In vivo intracellular signaling as a marker of antiangiogenic activity

Alterations in endothelial cell (EC) signaling could serve as a marker of effective antiangiogenic therapy. We determined the effect of an antiangiogenic tyrosine kinase inhibitor, SU6668, on tumor EC signaling in liver metastases in mice. In vitro immunofluorescence verified that pretreatment of ECs with SU6668 before exposure to VEGF decreased in vitro phosphorylation of Erk and Akt. Using double-fluorescence immunohistochemistry, phosphorylated Erk and Akt were constitutively expressed in ECs in liver metastases in untreated mice, but SU6668 blocked activation of these signaling intermediates. Determining the activation status of the Erk and Akt signaling pathways in tumor ECs may serve as a surrogate marker for the effectiveness of antiangiogenic regimens.     

In vitro plasminogen activator activity in human brain tumors

Cell cultures were prepared from nine human brain tumors. Fibrin plate assays showed plasminogen-dependent fibrinolytic activity in lysates and in material released by these neoplastic cells but not in those from normal adult human white matter. Antibodies against human urokinase caused catalytic inhibition of the urokinase and of the plasminogen activator from WI-38 cells, simian virus 40-transformed WI-38 cells, human prostatic cells, and human ovarian carcinoma cells. However, the anti-urokinase immunoglobulin G did not inhibit the plasminogen activator activity of any of the human brain tumor preparations. These studies indicate that the plasminogen activator produced by human brain tumor cells is antigenically different from the plasminogen activator of other human normal and neoplastic cells.     

In vitro procoagulant activity induced in endothelial cells by chemotherapy and antiangiogenic drug combinations: modulation by lower-dose chemotherapy

One of the emerging problems concerning the use of antiangiogenic drugs, when used in combination with certain chemotherapy regimens, is enhanced rates and severity of adverse clotting events. For as yet unknown reasons, certain drugs and particular combinations can induce an elevated incidence of thromboembolic events in treated cancer patients [e.g., SU5416, a vascular endothelial cell growth factor receptor-2 (VEGFR-2) antagonist, when combined with gemcitabine and cisplatin (CDDP)]. Such results highlight the need to develop assays capturing the essence of enhanced clot formation under such combination treatment and which may have predictive potential as well. Here, we report the possibility of such an assay (i.e., the ratio of tissue factor over tissue factor pathway inhibitor expression or activity in cultured human endothelial cells calculated as a coagulation index). A marked increase in coagulation index was observed after exposure to SU5416 and the CDDP/gemcitabine chemotherapy combination in contrast to either of these treatments used alone. Substitution of SU5416 with any one of ZD6474, SU6668, IMC-1121, a monoclonal antibody to VEGFR-2, or an antibody to VEGF (bevacizumab) did not cause a marked increase in the coagulation index, nor did the combination of SU5416 with 5-fluorouracil and leucovorin. Finally, we noted that reducing the concentrations of gemcitabine and CDDP (i.e., use of "metronomic dosing" in vitro) significantly attenuated the coagulation index increase induced by these drugs, suggesting that use of low-dose chemotherapy regimens might be an approach to consider for reducing the incidence of adverse clotting events associated with chemotherapy alone or in conjunction with antiangiogenic drug combination therapies.     

In vivo and in vitro antitumor activity expressed by cells of concomitantly immune mice

Growth of a primary tumor is often accompanied by the development of resistance to subsequent challenge implants of the same tumor, i.e., concomitant immunity. Using the P815 mastocytoma tumor, the kinetics of concomitant immunity was found to be governed by duration of exposure to the tumor and tumor mass. By implanting small "challenges" prior to the immunizing tumor, resistance to the growth of existing tumor foci was demonstrated. Winn-type assays revealed that antitumor activity was present in cell populations from the peritoneal exudate and lymph node draining the tumor. Peritoneal exudate cells, when infused systemically, were also able to confer protection against P815 mastocytoma challenge, suggesting their role as mediators of concomitant immunity. The 51Cr release technique indicated that cytolytic activity in lymph node cells, peritoneal exudate cells, and the spleen was present over a time course parallel to the kinetics of in vivo challenge. The peritoneal resident cell population was only slightly active; thus, effectors accumulated in the inflammatory exudate. Removal of specific subsets of cells from effector populations with antibody to surface markers and complement produced similar effects on both Winn and cytolytic assays. Anti-Thy 1.2 ablated measurable activity. It was substantially but not completely reduced by anti-Lyt 1.1 and only to a small degree by anti-Lyt 2.1.     

