scholarly journals Mammalian Target of Rapamycin Inhibitors Induce Tumor Cell ApoptosisIn VivoPrimarily by Inhibiting VEGF Expression and Angiogenesis

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Patrick Frost ◽  
Eileen Berlanger ◽  
Veena Mysore ◽  
Bao Hoang ◽  
YiJiang Shi ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Critical Role ◽  
Xenograft Model ◽  
Mtor Inhibitors ◽  
B Cell Lymphoma ◽  
G1 Arrest ◽  
Vegf Expression ◽  
Entry Site

We found that rapalog mTOR inhibitors induce G1 arrest in the PTEN-null HS Sultan B-cell lymphoma linein vitro, but that administration of rapalogs in a HS Sultan xenograft model resulted in significant apoptosis, and that this correlated with induction of hypoxia and inhibition of neoangiogenesis and VEGF expression. Mechanistically, rapalogs prevent cap-dependent translation, but studies have shown that cap-independent, internal ribosome entry site (IRES)-mediated translation of genes, such as c-myc and cyclin D, can provide a fail-safe mechanism that regulates tumor survival. Therefore, we tested if IRES-dependent expression of VEGF could likewise regulate sensitivity of tumor cellsin vivo. To achieve this, we developed isogenic HS Sultan cell lines that ectopically express the VEGF ORF fused to the p27 IRES, an IRES sequence that is insensitive to AKT-mediated inhibition of IRES activity and effective in PTEN-null tumors. Mice challenged with p27-VEGF transfected tumor cells were more resistant to the antiangiogenic and apoptotic effects of the rapalog, temsirolimus, and active site mTOR inhibitor, pp242. Our results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and underscore the importance of IRES activity as a resistance mechanism to such targeted therapy.

The Internal Ribosome Entry Site-Medaited Expression of VEGF Rescues HS Sultan Tumor Cells From Mammalian Target of Rapamycin (mTOR)-Inhibitors Induced Apoptosis in Vivo

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1659-1659
Author(s):  
Patrick Frost ◽  
Joseph Gera ◽  
Alan K. Lichtenstein
Keyword(s):  
Tumor Cells ◽  
Resistance Mechanism ◽  
Mtor Inhibitors ◽  
G1 Arrest ◽  
Vegf Expression ◽  
Tumor Cell Apoptosis ◽  
Safe Mechanism ◽  
Fail Safe

Abstract Abstract 1659 An important molecular target of mTOR inhibitors in cancer therapy is VEGF expression and neo-angiogenesis. In prior studies, we demonstrated that, although rapalog mTOR inhibitors only induce G1 arrest in B-cell tumor lines, their administration in vivo in xenograft models resulted in tumor cell apoptosis that correlated with inhibition of neo-angiogenesis and VEGF expression within the tumor bed. Other in vitro studies have shown that IRES-dependent translation of myc and D-cyclins can provide a fail-safe mechanism for expression when mTOR inhibitors prevent cap-dependent translation and may act as a resistance mechanism to induction of G1 arrest in vitro. We, thus, tested if VEGF IRES activity could likewise regulate induction of anti-tumor effects and apoptosis in vivo. To test if VEGF IRES activity can regulate anti-tumor responses in vivo, we utilized the HS-Sultan B cell lymphoma line that is PTEN null. Its heightened AKT activity disarms the VEGF IRES, preventing this fail-safe mechanism and sensitizing to mTOR inhibitors. We ectopically expressed a version of the VEGF ORF in these cells fused to the p27 IRES (p27-VEGF), an IRES which is insensitive to AKT and effective in PTEN-null tumors. p27-VEGF transfected tumor cells were used as subcutaneous challenges in immunodeficient mice and results of mTOR inhibitor treatment compared to control mice challenged with tumor cells transfected with the VEGF ORF but without an AKT-resistant IRES. The anti-tumor responses were enumerated by assessing tumor size and tumor apoptosis, neo-angiogenesis and VEGF expression assessed by immunohistochemistry, ELISA, and Western blot analysis of tumor lysate. Ectopic expression of VEGF fused to the p27 IRES specifically enhanced VEGF expression and neo-angiogenesis in tumors of mice treated with the rapalog temsirolimus or active site mTOR inhibitor pp242 and significantly reduced tumor cell apoptosis and anti-tumor responses. The results confirm the critical role of VEGF expression in tumors during treatment with mTOR inhibitors and, furthermore, underscore the importance of IRES activity as a resistance mechanism to such targeted therapy. Disclosures: No relevant conflicts of interest to declare.

