Tumor Tissue Of Tumor-bearing Mice Research Articles

ETS homologous factor, controlled by lysine-specific demethylase 5B, suppresses clear cell renal cell carcinoma by inducing Filamin-B

BackgroundClear cell renal cell carcinoma (ccRCC) remains a deadly disease with a poor prognosis. Here, we identified the ETS homologous factor (EHF) and its target Filamin-B (FLNB) as molecules related to immune evasion in ccRCC. We also explored the upstream modifier that manipulates EHF in ccRCC. DesignCell proliferation and apoptosis assay, wound healing assay, and Transwell assay were designed to analyze the effects of EHF or FLNB knockdown on the biological activity of ccRCC cells. The growth of differently treated ccRCC cells was assessed by orthotopic tumors. ccRCC cells with different treatments were co-cultured with macrophages, and the role of the lysine-specific demethylase 5B (KDM5B)/EHF/FLNB axis on macrophage polarization or ccRCC progression was characterized by detecting the expression of M2 macrophage markers in the co-culture system or tumor tissues of tumor-bearing mice. ResultsThe expression of EHF and FLNB was higher, while KDM5B was lower in HK2 cells than in ccRCC cells. EHF overexpression inhibited the biological behavior of ccRCC cells and tumor growth in mice. EHF activated FLNB transcription. Knockdown of FLNB supported the biological activity of ccRCC cells and tumor growth and reversed M2 macrophage polarization in tumor tissues of mice in the presence of EHF. KDM5B inhibited EHF expression by H3K4me3 demethylation, and EHF knockdown potentiated M2 macrophage polarization and tumor growth in vivo repressed by KDM5B knockdown. ConclusionsKDM5B inhibited the expression of EHF by repressing H3K4me3 modification and the transcription of FLNB by EHF to promote immune evasion and progression of ccRCC.

Establishment of a novel co-cultured liver cancer model based on tumor microenvironment

Objective: To construct a new co-cultured liver cancer research model composed of activated hepatic stellate cells (aHSC) and liver cancer cells, explore the efficacy difference between it and traditional model, so as to establish a liver cancer research model in vitro and in vivo that can reflect the real clinical efficacy. Methods: A new co-culture model of liver cancer consisting of aHSC and liver cancer cells was constructed. The differences in efficacy between the new co-culture model and the traditional single cell model were compared by cytotoxicity test, cell migration test, drug retention test and in vivo tumor inhibition test. Western blot was used to detect the drug-resistant protein P-gp and epithelial-mesenchymal transition-related proteins. Masson staining was used to observe the deposition of collagen fibers in tumor tissues of tumor-bearing mice. CD31 immunohistochemical staining was used to observe the microvessel density in tumor tissues of tumor-bearing mice. Results: The cytotoxicity of single cell model and co-culture model was dose-dependent. With the increase of curcumin (CUR) concentration, the cell viability decreased, but the cell viability of single cell model decreased faster than that of co-culture model. When the concentration of CUR was 10 μg/ml, the cell viability of the co-culture model was 62.3% and the migration rate was (28.05±3.68)%, which were higher than those of the single cell model [38.5% and (14.91±5.92)%, both P<0.05]. Western blot analysis showed that the expressions of P-gp and vimentin were up-regulated in the co-culture model, which were 1.55 and 2.04 fold changes of the single cell model, respectively. The expression of E-cadherin was down-regulated, and the expression level of E-cadherin in the single cell model was 1.17 fold changes of the co-culture model. Drug retention experiment showed that the co-culture model could promote drug efflux and reduce drug retention. In vivo tumor inhibition experiment showed that the m-HSC+ H22 co-transplantation model had faster tumor growth and larger tumor volume than those of the H22 single cell transplantation model. After CUR treatment, the tumor growths of m-HSC+ H22 co-transplantation model and H22 single cell transplantation model were inhibited. Masson staining showed that the deposition of collagen fibers in tumor tissues of m-HSC+ H22 co-transplantation model mice was more than that of H22 single cell transplantation model. CD31 immunohistochemical staining showed that the microvessel density in tumor tissue of m-HSC+ H22 co-transplantation model was higher than that of H22 single cell transplantation model. Conclusions: The aHSC+ liver cancer cell co-culture model has strong proliferation and metastasis ability and is easy to be resistant to drugs. It is a new type of liver cancer treatment research model superior to the traditional single cell model.

