Apatinib treatment efficiently delays biochemical-only recurrent ovarian cancer progression.

BACKGROUND: Biochemical recurrence is defined as only rising CA-125 but no radiographic evidence of disease; noteworthily, it generally precedes the onset of clinical evidence. Now treatment strategies of biochemical recurrence ovarian cancer (OC) remain controversial. Apatinib as monotherapy or in combination with other chemotherapeutic agents has shown its effect in the treatment of some advanced malignancies. In our study, we focused on the efficacy of apatinib in recurrent OC, especially its clinical activity in biochemical-only recurrent OC patients. METHODS: We retrospectively analyzed clinical material of 41 recurrent patients who had received apatinib monotherapy or apatinib plus chemotherapy between June 2016 and August 2018. Apatinib was administered at a 500mg daily dose. Response was determined according to measurable disease or serum carbohydrate antigen (CA)-125 levels. Progression-free survival (PFS) was estimated by Kaplan-Meier method. RESULTS: All patients were evaluable, 19 (46.34%) had biochemical relapse and 22 (53.66%) had clinical relapse. The objective response rate (ORR) and disease control rate (DCR) in the overall population were 31.71% and 78.05%, respectively. The median PFS was 7 months (95% confidence interval 5.43-8.57). And in patients with biochemical-only relapse, the median PFS was 6 months, with ORR of 26.32% and DCR of 89.47%. CONCLUSIONS: Apatinib is a well-tolerated and effective agent to delay clinical progression of patients with biochemical-only recurrent OC. More important, our study shows the promising prospect for treating OC patients with asymptomatic biochemical relapse.

TFRC promotes epithelial ovarian cancer cell proliferation and metastasis via up-regulation of AXIN2 expression.

Epithelial ovarian cancer (EOC) is the most common cause of gynecological cancer death. Recent studies have reported that iron overload could accelerate cancer progression. TFRC is an important participant in intracellular iron transport, and we noticed that it was abnormally overexpressed in EOC; however, its specific role in EOC remained unclear. Therefore, our study aimed to reveal the clinical significance and biological function of TFRC in human EOC. First, we detected dramatically increased TFRC expression in EOC tissues, which was associated with a worse prognosis for patients. Subsequently, we verified that TFRC knockdown significantly inhibited the proliferation and metastasis of EOC cells (SKOV3 and A2780) in vitro and in vivo. More significantly, we demonstrated that TFRC-mediated proliferation and metastasis of EOC cells resulted from its positive regulation of AXIN2 expression. In conclusion, our findings suggest that TFRC accelerates the progression of EOC by promoting cancer cell proliferation and metastasis via upregulation of AXIN2 expression, which highlights its potential as a novel therapeutic target for human EOC.

Aryl hydrocarbon receptor activation modulates γδ intestinal intraepithelial lymphocytes and protects against ischemia/reperfusion injury in the murine small intestine.

The pathogenesis of intestinal ischemia/reperfusion (I/R) is associated with dysregulation of the intestinal immune system. The aryl hydrocarbon receptor (AhR), a receptor expressed in gamma­delta (γδ) intraepithelial lymphocytes (IELs), is thought to regulate inflammation in the bowel. γδIELs are a key immunologic compartment with a capacity to modulate immune responses. In the present study, the function of the AhR in γδIELs in a mouse model of intestinal I/R injury was investigated to determine whether the AhR attenuates intestinal injury induced by intestinal I/R. Mice were assigned to three groups: sham, I/R and I/R+6­formylindolo(3,2­b)carbazole (FICZ). The sham group received no ischemia treatment, whereas the I/R and I/R+FICZ groups underwent upper mesenteric vessel ischemia for 30 min. The I/R group was injected intraperitoneally with 0.3 ml saline and the I/R+FICZ group was administered 1 µg of FICZ before a subsequent 6 h reperfusion. Then, the mice were sacrificed and the entire small intestinal tissues were collected for histologic examination. The phenotype and apoptosis of γδIELs and activation of CD4+ and CD8+ IELs were examined using flow cytometry. The cytokine mRNA and anti­apoptosis gene expression in IELs were measured by qPCR. FICZ increased the γδIEL population and anti­apoptosis genes in the γδIELs. FICZ reduced the percentage of activated CD4+ and CD8+ subpopulations and the expression of pro­inflammatory mediator genes in IELs. FICZ inhibited inflammation in the gastrointestinal tract of mice with I/R injury. These results suggest that the AhR plays an important role in protecting the small intestine from I/R and increasing the γδIEL population by decreasing apoptosis of γδIELs.

