Novel modulation on myeloid-derived suppressor cells (MDSCs) by methionine encephalin (MENK)

The purpose of this study was to identify the modulatory effect of MENK on MDSCs and to investigate the relationship between this modulation and the expression of opioid receptors. Our results showed that MENK could inhibit the proliferation of MDSCs from both marine bone marrow and spleen. MENK also could promote the expression of opioid receptors MOR and DOR. MENK suppressed the PMN-MDSCs generated from splenocyte while up-regulated M-MDSCs. The stimulation of MENK increased the production of IL-4 secreted by MDSCs from slpenocytes. Our currently data indicated that MENK could suppress the accumulation of MDSCs in tumor-bearing mice via binding to and up-regulating expressions of subunits of opioid receptors. Therefore, it is concluded that MENK, through triggering opioid receptors could exert inhibiting modulation on MDSCs.     

Inhibition of curcumin on myeloid-derived suppressor cells is requisite for controlling lung cancer

Lung cancer remains the leading cause of cancer mortality. Myeloid-derived suppressor cells (MDSCs) are potent immune-suppressive cells and present in most cancer patients. Recently, several studies have shown that curcumin inhibits the expansion of MDSCs in some cancers. However, it is not clear how curcumin modulates the suppressive function of MDSCs, and whether curcumin achieves anti-tumor effects via regulating the expansion of MDSCs in lung cancer. Here, our results showed that curcumin significantly inhibited tumor growth in a Lewis lung carcinoma (LLC) isogenic tumor model. Curcumin reduced the accumulation of MDSCs in spleen and tumor tissue in LLC isogenic model. And curcumin promoted the maturation and differentiation of MDSCs in tumor tissue. Notably, curcumin inhibited the expression level of immune suppressive factors of MDSCs, arginase-1 (Arg-1) and ROS, in purified MDSCs from tumor tissue in vivo. Expectedly, curcumin also inhibited the immunosuppressive function of isolated MDSCs from tumor tissue and spleen of tumor bearing mice in vitro. Moreover, curcumin decreased the level of IL-6 in the tumor tissue and serum from LLC-bearing mice. Taken together, curcumin indeed possesses anti-cancer effect and inhibits the accumulation and function of MDSCs. And curcumin reduces the level of IL-6 in tumor-bearing mice to impair the expansion and function of MDSCs. These results suggest that inhibition of MDSCs in tumor is requisite for controlling lung cancer.     

小鼠结肠癌肝转移模型的构建

目的建立适合基因表达谱研究的小鼠结肠癌肝转移模型。方法将小鼠结肠癌细胞(C26)悬液接种于BALB/C小鼠脾内,分为切脾组和保脾组,观察小鼠生存时间及腹腔内肿瘤生长情况。结果切脾小鼠自然生存期11～15d,平均(13.05±0.71)d,保脾小鼠自然生存期9～15d,平均(11.64±1.49)d。小鼠肝脏转移率均为100%。切脾小鼠肝各叶完全被肿瘤占据,保脾小鼠脾内肿瘤巨大,肝内转移多为散在的米粒大小瘤结节转移。结论用于基因表达谱研究的小鼠结肠癌肝转移模型应采用脾内注射切脾法。     

CD30L/CD30 signaling regulates the formation of the tumor immune microenvironment and inhibits intestinal tumor development of colitis-associated colon cancer in mice

Inflammatory bowel disease is one of the major causes of colitis-associated colon cancer (CAC). Therefore, it is necessary to explore new therapies to prevent colon cancer (CRC) in view of the relationship between chronic inflammation and tumor development. Previous studies on the correlation between CD30L/CD30 and cancer were mostly limited to lymphoid or homogenous tumors, while there have been only a few reports on the role of CD30L/CD30 signal transduction in the pathogenesis of CAC. In this study, we established an AOM/DSS-induced CAC model with CD30LKO mice to explore the effect of CD30L/CD30 signal transduction on the formation of the intestinal tumor immune microenvironment (TIME) during the development of intestinal tumors. Our results revealed that CD30L deficiency promoted the accumulation of myeloid derived suppressor cells (MDSCs), increased the expression of PD-L1 on MDSCs and tumor associated macrophages (TAMs), and enhanced the secretion of various inflammatory and immunosuppressive factors in the intestinal mucosa of CAC mice. Furthermore, CD30L gene deletion could selectively promote the upregulation of PD-1 expression on CD4⁺ and CD8⁺ T cells and inhibit their activation, differentiation and secretion of effector cytokines, which led to an attenuation of antitumor immune responses mediated by T_EM (CD44⁺CD62L^-) cells. Thus, our data suggest that CD30L/CD30 signaling might be a potential candidate target for immunological therapy in CAC.     

