OBJECTIVE: To analyze the effects of mangiferin combined with bortezomib on the proliferation, invasion, apoptosis and autophagy of human Burkitt lymphoma Raji cells, as well as the expression of CXC chemokine receptors (CXCRs) family, and explore the molecular mechanism between them to provide scientific basis for basic research and clinical work of Burkitt lymphoma. METHODS: Raji cells were intervened with different concentrations of mangiferin and bortezomib alone or in combination, then cell proliferation was detected by CCK-8 assay, cell invasion ability was detected by Transwell chamber method, cell apoptosis was detected by Annexin V/PI double-staining flow cytometry, apoptosis, autophagy and Akt/mTOR pathway protein expression were detected by Western blot, and the expression changes of CXCR family was detected by real-time quantitative PCR (RT-qPCR). RESULTS: Different concentrations of mangiferin intervened Raji cells for different time could inhibit cell viability in a concentration- and time-dependent manner (r =-0.682, r =-0.836). When Raji cells were intervened by combination of mangiferin and bortezomib, compared with single drug group, the proliferation and invasion abilities were significantly decreased, while the apoptosis level was significantly increased (P <0.01). Mangiferin combined with bortezomib could significantly up-regulate the expression of pro-apoptotic protein Bax and down-regulate the expression of anti-apoptotic protein Bcl-2 after intervention in Raji cells. Caspase-3 was also hydrolyzed and activated, and then induced the apoptosis of Raji cells. Mangiferin combined with bortezomib could up-regulate the expression of LC3Ⅱ protein in Raji cells, and the ratio of LC3Ⅱ/LC3Ⅰ in cells was significantly up-regulated compared with single drug or control group (P <0.01). Mangiferin combined with bortezomib could significantly inhibit the phosphorylation levels of Akt and mTOR, inhibit the proliferation and invasion of Raji cells by inhibiting Akt/mTOR pathway, and induce cell autophagy and apoptosis. Mangiferin and bortezomib could down-regulate the expressions of CXCR4 and CXCR7 mRNA after single-agent intervention in Raji cells, and the down-regulations of CXCR4 and CXCR7 mRNA expression were more significant when the two drugs were combined (P <0.01). Mangiferin alone or combined with bortezomib had no significant effect on CXCR5 mRNA expression in Raji cells (P >0.05), while the combination of the two drugs could down-regulate the expression of CXCR3 (P <0.05). CONCLUSION Mangiferin combined with bortezomib can synergistically inhibit the proliferation and invasion of Raji cells, and induce autophagy and apoptosis. The mechanism may be related to the inhibition of Akt/mTOR signaling pathway, down-regulation of anti-apoptotic protein Bcl-2 and up-regulation of pro-apoptotic protein Bax, and the inhibition of the expression of CXCR family.
Humans ; Antineoplastic Agents/therapeutic use* ; Apoptosis/drug effects* ; Apoptosis Regulatory Proteins/immunology* ; Autophagy/immunology* ; bcl-2-Associated X Protein/immunology* ; Bortezomib/therapeutic use* ; Burkitt Lymphoma/immunology* ; Cell Line, Tumor ; Cell Proliferation/drug effects* ; Drug Therapy, Combination ; Proto-Oncogene Proteins c-akt ; Proto-Oncogene Proteins c-bcl-2 ; Receptors, CXCR/immunology* ; RNA, Messenger ; TOR Serine-Threonine Kinases ; Xanthones/therapeutic use*

OBJECTIVEEscape from the body's immune response is a basic characteristic of lung cancer, and indoleamine-2,3-dioxygenase (IDO) plays a key role in mediating immune escape of non-small-cell lung cancer, which leads to recurrence and metastasis. Feiji Recipe, a compound Chinese herbal medicine, has the effect of stabilizing lesions and prolonging survival in patients with lung cancer. The purpose of this study was to investigate the mechanisms underlying the anticancer properties of Feiji Recipe.

