Chitinase induces lysis of MCF-7 cells in culture and of human breast cancer xenograft B11-2 in SCID mice.

BACKGROUND: It has long been known that the polycarbohydrates on the neoplastic cell surface are different from those on normal cells; differences which allow one to attack tumor cells selectively. Although the exact differences between tumor cells and normal cells are still not clearly known, research into these differences is ongoing for anticancer drug development. MATERIALS AND METHODS: The human breast cancer cell line MCF-7 in culture and human breast xenograft B(11)-2 in SCID mice were used in our observations. Two different samples of chitinase from different bacteria were tested in the experiments. Optical observation of regular H & E-stained tumor tissue sections and observations by transmission electron microscopic techniques were used in this study. RESULTS: MCF-7 breast cancer cells in culture showed structural damage within 7 hours after 1.3 unit/ml of chitinase was added to the medium, while normal mice spleen cells did not. The transplanted B(11)-2 xenograft tissue in mice started to lyse 12 hours after chitinase was injected; the size of the tumor gradually reduced and finally a scab was formed, which came off the skin a few days later. All the tested tumor-bearing mice survived and these cured mice had no tumor re-growth during the following 1-year observation period. CONCLUSION: Chitinase selectively lysed the tumor cells in vitro and in vivo. Injected chitinase destroyed the tumor tissue and cured the mice. The further development of this type of treatment and of the mechanisms of chitinase action are discussed.

In vivo antitumor activity of folate receptor-targeted liposomal daunorubicin in a murine leukemia model.

BACKGROUND: Folate receptor (FR) is selectively amplified among human tumors, including in 70% of myeloid leukemias. FR-targeted liposomal delivery is an attractive strategy for enhancing the therapeutic efficacy of anticancer agents against FR(+) tumors. In this study, FR-targeted liposomal daunorubicin was evaluated in an FR+ L1210JF murine ascites tumor model for therapeutic efficacy in vivo. MATERIALS AND METHODS: FR-targeted liposomal daunorubicin (F-L-DNR) and non-targeted liposomal daunorubicin (L-DNR) were prepared by polycarbonate membrane extrusion followed by remote loading of DNR. FR-targeted liposomal uptake by L1210JF cells was characterized in vitro using fluorescent liposomes entrapping calcein. For in vivo therapeutic study, B6D2F1 mice on a folate-free diet were intraperitoneally implanted with FR (+) L1210JF cells and treated with 4 intraperitoneal injections of 10 mg/kg liposomal DNR at 1, 5, 9 and 13 days following tumor cell inoculation. Animal survival was then monitored daily. RESULTS: LI210JF cells showed approximately 10(3) times greater uptake for FR-targeted liposomal calcein compared to the non-targeted control. Uptake of the targeted liposomes could be blocked by 1 mM folic acid. In the therapeutic study, mice treated with F-L-DNR showed significantly greater tumor inhibition and 40.7% greater increase in life-span compared to those that received identical doses of L-DNR. Meanwhile, free DNR given at the same dose failed to prolong the survival of the treated mice. CONCLUSION: F-L-DNR can effectively target FR(+) leukemia cells in vivo. Further preclinical evaluation is warranted to determine its potential application in leukemia therapy.

Penetration into solid tumor tissue of fluorescent latex microspheres: a mimic of liposome particles.

BACKGROUND: Using liposomes as a vehicle to transport anticancer drugs to cancer cells, to increase their effectiveness and decrease their toxicity, has been studied for many years. However, due to technical difficulties, the path of penetration for liposome particles into solid tumor tissue is still not clear. MATERIALS AND METHODS: In this report, rhodamine-labeled fluorescent latex microspheres were used as a model of liposome particles, combined with fluorescent staining of blood vessel CD31 and tumor cell nuclei. The penetration of microspheres from blood vessels in L1210JF solid tumors of mice was observed. After fluorescent latex microspheres were injected into tail vein, tumor tissue samples were collected at various times and cryosections were then made for fluorescent staining. RESULTS: Under fluorescence microscopy, the red fluorescent latex microspheres, the green fluorescent blood vessels and the blue tumor cells in the cancer tissue were seen clearly. The leaking of microspheres out from blood vessels was seen directly. CONCLUSION: The results confirmed that the tiny particles can only leak out through the holes of the broken blood vessels and spread out through the space in between the cells of the solid tumor.

Antitumor activity of folate receptor-targeted liposomal doxorubicin in a KB oral carcinoma murine xenograft model.

PURPOSE: The expression of folate receptor (FR) is amplified in many types of human cancers. Previously, FR-targeted liposomal doxorubicin (f-L-DOX) has been shown to exhibit superior and selective cytotoxicity against FR(+) tumor cells in vitro compared to nontargeted liposomal doxorubicin (L-DOX). This study further investigates f-L-DOX for its antitumor efficacy in vivo using a murine tumor xenograft model. METHODS: F-L-DOX composed of DSPC/cholesterol/PEG-DSPE/ folate-PEG-DSPE (65:31:3.5:0.5, mole/mole) was prepared by polycarbonate membrane extrusion followed by remote loading of DOX. Athymic mice on a folate-free diet were engrafted with FR(+) KB cells. Two weeks later, these mice were treated with f-L-DOX, L-DOX, or free DOX in a series of six injections (given intraperitoneally on every fourth day at 10 mg/kg DOX) and monitored for tumor growth and animal survival. The plasma clearance profiles of the DOX formulations and the effect of dietary folate on plasma folate concentration were also analyzed. RESULTS: Plasma folate level remained in the physiologic range relative to that in humans. F-L-DOX exhibited an extended systemic circulation time similar to that of L-DOX. Mice that received f-L-DOX showed greater tumor growth inhibition and a 31% higher (p < 0.01) increase in lifespan compared to those that received L-DOX. Meanwhile, free DOX given at the same dose resulted in significant toxicity and was less effective in prolonging animal survival. CONCLUSIONS: FR-targeted liposomes are a highly efficacious vehicle for in vivo delivery of anticancer agents and have potential application in the treatment of FR(+) solid tumors.

