Prostate cancer-specific monoclonal antibody 5D4 significantly enhances the cytotoxicity of doxorubicin-loaded liposomes against target cells in vitro

Long-circulating liposomes loaded with doxorubicin (Dox) were additionally modified with the prostate cell-specific monoclonal antibody 5D4 (mAb 5D4). The resultant Dox-loaded 5D4-immunoliposomes specifically recognized prostate cancer cell lines of several different types expressing the mAb 5D4 antigen, PSMA, and significantly enhanced cytotoxicity toward these cells compared with the non-targeted Dox-liposomes in vitro while no increased toxicity was observed toward non-prostate (lung) cancer cell line.     

Sulfonyl-containing aldophosphamide analogues as novel anticancer prodrugs targeted against cyclophosphamide-resistant tumor cell lines

A series of sulfonyl-group containing analogues of aldophosphamide (Aldo) were synthesized as potential anticancer prodrugs that liberate the cytotoxic phosphoramide mustards (PM, IPM, and tetrakis-PM) via beta-elimination, a nonenzymatic activation mechanism. Kinetic studies demonstrated that all these compounds spontaneously liberate phosphoramide mustards with half-lives in the range of 0.08-15.2 h under model physiological conditions in 0.08 M phosphate buffer at pH 7.4 and 37 degrees C. Analogous to Aldo, the rates of beta-elimination in all compounds was enhanced in reconstituted human plasma under same conditions. The compounds were more potent than the corresponding phosphoramide mustards against V-79 Chinese hamster lung fibroblasts in vitro (IC(50) = 1.8-69.1 microM). Several compounds showed excellent in vivo antitumor activity in CD2F1 mice against both P388/0 (Wild) and P388/CPA (CP-resistant) tumor cell lines.     

Selective cytotoxicity against human tumor cells by an anti-gastric cancer monoclonal antibody-mitomycin C conjugate

An anti-gastric cancer monoclonal antibody, MGb2, was chosen to prepare MGb2-mitomycin C (MMC) conjugate. Four to five molecules of MMC were introduced into each molecule of antibody with the antibody activity well retained. The conjugate showed a highly selective cytotoxicity upon human gastric cancer cells KATO-III. In the 48-h exposure test, the cytotoxic effect of MGb2-MMC upon target cells was similar to that of free MMC, but much greater than that of normal mouse immunoglobulin-MMC conjugate. Instead, the MGb2-MMC showed a statistically less cytotoxic effect upon non-target cells. Imaging and biodistribution studies indicated that the MGb2 was still well localized in tumor tissue after its conjugation with MMC.     

Monoclonal antibody 2C5-mediated binding of liposomes to brain tumor cells in vitro and in subcutaneous tumor model in vivo

This study aimed to investigate the monoclonal antibody (mAb) 2C5 with nucleosome-restricted specificity for its ability to specifically recognize human brain tumor cells and to serve as a specific ligand for liposome targeting to brain tumor cells in vitro and in vivo. The affinity of mAb 2C5 towards brain tumor cells was tested by flow cytometry. The interaction of 2C5-immunoliposomes (ILS) with brain tumor cells in vitro was studied by fluorescence microscopy. For in vivo accumulation studies, ¹¹¹In-ILS were administered i.v. into mice bearing subcutaneously grown brain tumor. mAb 2C5 was found to be reactive against several tested brain tumor cell lines. mAb 2C5 and 2C5-ILS demonstrated enhanced cell-surface binding with CCF-STTG1,U-87 MG and LN-18 cells in vitro. 2C5-ILS displayed significantly better accumulation in the subcutaneously grown brain tumor than non-specific control IgG-ILS. mAb 2C5 specifically recognizes brain tumor cells and can serve as a ligand to target drug carriers such as liposomes to brain tumor cells in vivo.     