Design and Evaluation of a Carrier-Free Prodrug-Based Palmitic-DEVD-Doxorubicin Conjugate for Targeted Cancer Therapy.

In the development of new drugs, typical polymer- or macromolecule-based nanocarriers suffer from manufacturing process complexity, unwanted systematic toxicity, and low loading capacity. However, carrier-free nanomedicines have made outstanding progress in drug delivery and pharmacokinetics, demonstrating most of the advantages associated with nanoparticles when applied in targeted anticancer therapy. Here, to overcome the problems of nanocarriers and conventional cytotoxic drugs, we developed a novel, carrier-free, self-assembled prodrug consisting of a hydrophobic palmitic (16-carbon chain n-hexadecane chain) moiety and hydrophilic group (or moiety) which is included in a caspase-3-specific cleavable peptide (Asp-Glu-Val-Asp, DEVD) and a cytotoxic drug (doxorubicin, DOX). The amphiphilic conjugate, the palmitic-DEVD-DOX, has the ability to self-assemble into nanoparticles in saline without the need for any carriers or nanoformulations. Additionally, the inclusion of doxorubicin is in its prodrug form and the apoptosis-specific DEVD peptide lead to the reduced side effects of doxorubicin in normal tissue. Furthermore, the carrier-free palmitic-DEVD-DOX nanoparticles could passively accumulate in the tumor tissues of tumor-bearing mice due to an enhanced permeation and retention (EPR) effect. As a result, the palmitic-DEVD-DOX conjugate showed an enhanced therapeutic effect compared with the unmodified DEVD-DOX conjugate. Therefore, this carrier-free palmitic-DEVD-DOX prodrug has great therapeutic potential to treat solid tumors, overcoming the problems of conventional chemotherapy and nanoparticles.

Delicaflavone Represses Lung Cancer Growth by Activating Antitumor Immune Response through N6-Methyladenosine Transferases and Oxidative Stress.

Our previous studies have shown that delicaflavone (DLL), a biocomponent extracted from Selaginella doederleinii Hieron, has antitumor activity. However, the role of DLL in the antitumor immune response is unknown. In this study, we tested the potential roles of DLL in antitumor immune response. An animal tumor model with Lewis lung cancer cell line (3LL) in C57BL/6 mice was established to determine whether DLL induced the tumor-bearing host's antitumor immune response. m6A-MeRIP-qPCR, western blot, and flow cytometry were performed to explore the underlying mechanisms. DLL inhibited the proliferation of 3LL lung cancer cells in vitro and in vivo and induced tumor cell oxidative stress. DLL significantly inhibited tumor growth in immunocompetent mice compared with nude mice. DLL treatment significantly increased Th1 cytokine production and CD8+ T cell infiltration into tumor tissues in tumor-bearing mice. DLL-mediated antitumor immune effects were reversed by overexpression of the N6-methyladenosine (m6A) transferase Mettl3/Mettl14. Mechanistically, DLL upregulated the expression of Stat1 and Irf1 and the secretion of cytokines by inhibiting Mettl3 and Mettl14 in lung cancer cells. In conclusion, DLL inhibited lung cancer cell growth by suppressing Mettl3/Mettl14 to activate antitumor immunity. These findings provided an opportunity to enhance lung cancer immunotherapy.

Icariside I – A novel inhibitor of the kynurenine-AhR pathway with potential for cancer therapy by blocking tumor immune escape

