Fas ligand-expressing tumors induce tumor-specific protective immunity in the inoculated hosts but vaccination with the apoptotic tumors suppresses antitumor immunity

The interaction between Fas and Fas ligand (FasL) is involved in the apoptotic death of a number of cells including lymphocytes. Forced expression of FasL in tumors can induce apoptosis of infiltrating Fas-positive T cells; accordingly, tumors can survive in the milieu of systemic immune responses. However, FasL-expressing murine lung carcinoma (A11) and melanoma (B16) cells did not develop subcutaneous tumors and FasL-expressing A11 (A11/FasL) cells produced few spontaneous lung metastatic foci in syngeneic mice. The mice that rejected A11/FasL cells were resistant to subsequent challenge of parent A11 but not irrelevant B16 cells. Vaccination of mice with UV-treated A11/FasL, but not UV-treated A11 cells, however, augmented the growth rate of A11 but not B16 tumors, both of which were subsequently inoculated. The number of lung metastatic foci of A11 cells was also increased in the mice that received UV-treated A11/FasL but not UV-treated A11 cells. Intraperitoneal injection of UV-treated A11/FasL cells resulted in the production of larger amounts of immunosuppressive TGF-beta in peritoneal exudate than that of UV-treated A11 cells. Expression of the CD80 costimulatory molecule in tissues where UV-treated A11/FasL cells were inoculated was lower than the expression at an untreated A11/FasL-injected site. Our results indicated that apoptotic FasL-expressing tumor cells could impair host immune responses against the tumors, in contrast to potent antitumor immunity generated by viable FasL-expressing tumors.     

Fas-related apoptosis in gastric adenocarcinoma

The aims of this study were to determine whether human gastric adenocarcinomas express FasL or Fas, whether FasL expression is associated with increased apoptotic induction, especially, tumor-infiltrating lymphocyte (TIL) and whether apoptotic induction is associated with the tumor stage and histologic type. Early gastric carcinoma (EGC) (n=38) and advanced gastric carcinoma (AGC) (n=61) cases were analyzed in patients who received gastric resection from 1997 to 1998. There were 38 diffuse type and 61 intestinal type cases. Immunohistochemical staining for Fas, FasL and CD45, TACS trade mark in situ apoptosis detection kit, and double staining with the observation under a confocal scanning laser microscope were employed. FasL was localized to neoplastic cells in 23 cases (61%) of EGC group and 40 cases (66%) of AGC group. The extent of FasL expression was variable, with both FasL-positive and -negative neoplastic regions occurring within tumors. TILs were detected by CD45, and apoptosis of TILs as detected by co-expression of CD45 and TACS on serial histologic sections. TILs adjacent to FasL expressing tumor regions were decreased in number and TILs adjacent to FasL-negative tumor regions were increased in number; apoptotic induction of TIL showed the opposite pattern (p<0.05). Fas was expressed homogeneously throughout the tumor masses independent of the tumor stage. Fas expression in 39 cases (64%) was intestinal type and in 26 cases (68%), diffuse type. Labeling indices for tumoral apoptosis in EGC and AGC were 6.72% and 7.13%, respectively. Co-expression of FasL and Fas, which occurrs over large areas of the tumors, did not result in an enhanced rate of tumor cell apoptosis. In addition, tumor stage and other prognostic factors were not associated with Fas and FasL expressions, number of TIL and apoptotic induction. A statistically significant reduction of TILs concomitant with significantly increased TIL apoptosis adjacent to FasL-expressing regions of gastric adenocarcinomas were demonstrated. This finding suggests Fas-mediated apoptotic depletion of TIL in response to FasL expression in stomach cancers, and provides evidence to support the Fas counterattack theory as a mechanism of immune escape in gastric cancer. Furthermore, gastric carcinoma cells of the intestinal and the diffuse type did not differ in their expression of the Fas-apoptotic system.     