Monoclonal antibody 2C5-mediated binding of liposomes to brain tumor cells in vitro and in subcutaneous tumor model in vivo

This study aimed to investigate the monoclonal antibody (mAb) 2C5 with nucleosome-restricted specificity for its ability to specifically recognize human brain tumor cells and to serve as a specific ligand for liposome targeting to brain tumor cells in vitro and in vivo. The affinity of mAb 2C5 towards brain tumor cells was tested by flow cytometry. The interaction of 2C5-immunoliposomes (ILS) with brain tumor cells in vitro was studied by fluorescence microscopy. For in vivo accumulation studies, (111)In-ILS were administered i.v. into mice bearing subcutaneously grown brain tumor. mAb 2C5 was found to be reactive against several tested brain tumor cell lines. mAb 2C5 and 2C5-ILS demonstrated enhanced cell-surface binding with CCF-STTG1,U-87 MG and LN-18 cells in vitro. 2C5-ILS displayed significantly better accumulation in the subcutaneously grown brain tumor than non-specific control IgG-ILS. mAb 2C5 specifically recognizes brain tumor cells and can serve as a ligand to target drug carriers such as liposomes to brain tumor cells in vivo.     

Differential cytotoxicity of anticancer agents in pre- and post-immortal lymphoblastoid cell lines

We studied the cytotoxic effect of various anticancer agents on lymphoblastoid cell lines transformed by Epstein-Barr virus. Post-immortal N0005 (post-N0005) is an immortalized cell line derived from pre-immortal N0005 (pre-N0005) accompanied by increased telomerase activity, short-telomere, abnormal karyotypes, mutation of p53 gene, down regulation of p16/Rb and the ability to grow in soft agar medium. Compared with pre-N0005 cells, post-N0005 cells were significantly (p<0.001 by the Student t test) more resistant to the killing activity of seven DNA-modifying agents: camptothecin, etoposide, bleomycin, fluorouracil, thioguanine, melphalan and actinomycin D. However, both pre-N0005 and post-N0005 cells showed similar levels of cytotoxicity against four DNA-non-modifying agents: colchicine, paclitaxel, vincristine and methotrexate. DNA-modifying and DNA-non-modifying agents are distinguished by their different sensitivities with pre-N0005 and post-N0005. Based on these results, we propose that pre-N0005 and post-N0005 cell lines be used as a new method to assess and screen anticancer agents.     

In vitro characterization of the anticancer activity of membrane-active cationic peptides. I. Peptide-mediated cytotoxicity and peptide-enhanced cytotoxic activity of doxorubicin against wild-type and p-glycoprotein over-expressing tumor cell lines

Cationic amphipathic peptides, such as the defensins and cecropins, induce cell death in prokaryotic and eukaryotic cells by increasing membrane permeability. Increased permeability may lead to cell lysis or, alternatively, may produce subtle changes in the membrane's barrier function that promote cell death. The in vitro cytotoxic and lytic activity of short mammalian-derived extended-helical cationic peptides and insect-derived alpha-helical peptides was measured in this study with the objective of establishing the anticancer potential of these agents. Two specific aims were addressed: (i) to assess the activity of peptides against non-malignant cells (sheep erythrocytes and human umbilical vein endothelial cells) versus tumor cells; and (ii) to characterize the cytotoxic activity using multidrug-resistant tumor cell lines in the presence and absence of the anthracycline doxorubicin. Cell lysis assays demonstrated that the lytic activity of the peptides tested was 2->50 times more cytotoxic to tumor cells than to non-malignant cells. Further, the cytotoxic activity of these peptides was equivalent when tested against sensitive and multidrug-resistant cell lines. In addition to their inherent cytotoxic activity, these membrane-active peptides can also augment the in vitro cytotoxic activity of doxorubicin against multidrug-resistant tumor cells.     

Enhanced cytotoxicity of doxorubicin encapsulated in liposomes with reconstituted Sendai F-proteins.

Sendai F-virosomes, a novel type of liposome with reconstituted Sendai F-proteins, have been tested as a delivery system for various bioactive materials. However, encapsulation limitations and difficulties in controlling their constituents were drawbacks for further application to therapeutic purposes. We have tried to control virosomal constituents and have enhanced drug encapsulation efficiency into the virosomes. In vitro cytotoxicity of doxorubicin encapsulated in the F-virosomes were compared with free doxorubicin and doxorubicin in conventional liposomes. The F-virosomes were spontaneously prepared by detergent dialysis, a reconstitution process of Sendai F-proteins into liposomes. The reconstitution density of F-proteins affected the vesicle size of virosomes prepared by detergent dialysis; the larger amount of F-proteins made a smaller size of virosomes. There was little variation of size with time at physiological conditions, whilst the vesicle size of virosomes increased at acidic storage conditions (pH 5.5). Doxorubicin encapsulated in the F-virosomes exhibited a lower IC50 against B16BL6 mouse melanoma cells and Chang human hepatocarcinoma cells than that in conventional liposomes. The F-virosomes also exhibited higher cellular uptake than conventional liposomes. Addition of dioleoylphophatidylethanolamine, a fusogenic phospholipid, into the F-virosome further increased the cellular uptake as well as in vitro cytotoxicity. These types of virosome formulations can be clinically applicable as versatile vesicles for the efficient delivery of various therapeutic drugs, including genetic materials.     

