Guoxing Zheng

The 4-1BB costimulation augments the proliferation of CD4+CD25+ regulatory T cells.

The thymus-derived CD4+CD25+ T cells belong to a subset of regulatory T cells potentially capable of suppressing the proliferation of pathogenic effector T cells. Intriguingly, these suppressor cells are themselves anergic, proliferating poorly to mitogenic stimulation in culture. In this study, we find that the 4-1BB costimulator receptor, best known for promoting the proliferation and survival of CD8+ T cells, also induces the proliferation of the CD4+CD25+ regulatory T cells both in culture and in vivo. The proliferating CD4+CD25+ T cells produce no detectable IL-2, suggesting that 4-1BB costimulation of these cells does not involve IL-2 production. The 4-1BB-expanded CD4+CD25+ T cells are functional, as they remain suppressive to other T cells in coculture. These results support the notion that the peripheral expansion of the CD4+CD25+ T cells is controlled in part by costimulation.

Depleting Intratumoral CD4+CD25+ Regulatory T Cells via FasL Protein Transfer Enhances the Therapeutic Efficacy of Adoptive T Cell Transfer

One strategy for improving adoptive therapy is preconditioning the host immune environment by depleting CD4(+)CD25(+) regulatory T cells (Treg) suppressive to antitumor responses. Given that Treg increase, or selectively accumulate, within tumors and are sensitive to FasL-mediated apoptosis, we test here the hypothesis that inducing apoptosis of intratumoral Treg using FasL may improve adoptive T cell therapy. We show that FasL applied intratumorally via protein transfer decreases intratumoral Treg via inducing apoptosis in these cells. Significantly, we show that the use of FasL prior to the infusion of tumor-reactive CD8(+) T cells enhances the therapeutic efficacy of adoptive T cell transfer against established tumors, which is mediated by persistent, systemic antitumor immunity. Intratumoral FasL protein transfer also results in neutrophil infiltration of tumor. However, we show that intratumoral immunodepletion of neutrophils does not abolish the effect of FasL on adoptive transfer. Rather, the effect of FasL is completely abolished by cotransfer of Treg, isolated from the tumor-draining lymph nodes. Hence, our study shows for the first time that using FasL to predeplete intratumoral Treg provides a useful means for optimizing adoptive therapy.

18α-glycyrrhetinic acid targets prostate cancer cells by down-regulating inflammation-related genes

Glycyrrhetinic acid is an active triterpenoid metabolite of glycyrrhizin abundantly present in licorice roots. Glycyrrhetinic acid exists as α and β stereo-isomeric forms. Both stereo-isomeric forms are known to have anti-inflammatory and anticancer activity. However, the effects and anticancer mechanism of α glycyrrhetinic acid in prostate cancer cells has not yet been evaluated. Therefore, we investigated the growth inhibition, induction of apoptosis and the anticancer mechanisms of 18α-glycyrrhetinic acid (AGA), on the androgen-independent metastatic prostate cancer cell line DU-145. Our results showed that AGA inhibited proliferation and growth of these cells by inducing apoptosis as determined by Annexin V and flow cytometry analyses. Our studies also showed that HUVEC tube formation was drastically reduced when cultured in conditioned medium of AGA-treated DU-145 cells. In addition, AGA treatment prevented the invasion of DU-145 prostate cancer cells on matrigel coated transwells via down-regulation of NF-κB (p65), VEGF and MMP-9 expression. Furthermore, AGA treatment also down-regulated the expression of pro-inflammatory cytokine/growth factor genes HMGB1, IL-6 and IL-8 in DU-145 cells. Interestingly, AGA simultaneously upregulated the expression of non-steroidal anti-inflammatory gene-1 (NAG-1) in DU-145 cells suggesting its anti-inflammatory activity on prostate cancer cells. Taken together, the results of this study suggest that AGA may be a promising anticancer agent that merits further investigation for the chemoprevention and treatment of prostate cancer.

