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Purpose: Human neutrophil peptides (HNP1-3), small molecular antimicrobial peptides, are expressed within tumors and associated with tumor necrosis and inhibition of angiogenesis. Recent investigations have suggested that HNP1-3 are likely to be involved in the host immune responses to tumors.Experimental Design: We used recombinant pSec-HNP1, which expresses a secretable form of HNP1, to obtain expression of HNP1 in the tumor milieu in immunocompetent mice to explore the possible roles of HNP1 in tumor immunity. The antitumor effects were investigated in established CT26 colon cancer and 4T1 breast cancer models.Results: HNP1-mediated chemotactic and activating effects on immature dendritic cells were detected both in vitro and in vivo. Intratumoral expression of HNP1 resulted in not only significant tumor growth inhibition but also increased CTL infiltration within tumors. Adoptive transfer of splenocytes and a 51Cr release assay revealed specific cellular immune responses. Furthermore, increased antibodies were also found in sera from pSec-HNP1treated mice supporting specific humoral immune responses. Increased apoptosis and decreased angiogenesis were also shown in treated tumors.Conclusions: These findings indicate that HNP1 can exert multiple antitumor effects through different mechanisms; more importantly, HNP1 mediates host immune responses to tumors in situ through the recruitment and subsequent activation of immature dendritic cells and thus shows promising potential in cancer therapy. (Clin Cancer Res 2009;15(22):690111)

Supplementary Data from Intratumoral Expression of Mature Human Neutrophil Peptide-1 Mediates Antitumor Immunity in Mice

In this study, we reported millepachine (MIL), a novel chalcone compound for the first time isolated from Millettia pachycarpa Benth (Leguminosae), induced cell cycle arrest and apoptosis in human hepatocarcinoma cells in vitro and in vivo. In in vitro screening experiments, MIL showed strong antiproliferation activity in several human cancer cell lines, especially in HepG2 cells with an IC50 of 1.51 µM. Therefore, we chose HepG2 and SK-HEP-1 cells to study MIL's antitumor mechanism. Flow cytometry showed that MIL induced a G2/M arrest and apoptosis in a dose-dependent manner. Western blot demonstrated that MIL-induced G2/M arrest was correlated with the inhibition of cyclin-dependent kinase 1 activity, including a remarkable decrease in cell division cycle (cdc) 2 synthesis, the accumulation of phosphorylated-Thr14 and decrease of phosphorylation at Thr161 of cdc2. This effect was associated with the downregulation of cdc25C and upmodulation of checkpoint kinase 2 in response to DNA damage. MIL also activated caspase 9 and caspase 3, and significantly increased the ratio of Bax/Bcl-2 and stimulated the release of cytochrome c into cytosol, suggesting MIL induced apoptosis via mitochondrial apoptotic pathway. Associated with those effects, MIL also induced the generation of reactive oxygen species. In HepG2 tumor-bearing mice models, MIL remarkably and dose dependently inhibited tumor growth. Treatment of mice with MIL (20mg/kg intravenous [i.v.]) caused more than 65% tumor inhibition without cardiac damage compared with 47.57% tumor reduction by 5mg/kg i.v. doxorubicin with significant cardiac damage. These effects suggested that MIL and its easily modified structural derivative might be a potential lead compound for antitumor drug.

Thermotherapy and thermochemotherapy have been used in clinics to treat patients with malignant diseases, including colon cancer, and their efficacy has been well proved. Heat shock proteins (HSPs), especially Hsp70, play important roles in neutralizing their efficacy. It has been reported that quercetin can suppress cancer by inhibiting the intratumoral expression of Hsp70. This study was designed to investigate whether quercetin could enhance sensitivity to thermotherapy and thermochemotherapy. Soluble quercetin liposome was used in this study. The effects of quercetin were investigated in vitro and in mouse colon cancer models of subcutaneous tumor and peritoneal carcinomatosis. The results showed that quercetin liposome inhibited the upregulation of Hsp70 and enhanced apoptosis induced by hyperthermia and thermochemotherapy. Systemic administration of quercetin liposome can sensitize CT26 cells to thermotherapy and chemothermotherapy. This study suggests that quercetin liposome might be potentially applied for clinical cancer therapy.

We previously reported the potential of a novel small molecule 3-amino-6-(3-methoxyphenyl)thieno[2.3-b]pyridine-2-carboxamide (SKLB70326) as an anticancer agent. In the present study, we investigated the anticancer effects and possible mechanisms of SKLB70326 in vitro . We found that SKLB70326 treatment significantly inhibited human hepatic carcinoma cell proliferation in vitro , and the HepG2 cell line was the most sensitive to its treatment. The inhibition of cell proliferation correlated with G 0 /G 1 phase arrest, which was followed by apoptotic cell death. The SKLB70326-mediated cell-cycle arrest was associated with the downregulation of cyclin-dependent kinase (CDK) 2, CDK4 and CDK6 but not cyclin D1 or cyclin E. The phosphorylation of the retinoblastoma protein (Rb) was also observed. SKLB70326 treatment induced apoptotic cell death via the activation of PARP, caspase-3, caspase-9 and Bax as well as the downregulation of Bcl-2. The expression levels of p53 and p21 were also induced by SKLB70326 treatment. Moreover, SKLB70326 treatment was well tolerated. In conclusion, SKLB70326, a novel cell-cycle inhibitor, notably inhibits HepG2 cell proliferation through the induction of G 0 /G 1 phase arrest and subsequent apoptosis. Its potential as a candidate anticancer agent warrants further investigation.

Inducing apoptosis is a promising therapeutic approach to overcome cancer. Here we described that a novel synthesized compound, 3-amino-N-(4-chlorobenzyl)-6-(3-methoxyphenyl)thieno[2,3-b]pyridine-2-carboxamide (SKLB703), exhibits antitumor activity via inducing apoptosis both invitro and invivo. Our results showed that SKLB703 inhibited the proliferation of a panel of human cancer cell lines, and human hepatocellular carcinoma cell line HepG2 was the most sensitive. The proliferation inhibitory effect of SKLB703 was associated with its apoptosis-inducing effect by activating caspase-3 and caspase-9 rather than caspase 8. Exposure of HepG2 to SKLB703 also resulted in Bax upregulation, Bcl-2 downregulation, cytochrome c release and mitochondrial transmembrane potential change in mitochondrial apoptotic pathway. Moreover, the decrease of phosphorylated p 44/42 mitogen-activated protein kinase and phosphorylated Akt was observed. SKLB703 suppressed the growth of established tumors in xenograft models in mice, whereas no toxicity was exhibited. TUNAL analysis showed that SKLB703 induced HepG2 tumor apoptosis. Taken together, the present study demonstrates that SKLB730 exhibits its antitumor activity through inducing apoptosis via mitochondrial apoptotic pathway. Its potential to be a candidate of anticancer agent is worth being further investigated.

In this article, MPEG/PCL-g-Chitosan (MPEG/PCL-g-CS) was successfully prepared via free‐radical polymerization method. MPEG/PCL copolymer was first synthesized from MPEG and ϵ‐caprolactone. Then, the double bond ended MPEG/PCL (MPEG/PCL macromonomer) was obtained by reaction with acryloyl chloride at 40oC in pre-dried dichloromethane. MPEG/PCL-g-CS copolymer was characterized by 1H-nuclear magnetic resonance (1H-NMR), Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), and differential scanning calorimeter (DSC), respectively. The obtained amphiphilic MPEG/PCL-g-CS copolymer could self-assemble into nanoparticles in aqueous solution. The critical micellar concentration (CMC) of MPEG/PCL-g-CS copolymer is about 1.78 mg/L determined by the fluorescence robe technique. In vitro cytotoxicity test revealed that MPEG/PCL-g-CS nanoparticles were low cytotoxicity against HEK293 cells after incubated for 24 h. The coupling of CS with MPEG/PCL endowed the pH sensitivity and cationic characteris...