In vivo assessment of antiangiogenic activity of SU6668 in an experimental colon carcinoma model.

PURPOSE: The purpose of this research was to assess in vivo by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) the antiangiogenic effect of SU6668, an oral, small molecule inhibitor of the angiogenic receptor tyrosine kinases vascular endothelial growth factor receptor 2 (Flk-1/KDR), platelet-derived growth factor receptor, and fibroblast growth factor receptor 1. EXPERIMENTAL DESIGN: A s.c. tumor model of HT29 human colon carcinoma in athymic mice was used. DCE-MRI with a macromolecular contrast agent was used to measure transendothelial permeability and fractional plasma volume, accepted surrogate markers of tumor angiogenesis. CD31 immunohistochemical staining was used for assessing microvessels density and vessels area. Experiments were performed after 24 h, and 3, 7, and 14 days of treatment. RESULTS: DCE-MRI clearly detected the early effect (after 24 h of treatment) of SU6668 on tumor vasculature as a 51% and 26% decrease in the average vessel permeability measured in the tumor rim and core (respectively). A substantial decrease was also observed in average fractional plasma volume in the rim (59%) and core (35%) of the tumor. Histological results confirmed magnetic resonance imaging findings. After 3, 7, and 14 days of treatment, postcontrast magnetic resonant images presented a thin strip of strongly enhanced tissue at the tumor periphery; histology examination showed that this hyperenhanced ring corresponded to strongly vascularized tissue adjacent but external to the tumor. Histology also revealed a strong decrease in the thickness of peripheral viable tissue, with a greatly reduced vessel count. SU6668 greatly inhibited tumor growth, with 60% inhibition at 14 days of treatment. CONCLUSIONS: DCE-MRI detected in vivo the antiangiogenic efficacy of SU6668.     

Prediction of in vivo synergistic activity of antiangiogenic compounds by gene expression profiling

Angiogenesis, an essential phenotype for tumor formation, requires the interaction of many cells within the tumor microenvironment. Therefore, successful antiangiogenic therapies must be able to block all of the different mechanisms tumors use to induce neovascularization. A major challenge for developing such protocols is determining which agents are likely to have the highest degree of synergistic activity in vivo. We treated human microvascular endothelial cells with six inhibitors of angiogenesis and used microarrays to seek divergent patterns of gene expression suggestive of potential synergies. The expression profiles of a thrombospondin-mimetic peptide (DI-TSPa) and TNP-470 (TNP) were very similar, whereas endostatin had a dramatically different profile. In vitro, endostatin was synergistically antiangiogenic with either TNP-470 or DI-TSPa. In vivo, mice bearing Lewis lung carcinoma cells treated with a combination of endostatin and either DI-TSPa or TNP-470, at doses that were ineffective when used alone, resulted in a marked inhibition of tumor growth and decreased tumor angiogenesis. Conversely, animals treated with both DI-TSPa and TNP-470 demonstrated a modest effect on both tumor growth and angiogenesis. These results suggest that even in the absence of a complete mechanistic understanding of how these inhibitors work, gene expression profiling may be used to predict synergistic antiangiogenic activity and thus maximize their antitumor efficacy.     

In vitro and in vivo anticancer activity of (+)-spongistatin 1

The marine natural product (+)-spongistatin 1 is an extremely potent growth inhibitory agent having activity against a wide variety of cancer cell lines, while exhibiting low cytotoxicity against quiescent human fibroblasts. Consistent with a microtubule-targeting mechanism of action, (+)-spongistatin 1 causes mitotic arrest in DU145 human prostate cancer cells. More importantly, (+)-spongistatin 1 exhibits significant in vivo antitumor activity in the LOX-IMVI human melanoma xenograft model. (+)-Spongistatin 1 is, thus, an important class of microtubule targeting anticancer agent that warrants further investigation.     

