Mengde Cao

Kinase inhibitor Sorafenib modulates immunosuppressive cell populations in a murine liver cancer model.

Accumulating evidence suggests that regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSC) are elevated in cancer patients and tumor-bearing hosts, and that depletion of Tregs and MDSC may enhance the anti-tumor immunity of the host. Sorafenib, a novel multi-kinase inhibitor, is approved for the treatment of several human cancers, including advanced hepatocellular carcinoma (HCC). Sorafenib is believed to inhibit tumor growth via anti-angiogenesis, cell cycle arrest, and inducing apoptosis. However, the impact of Sorafenib on immune cell populations in tumor-bearing hosts is unclear. In this report, we show that Tregs and MDSC are increased in the spleens and bone marrows of the BALB/c mice with liver hepatoma. The increase in Tregs and MDSC was positively correlated with tumor burden. Treatment of Sorafenib not only inhibited HCC cell growth in mice but also significantly decreased the suppressive immune cell populations: Tregs and MDSC. In conclusion, our study strongly suggests that Sorafenib can enhance anti-tumor immunity via modulating immunosuppressive cell populations in the murine liver cancer model.

Hepatocellular carcinoma cell supernatants increase expansion and function of CD4(+)CD25(+) regulatory T cells.

Dysfunction of the host immune system in cancer patients can be due to a number of factors, including suppression of tumor-associated antigen reactive lymphocytes by CD4+CD25+ regulatory T (Treg) cells. Several studies suggest that Tregs are elevated in cancer patients and that depletion of Tregs may enhance the antitumor immunity of host, but the pathogenic and mechanistic relationship between cancer and Tregs is still unclear. In this report, we show that Tregs are increased in peripheral blood mononuclear cells (PBMCs) from hepatocellular carcinoma (HCC) patients and positively correlate with tumor burden. When PBMCs are co-cultured with human hepatoma cell lines Huh7, HepG2, and Hclone5, CD4+CD25+-T cell populations increase in frequency and undergo phenotypic and functional changes. CD45RA, CD45RO, CD69, CD62L, GITR, CTLA-4, Ki67, granzyme A, granzyme B, and FOXP3 expression were upregulated in CD4+CD25+ cells after in vitro exposure to HCC cell lines. CD4+CD25+ T cells from PBMCs that were co-cultured with Huh7 cells also have higher suppressor ability compared to that of the CD4+CD25+ T cells from control PBMC. Huh7 culture supernatants appear to promote CD4+CD25+ T-cell proliferation and inhibit CD4+CD25− T-cell proliferation. In conclusion, these results strongly suggest that tumor-related factors not only induce and expand CD4+CD25+ cells, but also enhance their suppressor ability.

Gamma irradiation alters the phenotype and function of CD4+CD25+ regulatory T cells.

To examine the effects of gamma irradiation on Tregs, changes in phenotype and suppression function in Tregs treated with or without gamma ray were analyzed. Purified CD4+CD25+ regulatory T cells were irradiated at different dosages with a 137Cs source gamma ray at 4.8 Gy/min. After culture, the phenotype and function changes were determined by flow cytometry and [3H]‐thymidine incorporation, respectively. A dose‐dependent reduction of Tregs proliferation in response to gamma irradiation was noted, which paralleled the apoptosis induction of Tregs. Gamma irradiation downregulated the Tregs expression of CD45RO, CD62L, FOXP3, membrane TGF‐β, but upregulated Bax and GITR. High dose gamma irradiation (30 Gy) significantly abolished the suppression of Tregs on CD4+CD25− T cells proliferation. Thus Tregs not only influences the phenotype but also alters their suppressive capacities. Our findings suggest that radiotherapy may be an important strategy to alter the immunologic balance of Tregs and effector cells in cancer therapy.

Influence of serum and soluble CD25 (sCD25) on regulatory and effector T-cell function in hepatocellular carcinoma.