Methionine enkephalin (MENK) mounts antitumor effect via regulating dendritic cells (DCs)

MENK, an endogenous opioid peptide has been reported to have many immunological and antitumor activities. So far the detailed mechanisms of antitumor through regulating DCs by MENK have not been elucidated yet. The aim of this work was to investigate the antitumor mechanisms of MENK via regulating DC. The monitoring methods, such as ELISA, MTS assay, CFSE, Real-time PCR and Western blot were included in our research. We found bone marrow derived dendritic cells (BMDCs) in 36 female C57BL/6 mice treated with MENK enhanced expression of key surface molecules, increased production of critical cytokines reduced endocytosis of FITC-dextran, upregulated TLR4 through MyD88/NF-κB signaling pathway and mounted higher antitumor activity. These observations were further supported by an enhancement of nuclear translocation of the p65NF-κB subunit involved in this process. Surprisingly, mu-opioid receptors were the main participants of this kind of activation, not delta-opioid receptors nor kappa-opioid receptors, and these interactions could be partly blocked by Naltrexone (a kind of opioid antagonist). In vivo study the activated CD4⁺, CD8⁺ T cells and decreased ability to induce differentiation of Foxp3⁺ regulatory T cells were detected post treatment of MENK. Thus, it is concluded that MENK could exert antitumor effect through precisely regulating opioid receptor mediated functions of DCs. In addition, MENK treated DCs may serve as a new immunotherapy approach against tumor.     

IFN-γ producing T cells contribute to the increase of myeloid derived suppressor cells in tumor-bearing mice after cyclophosphamide treatment

It has been reported that treatment with cyclophosphamide (CTX) as a chemotherapeutic drug in cancer patients or tumor-bearing mice can result in an increase in the proportion of myeloid derived suppressor cells (MDSCs) in blood and lymphoid organs. Here we sought to clarify the possible mechanism of this unwanted increase in proportion of MDSCs in tumor-bearing mice after CTX treatment. We found that both CD4(+) T cells and CD8(+) T cells underwent an expansion and activation before the increase of MDSCs in the early period of CTX treatment in 4T1 breast tumor-bearing mice. The proportion of MDSCs in nude mice lacking T cells after CTX therapy was comparable to that in nude mice without CTX treatment. T cell transfer to 4T1-bearing nude mice enhanced the proportion of MDSCs in tumor-bearing mice after CTX therapy. The co-culture of MDSCs and T cells in vitro also showed that CD4(+) T cells and CD8(+) T cells could facilitate the expansion and survival of MDSCs, and this effect was mediated by IFN-γ released by T cells. These results gave an explanation of the unwanted consequence resulted from CTX treatment in tumor-bearing mice. It also provided some insights into the strategies for eliminating the bad side of CTX treatment and to make it more effective in cancer therapy.     

新疆紫草提取物对小鼠急性酒精性肝损伤的保护作用

目的研究新疆紫草提取物(AE-Ⅰ、AE-Ⅱ)对小鼠急性酒精性肝损伤的保护作用。方法灌胃35度二锅头白酒16 ml/kg造酒精性肝损伤模型,观察肝脏、脾脏系数,血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、胆固醇(CHO)、甘油三酯(TG)、肝组织超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、丙二醛(MDA)、一氧化氮(NO)的水平和肝脏病理组织学变化。结果AE-Ⅰ、Ⅱ各剂量组均可降低小鼠脾脏系数和血清ALT、TG活性,升高肝组织SOD、GSH-Px活性,并降低肝组织MDA和NO含量(P<0.05);而仅有AE-Ⅰ(0.8 g/kg)可以降低肝脏系数(P<0.05);AE-Ⅰ(0.8 g/kg)和AE-Ⅱ(2,4 g/kg)可降低小鼠血清AST活性(P<0.05);AE-Ⅰ(0.8 g/kg)和AE-Ⅱ(4 g/kg)可降低小鼠血清CHO活性(P<0.05);AE-Ⅰ、Ⅱ均可不同程度地改善肝脏病理组织性损伤。结论AE-Ⅰ、AE-Ⅱ对小鼠急性酒精性肝损伤具有保护作用。     