BackgroundAlthough therapeutic antibodies against immune checkpoints such as PD-1/PD-L1 have achieved unprecedented success in clinical tumor patients, there are still many patients who are ineffective or have limited responses to immune checkpoint blockade (ICB). Discovery of novel strategies for cancer immunotherapy including natural small molecules is needed. MethodsOwing to its extremely low content in Epimedium genus, we firstly constructed a microbial cell factory to enzymatically biosynthesize icariside I, a natural flavonoid monosaccharide from Herbal Epimedium. Using a combination of targeted MS-based metabolomics, flow cytometric analysis, and biological assays, the therapeutic potentials of icariside I were subsequently investigated in vivo and in vitro. ResultsWe find that icariside I markedly downregulates a series of intermediate metabolites such as kynurenine, kynurenic acid and xanthurenic acid and corresponding key enzymes involved in kynurenine-AhR pathway in both tumor cells and tumor-bearing mice. In vivo, oral administration of icariside I downregulates SLC7A8 and PAT4 transporters and AhR, thus inhibiting nuclear PD-1 in CTLs. Moreover, icariside I significantly upregulates CD8 + T cells in both peripheral blood and tumor tissues of tumor-bearing mice. Consequently, interferon-γ (IFN-γ) secreted by CD8 + T cells suppresses tumor growth through activation of JAK1-STAT1 signaling, thus inducing tumor cell apoptosis. ConclusionsThese results suggest that icariside I could be an effective small molecule drug for tumor immunotherapy by blocking kynurenine-AhR pathway and tumor immune escape.

Carbon spheres with high photothermal conversion efficiency for photothermal therapy of tumor

Development of high-performance photothermal agent plays a decisive role in photothermal therapy of tumor. Herein, carbon spheres are rapidly synthesized by pyrolysis of pyrogallol-formaldehyde spheres at 400–1000 °C under nitrogen atmosphere. As-prepared carbon spheres exhibit strong absorption in near-infrared region and good photothermal effect. The results reveal that the photothermal conversion efficiency of carbon spheres has a tight association with carbon defects caused by pyrolysis. The carbon defects serve as the nonradiative recombination centers of the excited electrons to promote photothermal conversion efficiency. Strikingly, carbon spheres after calcination at 700 °C exhibit the photothermal conversion efficiency of 54.2% under 808 nm laser irradiation, which is superior to the reported carbon-based photothermal agents. The prepared carbon spheres have good cell biocompatibility and can ablate the tumor tissues in tumor-bearing mice through the conversion of near-infrared laser energy into thermal energy.

Effect of fruquintinib on programmed death receptor-1 blockade antitumor immune responses in colorectal cancer.

150 Background: Programmed death receptor-1 (PD-1) blockade shows little benefit in the patients with microsatellite-stable colorectal cancer (MSS-CRC).Fruquintinib, a China-made anti-angiogenic drug, applied for third line therapy in mCRC. However, the effects of combination of fruquintinib and PD-1 blockade on MSS-CRC and its relative mechanisms are not well determined. Methods: Syngeneic xenograft mouse models were establish using murine MC38 and CT26 colon cancer cells to assess treatment efficacy. The percentages of immune cells were detected in peripheral blood, spleen and tumor tissues in tumor-bearing mice by flow cytometry analysis. Angiogenesis in tumor tissues was detected by immunofluorescence. The safety of drug treatment was evaluated by histopathology analysis in murine main organs. The efficacy of combination of fruquintinib and sintilimab were verified in the treatment of MSS-CRC patients. Results: Our results showed the combination of fruquintinib and sintilimab exhibited strongest inhibition of tumor growth and achieved longest survival time in mice bearing MC38 or CT26 xenograft tumors, compared to fruquintinib and sintilimab alone. Mechanistically, the combination of fruquintinib and sintilimab reduced angiogenesis, reprogramed the vascular structure, enhanced the infiltration of CD8+T cells, CD8+TNFα+T cells and CD8+IFNγ+T cells and reduced the ratios of MDSCs and macrophages in mice. No obvious damage was observed in main organs in tumor-bearing mice with the combined treatment. Moreover, the treatment of combination of fruquintinib and sintilimab anti-PD-1 antibodies achieved effective response in five refractory advanced MSS CRC patients. Conclusions: Our results show that combination of fruquintinib and sintilimab synergistically inhibits CRC growth by alterating antitumor immune microenvironment.