Our previous studies showed that high levels of soluble CD25 (sCD25) in the serum of patients with hepatocellular carcinoma (HCC) correlated with blunted effector T‐cells (Teff) responses, tumour burden and poor survival. Understanding the interactions between Teff, CD4+CD25+ regulatory T cells (Treg) and soluble factors can identify novel therapeutic targets. In this study, we characterize the mechanisms by which HCC serum and sCD25 mediate suppression of Teff and evaluate the effect of sCD25 on the suppression assays with normal healthy control cells (NHC) at a 1:1 Treg to Teff cell ratio to determine whether sCD25 has any impact on Treg suppression. HCC serum and sCD25 suppressed Teff proliferation and downregulated CD25 expression on HCC Teff in a dose‐dependent fashion with sCD25 doses above 3000 pg/ml. Treg from HCC and cirrhosis patients suppressed proliferation of target CD4+CD25− Teff in serum‐free medium (SFM). HCC Treg showed a higher degree of suppression than cirrhosis‐derived Treg. In contrast, Treg from NHC did not suppress target Teff in SFM. However, isolated Treg from all three study subjects (HCC, cirrhosis and NHC) suppressed CD4+CD25− Teff in serum conditions or in the presence of sCD25 in the range 6000–12,000 pg/ml. In conclusion, downregulation of CD25 cell surface expression on Teff is part of the overall suppressive mechanism of sCD25 and HCC serum on Teff responses. The observed sCD25 and HCC serum‐mediated suppression is further influenced via novel immune‐inhibitory interaction between CD4+CD25+ Treg and sCD25.

Hepatocellular Carcinoma Immunopathogenesis: Clinical Evidence for Global T Cell Defects and an Immunomodulatory Role for Soluble CD25 (sCD25)

BackgroundThe mechanisms involved in hepatocellular carcinoma (HCC) establishing an immunologically tolerogenic tumor environment remain poorly characterized.AimsThis study evaluates effector T cell responses and soluble IL-2 receptor alpha chains (sCD25) in relation to HCC stage/survival and characterizes the impact of sCD25 on effectors.MethodsEffector cell responses with serum from HCC patients and in serum free conditions were assessed by IFN-γ ELISpot, proliferation and ATP production assays at baseline, after depletion of sCD25, and after supplementation with recombinant sCD25. Sera sCD25 were measured by ELISA and any relationship with stage/survival was determined.ResultsHepatocellular carcinoma patients had marked global impairment in T cell responses at baseline which correlate with tumor burden and poor outcome. The impairment in immune responses is characterized by low IFN-γ production, cell proliferation, and ATP production. Effector responses are impaired by serum from HCC patients in a dose-dependent manner, implicating soluble factors in the observed immunosuppression. Significant elevations in serum levels of sCD25 are found in patients with HCC, which correlate with tumor burden and a worse survival. T cell reactivity is inversely proportional to serum level of sCD25. Impaired T cell responses improve with sCD25 depletion from HCC serum or IL-2 supplementation suggesting impairment in IL-2 signaling. In contrast, adding increasing doses of sCD25 suppresses effector T cells, which partly involves induction of apoptosis.ConclusionsThese findings show that HCC patients have blunted T cell immunity that is partly related to elevated levels of sCD25, supporting a novel immuno-inhibitory role for this soluble receptor.

Different radiosensitivity of CD4(+)CD25(+) regulatory T cells and effector T cells to low dose gamma irradiation in vitro.

Purpose: To determine the radiosensitivity difference of human Cluster of Differentiation (CD)4+CD25+ regulatory T cells (Treg) and effector T cells to low dose gamma ray and elucidate the underlying mechanisms in vitro. Materials and methods: Blood samples were collected from five health subjects and five patients with advanced hepatocellular carcinoma (HCC). Treg and CD4+CD25− T cells were selected using magnetic microbeads. The proliferative profiles, cytokine secretion, and differential expressions of apoptosis-related proteins in Treg and CD4+CD25− T cells were compared using [3H]-thymidine incorporation, Luminex assay and flow cytometry when treated with various low doses of γ-ray. Results: A dose-dependent reduction of proliferation in response to irradiation which paralleled the induction of apoptosis existed in Treg and CD4+CD25− T cells. Treg were more radiosensitive to low-dose irradiation (0.94 Gray [Gy]) than effector T cells. The interferon-γ (IFNγ) was significantly upregulated and interleukin 10 (IL-10) was significantly downregulated in irradiated Treg. An enhanced immune response to low dose gamma ray existed in the peripheral blood in patients with advanced HCC. Higher levels of active caspase-3, CD95, B cell lymphoma 2 (Bcl-2)-associated X protein (Bax) expression were observed in Treg compared to CD4+CD25− T cells. In addition, gamma irradiation activated CD4+CD25− T cells to express CD25. Conclusions: These studies revealed that Treg were more radiosensitive than CD4+CD25− T cells to low dose irradiation. Higher expressions of apoptosis-related proteins such as caspase-3, CD95 and Bax were observed in Treg when compared to CD4+CD25− T cells. Our results suggest that treatment with low doses of gamma irradiation may be a viable strategy to enhance immune response in patients with advanced HCC.