The Role of AKT and ERK in Regulating D-Cyclin Translation Inhibition and G1 Arrest in Multiple Myeloma Cells Treated with mTOR Inhibitors: Rationale for Combination Thereapy.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4795-4795
Author(s):  
Patrick J. Frost ◽  
YiJiang Shi ◽  
Carolyne Bardalaban ◽  
Bao Hoang ◽  
Alan Lichtenstein
Keyword(s):  
Multiple Myeloma ◽  
Mtor Inhibitors ◽  
G1 Arrest ◽  
Entry Site ◽  
Internal Ribosome Entry ◽  
Myeloma Cells ◽  
Transfected Cells ◽  
P38 Inhibitor

Abstract In a previous study, we showed that heightened AKT activity sensitized multiple myeloma (MM) cells to the in vivo anti-tumor effects of CCI-779. To test the mechanism of AKT’s regulatory role, we studied isogenic U266 MM cell lines transfected with an activated AKT allele or empty vector. The AKT-transfected cells were markedly more sensitive to cytostasis induced in vitro by rapamycin or in vivo by CCI-779. In contrast, cells with quiescent AKT were completely resistant. The ability of rapamycin and CCI-779 to inhibit D-cyclin expression was also significantly greater in AKT-transfected MM cells and this was, in part, due to a greater ability to curtail cap-independent translation and internal ribosome entry site (IRES) activity of D-cyclin transcripts. As ERK/p38 activity can facilitate IRES-mediated translation of some transcripts, we investigated ERK/p38 as regulators of rapamycin sensitivity. AKT-transfected cells demonstrated significantly decreased ERK and p38 activity, suggesting their involvement. However, only an ERK inhibitor prevented D-cyclin IRES activity in resistant “low AKT” myeloma cells while a p38 inhibitor had no effect. Furthermore, the combination of rapamycin and the ERK inhibitor successfully sensitized myeloma cells to rapamycin in terms of down regulated D-cyclin protein expression and G1 arrest. These data support a scenario where ERK facilitates D-cyclin IRES function and heightened AKT activity down regulates this ERK-dependent phenomenon. Thus ERK and AKT activity are potential predictors of responsiveness to mTOR inhibitors.

scholarly journals Trifluoperazine, an Antipsychotic Drug, Effectively Reduces Drug Resistance in Cisplatin-Resistant Urothelial Carcinoma Cells via Suppressing Bcl-xL: An In Vitro and In Vivo Study

2019 ◽  
Vol 20 (13) ◽  
pp. 3218 ◽  
Author(s):  
Kuan-Lin Kuo ◽  
Shing-Hwa Liu ◽  
Wei-Chou Lin ◽  
Fu-Shun Hsu ◽  
Po-Ming Chow ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Urothelial Carcinoma ◽  
Cisplatin Resistance ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
G1 Arrest ◽  
Antitumor Effects ◽  
In Vivo Models

Cisplatin-based chemotherapy is the primary treatment for metastatic bladder urothelial carcinoma (UC). Most patients inevitably encounter drug resistance and resultant disease relapse. Reduced apoptosis plays a critical role in chemoresistance. Trifluoperazine (TFP), an antipsychotic agent, has demonstrated antitumor effects on various cancers. This study investigated the efficacy of TFP in inhibiting cisplatin-resistant bladder UC and explored the underlying mechanism. Our results revealed that cisplatin-resistant UC cells (T24/R) upregulated the antiapoptotic factor, B-cell lymphoma-extra large (Bcl-xL). Knockdown of Bcl-xL by siRNA resensitized cisplatin-resistant cells to the cisplatin cytotoxic effect. TFP (10–45 μM) alone elicited dose-dependent cytotoxicity, apoptosis, and G0/G1 arrest on T24/R cells. Co-treatment of TFP potentiated cisplatin-induced cytotoxicity in T24/R cells. The phenomenon that TFP alleviated cisplatin resistance to T24/R was accompanied with concurrent suppression of Bcl-xL. In vivo models confirmed that TFP alone effectively suppressed the T24/R xenograft in nude mice. TFP co-treatment enhanced the antitumor effect of cisplatin on the T24/R xenograft. Our results demonstrated that TFP effectively inhibited cisplatin-resistant UCs and circumvented cisplatin resistance with concurrent Bcl-xL downregulation. These findings provide a promising insight to develop a therapeutic strategy for chemoresistant UCs.

