Development of β-amino-carbonyl compounds as androgen receptor antagonists

Aim： Androgen receptor （AR） antagonists have proven to be useful in the early control of prostate cancer. The aim of this study was to identify and characterize a novel β-amino-carbonyl-based androgen receptor antagonist. Methods Different isomers of the β-amino-carbonyl compounds were obtained by chiral separation. The bioactivities of the isomers were evaluated by AR nuclear translocation, mammalian two-hybrid, competitive receptor binding and cell proliferation assays. The expression of genes downstream of AR was analyzed with real-time PCR. The therapeutic effects on tumor growth in vivo were observed in male SClD mice bearing LNCaP xenografts. Results： Compound 21 was previously identified as an AR modulator by the high-throughput screening of a diverse compound library. In the present study, the two isomers of compound 21, termed compounds 21-1 and 2.1.-2, were characterized as partial AR agonists in terms of androgen-induced AR nuclear translocation, prostate-specific antigen expression and cell proliferation. Further structural modifications led to the discovery of a androgen receptor antagonist （compound 6012）, which blocked androgen receptor nuclear translocation, androgen-responsive gene expression and androgen-dependent LNCaP cell proliferation. Four stereoisomers of compound 6012 were isolated, and their bioactivities were assessed. The pharmacological effects of 6012, including AR binding, androgen-induced AR translocation, NH2- and COOH-terminal interaction, growth inhibition of LNCaP cells in vitro and LNCaP xenograft growth in nude mice, were mainly restricted to isomer 6012-4 （1R, 3S）. Conclusion： Compound 6012-4 was determined to be a novel androgen receptor antagonist with prostate cancer inhibitory activities comparable to bicalutamide both in vitro and in vivo.     

Plumbagin attenuates cancer cell growth and osteoclast formation in the bone microenvironment of mice

Aim： To investigate the effects of plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, on human breast cancer cell growth and the cancer cell-induced osteolysis in the bone microenvironment of mice.
Methods： Human breast cancer cell subline MDA-MB-231SA with the ability to spread and grow in the bone was tested. The cell proliferation was determined using the CCK-8 assay. Apoptosis was detected with Annexin V/PI double-labeled flow cytometry. Red fluorescent protein-labeled MDA-MB-231SArfp cells were injected into the right tibia of female BALB/c-nu/nu mice. Three days after the inoculation, the mice were injected with plumbagin （2, 4, or 6 mg/kg, ip） 5 times per week for 7 weeks. The growth of the tumor cells was monitored using an in vivo imaging system. After the mice were sacrificed, the hind limbs were removed for radiographic and histological analyses.

Results： Plumbagin （2.5–20 μmol/L） concentration-dependently inhibited the cell viability and induced apoptosis of MDA-MB-231SA cells in vitro （the IC50 value of inhibition of cell viability was 14.7 μmol/L）. Administration of plumbagin to breast cancer bearing mice delayed the tumor growth by 2–3 weeks and reduced the tumor volume by 44%–74%. The in vivo imaging study showed that plumbagin dose-dependently inhibited MDA-MB-231SArfp cell growth in bone microenvironment. Furthermore, X-ray images and micro-CT study demonstrated that plumbagin reduced bone erosion area and prevented a decrease in bone tissue volume. Histological studies showed that plumbagin dose-dependently inhibited the breast cancer cell growth, enhanced the cell apoptosis and reduced the number of TRAcP-positive osteoclasts.

Conclusion： Plumbagin inhibits the cell growth and induces apoptosis in human breast cancer cells in mice bone microenvironment, leading to significant reduction in osteolytic lesions caused by the tumor cells.     

Effect of Lulong Zaisheng Decoction II on chemotherapy-induced bone marrow suppression and its mechanism北大核心CSCD

