Kaoru Terai

Addition of receptor tyrosine kinase inhibitor to radiation increases tumour control in an orthotopic murine model of breast cancer metastasis in bone

The receptor tyrosine kinase inhibitor, SU11248, was added to localised radiation to evaluate the response of bone metastases and to define the basic mechanism of radiosensitisation. Treatment with SU11248 and radiation was assessed in vitro using cultured 4T1 breast cancer cells and in vivo using an orthotopic 4T1 murine mammary tumour model of breast cancer bone metastasis. Cultured 4T1 cells treated with SU11248 (1 microM) and radiation (10 Gy) showed an almost 7.5-fold increase in caspase-mediated apoptosis after 24 h of incubation, compared to either treatment alone. Mice treated with SU11248 (40 mg/kg/daily) and radiation (15 Gy/single-dose) had a relatively greater reduction in tumour growth, bone osteolysis, osteoclast maturation and microvessel density. Combined modality treatment resulted in improvements in behavioural pain assessment scores and normalisation of neurochemical changes in the spinal cord receiving primary afferent innervation from tumour-bearing femora. Our study demonstrates that SU11248 enhances the radiation control of metastatic breast tumours in bone and tumour-induced pain.

Protein kinase C-β inhibitor enzastaurin (LY317615.HCI) enhances radiation control of murine breast cancer in an orthotopic model of bone metastasis

SummaryRadiation therapy is a widely used treatment for metastatic bone cancer, but the rapid onset of tumor radioresistance is a major problem. We investigated the radiosensitizing effect of enzastaurin, a protein kinase Cβ (PKCβ) inhibitor, on bone tumor growth and tumor-related pain. We found that enzastaurin enhanced the effect of ionizing radiation on cultured murine 4T1 breast cancer and murine endothelial cells, suppressing their proliferation and colony formation. Enzastaurin and ionizing radiation also induced caspase-mediated apoptosis of 4T1 cells to a greater degree than radiation alone. Enzastaurin treatment of 4T1 cells blocked the phosphorylation of PKCβ, as well as Ras and two of its downstream effectors ERK1/2 and RAL-GTP. Using an orthotopic model of bone metastasis, we observed that a combination of enzastaurin and localized radiation treatment reduced tumor blood vessel density, bone destruction and pain compared to single modality treatment. In conclusion, we demonstrate that inhibition of PKCβ in combination with localized radiation treatment suppresses tumor growth and alleviates pain as compared to radiation-only treatment. We also show that the radiosensitizing effect of enzastaurin is associated with suppression of tumor cell proliferation and tumor-induced angiogenesis possibly through inhibition of the Ras pathway.

Antiangiogenesis therapy using a novel angiogenesis inhibitor, anginex, following radiation causes tumor growth delay

BackgroundThe present study investigated whether treatment with anginex, a novel antiangiogenic peptide, could block re-vascularization after radiation treatment.MethodsA squamous cell (SCCVII) xenograft tumor mouse model was employed to assess the effects of anginex given post-radiation on tumor growth, microvessel density (MVD), and oxygen levels. The oxygen status was determined by the partial pressure of O2.ResultsTumors in untreated mice increased threefold in 7.0 days, anginex-treated tumors (10 mg/kg intraperitoneal, twice) required 7.3 ± 0.9 days, and tumors exposed to 8-Gy radiation increased threefold over 11 days. Combination treatment of anginex and radiation caused the tumors to grow threefold in 16.1 ± 1.6 days, a delay which was significant and deemed supra-additive. Oxygen levels in tumors treated by stand-alone or combination therapies were significantly reduced; for example from 19.5 ± 4.9 mmHg in controls to 9.7 ± 1.9 mmHg in combination-treated, size-matched tumors. In addition, immunohistochemistry showed a decrease in MVD in the tumors treated with anginex, radiation, or the combination. These results suggest that a combination of anginex and radiation can greatly affect the amount of functional vasculature in tumors and prolong radiation-induced tumor regression.ConclusionAntiangiogenesis therapy with anginex, in addition to radiotherapy, will be useful by blocking angiogenesis-dependent regrowth of vessels.

