Zhiming Hu

A Novel Therapeutic Vaccine of Mouse GM-CSF Surface Modified MB49 Cells Against Metastatic Bladder Cancer

PURPOSE Immunotherapy is considered effective for muscle invasive bladder cancer mini metastasis. We developed what is to our knowledge a novel technology by which streptavidin tagged mouse GM-CSF was displayed on the surface of biotinylated bladder cancer cells to induce antitumor immunity. MATERIALS AND METHODS Mouse subcutaneous and lung metastasis bladder cancer models were established. Mice were injected subcutaneously with 1 × 10(6) mouse GM-CSF surface modified MB49 bladder cancer cells and monitored for tumor growth and survival. Immunohistochemical and flow cytometric assay were done to assess the proportion of T lymphocytes. The T-lymphocyte cytotoxicity assay was performed to assess MB49 specific cytotoxicity. On day 60 after MB49 implantation the vaccine cured mice were injected subcutaneously with MB49 or RM-1 cells in the left or right hind leg, respectively. They were monitored for survival and T-lymphocyte cytotoxicity. RESULTS Mouse GM-CSF surface modified vaccine significantly inhibited tumor growth in the subcutaneous model and extended survival in the lung model. More CD4 and CD8 T cells appeared at tumor sites and in peripheral blood in the vaccine treated group than in other control groups. Splenocytes from the vaccine treated group showed the most potent cytotoxicity on MB49 cells. Cured mice in the vaccine treated group resisted the second injection of MB49 bladder cancer cells but not the RM-1 prostate cancer cell challenge. CONCLUSIONS Mouse GM-CSF surface modified MB49 bladder cancer cell vaccine induced specific antitumor immunity and was efficient for metastatic bladder cancer.

Protective effects of a novel trimerized sTNFRII on acute liver injury

TNF α plays a central role in the pathogenesis of inflammatory diseases such as rheumatoid arthritis and murine acute liver injury induced by injection of D-galactosamine and subsequent LPS. Recombinant Fc-fused soluble TNF receptor II (sTNFRII-Fc) has been used in the treatment of rheumatoid arthritis for a decade. We have recently constructed a novel fusion protein sTNFRII-gAD, which is composed of a soluble TNF receptor II and a globular domain of adiponectin. Utilizing the inclination of gAD to form homologous trimer naturally, we sought to explore TNFα antagonism of the novel trimerized sTNFRII-gAD and meantime compare TNFα-neutralizing effects in vitro and in vivo between sTNFRII-Fc and sTNFRII-gAD. Here, we evaluated the TNFα-antagonizing activity of sTNFRII-gAD with TNFα-induced L929 cytotoxicity assay. Furthermore, sTNFRII-Fc or sTNFRII-gAD was administered simultaneously with d-galactosamine 1h prior to LPS injection in the murine model of acute liver injury. Serum TNFα and TNFα-sTNFRII-gAD complex were measured by ELISA and the liver injury was assessed through alanine transaminase measurement and liver histological analysis. sTNFRII-gAD was shown to have higher TNFα-neutralizing activity than sTNFRII-Fc (p<0.05) in the L929 cytotoxicity assay. With a significant attenuation of murine lethality (p<0.05), sTNFRII-gAD showed more protective effects than sTNFRII-Fc in the murine model of acute liver injury. These results demonstrated that sTNFRII-gAD was more efficacious than sTNFRII-Fc as a TNFα antagonist, highlighting the potential of sTNFRII-gAD for the treatment of diseases associated with excessive TNFα.

