EIF3B stabilizes PCNA by counteracting SYVN1-mediated ubiquitination to serve as a promotor in cholangiocarcinoma.

Cholangiocarcinoma, a prevalent hepatic malignancy, exhibits a progressively rising incidence. While Eukaryotic translation initiation factor 3 subunit B (EIF3B) has been implicated in the occurrence and development of various cancers, its specific roles in cholangiocarcinoma remain unexplored. Immunohistochemical (IHC) analysis was employed to detect EIF3B/PCNA expression in cholangiocarcinoma. Cells were manipulated using short hairpin RNA (shRNA)-mediated lentiviruses or overexpression plasmids. Statistical significance was assessed using the Student's t-test and one-way ANOVA, with P < 0.05 considered statistically significant. EIF3B exhibited robust expression in cholangiocarcinoma, demonstrating a significant correlation with the pathological grade of cholangiocarcinoma patients. Furthermore, modulation of EIF3B expression, either depletion or elevation, demonstrated the ability to inhibit or enhance cholangiocarcinoma cell survival and migration in vitro. Mechanistically, we identified Proliferating Cell Nuclear Antigen (PCNA) as a downstream gene of EIF3B, driving cholangiocarcinoma. EIF3B stabilized PCNA by inhibiting PCNA ubiquitination, a process mediated by E3 ligase SYVN1. Similar to EIF3B, PCNA levels were also abundant in cholangiocarcinoma, and knocking down PCNA impeded cholangiocarcinoma development. Intriguingly, silencing PCNA attenuated the promotion induced by EIF3B overexpression. Furthermore, the elevated P21 protein level in shEIF3B RBE cells was partially attenuated after UC2288 (P21 signaling pathway inhibitor) treatment. Our findings underscored the potential of EIF3B as a therapeutic target for cholangiocarcinoma. Unraveling its functions holds promise for the development of more specific and effective targeted therapy strategies.

The impact of liver fibrosis on the progression of hepatocellular carcinoma via a hypoxia-immune-integrated prognostic model.

The impact of liver fibrosis on the deterioration of hepatocellular carcinoma (HCC) remains controversial. We hope to explore this issue through establishing a fibrosis-hypoxia-glycolysis-immune related prognostic model. Liver fibrosis-related genes from Molecular Signatures Database were used to evaluate the degree of fibrosis in HCC patients from the TCGA database. The patients were divided into two groups using the fibrosis-related expression matrix based on the algorithm uniform manifold approximation and projection (UMAP) and evaluated for fibrosis by UMAP cluster and gene enrichment analysis. Prognostic model was constructed by differential analysis, LASSO, and multivariate regression analysis. Immune-infiltration analysis was performed by CIBERSORT. Quantitative PCR and immunohistochemistry were performed to measure the gene expression levels in HCC patients from our hospital. In 365 HCC patients from the TCGA database, 111 HCC patients with high fibrosis score have a worse prognosis than those with low fibrosis based on 129 genes related to liver fibrosis, which may be caused by the interaction between fibrosis, angiogenesis, hypoxia, glycolysis, inflammatory response, and high immune infiltration. We constructed a Fibrosis-Hypoxia-Glycolysis-Immune Prognostic Model (FHGISig), which could significantly predict disease progression in HCC patients. Furthermore, we revealed a close correlation between FHGISig and immune cell infiltration level as well as immune checkpoints. Finally, PCR results found TFF3 mRNA was significantly lower in cirrhotic HCC patients compared with non-cirrhotic ones. Liver fibrosis is a poor-prognostic factor for HCC, and our FHGISig could significantly predict disease progression, which could also be a potential predictive marker for immunotherapy in HCC patients.

The advanced lung cancer inflammation index predicts chemotherapy response and infection risk in multiple myeloma patients receiving induction chemotherapy.