Fibroblast growth factor receptors (FGFRs), overexpressed on the surface of a variety of tumor cells and on tumor neovasculature in situ, are potential targets for tumor- and vascular-targeting therapy. This study aimed to develop a FGFR-mediated drug delivery system to target chemotherapeutic agents to FGFR-overexpressed tumor cells and tumor neovasculature endothelial cells in vitro and in vivo. Here we designed a truncated human basic fibroblast growth factor peptide (tbFGF), which was attached to the surface of cationic liposomal doxorubicin (LPs-DOX) and paclitaxel (LPs-PTX) via electrostatic force. Then we characterized the tbFGF-modified liposome (tbFGF-LPs) and examined internalization of doxorubicin in tumor cells (TRAMP-C1, B16) and HUVEC cells in vitro. In vivo, we evaluated the biodistribution and antitumor efficacy of tbFGF-LPs-DOX and tbFGF-LPs-PTX in C57BL/6J mice bearing TRAMP-C1 prostate carcinoma and B16 melanoma, respectively. The tbFGF-LPs-DOX significantly improved the uptake of doxorubicin in TRAMP-C1, B16 and HUVEC cells, respectively. Biodistribution study in B16 tumor-bearing mice showed that tbFGF-LPs-PTX achieved 7.1-fold (72.827+/-7.321mgh/L vs 10.292+/-0.775mgh/L, mean+/-SD, P

Simple and efficient gene transfer to the skin would facilitate many local and systemic gene therapy applications. This study reports a novel approach that allows expression of plasmid DNA in epidermis and hair follicle cells with dimethyl sulfoxide (DMSO) after pre-treatment with depilation and retinoic acid (RA) for the purposes of gene therapy. This study investigated the transdermal efficacy of gene to mouse skin when utilizing DMSO after RA pre-treatment. Retinoic acid pre-treatment can increase the efficiency of transfection. This finding indicates that one can more effectively and much less expensively make use of genes therapy to treat diseases of the hair and skin.

Patients with localized prostate cancer can usually achieve initial response to conventional treatment. However, most of them will inevitably progress to advanced disease stage. There is a clear need to develop innovative and effective therapeutics for prostate cancer. Mouse survivin T34A (mS-T34A) is a phosphorylation-defective Thr34 → Ala dominant negative mutant, which represents a potential promising target for cancer gene therapy. This study was designed to determine whether mS-T34A plasmid encapsuled by DOTAP-chol liposome (Lip-mS) has the anti-tumor activity against prostate cancer, if so, to further investigate the possible mechanisms. In vitro, TRAMP-C1 cells were transfected with Lip-mS and examined for apoptosis by PI staining and flow cytometric analysis. In vivo, subcutaneous prostate cancer models were established in C57BL/6 mice, which were randomly assigned into three groups to receive i.v. administrations of Lip-mS, pVITRO2-null plasmid complexed with DOTAP-chol liposome (Lip-null) or normal saline every 2 days for eight doses. Tumor volume was measured. Tumor tissues were inspected for apoptosis by TUNEL assay. Microvessel density (MVD) was determined by CD31 immunohistochemistry. Alginate-encapsulated tumor cell test was conducted to evaluate the treatment effect on angiogenesis. Administration of Lip-mS resulted in significant inhibition in the growth of mouse TRAMP-C1 tumors. The anti-tumor response was associated with increased tumor cell apoptosis and decreased microvessel density. The present study may be of importance in the exploration of the potential application of Lip-mS in the treatment of a broad spectrum of tumors.

PNAS-4, a novel pro-apoptotic gene activated during the early response to DNA damage, can inhibit proliferation via apoptosis when overexpressed in some tumor cells. The objectives of this study were to determine whether PNAS-4 could enhance apoptosis induced by cisplatin besides its induction of apoptosis, and to evaluate the usefulness of combined treatment with mouse PNAS-4 (mPNAS-4) gene therapy and low-dose cisplatin chemotherapy in the inhibition of tumor growth in colon carcinoma (CT26) and Lewis lung carcinoma (LL/2) murine models.In this study, the in vitro growth-inhibitory and pro-apoptotic effects of PNAS-4 and/or cisplatin on CT26, LL/2, and SKOV3 cancer cells were assessed by MTT assay, flow cytometric analysis, DNA fragmentation, and morphological analysis, respectively. The in vivo antitumor activity of combined treatment with mPNAS-4 gene therapy and low-dose cisplatin were evaluated in the inhibition of tumor growth in colon carcinoma (CT26) and Lewis lung carcinoma (LL/2) murine models. Tumor volume and survival time were observed. Induction of apoptosis was also assessed in tumor tissues.In vitro, PNAS-4 inhibited proliferation of colon carcinoma (CT26), Lewis lung carcinoma (LL/2) and human ovarian cancer (SKOV3) cell lines via apoptosis, and significantly enhanced the apoptosis of CT26, LL/2, and SKOV3 cells induced by cisplatin. In vivo systemic administration of expression plasmid encoding mPNAS-4 (pcDNA3.1-mPS) and cisplatin, significantly decreased tumor growth through increased tumor cell apoptosis compared to treatment with mPNAS-4 or cisplatin alone.Our data suggests that the combined treatment with mPNAS-4 plus cisplatin may augment the induction of apoptosis in tumor cells in vitro and in vivo, and that the augmented antitumor activity in vivo may result from the increased induction of apoptosis. The present study may provide a novel way to augment the antitumor efficacy of cytotoxic chemotherapy.

Caspy2, a zebrafish protease, is an active caspase for inducing apoptosis in mammalian cells. To investigate whether caspy2‐mediated apoptosis could be used in cancer therapy, its cDNA was amplified and cloned into eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid was mixed with cationic liposome and introduced into various tumour cell lines in vitro. Our data showed that caspy2 induced remarkable apoptosis of cancer cells in vitro. Treatment of mice‐bearing CT26 colon carcinoma or MethA fibrosarcoma with intratumoral injection of liposome‐caspy2 plasmid complex resulted in substantial killing of neoplastic cells and long‐term survivors. Apoptotic cells were widely distributed in caspy2‐treated tumour tissue. Infiltration of CD8(+) T lymphocyte was also observed apparently in the tumour tissue after the treatment with caspy2. Tumour‐specific major histocompatibility complex (MHC) class I‐dependent CD8(+) cytotoxic T lymphocyte activity was found by means of (51)Cr release assay. In MethA model, the mice acquired a long‐time protective immunity against the parental tumour cell re‐challenge. These results indicated that caspy2 can act as both apoptosis inducer and immune response initiator, which may account for its extraordinary antitumour effect. Furthermore, in vivo depletion of CD8(+) T lymphocytes could completely abrogate the antitumour immune activity, whereas the depletion of CD4(+) cells showed partial abrogation. In this study, we developed a novel anticancer strategy that combines both induction of apoptosis and immune response in mice‐bearing tumours with a single caspy2 gene. This approach may provide an important way for treatment of cancer.

The present study was to investigate the anticancer effect of chloroquine on proliferation of mouse colon cancer cell line CT26 in vivo and in vitro and the possible mechanism. We found that chloroquine inhibited CT26 proliferation by concentration- and time-dependent manner. This effect was associated with apoptosis induction and decreased level of phosphorylated p42/44 mitogen-activated protein kinase and phosphorylated Akt. The in vivo study showed chloroquine-reduced tumor volume and prolonged survival time in CT26-bearing mice. These observations indicated chloroquine could inhibit CT26 proliferation by inducing apoptosis both in vitro and in vivo, providing its chemotherapeutic potential of human cancers.