Establishment of human colon cancer transplantation tumor model in normal immune mice

Objective: Establishment of a new model of human primary colon cancer transplantation tumor in normal immune mice and to provide a reliable experimental animal model for studying the pathogenesis of colon cancer under normal immunity. Methods: Human colon cancer cells come from colon cancer patients who underwent surgery in the Affiliated Hospital of Jining Medical College in 2017. The mice in the cell control group were inoculated with phosphate buffered solution (PBS) containing colon cancer cells, the microcarrier control group was inoculated with PBS containing microcarrier 6, and the cell-microcarrier complex group was inoculated with the PBS containing colon cancer cell-microcarrier complex. The cells of each group were inoculated under the skin of the right axilla of mice by subcutaneous injection, and the time, size, tumor formation rate and pathological changes under microscope were recorded. The transplanted tumor tissue was immunohistochemically stained with the EnVisiion two-step method, and the tumor formation rate of the transplanted tumor was judged according to the proportion of positive cells in the visual field. The polymerase chain reaction (PCR) method was used to detect the expression of human-specific Alu sequence in mice tumor tissue. Results: After inoculation with tumor cells, the mice in the cell control group and the microcarrier control group did not die and did not form tumors; the mice in the cell-microcarrier complex group had palpable subcutaneous tumors in the right axillary subcutaneously on the 5th to 7th days after inoculation, and tumor formation rate is 67% (10/15), and the tumor volume can reach about 500 mm(3) 2 to 3 weeks after vaccination. The immunohistochemistry results showed that CK20, CDX-2 and carcinoembryonic antigen were all positively expressed. The PCR results showed that the expression of human-specific Alu sequence can be detected in the transplanted tumor tissue of tumor-bearing mice. Conclusion: Human primary colon cancer cells used microcarrier 6 as a carrier to form tumors in normal immunized mice, and successfully established a new model of human colon cancer transplantation tumor in normal immune mice.

Near-Infrared Fluorescent Probe for Imaging and Evaluating the Role of Vanin-1 in Chemotherapy.

Pantetheinase (also known as Vanin-1) is highly expressed in the liver, kidneys, and intestine and is closely associated with a number of diseases. Vanin-1 can hydrolyze pantetheine to pantothenic acid (vitamin B5) and cysteamine and participate in the synthesis of glutathione (GSH). GSH is highly expressed in tumor cells and plays a major role in the resistance of tumor cells to cisplatin. Therefore, we urgently need a method to monitor the activity level of Vanin-1 in tumor cells and tissues and elucidate the relationship between the role of Vanin-1 in GSH synthesis and tumor resistance. Herein, we report a Cy-Pa fluorescent probe for imaging Vanin-1 in cells and in vivo that can qualitatively and quantitatively detect the fluctuation of Vanin-1 concentrations in HepG2 and HepG2/DDP cells or tumor tissues of tumor-bearing mice. This probe shows excellent potential in in situ real-time monitoring of endogenous Vanin-1. Moreover, we proved that Vanin-1 can inhibit GSH synthesis using the probe. When the Vanin-1 inhibitor RR6 was used in combination with cisplatin, HepG2 and HepG2/DDP cells showed increased resistance to cisplatin, while the therapeutic efficiency of cisplatin was reduced in HepG2 and HepG2/DDP xenografts. In this study, Vanin-1 was shown to play an important role in the treatment of cancer, and the study of Vanin-1 may provide an idea for the treatment of cancer in the future.

The Effect of Sirtuin 2 (Sirt2) Overexpressing Bone Marrow Mesenchymal Stem Cells on the Growth of Human Epidermal Growth Factor Receptor 2 (Her-2) Breast Cancer Cells and Its Mechanism

Our study investigates the effect of high expression of Sirt2 in MSCs (MSCs-Sirt2) on Her-2 breast cancer cell proliferation. A mouse subcutaneous xenograft tumor model was established and MSCssirt2 analysis was performed on nude mice. TUNEL staining, flow cytometry, western-blot, real-time PCR and immunohistochemistry were used to detect cancer cell apoptosis. The number of NK cells infiltrated by flow cytometry detected the tumor tissue of tumor-bearing mice, and its killing activity on tumor-bearing mice was detected by isotope labeling and release method. The levels of TNF-α, IFN-γ, IL-8, IL-6 and IL-10 were detected by ELISA. Caspase-3 level was decreased in the MSCs group (P &lt;0.01) while increased in the MSCs-sirt2 group (P &lt;0.001). However, PCNA expression showed an opposite profile in the Her-2 group and MSCs-sirt2 group compared to Caspase-3 level (P &lt;0.01). The tumor volume and weight in the MSCs-sirt2 group was significantly reduced (P &lt; 0.01), while increased in the MSCs group significantly (P &lt; 0.05). The number of Ki-67-positive tumor cells in MSCs-sirt2 group was significantly reduced (P &lt;0.01) and increased in MSCs group (P &lt; 0.001) with oppositive number of TUNEL-positive tumor cells in the MSCs-sirt2 group and MSCs group (P &lt;0.01). IFN-γ level showed an upward trend (P &lt;0.001). The NK cell toxicity of MSCs-Sirt2 group was significantly higher (P &lt;0.001). MSCs-Sirt2 has an inhibitory effect on Her-2 breast cancer cell growth by enhancing the local inflammatory response of NK cells.

