Celastrol inhibits migration, proliferation and transforming growth factor-β2-induced epithelial-mesenchymal transition in lens epithelial cells

AIM: To investigate the mechanism of celastrol in inhibiting lens epithelial cells(LECs) fibrosis, which is the pathological basis of cataract.METHODS: Human LEC line SRA01/04 was treated with celastrol and transforming growth factor-β2(TGF-β2). Wound-healing assay, proliferation assay, flow cytometry, real-time polymerase chain reaction(PCR), Western blot and immunocytochemical staining were used to detect the pathological changes of celastrol on LECs. Then, we cultured Sprague-Dawley rat lens in medium as a semi-in vivo model to find the function of celastrol further.RESULTS: We found that celastrol inhibited the migration of LECs, as well as proliferation(P<0.05). In addition, it induced the G2/M phase arrest by cell cyclerelated proteins(P<0.01). Moreover, celastrol inhibited epithelial-mesenchymal transition(EMT) by the blockade of TGF-β/Smad and Jagged/Notch signaling pathways.CONCLUSION: Our study demonstrates that celastrol could inhibit TGF-β2-induced lens fibrosis and raises the possibility that celastrol could be a potential novel drug in prevention and treatment of fibrotic cataract.

Inducement of ER Stress by PAD Inhibitor BB-Cl-Amidine to Effectively Kill AML Cells

Objective Acute myeloid leukemia(AML)is a highly heterogeneous and recurrent hematological malignancy.Despite the emergence of novel chemotherapy drugs,AML patients’complete remission(CR)remains unsatisfactory.Consequently,it is imperative to discover new therapeutic targets or medications to treat AML.Such epigenetic changes like DNA methylation and histone modification play vital roles in AML.Peptidylarginine deminase(PAD)is a protein family of histone demethylases,among which the PAD2 and PAD4 expression have been demonstrated to be elevated in AML patients,thus suggesting a potential role of PADs in the development or maintenance of AML and the potential for the identification of novel therapeutic targets.Methods AML cells were treated in vitro with the pan-PAD inhibitor BB-Cl-Amidine(BB-Cl-A).The AML cell lines were effectively induced into apoptosis by BB-Cl-A.However,the PAD4-specific inhibitor GSK484 did not.Results PAD2 played a significant role in AML.Furthermore,we found that BB-Cl-A could activate the endoplasmic reticulum(ER)stress response,as evidenced by an increase in phosphorylated PERK(p-PERK)and eIF2α(p-eIF2α).As a result of the ER stress activation,the BB-Cl-A effectively induced apoptosis in the AML cells.Conclusion Our findings indicated that PAD2 plays a role in ER homeostasis maintenance and apoptosis prevention.Therefore,targeting PAD2 with BB-Cl-A could represent a novel therapeutic strategy for treating AML.

Up-regulation of indoleamine 2,3-dioxygenase 1(IDO1)expression and catalytic activity is associated with immunosuppression and poor prognosis in penile squamous cell carcinoma patients

Background: Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan (Trp)catabolism have been demonstrated to play an important role in tumor immunosuppression. This study examined the expression and catalytic activity of IDO1 in penilesquamous cell carcinoma (PSCC) and explored their clinical significance.Methods: IDO1 expression level, serum concentrations of Trp and kynurenine (Kyn)were examined in 114 PSCC patients by immunohistonchemistry and solid-phaseextraction-liquid chromatography-tandem mass spectrometry. The survival was analyzed using Kaplan-Meier method and the log-rank test. Hazard ratio of death was analyzed via univariate and multivariate Cox regression. Immune cell types were definedby principal component analysis. The correlativity was assessed by Pearson’s correlation analysis.Results: The expression level of IDO1 in PSCC cells was positively correlatedwith serum Kyn concentration and Kyn/Trp radio (KTR;both P < 0.001) but negatively correlated with serum Trp concentration (P = 0.001). Additionally, IDO1 upregulation in cancer cells and the increase of serum KTR were significantly associated with advanced N stage (both P < 0.001) and high pathologic grade (P = 0.008and 0.032, respectively). High expression level of IDO1 in cancer cells and serumKTR were associated with short disease-specific survival (both P < 0.001). However, besides N stage (hazard radio [HR], 6.926;95% confidence interval [CI],2.458-19.068;P < 0.001) and pathologic grade (HR, 2.194;95% CI, 1.021-4.529;P = 0.038), only serum KTR (HR, 2.780;95% CI, 1.066-7.215;P = 0.036) was anindependent predictor for PSCC prognosis. IDO1 expression was positively correlated with the expression of interferon-𝛾 (IFN𝛾, P < 0.001) and immunosuppressivemarkers (programmed cell death protein 1, cytotoxic T-lymphocyte-associated protein 4 and programmed death-ligand 1 and 2;all P < 0.05), and the infiltration ofimmune cells (including cytotoxic T lymphocytes, regulatory T lymphocytes, tumorassociated macrophages, and myeloid-derived suppressor cells;all P < 0.001) inPSCC tissues. Furthermore, the expression of IDO1 was induced by IFN𝛾 in a dosedependent manner in PSCC cells.Conclusions: IFN𝛾-induced IDO1 plays a crucial role in immunoediting andimmunosuppression in PSCC. Additionally, serum KTR, an indicator of IDO1catabolic activity, can be utilized as an independent prognostic factor for PSCC.