Fas ligand expression is correlated with metastasis in colorectal carcinoma

The Fas/Fas ligand (FasL) system is one apoptotic pathway through which malignant tumors can evade immune surveillance. While FasL is expressed in malignant tumors, Fas is conversely downregulated to escape host immune attack, resulting in tumor invasion. The aim of the current study was to find out further clinicopathological significance of FasL expression in carcinoma of the colon and rectum. FasL expression was investigated using immunohistochemical staining in 143 consecutive patients with primary colorectal carcinomas. Seventy-nine carcinomas (55.2%) expressed FasL. The incidence of lymph node and distant metastases in carcinomas expressing FasL was significantly higher than in carcinomas that did not express FasL (p = 0.031 and p = 0.0003, respectively). Although univariate analysis showed that survival in patients with carcinomas expressing FasL was significantly poorer than that in patients with carcinomas without FasL expression (p = 0.001), only Dukes' stage was an independent prognosticator by multivariate analysis. FasL expression was found to be correlated with lymph node involvement and distant metastases in colorectal carcinoma.     

Angiogenesis and growth of murine colon carcinoma are dependent on infiltrating leukocytes

We determined whether the angiogenesis and growth of murine colon carcinomas growing in the wall of the cecum is dependent on infiltrating leukocytes. Syngeneic BALB/c or SCID mice were treated with a myelosuppressive, maximally tolerated dose of doxorubicin. Parental or multidrug resistant CT-26 colon carcinoma cells were implanted into the cecal wall 3 days after the second intravenous injection of doxorubicin. Control mice developed large, well-vascularized tumors, whereas doxorubicin-pretreated mice did not. Intravenous injection of spleen cells from normal BALB/c or SCID mice one day prior to tumor cell implantation reversed the decreased vascularity and tumorigenicity. The production of proangiogenic molecules and microvessel density in tumors directly correlated with the number of infiltrating leukocytes, suggesting that tumor-infiltrating leukocytes are essential to angiogenesis of murine colon carcinomas.     

Fas/FasL 在肺癌中的表达与淋巴结转移的关系

 目的　探讨 Fas/Fas L在肺癌中的表达及其与淋巴结转移的关系。方法　用免疫组化方法同时检测 42例非小细胞肺癌组织及肺癌转移淋巴结的 Fas/Fas L表达。结果　肺癌的 Fas表达率为 52 .3% (2 2 /4 2 ) ,其中鳞癌为 58.3% (1 4/2 4 ) ,腺癌为 44.4% (8/1 8) ,二者无显著差异 (P>0 .0 5)。肺癌的 Fas表达与有否淋巴结转移无明显相关 (P>0 .0 5)。肺癌的 Fas L表达为 57.1 % (2 4 /4 2 ) ,其中鳞癌 58.3% (1 4/2 4 )与腺癌 55.5% (1 0 /1 8)之间无显著差别 (P>0 .0 5)。肺癌伴淋巴结转移的 Fas L表达为 72 .7% (1 6/2 2 ) ,无淋巴结转移的 Fas L表达为 40 % (8/2 0 ) ,有显著差别 (P<0 .0 5)。1 6例 Fas L阳性肺癌的转移淋巴结均呈表达 Fas L。结论　肺癌表达 Fas L,并可能在肺癌转移机制中起免疫逃避作用。     

Antiangiogenic therapy with mammalian target of rapamycin inhibitor RAD001 (Everolimus) increases radiosensitivity in solid cancer.