MDSC as a mechanism of tumor escape from sunitinib mediated anti-angiogenic therapy

Sunitinib is a receptor tyrosine kinase inhibitor (TKI) that is front-line therapy for metastatic renal cell carcinoma (mRCC). Its antitumor activity is related to its ability to block tumor cell and tumor vasculature cell signaling via several TKI receptors (i.e. vascular endothelial growth factor receptors VEGFRs, platelet-derived growth factors (PDGFs), and stem cell factors). Sunitinib also targets myeloid derived suppressor cells (MDSCs) significantly reducing their accumulation in the peripheral blood and reversing T cell (IFNγ) suppression in both mRCC patients and in murine tumor models. This reduction in immune suppression provides a rationale for combining sunitinib with immunotherapy for the treatment of certain tumor types. Despite these encouraging findings, however, we have observed that sunitinib has variable impact at reducing MDSCs and restoring T cell function within the tumor microenvironment. Given the immunosuppressive and proangiogenic activities of MDSC, it seems plausible that their persistence may contribute to the resistance that develops in sunitinib-treated patients. While sunitinib reduced tumor infiltrating MDSCs in Renca and CT26-bearing mice, coinciding with strong to modest decreases in tumor size respectively, it was ineffective at reducing MDSCs (<35% reduction in Gr1+CD11b+) or tumor burden in 4T1-bearing mice. Persistence of intratumor MDSCs was paralleled by depressed intratumor T cell IFNγ response and increased GM-CSF expression. Additionally, in vitro and in vivo experiments showed that GM-CSF prolongs survival of MDSCs, thus protecting them from the effects of sunitinib via a pSTAT5-dependent pathway. Although preliminary, there is evidence of intratumor MDSC resistance in some mRCC patients following sunitinib treatment. Intratumor MDSC persistence and T cell IFNγ response post nephrectomy in patients receiving sunitinib in a neoadjuvant setting are being compared to RCC patients undergoing nephrectomy without prior sunitinib treatment. Tumors from untreated patients showed suppressed T cell IFNγ response along with substantial expression of MDSCs (5% of total digested cells). Thus far, tumors from 5/8 neoadjuvant patients showed persistence of intratumor MDSCs and low T cell IFNγ production post sunitinib treatment, findings that parallel results from untreated tumors. In the remaining 3 neoadjuvant patients, intratumor MDSCs were detected at low levels which coincided with a T cell IFNγ response similar to that observed with normal donor peripheral T cells. GM-CSF's role in promoting MDSC survival in patient tumors is supported by the observation that GM-CSF is produced in short-term RCC cultures at levels capable of protecting MDSCs from sunitinib-induced cell death. Additionally, persistence of MDSC also may be associated with increased expression of proangiogenic proteins, such as MMP9, MMP8, and IL-8 produced by tumor stromal cells or infiltrating MDSCs. Indeed our findings suggest that the most dominate MDSC subset in RCC patients is the neutrophilic population that produces proangiogenic proteins. We propose that the development of sunitinib resistance is partly mediated by the survival of MDSCs intratumorally, thereby providing sustained immune suppression and angiogenesis.     

l-arginine and docetaxel synergistically enhance anti-tumor immunity by modifying the immune status of tumor-bearing mice