scholarly journals Pancreatic tumor cell metastasis is restricted by MT1-MMP binding protein MTCBP-1

2018 ◽  
Vol 218 (1) ◽  
pp. 317-332 ◽  
Author(s):  
Li Qiang ◽  
Hong Cao ◽  
Jing Chen ◽  
Shaun G. Weller ◽  
Eugene W. Krueger ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Pancreatic Tumor ◽  
Binding Protein ◽  
Critical Role ◽  
Inverse Correlation ◽  
Pancreatic Tumors ◽  
Matrix Remodeling ◽  
Peripheral Tissues

The process by which tumor cells mechanically invade through surrounding stroma into peripheral tissues is an essential component of metastatic dissemination. The directed recruitment of the metalloproteinase MT1-MMP to invadopodia plays a critical role in this invasive process. Here, we provide mechanistic insight into MT1-MMP cytoplasmic tail binding protein 1 (MTCBP-1) with respect to invadopodia formation, matrix remodeling, and invasion by pancreatic tumor cells. MTCBP-1 localizes to invadopodia and interacts with MT1-MMP. We find that this interaction displaces MT1-MMP from invadopodia, thereby attenuating their number and function and reducing the capacity of tumor cells to degrade matrix. Further, we observe an inverse correlation between MTCBP-1 and MT1-MMP expression both in cultured cell lines and human pancreatic tumors. Consistently, MTCBP-1–expressing cells show decreased ability to invade in vitro and metastasize in vivo. These findings implicate MTCBP-1 as an inhibitor of the metastatic process.

scholarly journals NUSAP1 Promotes Gastric Cancer Progression Through Regulation of Yap Stability

2020 ◽  
Author(s):  
Hui Guo ◽  
Jianping Zou ◽  
Ling Zhou ◽  
Yan He ◽  
Miao Feng ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Cancer Progression ◽  
Target Genes ◽  
Hippo Pathway ◽  
Critical Role ◽  
Xenograft Model ◽  
Migration And Invasion

Abstract Background:Nucleolar and spindle associated protein (NUSAP1) is involved in tumor initiation, progression and metastasis. However, there are limited studies regarding the role of NUSAP1 in gastric cancer (GC). Methods: The expression profile and clinical significance of NUSAP1 in GC were analysed in online database using GEPIA, Oncomine and KM plotter, which was further confirmed in clinical specimens.The functional role of NUSAP1 were detected utilizing in vitro and in vivo assays. Western blotting, qRT-PCR, the cycloheximide-chase, immunofluorescence staining and Co-immunoprecipitaion (Co-IP) assays were performed to explore the possible molecular mechanism by which NUSAP1 stabilizes YAP protein. Results:In this study, we found that the expression of NUSAP1 was upregulated in GC tissues and correlates closely with progression and prognosis. Additionally, abnormal NUSAP1 expression promoted malignant behaviors of GC cells in vitro and in a xenograft model. Mechanistically, we discovered that NUSAP1 physically interacts with YAP and furthermore stabilizes YAP protein expression, which induces the transcription of Hippo pathway downstream target genes. Furthermore, the effects of NUSAP1 on GC cell growth, migration and invasion were mainly mediated by YAP. Conclusions:Our data demonstrates that the novel NUSAP1-YAP axis exerts an critical role in GC tumorigenesis and progression, and therefore could provide a novel therapeutic target for GC treatment.

scholarly journals Hydroxychloroquine Synergizes With The PI3K Inhibitor BKM120 to Exhibit Antitumor Efficacy Independent of Autophagy

2021 ◽  
Author(s):  
Xin Peng ◽  
Shaolu Zhang ◽  
Wenhui Jiao ◽  
Zhenxing Zhong ◽  
Yuqi Yang ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Cell Biology ◽  
Molecular Mechanisms ◽  
Critical Role ◽  
Single Agent ◽  
Antitumor Efficacy ◽  
Antitumor Effects ◽  
Autophagy Inhibitor