METHODSAn orthotopic transplant model of mouse Lewis lung cancer, with stable expression of IDO gene, was established in C57BL/6 mice. Optical imaging was used to observe the effects of Feiji Recipe in the treatment of lung cancer in vivo. The effects of Feiji Recipe on the proliferation of mouse Lewis lung cancer cell line 2LL, 2LL-enhanced green fluorescent protein (2LL-EGFP) and 2LL-EGFP-IDO were investigated, and the apoptosis of T-cells was examined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide using flow cytometry. Chemical composition of Feiji Recipe was validated by high-performance liquid chromatography.

RESULTSCompared to the control group, the survival of animals treated with Feiji Recipe was significantly prolonged (P = 0.0074), and the IDO protein level decreased (P = 0.0072); moreover, the percentages of CD4CD25 T-cells and Foxp3 T-cells were significantly decreased (P < 0.05). The molecular mechanism of Feiji Recipe against lung cancer may relate to the regulation of immune cells, such as T-cells and regulatory T-cells.

CONCLUSIONThe molecular mechanism of Feiji Recipe in treatment of lung cancer is to restore the function of T-cells in the cancer microenvironment through interfering with the IDO pathway.

Animals ; Apoptosis ; drug effects ; Carcinoma, Lewis Lung ; drug therapy ; enzymology ; immunology ; physiopathology ; Cell Proliferation ; drug effects ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Growth Inhibitors ; administration & dosage ; Humans ; Indoleamine-Pyrrole 2,3,-Dioxygenase ; genetics ; immunology ; Lung Neoplasms ; drug therapy ; enzymology ; immunology ; physiopathology ; Male ; Mice ; Mice, Inbred C57BL ; T-Lymphocytes, Regulatory ; drug effects ; immunology

OBJECTIVETo investigate the inhibitory effect of high concentration insulin on K562 cell proliferation and its underlying mechanism.

METHODSK562 cells were treated by different concentration of insulin and/or anti-IGF-1R antibody (IGF-1R-Ab), MTT assay and flow cytometry were used to detect the K562 cells proliferation and apoptosis, respectivety; Western blot was used to measure the expression and phosphorylation level of IGE-IR, Akt, Erk1/2 in K562 cells under the different concentration of insulin.

RESULTSMTT assay showed that less than 40 mU/ml insulin could promote K562 cell proliferation, while high concentration (> 40 mU/ml) insulin has been shown to inhibit K562 cell proliferation; Flow cytometry showed that 40 mU/ml insulin suppressed K562 cell apoptosis (P < 0.05), while 200 mU/ml insulin could significantly induce K562 cell apoptosis (P < 0.01); 0.01 to 1.0 µg/ml IGF-1R-Ab has significantly enhanced the inhibitory and inducing effects of high concentration (> 40 mU/ml) of insulin on K562 cell proliferation and apoptosis respectively (r = 0.962, P < 0.001); Western blot showed that after K562 cells were treated with different concentrations of insulin ERK, and the p-ERK expression did not change significantly, after K562 cells were treated with 200 mU/ml insulin, the expression of IGF-1R and AKT also not were changed obviously, while the phosphorylation level of IGF-1R and AKT increased.

CONCLUSIONHigh concentration (>40 mU/ml) of insulin inhibits K562 cell proliferation and induces its apoptosis, and its mechanism may be related with the binding IGF-1R by insulin, competitively inhibiting the binding of IGF-1 and IGF-1R, the blocking the transduction of PI3K/AKT signal pathway.

Antibodies ; pharmacology ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Culture Media ; chemistry ; Humans ; Insulin ; pharmacology ; Insulin-Like Growth Factor I ; metabolism ; K562 Cells ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Phosphatidylinositol 3-Kinases ; metabolism ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Receptors, Somatomedin ; immunology ; Signal Transduction ; drug effects

OBJECTIVETo investigate whether Shen-Fu Injection (, SFI) reduces post-resuscitation immune dysfunction in a porcine model of cardiac arrest by modulating apoptosis of regulatory T lymphocytes (Treg) in the spleen.

