Ya-Fang Chang

Antitumor effect of orlistat, a fatty acid synthase inhibitor, is via activation of caspase-3 on human colorectal carcinoma-bearing animal.

We established a HT-29/tk-luc human colorectal carcinoma-bearing animal model for the study of the inhibition effect and mechanism of orlistat, a fatty acid synthase (FASN) inhibitor. The results showed that orlistat caused cell cycle arrest at G1 phase, and triggered apoptosis through caspase-3 activation. The tumor inhibition effect of orlistat may also due to the inhibition of fatty acid synthesis without altering FASN activity. The tumor size of orlistat-treated mice in vivo was significantly smaller than that of the controls with 55% inhibition. The therapeutic efficacy was further confirmed with the bioluminescent imaging and nuclear molecular imaging with ¹³¹I-FIAU/gamma scintigraphy and ¹¹C-acetate/microPET. We suggest that FASN is a potential target for the treatment of human colorectal carcinoma.

Sorafenib sensitizes human colorectal carcinoma to radiation via suppression of NF-κB expression in vitro and in vivo.

Over-expression of transcription factor nuclear factor-κB (NF-κB) in the residual tumor after the treatment is often observed at the later period of cancer radiotherapy, results in tumor radioresistance and poor treatment outcome. In this study, we combined sorafenib, a multikinase inhibitor, with ionizing radiation to evaluate the therapeutic effect, and to elucidate the possible mechanism responsible for the radiosensitization of sorafenib on a human HT29/tk-luc colorectal carcinoma in vitro and in vivo. Clonogenic survival and cell cycle analysis were used to evaluate the cytotoxicity of sorafenib in vitro. The combination effect and the role of NF-κB in therapeutic efficacy with respects in apoptosis and tumor cell invasion were studied with HT29/tk-luc tumor-bearing animal model. The expression of NF-κB and its downstream-related proteins were assayed with electrophoretic mobility shift assay (EMSA) and Western blot. Sorafenib combined with radiation shows the synergistic cytotoxicity on HT29/tk-luc cells and increased tumor cell apoptosis. Both EMSA and Western blot show that the NF-κB activity induced by radiation is significantly suppressed by sorafenib. Combination of sorafenib and radiation shows the maximum tumor growth inhibition as compared to that of sorafenib alone or radiation alone in vivo (P<0.001). In conclusion, the effect of sorafenib combined with radiation for the treatment of human colorectal carcinoma is synergistic. The mechanism of synergism is through the inhibition of radiation-induced NF-κB expression and its regulated downstream gene products.

Targeted therapy with fatty acid synthase inhibitors in a human prostate carcinoma LNCaP/ tk-luc -bearing animal model

Background:Fatty acid synthase (FASN) is highly upregulated in human prostate carcinomas. Inhibition of FASN could arrest cell cycle and trigger apoptosis rapidly, implying the reliance of cancer cell survival on FASN. However, little is known about the effect of C75, a FASN inhibitor, and siFASN (that is, small interfering RNA targeted at FASN) on prostate cancer in living subjects.Methods:We used C75 and siFASN to mediate the endogenous fatty acid metabolism in LNCaP human prostate cancer cells stably expressing herpes simplex virus type 1 thymidine kinase (HSV1-tk) and luciferase (luc) reporter genes, and assessed the effect of FASN blockade with different schedules of administration on tumor growth using noninvasive molecular imaging.Results:FASN blockade exhibited the proliferative inhibition and induced G1-phase cell cycle arrest of LNCaP cells. For in vivo studies, the tumor growth inhibition by C75 (total 120 mg kg−1; 30 mg kg−1 once a week or 15 mg kg−1 twice a week for 4 weeks) and siFASN (1.4 mg kg−1 every alternate day up to 16 days) treatments were 80% and 70%, respectively, compared with that of the control.Conclusion:The results suggest that C75 may be superior to siFASN in anticancer effect on prostate cancer.

