Anti Tumor Effect of 2-Oxoglutarate through Inhibition of Angiogenesis in a Murine Tumor Model.

Abstract Hypoxia-inducible factor 1 (HIF-1) plays an essential role in tumor angiogenesis and growth by regulating the transcription of several genes in response to hypoxic stress and changes in growth factors. HIF-1 is a heterodimeric transcriptional activator and consists of inducible α and constitutive β subunits. In oxygenated cells, intracellular oxygen concentrations are directly sensed by proteins containing the prolyl hydroxylase domain (PHD), which tag HIF-1α subunits for polyubiquitination and proteasomal degradation by prolyl hydroxylation using 2-oxoglutarate (2-OX) and dioxygen. Our recent studies have shown that 2-OX reduces HIF-1α, erythropoietin, and vascular endothelial growth factor (VEGF) expression by hypoxia in the hepatoma cell line Hep3B (Matsumoto K et al.; J. Cell. Physiol., 2006). Similar results were obtained in Lewis lung cancer (LLC) cells in in vitro studies. Here, to address the clinical usefulness of 2-OX, we investigated its antitumor effect using a mouse dorsal air sac (DAS) assay and a murine tumor xenograft model. For the DAS assay, LLC cells suspended in PBS with 0, 7.5, or 15 mM 2-OX per Millipore chamber were implanted subcutaneously into C57BL/6J mice. The values of blood vessel area by LLC cells were 100 ± 20.8, 64.0 ± 9.4, and 45.6 ± 4.4%, respectively, by quantitative analysis with angiogenesis-measuring software. This result indicated that 2-OX clearly inhibited the growth of subcutaneous tumors. To elucidate the effect of 2-OX on tumor growth, 2-OX was administrated to C57BL/6J mice inoculated with LLC cells. LLC cells (1 × 106) in PBS were implanted into the right flank region of 7-week-old mice. Daily intraperitoneal (i.p.) injections of 2-OX were started on the next day after implantation. From 6 to 12 days after implantation, we measured tumors with calipers and calculated volumes as (length × width2) × 0.5. LLC tumors were removed from mice 12 days after implantation for quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) and immunohistochemical studies. Tumor volumes and weights 12 days after implantation were as follows: PBS alone, 330.8 ± 108.1 mm3 and 192.6 ± 66.4 mg; 50 mg/kg 2-OX, 128.8 ± 16.4 mm3 and 111.0 ± 64.5 mg; and 100 mg/kg 2-OX, 78.7 ± 43.7 mm3 and 88.8 ± 57.5 mg. Quantitative RT-PCR revealed that 2-OX treatment (100 mg/kg) decreased the expression levels of the VEGF gene 0.67-fold in tumor tissues compared with control. We observed quantitative differences in microvessel density (PBS alone, 100 ± 16.5%; 100 mg/kg 2-OX, 46.0 ± 13.5%) using immunostaining for the endothelial cell marker CD31. Intraperitoneal injection of 2-OX significantly inhibited tumor growth and angiogenesis in tumor tissues. Combination therapy is necessary for anti-angiogenic therapy in the human. To examine the effect of 2-OX in combination with 5-fluorouracil (5-FU) chemotherapy, we injected 5-FU i.p. on day 6 and/or 2-OX i.p. on each of days 6–15 in this mouse model. Tumor volumes and weights 15 days after implantation were as follows: PBS alone, 531.2 ± 144.9 mm3 and 306.3 ± 186.6 mg; 2-OX alone, 324.0 ± 156.5 mm3 and 308.7 ± 299.0 mg; 5-FU alone, 287.1 ± 155.2 mm3 and 204.7 ± 108.9 mg; and 5-FU + 2-OX, 98.7 ± 64.9 mm3 and 130.3 ± 113.5 mg. 5-FU combined with 2-OX significantly inhibited tumor growth in this model, which was accompanied by 53% reduction of VEGF gene expression in tumor tissues removed from mice 15 days after implantation, using quantitative RT-PCR analysis. These results suggest that 2-OX is a promising anti-angiogenic therapeutic agent. To examine whether the inhibitory effect of 2-OX is specific for PHD-containing proteins, an RNAi study will be performed.