Piperine, a Bioactive Component of Pepper Spice Exerts Therapeutic Effects on Androgen Dependent and Androgen Independent Prostate Cancer Cells.

Prostate cancer is the most common solid malignancy in men, with 32,000 deaths annually. Piperine, a major alkaloid constituent of black pepper, has previously been reported to have anti-cancer activity in variety of cancer cell lines. The effect of piperine against prostate cancer is not currently known. Therefore, in this study, we investigated the anti-tumor mechanisms of piperine on androgen dependent and androgen independent prostate cancer cells. Here, we show that piperine inhibited the proliferation of LNCaP, PC-3, 22RV1 and DU-145 prostate cancer cells in a dose dependent manner. Furthermore, Annexin-V staining demonstrated that piperine treatment induced apoptosis in hormone dependent prostate cancer cells (LNCaP). Using global caspase activation assay, we show that piperine-induced apoptosis resulted in caspase activation in LNCaP and PC-3 cells. Further studies revealed that piperine treatment resulted in the activation of caspase-3 and cleavage of PARP-1 proteins in LNCaP, PC-3 and DU-145 prostate cancer cells. Piperine treatment also disrupted androgen receptor (AR) expression in LNCaP prostate cancer cells. Our evaluations further show that there is a significant reduction of Prostate Specific Antigen (PSA) levels following piperine treatment in LNCaP cells. NF-kB and STAT-3 transcription factors have previously been shown to play a role in angiogenesis and invasion of prostate cancer cells. Interestingly, treatment of LNCaP, PC-3 and DU-145 prostate cancer cells with piperine resulted in reduced expression of phosphorylated STAT-3 and Nuclear factor-κB (NF-kB) transcription factors. These results correlated with the results of Boyden chamber assay, wherein piperine treatment reduced the cell migration of LNCaP and PC-3 cells. Finally, we show that piperine treatment significantly reduced the androgen dependent and androgen independent tumor growth in nude mice model xenotransplanted with prostate cancer cells. Taken together, these results support further investigation of piperine as a potential therapeutic agent in the treatment of prostate cancer.

HMGB1: A Promising Therapeutic Target for Prostate Cancer

High mobility group box 1 (HMGB1) was originally discovered as a chromatin-binding protein several decades ago. It is now increasingly evident that HMGB1 plays a major role in several disease conditions such as atherosclerosis, diabetes, arthritis, sepsis, and cancer. It is intriguing how deregulation of HMGB1 can result in a myriad of disease conditions. Interestingly, HMGB1 is involved in cell proliferation, angiogenesis, and metastasis during cancer progression. Furthermore, HMGB1 has been demonstrated to exert intracellular and extracellular functions, activating key oncogenic signaling pathways. This paper focuses on the role of HMGB1 in prostate cancer development and highlights the potential of HMGB1 to serve as a key target for prostate cancer treatment.

Modifying Dendritic Cells via Protein Transfer for Antitumor Therapeutics

Purpose: The modification of therapeutic dendritic cells (DC) with various immunostimulatory molecules represents a useful means for improving the antitumor efficacy of DC transfer–based immunotherapy. We have evaluated the feasibility of modifying therapeutic DCs with multiple immunostimulatory molecules using a time-efficient, protein transfer (or protein “painting”)–based method. Experimental Design: Bone marrow–derived DCs were painted with either control protein human IgG (hIgG) or three immunostimulatory molecules, SLC, 4-1BBL, and TRANCE (the triad protein). Painted DCs were injected intratumorally into mice bearing established tumors. Subsequently, the capacities of painted DCs to migrate to the draining lymph nodes, recruit the host T cells, promote Th1 cytokine responses, and elicit therapeutic antitumor responses were evaluated. Results: The triad protein transfer yields a uniform population of DCs that coexpress all three of the proteins. Compared with the hIgG-painted DCs, the triad protein–painted DCs migrate more efficiently to the draining lymph nodes and show enhanced capabilities to induce T cell infiltration of tumors and to promote Th1 cytokine responses in vivo. Furthermore, in both the EG.7 and TRAMP-C2 tumor models, compared with the DCs painted with hIgG or only one of the three proteins, the triad protein–painted DCs, upon adoptive transfer, elicit stronger therapeutic responses against established tumors. Importantly, the antitumor responses of the triad protein–painted DCs are mediated by systemic antitumor immunity. Conclusions: This study establishes, for the first time, the feasibility of optimizing DC transfer–based immunotherapy via combinatorial protein transfer of therapeutic DCs with an array of immunostimulatory molecules.

