (DDA-LDOX) had superior properties (cytotoxicity and DOX uptake into tumor cells). On the other hand, it was suggested that fixed aqueous layer thickness (FALT) around the liposomal membrane was concerned with long circulation in blood or antitumor effect. In 100th AACR meeting (2009), we reported the connection with the FALT and the biological factor (amount of DOX uptake / IC50 of cell cytotoxicity) in vitro. In this study, DDA-LDOX had been studied the antitumor activity on primary tumor in vivo. Moreover, another activity of DDA-LDOX was examined against liver metastasis as the risk factor in cancer therapy. [Methods] Liposome (DSPC/cholesterol/DSPG/PEG/DOX = 100:100:60:15:18 μmol) was prepared according to the method of Bangham. PEG2000 modified liposome and PEG2000/PEG500 modified liposome was expressed as Single-LDOX and Mixed-LDOX, respectively. Zeta potentials of liposomes were measured by the change of NaCl concentration in buffer and FALT was calculated. In the study of antitumor activity to primary tumor, M5076 ovarian sarcoma cells were transplanted into the backs of mice, and each liposomes were injected intravenously at a dose of 2.5 mg/kg on 19th, 22nd and 25th day after tumor inoculation. The mice were sacrificed after 48 hr from last administration, and the tumor was removed and weighed. In the study of anti-metastasis activity, M5076 ovarian sarcoma cells were inoculated in tail vein of mice. These mice were injected various liposomes at a dose of 2.5 mg/kg (i.v.) on 6th, 9th and 12th day after tumor inoculation. After 48 hr from last administration, the liver was removed and weighed, and score of liver metastasis was recorded (0-5 stage, 0: non-metastasis). [Results and discussion] In the result of antitumor activity, the tumor weight after Single-LDOX treatment was reduced to 65.5% of control level (1.13±0.45 g). In contrast, after DDA-LDOX treatment, it was reduced to 36.4% of control level. In anti-metastasis activity, the score of hepatic metastasis was DOX sol > plain-LDOX > Single-LDOX = DDA-LDOX. The inhibitory effect on liver metastasis by Single-LDOX and DDA-LDOX were obviously. The metastasis score correlated with the liver weight, namely, inhibition of metastasis by DDA-LDOX was supported from liver weight. DOX concentration in the liver was plain-LDOX > Single-LDOX = DDA-LDOX > DOX sol. Thus, we speculated that weak effect of plain-LDOX was caused by DOX accumulation into hepatic normal cell. In this study, the character of Single-LDOX was similar to DOXIL®, namely, it was suggested that DDA-LDOX increase the therapeutic efficacy, compared with DOXIL®. In conclusion, we expected that DDA-LDOX had the dual effect on antitumor activity and liver metastasis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1443.

Enhancement of doxorubicin concentration in the M5076 ovarian sarcoma cells by cucurbitacin E co-treatment

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.

Voreloxin, formerly SNS-595, has potent activity against a broad panel of cancer cell lines and in vivo tumor models

Voreloxin, formerly known as SNS-595 or AG-7352, is a novel naphthyridine analog currently under investigation for the treatment of ovarian and hematologic malignancies. Voreloxin mechanism of action includes DNA intercalation and inhibition of topoisomerase II that causes selective DNA damage. In this study, we describe the anti-proliferative activity of voreloxin in a wide range of in vitro and in vivo models of human cancers. The cytotoxicity of voreloxin in vitro was examined by MTT assay in 15 cell lines, including 4 drug-resistant lines. Activation of caspase in cell lines and tumors was evaluated by immunohistochemistry. Anti-tumor activity was assessed in 16 xenograft and 3 syngeneic tumor models in mice. Tumors were allowed to grow to approximately 150 mm(3) prior to treatment with voreloxin or comparator drugs. Activity of the anti-cancer agents was determined by calculating the inhibition rate (IR = [1 - (average tumor weight treated/average tumor weight control)] x 100%) and survival ratio (number surviving mice/number of mice per group at start of study) for each agent and dose and schedule tested. In vitro studies demonstrated voreloxin has broad anti-proliferative activity in 11 tumor cell lines, with IC(50) values ranging from 0.04 to 0.97 muM. Similar activity was observed in vitro in drug-resistant cell lines, including those that overexpress P-glycoprotein and have reduced topoisomerase levels. After a single intravenous dose, voreloxin concentrations in tumor were correlated with induction of the apoptosis marker caspase-3. The optimal dose and schedule was established using a KB nasopharyngeal carcinoma xenograft model. Administration of voreloxin at 20 mg/kg weekly for five doses effectively inhibited tumor growth (86%). Voreloxin demonstrated strong dose-dependent tumor growth inhibition (63-88%) in 10 of 11 solid tumor (breast, ovarian, colon, lung, gastric, and melanoma) xenograft models, 2 hematologic tumor xenograft models, 3 multidrug resistant tumor models and 3 murine syngeneic tumor models (Colon 26, Lewis Lung carcinoma, M5076 Ovarian Sarcoma). These data demonstrate that voreloxin is a broadly active anti-tumor agent in vitro and in vivo, with potent activity in aggressive and drug-resistant tumor models.