Aim To investigate the effect of Lulong Zaisheng Decoction II on chemotherapy-induced bone marrow suppression in nude mice bearing colorectal cancer. Methods Male BALB/C nude mice were inoculated with human colon cancer cell HT-29 under the armpit. The tumor bearing nude mice were randomly divided into five groups: control group, chemotherapy group, positive drug group, Lulong Zaisheng Decoction II groups with high and low doses. The mice were given drugs by gavage once a day for 10 consecutive days. From the fourth day of the experiment, except for the control group, the nude mice were intraperitoneally injected with 5-FU at dose of 25 mg • kg-1 for 7 consecutive days. The mice in control group were injected with the same volume of normal saline. On the 11th day, blood was collecled from the orbit to measure peripheral hemogram. The mice were killed after cervical dislocation, then the morphology of bone marrow cells was observed - Bone marrow cell cycle, apoplosis rate and CD34+ were detected by flow cytometiy, mRNA expressions of granulocyle-macrophage colony stimulating factor (GM-CSF), granulocyte-macrophage colony stimulating factor receptor (GM-CSFR), inlerleukin-1 p (IL-1 (3) and inlerleukin-3 (IL-3) were tested by quantitative real-lime PCR, and the protein expressions of vascular endothelial growth factor (VEGF) and intercellular cell adhesion molecule-1 (VCAM-1) were tested by Western blot. Results Lulong Zaisheng Decoction II significantly elevated the number of white cells, increased the proportion of CD34 positive cells and the percentage of S cells, and reduced the apoplosis rate of bone marrow cells. Furthermore, it obviously up-regulated the mRNA expressions of GM-CSF, GM-CSFR, IL-1 (3 and IL-3, and promoted the protein expressions of VEGF and VCAM-1 in bones. Conclusions Lulong Zaisheng Decoction II can significantly alleviate bone marrow suppression caused by chemotherapy. Its mechanism is closely related to regulating cell cycle, preventing bone marrow cell apoptosis, promoting the secretion and expression of hematopoietic growth factors, and improving bone marrow hematopoietic microenvironment. © Food and Fermentation Industries. All rights reserved.     

Evaluation of doxorubicin-loaded pH-sensitive polymeric micelle release from tumor blood vessels and anticancer efficacy using a dorsal skin-fold window chamber model

Aim： To evaluation the doxorubicin （DOX）-loaded pH-sensitive polymeric micelle release from tumor blood vessels into tumor interstitium using an animal vessel visibility model, the so-called dorsal skin-fold window chamber model.
Methods： DOX-loaded pH-sensitive polyHis-b-PEG micelles and DOX-loaded pH-insensitive PLLA-b-PEG micelles were prepared. The uptake of the micelles by MDA-MB-231 breast cancer cells in vitro and in vivo was examined using flow cytometry. The pharmacokinetic parameters of the micelles were determined in SD rats after intravenous injection of a DOX dose （6 mg/kg）. The release of the micelles from tumor vasculature and the antitumor efficacy were evaluated in MDA-MB-231 breast cancer xenografted in nude mice using a dorsal skin-fold window chamber.
Results： The effective elimination half-life t1/2 of the pH-sensitive, pH-insensitive polymeric micelles and DOX-PBS in rats were 11.3 h, 9.4 h, and 2.1 h, respectively. Intravital microscopy in MDA-MB-231 breast cancer xenografted in nude mice showed that the pH-sensitive polymeric micelles rapidly extravasated from the tumor blood vessels, and DOX carried by the pH-sensitive micelles was preferentially released at the tumor site as compared to the pH-insensitive polymeric micelles. Furthermore, the pH-sensitive polymeric micelles exhibited significant greater efficacy in inhibition of tumor growth in the nude mice.
Conclusion： When DOX is loaded into pH-sensitive polymeric micelles, the acidity in tumor interstitium causes the destabilization of the micelles and triggers drug release, resulting in high local concentrations within the tumor, thus more effectively inhibiting the tumor growth in vivo.     

Pharmacokinetic-pharmacodynamic modeling of the anticancer effect of erlotinib in a human non-small cell lung cancer xenograft mouse model