PURPOSE: Radiotherapy exerts direct antivascular effects in tumors and also induces a proangiogenic stress response in tumor cells via the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (mTOR) pathway. Therefore, the combination of radiotherapy and antiangiogenic therapy with mTOR inhibitor RAD001 (Everolimus) might exert additive/synergistic effects on tumor growth. EXPERIMENTAL DESIGN: Effects of radiation combined with mTOR inhibitor RAD001 were studied on proliferation of murine colon cancer CT-26, human pancreatic cancer L3.6pl, and human umbilical vascular endothelial cells in vitro. In vivo tumor growth of subcutaneous colon cancer CT 26 and orthotopic pancreatic cancer L3.6pl was assessed after fractionated radiotherapy (5 x 2 or 5 x 4 Gy) with or without the addition of the mTOR inhibitor RAD001. RAD001 (1.5 mg/kg/d) was administered until the end of experiments beginning before or after radiotherapy. RESULTS: A single dose of 2 Gy reduced in vitro proliferation of L3.6pl (-16%), CT-26 (-70%), and human umbilical vascular endothelial cells (HUVEC; -72%). The mTOR inhibitor RAD001 (10 ng/mL) suppressed proliferation of HUVEC (-83%), L3.6pl (-8%), and CT-26 (-82%). Combination of even low concentrations of 0.01 ng/mL RAD001 and 0.25 Gy radiation significantly reduced proliferation of HUVECs (-57%), whereas additive effects of RAD001 and radiation on tumor cells were seen only at the highest concentrations tested. In vivo, RAD001 introduced before radiotherapy (5 x 2 Gy) improved tumor growth control in mice (L3.6pl: 326 mm(3) versus 1144 mm(3); CT-26: 210 mm(3) versus 636 mm(3); P < 0.05 versus control). RAD001 turned out to possess a dose-modifying effect on radiotherapy. CONCLUSION: Endothelial cells seem to be most sensitive to combination of mTOR inhibition and radiotherapy. Additive tumor growth delay using the mTOR inhibitor RAD001 and radiotherapy in vivo therefore might rely on combined antiangiogenic and antivascular effects.     

FasL EXPRESSION IN HUMAN COLON CARCINOMAS

Fas and FasL are the members of the tumor necrosis factor (TNF) receptor and TNF families, respectively[1, 2]. Upon oligomerization of Fas either with FasL[3] or with agonistic antibodies[4], it confers an apoptotic signal to Fas-sensitive cells to induce apoptosis of the target cells[5]. The Fas/FasL system plays an important role for tumor cells to escape the host's immune surveillance during tumor formation. On one hand, tumor cells down-regulate expression of Fas on the tumor cell…     

醋酸甲羟孕酮对人结肠癌LS174T细胞体内生长抑制的作用

目的检测醋酸甲羟孕酮(MPA)对人结肠癌细胞LS174T移植瘤生长的抑制作用并初步分析其机制。方法建立人结肠癌裸鼠皮下移植瘤模型。随机分为3组,每组10只,对照组:静脉注射或以同等容量的生理盐水灌胃;处理组分为MPA治疗组和MPA+5-Fu治疗组,MPA治疗组:每只每次灌胃MPA30mg/kg;MPA+5-Fu治疗组:每只每次静脉注射5-Fu30mg/kg;每只每次MPA30mg/kg灌胃,MPA灌胃每周5次,5-Fu静脉注射每周3次,共用3周。5周后处死动物,切除瘤灶、称瘤重、测瘤体积、计算抑瘤率;标本用流式细胞仪分析移植瘤细胞周期、凋亡率和细胞表面Fas/FasL表达;免疫组织化学SABC法检测移植瘤组织Fas/Fas-L表达。结果对照组、MPA治疗组、MPA+5-Fu合用组肿瘤体积分别为(1.65±0.68)cm3、(0.78±0.31)cm3、(0.23±0.12)cm3;抑瘤率分别为0、52.7%、86.1%。流式细胞仪分析细胞周期,移植瘤LS174T细胞经MPA处理后阻止G1期细胞向S期的进程,S期和G2/M期细胞相对减少,MPA+5-Fu合用组作用更明显。对照组、MPA治疗组、MPA+5-Fu合用组细胞凋亡率分别为3.6%、13.7%、28.6%,MPA作用后移植瘤LS174T细胞表面Fas和FasL表达显著增强。结论MPA对人结肠癌移植瘤LS174T细胞生长具有显著的抑制作用和诱导凋亡作用,其机制可能与MPA使LS174T细胞停滞于G1期及细胞表面Fas和FasL表达上调有关。     