METHODSAfter 8-min untreated ventricular fibrillation and 2-min basic life support, 24 pigs were divided into 3 groups with a random number table, i.e. SFI group, epinephrine (EP) group, and saline (SA) group (8 in each group), which received central venous injection of SFI (1.0 mL/kg), EP (0.02 mg/kg) and SA, respectively. The same procedure without CA initiation was achieved in the sham-operated (sham) group (n=6). After successful return of spontaneous circulation (ROSC), apoptosis rate of splenic Treg was detected by flow cytometry; and the mRNA expression of forkhead/winged helix transcription factor (Foxp3) of splenic Treg was detected by real time-polymerase chain reaction; and the levels of interleukin-4 (IL-4) and interferon-γ (IFN-γ) in porcine splenic Treg were detected by using enzyme-linked immunosorbent assay (ELISA).

RESULTSCompared with the sham group, the apoptosis rate of Treg was significantly decreased, and the levels of Foxp3 mRNA expression, IFN-γ, IL-4 and IFN-γ/IL-4 were increased in the SA group (P<0.05 or P<0.01). Compared with the EP and SA groups, SFI treatment increased the apoptosis rate of Treg and reduced the levels of Foxp3 mRNA expression, IFN-γ and IFN-γ/IL-4 (P<0.05).

CONCLUSIONSSFI has signifificant effects in attenuating post-resuscitation immune dysfunction by modulating apoptosis of Treg in the spleen.

Animals ; Apoptosis ; drug effects ; Cardiopulmonary Resuscitation ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Forkhead Transcription Factors ; genetics ; metabolism ; Heart Arrest ; drug therapy ; immunology ; pathology ; physiopathology ; Hemodynamics ; drug effects ; Injections ; Interferon-gamma ; metabolism ; Interleukin-4 ; metabolism ; Lymphocyte Subsets ; drug effects ; metabolism ; Male ; Oxygen ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Spleen ; immunology ; Survival Analysis ; Swine ; Swine, Miniature ; T-Lymphocytes, Regulatory ; drug effects ; immunology

Pentraxin 3 (PTX3) was identified as a marker of the inflammatory response and overexpressed in various tissues and cells related to cardiovascular disease. Honokiol, an active component isolated from the Chinese medicinal herb Magnolia officinalis, was shown to have a variety of pharmacological activities. In the present study, we aimed to investigate the effects of honokiol on palmitic acid (PA)-induced dysfunction of human umbilical vein endothelial cells (HUVECs) and to elucidate potential regulatory mechanisms in this atherosclerotic cell model. Our results showed that PA significantly accelerated the expression of PTX3 in HUVECs through the IkappaB kinase (IKK)/IkappaB/nuclear factor-kappaB (NF-kappaB) pathway, reduced cell viability, induced cell apoptosis and triggered the inflammatory response. Knockdown of PTX3 supported cell growth and prevented apoptosis by blocking PA-inducted nitric oxide (NO) overproduction. Honokiol significantly suppressed the overexpression of PTX3 in PA-inducted HUVECs by inhibiting IkappaB phosphorylation and the expression of two NF-kappaB subunits (p50 and p65) in the IKK/IkappaB/NF-kappaB signaling pathway. Furthermore, honokiol reduced endothelial cell injury and apoptosis by regulating the expression of inducible NO synthase and endothelial NO synthase, as well as the generation of NO. Honokiol showed an anti-inflammatory effect in PA-inducted HUVECs by significantly inhibiting the generation of interleukin-6 (IL-6), IL-8 and monocyte chemoattractant protein-1. In summary, honokiol repaired endothelial dysfunction by suppressing PTX3 overexpression in an atherosclerotic cell model. PTX3 may be a potential therapeutic target for atherosclerosis.
Apoptosis/drug effects ; Atherosclerosis/chemically induced/*drug therapy/immunology/pathology ; Biphenyl Compounds/chemistry/isolation & purification/*pharmacology ; C-Reactive Protein/*genetics/immunology ; Down-Regulation/drug effects ; Drugs, Chinese Herbal/chemistry/isolation & purification/*pharmacology ; Human Umbilical Vein Endothelial Cells ; Humans ; I-kappa B Kinase/*immunology ; Lignans/chemistry/isolation & purification/*pharmacology ; Magnolia/chemistry ; Palmitic Acid ; Protein-Serine-Threonine Kinases/*immunology ; Serum Amyloid P-Component/*genetics/immunology ; Signal Transduction/drug effects