Zinc finger and BTB domain-containing protein 46 is essential for survival and proliferation of acute myeloid leukemia cell line but dispensable for normal hematopoiesis

Background:Zinc finger and BTB domain-containing protein 46(Zbtb46)is a transcription factor identified in classical dendritic cells,and maintains dendritic cell quiescence in a steady state.Zbtb46 has been reported to be a negative indicator of acute myeloid leukemia(AML).We found that Zbtb46 was expressed at a relatively higher level in hematopoietic stem and progenitor cells(HSPCs)compared to mature cells,and higher in AML cells compared to normal bone marrow(BM)cells.However,the role of Zbtb46in HSPCs and AML cells remains unclear.Therefore,we sought to elucidate the effect of Zbtb46 in normal hematopoiesis and AML cells.Methods:We generated Zbtb46^fl/fl and Zbtb46^fl/fl Mx1-Cre mice.The deletion of Zbtb46 in Zbtb46^fl/fl Mx1-Cre mice was induced by intraperitoneal injection of double-stranded poly(I).poly(C)(poly(I:C)),and referred as Zbtb46 cKO.After confirming the deletion of Zbtb46,the frequency and numbers of HSPCs and mature blood cells were analyzed by flow cytometry.Serial intraperitoneal injection of 5-fluorouracil was administrated to determine the repopulation ability of HSCs from Zbtb46^fl/fl and Zbtb46 cKO mice.The correlation between Zbtb46 expression and prognosis was analyzed using the data from the Cancer Genome Atlas.To investigate the role of Zbtb46 in AML cells,we knocked down the expression of Zbtb46 in THP-1 cells using lentiviral vectors expressing small hairpin RNAs targeting Zbtb46.Cell proliferation rate was determined by cell count assay.Cell apoptosis and bromodeoxyuridine incorporation were determined by flow cytometry.Results:The percentages and absolute numbers of HSPCs and mature blood cells were comparable in Zbtb46 cKO mice and its Zbtb46^fl/fl littermates(Zbtb46^fl/fl vs.Zbtb46 cKO,HPC:801,310±84,282 vs.907,202±97,403,t=0.82,P=0.46;LSK:86,895±7802 vs.102,210±5025,t=1.65,P=0.17;HSC:19,753±3116 vs.17,608±3508,t=0.46,P=0.67).The repopulation ability of HSCs from Zbtb46^fl/fl Mx1-Cre mice was similar to those from Zbtb46^fl/fl control(P=0.26).Zbtb46 had elevated expression in AML cells compared to total BM cells from normal control.Knockdown of Zbtb46 in THP-1 cells led to a significant increase in cell apoptosis and reduced cell growth and proliferation.Conclusion:Collectively,our data indicate that Zbtb46 is essential for survival and proliferation of AML cells,but dispensable for normal hematopoiesis.

A single copy of large tumor suppressor 1 or large tumor suppressor 2 is sufficient for normal hematopoiesis

Background:Hematopoietic stem cells(HSCs)have the ability to differentiate into all subsets of blood cells and self-renew.Large tumor suppressor 1(LATS1)and large tumor suppressor 2(LATS2)kinases are essential for cell cycle regulation,organism fitness,genome integrity,and cancer prevention.Here,we investigated whether Lats1 and Lats2 are critical for the maintenance of the selfrenewal and quiescence capacities of HSCs in mice.Methods:Quantitative reverse transcription-polymerase chain reaction was used to determine the expression levels of Lats1 and Lats2 in subsets of progenitor cells and mature bone marrow cells.A clustered regularly interspaced short palindromic repeats system was used to generate Lats1 or Lats2 knockout mice.Complete blood cell counts were used to compare the absolute number of white blood cells,lymphocytes,monocytes,neutrophils,and platelets between Lats1 or Lats2 heterozygotes and littermates.Flow cytometry was used to assess the size of hematopoietic progenitor cells(HPCs)and HSC pools in Lats1 or Lats2 heterozygotes and littermates.The comparison between the two groups was analyzed using Student’s t test.Results:Lats1 and Lats2 were widely expressed in hematopoietic cells with higher expression levels in primitive hematopoietic cells than in mature cells.Lats1 or Lats2 knockout mice were generated,with the homozygotes showing embryonic lethality.The size of the HPC and HSC pools in Lats1(HPC:wild-type[WT]vs.heterozygote,220,426.77±54,384.796 vs.221,149.4±42,688.29,P=0.988;HSC:WT vs.heterozygote,2498.932±347.856 vs.3249.763±370.412,P=0.105)or Lats2(HPC:WT vs.heterozygote,425,540.52±99,721.86 vs.467,127.8±89,574.48,P=0.527;HSC:WT vs.heterozygote,4760.545±1518.01 vs.5327.437±873.297,P=0.502)heterozygotes were not impaired.Moreover,the depletion of Lats1 or Lats2 did not affect the overall survival of the heterozygotes(Lats1:P=0.654;Lats2:P=0.152).Conclusion:These results indicate that a single allele of Lats1 or Lats2 may be sufficient for normal hematopoiesis.