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Gamma-radiated immunosuppressed tumor xenograft mice can be a new ideal model in cancer research

Scientific Reports ◽

10.1038/s41598-020-80428-5 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Hamid Khodayari ◽

Saeed Khodayari ◽

Solmaz Khalighfard ◽

Arash Tahmasebifar ◽

Mahboubeh Tajaldini ◽

...

Keyword(s):

Gamma Radiation ◽

High Capacity ◽

Xenograft Model ◽

Tumor Xenograft ◽

Whole Body ◽

Cell Injection ◽

Tumor Tissues ◽

Post Radiation ◽

Xenograft Models ◽

Xenograft Mice

AbstractTumor xenograft models can create a high capacity to study human tumors and discover efficient therapeutic approaches. Here, we aimed to develop the gamma-radiated immunosuppressed (GIS) mice as a new kind of tumor xenograft model for biomedical studies. First, 144 mice were divided into the control and treated groups exposed by a medical Cobalt-60 apparatus in 3, 4, and 5 Gy based on the system outputs. Then, 144 BALB/c mice were divided into four groups; healthy, xenograft, radiation, and radiation + xenograft groups. The animals in the xenograft and radiation + xenograft groups have subcutaneously received 3 × 106 MCF-7 cells 24 h post-radiation. On 3, 7, 14, and 21 days after cell injection, the animals were sacrificed. Then, the blood samples and the spleen and tumor tissues were removed for the cellular and molecular analyses. The whole-body gamma radiation had a high immunosuppressive effect on the BALB/c mice from 1 to 21 days post-radiation. The macroscopic and histopathological observations have proved that the created clusters' tumor structure resulted in the xenograft breast tumor. There was a significant increase in tumor size after cell injection until the end of the study. Except for Treg, the spleen level of CD4, CD8, CD19, and Ly6G was significantly decreased in Xen + Rad compared to the Xen alone group on 3 and 7 days. Unlike IL-4 and IL-10, the spleen level of TGF-β, INF-γ, IL-12, and IL-17 was considerably decreased in the Xen + Rad than the Xen alone group on 3 and 7 days. The spleen expressions of the VEGF, Ki67, and Bax/Bcl-2 ratio were dramatically increased in the Xen + Rad group compared to the Xen alone on 3, 7, 14, and 21 days. Our results could confirm a new tumor xenograft model via an efficient immune-suppressive potential of the whole-body gamma radiation in mice.

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Review of: Tamoxifen and TRAIL synergistically induce apoptosis in breast cancer cells

Breast Cancer Online ◽

10.1017/s1470903108006676 ◽

2008 ◽

Vol 11 (2) ◽

Cited By ~ 1

Author(s):

Alison J. Butt

Keyword(s):

Breast Cancer ◽

Tumor Growth ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Single Agent ◽

Xenograft Model ◽

Estrogen Receptor Α ◽

Tumor Xenograft ◽

Breast Cancers ◽

Induced Apoptosis

Citation of original article:C. Lagadec, E. Adriaenssens, R. A. Toillon, V. Chopin, R. Romon, F. Van Coppenolle, H. Hondermarck, X. Le Bourhis. Oncogene advance online publication, 3 September 2007; doi:10.1038/sj.onc.1210749.Abstract of the original article:Tamoxifen (TAM), is widely used as a single agent in adjuvant treatment of breast cancer. Here, we investigated the effects of TAM in combination with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in estrogen receptor-α (ER-α)-positive and -negative breast cancer cells. We showed that cotreatment with TAM and TRAIL synergistically induced apoptosis regardless of ER-α status. By contrast, cotreatment did not affect the viability of normal breast epithelial cells. Cotreatment with TAM and TRAIL in breast cancer cells decreased the levels of antiapoptotic proteins including FLIPs and Bcl-2, and enhanced the levels of proapoptotic proteins such as FADD, caspase 8, tBid, Bax and caspase 9. Furthermore, cotreatment-induced apoptosis was efficiently reduced by FADD- or Bid-siRNA, indicating the implication of both extrinsic and intrinsic pathways in synergistic apoptosis induction. Importantly, cotreatment totally arrested tumor growth in an ER-α-negative MDA-MB-231 tumor xenograft model. The abrogation of tumor growth correlated with enhanced apoptosis in tumor tissues. Our findings raise the possibility to use TAM in combination with TRAIL for breast cancers, regardless of ER-α status.