This study was aimed to elucidate the expression of costimulatory molecule CD80 and CD86 in HL-60 cells induced by proteasome inhibitor MG132 and its effect on allogeneic mixed lymphocyte reaction. Acute myelocytic leukemia cell line HL-60 and chronic myelocytic leukemia cell line K562 were cultured. The viability of the cells was measured by flow cytometry. Proteasome inhibitor MG132 at the concentrations of 2 or 3 µmol/L was used to stimulate the HL-60 cell cultured for 24 h and 48 h respectively, and the Annexin V/7-AAD staining and flow cytomotry were used to detect the apoptosis of the HL-60 cells. HL-60 and K562 cells were treated with 1 µmol/L MG132 for 24 h and 48 h respectively, then CD80 and CD86 antibodies were added, finally the expression of CD80 and CD86 was analysed by flow cytomery. The mRNA expression of CD86 in the HL-60 cells treated with 1 µmol/L MG132 was detected by RT-PCR. HL-60 and K562 cells were treated by 1 µmol/L MG132 and then underwent irradiation of 75 Gy (60)Co to kill the cells with their antigenicity preserved. Peripheral blood mononuclear cells (PBMNCs) of healthy volunteers, as reactive cells, were isolated and inoculated into the (60)Co irradiated HL-60 cells of different concentrations, as stimulating cells, CCK-8 was added and then the A value of absorbance was measured at the wave length of 450 nm in an enzyme labeling instrument. The results showed that the cell viability of the HL-60 cells treated with 1 µmol/L MG132 for 24 h an d 48 h was 92.95% and 85.87% respectively. The apoptotic rates of the HL-60 cells treated with MG132 increased in dose-and time-dependent manner. High-concentration of MG132 directly killed HL-60 cells. Before MG132 treatment K562 cells did not express CD86, but the CD86 expression of the HL-60 cells was up-regulated time-dependently after MG132 treatment (P < 0.01). The mRNA expression of CD86 in the HL-60 treated with MG132 was up-regulated time-dependently (P < 0.01). CCK-8 test showed that the proliferation level of PBMNC gradually increased along with the concentration of HL-60 cells treated with MG132 and reached its peak when the concentration of the HL-60 cells was 1×10(5) (P < 0.01). No remarkable proliferation of PBMNC was observed in the K562 groups no matter if the HL-60 cells had been treated with MG132. It is concluded that the high concentration of MG132 can directly kill HL-60 cells, low-concentration of MG132 can induce the expression of costimulatory molecule CD86 in HL-60 cells, also can improve the proliferation of PBMNC.
Apoptosis ; B7-2 Antigen ; immunology ; Cell Survival ; Flow Cytometry ; HL-60 Cells ; Humans ; K562 Cells ; Leukocytes, Mononuclear ; drug effects ; Leupeptins ; pharmacology ; Lymphocyte Culture Test, Mixed ; Proteasome Inhibitors ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Up-Regulation

OBJECTIVETo study the inhibitory effect of CD133 monoclonal antibody labeled with ¹³¹I (¹³¹I-CD133mAb) on Huh-7 human liver cancer cell line overexpressing CD133 antigen in vitro and in mouse models bearing the tumor cell xenograft.