In vitro and in vivo pharmacokinetic/pharmacodynamic activity of clofoctol

Abstract

The aim of the study was to examine the in vitro susceptibility of clinical isolates of respiratory pathogens to clofoctol compared with amoxicillin and erythromycin, and to characterize the pharmacokinetic/pharmacodynamic (PK/PD) relationships of clofoctol using a murine pneumonia infection model. Strains clinically isolated from patients between 2005 and 2009 were used to examine susceptibility: penicillin-susceptible Streptococcus pneumoniae, penicillin-resistant S. pneumoniae, Streptococcus pyogenes, methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, and Haemophilus influenzae. The in vitro activity of clofoctol against clinical isolates has essentially remained unchanged over recent years. The MIC₅₀ and MIC₉₀ of clofoctol against penicillin-resistant S. pneumoniae are lower than that of amoxicillin and erythromycin. The area under curve/minimum inhibitory concentration (AUC/MIC) ratio is the PK/PD parameter that best correlates with in vivo clofoctol efficacy; the value of AUC/MIC required to achieve the maximum effect in this study was 75·5.     

In vitro and in vivo antitumor activity of lymphokine-induced cytotoxic cells

The present study demonstrates that LICC possess both in vitro and in vivo antitumor activity. The LICC were generated by culturing normal spleen cells with syngeneic peritoneal cells and indomethacin, or with a conditioned medium containing IL 2 with or without a putative new lymphokine, the CCDF. The LICC thus generated selectively killed the lymphoid or solid tumor targets of different H-2 haplotypes and of different etiological origins. The precursors of LICC were probably NK-like cells. The effectors were neither classical NK nor classical cytotoxic T lymphocytes. The LICC were very effective in preventing growth of both lymphoid and solid tumors in vivo, and Thy I+ cells were essential for the anti-tumor effect. The ability to generate LICC was preserved in the tumor-bearing hosts until the terminal stage of tumor growth, when the generation of suppressor T-cells interfered with LICC induction. LICC seem to play an important role in defense against non-immunogenic tumors.     

In vivo,ex vivo and in vitro dasatinib activity in chronic lymphocytic leukemia

Dasatinib inhibits the breakpoint cluster region-Abelson murine leukemia 1 (BCR-ABL1) gene along with other kinases known to be overexpressed and abnormally active in patients with chronic lymphocytic leukemia (CLL). The current study used primary leukemic cells obtained from 53 patients with CLL that were treated with dasatinib. A 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay and Annexin V staining was performed to assess the cytotoxic effects of dasatinib treatment. The XTT assay revealed that the median cytotoxicity of dasatinib was 8.30% (range, 0.00–77.89%). Due to high dispersion of dasatinib activity, patients were divided into sensitive (n=27; 50.94%; median cytotoxicity, 22.81%) and resistant groups (n=26; 49.06%; median cytotoxicity, 0.00%). A median cytotoxicity of 8.30% was selected as a cut off value. Using Annexin V staining and flow cytometry on exemplary sensitive and resistant CLL samples, it was revealed that 17.71 and 1.84% of cells were apoptotic, respectively. The current study presented a case of a patient with concomitant occurrence of CLL and chronic myeloid leukemia (CML) with a major molecular response after dasatinib treatment. A simultaneous reduction of circulating CLL cells indicated in vivo anti-CLL activity induced by dasatinib. After an in vitro culture of the patient''s mononuclear cells with subsequent dasatinib treatment, a higher percentage of CLL cells undergoing apoptosis was obsevered when compared with untreated samples (38.19 vs. 21.99%, respectively). Similarly, the percentage of CLL apoptotic cells (ΔΨm^low) measured by chloromethyl-X-rosamine was higher after incubation with dasatinib (7.28%) than in the negative control (2.86%). In conclusion, dasatinib induced antileukemic effects against CML and CLL cells. The results of the current study indicated that dasatinib may induce apoptosis ex vivo, in vitro and in vivo in CLL.     

In vitro and in vivo multidrug resistance reversal activity by a Betti-base derivative of tylosin

Background:

The multidrug resistance (MDR) proteins are present in a majority of human tumours. Their activity is important to understand the chemotherapeutic failure. A search for MDR-reversing compounds was conducted among various Betti-base derivatives of tylosin.