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Lanreotide promotes apoptosis and is not radioprotective in GH3 cells

Endocrine Related Cancer ◽

10.1677/erc-09-0003 ◽

2009 ◽

Vol 16 (3) ◽

pp. 1045-1055 ◽

Cited By ~ 7

Author(s):

Shoucheng Ning ◽

Susan J Knox ◽

Griffith R Harsh ◽

Michael D Culler ◽

Laurence Katznelson

Keyword(s):

Tumor Growth ◽

Survival Curve ◽

Somatostatin Analogs ◽

Xenograft Model ◽

Somatostatin Analog ◽

Tumor Xenograft ◽

Gh3 Cells ◽

Growth Delay ◽

Human Pituitary ◽

The Impact

Somatostatin analogs are a mainstay of medical therapy in patients with GH producing human pituitary tumors, and it has been suggested that somatostatin analogs may be radioprotective. We utilized GH secreting rat GH3 cells to investigate whether a somatostatin analog may limit the effects of radiation on proliferation and apoptosis in vitro and on tumor growth in vivo. Treatment with lanreotide alone at doses of either 100 or 1000 nM for 48 h reduced clonogenic survival by 5–10%. Radiation alone produced a dose-dependent survival curve with a SF2 of 48–55%, and lanreotide had no effect on this curve. The addition of lanreotide resulted in a 23% increase in the proportion of apoptotic sub-G1 cells following irradiation (P<0.01). In a mouse GH3 tumor xenograft model, lanreotide 10 mg/kg moderately inhibited the growth of GH3 tumors, with a 4× tumor growth delay (TGD) time that ranged from 4.5 to 8.3 days. Fractionated local tumor radiation alone significantly inhibited tumor growth and produced a TGD of 35.1±5.7 days for 250 cGy fractions. The combination of lanreotide, either antecedent to or concurrent, with radiation of 250, 200 or 150 cGy/fraction for 5 days inhibited tumor growth and produced the TGD times that were similar to radiation alone (P>0.05). Pretreatment with lanreotide had the most significant radiosensitizing effect. These studies demonstrate that the somatostatin analog lanreotide is not radioprotective in GH3 cells, and further studies are necessary to determine the impact of lanreotide on apoptosis.

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Analysis of gene expression in Lassa virus-infected HuH-7 cells

Journal of General Virology ◽

10.1099/vir.0.82529-0 ◽

2007 ◽

Vol 88 (5) ◽

pp. 1568-1575 ◽

Cited By ~ 18

Author(s):

Stefanie Müller ◽

Robert Geffers ◽

Stephan Günther

Keyword(s):