Addressing the "Fas counterattack" controversy: blocking fas ligand expression suppresses tumor immune evasion of colon cancer in vivo

Fas ligand (FasL/CD95L) is a transmembrane protein belonging to the tumor necrosis factor superfamily that can trigger apoptotic cell death following ligation to its receptor, Fas (CD95/APO-1). Expression of FasL may help to maintain tumor cells in a state of immune privilege by inducing apoptosis of antitumor immune effector cells-the "Fas counterattack." However, the ability of FasL to mediate tumor immune privilege is controversial due to studies that indicate FasL has both pro- and anti-inflammatory activities. To resolve this controversy and functionally define the role of FasL in tumor immune evasion, we investigated if suppression of endogenously expressed FasL in colon tumor cells resulted in reduced tumor development and improved antitumor immune challenge in vivo. Specifically, FasL expression in CMT93 colon carcinoma cells was down-regulated following stable transfection with a plasmid encoding antisense FasL cDNA. Down-regulation of FasL expression had no effect on tumor growth in vitro but significantly reduced tumor development in syngeneic immunocompetent mice in vivo. Tumor size was also significantly decreased. Reduced FasL expression by tumor cells led to increased lymphocyte infiltration. The overall level of neutrophils present in all of the tumors examined was low, with no difference between the tumors, irrespective of FasL expression. Thus, down-regulation of FasL expression by colon tumor cells results in an improved antitumor immune challenge in vivo, providing functional evidence in favor of the "Fas counterattack" as a mechanism of tumor immune evasion.     

Postpromotional effects of dietary marine or safflower oils on large bowel or pulmonary implants of CT-26 in mice

Marine oils containing n-3 fatty acids exhibit variable antineoplastic effects. Diets containing low (11.6% of kcal) or high (46.5% of kcal) levels of marine oils as the exclusive fat source were compared to diets containing identical amounts of safflower oil (n-6) in weanling, male BALB/c ByJ mice. All diets provided approximately 90 kcal/100 g body weight/day, and contained identical quantities of vitamins, minerals, protein, and fiber. The growth of transplantable colon carcinoma, CT-26, (10(6) cells/animal) implanted, subserosally, into the descending colon via laparotomy, was observed weekly over 28 days by necropsy in all dietary groups. At each time period animals fed safflower oil had larger tumors than those fed marine oil. Tumor volumes at 21 days postimplantation were as follows: low fat marine, 55 mm3 (5-196 mm3) [median (range)]; high fat marine, 70 (26-194); low fat safflower, 216 (32-800); high fat safflower, 247 (70-1352). Marine oil tumors were smaller than safflower oil tumors (P less than 0.005 by analysis of variance; P less than 0.01 by Scheffe test). Metastatic potential was assessed by pulmonary colonization. CT-26 was injected i.v. in tail veins (10(5) cells/animal). Mice were sacrificed and colonies were counted after 21 days. Mice fed low fat marine, high fat marine, and low fat safflower oil diets, 10-14 colonies; high fat safflower, 55 colonies (P less than 0.001 by analysis of variance). Hence, dietary marine oil significantly suppressed growth of this colon carcinoma at all intake levels studied and inhibited pulmonary colonization at higher intakes relative to safflower oil.     