Effects of a single-dose administration of Bowman-Birk inhibitor concentrate on anti-proliferation and inhabitation of metastasis in M5076 ovarian sarcoma-bearing mice

The present study was designed to investigate the effects of Bowman-Birk inhibitor concentrate (BBIC) on anti-proliferation, up-regulation of Connexin 43 (Cx43) expression and inhabitation of hepatic metastasis in mice with M5076 ovarian sarcoma. M5076 ovarian sarcomas (1x106 cells/animal) were subcutaneously transplanted into the back of BDF1 mice. The 'pre-treated' (n=10) and 'post-treated' (n=10) groups were fed a standard diet (CE-2) compounded with 0.5% BBI from 3 weeks before and from the day of tumor inoculation, respectively, until 4 weeks after tumor inoculation. The 'control group' (n=10) was fed CE-2 alone. Relative tumor weights in the pre- (0.013±0.010) and post- (0.012±0.013) treated groups were significantly reduced by 30.0 and 32.5%, respectively, as compared to the control group (0.040±0.022, p<0.01). The relative densities of Cx43 mRNA and Cx43 protein were significantly higher in the BBIC-treated groups than in the control group. The median numbers of macroscopic spontaneous metastases were significantly lower in the pre- (1.0±2.3) and post- (1.9±3.6) treated groups than in the control group (71.4±97.3). These results suggest that BBIC reduces proliferation as a result of increased expression of Cx43 genes in mice with M5076 ovarian sarcoma. In addition, BBIC inhibits hepatic metastasis in M5076-bearing mice.

Connexin 43-dependent tumor-suppressing effect of the Bowman-Birk protease inhibitor on M5076 ovarian sarcoma-bearing mice

The present study was designed to confirm whether the Bowman-Birk inhibitor (BBI) induces an increase in p27 accumulation without S phase kinase-associated protein 2 (skp2) degradation by means of the expression of connexin (Cx) 43 as a gap junctional intercellular communication (GJIC)-dependent pathway in mice with M5076 ovarian sarcoma. M5076 ovarian sarcomas (1x105 cells/animal) were subcutaneously transplanted onto the backs of BDF1 mice receiving 10, 20 or 40 mg/kg of purified BBI intraperitoneally. Relative tumor weight (p<0.01, r=0.503) was negatively correlated with the dose of BBI. In contrast, the relative density of Cx43 mRNA (p<0.01, r=0.570) and Cx43 (p<0.01, r=0.718) was positively correlated with the dose of BBI, as were p21 (p<0.01, r=0.633), p27 (p<0.01, r=0.561) and skp2 (p<0.01, r=0.733). We therefore suggest that the anti-carcinogenic effects of BBI induce negative growth control by means of an increase in p27 accumulation caused by the expression of Cx43 as a GJIC pathway.

Screening of biochemical modulator by tumor cell permeability of doxorubicin

We screened various food components for their ability to inhibit doxorubicin (DOX) permeability in tumor cells in vitro with the aim of finding novel modulators. Capsaicin did not change DOX permeability in the tumor cells, although the capsaicin derivatives gingerol and ferulic acid tended to promote DOX efflux. Combinations of these components with DOX were also not effective. In contrast, cucurbitacin E significantly promoted DOX influx into tumor cells and increased DOX concentration in tumor cells. Furthermore, combined cucurbitacin E significantly suppressed DOX efflux from tumor cells and was shown to maintain the DOX level in tumor cells. It was also confirmed that the combination of cucurbitacin E with DOX resulted in effective cytotoxicity for tumor cells in culture. Additionally, the combination of cucurbitacin E and DOX showed increased cytotoxicity when compared to each treatment alone. In vivo, DOX alone treatment did not change the time course of tumor size or tumor weight of M5076 ovarian sarcoma, compared to control levels. In contrast, the combination of cucurbitacin E with DOX resulted in decreased tumor size and tumor weight, compared to that in DOX alone group, indicating effective antitumor activity. In conclusion, the combination of cucurbitacin E with DOX may be an effective tool with treated application in the cancer chemotherapy.