Gene Expression ◽

Virus Infection ◽

Real Time ◽

Hepatoma Cell ◽

Hepatoma Cell Line ◽

Lassa Virus ◽

Mrna Levels ◽

Rt Pcr ◽

Array Data ◽

U133a Array

The pathogenesis of Lassa fever is poorly understood. As the liver is a major target organ of Lassa virus, gene expression in Lassa virus-infected HuH-7 cells, a differentiated human hepatoma cell line, was studied. Cellular mRNA levels were measured at the late phase of acute infection, when virtually all cells expressed large amounts of nucleoprotein, and virus RNA concentration had reached >108 copies (ml supernatant)−1. Two types of transcription array were used: cDNA-based macroarrays with a set of 3500 genes (Atlas Human 1.2 arrays; Clontech) and oligonucleotide-based microarrays covering 18 400 transcripts (Human Genome U133A array; Affymetrix). Data analysis was based on statistical frameworks controlling the false-discovery rate. Atlas array data were considered relevant if they could be verified by U133A array or real-time RT-PCR. According to these criteria, there was no evidence for true changes in gene expression. Considering the precision of the U133A array and the number of replicates tested, potential expression changes due to Lassa virus infection are probably smaller than twofold. To substantiate the array data, beta interferon (IFN-β) gene expression was studied longitudinally in Lassa virus-infected HuH-7 and FRhK-4 cells by using real-time RT-PCR. IFN-β mRNA levels increased only twofold upon Lassa virus infection, although there was no evidence that the virus inhibited poly(I : C)-induced IFN-β gene expression. In conclusion, Lassa virus interferes only minimally with gene expression in HuH-7 cells and poorly induces IFN-β gene transcription.

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Baicalin Promotes Mammary Gland Development via Steroid-Like Activities

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.682469 ◽

2021 ◽

Vol 9 ◽

Author(s):

Weizhen Chen ◽

Wei Wei ◽

Liya Yu ◽

Xin Zhang ◽

Fujing Huang ◽

...

Keyword(s):

Mammary Gland ◽

Tumor Growth ◽

Cell Line ◽

Cancer Progression ◽

Mammary Gland Development ◽

Biological Activities ◽

Xenograft Model ◽

Mammary Development ◽

Tumor Xenograft ◽

Gland Development

Baicalin, the main flavonoid component extracted from Scutellaria roots, has a variety of biological activities and is therefore used in the treatment of many kinds of diseases. However, whether baicalin affects the normal development of tissues and organs is still unclear. Here, using a mouse mammary gland model, we investigated the effects of baicalin on the expansion of mammary stem cells (MaSCs) and mammary development, as well as breast cancer progression. Interestingly, we found that baicalin administration significantly accelerates duct elongation at puberty, and promotes alveolar development and facilitates milk secretion during pregnancy. Furthermore, self-renewal of MaSCs was significantly promoted in the presence of baicalin. Moreover, in a tumor xenograft model, baicalin promoted tumor growth of the MDA-MB-231 cell line, but suppressed tumor growth of the ZR-751 cell line. Mechanistically, baicalin can induce expression of the protein C receptor, while inhibiting the expression of the estrogen receptor. Transcriptome analysis revealed that baicalin is involved in signaling pathways related to mammary gland development, immune response, and cell cycle control. Taken together, our results from comprehensive investigation of the biological activity of baicalin provide a theoretical basis for its rational clinical application.

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Effects of novel heparin-derived compounds on tumor uptake of chemotherapeutics and chemoresponse

Journal of Clinical Oncology ◽

10.1200/jco.2009.27.15_suppl.2537 ◽

2009 ◽

Vol 27 (15_suppl) ◽

pp. 2537-2537 ◽

Cited By ~ 1

Author(s):

P. Phillips ◽

M. Yalcin ◽

H. Cui ◽

H. H. Abdel-Nabi ◽

M. Sajjad ◽

...

Keyword(s):