Cytokines and chemokines are expressed at different levels in small and large murine colon-26 tumors following intratumoral injections of CpG ODN

Direct tumor injections of (CpG ODN) into murine colon tumor 26 (CT-26) tumors can induce a potent antitumor response. Tumor size at the beginning of treatment determines the final therapeutic outcome, with smaller tumors responding favorably to CpG ODN therapy whereas large tumors do not. CpG ODN injections in small tumors resulted in tumor necrosis and extensive inflammatory cell infiltration, with average survival that is significantly higher (48.1 +/- 34 days) when compared to control ODN-treated mice (16.1 +/- 3.5 days). Cytokines and chemokines are expressed at different levels in small and large CT-26 tumors following intratumoral injections of CpG ODN. We observed that granulocyte-macrophage colony-stimulating factor and interleukin (IL) 6 are the major cytokines that were overexpressed in CpG ODN-treated small tumors but not in large tumors. Similarly, several chemokines (CXCL1, CCL2, and CCL3) were also significantly higher in CpG ODN-treated small tumors compared to control ODN-treated tumors.     

顺铂以T细胞依赖性方式增强ClK细胞的抗肿瘤作用

目的 观察顺铂预处理化疗联合细胞因子诱导的杀伤细胞( cytokine - induced killer cells,CIK cells)对小鼠CT - 26结肠癌的抑制作用,并探讨介导顺铂免疫调节效作用的机制.方法 分别建立BALB/c野生鼠或BALB/c nu/nu裸鼠CT - 26结肠癌模型,以顺铂(Cispla...     

Effects of the synthetic vasopressin analog desmopressin in a mouse model of colon cancer

Experimental and clinical data indicated that perioperative administration of the hemostatic peptide desmopressin (DDAVP) can inhibit progression of residual metastatic cells. The compound seems to act by inducing an agonist effect on specific V2 vasopressin membrane receptors present in both tumor cells and endothelial cells. Here we explored the antitumor effects of DDAVP in cultured colon carcinoma cells and in a syngeneic Balb/c mouse model. Both human Colo-205 and mouse CT-26 colon carcinoma cell lines expressed the V2 receptor, as revealed by immunofluorescence. DDAVP (at doses ranging from 100 ng/ml to 1 μg/ml) exerted a modest but significant antiproliferative effect on cultured CT-26 and Colo-205 cells. In vivo, DDAVP (2 intravenous doses of 2 μg/kg) reduced accumulation of ascites and formation of intestinal tumor nodules in mice intraperitoneally inoculated with CT-26 cells. Perioperative administration of DDAVP significantly inhibited tumor progression in animals surgically implanted in the spleen with CT-26 cells, and caused some reduction in liver metastasis. Although DDAVP and 5-fluorouracil demonstrated additive cytostatic effects in vitro, no antitumor effects were observed in this study in mice receiving a single cycle of chemotherapy (25 mg/kg) in combination with the peptide. Our data suggest that DDAVP may be potentially used to minimize spread or survival of residual malignant cells during surgical procedures for colon and other gastrointestinal tumors.     

白细胞介素18体外促进表达FasL的结肠癌细胞对肝细胞的杀伤

目的 探讨内源性细胞因子白细胞介素18(IL-18)和结肠癌细胞自身表达FasL的相互作用,及其对大肠癌肝浸润转移的影响.方法 采用免疫组织化学技术,检测结肠癌SW620细胞和Chang肝细胞Fas和Fas配体(FasL)的表达.采用非放射性细胞毒分析,测定SW620细胞与Chang细胞共培养后乳酸脱氢酶(LDH)的释放值,计算效应细胞的杀伤效应.将IL-18刺激36 h和未刺激的大肠癌SW620细胞(效应细胞)与干扰素γ刺激后的Chang细胞(靶细胞)共培养6 h,观察IL-18对杀伤效应的影响.结果 结肠癌SW620细胞FasL染色呈阳性反应,阳性反应物主要定位于细胞膜及核周区,而Fas染色几乎呈阴性反应.Chang细胞Fas免疫组化染色呈阳性反应,阳性反应物主要定位于细胞膜,而FasL染色呈阴性反应.在效靶比为10:1、5:1、2.5:1和1.25:1时,IL-18处理组的细胞毒杀伤效应分别为68.3%、49.8%、21.1%和9.7%,明显高于对照组的细胞毒杀伤效应(分别为32.7%、21.8%、11.1%和6.7%,均P＜0.05).结论 IL-18通过上调结肠癌细胞的FasL表达,能显著增强表达FasL的大肠癌细胞对肝细胞的毒性,有助于结肠癌细胞在肝脏形成转移灶.     