Cytidine is a novel substrate for wild-type concentrative nucleoside transporter 2

Nucleoside transporter (NT) plays key roles in the physiology of nucleosides and the pharmacology of its analogues in mammals. We previously cloned Na +/nucleoside cotransporter CNT2 from mouse M5076 ovarian sarcoma cells, the peptide encoded by it differing from that by the previously reported mouse CNT2 in five substitutions, and observed that the transporter can take up cytidine, like CNT1 and CNT3. In the present study, we examined which of the two aforementioned CNT2 is the normal one, and whether or not cytidine is transported via the previously reported CNT2. The peptide encoded by CNT2 derived from mouse intestine, liver, spleen, and ovary was identical to that previously reported. The uptake of [ 3H]cytidine, but not [ 3H]thymidine, by Cos-7 cells transfected with CNT2 cDNA obtained from mouse intestine was much greater than that by mock cells, as in the case of [ 3H]uridine, a typical substrate of NT. [ 3H]Cytidine and [ 3H]uridine were taken up via CNT2, in temperature-, extracellular Na +-, and substrate concentration-dependent manners. The uptake of [ 3H]cytidine and [ 3H]uridine mediated by CNT2 was significantly inhibited by the variety of nucleosides used in this study, except for thymidine, and inhibition of the [ 3H]uridine uptake by cytidine was competitive. The [ 3H]uridine uptake via CNT2 was significantly decreased by the addition of cytarabin or gemcitabine, antimetabolites of cytidine analogue. These results indicated that the previously reported mouse CNT2 is the wild-type one, and cytidine is transported mediated by the same recognition site on the CNT2 with uridine, and furthermore, cytidine analogues may be substrates for the transporter.

Contribution of an unidentified sodium-dependent nucleoside transport system to the uptake and cytotoxicity of anthracycline in mouse M5076 ovarian sarcoma cells

In the present study, we investigated whether an unidentified system for Na +-dependent nucleoside transport is expressed by mouse M5076 ovarian sarcoma cells, besides concentrative nucleoside transporter 2 (CNT2 M), and is involved in the uptake and cytotoxicity of anthracyclines. In a transport assay involving CNT2 M-transfectants, CNT2 M was found to transport [ 3H]cytidine in a Na +-dependent manner, and 500 μM cytidine completely inhibited the Na +-dependent uptake of [ 3H]uridine via the transporter. In contrast, the Na +-dependent [ 3H]uridine uptake by M5076 cells decreased with 500 μM cytidine only to 70% of the control level. Furthermore, transfection of CNT2 M-specific siRNAs into M5076 cells resulted in a reduction in the Na +-dependent uptake of [ 3H]uridine by only 23%, although the expression of CNT2 M mRNA and Na +-dependent uptake of [ 3H]cytidine disappeared in the cells. The uptake of pirarubicin (THP), an anthracycline, by M5076 cells requiring extracellular Na + was significantly inhibited by 500 μM uridine, but not 500 μM cytidine. The Na +-dependent and cytidine-insensitive uptake of [ 3H]uridine and the that of THP by M5076 cells significantly increased on cotreatment with both cholate and taurocholate, and the enhancement of THP uptake by the bile acids was reversed by cotreatment with 500 μM uridine. Furthermore, the cytotoxicity of THP and doxorubicin, which were previously reported to be taken up via the same transporter, toward M5076 cells was enhanced by cotreatment with both the bile acids. Therefore, it was indicated that an unidentified Na +-dependent transport system for nucleosides is expressed by M5076 cells, and contributes to the uptake and cytotoxicity of the anthracyclines.

Restoration of connexin 43 by Bowman-Birk protease inhibitor in M5076 bearing mice

The present study was designed to investigate the effects of Bowman-Birk inhibitor (BBI) on up-regulation of connexin (Cx) expression to estimate BBI's tumor-suppressor effect in mice with M5076 ovarian sarcoma. The relative tumor weight (p<0.05, r(2)=0.301) and proliferating cell nuclear antigen (PCNA, p<0.01, r(2)=0.493) were negatively correlated with the doses of BBI. In contrast, the relative density of Cx43 was positively correlated with the doses of BBI (p<0.05, r(2)=0.351). Therefore, it suggests that the anti-carcinogenic effects of BBI induced negative growth control caused by the expression of Cx43 genes in mice with M5076 ovarian sarcoma.