Tumor Growth ◽

Human Lung ◽

Lung Tumor ◽

Xenograft Model ◽

Chemotherapeutic Agents ◽

Tumor Xenograft ◽

Tumor Uptake ◽

Human Lung Tumor ◽

Biodistribution Studies ◽

Human Lung Carcinoma

2537 Thrombotic complications are the second most common cause of mortality in cancer patients and fibrin deposition in the tumor microenvironment might play a key role in tumor progression and inference with tumor chemotherapeutic uptake. Treatments that target these processes may result in improved uptake of chemotherapeutic agents and subsequent inhibition of tumor growth and metastasis. Tissue Factor (TF) is frequently associated with aggressive behavior and poor outcome in tumors. We have previously demonstrated potent anti-tumor efficacy for various mechanisms that interfere with TF/VIIa. The purpose of this study was to investigate the effects of low molecular weight heparins (LMWH) and sulfated non-anticoagulant LMWH (S-NACH) on tumor chemotherapeutic uptake. Studies: (1) Nude mice xenograft A549 human lung carcinoma: LMWH or S-NACH at 10 mg/kg S.C. daily effectively limited tumor growth. (2) LCC6 human lung tumor xenograft model: Paclitaxel alone or in combination with Tinzaparin or S-NACH on tumor re-growth after discontinuation of treatment: Paclitaxel + S-NACH treatment showed significant (P<0.01) tumor growth suppression and improved survival when compared to Paclitaxel. (3) Biodistribution studies: animals were injected with LMWH S.C. daily for 5 days (10 mg/kg) then injected i.v. with [124-I]-Paclitaxel. LMWH increased [124-I]-Paclitaxel uptake into LCC6 tumors with tumor: muscle ratios several fold greater than that of [124-I]-Paclitaxel alone at 24 hrs post injection. This is a highly significant result in the light of the fact that the FDA criterion for a clinically meaningful effect is a 15% increase in uptake. (4) HPLC studies of tumor uptake of Doxorubicin (DOX in mice treated with 10 mg/kg of LMWH or S-NACH for 10 days followed by Doxorubicin (2.5 mg/kg). Both LMWH and S-NACH significantly (P<0.01) increased the uptake of chemotherapeutic agent DOX in MCF7 DOX resistant tumors by 1.5–2 folds but not in heart or lung tissues, confirming the findings obtained with another agent [124-I]-Paclitaxel. Conclusions: LMWH or S-NACH increased chemotherapeutics uptake and hence chemoresponse. Protocols utilizing adjuvant or neo-adjuvant therapy with LMWH or S-NACH could lead to increase tumor chemo responsiveness and overcoming tumor chemo resistance. No significant financial relationships to disclose.

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Overexpression of Long Non-Coding RNA NNT-AS1 Correlates with Tumor Progression and Poor Prognosis in Osteosarcoma

Cellular Physiology and Biochemistry ◽

10.1159/000487966 ◽

2018 ◽

Vol 45 (5) ◽

pp. 1904-1914 ◽

Cited By ~ 18

Author(s):

Hui Ye ◽

Jinkuang Lin ◽

Xuedong Yao ◽

Yizhong Li ◽

Xiaobin Lin ◽

...

Keyword(s):

Tumor Growth ◽

Poor Prognosis ◽

Significant Risk ◽

Xenograft Model ◽

Significant Risk Factor ◽

Tumor Xenograft ◽

Migration And Invasion ◽

Mouse Xenograft Model

Background/Aims: Increasing evidence demonstrates that long non-coding RNAs (lncRNAs) play critical regulatory roles in cancers, including osteosarcoma. A previous study showed that Nicotinamide Nucleotide Transhydrogenase-antisense RNA1 (NNT-AS1) was aberrantly expressed in several types of cancer. However, the potential biological roles and regulatory mechanisms of NNT-AS1 in osteosarcoma progression remain unknown. Methods: Quantitative RT-PCR was performed to examine the expression of NNT-AS1 in human tissues and cells. The biological functions of NNT-AS1 were determined by CCK-8, colony formation, Flow cytometry and Transwell assays in vitro. A mouse xenograft model was performed to investigate the effect of NNT-AS1 on tumor growth in vivo. Results: In this study, we found the expression of NNT-AS1 was significantly increased in tumor tissues compared to adjacent normal tissues. Furthermore, upregulated NNT-AS1 expression predicted poor prognosis and was an independent and significant risk factor for osteosarcoma patient survival. Further experiments revealed that NNT-AS1 knockdown significantly inhibited cell proliferation by inducing cell cycle arrest and promoting apoptosis in osteosarcoma cells. Moreover, NNT-AS1 silencing suppressed cell migration and invasion in vitro. In a tumor xenograft model, knockdown of NNT-AS1 suppressed tumor growth of OS-732 cells in vivo. Conclusions: Taken together, these findings indicate that NNT-AS1 functions as an oncogene in osteosarcoma and could be a novel diagnostic and therapeutic target for osteosarcoma.