Vitamin K and its analogs: Potential avenues for prostate cancer management

Epidemiological studies have demonstrated a relationship between cancer incidence and dietary habits. Especially intake of certain essential nutrients like vitamins has been shown to be beneficial in experimental studies and some clinical trials. Vitamin K (VK) is an essential nutrient involved in the blood clotting cascade, and there are considerable experimental data demonstrating its potential anticancer activity in several cancer types including prostate cancer. Previous in vitro and in vivo studies have focused mainly on anti-oxidative effects as the underlying anticancer mechanism of VK. However, recent studies reveal that VK inhibits the growth of cancer cells through other mechanisms, including apoptosis, cell cycle arrest, autophagy, and modulation of various transcription factors such as Myc and Fos. In the present review, we focus on the anticancer effect of dietary VK and its analogs on prostate cancer, with an emphasis on the signaling pathways that are activated following exposure to these compounds. This review also highlights the potential of VK and its derivatives as an adjuvant treatment in combination with other vitamins or with chemotherapeutic drugs. Based on our recent results and a review of the existing literature, we present evidence that VK and its derivatives can potentially be explored as cancer therapy, especially for prostate cancer.Epidemiological studies have demonstrated a relationship between cancer incidence and dietary habits. Especially intake of certain essential nutrients like vitamins has been shown to be beneficial in experimental studies and some clinical trials. Vitamin K (VK) is an essential nutrient involved in the blood clotting cascade, and there are considerable experimental data demonstrating its potential anticancer activity in several cancer types including prostate cancer. Previous in vitro and in vivo studies have focused mainly on anti-oxidative effects as the underlying anticancer mechanism of VK. However, recent studies reveal that VK inhibits the growth of cancer cells through other mechanisms, including apoptosis, cell cycle arrest, autophagy, and modulation of various transcription factors such as Myc and Fos. In the present review, we focus on the anticancer effect of dietary VK and its analogs on prostate cancer, with an emphasis on the signaling pathways that are activated following exposure to these compounds. This review also highlights the potential of VK and its derivatives as an adjuvant treatment in combination with other vitamins or with chemotherapeutic drugs. Based on our recent results and a review of the existing literature, we present evidence that VK and its derivatives can potentially be explored as cancer therapy, especially for prostate cancer.

Hepatoma-derived growth factor: A survival-related protein in prostate oncogenesis and a potential target for vitamin K2

Hepatoma-derived growth factor (HDGF) is a heparin-binding growth factor, which has previously been shown to be expressed in a variety of cancers. HDGF overexpression has also previously been correlated with a poor prognosis in several cancers. The significance of HDGF in prostate cancer, however, has not been investigated. Here, we show that HDGF is overexpressed in both androgen-sensitive LNCaP cells and androgen-insensitive DU145, 22RV1, and PC-3 cells. Forced overexpression enhanced cell viability of RWPE-1 cells, whereas HDGF knockdown reduced cell proliferation in human prostate cancer cells. We also show that HDGF may serve as a survival-related protein as ectopic overexpression of HDGF in RWPE cells up-regulated the expression of antiapoptosis proteins cyclin E and BCL-2, whereas simultaneously down-regulating proapoptotic protein BAX. Western blot analysis also showed that HDGF overexpression modulated the activity of phospho-AKT as well as NF-kB, and these results correlated with in vitro migration and invasion assays. We next assessed the therapeutic potential of HDGF inhibition with a HDGF monoclonal antibody and vitamin k2, showing reduced cell proliferation as well as inhibition of NF-kB expression in HDGF overexpressed RWPE cells treated with a HDGF monoclonal antibody and vitamin K2. Collectively, our results suggest that HDGF is a relevant protein in prostate oncogenesis and may serve as a potential therapeutic target in prostate cancer.