l-arginine (l-Arg) supplementation has been reported to enhance the function of immune cells, including dendritic cells (DCs) and T lymphocytes, in cancer models thereby countering the suppressive effects of myeloid-derived suppressor cells (MDSCs). The balance of the active immune cells is one factor that determines the progression of cancers in vivo. Docetaxel (DTX), an immunomodulatory chemotherapeutic agent, is now widely used in several types of malignancies including breast cancer. We hypothesized that the combination of DTX and l-Arg would elicit a more robust antitumor response than either molecule alone. To test this hypothesis we utilized BALB/c mice inoculated with 4T1 mammary carcinoma cells. DTX and l-Arg synergistically limited tumor growth in vivo and moderately increased the life span of tumor bearing mice. The anti-tumor effects were associated with the proliferation of splenic CD8⁺ CTL and CD4⁺ Th1 effector cells, as well as increased serum levels of interferon gamma. More importantly, DTX + l-Arg effectively increased anti-tumor immunity within the tumor microenvironment. Furthermore, the combined therapy increased the number of myeloid (mDCs) and plasmacytoid (pDCs) dendritic cells, potent activators of the T cell response, and enhanced expression of the maturation markers CD86 and MHC II (required for antigen presentation). The combination therapy also reduced the proliferation of MDSCs. These data suggest that DTX + l-Arg may be a novel therapeutic strategy for breast cancer patients.     

结肠癌术后辅助免疫疗法抑制肿瘤腹腔转移的免疫机制

目的探讨辅助免疫疗法促进小鼠腹腔巨噬细胞TRAIL表达,增强肿瘤细胞对化疗药物的敏感性,抑制肿瘤腹腔转移的免疫机制。方法用氟脲嘧啶(5-FU)治疗小鼠结肠癌转移模型,辅助免疫治疗,观察各组小鼠腹腔肿瘤生长情况,ELISA方法检测脾细胞培养物上清中分泌IFN-γ的水平,FSCA分析各组小鼠脾细胞内T淋巴细胞和NK细胞比例和腹腔巨噬细胞表面TRAIL的表达。结果化疗联合免疫治疗组小鼠腹腔未见肿瘤细胞生长,脾细胞内CD3+、CD4+、CD8+的T淋巴细胞和NK细胞比例明显增高,脾细胞培养物上清中分泌高水平IFN-γ,巨噬细胞表面诱导肿瘤细胞凋亡的TRAIL分子表达明显增强。结论结肠癌术后辅助性免疫治疗,能活化特异性CTL分泌IFN-γ,改善腹腔抗原提呈微环境,诱导肿瘤细胞凋亡,对化疗药物的敏感性显著增强,是有效抑制小鼠结肠癌腹腔转移的重要免疫机制。     

牛膝多糖对链佐星诱导性糖尿病小鼠的保护作用(英文)

目的研究牛膝多糖(ABP)对链佐星诱导性糖尿病小鼠的保护作用。方法将雄性ICR小鼠分为正常对照组、糖尿病模型组、ABP50和100mg.kg-1(ip,每天1次,共15d)治疗组。分别于给药前,给药8和15d时用血糖测量仪检测血糖水平,并进行血糖耐受试验。末次给药第2天测量小鼠体重,处死,测量心、肝、脾和肾脏器官重量,同时制备血清,采用放射免疫分析试剂盒检测血清胰岛素水平,比色法测定血清甘油三酯(TG)、总胆固醇(TC)、钙(Ca)和磷(P)含量及谷草转氨酶(GOT)、谷丙转氨酶(GPT)和碱性磷酸酶(ALP)活性。采用ELISA检测血清瘦素、脂联素、肿瘤坏死因子α(TNF-α)和白细胞介素6(IL-6)的浓度。结果与正常对照组相比,糖尿病模型组小鼠体重下降,血清胰岛素水平降低,血糖升高(P<0.05,P<0.01)。ABP50和100mg.kg-1在血糖耐受试验中能较好地调节血糖水平,其血糖在15d时分别比模型组下降了27.4%和16.3%(P<0.05,P<0.01)。ABP50mg.kg-1治疗组小鼠脾和肾脏指数,血糖、血清TG和TC浓度,GOT,GPT和ALP活性均显著低于糖尿病模型组(P<0.05),血清瘦素水平下降并接近正常水平,血清脂联素、TNF-α和IL-6水平均显著高于模型组(P<0.05,P<0.01);ABP100mg.kg-1对血清TC和TG水平及ALP和GPT活性无明显作用,对上述其他指标的作用与ABP50mg.kg-1相似。各组血清胰岛素浓度及Ca和P含量均无明显变化。结论ABP对链佐星诱导的糖尿病小鼠具有一定的保护作用。     