Aim： Erlotinib is used to treat non-small-cell lung cancer （NSCLC）, which targets epidermal growth factor receptor （EGFR） tyrosine kinase. The aim of this study was to investigate the relationship between erlotinib plasma concentrations and phosphorylated EGFR （pEGFR） levels, as well as the relationship between pEGFR levels and tumor growth inhibition in a human non-small-cell lung cancer xenograft mouse model. Methods： Female BALB/c nude mice were implanted with the human NSCLC cell line SPC-A-1. The animals were given via gavage a single dose of erlotinib （4, 12.5, or 50 mg/kg）. Pharmacokinetics of erlotinib was determined using LC-MS/MS. Tumor volume and pEGFR levels in tumor tissues were measured at different time points after erlotinib administration, The levels of pEGFR in tumor tissues was detected using Western blotting and ELISA assays. Results： The pharmacokinetics of erlotinib was described by a two-compartment model with first order extravascular absorption kinetics. There was a time delay of approximately 2 h between erlotinib plasma concentrations and pEGFR degradation. The time course of pEGFR degradation was reasonably fit by the indirect response model with a calculated IC~o value of 1.80 pg/mL. The relationship between pEGFR levels and tumor volume was characterized by the integrated model with a Kbio value of 0.507 cm3/week which described the impact of pEGFR degradation on tumor growth. Conclusion： The pharmacokinetic/pharmacodynamic properties of erlotinib in a human tumor xenograft model were described by the indirect response model and integrated model, which will be helpful in understanding the detailed processes of erlotinib activity and determining an appropriate dosing regimen in clinical studies.     

Pharmacokinetic evaluation of the anticancer prodrug Simmitecan in different experimental animals

Aim： To investigate the pharmacokinetics and disposition of simmitecan （L-P） that was a water-soluble ester prodrug of chimmitecan （L-2-Z） with potent anti-tumor activities in different experimental animals, and to assess its drug-drug interaction potential. Methods： SD rats were injected with a single iv bolus doses of L-P （3.75, 7.5 and 15 mp=/kg）. The pharmacokinetics, tissue distribution excretion and metabolism of L-P and its active metabolite L-2-Z were studied through quantitative measurements and metabolite profiling with LC/MS. The binding of L-P and L-2-Z to rat plasma proteins was examined using an ultrafiltration method. Systemic exposures of beagle dogs to L-P as well as drug distribution in tumors of the nude mice xenograft model of human hepatic cancer SMMC-7721 cells were also examined. The metabolism of L-P by liver mcirosomal carboxylesterase in vitro was investigated in different species. The effects of L-P and L-2-Z on cytochrome P450 enzymes were examined using commercial screening kits. Results： The in vivo biotransformation of L-P to L-2-Z showed a significant species difference, with a mean elimination half-life tl/2 of approximately 1.4 h in rats and 1.9 h in dogs. The systemic exposure levels of L-P and L-2-Z were increased in a dose-dependent manner. In rats, approximately 66% of L-P and 79% of L-2-Z were bound to plasma proteins. In rats and the nude mice bearing human hepatic cancers, most organ tissues had significantly higher concentrations of L-P than the corresponding plasma levels. In the tumor tissues, the L-P levels were comparable to those of plasma, whereas the L-2-Z levels were lower than the L-P levels. In rats, L-P was eliminated mainly via biliary excretion, but metabolism played an important role in elimination of the intact L-P. Finally, L-P and L-2-Z exerted moderate inhibition on the activity of CYP3A4 in vitro. Conclusion： L-P and L-2-Z have relatively short elimination half-lives and L-P is mainly eliminated via biliary excretion. The species difference in the conversion of L-P to L-2-Z and potential drug-drug interactions due to inhibition of CYP3A4 should be considered in further studies.     

Overexpression of coxsackie and adenovirus receptor inhibit growth of human bladder cancer cell in vitro and in vivo

Aim： To study the effect of the overexpression of coxsackie and the adenovirus receptor （CAR） on the growth of the human bladder cancer cell in vitro and in vivo. Methods： A retroviral vector pLXSN-CAR expressing CAR was constructed and confirmed by restriction enzyme mapping. The pLXSN-CAR vector and con- trol vector pLXSN were transfected into the PT67 packaging cell line to generate retrovirus with high titer. The CAR-negative T24 cell was infected with the pLXSNCAR and the pLXSN retrovirus, respectively. The positive clone cells were selected with G418 for 2 weeks. The expression level of the CAR protein was detected by Western blot assay. T24 cell growth in vitro was determined by 3-（4, 5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay. Anchorage-independent growth was measured by soft-agar colony formation assay. In vivo cell growth was determined by a nude mice xenograft model. Results： The pLXSN-CAR vector containing full-length CAR cDNA was successfully constructed. Western blot analysis showed that a 46 kDa specific band was found in pLXSN-CA-transfected T24 cells. MTT assay identified the growth inhibition of T24/pLXSN-CAR cells. The cell colony forming ability of T24/pLXSNCAR cells was significantly lower than that of T24/pLXSN and parental T24 cells. There was a reduction in the tumor size in the T24/pLXSN-CAR group as compared with that of the T24/pLXSN group and parental T24 group. Conclusion： The overexpression of CAR in T24 bladder cancer cells can inhibit cell growth both in vitro and in vivo.     