Methods:

Here, we evaluate the in vitro and in vivo P-glycoprotein (P-gp)-modulating activity of the most promising compound N-tylosil-1-α-amino-(3-bromophenyl)-methyl-2-naphthol (TBN) using human MDR1 gene-transfected and parental L5178 mouse lymphoma cell lines.

Results:

In vitro experiments showed that TBN dramatically increased the P-gp-mediated cellular uptake of the fluorescent substrate rhodamine 123. Similarly, TBN was found to act as a very potent enhancer of the cytotoxicity of doxorubicin on the resistant cell line. We also provide in vivo evidence using DBA/2 mice in support for an increased tumoural accumulation of doxorubicin, without affecting its tissue distribution, resulting in an enhanced antitumoural effect.

Conclusion:

Our results suggest that TBN is a potent modulator of the P-gp membrane pump and that the compound could be of clinical relevance to improve the efficacy of chemotherapy in MDR cancers.     

In vitro and in vivo activity of 12-O-3-N-dansylamino TPA

Dansyl-TPA, a fluorescent TPA analogue, which is a label with a high affinity for C3H/10T1/2 cells and induces ³H-choline release from these cells (Tran et al. Nouv. J. Chim 1984, 8, 751–757), has been studied for in vitro promoting activity in the same cell line initiated by a carcinogen MNNG and in vivo short-term mouse skin tests. In vitro, dansyl-TPA expresses transforming effect in its own (without MNNG pretreatment) as well as increases the production of transformed foci in MNNG-treated cells. In in vivo skin tests, dansyl-TPA displays lower effects than TPA on mouse skin. These results indicate a low promoting potential of dansyl-TPA.     

The chemopreventive agent oltipraz possesses potent antiangiogenic activity in vitro, ex vivo, and in vivo and inhibits tumor xenograft growth.

Angiogenesis plays a pivotal role in tumor growth and represents a key target for chemopreventive intervention. On the basis of the structural features and lack of target organ specificity of the synthetic dithiolethione oltipraz, inhibition of angiogenesis was assessed as a potential mechanism for its broad-based chemopreventive activity. The effects of oltipraz on the development and maturation of a vascular network was determined in vitro using two-dimensional capillary tube formation assays with human umbilical vein endothelial cells plated on Matrigel and ex vivo using primary rat aortic ring explant cultures in three-dimensional collagen gels, respectively. The antiangiogenic and antitumor efficacy of oltipraz administration in vivo in nude mice was evaluated by determining its effects on neovascularization in s.c. Matrigel implants seeded with vascular endothelial growth factor and basic fibroblast growth factor-stimulated porcine aortic endothelial cells and on tumor growth and angiogenesis in SVR murine angiosarcoma xenografts implanted s.c. A dose-dependent reduction (0.4-100 microM) in microvessel formation was observed in both human and rodent bioassays after oltipraz exposure, with inhibition approaching 100% in the rat aortic ring assay at the highest concentration (P < 0.01). Similarly, oltipraz (40 microM) inhibited complete capillary tube formation by human umbilical vein endothelial cells by 62% (P < 0.05) relative to control cultures. p.o. administration of oltipraz (250 mg/kg/day for 6 days) to nude mice implanted with porcine aortic endothelial cell-Matrigel plugs resulted in a 42% reduction in neovascularization (P < 0.05) relative to vehicle-treated control mice. Administration of the same dose of oltipraz to athymic mice bearing established s.c. SVR angiosarcoma xenografts for 10 days resulted in a significant inhibition of tumor growth as early as day 4 of dosing (P < 0.005), with a maximum inhibition of tumor growth (81%, P < 0.001) relative vehicle-treated mice by day 10. The observed efficacy of oltipraz in this model is comparable with that of SU 5416 and TNP-470, known antiangiogenic agents currently under clinical development. Plasma levels of oltipraz at the termination of in vivo efficacy studies were 66.4 +/- 7 microM as determined by reversed phase high-performance liquid chromatography, a concentration range associated with significant antiangiogenic activity of oltipraz in vitro and ex vivo. These data suggest that the chemopreventive agent oltipraz may be effective in the treatment of advanced stage cancers and metastases, in part, because of its antiangiogenic activity in vivo.     