Cisplatin enhances the anticancer effect of β-lapachone by upregulating NQO1

NAD(P)H:quinone oxidoreductase (NQO1) has been reported to play an important role in cell death caused by &bgr;-lapachone (&bgr;-lap), 3,4-dihydro-22,2-dimethyl-2H-naphthol[1,22b]pyran-5,6-dione. This study investigated whether cisplatin (cis-diamminedichloroplatinum) sensitizes cancer cells to &bgr;-lap by upregulating NQO1. The cytotoxicity of cisplatin and &bgr;-lap alone or in combination against FSaII fibrosarcoma cells of C3H mice in vitro was determined with a clonogenic survival assay and assessment of &ggr;-H2AX foci formation, a hallmark of DNA double-strand breaks. The cellular sensitivity to &bgr;-lap progressively increased during the 24 h after cisplatin treatment. The expression and enzymatic activity of NQO1 also increased during the 24 h after cisplatin treatment, and dicoumarol, an inhibitor of NQO1, was found to nullify the cisplatin-induced increase in &bgr;-lap sensitivity. The role of NQO1 in the cell death caused by &bgr;-lap alone or in combination with cisplatin was further elucidated using NQO1-positive and NQO1-negative MDA-MB-231 human breast cancer cells. Cisplatin increased the sensitivity of the NQO1-positive but not the NQO1-negative MDA-MB-231 cells to &bgr;-lap treatment. Combined treatment with cisplatin and &bgr;-lap suppressed the growth of FSaII tumors in the legs of C3H mice in a manner greater than additive. It is concluded that cisplatin markedly increases the sensitivity of cancer to &bgr;-lap in vitro and in vivo by upregulating NQO1.

Blood outgrowth endothelial cell-based systemic delivery of antiangiogenic gene therapy for solid tumors

Endothelial cells and endothelial cell precursors encoding a therapeutic gene have induced antitumor responses in preclinical models. Culture of peripheral blood provides a rich supply of autologous, highly proliferative endothelial cells, also referred to as blood outgrowth endothelial cells (BOECs). The aim of this study was to evaluate a novel antiangiogenic strategy using BOECs expressing fms-like tyrosine kinase-1 (sFlt1) and/or angiostatin–endostatin (AE) fusion protein. Conditioned medium from BOECs expressing sFlt1 or AE suppressed in vitro growth of pulmonary vein endothelial cells by 70% compared with conditioned medium from non-transduced BOEC controls. Reverse transcriptase-PCR analysis indicated that systemically administered BOECs proliferated in tumor tissue relative to other organs in C3(1)SV40 TAG transgenic (C3TAG) mice with spontaneous mammary tumors. Tumor volume was reduced by half in C3TAG mice and in mice bearing established lung or pancreatic tumors in response to the treatment with sFlt1-BOECs, AE-BOECs or their combination. Studies of tumor vascular density confirmed that angiogenic inhibition contributed to slowed tumor growth. In an orthotopic model of glioma, the median survival of mice treated with sFlt1-BOECs was double that of mice receiving no BOEC treatment (P=0.0130). These results indicate that further research is warranted to develop BOECs for clinical application.

Novel Ras pathway inhibitor induces apoptosis and growth inhibition of K-ras-mutated cancer cells in vitro and in vivo

MT477 is a novel quinoline with potential activity in Ras-mutated cancers. In this study, MT477 preferentially inhibited the proliferation of K-ras-mutated human pulmonary (A549) and pancreatic (MiaPaCa-2) adenocarcinoma cell lines, compared with a non-Ras-mutated human lung squamous carcinoma cell line (H226) and normal human lung fibroblasts. MT477 treatment induced apoptosis in A549 cells and was associated with caspase-3 activation. MT477 also induced sub-G1 cell-cycle arrest in A549 cells. Although we found that MT477 partially inhibited protein kinase C (PKC), it inhibited Ras directly followed in time by inhibition of 2 Ras downstream molecules, Erk1/2 and Ral. MT477 also caused a reorganization of the actin cytoskeleton and formation of filopodias in A549 cells; this event may lead to decreased migration and invasion of tumor cells. In a xenograft mouse model, A549 tumor growth was inhibited significantly by MT477 at a dose of 1 mg/kg (P < 0.05 vs vehicle control). Taken together, these results support the conclusion that MT477 acts as a direct Ras inhibitor. This quinoline, therefore, could potentially be active in Ras-mutated cancers and could be developed extensively as an anticancer molecule with this in mind.

Novel insulin-like growth factor-methotrexate covalent conjugate inhibits tumor growth in vivo at lower dosage than methotrexate alone