Lapatinib-incorporated lipoprotein-like nanoparticles： preparation and a proposed breast cancer-targeting mechanism

Aim： Lapatinib is a dual inhibitor of EGFR and human epidermal growth factor receptor 2 （HER2）, and used to treat advanced breast cancer. To overcome its poor water solubility, we constructed lapatinib-incorporated lipoprotein-like nanoparticles （LTNPs）, and evaluated the particle characteristics and possible anti-breast cancer mechanisms.
Methods： LTNPs （lapatinib bound to albumin as a core, and egg yolk lecithin forming a lipid corona） were prepared. The particle characteristics were investigated using transmission electron microscopy （TEM） and atomic force microscopy （AFM）. The uptake and subcellular localization of LTNPs, as well as the effects of LTNPs on cell cycle were examined in BT-474 human breast cancer cells in vitro. Mice bearing BT-474 subcutaneous xenograft were intravenously injected with coumarin-6 loaded LTNPs （30 mg/kg） to study the targeting mechanisms in vivo.
Results： The LTNPs particles were generally spherical but flexible under TEM and AFM, and approximately 62.1 nm in size with a zeta potential of 22.80 mV. In BT-474 cells, uptake of LTNPs was mediated by endosomes through energy-dependent endocytosis involving clathrin-dependent pinocytosis and macropinocytosis, and they could effectively escape from endosomes to the cytoplasm. Treatment of BT-474 cells with LTNPs （20 μg/mL） induced a significant cell arrest at G0/G1 phase compared with the same concentration of lapatinib suspension. In mice bearing BT-474 xenograft, intravenously injected LTNPs was found to target and accumulate in tumors, and colocalized with HER2 and SPRAC （secreted protein, acidic and rich in cysteine）.
Conclusion： LTNPs can be taken up into breast cancer cells through specific pathways in vitro, and targeted to breast cancer xenograft in vivo via enhanced permeability and retention effect and SPARC.     

Berbamine exhibits potent antitumor effects on imatinib-resistant CML cells in vitro and in vivo

Aim The aim of this study was to explore the effects and mechanism of berbamine on imatinib-resistant BCR-ABL-positive human leukemia K562 （K562-r） cells in vitro and in vivo. Methods：Cell viability was measured by MTT assay, and apoptotic morphology changes were detected by fluorescence microscopy. The apoptosis rate was measured by flow cytometric assay, mdr-1 mRNA levels were determined by RT-PCR. Bcl-2 family proteins, cytochrome c（ cyt C） , poly （ADP-ribose） polymerase （PARP）, and P-glycoprotein were detected by Western blot. BALB/c nu/nu mice were injected with K562-r cells subcutaneously. Tumor-bearing mice were treated intravenously with berbamine.
Results： MTT tests revealed that berbamine significantly inhibited K562-r cell proliferation and increased the chemosensitivity of K562-r cells to imatinib. The apoptosis rate was significantly increased following treatment with 21.2μmol/L berbamine; formation of typical apoptotic blebs was apparent, as observed by fluorescence microscopy. Expression levels of mdr-I mRNA and P-gp protein were high in untreated K562-r cells and significantly down-regulated by berbamine treatment. Berbamine-treated K562-r cells also exhibited down-regulated expression of the anti-apoptotic proteins Bcl-2 and Bcl-XL up-regulated expression of the apoptotic proteins Bax and cytoplasmic cyt C, and stimulated proteolytic cleavage of PARP. In addition, berbamine also suppressed the growth of K562-r xenotransplanted tumors in vivo.
Conclusion Berbamine inhibited proliferation of K562-r ceils both in vitro and in vivo. Berbamine-induced apoptosis in K562-r cells appeared to occur through a mechanism involving Bcl-2 family proteins, as well as mdr-1 mRNA and P-gp pro- tein. Berbamine in combination with imatinib restored the chemo-sensitivity of K562-r cells to imatinib. Our findings suggest that berbamine may be useful in treating imafinib-resistant CML patients.     