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Differential Angiogenic Potential of 3-Dimension Spheroid of HNSCC Cells in Mouse Xenograft

International Journal of Molecular Sciences ◽

10.3390/ijms22158245 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8245

Author(s):

So-Young Choi ◽

Soo Hyun Kang ◽

Su Young Oh ◽

Kah Young Lee ◽

Heon-Jin Lee ◽

...

Keyword(s):

Culture Media ◽

3D Culture ◽

Xenograft Model ◽

Fluorescent Imaging ◽

Tumor Xenograft ◽

In Vivo Model ◽

Mouse Tumor ◽

Tumor Tissues ◽

3D Spheroids

The experimental animal model is still essential in the development of new anticancer drugs. We characterized mouse tumors derived from two-dimensional (2D) monolayer cells or three-dimensional (3D) spheroids to establish an in vivo model with highly standardized conditions. Primary cancer-associated fibroblasts (CAFs) were cultured from head and neck squamous cell carcinoma (HNSCC) tumor tissues and co-injected with monolayer cancer cells or spheroids into the oral mucosa of mice. Mice tumor blood vessels were stained, followed by tissue clearing and 3D Lightsheet fluorescent imaging. We compared the effect of exosomes secreted from 2D or 3D culture conditions on the angiogenesis-related genes in HNSCC cells. Our results showed that both the cells and spheroids co-injected with primary CAFs formed tumors. Interestingly, vasculature was abundantly distributed inside the spheroid-derived but not the monolayer-derived mice tumors. In addition, cisplatin injection more significantly decreased spheroid-derived but not monolayer-derived tumor size in mice. Additionally, exosomes isolated from co-culture media of FaDu spheroid and CAF upregulated angiogenesis-related genes in HNSCC cells as compared to exosomes from FaDu cell and CAF co-culture media under in vitro conditions. The mouse tumor xenograft model derived from 3D spheroids of HNSCC cells with primary CAFs is expected to produce reliable chemotherapy drug screening results given the robust angiogenesis and lack of necrosis inside tumor tissues.

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The effect of lncRNA HOTAIR on chemoresistance of ovarian cancer through regulation of HOXA7

Biological Chemistry ◽

10.1515/hsz-2017-0274 ◽

2018 ◽

Vol 399 (5) ◽

pp. 485-497 ◽

Cited By ~ 14

Author(s):

Siwei Liu ◽

Huajiang Lei ◽

Fangyuan Luo ◽

Yilin Li ◽

Lan Xie

Keyword(s):

Ovarian Cancer ◽

Tumor Growth ◽

Cancer Cells ◽

Nude Mice ◽

Tumor Development ◽

Xenograft Model ◽

Tumor Xenograft ◽

Migration And Invasion ◽

Ovarian Cancer Cells ◽

Qrt Pcr

AbstractThis study aimed at investigating the biological functions of long non-coding RNAs (lncRNAs) hox transcript antisense intergenic RNA (HOTAIR) in resistant ovarian cancer cells, exploring the regulation effect of HOTAIR onHOXA7, and investigating their influence on the chemosensitivity of ovarian cancer cells. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied for the verification of HOTAIR expression in resistant and sensitive groups. How HOTAIR downregulation affected cell proliferation, migration and invasion, and apoptosis were determined using the MTT assay and the colony formation assay, the Transwell assay and flow cytometry analysis, respectively. Immunohistochemistry was used to inspect the protein expression of HOXA7 in resistant and sensitive ovarian cancer tissues. The regulation relationship between HOTAIR andHOXA7was investigated by qRT-PCR and Western blot. The effect of HOTAIR andHOXA7on tumor growth was confirmed by the tumor xenograft model of nude mice. By knocking downHOXA7, HOTAIR downregulation restrained the ovarian cancer deterioration in functional experiments. Silencing of HOTAIR andHOXA7could effectively inhibit tumor growth and increase chemosensitivity of ovarian tumors in nude mice. Downregulation of HOTAIR negatively affected the survival and activity of resistant ovarian cancer cells, and suppressed the expression ofHOXA7. Silencing of HOTAIR andHOXA7could increase the chemosensitivity of ovarian cancer cells, thus suppressing tumor development.

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