Preparation and Evaluation of 99mTc‐labeled anti‐CD11b Antibody Targeting Inflammatory Microenvironment for Colon Cancer Imaging

CD11b, an active constituent of innate immune response highly expressed in myeloid‐derived suppressor cells (MDSCs), can be used as a marker of inflammatory microenvironment, particularly in tumor tissues. In this research, we aimed to fabricate a ^99mTc‐labeled anti‐CD11b antibody as a probe for CD11b⁺ myeloid cells in colon cancer imaging with single‐photon emission computed tomography (SPECT). In situ murine colon tumor model was established in histidine decarboxylase knockout (Hdc^?/?) mice by chemicals induction. ^99mTc‐labeled anti‐CD11b was obtained with labeling yields of over 30% and radiochemical purity of over 95%. Micro‐SPECT/CT scans were performed at 6 h post injection to investigate biodistributions and targeting of the probe. In situ colonic neoplasma as small as 3 mm diameters was clearly identified by imaging; after dissection of the animal, anti‐CD11b immunofluorescence staining was performed to identify infiltration of CD11b+ MDSCs in microenvironment of colonic neoplasms. In addition, the images displayed intense signal from bone marrow and spleen, which indicated the origin and migration of CD11b⁺ MDSCs in vivo, and these results were further proved by flow cytometry analysis. Therefore, ^99mTc‐labeled anti‐CD11b SPECT displayed the potential to facilitate the diagnosis of colon tumor in very early stage via detection of inflammatory microenvironment.     

Differential effects of low-dose fludarabine or 5-fluorouracil on the tumor growth and myeloid derived immunosuppression status of tumor-bearing mice

Myeloid-derived suppressor cells (MDSCs) accumulate in tumor-bearing hosts and play a key role in the suppression of the innate and adaptive immunity. Chemotherapeutic strategies have been developed to deplete or deactivate MDSCs in different tumor models. The pyrimidine analog, 5-fluorouracil (5-FU) is found to reduce the tumor size by depleting MDSCs. Here, we asked whether the purine analog, fludarabine (Flu), could exert similar effects. Employing a lymphoma model, we demonstrated that in mice with advanced tumors (where MDSC-induced suppression was present), treatment with a single low-dose Flu (25, 50, 100 mg/kg) elevated the numbers of splenic MDSCs and serum arginase activity, and simultaneously, increased the tumor growth (only the highest dose). On the other hand, in mice with palpable tumors (where the MDSC-induced suppression was in progress), treatment with Flu had no significant effects on the tumor growth or the number of splenic MDSCs. In contrast to Flu, treatment with low-dose 5-FU, irrespective of tumor stage, caused tumor regression which coincided with significant reductions in the numbers of splenic MDSCs and blood neutrophils, but increases in the ratios of splenic CD4⁺ T and CD8⁺ T cells to suppressive MDSCs. Finally, in healthy mice (where MDSC-induced immuosuppression did not exist), 5-FU, but not Flu induced significant decreases in the number of myeloid cells in the bone marrow, naturally occurring splenic MDSCs and thymocytes. In conclusion, Flu exacerbates MDSC-induced immunosuppression in a tumor stage-dependent manner, whereas 5-FU alleviates the suppressive effects of MDSC at all stages of tumor development.     

PID1,a new tumor-promoting gene in insulin resistance mediated acceleration of hepatocellular carcinoma development and progression