Aryl hydrocarbon receptor inhibits inflammation in DSS­induced colitis via the MK2/p­MK2/TTP pathway.

The pathogenesis of inflammatory bowel disease (IBD) is believed to be associated with the abnormal expression of inflammatory factors. The aryl hydrocarbon receptor (AhR) is a ligand­dependent transcription factor, which can suppress the inflammatory response and attenuate experimental colitis. However, the detailed mechanism underlying the effects of AhR remains unclear. The present study investigated the role of AhR in the pathogenesis of IBD. Colitis was induced in mice by administration of 3% dextran sulphate sodium (DSS) for 7 days. The mice were also administered injections of the AhR agonist, 6­formylindolo(3,2­b)carbazole (FICZ), starting 2 days after the first administration of DSS. Furthermore, LoVo cells were treated with lipopolysaccharide (LPS) in the presence or absence of FICZ for 8 h. The protein expression levels of AhR, cytochrome P450 1A1 (CYP1A1) and tristetraprolin (TTP) were assessed by western blotting and immunofluorescence, whereas mRNA expression levels were assessed by reverse transcription­quantitative polymerase chain reaction. The results indicated that injection of mice with FICZ significantly attenuated DSS­induced colitis; in addition, the expression levels of inflammatory cytokines were markedly downregulated. Conversely, the expression levels of AhR and TTP were upregulated. In addition, mice in the AhR­knockout + DSS group exhibited elevated inflammatory cytokine production and developed more severe colitis. In LoVo cells, incubation with FICZ decreased the expression levels of inflammatory cytokines, whereas AhR and TTP expression was increased. In addition, the levels of phosphorylated­mitogen­activated protein kinase­activated protein kinase 2 (p­MK2) were decreased. These results suggested that AhR deficiency resulted in increased susceptibility to colitis, whereas activation of AhR by FICZ could ameliorate DSS­induced colitis via the MK2/p­MK2/TTP pathway.

AhR­E2F1­KGFR signaling is involved in KGF­induced intestinal epithelial cell proliferation.

Keratinocyte growth factor (KGF) stimulates intestinal epithelial cell proliferation upon binding to the KGF receptor (KGFR). The activated aryl hydrocarbon receptor (AhR) serves an important role in the development of tissues by promoting the expression of AhR receptors, which can regulate cell proliferation. In the present study, the signaling pathway between AhR and KGFR in investigated with regards to KGF­induced intestinal epithelial cell proliferation. Male C57BL/6J wild type and AhR­/­ mice, were randomized into four groups: Control, KGF, AhR­/­ + KGF and AhR­/­ (n=6 per group). The small bowel was harvested on day 5 post­treatment. LoVo cells were used to study signaling pathways in vitro and were divided into the following four treatment groups: DMSO, KGF, KGF + small­interfering (si)AhR and siAhR. In vivo, knockdown of AhR mRNA transcripts may abolish KGF­induced intestinal epithelial cell proliferation. Furthermore, KGFR expression was downregulated following knockdown or silencing of AhR expression in vivo and in vitro. The present study identified that the transcription factor E2F1 could regulate KGFR expression, and that siAhR treatment led to reduced expression of E2F1 in the nucleus and inhibited KGF­induced cell proliferation. In conclusion, the current results demonstrated that the AhR­E2F1­KGFR pathway is involved in KGF­induced intestinal epithelial cell proliferation.