In vitro apoptotic induction of human glioblastoma cells by Fas ligand plus etoposide and in vivo antitumour activity of combined drugs in xenografted nude rats

Human glioblastomas that express Fas/CD95 receptor are highly resistant to conventional brain tumour therapies. The aim of this study is to evaluate anti-tumour properties of a combination of Fas ligand (FasL) plus etoposide with or without dexamethasone on intracerebral experimental glioblastomas. The human Fas-expressing glioblastoma cell line, U-87 MG, was firstly studied in vitro for apoptosis and proliferation assays in the presence of FasL and etoposide, separately or associated, in order to detect a supra-additive effect on FasL or etoposide-induced apoptosis. The tumourigenicity of the U-87 MG cell line and therapeutic effects of FasL-etoposide alone or combined with dexamethasone were next studied on U-87 MG cells xenografted to nude-rat brain and tumour size was hence examined by histological and immunohistochemical stainings. We demonstrated in vitro that the combination of both molecules strongly inhibited the proliferation rate and increased the sensitivity of glioblastoma cells to Fas or etoposide-mediated apoptosis. Moreover, analysis of rat brains was performed 30 days after xenograft of glioblastoma cells. These data show that the combination of FasL and etoposide could reduce significantly the tumour size and that the addition of dexamethasone enhanced the inhibiting effect of FasL and etoposide on tumour growth in vivo.     

Expression of heterogeneous profiles of plasminogen activators and plasminogen activator inhibitors by human glioma lines

Expression of plasminogen activator (PA) enzyme activity is believed to be one of the mechanisms by which malignant cells cause pericellular proteolysis of stromal structures during implantation and tissue invasion. In this study, four cell lines derived from human gliomas were studied to ascertain which PA enzymes and PA inhibitors determine the level of secreted PA activity. A plasminogen-dependent esterolytic assay was used, and two lines (U251 and U373) were found to secrete high levels of PA activity, while PA activity was undetectable in the conditioned media from the remaining two lines (U138 and LM). The PA produced by U251 and U373 resolved as single bands comigrating with high molecular weight urokinase (Mr 54,000) on casein-plasminogen zymography. Northern blot analysis demonstrated high levels of mRNA for urokinase-type PA (uPA) in U251 and U373, as well as a considerably lower level of uPA message in LM. U251 and U373 also contained mRNA for tissue-type PA (tPA), although secreted tPA activity was not demonstrated by zymography. The U138 line contained essentially undetectable levels of mRNA for either uPA or tPA. U138 was also unique in secreting PA inhibitor activity and contained high levels of mRNA for PA inhibitor 2, which was not seen in any other line. All cell lines contained PA inhibitor 1 mRNA, with substantially more expression in the U138 and LM lines than in U251 and U373. None of the lines secreted measureable anti-plasmin activity. We conclude that there is considerable heterogeneity among human glioma cells in expression of PA enzymes and PA inhibitors. The coordinated regulation of these proteins likely determines secreted PA activity and the resultant role of plasminogen activation in tumor implantation and invasion.     

Localization of plasminogen activators in human colon cancer by immunoperoxidase staining

The immunoperoxidase technique, using antibodies against human urinary urokinase (Mr 55,000), was used for the localization of this enzyme in histological preparations of human colon tumors and normal colon tissue. The localization of tissue (vascular) activator was also investigated using antibodies against enzyme purified from human malignant melanoma. Both the "indirect method" and the peroxidase-antiperoxidase technique were found to be useful. Urokinase-reactive material was found in all tissues examined (33 primary cancers, 11 metastases, and 8 adenomas). In the normal colon, urokinase was found only in some of the goblet cells of the mucosal epithelium. In colon cancer, diffuse specific staining was observed in the cytoplasm, but the most intense staining was localized at the edge of the cancer cells bordering the lumen of the glands. In some cases, intense supranuclear staining could be observed in a location corresponding to the Golgi apparatus. In a few instances, urokinase could be seen associated with fibroblasts near the advancing front of an invading tumor. Adenoma, a benign tumor but often a precursor of cancer, also showed the presence of urokinase. Most significant were the observations showing that, in regions of the mucosal glands where normal epithelial cells were abruptly replaced by cancer cells, the appearance of cytoplasmic urokinase showed strict and exclusive association with the malignant cells, and the same was the case in transitions from normal epithelium to adenoma. In contrast to urokinase, tissue plasminogen activator was not associated with cancer cells, but was consistently present in the stroma which separates the cancer glands and was localized in the endothelium of the blood vessels. This visual evidence was supported by results of extraction of plasminogen activators from tumors, and from the separated mucosal and submucosal layers of the normal colon of the same patients, which showed that urokinase is most abundant in the tumor tissue and least abundant in the submucosa, while tissue activator is most prevalent in the well-vascularized mucosa and submucosa and scarce in the usually poorly vascularized adenocarcinomas.     