Cdc6 contributes to cisplatin-resistance by activation of ATR-Chk1 pathway in bladder cancer cells

High activation of DNA damage response is implicated in cisplatin (CDDP) resistance which presents as a serious obstacle for bladder cancer treatment. Cdc6 plays an important role in the malignant progression of tumor. Here, we reported that Cdc6 expression is up-regulated in bladder cancer tissues and is positively correlated to high tumor grade. Cdc6 depletion can attenuate the malignant properties of bladder cancer cells, including DNA replication, migration and invasion. Furthermore, higher levels of chromatin-binding Cdc6 and ATR were detected in CDDP-resistant bladder cancer cells than in the parent bladder cancer cells. Intriguingly, down-regulation of Cdc6 can enhance sensitivity to CDDP both in bladder cancer cells and CDDP-resistant bladder cancer cells. Cdc6 depletion abrogates S phase arrest caused by CDDP, leading to aberrant mitosis by inactivating ATR-Chk1-Cdc25C pathway. Our results indicate that Cdc6 may be a promising target for overcoming CDDP resistance in bladder cancer.

Novel Immunotherapy for Metastatic Bladder Cancer Using Vaccine of Human Interleukin-2 Surface-modified MB 49 Cells

OBJECTIVE To develop a novel protein-anchor technology to immobilize human interleukin-2 on tumor cells to induce antitumor immunity. METHODS Interleukin-2 surface-modified MB49 cells were prepared as a vaccine. Subcutaneous and pulmonary metastatic mouse models of MB49 bladder cancer were used to evaluate the antitumor efficiency of the vaccine. Immunohistochemistry, flow cytometric, and cytotoxic T-lymphocyte assay were performed to assess the proportion and cytotoxicity of the T lymphocytes. RESULTS The IL-2 surface-modified MB49 cell vaccine inhibited tumor growth and extended the survival of the mice, and the vaccine-cured mice effectively resisted the second MB49 but not the RM-1 prostate cancer cell challenge. Furthermore, more cytotoxicity on the MB49 cells and more CD4-positive, CD8-positive T cells appeared in the vaccine-treated group. CONCLUSION The results of our study have demonstrated that the human interleukin-2 surface-modified MB49 bladder cancer cell vaccine induced specific antitumor immunity and was efficient against metastatic bladder cancer.

Norcantharidin inhibits pre-replicative complexes assembly of HepG2 cells.

Norcantharidin (NCTD) is currently used for anticancer therapy but the exact mechanism of action remains unknown. Pre-replicative complexes (pre-RCs) are essential for cell DNA replication and highly related to malignant proliferation. Here, we examined the inhibitory effect of NCTD on pre-RC components in HepG2 cells. We showed that NCTD induced degradation of Cdc6 and Mcm2 in a dose-dependent manner. Under 100 μM NCTD concentration, about 70% of Cdc6 and 50% of Mcm2 were degraded. In addition, the nuclear translocation of Mcm6 was inhibited by NCTD. Further studies aiming at G1 synchronous cells showed that, NCTD reduced the chromatin-bound Cdc6, Mcm2 and Mcm6. Moreover, the cells were blocked from entering the S phase and accumulated at the G1 phase when released synchronously into the cell cycle. Consistently, the DNA replication was inhibited by NCTD. Finally, the combination NCTD with Cdc6 depletion lead to more severe cytotoxicity (88%) than NCTD (52%) and Cdc6 depletion (39%) alone. A synergic cytotoxicity was observed between Cdc6 depletion and NCTD. In conclusion, our results demonstrate that NCTD inhibits pre-RC assembly; subsequently blocks the G1 to S transition; and inhibits DNA replication in HepG2 cells. Pre-RCs are an intriguing target for cancer therapy, which merits further investigations for anticancer development.