OPA1 downregulation is involved in sorafenib-induced apoptosis in hepatocellular carcinoma

Sorafenib has been used to treat advanced hepatocellular carcinoma (HCC), but the underlying molecular mechanisms remain controversial and why some patients do not respond to this therapy is poorly understood. In this study, we show that sorafenib triggers cell growth inhibition and apoptosis in HCC cells by directly targeting the mitochondria. Treatment with sorafenib induces rapid mitochondrial fragmentation, which is associated with the deregulation of mitochondria fusion-related protein optic atrophy 1 (OPA1). Exposure of cells or isolated mitochondria to sorafenib substantially induces cytochrome c release. Our data indicate that siRNA-mediated OPA1 knockdown significantly sensitizes HCC cells to sorafenib-induced apoptosis. Furthermore, sorafenib has no apparent apoptotic toxicity to normal human primary hepatocytes. Sorafenib inhibits HCC xenograft tumor growth in vivo and murine xenograft tumor tissue analysis reveals mitochondria fusion protein. OPA1 expression levels are strongly downregulated by sorafenib treatment. Western blotting evaluation of patient HCC with matched non-tumor tissue samples demonstrates that OPA1 expression is decreased in up to 40% of HCC patients. Taken together, we have shown that sorafenib suppresses the tumorigenesis of HCC through the induction of mitochondrial injury via OPA1. Our results provide new insights into the pathogenesis of HCC and suggest that OPA1 is a novel therapeutic target in patients with HCC.

Argininosuccinate synthetase as a plasma biomarker of liver injury after acetaminophen overdose in rodents and humans.

Abstract Context: New biomarkers are needed in acetaminophen (APAP) hepatotoxicity. Plasma argininosuccinate synthetase (ASS) is a promising candidate. Objective: Characterize ASS in APAP hepatotoxicity. Methods: ASS was measured in plasma from rodents and humans with APAP hepatotoxicity. Results: In mice, ASS increased before injury, peaked before alanine aminotransferase (ALT) and decreased rapidly. Fischer rats had a greater increase in ASS relative to ALT. Patients with abnormal liver test results had very high ASS compared to controls. ASS appeared to increase early in some patients, and declined rapidly in all. Conclusions: ASS may be a useful biomarker of acute cell death in APAP hepatotoxicity.

ASS and SULT2A1 are Novel and Sensitive Biomarkers of Acute Hepatic Injury-A Comparative Study in Animal Models

Liver and kidney damage associated with polytrauma, endotoxic shock/sepsis, and organ transplantation, are among the leading causes of the multiple organ failure. Development of novel sensitive biomarkers that detect early stages of liver and kidney injury is vital for the effective diagnostics and treatment of these life-threatening conditions. Previously, we identified several hepatic proteins, including Argininosuccinate Synthase (ASS) and sulfotransferases which were degraded in the liver and rapidly released into circulation during Ischemia/Reperfusion (I/R) injury. Here we compared sensitivity and specificity of the newly developed sandwich ELISA assays for ASS and the sulfotransferase isoform SULT2A1 with the standard clinical liver and kidney tests Alanine Aminotransferase (ALT) and Aspartate Transaminase (AST) in various pre-clinical models of acute injury. Our data suggest that ASS and SULT2A1 have superior characteristics for liver and kidney health assessment in endotoxemia, Ischemia/Reperfusion (I/R), chemical and drug-induced liver injury and may be of high potential value for clinical applications.

Reactive oxygen species is essential for cycloheximide to sensitize lexatumumab-induced apoptosis in hepatocellular carcinoma cells.

This study aims to investigate apoptosis induced by lexatumumab (Lexa) in hepatocellular carcinoma (HCC) cells. We assessed the sensitivity of HCC cell lines and normal human hepatocytes to Lexa and explored the sensitization of HCC cells to Lexa-induced apoptosis by cycloheximide (CHX). Our data indicated that CHX sensitized HCC cell lines to Lexa-induced apoptosis, whereas treatment using solely CHX or Lexa was ineffective. The sequential treatment of CHX followed by Lexa dramatically induced caspase-dependent apoptosis in HCC cells and had synergistically increased intracellular rates of reactive oxygen species (ROS). Additionally, when ROS production was blocked by N-acetyl-L-cysteine (NAC), HCC cells were protected against Lexa and CHX combination treatment-induced apoptosis. ROS generation induced by combination treatment of Lexa and CHX triggered pro-apoptotic protein Bax oligomerization, conformation change, and translocation to mitochondria, which resulted in the release of cytochrome c and subsequent cell death. Furthermore, HSP90 was involved in mediating Lexa and CHX combination treatment-induced ROS increase and apoptotic death. More importantly, we observed that combination treatment of Lexa and CHX did not cause apoptotic toxicity in normal human primary hepatocytes. These results suggest that Lexa and CHX combination treatment merits investigation for the development of therapies for patients with HCC.