In vitro and in vivo radiosensitization induced by hydroxyapatite nanoparticles

Background

Previous study showed that hydroxyapatite nanoparticles (nano-HAPs) inhibited glioma growth in vitro and in vivo; and in a drug combination, they could reduce adverse reactions. We investigated the possible enhancement of radiosensitivity induced by nano-HAPs.

Methods

In vitro radiosensitization of nano-HAPs was measured using a clonogenic survival assay in human glioblastoma U251 and breast tumor brain metastatic tumor MDA-MB-231BR cells. DNA damage and repair were measured using γH2AX foci, and mitotic catastrophe was determined by immunostaining. The effect of nano-HAPs on in vivo tumor radiosensitivity was investigated in a subcutaneous and an orthotopic model.

Results

Nano-HAPs enhanced each cell line''s radiosensitivity when the exposure was 1 h before irradiation, and they had no significant effect on irradiation-induced apoptosis or on the activation of the G₂ cell cycle checkpoint. The number of γH2AX foci per cell was significantly large at 24 h after the combination modality of nano-HAPs + irradiation compared with single treatments. Mitotic catastrophe was also significantly increased at an interval of 72 h in tumor cells receiving the combined modality compared with the individual treatments. In a subcutaneous model, nano-HAPs caused a larger than additive increase in tumor growth delay. In an orthotopic model, nano-HAPs significantly reduced tumor growth and extended the prolongation of survival induced by irradiation.

Conclusions

These results show that nano-HAPs can enhance the radiosensitivity of tumor cells in vitro and in vivo through the inhibition of DNA repair, resulting in an increase in mitotic catastrophe.     

Norcantharidin: a potential antiangiogenic agent for gallbladder cancers in vitro and in vivo

Our objective was to explore the antiangiogenic activity of norcantharidin (NCTD) as an angiogenic inhibitor for gallbladder cancers. In vitro and in vivo experiments to determine the effects of NCTD on HUVECs, chicken CAM capillaries and gallbladder cancer xenograft angiogenesis in nude mice were respectively done. The MTT method was used to assay the cytotoxicity of NCTD on HUVECs. Immunofluorescence was used to evaluate HUVEC apoptosis. The scraping line method, matrigel invasion assay and tube formation assay were used to detect the migration, invasion and tube formation of HUVECs. A digital camera was used to observe chicken CAM capillaries. Experiments with NCTD in a xenograft model were used to observe the effect of NCTD on xenograft growth and survival of mice with xenografts. CD?? immunohistochemistry, flow cytometry and micro-MRA were used, respectively, to determine MVD, cell apoptosis and hemodynamic analysis of the xenografts. Immunohistochemistry and RT-PCR were used, respectively, to detect the expression of VEGF, Ang-2, TSP, TIMP-2 proteins/mRNAs of the xenografts. The xenograft MVD associated with tumor volume, the PCNA/apoptosis ratio and related-protein expression was evaluated simultaneously. We found that NCTD effectively inhibited the proliferation, migration, invasion and capillary-like tube formation of HUVECs in vitro; it reduced angiogenesis and directly destroyed the formed CAM capillaries in vivo. In the experiments in mice, NCTD not only inhibited significantly xenograft proliferation and growth, prolonged survival time of mice with xenografts, decreased the xenograft MVD and vascular perfusion, but also, similarly to ES, decreased significantly the expression of VEGF or Ang-2 protein/mRNA, increased the expression of TSP or TIMP-2 protein/mRNA. Moreover, the xenograft MVD was positively related with tumor volume, PCNA/apoptosis ratio, and VEGF or Ang-2 expression, respectively (all P<0.05), but negatively correlated with TSP or TIMP-2 expression (both P<0.05). These data showed that NCTD could serve as a potential antiangiogenic agent for gallbladder cancers.     