The matrix (M) protein of vesicular stomatitis virus (VSV) plays a key role in inducing cell apoptosis during infection. To investigate whether M protein-mediated apoptosis could be used in cancer therapy, its cDNA was amplified and cloned into eukaryotic expression vector pcDNA3.1(+). The recombinant plasmid or the control empty plasmid pcDNA3.1(+) was mixed with cationic liposome and introduced into various tumor cell lines in vitro, including lung cancer cell LLC, A549, colon cancer cell CT26 and fibrosarcoma cell MethA. Our data showed that the M protein induced remarkable apoptosis of cancer cells in vitro compared with controls. Fifty micrograms of plasmid in a complex with 250 microg cationic liposome was injected intratumorally into mice bearing LLC or MethA tumor model every 3 days for 6 times. It was found that the tumors treated with M protein plasmid grew much more slowly, and the survival of the mice was significantly prolonged compared with the mice treated with the control plasmid. In MethA fibrosarcoma, the tumors treated with M protein plasmid were even completely regressed, and the mice acquired longtime protection against the same tumor cell in rechallenge experiments. Both apoptotic cells and CD8(+) T cells were widely distributed in M protein plasmid-treated tumor tissue. Activated cytotoxic T lymphocytes (CTLs) were further detected by means of (51)Cr release assay in the spleen of the treated mice. These results showed that M protein of VSV can act as both apoptosis inducer and immune response initiator, which may account for its extraordinary antitumor effect and warrant its further development in cancer gene therapy.

Dendritic cells (DCs) are important cells in initiating an immune response. A generation of functional DCs has potential clinical use in treating cancer. However, the source of DCs and patient immunodeficiency with cancer have been hindrances in clinical therapy. We generated DCs from human umbilical cord blood mononuclear cells (UBMCs) with recombinant human granulocyte-macrophage colony stimulating factor, recombinant human interleukin-4, and recombinant human tumor necrosis factor-alpha. The mature DC-A549 lung cancer vaccine (AgL-DC) was prepared through loading A549 lysate, treating with lipopolysaccharide (LPS) and positive selecting with CD83 magnetic beads. AgL-DC can secrete interleukin (IL)-12 and IL-1. Further in vitro analysis showed that AgL-DC notably induced human UBMC lymphocyte proliferation (p0.01) by 3-(4,5-dimethylthiazol-z-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, increased the cytotoxic T-lymphocyte (CTL) activity of UBMC lymphocytes against A549 cells (p0.05, at effector cells:target cells ratios of 50:1 and 100:1) by lactate dehydrogenase (LDH) cytotoxic assay, and improved production of IL-6 and tumor necrosis factor-beta (p0.01, p0.05) by enzyme-linked immunosorbent assay. Subsequently, the reconstitute immunity model in severe combined immunodeficiencies (SCID) mice has been established using human UBMC transplantation, and similar trends to results of UBMC in vitro experiments have been shown in lymphocyte proliferation, CTL activity, and IL-6 and tumor necrosis factor-beta secretion levels in these models. AgL-DC also significantly (p0.01) increased the antitumor effect in vivo. The tumor infiltrating immunocytes were positively expressed human CD83 and CD3 molecules, and they were negatively expressed in tumor tissue treated with control. These results have demonstrated that umbilical cord DCs are a useful source of vaccine cells for augmenting CTL-mediated cytotoxicity and have potential usefulness in cellular therapy for human cancer in a new vaccination strategy.

Chloroquine is an antimalarial drug that has been used in the treatment and prophylaxis of malaria since the 1950s. The present study was undertaken to examine the effects of chloroquine on Bcap-37 human breast cancer cells' growth, cell cycle modulation, apoptosis induction, and associated molecular alterations in vitro. The chloroquine treatment decreased the viability of Bcap-37 cells in a concentration- and time-dependent manner, which correlated with G(2)/M phase cell cycle arrest. The chloroquine-mediated cell cycle arrest was associated with a decrease in protein levels/activity of polo-like kinase 1 (Plk1), phosphorylated cell division cycle 25C (Cdc25C), phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated Akt. The chloroquine-treated Bcap-37 cells exhibited a marked decrease in the level of mitochondrial transmembrane potential (DeltaPsim), which was accompanied by the activation of caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). Exposure of Bcap-37 cells to chloroquine also resulted in the induction of spindle abnormalities. In conclusion, the findings in this study suggested that chloroquine might have potential anticancer efficacy, which could be attributed, in part, to its proliferation inhibition and apoptosis induction of cancer cells through modulation of apoptosis and cell cycle-related proteins expressions, down-regulation of mitochondrial transmembrane potential (DeltaPsim), and induction of spindle abnormalities.

Quercetin, a widely distributed bioflavonoid, inhibits the growth of various tumor cells. The present study was designed to investigate whether a novel quercetin derivative [phenylisocyanate of quercetin (PHICNQ)] exerts antitumor activity against K562 and CT26 tumor cell lines by inducing apoptosis, and to examine the possible mechanism in the phenomenon. The cell proliferation assay of K562 and CT26 tumor cells was determined by the trypan blue dye exclusion test. Apoptosis of PHICNQ-treated cells was determined by morphological analysis, agarose gel DNA electrophoresis and quantitated by flow cytometry after staining with propidium iodide. Cell cycle was evaluated by flow cytometry. The expression of heat shock protein 70 was checked by Western blot analysis. Our results showed that PHICNQ inhibited the proliferation of K562 and CT26 cells in a dose-dependent and time-dependent manner. PHICNQ was 308- and 73-fold more active on CT26 and K562 cells than quercetin, respectively. In addition to this cytostatic effect, treatment of K562 and CT26 tumor cells with PHICNQ induced apoptosis. PHICNQ treatment downregulated the expression of heat shock protein 70 more dramatically than quercetin treatment. These results suggest that PHICNQ is a more powerful antiproliferative derivative than quercetin, with cytostatic and apoptotic effects on K562 and CT26 tumor cells. PHICNQ may trigger apoptosis in tumor cells through inhibition of heat shock protein 70 synthesis and expression.

Purpose: Tie-2 is an endothelium-specific receptor tyrosine kinase known to play a key role in tumor angiogenesis. The present study explores the feasibility of immunotherapy of tumors by using a protein vaccine based on chicken Tie-2 as a model antigen to break the immune tolerance against Tie-2 in a cross-reaction between the xenogeneic homologous and self-Tie-2.Experimental Design and Results: In this study, a chicken homologous Tie-2 protein vaccine (chTie-2) and a corresponding mouse Tie-2 vaccine as a control were prepared and the antitumor effect of these vaccines was tested in two tumor models (murine B16F10 melanoma and murine H22 hepatoma). Immunotherapy with chTie-2 was found effective in two tumor models. Autoantibodies against mouse Tie-2 were detected in sera of mice immunized with chTie-2 through Western blot analysis and ELISA assay. Anti-Tie-2 antibody-producing B cells were detectable by ELISPOT. Histologic examination revealed that autoantibodies were deposited on the endothelial cells of tumor tissues. Purified immunoglobulins from chTie-2-immunized mice could induce the apoptosis of human umbilical vein endothelial cells in vitro. Importantly, adoptive transfer of purified immunoglobulins led to antitumor effect in vivo; apparently, angiogenesis was significantly inhibited in these tumors. Furthermore, the antitumor activity and production of autoantibodies could be abrogated by depletion of CD4+ T lymphocytes.Conclusions: Our findings may provide a vaccine strategy for cancer therapy and show the potential utilization of interference with Tie-2 pathway.

Vascular endothelial growth factor receptor-2 (VEGFR-2) has been shown to play a major role in inducing the full spectrum of VEGF biological response which is essential for tumor angiogenesis. We have demonstrated that immunotherapy of tumors with a vaccine based on quail homologous VEGFR-2 (qVEGFR) was effective in providing both protective and therapeutic antitumor immunity in several tumor models in mice. The purpose of this study was to determine whether the combination therapy of low-dose gemcitabine with qVEGFR as a vaccine could inhibit tumor growth to a greater extent. To test this concept, H22 hepatoma and Lewis lung carcinoma models were established in BALB/c mice and C57BL/6 mice, respectively. Mice were treated with either qVEGFR as a protein vaccine, gemcitabine, or both agents together. qVEGFR or low-dose chemotherapy treatment individuallyresulted in tumor inhibition to a certain extent.Remarkably, the combination therapy resulted in synergistic antitumor activity. Histological examination revealed that there was endothelial deposition of immunoglobulins within tumor tissues from mice treated with vaccine or combination therapy, especially intratumor angiogenesis was suppressed more significantly for the combination group. Also, ELISPOT analysis showed that mice treated with either qVEGFR alone or in combination with low-dose chemotherapy produced similar amount of anti-VEGFR antibody-producing B cells, which suggested that low-dose gemcitabine did not suppress the host’s immune response, but potentiated the antitumor activity of the qVEGFR vaccine. Furthermore, TUNEL staining demonstrated a significant increase in the number of TUNEL-positive cells in the combination group compared with those of other groups. The observations may provide a new bio-chemotherapeutic approach for cancer.