Inhibition of EGFR autophosphorylation plays an important role in the anti-breast cancer efficacy of the dithiocarbamate derivative TM208

Aim： To investigate the effects of a novel dithiocarbamate derivative TM208 on human breast cancer cells as well as the pharmacoki- netic characteristics of TM208 in human breast cancer xenograft mice. Methods： Human breast cancer MCF-7 and MDA-MB-231 cells were treated with TM208 or a positive control drug tamoxifen. Cell pro- liferation was examined using SRB and colony formation assays. Cell apoptosis was analyzed with Annexin V-FITC/PI staining assay. Protein expression was examined with Western blot, ELISA and immunohistochemical analyses. MCF-7 breast cancer xenograft nude mice were orally administered TM208 （50 or 150 mg.k$1〈1-1） or tamoxifen （50 mg.kgl〈t-~） for 18 d. On d 19, the tumors were collected for analyses. Blood samples were collected from the mice treated with the high dose of TM208, and plasma concentrations of TM208 were measured using LC-MS/MS. Results： Treatment of MCF-7 and MDA-MB-231 cells with TM208 dose-dependently inhibited the cell proliferation and colony formation in vitro （the IC~o values were 36.38＋3.77 and 18.13＋0.76 pmol/L, respectively）. TM208 （20-150pmol/L） dose-dependently induced apoptosis of both the breast cancer cells in vitro. In MCF-7 breast cancer xenograft nude mice, TM208 administration dose-depend- ently reduced the tumor growth, but did not result in the accumulation of TM208 or weight loss. TM208 dose-dependently inhibited the phosphorylation of EGFR and ERK1/2 in both the breast cancer cells in vitro as well as in the MCF-7 xenograft tumor. Conclusion： Inhibition of EGFR autophosphorylation plays an important role in the anticancer effect of TM208 against human breast cancer.     

ntranasal co-delivery of IL-6 gene enhances the mmunogenicity of anti-caries DNA vaccine

Aim： To investigate the effects of co-delivering 11_-6 expressing plasmid pCI-IL-6 on the immunogenicity of the anti-caries DNA vaccine pClA-P, which encodes the surface protein antigen PAc of Streptococcus mutans. Methods： Plasmid pCI-IL-6 was constructed by inserting the murine IL-6 gene into the pCI vector. Expression of IL-6 in vitro was assessed using Western blot analysis. BALB/c mice were intranasally co-immunized with pClA-P plus pCI-IL-6 on d 0 and 14. Anti-PAc IgG and secretory IgA （slgA） were assessed by ELISA. Splenocytes from the mice were re-stimulated with the PAc protein, and IFN-y and IL-4 production was measured using ELISA. Splenocyte proliferation was analyzed with flow cytometry. Rats were similarly immunized, and dental caries scores were determined using the Keyes method. Results： Marked expression of IL-6 was found in C0S-7 cells transfected with pCI-IL-6. In the pCI-IL-6 co-immunized mice, the specific IgG antibodies in serum and slgA antibodies in saliva were significantly higher than those in the control mice at weeks 4 and 8. Moreover, the secretion of IFN-y from splenocytes in response to re-stimulation with PAc protein was significantly higher in the pCI-IL-6 co-immunized mice than that in the control mice, whereas the secretion of IL-4 had no significant difference. The proliferation of splenocytes from the pCI-IL-6 co-immunized mice was significantly higher than that from the mice immunized with pClA-P and pCl vector. In the rat caries model, the pCI-IL-6 co-immunization rats displayed lower caries scores than the control rats. Conclusion： Intranasal co-delivery of IL-6 gene significantly enhances the immunogenicity of the anti-caries DNA vaccine.     