In vitro and in vivo enhancement of ricin-A chain immunotoxin activity by novel indolizine calcium channel blockers: delayed intracellular degradation linked to lipidosis induction.

With regard to increasing the clinical potential of ricin A-chain immunotoxins (RTA-ITs), a novel class of calcium channel blockers, indolizines SR33557 [2-isopropyl-1-[4-(3-N-methyl-N-(3,4-dimethoxy-beta- phenethyl)amino)propyloxy)benzenesulfonyl))indolizine] and SR33287 [isopropyl-2-((1-butylamino-3-propyl)oxy-4-benzoyl)-3-indolizine], were evaluated for their ability to enhance RTA-IT activity in vitro and in vivo. Five microM SR33287 and 5 microM SR33557 were potent enhancers of both anti-Thy 1.2 AT15E RTA-IT (84- and 64-fold, respectively) on T2 cells and anti-CD5 T101 (622- and 538-fold) and T101 F(ab')2 RTA-IT (34- and 28-fold) on CEM III cells. This was superior to the effect achieved by both 10 microM verapamil and 10 mM NH4Cl, albeit slightly inferior to that of 50 nM monensin and 5 microM perhexiline. Murine T2 lymphoma cells bearing the Thy 1.2 antigen were injected i.v. in Thy 1.2 (-) BL. 1.1 mice (median survival time, 17.7 days). Intravenous treatment with 10 micrograms of AT15E RTA-IT prolonged the survival of mice (median survival time, 26.8 days). When 400 micrograms of SR33287 were coinjected i.v. with 10 micrograms of AT15E RTA-IT, mouse survival was further increased, with 5 of 6 mice surviving, disease free, over 42 days. SR33287 had a significant impact on the intracellular routing of 125I-AT15E RTA-IT, which induced a greater than 2-fold increase in intracellular intact AT15E RTA-IT at 90 min. This effect on RTA-IT half-life was distinctly different from that observed with either NH4Cl or monensin and may be linked to the inhibition of acid lysosomal sphingomyelinase by SR33287, leading to cellular lipidosis. In conclusion, indolizines appear to be promising agents not only for immunotoxin enhancement but also for increasing the activity of any number of targeted therapeutic agents where modifying either the intracellular routing or increasing the intracellular half-life of the ligand would be beneficial to its cytotoxic activity.     

Shikonin exerts antitumor activity via proteasome inhibition and cell death induction in vitro and in vivo

Dysregulation of the ubiquitin‐proteasome pathway plays an essential role in tumor growth and development. Shikonin, a natural naphthoquinone isolated from the traditional Chinese medicine Zi Cao (gromwell), has been reported to possess tumor cell‐killing activity, and results from a clinical study using a shikonin‐containing mixture demonstrated its safety and efficacy for the treatment of late‐stage lung cancer. In this study, we reported that shikonin is an inhibitor of tumor proteasome activity in vitro and in vivo. Our computational modeling predicts that the carbonyl carbons C₁ and C₄ of shikonin potentially interact with the catalytic site of β5 chymotryptic subunit of the proteasome. Indeed, shikonin potently inhibits the chymotrypsin‐like activity of purified 20S proteasome (IC₅₀ 12.5 μmol/L) and tumor cellular 26S proteasome (IC₅₀ between 2–16 μmol/L). Inhibition of the proteasome by shikonin in murine hepatoma H22, leukemia P388 and human prostate cancer PC‐3 cultures resulted in accumulation of ubiquitinated proteins and several proteasome target proapoptotic proteins (IκB‐α, Bax and p27), followed by induction of cell death. Shikonin treatment resulted in tumor growth inhibition in both H22 allografts and PC‐3 xenografts, associated with suppression of the proteasomal activity and induction of cell death in vivo. Finally, shikonin treatment significantly prolonged the survival period of mice bearing P388 leukemia. Our results indicate that the tumor proteasome is one of the cellular targets of shikonin and inhibition of the proteasome activity by shikonin contributes to its antitumor property. © 2008 Wiley‐Liss, Inc.