IL-30, the p28 subunit of IL-27, interacts with the Epstein-Barr virus-induced gene 3 (EBI3) to form IL-27, which modulates the inflammatory responses in autoimmune and infectious diseases. Several previous studies have provided evidence for the role of IL-30 in the anti-inflammatory process. However, the effect of IL-30 in macrophage-mediated immune responses is not well understood. With the recent observation in our experiment, we found that IL-30 exerted potent anti-inflammatory effects in the RAW 264.7 macrophages and in a lipopolysaccharide/D-galactosamine (LPS/D-GalN)-induced mouse model. IL-30 decreased the production of tumor necrosis factor (TNF)-α and IL-6 in LPS-stimulated RAW 264.7 macrophages in a dose-dependent manner. In the macrophage-mediated GalN and LPS model of acute liver injury, IL-30 prevented liver injury by suppressing serum enzyme activity and down-regulating the pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and interferon (IFN)-γ. IL-30 treatment decreased apoptosis in liver tissue and increased glutathione (GSH) levels. We postulated that IL-30 might function through gp130-mediated signaling pathways and then demonstrated that IL-30 affects LPS-induced inflammation through the STAT1, STAT3, and ERK signaling pathways. These data indicate that IL-30 can provide critical protection against macrophage-mediated liver inflammation through anti-apoptotic, anti-oxidant, and anti-inflammatory activities.

To investigate the therapeutic effect of the plasmid pcDNA3.1-IL15 complexed with cationic liposome (CL-IL15) in the B16-F10 melanoma lung metastasis model.A plasmid with high secretive efficiency of IL-15 was constructed and the optimum mix ratio was determined to formulate cationic liposome-plasmid complex with the optimal encapsulation. The CHO-K1 cell line was transfected by CL-IL15. The secretion of transfected IL-15 gene was detected by Western blot and its biological function was measured through the proliferation response of CTLL-2 cytotoxic T cell line of murine by MTT assay. The C57BL/6 mice were inoculated intravenously (i.v.) with B16-F10 melanoma lung metastasis cells then treated (i.v.) by CL-IL15 in a therapeutic setting to determine the tumorigenesis and research the corresponding mechanisms.The pcDNA3.1-IL15 plasmid was successfully constructed and the mass-ratio of optimal condition of cationic liposome-plasmid with perfect entrapment was 1:5 (plasmid: cationic liposome). Western blot analysis displayed the detection of IL-15 both in the medium and the pcDNA3.1-IL15 transfected cells. MTT assay showed that CTLL-2 cells could proliferate with the medium obtained from CHO-K1 cells transfected by CL-IL15. And the administration of CL-IL15 complexes led to the significant inhibition lung metastasis of malignant melanoma (P0.05).CL-IL15 could inhibit the metastasis of malignant melanoma and the cationic liposome delivered plasmid pcDNA3.1-IL-15 complexes may be an efficient therapeutic strategy for the treating of lung metastasis. And the effective splenic cell-mediated cytotoxicity and the obvious NK cells recruitment may be involved.

Hepatocellular carcinoma is one of the most common cancers in worldwide. We previously reported a novel thienopyridine derivative 3-amino-6-(3,4-dichlorophenyl) thieno[2,3-b]pyridine-2-carboxamide (SKLB70359) which possesses anticancer activity against hepatocellular carcinoma. In present study, we further investigated its anticancer activity and possible mechanism. The SKLB70359 treatment decreased the viability of a panel of hepatocellular carcinoma cell lines in a concentration- and time-dependent manner with IC(50) 0.4 ~ 2.5 μM. The mechanism study showed that SKLB70359 induced G0/G1 cell cycle arrest and then led to apoptotic cell death of HepG2 cell. The SKLB70359 induced G0/G1 cell cycle arrest was characterized by down-regulation of cyclin-dependent kinase 2 (CDK2), CDK4, CDK6 expression and up-regulation of p53, p21(WAF1). Activating of caspase-3 and caspase-9 was also observed. Meanwhile, proliferation inhibitory effect of SKLB70359 was associated with decreased level of phosphorylated p44/42 mitogen activated protein kinase (p44/42 MAPK) and phosphorylated retinoblastoma protein (Rb). Moreover, SKLB70359 exhibit less toxicity to non-cancer cells than tumor cells. In conclusion, the findings in this study suggested that SKLB70359 have potential anticancer efficacy via G0/G1 cell cycle arrest and apoptosis induction. Its potential to be a candidate of anticancer agent is worth being further investigated.

Antagonizing angiogenesis-related receptor tyrosine kinase is a promising therapeutic strategy in oncology. In present study, we designed and synthesized a novel vascular endothelial growth factor receptor (VEGFR) inhibitor N-methyl-4-(4-(3-(trifluoromethyl) benzamido) phenoxy) picolinamide SKLB610 that potently suppresses human tumor angiogenesis. SKLB610 inhibited angiogenesis-related tyrosine kinase VEGFR2, fibroblast growth factor receptor 2 (FGFR2) and platelet-derived growth factor receptor (PDGFR) at rate of 97%, 65% and 55%, respectively, at concentration of 10μM in biochemical kinase assays. In vitro, SKLB610 showed more selective inhibition of VEGF-stimulated human umbilical vein endothelial cells (HUVECs) proliferation, and this proliferation inhibitory effect was associated with decreased phosphorylation of VEGFR2 and p42/44 mitogen-activated protein kinase (p42/44 MAPK). Antiangiogenic evaluation showed that SKLB610 inhibited the HUVECs capillary-tube formation on Matrigel in vitro and the sub-intestinal vein formation of zebrafish in vivo. Moreover, SKLB610 inhibited a panel of human cancer cells proliferation in a concentration-dependent manner and human non-small cell lung cancer cell line A549 and human colorectal cancer cell line HCT116 were most sensitive to SKLB610 treatment. In vivo, chronic intraperitoneally administration of SKLB610 at dose of 50mg/kg/d resulted in significant inhibition in the growth of established human A549 and HCT116 tumor xenografts in nude mice without exhibit toxicity. Histological analysis showed significant reductions in intratumoral microvessel density (CD31 staining) of 43-55% relative to controls depending on the specific tumor xenografts. In conclusion, the present study demonstrated that SKLB610 exhibited its antitumor activity as a multi-targeted inhibitor with more potent inhibition of VEGFR2 activity. Its potential to be a candidate of anticancer agent is worth being further investigated.

Colon carcinoma is one of the common malignant tumors and has high morbidity and mortality in the world. Pigment epithelial-derived factor (PEDF) has been found to be the most potent natural inhibitor of angiogenesis and PEDF gene has been extensively used for the therapy of tumors, which suggests a potential approach to the therapy of colon carcinoma. However, the transfer of PEDF gene largely depends on the effective gene delivery systems. Poly (lactic-co-glycolic acid) nanoparticles (PLGANPs) have been extensively used for gene therapy due to its low-toxicity, biocompatibility and biodegradability, due to its potential to be an excellent carrier of the PEDF gene. We investigated the effect of PEDF gene loaded in PLGA nanoparticles (PEDF-PLGANPs) on the mouse colon carcinoma cells (CT26s) in vitro and in vivo. Blank PLGANPs (bPLGANPs) showed lower cytotoxicity than PEI to the CT26s. In vitro, PEDF-PLGANPs directly induced CT26 apoptosis and inhibit human umbilical vein endothelial cell (HUVEC) proliferation. In vivo, PEDF-PLGANPs inhibited CT26 tumors growth by inducing CT26 apoptosis, decreasing MVD and inhibiting angiogenesis. Our present study demonstrates the inhibitory effect of PEDF-PLGANPs on the growth of CT26s in vitro and in vivo for the first time. PLGANP-mediated PEDF gene could provide an innovative strategy for the therapy of colon carcinoma.