OBJECTIVE To investigate the effect of phosphotyrosine interaction domain containing 1(PID1,NYGGF4)on promotion of IR and HCC,and explore its underlying mechanisms.METHODS Lentivirus were used to mediate the knockdown of PID1 in HFD induced IR mouse model as well as ob/ob mice.Intraperitoneal glucose and insulin tolerance were performed 4 weeks after lentivirus injection.Hydrodynamics-based transfection was applied to inducethe liver specific overexpression of PID1.Flow cytometry was exerted to detect the proportion and function of immune cells.qR T-PCR and Western blot were used to detect the expression of downstream pathways of PID1.Immunoprecipitation was used to determine the receptor of PID1.Chromatin immunoprecipitation(ChI P)was operated to measure the modification of H3K4me3 of PID1 promoter.RESULTS PID1 restriction improved insulin resistance,hyperglycemia and fatty liver.Conversely,hepatic knockdown of PID1 attenuated liver xenografted tumor growth.Moreover,PID1 liver-specific protooncogenes via hydrodynamics-based transfection established a primary hepatocellular carcinoma mouse model,induced an immunosuppressive environment,with the reduction of CD3~+,CD4~+,CD8~+T cel s,retarded maturation of dendritic cel s(DCs),pronounced differentiation of regulatory T cells(Tregs),and recruitment of MDSC.In addition,PID1 overexpression activated proliferation related genes,promoted anti-inflammatory genes,suppressed pro-inflammatory genes,induced glycolysis and lipid metabolism genes to facilitate tumorigenesis in liver.Importantly,PID1 exerted its tumor-promoting function through binding to epidermal growth factor receptor(EGFR)and activation of downstream MAPK pathway.As such,PID1 exist trimethylation of histone H3 at lysine 4(H3K4me3)modification and IR up-regulated the expression of PID1 by activation the H3K4me3 modification.CONCLUSION PID1 is a new gene that exerts both liver cancer-promoting and insulin resistance inducing function.IR accelerates liver cancer development and progressionpartially dependent on the activation of PID1.     

Methionine enkephalin,its role in immunoregulation and cancer therapy

Methionine enkephalin (MENK), an endogenous neuropeptide has a crucial role in both neuroendocrine and immune systems. MENK is believed to have an immunoregulatory activity to have cancer biotherapy activity by binding to the opioid receptors on immune and cancer cells. Clinical trial studies in cancer patients have shown that MENK activates immune cells directly and by inhibiting regulatory T-cells (Tregs). MENK may also change the tumor microenvironment by binding to opioid receptor on or in cancer cells. All of these mechanisms of action have biologic significance and potential for use in cancer immunotherapy. Furthermore, they reveal a relationship between the endocrine and immune systems. Due to the apparent role of MENK in cancer therapy we reviewed herein, the research undertaken with MENK in recent years; which has advanced our understanding of the role MENK has in cancer progression and its relationship to immunity, supporting MENK as a new strategy for cancer immunotherapy.     

金银花总黄酮对免疫性肝损伤小鼠的影响

目的观察金银花总黄酮（LJTF）对卡介苗（BCG）联合脂多糖（LPS）所致小鼠免疫性肝损伤炎症因子的影响。方法采用BCG2．5mg静脉注射致敏小鼠,d10给予LPS7．5μg静脉攻击造成小鼠免疫性肝损伤模型,ig给予IJTF（100、200、400mg·kg。）连续10d。观察肝组织形态学改变,称量体重,肝脏、脾脏重量,计算肝、脾脏器指数,分光光度法测定肝匀浆中NO、iNOS的含量,免疫组化测定肝脏TNF—d的表达。结果LJTFig能提高免疫性肝损伤小鼠肝、脾脏器指数,改善免疫性肝损伤小鼠肝脏组织学改变,降低肝匀浆中NO、iNOS的水平,LJTF也能降低免疫性肝损伤小鼠TNF-α在肝脏中的强烈表达。结论LJTFig可以减少炎症介质的释放,对免疫性肝损伤小鼠有保护作用。     

槐属植物生物碱衍生物施普睿达抗肿瘤作用的研究

目的:对槐属植物生物碱衍生物施普睿达体内抗肿瘤作用进行研究,并通过肝、脾、胸腺指数等多项指标综合考查施普睿达对荷瘤动物免疫机能的影响。方法:将昆明种小鼠移植宫颈癌U14、肉瘤S180和肝癌H22细胞后,腹腔注射施普睿达,观察施普睿达对实体瘤的抑瘤率。结果:施普睿达对3种移植瘤均有明显的抑制作用,最佳抑瘤率分别为U1474.3%、S18073.9%和H2279.3%,对小鼠的肝、脾指数和胸腺指数几乎无影响;而阳性对照药顺铂使小鼠的肝重、脾指数和胸腺指数明显下降。结论:施普睿达具有抑制S180、U14及H22等多种小鼠移植性肿瘤生长的作用,而且不影响荷瘤动物体重及免疫功能,属于低毒的抗肿瘤新药。