METHODS¹³¹I-CD133mAb was prepared by chloramines-T method and evaluated for its stability. Flow cytometry and immunohistochemistry were used to detect the expression of CD133 in Huh-7 cells and in Huh-7 cell-derived tumors, respectively. Huh-7 cells treated with ¹³¹I-CD133mAb plus cisplatin (DDP), ¹³¹I -CD133mAb, DDP, or no treatment (blank control) were examined for cell proliferation suppression by MTT assay with the IC₅₀ calculated. BALB/c mice bearing subcutaneous Huh-7 cell xenograft in the right forelegs were treated with ¹³¹I -CD133mAb, DDP, or both every two days for two weeks. The tumor size and volume were measured twice a week, and pathological examination of the tumor was carried out after the treatments. The tumor inhibition rate was calculated and tumor cell apoptosis observed with HE staining.

RESULTSThe labeling ratio of ¹³¹I-CD133mAb was 90.25% and the radiochemical purity was 97.78%. Huh-7 cells showed obviously higher CD133 expression than HepG2 cells. ¹³¹I-CD133mAb combined with DDP group resulted in a significantly higher tumor inhibition rate than other treatments in the tumor-bearing mice.

CONCLUSION¹³¹I-CD133mAb can inhibit the growth of liver cancer cells with a high CD133 expression both in vivo and in vitro.

AC133 Antigen ; Animals ; Antibodies, Monoclonal ; pharmacology ; Antigens, CD ; immunology ; Apoptosis ; Carcinoma, Hepatocellular ; drug therapy ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Cisplatin ; pharmacology ; Glycoproteins ; immunology ; Hep G2 Cells ; Humans ; Liver Neoplasms ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Peptides ; immunology ; Xenograft Model Antitumor Assays

OBJECTIVETo explore the enhanced sensitivity of leukemia cell line KG-1a to activated immune cell-mediated cytolysis after treated with resveratrol.

METHODSThe value of 50% inhibition concentration (IC₅₀) for KG-1a by resveratrol was analyzed using trypan blue staining. Peripheral blood mononuclear cells were separated, and then activated by interleukin (IL)-2 and IL-15. The sensitivity of KG-1a treated with and without resveratrol to activated immune cell-mediated cytolysis was assayed by lactate dehydrogenase (LDH) -releasing assay. The expression of tumor necrosis factor related apoptosis inducing ligand (TRAIL) on the surface of activated immune cells and its receptors (DR4/5 and DcR1/2) on the surface of KG-1a were detected by flow cytometry.

RESULTSResveratrol could inhibit the proliferation of KG-1a and IC50 at 24 h was 25 mmol/L. At a ratio of 10:1 or 20:1 between effect and target, the cytolytic rates of treated KG-1a by activated immune cells were (55.80 ± 10.88)% and (72.31 ± 13.06)%, significantly higher than (24.96 ± 9.25)% and (37.93 ± 5.21)% of untreated KG-1a (P<0.05). The expression of DR5 on the surface of KG-1a treated with resveratrol was (9.05 ± 3.57)%, significantly higher than (3.11 ± 0.54)% of untreated KG-1a (P<0.05). Conversely, the expression of DcR1 on the surface of treated KG-1a was (13.23 ± 3.56)%, lower than (53.75 ± 10.51)% of KG-1a (P<0.05). When TRAIL pathway on the surface of activated immune cells was blocked, the cytolytic rates of treated KG-1a were (35.97 ± 6.36)% and (49.80 ± 10.68)%, significantly lower than (52.92 ± 6.98)% and (70.73 ± 9.79)% of untreated KG-1a (P<0.05) at the same ratio of effector and target.

CONCLUSIONResveratrol could enhance cytolytic sensitivity of KG-1a by activated immune cells through TRAIL pathway.

Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Leukemia ; metabolism ; pathology ; Leukocytes, Mononuclear ; drug effects ; immunology ; metabolism ; Male ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; metabolism ; Receptors, Tumor Necrosis Factor, Member 10c ; metabolism ; Stilbenes ; pharmacology ; TNF-Related Apoptosis-Inducing Ligand ; metabolism

BACKGROUNDProtectin D1 (PD1), derived from docosahexaenoic acid, has been shown to control and resolve inflammation in some experimental models of inflammatory disorders. We investigated the protective roles of protectin D1 in pulmonary inflammation and lung injury induced by lipopolysaccharide (LPS).

METHODSMice were randomly assigned to six groups (n = 6 per group): sham-vehicle group, sham-PD1 group, sham-zVAD-fmk group, LPS-vehicle group, LPS-PD1 group, and LPS-PD1-zVAD-fmk group. Mice were injected intratracheally with 3 mg/kg LPS or saline, followed 24 hours later by intravenous injection of 200 µg/mouse PD1 or vehicle. At the same time, some mice were also injected intraperitoneally with the pan-caspase inhibitor zVAD-fmk. Seventy-two hours after LPS challenge, samples of pulmonary tissue and bronchoalveolar lavage fluid were collected. Optical microscopy was used to examine pathological changes in lungs. Cellularity and protein concentration in bronchoalveolar lavage fluid were analyzed. Lung wet/dry ratios and myeloperoxidase activity were measured. Apoptosis of neutrophils in bronchoalveolar lavage fluid (BALF) was also evaluated by flow cytometry.

RESULTSIntratracheal instillation of LPS increased neutrophil counts, protein concentration in bronchoalveolar lavage fluid and myeloperoxidase activity, it induced lung histological injury and edema, and also suppressed apoptosis of neutrophils in BALF. Posttreatment with PD1 inhibited LPS-evoked changes in BALF neutrophil counts and protein concentration and lung myeloperoxidase activity, with the outcome of decreased pulmonary edema and histological injury. In addition, PD1 promoted apoptosis of neutrophils in BALF. The beneficial effects of PD1 were blocked by zVAD-fmk.

CONCLUSIONPosttreatment with PD1 enhances resolution of lung inflammation during LPS-induced acute lung injury by enhancing apoptosis in emigrated neutrophils, which is, at least in part, caspase-dependent.

Acute Lung Injury ; chemically induced ; drug therapy ; immunology ; Animals ; Apoptosis ; drug effects ; Docosahexaenoic Acids ; therapeutic use ; Inflammation ; drug therapy ; Lipopolysaccharides ; toxicity ; Male ; Mice ; Mice, Inbred BALB C ; Neutrophils ; cytology ; drug effects ; Peroxidase ; metabolism

The purpose of this paper is to study the effect of kinetin (Kn) on immunity and splenic lymphocyte proliferation in vitro of aging rats induced by D-galactose (D-gal). Fifty SD rats were randomly divided into five groups: control group, aging model group, Kn low dose group, Kn middle dose group and Kn high dose group. The aging model group was proposed by napes subcutaneous injection of D-gal (125 mg/kg) for 45 d, and anti-aging groups were intragastrically administered with 5, 10, 20 mg/kg of Kn respectively from day 11. IgG, IgA, IgM contents of serum, the apoptosis percentage, stimulation index (SI) and proliferation index (PI) of splenic lymphocyte in vitro were evaluated. The results showed that the apoptosis percentage of splenic lymphocyte in aging model rats was higher, the serum IgG, IgA and IgM contents, SI and PI were lower than control group. Kn significantly decreased the apoptosis percentage of splenic lymphocyte, while increased the serum IgG, IgA and IgM contents, SI and PI in aging model group. These results suggest that Kn could inhibit the apoptosis, while promote the proliferation of splenic lymphocyte, and then effectively enhance the immune power of the aging rats and slow down the aging process.
Aging ; drug effects ; immunology ; Animals ; Antibodies ; blood ; Apoptosis ; Cell Proliferation ; drug effects ; Galactose ; adverse effects ; Kinetin ; pharmacology ; Lymphocytes ; cytology ; Rats ; Rats, Sprague-Dawley ; Spleen ; cytology