Targeted Photodynamic Therapy (PDT) of Lung Cancer with Biotinylated Silicon (IV) Phthalocyanine.

Lung cancer is the leading cause of cancer-associated mortality in the world. Traditional cancer therapies prolong the life expectancy of patients but often suffer from adverse reactions. Photodynamic Therapy (PDT) has been recommended as a treatment option for lung cancer in several countries, due to its non-invasive procedures, high selectivity and weak side effects. We have designed and synthesized a biotin receptor-targeted silicon phthalocyanine (IV) (compound 1) which showed a good therapeutic effect on biotin receptor-positive tumors. Since the overexpression of Biotin Receptor (BR) is also present in human lung cancer cells (A549), we explored the therapeutic properties of compound 1 on A549 xenograft tumor models. The selectivity of compound 1 toward A549 cells was studied with a fluorescence microscope and IVIS Spectrum Imaging System. The cytotoxicity was measured using the MTT assay. In vivo anti-tumor activity was investigated on the nude mice bearing A549 xenografts. In vitro assays proved that compound 1 could selectively accumulate in A549 cells via the BR-mediated internalization. In vivo imaging and distribution experiments showed that compound 1 could selectively accumulate in tumor tissues of tumor-bearing mice. After 16 days of the treatment, the volumes of tumor in the PDT group were obviously smaller than that in other groups. This study demonstrates that compound 1 is a promising photosensitizer and has broad application prospects in clinical PDT of lung cancers.

Immune Responses Raised in an Experimental Colon Carcinoma Model Following Oral Administration of Lactobacillus casei.

The role of dietary probiotic strains on host anti-cancer immune responses against experimental colon carcinoma was investigated. We have previously shown that Lactobacillus casei administration led to tumor growth suppression in an experimental colon cancer model. Here, we investigated the underlying immune mechanisms involved in this tumor-growth inhibitory effect. BALB/c mice received daily live lactobacilli per os prior to the establishment of a syngeneic subcutaneous CT26 tumor. Tumor volume, cytokine production, T cell differentiation and migration, as well as tumor cell apoptosis were examined to outline potential immunomodulatory effects following L. casei oral intake. Probiotic administration in mice resulted in a significant increase in interferon gamma (IFN-γ), Granzyme B and chemokine production in the tumor tissue as well as enhanced CD8+ T cell infiltration, accompanied by a suppression of tumor growth. Cytotoxic activity against cancer cells was enhanced in probiotic-fed compared to control mice, as evidenced by the elevation of apoptotic markers, such as cleaved caspase 3 and poly (ADP-ribose) polymerase 1 (PARP1), in tumor tissue. Oral administration of Lactobacillus casei induced potent Th1 immune responses and cytotoxic T cell infiltration in the tumor tissue of tumor-bearing mice, resulting in tumor growth inhibition. Thus, the microorganism may hold promise as a novel dietary immunoadjuvant in raising protective anti-cancer immune responses.