Abstract Background: The critical role of phosphoinositide 3-kinase (PI3K) activation in tumor cell biology has prompted massive efforts to develop PI3K inhibitors (PI3Kis) for cancer therapy. However, recent results from clinical trials have shown only a modest therapeutic efficacy of single-agent PI3Kis in solid tumors. Targeting autophagy has controversial context-dependent effects in cancer treatment. As a FDA-approved lysosomotropic agent, hydroxychloroquine (HCQ) has been well tested as an autophagy inhibitor in preclinical models. Here, we elucidated the novel mechanism of HCQ alone or in combination with PI3Ki BKM120 in the treatment of cancer.Methods: The antitumor effects of HCQ and BKM120 on three different types of tumor cells were assessed by in vitro PrestoBlue assay, colony formation assay and in vivo zebrafish and nude mouse xenograft models. The involved molecular mechanisms were investigated by MDC staining, LC3 puncta formation assay, immunofluorescent assay, flow cytometric analysis of apoptosis and ROS, qRT-PCR, Western blot, comet assay, homologous recombination (HR) assay and immunohistochemical staining. Results: HCQ significantly sensitized cancer cells to BKM120 in vitro and in vivo. Interestingly, the sensitization mediated by HCQ could not be phenocopied by treatment with other autophagy inhibitors (Spautin-1, 3-MA and bafilomycin A1) or knockdown of the essential autophagy genes Atg5/Atg7, suggesting that the sensitizing effect might be mediated independent of autophagy status. Mechanistically, HCQ induced ROS production and activated the transcription factor NRF2. In contrast, BKM120 prevented the elimination of ROS by inactivation of NRF2, leading to accumulation of DNA damage. In addition, HCQ activated ATM to enhance HR repair, a high-fidelity repair for DNA double-strand breaks (DSBs) in cells, while BKM120 inhibited HR repair by blocking the phosphorylation of ATM and the expression of BRCA1/2 and Rad51. Conclusions: Our study revealed that HCQ and BKM120 synergistically increased DSBs in tumor cells and therefore augmented apoptosis, resulting in enhanced antitumor efficacy. Our findings provide a new insight into how HCQ exhibits antitumor efficacy and synergizes with PI3Ki BKM120, and warn that one should consider the “off target” effects of HCQ when used as autophagy inhibitor in the clinical treatment of cancer.

MTOR Inhibitors Activate the AKT Kinase in Multiple Myeloma Cells by Upregulating the IGF-1/IRS-1/PI-3 Kinase Cascade.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3350-3350 ◽  
Author(s):  
Yijiang Shi ◽  
HuaJun Yan ◽  
Patrick Frost ◽  
Bao Hoang ◽  
Joseph Gera ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Proteasome Inhibitors ◽  
Xenograft Model ◽  
Mtor Inhibitors ◽  
Pi3 Kinase ◽  
Serine Phosphorylation ◽  
G1 Arrest ◽  
Akt Activation ◽  
Induced Apoptosis

Abstract MTOR inhibitors, such as rapamycin and CCI-779, have shown pre-clinical potential as therapy for multiple myeloma (MM). By inhibiting expression of cell cycle proteins, these agents induce G1 arrest. However, by also inhibiting an mTOR-dependent phosphorylation of insulin receptor substrate-1 (IRS-1), they may alter its subcellular localization and/or prevent its degradation which could enhance IGF-1 signaling and downstream PI3-kinase/AKT activation. This may be a particular problem in MM where IGF-1-induced activation of AKT is an important anti-apoptotic cascade. We, thus, studied PI3-kinase/AKT activation in MM cells treated with mTOR inhibitors. Rapamycin enhanced basal AKT activity, AKT phosphorylation and PI3-kinase activity in MM cell lines. Both PTEN-null as well as PTEN-wild type myeloma lines were similarly affected. Rapamycin also significantly prolonged activation of AKT induced by exogenous IGF-1. CCI-779, used in a xenograft model, also resulted in MM cell AKT activation in vivo. Blockade of IGF-1 receptor function prevented rapamycin’s activation of AKT. Furthermore, rapamycin prevented serine phosphorylation of IRS-1 and IRS-1 degradation. Though similarly blocking IRS-1 degradation, proteasome inhibitors did not activate MM cell AKT. Although rapamycin sensitized MM cells for dexamethasone-induced apoptosis, it protected against PS-341-induced apoptosis. Thus, mTOR inhibitors activate PI3-K/AKT in MM cells and activation depends on basal IGF-1/IGF-R signaling. As activated AKT may protect against apoptosis, future use of mTOR inhibitors in myeloma patients will have to carefully consider the types of anti-myeloma agents used in combination.