Boron delivery to a murine lung carcinoma using folate receptor-targeted liposomes.

BACKGROUND: Folate receptor (FR) is amplified in a variety of human tumors. Folate-derived liposomes have been shown to selectively deliver entrapped agents into tumor cells via receptor-mediated endocytosis. In this preliminary study, the biodistribution of FR-targeted liposomes were evaluated as a potential delivery agent for Na3 (B20H17NH3) for boron neutron capture therapy (BNCT) of FR(+) tumors. MATERIALS AND METHODS: Na3 (B20H17NH3) was incorporated into liposomes by passive entrapment, following which they were administered intravenously into BALB/c mice bearing subcutaneous implants of FR(+) M109 murine lung carcinoma. Tumor and normal tissue boron content was measured by direct current plasma atomic emission spectroscopy. RESULTS: Mice that received FR-targeted and non-targeted control liposomes showed indistinguishable levels of tumor boron uptake (up to 85 microg/g tumor), which reached a maximum at the 24 hour time-point, while the tumor-to-blood (T/B) ratio continued to rise until the 72 hour time-point. CONCLUSION: High-level boron delivery is possible using liposomes as a delivery agent. FR targeting does not significantly enhance overall tumor localization but may improve boron delivery at the cellular and subcellular levels, which warrant further investigation.

Strategy for the treatment of acute myelogenous leukemia based on folate receptor beta-targeted liposomal doxorubicin combined with receptor induction using all-trans retinoic acid.

Up-regulation of folate receptor (FR) type-beta in acute myelogenous leukemia (AML) by all-trans retinoic acid (ATRA) and its restricted normal tissue distribution makes it a potential target for therapeutic intervention. The FR-beta in peripheral blood granulocytes was unable to bind folate and appeared to have a variant GPI membrane anchor, evident from its insensitivity to phosphatidylinositol-specific phospholipase C but not nitrous acid. Granulocyte FR-beta lacked mutations, and neither deglycosylation nor detergent solubilization restored folate binding. The posttranslational modification causing its nonfunctionality was evidently absent in FR-beta from AML cells from patient marrow, which bound folate. From flow cytometric analysis of 78 AML bone marrow specimens of different subtypes, 68% expressed FR-beta, most of which were also CD34+. In model cell lines that are FR - (KG-1a, L1210, and Chinese hamster ovary [CHO]) or FR + (KG-1, L1210 JF, and recombinant CHO-FR-beta), selective FR-mediated binding and cytotoxicity was obtained using folate-coated liposomes encapsulating fluorescent calcein (f-L-calcein) and doxorubicin (f-L-DOX), respectively, which could be blocked by 1 mM free folic acid. In the FR-beta-expressing KG-1 human AML cells, treatment with ATRA further increased this specificity. In mouse ascites leukemia models generated using L1210JF or KG-1 cells, increased median survival times were obtained with f-L-DOX treatment compared to nontargeted L-DOX. In the KG-1 model, ATRA treatment increased the cure rate with f-L-DOX from 10% to 60%. The above combined data from our 2 laboratories further support the feasibility and potential usefulness of selective ATRA-facilitated liposomal drug delivery in FR-beta + AMLs.

Boron-containing folate receptor-targeted liposomes as potential delivery agents for neutron capture therapy.

Boron neutron capture therapy (BNCT) depends on the selective delivery of a sufficient number of (10)B atoms to tumor cells to sustain a lethal (10)B(n,alpha)(7)Li reaction. Expression of FR frequently is amplified among human tumors. The goal of the present study was to investigate folate receptor (FR)-targeted liposomes as potential carriers for a series of boron-containing agents. Two highly ionized boron compounds, Na(2)[B(12)H(11)SH] and Na(3) (B(20)H(17)NH(3)), were incorporated into liposomes by passive loading with encapsulation efficiencies of 6% and 15%, respectively. In addition, five weakly basic boronated polyamines were investigated. Two were the spermidine derivatives: N(5)-(4-carboranylbutyl)spermidine.3HCl (SPD-5), N(5)-[4-(2-aminoethyl-o-carboranyl)butyl]spermidine.4HCl (ASPD-5). Three were the spermine derivatives: N(5)-(4-carboranylbutyl)spermine.4HCl (SPM-5), N(5)-[4-(2-aminoethyl-o-carboranyl)butyl]spermine.5HCl (ASPM-5), and N(5),N(10)-bis(4-carboranylbutyl)spermine.4 HCl (SPM-5,10). These were incorporated into liposomes by a pH-gradient-driven remote-loading method with varying loading efficiencies, which were influenced by the specific trapping agent and the structure of the boron compound. Greater loading efficiencies were obtained with lower molecular weight boron derivatives, using ammonium sulfate as the trapping agent, compared to those obtained with sodium citrate. The in vitro uptake of folate-derivatized, boronated liposomes was investigated using human KB squamous epithelial cancer cells, which have amplified FR expression. Higher cellular boron uptake (up to 1584 microg per 10(9) cells) was observed with FR-targeted liposomes than with nontargeted control liposomes (up to 154 microg per 10(9) cells), irrespective of the chemical form of the boron and the method used for liposomal preparation. KB cell binding of the FR-targeted liposomes was saturable and could be blocked by 1 mM free folic acid. Our findings suggest that further evaluation of FR-targeted liposomes is warranted to assess their potential as boron carriers for neutron capture therapy.