Abstract 1109: Characterization of hepatoma-derived growth factor (HDGF) as a novel survival related protein in prostate cancer cells

Prostate cancer (PCa) is a leading cause of death by cancer in men, resulting in approximately 32,000 deaths each year. While the cause of PCa is not clearly known at this time, some risk factors include age, diet, race, and activation of certain oncogenes. Hepatoma-derived growth factor (HDGF) is a recently discovered oncogene and a key mitogenic growth factor which is activated in lung, liver, and skin cancers, however, its role in the development of PCa is unknown. Therefore, the goal of this study is to characterize the role of HDGF in prostate carcinogenesis. A normal prostate cell line, RWPE-1, and four different PCa cell lines, LNCaP, 22RV1, DU145, and PC-3, were analyzed for their expression level of HDGF by western blot analysis. Results of this evaluation revealed that HDGF is over-expressed in multiple PCa cell lines while minimally expressed in RWPE-1 cells. When the HDGF gene is ectopically over-expressed in normal RWPE-1 cells, proliferative rate was increased by 1.5 fold compared to the normal cells, suggesting that HDGF has growth stimulating activity. On the contrary, down regulation of HDGF expression by siRNA significantly inhibited the survival of both androgen dependent (LNCaP) and androgen independent (PC-3) PCa cells. DNA content analysis of RWPE-1 cells stably expressing HDGF by flow cytometry showed that the majority of the cells were in the G2/M phase (55%) of the cell cycle while only 28% of the control RWPE-1 cells were at G2/M phase implying HDGF may have a mitotic function in prostate cells. Furthermore, AKT (phosphorylated form) and NFκB (p65 domain) signaling molecules were found to be activated in RWPE-1 cells expressing HDGF. This activation resulted in the increase of anti-apoptotic molecules Cyclin-E and BCL-2 and the simultaneous decrease in the pro-apoptotic molecule Bax. Activation of AKT-NFκB pathway plays a vital role in the carcinogenesis of several cancers, including PCa. Thus, HDGF over-expression may play a role in regulating this survival pathway in prostate carcinogenesis. Collectively, the results of this study suggest that HDGF is a key player in the development of PCa and may serve as a target for chemopreventive or chemotherapeutic strategies in PCa. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1109. doi:10.1158/1538-7445.AM2011-1109

Abstract 573: 18alpha- glycyrrhetinic acid (AGA) inhibits advanced prostate cancer cell proliferation and induces apoptosis by downregulating inflammation-related cytokine growth factor genes

Glycyrrhetinic acid is an active triterpenoid metabolite of glycyrrhizin abundantly present in licorice roots. Glycyrrhetinic acid exists as alpha and beta stereo-isomeric forms. Both stereo-isomeric forms are known to have anti-inflammatory and anti-cancer activity. However, the effects and anti-cancer mechanism of alpha glycyrrhetinic acid in prostate cancer cells is not yet evaluated. Therefore in this study, we have investigated the growth inhibition, induction of apoptosis and the anti-cancer mechanisms of 18α-glycyrrhetinic acid (AGA) on androgen independent metastatic prostate cell line DU-145. Our results showed that AGA inhibited proliferation and growth of these cells in a time and dose dependent manner. Induction of apoptosis was confirmed by Annexin V-FITC and propidium iodide staining. Further analysis showed that AGA treatment activated caspase- 8 and −3 death related enzymes in DU-145 cells. Interestingly, AGA treatment also simultaneously down regulated the expression of proinflammatory cytokine/growth factor genes HMGB1, IL-6 and IL-8. This is the first report demonstrating that AGA could play a dual role as an inducer of apoptosis and suppressor of pro-inflammatory gene expression in DU-145 prostate cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 573.