Basic fibroblast growth factor (bFGF) is a mitogen for endothelial cells, which participates in tumor angiogenesis. Active immunity against bFGF could be a promising approach for the biotherapy of cancer. Because bFGF is abundant in normal and malignant tissues, it is presumably difficult for normal bFGF to induce immunity due to self-tolerance. In addition, previous studies have shown that a complex consisting of a cationic liposome and a non-coding plasmid DNA can be used to stimulate innate immunity. This stimulation initiates a potent cytokine response, which can inhibit tumor growth. To investigate the effects of immunity against bFGF on murine colon carcinomas, we employed an N-, C-terminally truncated basic fibroblast growth factor (tbFGF, of human origin) as an antigen and a liposome-DNA complex as an adjuvant. After six immunizations, a robust bFGF-specific immune response was elicited. Subsequently, inhibition of tumor growth and a significant reduction in tumor vasculature were observed. The antitumor effect was confirmed by adoptive therapy of activated spleen cells from the immunized mice. In vitro, a CTL assay revealed that bFGF-specific cytotoxic T lymphocytes (CTL) resulted in the lysis of mouse microvascular endothelial cells (MS1) rather than that of the CT26 colorectal cancer cells. These results suggest that anti-angiogenesis treatment induced by a bFGF-specific CTLs against microvascular endothelial cells may be a useful method for cancer therapy.

Metastatic breast cancers are hard to treat and almost always fatal. Chloroquine diphosphate, a derivative of quinine, has long been used as a potent and commonly used medicine against different human diseases. We therefore investigated the effects of chloroquine diphosphate on a highly metastatic mouse mammary carcinoma cell line. In vitro treatment of 4T1 mouse breast cancer cells with chloroquine diphosphate resulted in significant inhibition of cellular proliferation and viability, and induction of apoptosis in 4T1 cells in a time- and dose-dependent manner. Further analysis indicated that induction of apoptosis was associated with the loss of mitochondrial membrane potential, release of cytochrome c, and activation of caspase-9 and caspase-3, and cleavage of poly(ADP-ribose) polymerase. The effect of chloroquine diphosphate was then examined using a mice model in which 4T1 cells were implanted subcutaneously. Chloroquine diphosphate (25mg/kg and 50mg/kg, respectively) significantly inhibited the growth of the implanted 4T1 tumor cells and induced apoptosis in the tumor microenvironment. Moreover, the metastasis of tumor cells to the lungs was inhibited significantly and the survival of the mice enhanced. These data suggested that chloroquine diphosphate might have chemotherapeutic efficacy against breast cancer including inhibition of metastasis.

To explore the application of MHC-Ig/peptide polymer technique for detecting antigen-specific cytotoxic T lymphocytes (CTLs) in mice with experimental autoimmune uveitis (EAU).B10RIII mice were immunized with an interphotoreceptor retinal-binding protein (IRBP) synthetic peptide (IRBP161-180). The in vivo primed T cells were separated and stained with MHC-Ig polymer combined with a panel of truncated peptides derived from IRBP161-180. The level of IRBP-specific CTLs cells was determined by FACS analysis. The CD8+ T cells were isolated from the primed T cells and stimulated with complex polymers containing MHC-Ig and various IRBP-derived peptides. The proliferation of CD8+ T cells was measured by H thymidine incorporation. The production of interferon- (IFN-) in the cell suspensions was measured by ELISA.The IRBP-specific CTLs were detected by MHC-Ig/peptide polymers. The MHC-Ig/IRBP168-177 peptide polymer dtected 12.3% specific CTLs, showing greater ability in stimulating proliferation of CTLs and production of IFN--than the other MHC-Ig/ peptide polymers (P0.01). The truncated 10-mer peptide, IRBP168-177, was the major antigenic epitope for the IRBP-specific CTLs. The MHC-Ig/IRBP168-177 peptide polymer detected the highest level(4.9% +/- 1.1%) of specific CTLs from peripheral blood mononuclear cells (PBMCs) at the acute stage of EAU.The MHC-Ig polymer technique is an effective instrument for detecting antigen-specific CTLs, with good sensitivity and specificity in EAU studies.

Lung cancer is one of the most highly malignant tumors, and a significant threat to human health. Lung cancer patients often exhibit tumor cell invasion and metastasis, which often render current treatments ineffective. Recently, the beneficial effects of low molecular weight heparin (LMWH) on cancer metastasis were reported in pre-clinical research studies. LMWH may be a potential drug for cancer therapy. However, the mechanism of LMWH on the invasion and metastasis of cancer has yet to be determined. This study investigated the effects of Fraxiparine on the proliferation, invasion and metastasis of the human lung adenocarcinoma A549 cell line. MTT assay and flow cytometry showed that Fraxiparine slightly inhibited the cell viability dose- and time-dependently, but did not arrest the A549 cells in the G1 phase nor induce early apoptosis. The transwell chamber assay showed that Fraxiparine significantly suppressed the invasion and migration of the A549 cells in vitro. Fraxiparine also markedly inhibited the adhesion of the A549 cells to Matrigel. The RT-PCR assay demonstrated that the reduction in invasion and metastasis may be related to the up-regulation of nm23-H1 and the down-regulation of the heparanase expression. Moreover, the RT-PCR assay and Western blot analysis demonstrated that down-regulation of the expression of integrin β1 and β3, as well as that of matrix metalloproteinase-2 and −9 may be responsible for the inhibition of the invasion and metastasis of A549 cells by Fraxiparine.

To track T lymphocytes proliferation with vital dye CFSE in experimental autoimmune uveitis (EAU).B10RIII mice were immunized with synthetic peptide of interphotoreceptor retinal-binding protein (IRBP161-180) to develop EAU. The proliferation of IRBP-specific T cell subsets was detected by CFSE staining, fluorescent antibody labeling, and flow cytometry.The IRBP-specific T cells divided after 4 days of stimulation with IRBP161-180, halving the fluorescence intensity. The proliferations of CD4+ and CD8+ T cells were asynchronous, with CD8+ T cells dividing more vigorously and having more drop in percentage of parent cells (P0.01).CFSE-Labeling can detect the proliferation of autoreactive T cells and their subsets in EAU effectively.

Background: Psoriasis is a chronic and relapsing inflammatory skin disease associated with various immunologic abnormalities. Repeated subcutaneous injection of interleukin-4 (IL-4) has been established as an effective treatment to counteract psoriasis. Objective: We investigated whether gene therapy using IL-4 expression plasmid (pIL-4) via transdermal delivery was an alternative treatment for psoriasis. In our experiment, dimethylsulfoxide (DMSO) was used as a penetration enhancer. Methods: At first, the penetration efficiency of the complex of reporter plasmid accompanied by DMSO was investigated both in vitro and in vivo. Then, the antipsoriasis efficiency of the treatment with pIL-4-DMSO was tested in mice. Results: The expression of the reporter gene was detected in epidermis and dermis both in vitro and in vivo. More importantly, the psoriasis symptoms were relieved, and significant reductions in some psoriasis-associated factors were observed after pIL-4-DMSO treatment. Conclusion: We conclude that the topical application of pIL-4-DMSO can treat psoriasis to a significant extent.

Radiosensitivity of tumors is due to a complex interaction of various factors, it has been reported that survivin also acts as a constitutive and inducible radioresistance factor in a panel of tumor cells and approaches designed to inhibit survivin expression or function may lead to tumor sensitisation to chemical and physical agents. Previously, we found that the plasmid encoding the phosphorylation-defective mouse survivin threonine 34-->alanine mutant complexed to DOTAP-chol liposome (Lip-mS) can suppress murine primary breast carcinoma. However, little is known regarding the biological effect of Lip-mS combined with radiation. The present study was designed to determine whether Lip-mS could enhance the anti-tumor activity of radiation. The Lewis Lung Carcinoma (LLC) cells treated with a combination of Lip-mS and radiation displayed apparently increased apoptosis compared with those treated with Lip-mS or radiation alone. Mice bearing LLC tumors were treated with intravenous injections of Lip-mS and radiation, the combined treatment significantly reduced mean tumor volume compared with either treatment alone. Moreover, the anti-tumor effect of Lip-mS combined with radiation was greater than their additive effect when compared with the expected effect of the combined treatment. These data suggest that inhibition of survivin using a dominant-negative mutant, survivin T34A, could sensitize LLC cells to radiation efficiently and the synergistic anti-tumor activity may in part result from increasing the apoptosis of tumor cells, inhibiting tumor angiogenesis and inducing a tumor-protective immune response in the combined treatment.