Immunomodulation of Curcumin on Adoptive Therapy with T Cell Functional Imaging in Mice

Adoptive T-cell therapy involves the ex vivo expansion and subsequent transfusion of tumor-specific T lymphocytes to eliminate tumors. Using immune modulators to block immunosuppressive factors in the tumor microenvironment has emerged as a promising strategy to enhance T-cell–mediated tumor regression. Curcumin, a major component of turmeric, has been shown to possess antitumor and immunomodulatory effects by regulating a diverse range of molecular targets. Thus, we hypothesize that these beneficial effects of curcumin may improve the therapeutic efficacy of adoptive therapy. Here, we have shown that curcumin enhances cytotoxicity of CD8+ T cells toward tumors via alteration of the tumor microenvironment when combined with adoptive therapy. We found that T-cell accumulation and function were increased in combined treatment due to the blockade of different immunosuppressors, including TGF-β, indoleamine 2,3-dioxygenase, and regulatory T cells. Furthermore, bioluminescent imaging with a granzyme B promoter–conjugated optical reporter also reflected improved cytotoxicity of antigen-specific CD8+ T cells in tumor-bearing mice during treatment. These findings suggest that combination of multitargeting drugs, such as curcumin, with adoptive therapy may have potential for clinical application. In addition, using a granzyme B–specific imaging reporter to assess T-cell function may also be applied for the development and therapeutic evaluation of new immunotherapy in preclinical studies. Cancer Prev Res; 5(3); 444–52. ©2011 AACR.

Serial Low Doses of Sorafenib Enhance Therapeutic Efficacy of Adoptive T Cell Therapy in a Murine Model by Improving Tumor Microenvironment

Requirements of large numbers of transferred T cells and various immunosuppressive factors and cells in the tumor microenvironment limit the applications of adoptive T cells therapy (ACT) in clinic. Accumulating evidences show that chemotherapeutic drugs could act as immune supportive instead of immunosuppressive agents when proper dosage is used, and combined with immunotherapy often results in better treatment outcomes than monotherapy. Controversial immunomodulation effects of sorafenib, a multi-kinases inhibitor, at high and low doses have been reported in several types of cancer. However, what is the range of the low-dose sorafenib will influence the host immunity and responses of ACT is still ambiguous. Here we used a well-established E.G7/OT-1 murine model to understand the effects of serial low doses of sorafenib on both tumor microenvironment and transferred CD8+ T cells and the underlying mechanisms. Sorafenib lowered the expressions of immunosuppressive factors, and enhanced functions and migrations of transferred CD8+ T cells through inhibition of STAT3 and other immunosuppressive factors. CD8+ T cells were transduced with granzyme B promoter for driving imaging reporters to visualize the activation and distribution of transferred CD8+ T cells prior to adoptive transfer. Better activations of CD8+ T cells and tumor inhibitions were found in the combinational group compared with CD8+ T cells or sorafenib alone groups. Not only immunosuppressive factors but myeloid derived suppressive cells (MDSCs) and regulatory T cells (Tregs) were decreased in sorafenib-treated group, indicating that augmentation of tumor inhibition and function of CD8+ T cells by serial low doses of sorafenib were via reversing the immunosuppressive microenvironment. These results revealed that the tumor inhibitions of sorafenib not only through eradicating tumor cells but modifying tumor microenvironment, which helps outcomes of ACT significantly.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part I. Reporter Gene Design, Characterization, and Optical in Vivo Imaging of Bone Marrow Stromal Cells after Myocardial Infarction.

Purpose To use multimodality reporter-gene imaging to assess the serial survival of marrow stromal cells (MSC) after therapy for myocardial infarction (MI) and to determine if the requisite preclinical imaging end point was met prior to a follow-up large-animal MSC imaging study. Materials and Methods Animal studies were approved by the Institutional Administrative Panel on Laboratory Animal Care. Mice (n = 19) that had experienced MI were injected with bone marrow-derived MSC that expressed a multimodality triple fusion (TF) reporter gene. The TF reporter gene (fluc2-egfp-sr39ttk) consisted of a human promoter, ubiquitin, driving firefly luciferase 2 (fluc2), enhanced green fluorescent protein (egfp), and the sr39tk positron emission tomography reporter gene. Serial bioluminescence imaging of MSC-TF and ex vivo luciferase assays were performed. Correlations were analyzed with the Pearson product-moment correlation, and serial imaging results were analyzed with a mixed-effects regression model. Results Analysis of the MSC-TF after cardiac cell therapy showed significantly lower signal on days 8 and 14 than on day 2 (P = .011 and P = .001, respectively). MSC-TF with MI demonstrated significantly higher signal than MSC-TF without MI at days 4, 8, and 14 (P = .016). Ex vivo luciferase activity assay confirmed the presence of MSC-TF on days 8 and 14 after MI. Conclusion Multimodality reporter-gene imaging was successfully used to assess serial MSC survival after therapy for MI, and it was determined that the requisite preclinical imaging end point, 14 days of MSC survival, was met prior to a follow-up large-animal MSC study. (©) RSNA, 2016 Online supplemental material is available for this article.