盐酸米托蒽醌聚乙二醇化脂质体的制备及对A549细胞体外抗肿瘤作用的研究

目的 研究盐酸米托蒽醌聚乙二醇化脂质体(DHAD-PEG-L)的制备及其抗肿瘤细胞增殖作用,比较DHAD-PEG-L与常规盐酸米托蒽醌(DHAD)制剂的抗肿瘤活性差异.方法 以主动载药法制备获得DHAD-PEG-L;以人非小细胞肺癌A549细胞为模型,采用MTT法考察DHAD-PEG-L和DHAD对肿瘤细胞的抑制效果,测定制剂作用肿瘤细胞48 h后的IC50值;以方差分析法比较DHAD-PEG-L与DHAD抗肿瘤活性的统计学差异.结果 DHAD-PEG-L和DHAD对A549细胞的IC50分别是(1.561±0.09)mg·L-1和(0.862±0.02)mg·L-1;药物经聚乙二醇(PEG)脂质体包封后,与常规制剂相比,高浓度时肿瘤抑制作用略有下降,低浓度时抑制作用增强.结论 PEG化技术可运用于脂质体,对DHAD进行减毒增效改造,预期可降低药物不良反应.     

Cytotoxic activity of styrylchromones against human tumor cell lines

A total of 6 newly-synthesized styrylchromones (SC-1 approximately SC-6) were compared for their cytotoxic activity against three normal oral human cells (gingival fibroblast HGF, pulp cell HPC, periodontal ligament fibroblast HPLF) and four human tumor cell lines (squamous cell carcinoma HSC-2, HSC-3, submandibular gland carcinoma HSG, promyelocytic leukemia HL-60). All compounds showed higher cytotoxic activity against tumor cell lines than against normal cells. Among the 6 compounds, SC-3, SC-4 and SC-5, which have one to three methoxy groups, showed higher tumor specificity and water solubility. The cytotoxic activity of SC-3 and SC-5 was slightly reduced by a lower concentration of NADH, a quinone reductase, but that of SC-3 was enhanced by higher concentrations of NADH, possibly due to demethylation of the methoxy groups. Agarose gel electrophoresis demonstrated that SC-3 and SC-5 induced intemucleosomal DNA fragmentation in HL-60 cells and production of large DNA fragment in HSC-2 cells. Both SC-3 and SC-5 enhanced the enzymatic activity to cleave the substrates for caspases 3, 8 and 9, suggesting the activation of both extrinsic and intrinsic apoptosis pathways. ESR spectroscopy showed that these compounds produced no detectable amount of radical and did not scavenge superoxide anion generated by the hypoxanthine-xanthine oxidase reaction. The highly tumor-specific cytotoxic action and apoptosis-inducing capability of SC-3 and SC-5 suggest their applicability for cancer chemotherapy.     

Protodioscin (NSC-698 796): its spectrum of cytotoxicity against sixty human cancer cell lines in an anticancer drug screen panel

Protodioscin (NSC-698 796) is a furostanol saponin isolated from the rhizome of Dioscorea collettii var. hypoglauca (Dioscoreaceae), a Chinese herbal remedy for the treatment of cervical carcinoma, carcinoma of urinary bladder and renal tumor for centuries. To systematically evaluate its potential anticancer activity, protodioscin was tested for cytotoxicity in vitro against 60 human cancer cell lines in the NCI's (National Cancer Institute, USA) anticancer drug screen. As a result, protodioscin was cytotoxic against most cell lines from leukemia and solid tumors in the NCI's human cancer panel, especially selectively against one leukemia line (MOLT-4), one NSCLC line (A549/ATCC), two colon cancer lines (HCT-116 and SW-620), one CNS cancer line (SNB-75), one melanoma line (LOX IMVI), and one renal cancer line (786 - 0) with GI50 < or = 2.0 microM. In the general view of mean graphs, leukemia, colon cancer and prostate cancer are the most sensitive subpanels, while ovarian cancer is the least sensitive subpanel. Based on an analysis of COMPARE computer program with protodioscin as a seed compound, no compounds in the NCI's anticancer drug screen database have cytotoxicity patterns (mean graphs) similar to those of protodioscin, indicating that a potential novel mechanism of anticancer action is involved.     