Depletion of regulatory T cells by anti-ICOS antibody enhances anti-tumor immunity of tumor cell vaccine in prostate cancer

ICOS+Treg cells exert important immunosuppressive effects in tumor immunity. We adopt a combination approach of ICOS+Treg cells depletion with tumor cell vaccine to evaluate anti-tumor immunity in mouse prostate cancer model. Streptavidin (SA)-mGM-CSF surface-modified RM-1 cells were prepared as the vaccine and the mouse subcutaneous prostate tumor model was used to evaluate the immunity. Tumor growth, flow cytometry, immunohistochemistry, immunofluorescence and enzyme linked immunosorbent assay (ELISA) were performed to evaluate the therapeutic effects. Our results demonstrated that SA-mGM-CSF vaccine was prepared successfully and tumor growth was inhibited. The tumor size in the combination group was much smaller than that in the vaccine with IgG mAb group. The portions of dendritic cells, CD8+ and CD4+T cells in the mice blood and tumor tissues were increased after treatment with vaccine. There were more immune-suppressing Tregs infiltrated into tumor after treatment with tumor cell vaccine, and ICOS blocking could deplete the infiltrated Tregs, and T lymphocytes increased more dramatically in the combination therapy group. The concentrations of interferon-γ were increased in all vaccine group, the concentrations of Interleukin-10 and Interleukin-4 were much lower in the combination group. Our study demonstrated that ICOS blocking could deplete the tumor-infiltrated ICOS+Treg cells. Combining GM-CSF surface-modified RM-1 cell vaccine with Anti-ICOS antibody could induce better antitumor immunity than a vaccine alone.

Sequential administration of GM-CSF and IL-2 surface-modified MB49 cells vaccines against the metastatic bladder cancer

OBJECTIVES Many strategies are pursued to enhance tumor vaccine immune response, including the utilization of cytokines. We have developed a novel protein-anchor technology to immobilize cytokines on tumor cell surface. Here we reported the preparation of tumor cell vaccines by immobilizing GM-CSF or IL-2 on MB49 bladder cancer cells and evaluated their antitumor efficacy (administrated alone or sequentially) in a metastatic mouse model. MATERIALS AND METHODS SA-mGM-CSF or SA-hIL-2 surface-modified MB49 cells were prepared as vaccine. Mice were treated with MB49 cell vaccines (administrated alone or sequentially). Survival time, tumor growth, flow cytometry, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and cytotoxic T lymphocytes (CTL) assay were used to evaluate the antitumor efficiency of the vaccines in the pulmonary metastatic model of bladder cancer. RESULTS GM-CSF vaccine induced more mature dendritic cells in the mice spleen. Combination with subsequent IL-2 vaccine significantly increased CD4(+), CD8(+), and IFN-γ(+)CD8(+) T but not CD4(+)Foxp3(+) T cell population and induced the highest production of IFN-γ, IL-12, but not IL-10. Furthermore, the splenocytes from the sequentially combined vaccines group showed the most potent cytotoxicity on MB49 cells. Finally, the sequentially combined vaccines evidently extended the survival time of mice (the median survival time of PBS, ethanol-fixed, anchored GM-CSF, anchored IL-2, and anchored GM-CSF + anchored IL-2 groups were 34, 37, 45, 47, and 59 days, respectively) and effectively protected the mice against a second MB49 cells but not RM-1 cells challenge. CONCLUSIONS This study demonstrated that sequential administration of GM-CSF and IL-2 surface-modified MB49 cells vaccines could effectively induce specific antitumor immune response.

Acute liver injury attenuation of a novel recombinant sTNFR through blocking hepatic apoptosis.

Abstract Context: Tumor necrosis factor (TNF) α plays a key role in acute liver injury (ALI) induced by injection of d-galactosamine (D-Gal)/lipopolysaccharide (LPS). A novel recombinant trimeric sTNFRII, sTNFRII-gAD, has been tested to be effective in ameliorating ALI, when administered prior to ALI establishment. This study aims to validate the protective effect of sTNFRII-gAD when given after ALI setup and further explore its effect on hepatic apoptosis. Materials and methods: The treatments were carried out concomitantly with ALI establishment with clinically approved sTNFRII-Fc (the dimeric sTNFRII) as a positive control. Lethality, liver weight, and serum alanine transaminase were measured, and histological analysis was performed to evaluate liver injury induced by D-Gal/LPS. Additionally, Terminal-deoxynucleoitidyl transferase-mediated nick end labeling (TUNEL) and Western blot analyses of caspase-3 were used to examine hepatocellular apoptosis. Results: sTNFRII-gAD given after D-Gal/LPS injection turned out to attenuate animal mortality significantly (p < 0.01), and had better hepatic protection. In terms of apoptosis, both sTNFRII-gAD and sTNFRII-Fc displayed noticeable improvement of apoptosis evidenced by dramatic decline of active caspase-3 compared to the control group. Conclusions: The results demonstrated that sTNFRII-gAD therapeutically diminished the lethality induced by D-Gal/LPS, possibly through blocking hepatic apoptosis initiated by TNFα. Of note, sTNFRII-gAD was superior to sTNFRII-Fc in some respects, indicating a promising alternative for the therapeutic strategy against the diseases associated with excessive TNFα.