The Monoclonal Antibody nBT062 Conjugated to Cytotoxic Maytansinoids Has Potent and Selective Cytotoxicity against CD138 Positive Multiple Myeloma Cells in Vitro and in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1716-1716 ◽  
Author(s):  
Hiroshi Ikeda ◽  
Teru Hideshima ◽  
Robert J. Lutz ◽  
Sonia Vallet ◽  
Samantha Pozzi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Monoclonal Antibody ◽  
Tumor Cells ◽  
Cell Lines ◽  
Xenograft Model ◽  
Growth Delay ◽  
Selective Cytotoxicity ◽  
Bystander Killing

Abstract CD138 is expressed on differentiated plasma cells and is involved in the development and/or proliferation of multiple myeloma (MM), for which it is a primary diagnostic marker. In this study, we report that immunoconjugates comprised of the murine/human chimeric CD138-specific monoclonal antibody nBT062 conjugated with highly cytotoxic maytansinoid derivatives (nBT062-SMCC-DM1, nBT062-SPDB-DM4 and nBT062-SPP-DM1) showed cytotoxic activity against CD138-positive MM cells both in vitro and in vivo. These agents demonstrated cytotoxicity against OPM1 and RPMI8226 (CD138-positive MM cell lines) in a dose and time-dependent fashion and were also cytotoxic against primary tumor cells from MM patients. Minimal cytotoxicity was noted in CD138-negative cell lines and no activity was observed against peripheral blood mononuclear cells from healthy volunteers, suggesting that CD138-targeting is important for immunoconjugate-mediated cytotoxicity. Examination of the mechanism of action whereby these immunoconjugates induced cytotoxicity in MM cells demonstrated that treatment triggered G2/M cell cycle arrest, followed by apoptosis associated with cleavage of PARP and caspase-3, -8 and -9. Neither interleukin-6 nor insulin-like growth factor-I could overcome the apoptotic effect of these agents. The level of soluble (s)CD138 in the BM plasma from 15 MM patients was evaluated to determine the potential impact of sCD138 on immunoconjugate function. The sCD138 level in BM plasma was found to be significantly lower than that present in MM cell culture supernatants where potent in vitro cytotoxicity was observed, suggesting that sCD138 levels in MM patient BM plasma would not interfere with immunoconjugate activity. Because adhesion to bone marrow stromal cells (BMSCs) triggers cell adhesion mediated drug resistance to conventional therapies, we next examined the effects of the conjugates on MM cell growth in the context of BMSC. Co-culture of MM cells with BMSCs, which protects against dexamethasoneinduced death, had no impact on the cytotoxicity of the immunoconjugates. The in vivo efficacy of these immunoconjugates was also evaluated in SCID mice bearing established CD138-positive MM xenografts and in a SCID-human bone xenograft model of myeloma. Significant tumor growth delay or regressions were observed at immunoconjugate concentrations that were well tolerated in all models tested. The ability of these agents to mediate bystander killing of proximal CD138-negative cells was also evaluated. While nBT062-SPDB-DM4 was inactive against CD138-negative Namalwa cells cultured alone, significant killing of these CD138-negative cells by nBT062-SPDB-DM4 was observed when mixed with CD138-positive OPM2 cells. This bystander killing may contribute to the eradication of MM tumors by disrupting the tumor microenvironment and/or killing CD138-negative MM tumor cells, such as the putative CD138 negative myeloma stem cells. These studies demonstrate strong evidence of in vitro and in vivo selective cytotoxicity of these immunoconjugates and provide the preclinical framework supporting evaluation of nBT062-based immunoconjugates in clinical trials to improve patient outcome in MM.

Loss of IRF8 Inhibits the Growth of Diffuse Large B-Cell Lymphoma

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3004-3004
Author(s):  
Yulian Xu ◽  
Lei Jiang ◽  
Rachel R. Fang ◽  
Jeff Xiwu Zhou ◽  
Herbert Morse
Keyword(s):  
Cell Proliferation ◽  
B Cell ◽  
Cell Lymphoma ◽  
Critical Role ◽  
B Cell Lymphoma ◽  
Expression Levels ◽  
Large B Cell Lymphoma ◽  
Large B Cell