Multimodality Molecular Imaging of Cardiac Cell Transplantation: Part II. In Vivo Imaging of Bone Marrow Stromal Cells in Swine with PET/CT and MR Imaging

Purpose To quantitatively determine the limit of detection of marrow stromal cells (MSC) after cardiac cell therapy (CCT) in swine by using clinical positron emission tomography (PET) reporter gene imaging and magnetic resonance (MR) imaging with cell prelabeling. Materials and Methods Animal studies were approved by the institutional administrative panel on laboratory animal care. Seven swine received 23 intracardiac cell injections that contained control MSC and cell mixtures of MSC expressing a multimodality triple fusion (TF) reporter gene (MSC-TF) and bearing superparamagnetic iron oxide nanoparticles (NP) (MSC-TF-NP) or NP alone. Clinical MR imaging and PET reporter gene molecular imaging were performed after intravenous injection of the radiotracer fluorine 18-radiolabeled 9-[4-fluoro-3-(hydroxyl methyl) butyl] guanine ((18)F-FHBG). Linear regression analysis of both MR imaging and PET data and nonlinear regression analysis of PET data were performed, accounting for multiple injections per animal. Results MR imaging showed a positive correlation between MSC-TF-NP cell number and dephasing (dark) signal (R(2) = 0.72, P = .0001) and a lower detection limit of at least approximately 1.5 × 10(7) cells. PET reporter gene imaging demonstrated a significant positive correlation between MSC-TF and target-to-background ratio with the linear model (R(2) = 0.88, P = .0001, root mean square error = 0.523) and the nonlinear model (R(2) = 0.99, P = .0001, root mean square error = 0.273) and a lower detection limit of 2.5 × 10(8) cells. Conclusion The authors quantitatively determined the limit of detection of MSC after CCT in swine by using clinical PET reporter gene imaging and clinical MR imaging with cell prelabeling. (©) RSNA, 2016 Online supplemental material is available for this article.

Abstract 1099: Sorafenib increases numbers and functions of tumor-infiltrated T cells and enhances therapeutic outcomes of adoptive T cell therapy by modifying tumor microenvironment

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: Many chemotherapeutic agents are known to have immunomodulatory activity to improve the effectiveness of adoptive T-cell therapy. We hypothesized that sorafenib could reverse the immunosuppression of tumor microenvironment to enhance T-cell mediated tumor regression. Experimental design: OVA-specific CD8+ T cells were co-cultured with sorafenib-pretreated OVA-overexpressing E.G7 mouse lymphoma cells to evaluate their function using flow cytometry, 51Cr-release assay, and transwell assay. The underlying mechanism of sorafenib-mediated immunomodulation was also explored by Western blotting. In animal studies, the activation of OVA-specific CD8+ T cells transduced with a granzyme B promoter-driven imaging reporter was monitored by bioluminescence imaging and the levels of regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) were assessed by flow cytometry. Results: We found that sorafenib suppressed the expression of critical immunosuppressive components, including TGF-β , IL-10, VEGF, indoleamine 2,3-dioxygenase (IDO) and CCL2/MCP-1, in E.G7 cells through the inhibition of STAT3 signaling pathway and decreased the percentage of Tregs and MDSCs in tumors. Surprisingly, the anti-tumor effect of small numbers of CD8+ T cells was substantially enhanced when combined with sorafenib in vivo. Citation Format: Jeng-Jong Hwang, Hui-Yen Chuang, Ya-Fang Chang. Sorafenib increases numbers and functions of tumor-infiltrated T cells and enhances therapeutic outcomes of adoptive T cell therapy by modifying tumor microenvironment. [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 1099. doi:10.1158/1538-7445.AM2014-1099