The insulin-like growth factor receptor is overexpressed on many types of cancer cells and has been implicated in metastasis and resistance to apoptosis. We report here the development of a novel covalent conjugate that contains the antifolate drug methotrexate coupled to an engineered variant of insulin-like growth factor-1 (IGF-1), long-R3-IGF-1, which was designed to target methotrexate to tumor cells that overexpress the membrane IGF-1 receptor. The IGF-methotrexate conjugate was found to contain at least 4 methotrexate molecules per IGF-1 protein. The IGF-methotrexate conjugate bound to MCF7 breast cancer cells with greater than 3.3-fold higher affinity than unconjugated long-R3-IGF-1 in a competition binding assay against radiolabeled wild-type IGF-1. Compared with free methotrexate, the IGF-methotrexate conjugate required slightly higher concentrations to inhibit the in vitro growth of the human prostate cancer cell line LNCaP. In vivo, however, in a mouse xenograft model using LNCaP cells, the IGF-methotrexate conjugate was more effective than free methotrexate even at a 6.25-fold lower molar dosage. Similarly, MCF7 xenografts were inhibited more effectively by the IGF-methotrexate conjugate than free methotrexate, even at a 4-fold lower molar dosage. Our results suggest that the targeting of the IGF receptor on tumor cells and tumor-related tissues with IGF-chemotherapy conjugates may substantially increase the specific drug localization and therapeutic effect in the tumor.

Enzastaurin renders MCF-7 breast cancer cells sensitive to radiation through reversal of radiation-induced activation of protein kinase C

Enzastaurin (LY317615.HCI), a protein kinase C (PKC)-beta inhibitor, has a radiosensitising effect on 4T1 murine breast cancer and human glioma cells; however, the exact mechanism of this action has not been evaluated. The present study investigated the effects of enzastaurin and gamma irradiation on PKC activity in MCF-7 human breast cancer cells in vitro and in vivo. Enzastaurin (5 microM) in combination with irradiation (2-8 Gy) produced a synergistic decline in MCF-7 clonogenic cell survival. Analysis of MCF-7 cells stained with Annexin V and 7-aminoactinomycin D showed a dose-dependent increase in apoptosis in response to enzastaurin (3, 5 and 7 microM) and irradiation (10 Gy) compared to irradiation alone. This pro-apoptotic effect was confirmed by increases in caspase-3 and -9 activity. In a MCF-7 xenograft model, irradiation with 25 Gy increased PKC-alpha activity by 2.5-fold compared to untreated controls, whereas PKC-epsilon and -betaII activity was increased by 1.8-fold. Radiation-induced activation of all three anti-apoptotic isoforms of PKC was reversed by pre-treatment with enzastaurin (75 mg/kg, twice daily for 3 days). We conclude that enzastaurin has a radiosensitising effect on MCF-7 human xenograft tumours through the reversal of anti-apoptotic activation of PKC isoforms.

Correction: Endothelial Expression of Endothelin Receptor A in the Systemic Capillary Leak Syndrome.

The captions for Figs ​Figs11 and ​and22 are switched. The caption that appears with Fig 1 corresponds to Fig 2, and the caption that appears with Fig 2 corresponds to Fig 1. Please see the figures with the appropriate captions below. Fig 1 Gene expression in BOEC from individual subjects. Fig 2 Morphology and gene expression in BOEC from SCLS patients and controls.

SU‐FF‐T‐275: Invitro Dose and Dose Rate Feasibility Study for Effective Tomotherapy TMI Treatment

Purpose: We reported the successful use of Tomotherapy for the total marrow irradiation (TMI) treatment. However, Tomotherapy delivers treatment at a much higher dose rate (400–900 cGy/min) than current TBI treatment (10 – 25 cGy/min) delivered using a standard medical accelerator. Even with the more accurate dosimetry possible with Tomotherapy, the effect of high dose rates even at limited doses is unclear at best. We aim to study the effect of high dose and dose rate on cell survival. Method and Materials: A549 human lungcells, BE(2)‐M17 human neuroblastoma cells and N2A mouse neuroblastoma cells were used. Cells were plated in T‐25 flasks with appropriate medium. After 8 hours attachment period, each flask of cells were irradiated with a different dose: 2, 4, 6, 9 and 12 Gy. As the dose rate, 112 cGy/min was used in A549 and BE(2)‐M17 cells and 400 cGy/min was used in N2A cells.Cells were cultured for 10–15 days and survived cells were making colonies. Colonies were fixed with a mixture of methanol and acetic acid (10:1 v/v) and stained with 1% crystal violet. Colonies containing more than 30 cells were scored. Results: BE(2)‐M17 cells were more sensitive to radiation than A549 and N2A cells. Mean lethal dose, D0, were 89, 136 and 188 rad in BE(2)‐M17, A549 and N2A cells in each. Plating efficiency at 6Gy were 0.50, 6.23 and 6.97 % in BE(2)‐M17, A549 and N2A cells in each. Extrapolation number were 8.17, 5.20 and 2.51 in BE(2)‐M17, A549 and N2A cells in each. Conclusions: We show that different cell lines have different dose response invitro. Currently we are continuing the survival study for dose rate from 10 cGy/min to 800 cGy/min which will be reported in the conference.