To study the mechanisms of nongenomic effect of 17beta-estradiol on human spermatozoa.The intracellular calcium ([Ca2+]i) in the spermatozoa was measured by flow cytometry after the spermatozoa was treated with the inhibitors of trans-membrane signaling transduction pathways and impermeable 17beta-estradiol (E2-BSA). Western blot was used to detect the activation of the signal proteins after the spermatozoa was treated with 1 x 10(-6) mol/L E2-BSA and tamoxifen, an estrogen receptor inhibitor.Adenylyl cyclase (AC) inhibitor SQ22536, phospholipase C (PLC) inhibitor U73122 and protein tyrosine kinase (TPK) inhibitor Genistein all deterred the increase of [Ca2+]i caused by E2-BSA. E2-BSA also increased the PLC protein and PKC protein significantly. Tamoxifen, an antagonist of estrogen receptor, did not inhibit the activation of PLC caused by E2-BSA.The E2-BSA has an effect on human spermatozoa in a nongenomic pathway, possibly through the transmembrane signal transduction in relation to AC, PLC and TPK.

Purpose: Human neutrophil peptides (HNP1-3), small molecular antimicrobial peptides, are expressed within tumors and associated with tumor necrosis and inhibition of angiogenesis. Recent investigations have suggested that HNP1-3 are likely to be involved in the host immune responses to tumors.Experimental Design: We used recombinant pSec-HNP1, which expresses a secretable form of HNP1, to obtain expression of HNP1 in the tumor milieu in immunocompetent mice to explore the possible roles of HNP1 in tumor immunity. The antitumor effects were investigated in established CT26 colon cancer and 4T1 breast cancer models.Results: HNP1-mediated chemotactic and activating effects on immature dendritic cells were detected both in vitro and in vivo. Intratumoral expression of HNP1 resulted in not only significant tumor growth inhibition but also increased CTL infiltration within tumors. Adoptive transfer of splenocytes and a 51Cr release assay revealed specific cellular immune responses. Furthermore, increased antibodies were also found in sera from pSec-HNP1treated mice supporting specific humoral immune responses. Increased apoptosis and decreased angiogenesis were also shown in treated tumors.Conclusions: These findings indicate that HNP1 can exert multiple antitumor effects through different mechanisms; more importantly, HNP1 mediates host immune responses to tumors in situ through the recruitment and subsequent activation of immature dendritic cells and thus shows promising potential in cancer therapy. (Clin Cancer Res 2009;15(22):690111)

In our previous study, we developed a novel cationic liposome, which was modified with truncated human basic fibroblast growth factor (tbFGF) peptide. This tbFGF-mediated cationic liposome could deliver chemotherapeutic agents or gene specifically to FGFRs on tumors and obtained higher transfection efficiency than plain cationic liposomes. In order to investigate whether this novel cationic liposome could achieve a synergistic/combined anti-tumor effect as a co-delivery system, we simultaneously delivered doxorubicin (DOX) and the plasmid encoding the phosphorylation-defective mouse survivin threonine 34--alanine mutant (Msurvivin T34A plasmid) to the same cells through this cationic liposome. As a result, an enhanced antiproliferative activity in vitro has been achieved by delivering DOX and DNA simultaneously to the Lewis lung carcinoma cells (LLC) using this liposome. The concentration of DOX in the co-delivery system which caused 50% killing was nearly 3-fold lower than that of the free DOX. Furthermore, the co-delivery system suppressed tumor growth more efficiently than either DOX or the Msurvivin T34A plasmid alone in the Lewis lung carcinoma-bearing C57BL/6 mice. After 18 days of treatment with the co-delivery system, the average tumor volume in mice was decreased by 80%, which was higher than liposomal DOX (70%, P0.05) and Msurvivin T34A plasmid (41%, P0.01). The co-delivery system also caused 15 days delay of tumor growth, which was longer than the other treatment groups. In conclusion, this novel cationic liposome is an efficient vector to simultaneously deliver drugs and DNA to the same cells in vitro and in vivo.

Hepatitis B virus X protein (HBx) is a multi-functional regulatory protein that is known to be involved in viral proliferation, transcriptional activation and cell growth control. However, the actual role of HBx in cell growth control remains controversial. In this study, the impact of HBx on cell growth in vitro and in vivo was further investigated. HBx was able to inhibit the growth of hepatocellular carcinoma (HCC) cells and induce G2/M arrest in vitro. Moreover, unlike many other G2/M arrest mechanisms, HBx did not inhibit cyclin B1-CDK1 kinase activity, but it persistently activated the cyclin B1-CDK1 kinase. In vivo, HBx inhibited tumor cell growth and induced apoptosis as well as inhibited the growth of vascular endothelial cells. In conclusion, HBx induced G2/M arrest and apoptosis through sustained activation of cyclin B1-CDK1 kinase, and negatively regulated cell growth in vitro and in vivo.

PNAS-4, a novel pro-apoptotic gene activated during the early response to DNA damage, can inhibit proliferation via apoptosis when overexpressed in some tumor cells. Recent studies have indicated that honokiol can induce apoptosis, inhibit angiogenesis, and suppress tumor growth. In the present study, we investigated whether mouse PNAS-4 (mPNAS-4) could augment the apoptosis of tumor cells induced by honokiol in vitro, and whether the antiangiogenic activity of honokiol and induction of apoptosis by mPNAS-4 could work cooperatively to improve the antitumor efficacy in vivo. In vitro, mPNAS-4 inhibited proliferation of murine colorectal carcinoma CT26 and Lewis lung carcinoma LL2 cells through induction of apoptosis, and significantly augmented the apoptosis of CT26 and LL2 cells induced by honokiol. Compared with treatment with mPNAS-4 or honokiol alone, in vivo systemic administration of an expression plasmid encoding mPNAS-4 and low-dose honokiol significantly suppressed tumor growth through the enhanced induction of apoptosis and the augmented inhibition of angiogenesis. Our data suggest that the combined treatment with mPNAS-4 plus honokiol augments antitumor effects in vitro and in vivo, and that the improved antitumor activity in vivo may be associated with enhanced induction of apoptosis and augmented inhibition of angiogenesis. The present study may provide a novel and effective method for the treatment of cancer.

Barbigerone is a naturally occurring isoflavone with antioxidant activity. In present study, we investigated the antitumor activity of barbigerone against murine lung cancer cells LL/2 and the possible mechanism in vitro. Our results showed that barbigerone inhibited LL/2 cells proliferation in a concentration- and time-dependent manner and caused apoptotic death of LL/2 cells. Barbigerone-induced apoptosis was characterized by enhanced mitochondrial cytochrome c release, activation of caspase-3,-9, but not caspase-8. Exposure of LL/2 cells to barbigerone resulted in upregulation of Bcl-2-associated protein (Bax) and down-regulation of Bcl-2. In addition, proliferation inhibitory effect of barbigerone was associated with decreased level of phosphorylated p42/44 mitogen-activated protein kinase (p42/44 MAPK) and phosphorylated Akt. Moreover, barbigerone exhibit less toxicity to non-cancer cells than tumor cells. In conclusion, our results indicated that barbigerone can inhibit murine lung cancer cell proliferation by inducing apoptosis via mitochondrial apoptotic pathway and by decreasing phosphorylated p42/44 MAPK and Akt. Its potential to be a candidate of anti-cancer agent is worth being further investigated.