Role of Sulforaphane on Histone Deacetylase Activity in Solid Ehrlich Carcinoma

Histone acylation is one in every of the posttranslational modification that incorporates a role within the regulation of sequence expression. This study was aimed to ameliorate neoplasm cell model Solid Ehrlich malignant neoplastic disease with sulforaphane extracted from cabbage alone and together with immune suppressant drug (MTX) by study the activity of simple protein deacetylase accelerator (HDAC). In this study, sulforaphane was extracted from cabbage leaves and evaluated victimization GC-MS and ultraviolet illumination spectrophotometry. 60 white male rats were divided into six equal groups. Group I: management ordinarily. The remaining mice were subjected to Ehrlich neoplasm cells. Group II: Tumor-bearing group. Group III: immune suppressant drug “MTX” treated group. Group IV, V treated with SFN before and when Paul Ehrlich cells implantation group VI (Methotrexate and sulforaphane-treated group). This result showed that sulforaphane was extracted from cabbage (Brassica oleracea) in concentration of 833 μg/g leave. HDAC was attenuated when treatment with sulforaphane after treatment with methotrexate or sulforaphane alone. The desoxyribonucleic acid injury was attenuated in neoplasm tissue of tumor-bearing mice when treatment with immune suppressant drug and hyperbolic considerably in tumor tissue of tumor-bearing mice treated with sulforaphane before and after carcinogenesis and together with methotrexate treatment after carcinogenesis.

Magnetic And pH Dual-Responsive Nanoparticles For Synergistic Drug-Resistant Breast Cancer Chemo/Photodynamic Therapy.

BackgroundDrug resistance is one of the prime reasons of chemotherapy failure in breast cancer and is also an important factor affecting prognosis.PurposeIn this study, we constructed a functional magnetic mesoporous silica-based nanocomposite (MMSN) for breast cancer chemotherapy/photodynamic therapy.MethodsMMSN was characterized by scanning electron microscopy and transmission electron microscopy to observe the morphology. The size distribution and zeta potential of the MSNs were determined using Malvern Particle Size Analyzer. Anti-tumor activity in vitro was investigated by CCK-8 assay, flow cytometry and transwell experiment, and the anti-tumor activity in vivo was probed into by magnetic targeting, toxicity, and antitumor effects in breast cancer-bearing BABL/c nude mice.ResultsThe results showed that the release of doxorubicin in the nanocomposites was pH sensitive, and the cumulative release rate reached 80.53% at 60 h under acidic conditions. The nanocomposites had a high cellular uptake ability in MCF-7/ADR cells, and the IC50 value of the nanocomposites on MCF-7/ADR cells was 4.23 μg/mL, much smaller than that of free DOX (363.2 μg/mL). The nanocomposites could effectively reverse resistance and induce apoptosis of MCF-7/ADR cells. The blood biochemistry parameters and H&E staining results showed no serious adverse effects after treatment with the nanocomposites. Prussian blue staining showed that the nanocomposites were able to target tumor tissues in tumor-bearing mice under a magnetic field. The combined chemical/photodynamic therapy significantly inhibited tumor growth in vivo.ConclusionNanocomposites with magnetic and pH dual-responsive performance has shown a promising platform for enhanced drug-resistant breast cancer treatment.

Effect of Sulforaphane and Methotrexate combined treatment on Histone Deacetylase Activity in Solid Ehrlich Carcinoma

Histone acetylation is one of the posttranslational modification that plays a role in the regulation of gene expression, also modulation of acetylation with histone deacetylase inhibitors (HDACi) had been considered a novel strategy for cancer chemoprevention. This study was aimed to investigate the activity of histone deacetylase enzyme (HDAC) in tumor cell model Solid Ehrlich carcinoma under the effect of the antitumor drug methotrexate in combination with sulforaphane (SFN) which is a natural compound found in cruciferous vegetables. In the present study, sulforaphane was extracted from cabbage outer leaves. The HDAC activity significantly increased in tumor-bearing mice, non-significantly decreased after treatment with methotrexate, and significantly decreased after treatment with methotrexate with oral supplement by sulforaphane. However, The DNA damage decreased significantly in tumor tissue of tumor-bearing mice after treatment with methotrexate and increased significantly in tumor tissue of tumor-bearing mice treated with oral sulforaphane in combination with methotrexate treatment after carcinogenesis. In conclusion, sulforaphane has an antitumor effect via its pro-oxidant activity and affects the HDAC activity via its metabolites in an inhibitory manner that enhanced the effect of methotrexate.