Abstract IRF8 is a transcription factor with a critical role in B lymphocyte development and biological functions. Although it has been reported that IRF8 is highly expressed in human diffuse large B-cell lymphoma (DLBCL) and the translocation of IRF8-IgH loci occurs in DLBCL, little information is available regarding the function and mechanisms for the role of IRF8 in DLBCL. In this study, by using several human DLBCL cell lines with shRNA-mediated decrease in IRF8 expression levels, we found that the loss of IRF8 significantly reduced the proliferation of lymphoma cells (Figure 1). Mechanistically, decreasing the levels of IRF8 led to a decrease in p38 and ERK phosphorylation (Figure 2), molecular events critical for B cell proliferation. Furthermore, using a xenograft lymphoma mice model, we found that the loss of IRF8 significantly inhibited the growth of lymphomas in vivo (n=5 for each group) (Figure 3). Analysis of public available data also suggested that the expression levels of IRF8 mRNA in human DLBCL tissues were inversely correlated patientsÕ overall survival time. Taken together, this study showed that IRF8 may play an oncogenic role in human DLBCL by promoting cell proliferation. Figure 1. Loss of IRF8 decreased the proliferation of DLBCL cells in vitro. Figure 1. Loss of IRF8 decreased the proliferation of DLBCL cells in vitro. Figure 2. Loss of IRF8 decreased the phosphorylation of p38 and ERK in DLBCL cells. Figure 2. Loss of IRF8 decreased the phosphorylation of p38 and ERK in DLBCL cells. Figure 3. Loss of IRF8 decreased the growth of DLBCL in vivo. Figure 3. Loss of IRF8 decreased the growth of DLBCL in vivo. Disclosures No relevant conflicts of interest to declare.

Chemoprotection by VEGF: Rationale for combination nab-paclitaxel and bevacizumab

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 1064-1064 ◽  
Author(s):  
V. Trieu ◽  
S. Ran ◽  
C. Bivens ◽  
N. Desai
Keyword(s):  
Protective Effect ◽  
Tumor Cells ◽  
Breast Tumor ◽  
T Test ◽  
Receptor Expression ◽  
Vegf Receptor ◽  
Vegf Expression ◽  
Clonogenic Assays

1064 Background: Nanoparticle albumin-bound (nab-) paclitaxel (ABX) has shown greater efficacy and less toxicity than solvent-based paclitaxel (TAX) in xenograft models and clinical trials. The goal of this study was to determine the effects of VEGF modulation in human MDA-MB-231 breast tumor cell line and the effects of ABX and VEGF-neutralizing antibody bevacizumab (AVA) combination on the growth and metastasis of orthotopically implanted MDA-MB-231 tumors. Methods: VEGF expression was evaluated by ELISA in MDA-MB-231 tumor extract one week after treatment (qdx5) with saline, doxorubicin (10 mg/kg), TAX (10 mg/kg), or ABX (15 mg/kg). VEGF-receptor expression in MDA-MB-231 was quantitated by RT-PCR. MDA-MB-231 cytotoxicity with ABX, VEGF, AVA alone or in combination was measured by cytotoxicity and clonogenic assays. Implanted MDA-MB-231 tumors expressing luciferase were treated with saline, 2 cycles of ABX (10 mg/kg, two qdx5 cycles separated by 1 week, N=5) alone or in combination with AVA (2, 4 and 8 mg/kg, 2/wkx6). Tumor lymph node and pulmonary metastasis was determined by measuring luciferase activity. Results: Compared with saline, MDA-MB-231 tumors following chemotherapies exhibited significant tumor shrinkage (p≤0.006, t-test) and VEGF induction (p<0.0001, t-test). MDA-MB-231 was shown to express VEGFR2. Exogenous VEGF had a protective effect on MDA-MB-231 tumor cells by reducing cytotoxicity of ABX in both cytotoxicity and clonogenic assays. Sequestration of VEGF with AVA increased cytotoxicity of ABX in vitro. Treatment of MDA-MB-231 breast tumors with ABX and AVA combination resulted in greater than additive antitumor response and significantly reduced metastasis to the lungs (p=0.025 vs control) and LN (p=0.022) at the highest AVA dose. Conclusions: Chemotherapies induced VEGF expression in MDA-MD-231 breast tumor in vivo. In vitro, VEGF exerted a protective effect against ABX chemotherapy in VEGFR2-expressing MDA-MD-231 cells, which was abrogated by addition of AVA. In vivo, ABX and AVA combination significantly inhibited the metastatic potential of MDA-MB-231 tumor cells. These data provide a rational basis for the combination of nab- paclitaxel and bevacizumab in VEGF-receptor expressing tumors. [Table: see text]

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