To investigate the anti-neoplastic effects of Valproic acid (VPA) on leukemic cells, especially drug-resistant lines, and to investigate whether modulation of GSH-redox status is involved in VPA-induced apoptosis.After the treatment of VPA at various concentrations for indicated times, cellular proliferation of the Jurkat, CEM, HL-60, K562, K562/AO2 cells were evaluated via MTT assay; and the activities of Caspase-3, Caspase-8 and Caspase-9 were quantitatively analyzed by colorimetric assay. The morphological change and cell cycle distribution were also examined on Jurkat (Dexamethasone-resistant) and K562/AO2 (Doxorubicin-resistant) cell lines. The levels of intracellular glutathione/glutathione disulfide (GSH/GSSG) and the activities of the typical antioxidant enzymes, i.e., glutathione reductase (GSH-Rd) and glutathione peroxidase (GSH-Px), were measured on cell lysates of Jurkat and K562/AO2 cell lines prior to and after VPA treatment. Apoptosis rates of Jurkat and K562/AO2 cells treated with VPA along or in combination with N-acety-l-cysteine (NAC), catalase (CAT) or DL-buthionine-(S,R)-sulfoximine (BSO) were determined by Annexin V/propidium iodide (PI) staining with flow cytometry analysis.At concentrations comparable with that achieved at clinical settings, VPA inhibited cell proliferation, activated Caspase-3, 8, and 9, and induced cell cycle arrest in Jurkat and K562/AO2. A rapid decrease in GSH-Rd and GSH-Px activities and GSH content in Jurkat and K562/AO2 were detected after VPA treatment. Co-administration of NAC or CAT attenuated VPA-induced apoptosis.VPA inhibit cell proliferation, induce cell cycle arrest and apoptosis in drug-resistant leukemic cells. Apoptosis correlates with down-regulation of intracellular GSH and disruption of intracellular GSH-redox balance, possibly through inhibition of glutathione reductase and glutathione peroxidase.

Background Metastasis in breast cancer is a vital concern in treatment because most women with primary breast cancer have micrometastases to distant sites at diagnosis. As a member of the inhibitor of apoptosis protein (IAP) family, survivin has been proposed as an attractive target for new anticancer interventions. In this study, we investigated the role of the plasmid encoding the phosphorylation-defective mouse survivin threonine 34→alanine mutant (Msurvivin T34A plasmid) in suppressing both murine primary breast carcinomas and pulmonary metastases. Methods In vitro study, induction of apoptosis by Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol) was examined by PI staining fluorescence microscopy and flow cytometric analysis. The anti-tumor and anti-metastases activity of Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol) was evaluated in female BALB/c mice bearing 4T1 s.c. tumors. Mice were treated twice weekly with i.v. administration of Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol), PORF-9 null plasmid complexed with cationic liposome (DOTAP/Chol), 0.9% NaCl solution for 4 weeks. Tumor volume was observed. After sacrificed, tumor net weight was measured and Lung metastatic nodules of each group were counted. Assessment of apoptotic cells by TUNEL assay was conducted in tumor tissue. Microvessel density within tumor tissue was determined by CD31 immunohistochemistry. Alginate-encapsulated tumor cells test was conducted to evaluate the effect on angiogenesis. By experiment of cytotoxicity T lymphocytes, we test whether Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol) can induce specific cell immune response. Results Administration of Msurvivin T34A plasmid complexed with cationic liposome (DOTAP/Chol) resulted in significant inhibition in the growth and metastases of 4T1 tumor model. These anti-tumor and anti-metastases responses were associated with triggering the apoptosis of tumor cells directly, inhibiting angiogenesis and inducing specific cellular immune response. Conclusion The present findings suggest that the Msurvivin T34A plasmid complexed with cationic liposome may provide an effective approach to inhibit the growth and metastases of a highly metastatic mouse breast cancer model with minimal side effects.

Chloroquine is an antimalarial drug that has been used in the treatment and prophylaxis of malaria since the 1950s. The present study was undertaken to examine the effects of chloroquine on Bcap-37 human breast cancer cells' growth, cell cycle modulation, apoptosis induction, and associated molecular alterations in vitro. The chloroquine treatment decreased the viability of Bcap-37 cells in a concentration- and time-dependent manner, which correlated with G(2)/M phase cell cycle arrest. The chloroquine-mediated cell cycle arrest was associated with a decrease in protein levels/activity of polo-like kinase 1 (Plk1), phosphorylated cell division cycle 25C (Cdc25C), phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated Akt. The chloroquine-treated Bcap-37 cells exhibited a marked decrease in the level of mitochondrial transmembrane potential (DeltaPsim), which was accompanied by the activation of caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP). Exposure of Bcap-37 cells to chloroquine also resulted in the induction of spindle abnormalities. In conclusion, the findings in this study suggested that chloroquine might have potential anticancer efficacy, which could be attributed, in part, to its proliferation inhibition and apoptosis induction of cancer cells through modulation of apoptosis and cell cycle-related proteins expressions, down-regulation of mitochondrial transmembrane potential (DeltaPsim), and induction of spindle abnormalities.

Genes involved in tumorigenesis may be targets for cancer immunotherapy. For many tumor types, molecular signatures do not yet exist. Methods that identify novel, tissue-specific tumor-related genes therefore have potentially important implications for developing cancer immunotherapy. Here, we used cross-reactive serological analysis of recombinant cDNA expression (CR-SEREX) to identify the novel testicular cancer-related gene, rat testicular antigenic protein 2 (RTAP2). We identified two RTAP2 splice variants (RTAP2a and RTAP2b) encoding 191 and 358 amino acid proteins, respectively. RTAP2a and RTAP2b proteins have identical N-termini but different C-terminal regions arising from alternative splicing of exon 4. Bioinformatics and reverse transcription-polymerase chain reaction (RT-PCR) revealed that the RTAP2a transcript was expressed selectively in adult testis and several tumor cell lines, while RTAP2b was ubiquitous. Our results suggest that RTAP2a may contribute to testicular development and tumorigenesis, and may be a candidate gene for cancer immunotherapy.

The purpose of this study was to investigate the effects of chloroquine diphosphate on the proliferation and apoptosis of human leukemic K562 cells, and to elucidate its possible mechanism of activity. The inhibitory effect of chloroquine diphosphate with different concentrations on K562 cell proliferation was detected by MTT method. Apoptosis was measured by flow cytometry (FCM); morphological analysis of apoptosis was performed after staining with propidium iodide (PI) under fluorescence microscope; cell apoptosis was assessed by the DNA ladder shown agarose gel electrophoresis. After treatment with chloroquine diphosphate, K562 cells were stained by Rhodamine 123 to detect changes in mitochondrial transmembrane potential (DeltaPsim) by FCM. The results showed that the cell viability decreased in dose-dependent manner, following chloroquine diphosphate treatment at different concentrations (1.5625, 3.125, 6.25, 12.5, 25, 50 and 100 micromol/L) for 24, 48 and 72 hours. By FCM analysis, the significant increases of sub-G(1) were observed. DNA ladder was detected and apoptotic nuclei were observed. DeltaPsim decreased in K562 cells after chloroquine diphosphate treatment. It is concluded that the chloroquine diphosphate can inhibit the proliferation of K562 cells and induce cell apoptosis, which may relate to down-regulation of mitochondrial transmembrane potential (DeltaPsim).

Honokiol, a well-tolerated natural product, can inhibit the proliferation of cancer cells. But its water insolubility hampers its systemic administration for therapy of cancer. As a drug delivery system, the pegylated liposome (PEGL) can increase the water solubility and targeting of the drug. Honokiol has been successfully encapsulated by PEGL in our laboratory. We wondered whether the combination treatment with pegylated liposomal honokiol (H-PEGL) and cisplatin (DDP) could improve the antitumor efficacy in ovarian carcinoma. H-PEGL could introduce apoptosis of SKOV3 cells in vitro, which was quantified by flow cytometric analysis, and the cellular morphologic changes were determined by propidium iodide staining. In a human ovarian carcinoma mouse model, combination treatment with H-PEGL (0.4 mg/day for 30 days; intraperitoneal) and DDP (5 mg/kg on days 7, 11, 15, 19; intraperitoneal) acted synergistically to inhibit tumor growth by 91.48% without notable toxicity, but H-PEGL and DDP alone only inhibit tumor growth by 66.83% and 52.5% as compared to the NaCl solution control, respectively. Assessment of microvessel density and apoptosis index by CD31 and terminal deoxynucleotidyl transferase–mediated nick end labeling immunohistochemistry respectively suggested that the antitumor activity of H-PEGL is mediated by angiogenesis inhibition and introduction of apoptosis. Our results showed us a splendid prospect of the clinical application of combination treatment on patients suffering from ovarian cancer with H-PEGL and DDP.