Uptake of the anthracycline pirarubicin into mouse M5076 ovarian sarcoma cells via a sodium-dependent nucleoside transport system

We have previously demonstrated that the cytotoxicity of anthracyclines, pirarubicin (THP) and doxorubicin (DOX), is partially dominated by their intracellular amounts, which depend on the uptake efficacy of transporter(s). To clarify their transport mechanism, we examined whether or not Na+/nucleoside cotransporter (CNT) is involved in the uptake of THP by M5076 cells. Expression of the CNT isoforms was determined by reverse-transcription PCR. We used two cell lines, intact M5076 and CNT2-transfected Cos-7 cells, to characterize the uptake of THP and [3H]uridine. The mRNA for CNT2, but not that for CNT1 or CNT3, was expressed in M5076 cells, and [3H]uridine uptake by the cells required a Na+ gradient as a driving force. THP uptake by M5076 cells depended on a Na+ gradient, and furthermore, formycin B and AZT had cis-inhibitory and trans-stimulatory effects on the uptake. The efflux of [3H]uridine from M5076 cells was stimulated by the addition of THP extracellularly, which constituted definite evidence of CNT-mediated uptake of THP. However, THP uptake by CNT2 transfectant was almost the same as that by mock cells, indicating that an unidentified CNT isoform contributes to THP uptake by M5076 cells, this being supported by the differences in transport characteristics of [3H]uridine between M5076 and CNT2-transfected cells. THP is partially taken up into M5076 cells via a novel Na+-dependent transport system common to nucleosides.

Relationships between the in vitro cytotoxicity and transport characteristics of pirarubicin and doxorubicin in M5076 ovarian sarcoma cells, and comparison with those in Ehrlich ascites carcinoma cells.

We sought to determine whether the de novo resistance of M5076 ovarian sarcoma cells, which show sensitivity to pirarubicin (THP), to doxorubicin (DOX) is due to differences in the transport characteristics between THP and DOX, and the results were compared with those for drug-sensitive Ehrlich ascites carcinoma cells. The in vitro cytotoxicity of the drugs was assessed by means of the tetrazolium dye assay. Transport experiments were performed by the rapid centrifugation method. In an in vitro cytotoxicity experiment, M5076 cells showed lower sensitivity to DOX than to THP, and the cytotoxicity of THP and DOX toward M5076 cells was lower than toward Ehrlich cells, and these results were similar to those of an in vivo experiment. This was due to the much lower expression of topoisomerase II in M5076 cells than in Ehrlich cells. The amount of intracellular DOX was found to be significantly lower than that of THP in both cell types, and furthermore, little free intracellular DOX was observed in M5076 cells, indicating that the low sensitivity of M5076 cells to DOX was partially a result of the low amount of intracellular DOX. There was no difference in the efflux rate, but there was an apparent difference in the uptake efficiency of the carrier between THP and DOX. These findings suggest that the cytotoxicities of THP and DOX toward M5076 and Ehrlich cells depend, at least in part, on the uptake efficiency of the carrier.

Inhibition of glutamate transporter by theanine enhances the therapeutic efficacy of doxorubicin

Theanine, a major amino acid existing in green tea, enhanced the antitumor activity of doxorubicin (DOX) due to inhibition of DOX efflux from tumor cells. In order to clarify the mechanism, we have investigated the contribution of glutamate transporters to the action of theanine, because theanine is a glutamate analogue. In M5076 ovarian sarcoma cells, glutamate transport inhibitors reduced the efflux of DOX, as well as theanine. Incidentally, theanine significantly inhibited the glutamate uptake by M5076 cells in a concentration-dependent manner similar to specific inhibitors. These results suggested that the inhibition of DOX efflux was induced by the inhibition of glutamate transport by theanine. In addition, RT-PCR and Western blot analysis revealed the expression of GLAST and GLT-1, astrocytic high-affinity glutamate transporters, in M5076 cells. Thus, theanine was shown to competitively inhibit the glutamate uptake by acting on these glutamate transporters. This action suggested the contribution of glutamate transporters to the inhibition of DOX efflux by theanine. We revealed the novel mechanism of enhancement of the antitumor efficacy of DOX via the inhibition of glutamate transporters by theanine.