Aryl hydrocarbon receptor activation modulates CD8αα+TCRαß+ IELs and suppression of colitis manifestations in mice.

BACKGROUND: This research is dedicated to investigating the effects and potential mechanism of action of the aryl hydrocarbon receptor on the intestinal mucosal immune system in dextran sulfate sodium (DSS)-induced colitis. METHODS: Colitis was induced by the administration of 3% DSS to wild-type C57BL/6J mice for 7days. 6-formylindolo(3, 2-b)carbazole (FICZ), an endogenous agonist of the aryl hydrocarbon receptor (AhR), was given intraperitoneally on a daily basis beginning 2days after the start of DSS administration. The mice were weighed and assessed, and colon tissues were measured. Intraepithelial lymphocytes (IELs) were isolated from the colon and examined by flow cytometry and quantitative real-time PCR. RESULTS: FICZ ameliorated DSS-induced colitis, resulting in a reduced disease activity index and improvement in the histology and length of the colon. Colitis reduced the percentage and number of CD8αα+TCRαß+ IELs. FICZ prevented the reduction in the numbers of CD8αα+TCRαß+ IELs by upregulating the expression of the IL-15 receptor and the aryl hydrocarbon receptor (AhR), and attenuating the apoptotic rate of CD8αα+TCRαß+ IELs. Finally, IL-10 was increased and IFN-γ was decreased in CD8αα+TCRαß+ IELs by FICZ administration in DSS-induced colitis. CONCLUSIONS: The results suggest that AhR activation ameliorated DSS-induced acute colitis, in a manner that is associated with the local expansion and functions of CD8αα+TCRαß+ IELs in acute colitis. The findings indicate that AhR-related ligands might be targeted as novel drug targets for IBD.

TLR2-Dependent Signaling for IL-15 Production Is Essential for the Homeostasis of Intestinal Intraepithelial Lymphocytes.

TLR2 signaling is related to colitis and involved in regulation of innate immunity in the intestinal tract, but the mechanisms remain unclear. The aim of this study is to investigate how TLR2 affects differentiation of intraepithelial lymphocytes (IELs) and regulates the susceptibility of colitis. IELs were isolated from the small intestine and colon of mice, respectively. The IEL phenotype, activation, and apoptosis were examined using flow cytometry and RT-PCR. IL-15 expression and IEL location were detected through immunohistochemistry. The experimental colitis was induced by administration of dextran sulfate sodium (DSS). We found that the numbers of CD8αα (+), CD8αß (+), and TCRγδ (+) IELs were significantly decreased in TLR2-deficient mice and the residual IELs displayed reduced activation and proliferation and increased apoptosis, accompanied with impaired IL-15 expression by intestinal epithelial cells (IECs). Further study showed that TLR2 signaling maintained the expression of IL-15 in IEC via NF-κB activation. Moreover, TLR2-deficient mice were found to be more susceptible to DSS-induced colitis as shown by the increased severity of colitis. Our results demonstrate that IECs contribute to the maintenance of IELs at least partly via TLR2-dependent IL-15 production, which provides a clue that may link IECs to innate immune protection of the host via IELs.

Aryl Hydrocarbon Receptor Activation in Intestinal Obstruction Ameliorates Intestinal Barrier Dysfunction Via Suppression of MLCK-MLC Phosphorylation Pathway.