Selective imaging of cancer cells with a pH-activatable lysosome-targeting fluorescent probe

Fluorescence imaging with tumor-specific fluorescent probe has emerged as a tool to aid surgeons in the identification and removal of tumor tissue. We report here a new lysosome-targeting fluorescent probe (NBOH) with BODIPY fluorephore to distinguish tumor tissue out of normal tissue based on different pH environment. The probe exhibited remarkable pH-dependent fluorescence behavior in a wide pH range from 3.0 to 11.0, especially a sensitive pH-dependent fluorescence change at pH range between 3.5 and 5.5, corresponding well to the acidic microenvironment of tumor cells, in aqueous solution. The response time of NBOH was extremely short and the photostability was proved to be good. Toxicity test and fluorescence cell imaging together with a sub-cellular localization study were carried out revealing its low biotoxicity and good cell membrane permeability. And NBOH was successfully applied to the imaging of tumor tissue in tumor-bearing mice suggesting potential application to surgery as a tumor-specific probe.

Nucleolin-targeted Extracellular Vesicles as a Versatile Platform for Biologics Delivery to Breast Cancer

Small interfering RNAs (siRNA)/microRNAs (miRNA) have promising therapeutic potential, yet their clinical application has been hampered by the lack of appropriate delivery systems. Herein, we employed extracellular vesicles (EVs) as a targeted delivery system for small RNAs. EVs are cell-derived small vesicles that participate in cell-to-cell communication for protein and RNA delivery. We used the aptamer AS1411-modified EVs for targeted delivery of siRNA/microRNA to breast cancer tissues. Tumor targeting was facilitated via AS1411 binding to nucleolin, which is highly expressed on the surface membrane of breast cancer cells. This delivery vesicle targeted let-7 miRNA delivery to MDA-MB-231 cells in vitro as confirmed with fluorescent microscopic imaging and flow cytometry. Also, intravenously delivered AS1411-EVs loaded with miRNA let-7 labeled with the fluorescent marker, Cy5, selectively targeted tumor tissues in tumor-bearing mice and inhibited tumor growth. Importantly, the modified EVs were well tolerated and showed no evidence of nonspecific side effects or immune response. Thus, the RNAi nanoplatform is versatile and can deliver siRNA or miRNA to breast cancer cells both in vitro and in vivo. Our results suggest that the AS1411-EVs have a great potential as drug delivery vehicles to treat cancers.

Endostatin enhances antitumor effect of tumor antigen-pulsed dendritic cell therapy in mouse xenograft model of lung carcinoma.

Objective To investigate the antitumor effect of endostatin combined with tumor antigen-pulsed dendritic cell (DC)-T cell therapy on lung cancer. Methods Transplanted Lewis lung cancer (LLC) models of C57BL/6 mice were established by subcutaneous injection of LLC cells in left extremity axillary. Tumor antigen-pulsed DC-T cells from spleen cells and bone of mice were cultured in vitro. Tumor-bearing mice were randomly divided into three groups, including DC-T+endostatin group, DC-T group, and phosphate-buffered saline (PBS) control group. Microvessel density (MVD) of tumor tissue in tumor-bearing mice was determined by immunohistochemistry (IHC). The expressions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were determined by Western blotting and IHC staining. The proportions of CD8+ T cells, mature dendritic cells (mDC), tumor-associated macrophages [TAM (M1/M2)], and myeloid-derived suppressor cells (MDSC) in suspended cells of tumor tissue were determined by flow cytometry. The expressions of interleukin (IL)-6, IL-10, IL-17, transforming growth factor-β (TGF-β) and interferon-γ (IFN-γ) in suspended cells of tumor tissue were detected by enzyme-linked immune sorbent assay (ELISA). Results DC-T cells combined with endostatin remarkably suppressed tumor growth. MVD of mice in DC-T+endostatin group was significantly lower than that of the control group and DC-T monotherapy group. The expressions of VEGF, IL-6 and IL-17 in tumors were markedly decreased, but IFN-γ and HIF-1α increased after treating with DC-T cells combined with endostatin, compared to control group and DC-T group. In the DC-T+endostatin group, the proportions of MDSC and TAM (M2 type) were significantly decreased, mDC and TAM (M1 type) were up-regulated, and CD8+ T cells were recruited to infiltrate tumors, in contrast to PBS control and DC-T monotherapy. DC-T cells combined with endostatin potently reduced the expressions of IL-6, IL-10, TGF-β and IL-17 in tumor tissue, and enhanced the expression of IFN-γ. Conclusions The study indicated the synergic antitumor effects between endostatin and tumor antigen-pulsed DC-T cells, which may be a prospective therapy strategy to achieve potent antitumor effects on lung cancer.