大肠癌组织中凋亡相关蛋白Fas、FasL和bcl-2的表达及其意义

目的：探讨凋亡相关蛋白Fas、FasL和bcl-2在大肠癌组织中的表达及其意义。方法：应用S－P免疫组化法检测80例大肠癌组织及20例正常大肠黏膜组织中Fas、FasL和bcl-2蛋白的表达情况。结果：大肠癌组织Fas蛋白阳性表达率显著低于正常大肠黏膜组织，淋巴结转移和肝转移者Fas蛋白阳性表达率低于无转移者，Ⅱ、Ⅲ级者低于I级者（P＜0．05），其表达与临床分期、组织学类型及病理分级相关（P＜0．05）。大肠癌组织中FasL、bcl-2蛋白阳性表达率均显著高于正常大肠黏膜组织，其表达与临床分期、组织学类型及病理分级均无关（P＞0．05）。大肠癌组织中Fas、FasL蛋白表达与bcl-2蛋白表达无关（P＞0．05）。结论:细胞凋亡是多因素共同调控的结果，在大肠癌发生、发展中，Fas/FasL系统与bcl-2可能起协调抑制肿瘤细胞凋亡的作用。     

Mouse macrophage metalloelastase gene delivery by HVJ-cationic liposomes in experimental antiangiogenic gene therapy for murine CT-26 colon cancer

We previously demonstrated that gene replacement of mouse macrophage metalloelastase (MME) into murine melanoma cells that grow rapidly and are MME deficient suppresses the primary tumor growth in vivo by halting angiogenesis. The aim of the present study was to evaluate the effectiveness of gene therapy against cancer using a cDNA-encoding MME gene. In a subcutaneous tumor model of CT-26 mouse colon cancer cells that are MME deficient, syngeneic mice repetitively treated with direct injections into the tumors of MME- hemagglutinating virus of Japan (HVJ), a type of HVJ-cationic liposome encapsulating a plasmid expressing MME, developed smaller tumors (210 +/- 47.2 mm(3) versus 925 +/- 156 mm(3) mean +/- SEM; p = 0.0004) with fewer microvessels (10.25 +/- 1.03 vs. 17.25 +/- 2.14; p = 0.03) than control mice. TUNEL staining revealed a significant increase of apoptotic cells in the MME-HVJ liposomes-treated tumors compared with control tumors. MME was effectively expressed in the s.c. tumors treated with MME-HVJ liposomes, inducing angiostatin generation in those tumors, as demonstrated by Western blot analysis. In conclusion, our study demonstrated that repeated in vivo transduction of the MME gene directly into the tumors using HVJ-cationic liposomes suppressed the tumor growth by an antiangiogenic mechanism, providing, then, a feasible strategy for gene therapy of cancer.     

Fas ligand expressed in colon cancer is not associated with increased apoptosis of tumor cells in vivo

Expression of Fas ligand (FasL/CD95L) may help to maintain colon cancers in a state of immune privilege by inducing apoptosis of antitumor immune effector cells. Colon tumor-derived cell lines appear to be relatively insensitive to apoptosis mediated by their own or exogenous FasL in vitro, despite expression of cell surface Fas. In our present study, we sought to investigate if FasL upregulated in human colon cancers leads to any increase in apoptosis of the tumor cells in vivo. FasL and Fas receptor (APO-1/CD95) expression by tumor cells were detected immunohistochemically. Apoptotic tumor cell death was detected by immunohistochemistry for caspase-cleaved cytokeratin-18. FasL expression did not correlate with the extent of apoptosis of tumor cells. There was no significant local difference in the frequency of apoptosis of tumor cells between tumor nests that expressed FasL (mean = 2.4%) relative to those that did not (mean = 2.6%) (p = 0.625, n = 10; Wilcoxon signed rank). FasL expressed by the tumor cells appeared to be functional, since FasL expression in tumor nests correlated with diminished infiltration of tumor-infiltrating lymphocytes (TILs). TILs were detected using immunohistochemistry for CD45. Expression of FasL by tumor nests was associated with a mean 4-fold fewer TILs relative to FasL-negative nests (range 2.4-33-fold, n = 10, p < 0.003). Together, our results indicate that colon tumors are insensitive to FasL-mediated apoptosis in vivo.     