BACKGROUND: Accumulating evidence suggests that the aryl hydrocarbon receptor (AhR) plays an important role in the maintenance of the function of the intestinal barrier in patients with inflammatory bowel disease and in mouse models. Intestinal obstruction (IO) is a clinical emergency consisting of severe dysfunction of intestinal barrier function, and whether AhR plays a role in the pathogenesis of IO remains unknown but would be highly significant. METHODS: Male C57BL/6 mice were subjected to IO and either treated with AhR endogenous agonist 6-formylindolo [3, 2-b] carbazole (FICZ) or left untreated. Intestinal tissue was harvested after 24 h. Correspondingly, Caco-2 monolayers were treated with FICZ in the absence or presence of hypoxia in vitro or left untreated. The cells were used after 12 h. RESULTS: Damage to the intestinal mucosa was anabatic and intestinal permeability was significantly higher in murine IO and hypoxia-induced Caco-2 models than in controls. Under these conditions the activity of AhR was lower and the fluorescence of zonula occludens-1 (ZO-1) was absent. The increased expression of myosin light chain kinase (MLCK) and phosphorylated MLC (pMLC) indicated that this pathway was open. However, treatment with FICZ caused retention of the tight junction protein ZO-1, alleviated the increase of intestinal permeability, and mitigated epithelial injury. Depletion of AhR by AhR small interfering RNA facilitated the unblocking of the MLCK-pMLC signaling pathway and repressed the protein expression of ZO-1 in vitro. CONCLUSION: AhR activation can ameliorate epithelial barrier dysfunction induced by IO through the suppression of MLCK-pMLC signaling, suggesting that AhR agonist may be a suitable means of addressing this condition.

The AhR is involved in the regulation of LoVo cell proliferation through cell cycle-associated proteins.

Some ingredients in foods can activate the aryl hydrocarbon receptor (AhR) and arrest cell proliferation. In this study, we hypothesized that 6-formylindolo [3, 2-b] carbazole (FICZ) arrests the cell cycle in LoVo cells (a colon cancer line) through the AhR. The AhR agonist FICZ and the AhR antagonist CH223191 were used to treat LoVo cells. Real-time PCR and Western blot analyses were performed to detect the expression of the AhR, CYP1A1, CDK4, cyclinD1, cyclin E, CDK2, P27, and pRb. The distribution and activation of the AhR were detected with immunofluorescence. A 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay and flow cytometric analysis were performed to measure cell viability, cell cycle stage, and apoptosis. Our results show that FICZ inhibited LoVo cell proliferation by inducing G1 cell cycle arrest but had no effect on epithelial apoptosis. Further analysis found that FICZ downregulated cyclinD1 and upregulated p27 expression to arrest Rb phosphorylation. The downregulation of cyclinD1 and upregulation of p27 were abolished by co-treatment with CH223191. We conclude that the AhR, when activated by FICZ (an endogenous AhR ligand), can arrest the cell cycle and block LoVo cell proliferation.

Keratinocyte Growth Factor Regulation of Aryl Hydrocarbon Receptor Activation in Colorectal Cancer Cells.

BACKGROUND: Keratinocyte growth factor (KGF) stimulates normal growth, development and intestinal epithelial cell proliferation. Cyclin D1 promotes the cell cycle by inhibiting retinoblastoma protein (RB1). The activated aryl hydrocarbon receptor (AhR) has an important influence on the development of tumors through its interactions with the cell cycle. AIM: The aim of the present study was to explore a new role for AhR in KGF-induced colon cancer cell growth. MATERIALS AND METHODS: Real-time PCR, western blot or immunofluorescence analysis were used to detect the expression of KGF, AhR, cyclin D1 and CYP1A1. Immunohistochemistry was used to observe the localization of AhR. MTT assay and flow cytometric analyses were performed to measure cell viability and the cell cycle. RESULTS: Real-time PCR analysis revealed that KGF, AhR, and CYP1A1 mRNAs were overexpressed in colorectal cancer tissues. Meanwhile, overexpression of AhR was primarily observed in epithelial cells. In in vitro assay, KGF promoted colon cancer cell growth, as well as up-regulated and activated AhR. At the same time, AhR-knockdown colon cancer cells were less responsive to KGF. Western blot analysis, real-time PCR, or immunofluorescence data indicated that cyclin D1 expression was up-regulated by KGF but this up-regulation was compromised when AhR was silenced, and the cell cycle was arrested in the G0/G1 stage in these cells. CONCLUSIONS: Our study suggests that KGF, AhR, and CYP1A1 are overexpressed in colorectal cancer tissues. Moreover, we reveal a new mechanism by which KGF promotes cell proliferation through the AhR-cyclin D1 pathway in colon cancer cells.