In-vitro toxicity of Ukrain against human Ewing tumor cell lines

Ukrain is advertised by the manufacturer as a drug for alternative cancer cures with high activity against progressive Ewing tumors. Using the MTT assay, we compared the cytotoxicity of Ukrain with the cytotoxicity of N,N',N'-triethylenethiophosphoramide (thioTEPA), Chelidonium majus L. alkaloids, doxorubicin, cyclophosphamide and etoposide against four human Ewing tumor cell lines. In addition, we studied the cytotoxicity of thioTEPA combined with C. majus L. alkaloids after 48, 72 and 96 h. All compounds reduced the growth of Ewing tumor cell lines in a dose-dependent manner. The concentrations that reduced cell growth by 50% ranged between 6.2 and 31.1 micromol/l for Ukrain, 1.9 and 26.1 micromol/l for C. majus L. extract, and 1.7 and 448 micromol/l for thioTEPA. The sensitivity profile of Ukrain was comparable to that of the C. majus L. alkaloids, and different from that of thioTEPA, cyclophosphamide, etoposide and doxorubicin. Overall, doxorubicin was the most cytotoxic drug followed by cyclophosphamide. Ukrain and the C. majus L. alkaloids were slightly more cytotoxic than etoposide, while thioTEPA showed the lowest cytotoxicity. Co-exposure of thioTEPA with C. majus L. alkaloids resulted in additive but not in synergistic cytotoxicity. The in-vitro results indicate that the cytotoxicity of Ukrain against Ewing tumors is comparable to that of etoposide. While the latter can be used on the basis of broad clinical experience and known risk-benefit ratio, Ukrain for the present might be considered as a candidate for subsequent drug development by xenograft studies followed by systematic clinical trials.     

Construction of an immunotoxin with the pore forming protein StI and ior C5, a monoclonal antibody against a colon cancer cell line

Sticholysin I (StI), a potent cytolysin isolated from the sea anemone Stichodactyla helianthus, was linked to the monoclonal antibody (mAb) ior C5. StI acts by forming hydrophilic pores in the membrane of the attacked cells leading to osmotic lysis. ior C5 is a murine IgG1, which recognizes the tumor associated antigen (TAA) ior C2. The cytolysin and the mAb were coupled by using the heterobifunctional cross-linking reagent sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Two hybrid molecules composed by one ior C5 and one or two StI molecules were obtained (named conjugated I and II, respectively). The purified conjugates were evaluated by a binding affinity assay against an ior C2-positive colon cancer cell line (SW948). Both molecules were able to recognize the antigen (Ag) in the same way that unconjugated ior C5 does. The activity of both conjugates against human erythrocytes and SW948 cells was assessed. They lost most of their hemolytic activity but their residual activity was very similar. Nevertheless, when their cytotoxicity was studied on the SW948 cell line, only conjugate II killed efficiently the cells, indicating a specific mAb-Ag interaction. In this chimeric molecule the ratio between the cytotoxic and the hemolytic activity was larger than that of the free cytolysin. This fact indicates an increase of the specificity of the toxic effect toward the SW948 cell line and consequently an increase of the difference between its hemolytic and cytotoxic doses. The results herein support the feasibility of directing StI to the surface of cancer cells expressing ior C2 Ag via the mAb ior C5.     

Harzianums A and B produced by a fungal strain, Hypocrea sp. F000527, and their cytotoxicity against tumor cell lines

A new compound, harzianum B (1), was isolated from the culture broth of a fungal strain, Hypocrea sp. F000527, together with a known trichothecene, harzianum A (2). Its structure was determined by spectroscopic analyses including HRFAB-MS and various (1)H NMR and (13)C NMR spectral data. Harzianum B (1) was characterised as (E,Z,E)-2', 4', 6'-octatriendioic acid esterified on the 4beta-hydroxyl group of trichodermol. Harzianums A (2) and B (1) showed cytotoxicity against several human cancer cell lines.     