Antitumor effects of concanavalin A and Sophora flavescens lectin in vitro and in vivo

Aim： Proteins with legume lectin domains are known to possess a wide range of biological functions. Here, the antitumor effects of two representative legume lectins, concanavalin A （ConA） and Sophora flavescens lectin （SFL）, on human breast carcinoma cells were investigated in vitro and in vivo. Methods： Human breast carcinoma MCF-7 cells and human normal mammary epithelial MCF-IOA cells were examined. Cell viability was detected using WST-1 and CCK-8 assays. Cell apoptosis was analyzed with Hoechst 33258 staining. Cell cycle was investigated using flow cytometry. The expression of relevant proteins was measured using Western blotting. Breast carcinoma MCF-7 bearing nude mice were used to study the antitumor effects in vivo. The mice were injected with ConA （40 mg/kg, ip） and SFL （55 mg/kg, ip） daily for 14 d. Results： ConA and SFL inhibited the growth of MCF-7 cells in dose- and time-dependent manners （ICso values were 15 and 20 pg/mL, respectively）. Both ConA and SFL induced apoptotic morphology in MCF-7 cells without affecting MCF-IOA cells. ConA and SFL dose- dependently increased the sub-G1 proportion in MCF-7 cells, while SFL also trip=~ered the G2/M phase cell cycle arrest. Both ConA and SFL dose-dependently increased the activities of caspase-3 and caspase-9 and release of cytochrome c from mitochondria into cytoplasm, up-regulated Bax and Bid, and down-regulated Bcl-2 and BcI-XL in MCF-7 cells. ConA reduced NF-KB, ERK, and JNK levels, and increased p53 and p21 levels, while SFL caused similar changes in NF-KB, ERK, p53, and p21 levels, but did not affect JNK expression. Administration of ConA and SFL significantly decreased the subcutaneous tumor mass volume and weight in MCF-7 bearing nude mice. Conclusion： ConA and SFL exert anti-tumor actions against human breast carcinoma MCF-7 cells both in vitro and in vivo.     

Diosgenin relieves goiter via the inhibition of thyrocyte proliferation in a mouse model of Graves＇ disease

Aim： To investigate the effects of diosgenin （Dio）, a naturally occurring steroid saponin, on goiter formation in a mouse model of Graves’ disease （GD） and the underlying mechanisms.
Methods： Female BALB/c mice were injected with adenovirus expressing the A subunit of thyrotropin receptor to induce GD. The mice were treated with Dio （20, 100 mg·kg-1·d-1, ip） for 12 or 24 d. The serum levels of TT4 and TRAb were examined using radioimmunoassay and electrochemiluminescence. The size and morphology of thyroid glands were examined. Thyrocyte proliferation was determined using BrdU incorporation assay. The expression of proliferation-associated proteins IGF-1, NF-κB, cyclin D1, and PCNA in thyroids was analyzed using immunohistochemistry and real-time PCR.

Results： The GD mice showed significantly high serum levels of TRAb and TT4 compared to the normal mice. Treatment of the GD mice with Dio for 24 d dose-dependently reduced the TT4 level and thyroid size, but did not affect the abnormal level of TRAb. Furthermore, Dio treatment dose-dependently reversed the morphological changes and reduced excessive thyrocyte proliferation in thyroids of the GD mice. Dio treatment also dose-dependently reduced the mRNA and protein levels of IGF-1, NF-κB, cyclin D1, and PCNA in thyroids of the GD mice.

Conclusion： Dio relieves goiter in a mouse model of GD through the inhibition of thyrocyte proliferation. The mechanisms involve the suppression of IGF-1, NF-κB, cyclin D1, and PCNA expression.     

CMV-hFasL transgenic mice are sensitive to low doses of streptozotocin-induced type Ⅰ diabetes mellitus

AIM: To investigate the role of Fas-FasL pathway in the pathogenesis of streptozotocin (STZ)-induced type Ⅰ diabetes mellitus. METHODS: Low dose injections of STZ were used to induce type Ⅰ diabetes mellitus in the CMV-hFasL transgenic mice. Blood glucose concentration was measured with Glucotrand Plus blood glucose test strips. Expression of hFasL was detected by RT-PCR and Western blotting. The severity of insulitis was determined by histological examination. Expressions of IL-1β and TNF-α mRNA in the pancreas were detected by semi-quantitative RT-PCR analysis. Fas expression in apoptotic RIN-5F cells was also confirmed by RT-PCR in vitro. RESULTS: hFasL was expressed in the islets of CMV-hFasL transgenic mice. The transgenic mice were sensitive to diabetic induction than the control WT mice. IL-1β and TNF-α expressions in the pancreas of CMV-hFasL transgenic mice were far more than that in WT mice. We also found STZ and IL-1β could both induce higher expression of Fas in RIN-5F. The combining     

Positive reaction of direct popliteal lymph node assay isn't induced in humanized NPG mice with reconstituted immune system北大核心CSCD