The therapeutic potential of SA-sCD40L in the orthotopic model of superficial bladder cancer.

Abstract Background. Intravesical administration is an important treatment against superficial bladder cancer and CD40L is essential for the protective anti-tumor immunity. In situ gene therapy with CD40L was demonstrated to successfully inhibit tumor cell growth in the orthotopic mouse model of bladder cancer. In the present study, we prepared streptavidin (SA)-tagged sCD40L and developed a novel immunotherapy for superficial bladder cancer based on the strong interaction between streptavidin and biotin. Material and methods. The SA-sCD40L fusion protein was expressed in E. coli and purified on the Ni-NTA column. After refolding with dialysis, the bi-function of the fusion protein was determined by flow cytometric analysis for streptaidin-mediated surface modification of MB49 bladder cancer cells and a mouse B cell CD40L-dependent proliferation assay. The mouse orthotopic model of MB49 superficial bladder cancer was used to evaluate the efficacy of SA-sCD40L immunotherapy. Results. The SA-sCD40L fusion protein exhibited both full biotin-binding property and CD40L bioactivity. After intravesical instillation, the SA-sCD40L bi-functional fusion protein was durably immobilized on the biotinylated mucosal surface of bladder wall for up to four days. The SA-sCD40L treatment significantly prolonged the survival of MB49 tumor-bearing mice and cured 50% of mice with MB49 superficial bladder cancer without significant adverse effects. In addition, more tumor-infiltrating CD4+or CD8+ T cells were observed in SA-sCD40L–treated group. Conclusion. Intravesical immobilization of SA-sCD40L elicited a strong and long-lasting immunity against the MB49 bladder cancer.

PD-1 blockade enhances the antitumor efficacy of GM-CSF surface-modified bladder cancer stem cells vaccine: Anti-PD-1 combined with CSCs vaccine against bladder cancer

Eliminating cancer stem cells (CSCs) is a key issue in eradicating tumor. The streptavidin–granulocyte‐macrophage‐colony stimulating factor (SA–GM‐CSF) surface‐modified bladder CSCs vaccine previously developed using our protein–anchor technology could effectively induce specific immune response for eliminating CSCs. However, program death receptor‐1 (PD‐1)/program death ligand 1 (PD‐L1) signaling in tumor microenvironment results in tumor‐adaptive immune resistance. Although the CSCs vaccine could increase the number of CD8+T cells, a part of these CD8+T cells expressed PD‐1. Moreover, the CSCs vaccine upregulated the PD‐L1 expression of tumor cells, resulting in immune resistance. Adding PD‐1 blockade to the CSCs vaccine therapy increased the population of CD4+, CD8+ and CD8+IFN‐γ+ but not CD4+ Foxp3+T cells and induced the highest production of IFN‐γ. PD‐1 blockade could effectively enhance the functions of tumor‐specific T lymphocytes generated by the CSCs vaccine. This combination therapy improved the cure rate among mice and effectively protected the mice against a second CSCs cell challenge, but not a RM‐1 cell challenge. These results indicate that PD‐1 blockade combined with the GM‐CSF‐modified CSCs vaccine effectively induced a strong and specific antitumor immune response against bladder cancer.