Prognostic significance of Fas and Fas ligand expressions in human esophageal cancer.

Esophageal carcinomas have recently been shown to express Fas ligand (FasL) and down-regulate Fas to escape from host immune surveillance. However, the prognostic importance of Fas/FasL and their correlation with clinicopathological characteristics are yet to be delineated in this highly malignant carcinoma. Specimens from 106 esophageal squamous cell carcinoma patients were used for immunohistochemical evaluation of Fas, FasL, and CD8 expressions. Fifty-two (49%) and 34 (32%) patients were positive for FasL and Fas, respectively. There were no associations between FasL expression and clinicopathological characteristics except lymph vessel invasion. Strong FasL expression correlated with significant (P = 0.0011) decrease in tumor nest CD8+ cells. However, neither FasL nor CD8+ had any impact on patient survival. Strong Fas expression was correlated with depth of invasion (40.3% in pT1,T2 versus 20.5% in pT3,T4; P = 0.0308), histological differentiation (45.7% in well versus 25.4% in nonwell; P = 0.0347), and lymph node metastasis (22.6% in positive versus 45.5% in negative; P = 0.0129). Fas expression was one of the independent favorable prognosticators for patients' survival (risk ratio, 3.26; P = 0.0103) in esophageal SCC. Fas expression was an independent prognosticator for recurrence-free survival, whereas FasL expression did not influence the survival in esophageal squamous cell carcinoma. Down-regulation of tumor Fas may be the hallmark of immune privilege for the tumor, thus causing the patients' poorer outcome. Tumor FasL may counterattack the host immune cells to such an extent that the prognosis is not affected.     

Inhibition of primary colon carcinoma growth and liver metastasis by the A3 adenosine receptor agonist CF101

Adenosine is a purine nucleoside that acts as a regulatory molecule by binding to specific G-protein-coupled A1, A(2A), A(2B), and A3 cell surface receptors. We have recently demonstrated that adenosine inhibits tumour cell growth and concomitantly stimulates bone marrow cell proliferation via activation of the A3 adenosine receptor (A3AR). In the present study, we show that a synthetic agonist to the A3AR, CF101, at the low nanomolar concentration range, inhibits HCT-116 human colon carcinoma cell growth. This effect was reversed by the selective A3AR antagonist MRS1523, demonstrating the specificity of the response. CF101 (given orally) was efficacious in inhibiting the development of primary tumours in xenograft and syngeneic models in which mice were inoculated subcutaneously with human HCT-116 or murine CT-26 colon carcinoma cells, respectively. Moreover, CF101 suppressed (50%, P<0.01) colon cancer liver metastases in syngeneic mice inoculated to the spleen with CT-26 cells. The mechanism of action entailed upregulation of interleukin-12 production in the CF101-treated groups and potentiation of NK cell activity. In the HCT-116 xenograft model in which a combined therapy of CF101 and 5-fluorouracyl (5-FU) was examined, an additive antitumour effect was demonstrated. Moreover, CF101 prevented the 5-FU-induced myelotoxicity, resulting in normal values of white blood cell and neutrophil counts. We conclude that the A3AR agonist CF101, a small orally bioavailable molecule, exerts systemic anticancer, antimetastatic, and myeloprotective effects in colon carcinoma-bearing mice, and may serve as an adjuvant treatment to enhance the chemotherapeutic index and prevent myelotoxicity.