CCL19 [chemokine (C-C motif) ligand 19; also known as MIP-3beta (macrophage inflammatory protein-3beta) or ELC (Epstein-Barr-virus-induced molecule 1 ligand chemokine)], one of the immunostimulatory cytokines, chemoattracts both DCs (dendritic cells) and T-lymphocytes. Adenoviral vector is one of the most used gene delivery vectors for cancer therapy because of its high gene-transfection efficiency. However, its wider application is limited, owing to immune responses that reduce transgene expression and decrease the efficacy of repeated administration. We constructed the recombinant replication deficient adenoviral vectors containing the CCL19 gene (Ad-CCL19) and combined them with PEG-PE [poly(ethylene glycol)-phosphatidylethanolamine]-modified cationic liposomes (Ad-CCL19/PEG-PE) for immunotherapy against murine fibrosarcoma. Although there were hardly any therapeutic differences between Ad-CCL19- and Ad-CCL19/PEG-PE-treated mice that were observed at the second administration, the final results demonstrated that Ad-CCL19/PEG-PE-treated mice survived much longer. The antitumour efficacy may be related to the high level of CCL19 after the final administration and lasting expression of IFN-gamma (interferon-gamma) and IL-12 (interleukin-12) in the Ad-CCL19/PEG-PE-treated group, which were measured by reverse-transcription PCR and ELISA. The results demonstrated that PEG-PE-cationic-liposome-conjugated adenovirus could prolong the expression of the therapeutic gene in vivo and may enhance the antitumour efficacy.

Background Psoriasis is an autoimmune disease that is caused by a shift in the Th1/Th2 balance toward Th1-dominant immunity. It has been established as an effective treatment to counteract psoriasis by subcutaneous injection of recombinant interleukin (IL)-4, and IL-4 gene therapy by topical transdermal penetration has shown its antipsoriatic effect in mice. Retinoic acid (RA) and dimethylsulfoxide can increase the efficiency of gene transfection in the topical transdermal delivery system. Objective We investigated whether RA could improve anti-psoriasis efficiency using IL-4 expression plasmid pORF-mIL-4 (pIL-4) via transdermal delivery system in K14-vascular endothelial growth (K14-VEGF) factor transgenic mice. Methods After pretreatment with RA, plasmid pIL-4 in 10% dimethylsulfoxide was applied to the ear skin by topical transdermal penetration. Hematoxylin- eosin staining and immunohistochemistry were performed with ear samples to evaluate anti-psoriasis efficiency in mice. Results The psoriasis pathological features were relieved and psoriasis-associated factors were significantly reduced. Conclusion Our results reveal that topical application of pIL-4 in dimethylsulfoxide by transdermal delivery with RA pretreatment can improve psoriasis significantly.

Cisplatin (CDDP), homoharringtonine (HHT), mitoxantrone (MIT) and hydroxycamptothecin (HCPT) are highly effective anti-tumor drugs. To evaluate their effects in the therapy of leukemia and establish a valuable method to estimate anti-tumor drugs, Annexin V/PI double parameter flow cytometry was used to detect the effects of these drug inducing apoptosis and death in Jurkat cell line. The results showed that MIT and HCTP-induced apoptosis effects on Jurkat cell line were obvious at 4 hours in early phase after adding drug (P0.05) and at 8 hours in late phase after adding drug (P0.05). HHT had obvious effect on inducing apoptosis of Jurkat cells, but no significant difference from low to high doses. The effect of CDDP on inducing apoptosis of Jurkat cell line was obviously weaker than that of HHT, MIT and HCPT, its weak effect on apoptosis of Jurkat cell line was found only at high concentration of drug for long time. Death effects on Jurkat cell line can not be observed in every experimental group. It is concluded that low dose of MIT can effectively induced apoptosis of Jurkat cell line. Annexin V/PI double parameter flow cytometry can be used as a reliable method for clinical screening anti-tumor drugs.

Mesenchymal stem cells (MSCs) were adenovirally engineered to secrete interleukin-12 (AdIL-12-MSCs) and evaluated for their anticarcinogenesis efficacy against three kinds of unestablished tumor models including B16 melanoma, LLC Lewis lung cancer and HCC hepatoma. Injection of AdIL-12-MSCs into protected mice before tumor inoculation prevented all of 12 mice in B16 preventive groups, 10 out of 12 in LLC lung cancer model and 11 out of 12 mice in HCC hepatoma model from developing tumors, whereas the control groups pre-receiving PBS were validated for 100% carcinogenesis; the tumor formation rates in free-AdIL-12 and vacant MSC groups were unveiled between approximately 83 and 100% even with plentiful angiogenesis and newborn lymphatic vessels, as well as distant metastases. As a novel approach, AdIL-12-MSC has revealed expected preventive effects on carcinogenesis (P

To establish a mouse model of systemic lupus erythematosus(SLE) and to discuss the role of environmental factors in the pathogenesis of SLE.Thirty 10-week-old female BALB/c mice were divided into the model group and control group. The model mice received a single intraperitoneal injection of 0. 5 mL of pristane, while control mice received a single i.p. injection of 0. 5 mL of 0.9% NS. Autoantibodies and proteinuria were detected before injection and monthly thereafter. 8 months after injection, all mice were bled to death. Kidneys were excised to observe the histopathologic evidence of glomerulonephritis.After pristane injection, antinuclear antibody(ANA) and anti-dsDNA antibody appeared in sera as early as 3 months and 4 months respectively. At 8 months, the incidence rates of ANA and anti-dsDNA antibody were 87.5% and 47.8% respectively. The protein concentration of urine in most mice wasor =1+ (300 mg/L). Light microscopy and immunofluorescence of kidney sections indicated glomerulonephritis. In control group, only one serum was ANA low-titer positive and another was anti-dsDNA antibody low-titer positive. The protein concentration of urine wasor = 1+. The histopathology and immunofluorescence showed no evidence of glomerulonephritis. Spearman correlation analysis indicated that anti-dsDNA antibody was correlated with proteinuria (r = 0.538, P = 0.003).The murine lupus model can be successfully established in female BALB/c mouse with a single i. p. injection of 0. 5 mL of pristane and the specific autoantibody anti-dsDNA for SLE has appeared in sera of BALB/c mice.

Single administration of low dose cyclophosphamide (CTX) was previously reported to enhance the antitumor efficacy of immunotherapies. To investigate the possible mechanisms for this effect, we examined whether a single administration of low dose CTX could augment the immunogenicity of dendritic cell (DC) vaccines. Fifty milligrams per kilogram body weight dose of CTX was administrated intraperitoneally to mice after B16 melanoma or C26 colon carcinoma tumor models were established, DC vaccine generated from mouse bone marrow and pulsed with B16 or C26 tumor cells lysates were vaccinated 4 days later. CTX treatment potentiated the antitumor effects of the DC vaccine, and increased the proportion of IFN-gamma secreting lymphocytes in spleens. Furthermore, a significantly reduced proportion of CD4+CD25+FoxP3+ regulatory T (Treg) cells was detected by flow cytometry in spleen lymphocytes from tumor-bearing mice treated with CTX. Thus, a single administration of low dose CTX could augment antitumor immune responses of DC vaccine by reducing the proportion of CD4+CD25+FoxP3+ Treg cells in tumor-bearing mice. Our results suggested a possible mechanism of CTX-induced immunopotentiation and provided a strategy of immunotherapy combining a low dose CTX with DC vaccine.