In vitro cytotoxicity of berberine against HeLa and L1210 cancer cell lines

Previous studies on anti-cancer activity of protoberberine alkaloids against a variety of cancer cell lines were extended to human uterus HeLa nad murine leukemia L1210 cell lines. Cytotoxicity was measured using in vitro techniques and cell morphology changes were examined by light microscopy in both cytostatic and cytocidal concentration ranges. The IC50 was found to be less than 4 microg/ml, a limit put forward by NCI for classification of the compound as a potential anti-cancer drug. The microscopy examination indicated that at cytocidal concentrations the HeLa and L120 cells died apoptotically. The comparative analysis revealed that berberine belongs to the camptothecin family of drugs characterized by the ability to induce DNA topoisomerase poisoning and hence apoptotic cell death. Although the cytotoxic potency of berberine was found to be several orders of magnitude lower compared to camptothecin, its significance may increase in future in view of the lack of unwanted side effects characteristic for camptothecin compounds currently in clinical use for treatment of cancer.     

Harzianums A and B produced by a fungal strain,Hypocrea sp. F000527, and their cytotoxicity against tumor cell lines

A new compound, harzianum B (1), was isolated from the culture broth of a fungal strain, Hypocrea sp. F000527, together with a known trichothecene, harzianum A (2). Its structure was determined by spectroscopic analyses including HRFAB-MS and various ¹H NMR and ¹³C NMR spectral data. Harzianum B (1) was characterised as (E,Z,E)-2′, 4′, 6′-octatriendioic acid esterified on the 4β-hydroxyl group of trichodermol. Harzianums A (2) and B (1) showed cytotoxicity against several human cancer cell lines.     

Harzianums A and B produced by a fungal strain, Hypocrea sp. F000527, and their cytotoxicity against tumor cell lines

A new compound, harzianum B (1), was isolated from the culture broth of a fungal strain, Hypocrea sp. F000527, together with a known trichothecene, harzianum A (2). Its structure was determined by spectroscopic analyses including HRFAB-MS and various ¹H NMR and ¹³C NMR spectral data. Harzianum B (1) was characterised as (E,Z,E)-2', 4', 6'-octatriendioic acid esterified on the 4β-hydroxyl group of trichodermol. Harzianums A (2) and B (1) showed cytotoxicity against several human cancer cell lines.     

Methyl protogracillin (NSC-698792): the spectrum of cytotoxicity against 60 human cancer cell lines in the National Cancer Institute's anticancer drug screen panel.

Methyl protogracillin (NSC-698792) was a furostanol saponin isolated from the rhizome of Dioscorea collettii var. hypoglauca (Dioscoreaceae), a Chinese herbal remedy for the treatment of cervical carcinoma, carcinoma of urinary bladder and renal tumor for centuries, in our previous studies. In order to systematically evaluate its potential anticancer activity, methyl protogracillin was tested for its cytotoxicity in vitro against 60 human cancer cell lines in the National Cancer Institute (NCI)'s anticancer drug screen. As a result, it was found that methyl protogracillin was cytotoxic against all the tested cell lines from leukemia and solid tumors in the NCI's human cancer panel; it showed particular selectivity against one colon cancer line (KM12), one central nervous system (CNS) cancer line (U251), two melanoma lines (MALME-3M and M14), two renal cancer lines (786-0 and UO-31) and one breast cancer line (MDA-MB-231) with GI50< or =2.0 microM. The selectivity between these seven most sensitive lines and the least sensitive line (CCRF-CEM) ranged from 26- to 56-fold. In the same cancer subpanel, selectivity more than 15-fold was observed between MDA-MB-231 and MCF-7, NCI-ADR-RES, BT-549 in breast cancer. From a general view of the mean graph, CNS cancer is the most sensitive subpanel, while ovarian cancer and renal cancer are the least sensitive subpanels. Based on an analysis of the COMPARE computer program with methyl protogracillin as a seed compound, no compounds in the NCI's anticancer drug screen database have similar cytotoxicity patterns (mean graph) to that of methyl protogracillin, indicating a potential novel mechanism of the anticancer action involved.