OBJECTIVE: To perform direct popliteal lymph node assay (d-PLNA) induced by D-penicillamine hydrochloride (D-pen) or streptozotocin (STZ) in humanized NPG (hu-NPG) mice with a reconstituted immune system, NPG mice and BALB/c mice respectively, and to compare the responses in these mice to elucidate the mechanism of d-PLNA. METHODS: Female NPG mice were irradiated and transplanted with human hematopoietic stem cells isolated from cord blood to form hu-NPG mice. The number and function of peripheral blood lymphocytes in hu-NPG mice were detected by flow cytometry and lymphocyte proliferation test. The hu-NPG, NPG and BALB/c mice were randomly divided into D-pen group and STZ group (5 mice per group), respectively. Then, the positive chemical D-pen (1 mg per mouse) or STZ (0.75 mg per mouse) was injected subcutaneously into the right hind footpad of these mice using the established d-PLNA protocol. On the 7th day after injection, the mice were sacrificed. The bilateral popliteal lymph nodes (PLNs) were weighed. The difference of d-PLNA response was evaluated at 7 d after the injection of positive drugs. RESULTS: All the NPG, hu-NPG and BALB/c mice behaved normally after the injection of positive drugs. No systemic toxicity was observed. The symptom of local irritation disappeared within 7 d. As in previous studies, the BALB/c mice showed a typical positive response of d-PLNA, as evidenced by the increase in both mass and cell count of the PLNs in the treated lateral (mass index >2 and cellularity index >5), while NPG mice and hu-NPG mice showed a negative resoponse. Pathological examinations demonstrated that the PLNs of NPG mice and hu-NPG mice had developmental defect in lymphoid tissue and were smaller in size than BALB/c mice. Phenotypic analysis of the peripheral blood lymphocytes in hu-NPG mice showed that the majority of human lymphocytes expressed B cell markers CD45+CD19+ (75.5±6.6)%, and only about (17.1±6.6)% of the lymphocytes expressed T cell markers CD45+CD3+. There were few lymphocytes in NPG mice. In addition, these cells did not proliferate with mitogen stimulation in the lymphocyte proliferation test. CONCLUSION: The d-PLNA reaction induced by D-pen or STZ in mice is closely related to the number and normal function of lymphocytes in vivo. Negative d-PLNA reaction results from a lack of normal lymphocytes in NPG mice or from defected human lymphocyte function in hu-NPG mice. Thus hu-NPG mice are currently not suitable for d-PLNA.     

The immunosuppressive effect of gossypol in mice is mediated by inhibition of lymphocyte proliferation and by induction of cell apoptosis

Aim： To investigate the immunosuppressive effect of gossypol in mice both in vitro and in vivo. Methods： The in vitro effect of gossypol on the proliferation of lymphocytes isolated from lymph nodes of BALB/c mice was determined by CFSE staining and by an MTS assay. Lymphocyte activation and lymphoblastic transformation were evaluated with immunostaining. Cell apoptosis was detected by Annexin-V and Hoechst 33342 staining. The in vivo immunosuppressive effect of gossypol on the DTH reaction was evaluated using a mouse DTH model induced by 2,4-dinitro-1- fluorobenzene （DNFB）. The thickness of the ears was measured, and the histological changes of the mouse auricles were observed after hematoxylin-eosin staining. The proliferation capacity of lymphocytes from DTH mice was also assayed. Results： In vitro, gossypol could significantly inhibit the proliferation of mouse lymphocytes stimulated with phorbol ester plus ionomycin in a dose-dependent manner. Although the expression of the early activation antigen CD69 was not affected, the lymphoblastic transformation of both T and B lymphocyte subsets was significantly suppressed by gossypol. Moreover, gossypol could induce apoptosis of lymphocytes, and the effect was time- and dose-dependent. In vivo, the DTH reaction in mice was markedly alleviated by gossypol injected intraperitoneally. Lymphocytes from drug-treated DTH mice had a reduced proliferation capacity as compared with lymphocytes from untreated DTH mice. Gossypol treatment also markedly reduced the number of infiltrated lymphocytes in the auricles of DTH mice. Conclusion： Gossypol exhibited immunosuppressive effects in mice, probably by inhibition of lymphocyte proliferation and by induction of cell apoptosis.