A Rapid and Sensitive HPLC Method for Quantitation of Paclitaxel in Biological Samples using Liquid-Liquid Extraction and UV Detection: Application to Pharmacokinetics and Tissues Distribution Study of Paclitaxel Loaded Targeted Polymeric Micelles in Tumor Bearing Mice.

A simple, rapid, and sensitive reversed-phase HPLC method was developed and validated for determination of paclitaxel (PTX) in plasma, various organs and tumor tissues of tumor-bearing mice. Tissue specimens of liver, kidneys, spleen, lungs, heart and tumor were separately homogenized in normal saline. Plasma or tissue homogenate (250 µl) containing PTX and internal standard (diazepam) were extracted by diethyl ether (6 ml). The separation was achieved on a µ-Bondapak C18 HPLC column using sodium acetate buffer solution (0.01 M)/acetonitrile (58/42 v/v) at pH 5 ± 0.1 and flow rate of 1.9 mL/min. The effluent was monitored at 227 nm and column temperature was adjusted at 58ºC. The internal standard and PTX were eluted at 4.2 and 5.2 min, respectively and no interfering peaks were observed. Calibration curves were linear over the concentration range of 0.25-10 µg/ml of PTX in plasma and 0.3-20 µg/ml PTX in tissue homogenates with acceptable precision and accuracy (<15%). The mean recoveries of the drug after plasma extraction was 87.4% ± 3.6 while those of tissue homogenates ranged from 62.1± 4.5 to 75.5± 3.2 depending on the type of tissues studied. PTX was stable in samples with no evidence of degradation during 3 freeze-thaw cycles and 3 months storage at -70 °C. The developed HPLC method was applied to quantify PTX in the mouse plasma and tissues after intravenous administration of 10 mg equivalent PTX/Kg dose of PTX-loaded tocopherol succinate-chitosan-polyethylene glycol-folate (TS-CS-PEG-FA) micelles formulation or Anzatax® (Cremophor® EL- based formulation of PTX) to female Balb/c mice.

Transcriptional Activation of Human CDCA8 Gene Regulated by Transcription Factor NF-Y in Embryonic Stem Cells and Cancer Cells

The cell division cycle associated 8 (CDCA8) gene plays an important role in mitosis. Overexpression of CDCA8 was reported in some human cancers and is required for cancer growth and progression. We found CDCA8 expression was also high in human ES cells (hESCs) but dropped significantly upon hESC differentiation. However, the regulation of CDCA8 expression has not yet been studied. Here, we characterized the CDCA8 promoter and identified its cis-elements and transcription factors. Three transcription start sites were identified. Reporter gene assays revealed that the CDCA8 promoter was activated in hESCs and cancer cell lines. The promoter drove the reporter expression specifically to pluripotent cells during early mouse embryo development and to tumor tissues in tumor-bearing mice. These results indicate that CDCA8 is transcriptionally activated in hESCs and cancer cells. Mechanistically, two key activation elements, bound by transcription factor NF-Y and CREB1, respectively, were identified in the CDCA8 basic promoter by mutation analyses and electrophoretic motility shift assays. NF-Y binding is positively correlated with promoter activities in different cell types. Interestingly, the NF-YA subunit, binding to the promoter, is primarily a short isoform in hESCs and a long isoform in cancer cells, indicating a different activation mechanism of the CDCA8 transcription between hESCs and cancer cells. Finally, enhanced CDCA8 promoter activities by NF-Y overexpression and reduced CDCA8 transcription by NF-Y knockdown further verified that NF-Y is a positive regulator of CDCA8 transcription. Our study unearths the molecular mechanisms underlying the activation of CDCA8 expression in hESCs and cancer cells, which provides a better understanding of its biological functions.