Objective: This study aims to determine the clinical significance of the advanced lung cancer inflammation index (ALI) in predicting prognosis, chemotherapy response, and infection risk in newly diagnosed multiple myeloma (MM) patients receiving induction therapy. Methods: A retrospective analysis of the clinical characteristics and laboratory data of 111 newly diagnosed MM patients from the Haematology Department of the Third Xiangya Hospital of Central South University from January 2014 to March 2020 was performed. We first determined the relationship between ALI and overall survival (OS), as well as clinical and laboratory parameters. Second, predictive factors for chemotherapy response were analysed by univariate and multivariate regression analyses. Third, univariate regression analysis of risk factors was performed using infection as the evaluable outcome. Results: Of the 111 evaluable patients, the low ALI group (<32.7) exhibited significantly poorer survival than the high ALI group (51 months versus 77 months). Multivariable analysis showed that advanced age, chemotherapy response and serum calcium level were independent prognostic factors for OS. Better chemotherapy efficacy in the high ALI group (89.3%) than in the low ALI group (42.2%) (p < 0.001) was noted. Multivariate analysis suggested that only ALI [HR: 0.110, 95% CI (0.035-0.350), p = 0.000] is an independent predictive factor in evaluating the efficiency of induction chemotherapy. Forty patients (36.04%) presented with infection after induction chemotherapy. Univariate analysis suggested that low ALI and abnormal renal function increase risk of infection in newly diagnosed MM patients. Conclusion: Our study confirmed that ALI is not only a prognostic biomarker for newly diagnosed patients, but also predicts chemotherapy efficacy in newly diagnosed MM patients receiving induction therapy.

Identification and validation of an immunological microenvironment signature and prediction model for epstein-barr virus positive lymphoma: Implications for immunotherapy.

Background: Epstein-Barr virus (EBV) is considered a carcinogenic virus, which is associated with high risk for poor prognosis in lymphoma patients, and there has been especially no satisfying and effective treatment for EBV+ lymphoma. We aimed to identify the immunological microenvironment molecular signatures which lead to the poor prognosis of EBV+ lymphoma patients. Methods: Differential genes were screened with microarray data from the GEO database (GSE38885, GSE34143 and GSE13996). The data of lymphoid neoplasm diffuse large B-cell lymphoma (DLBC) from the TCGA database and GSE4475 were used to identify the prognostic genes. The data of GSE38885, GSE34143, GSE132929, GSE58445 and GSE13996 were used to eluate the immune cell infiltration. Formalin-fixed, paraffin-embedded tissue was collected for Real Time Quantitative PCR from 30 clinical samples, including 15 EBV+ and 15 EBV- lymphoma patients. Results: Four differential genes between EBV+ and EBV- lymphoma patients were screened out with the significance of the survival and prognosis of lymphoma, including CHIT1, SIGLEC15, PLA2G2D and TMEM163. Using CIBERSORT to evaluate immune cell infiltration, we found the infiltration level of macrophages was significantly different between EBV+ and EBV- groups and was closely related to different genes. Preliminary clinical specimen verification identified that the expression levels of CHIT1 and TMEM163 were different between EBV+ and EBV- groups. Conclusions: Our data suggest that differences in expression levels of CHIT1 and TMEM163 and macrophage infiltration levels may be important drivers of poor prognosis of EBV+ lymphoma patients. These hub genes may provide new insights into the prognosis and therapeutic target for EBV+ lymphoma.

Detection of Pathogenic Isoforms of IKZF1 in Leukemic Cell Lines and Acute Lymphoblastic Leukemia Samples: Identification of a Novel Truncated IKZF1 Transcript in SUP-B15.

Leukemia-associated alternative splicing of IKZF1 can result in proteins with loss of one to four copies of its N-terminal zinc finger domains (N-ZnF). The best characterized pathogenic splice isoforms, Ik-6 and Ik-8, have been commonly found in BCR-ABL1+ acute lymphoblastic leukemia (ALL) and a subset of BCR-ABL1-like ALL. Infantile and childhood ALL that express these pathogenic IKZF1 isoforms have shown inferior clinical outcomes and can be resistant to tyrosine kinase inhibitors. Using ALL cell lines, we designed and validated a method to detect abnormal IKZF1 transcripts. In the SUP-B15 leukemia cell line, we noted novel IKZF1 transcripts that include both an Ik-6 splice and a transcript with a 14 base pair insertion at the C-terminus. There was also increased IKZF2 protein in SUP-B15 as compared to other ALL lines. Expression of Ik-6 could be suppressed by treatment with the pro-apoptotic type II histone deacetylase inhibitor givinostat. In 17 adult ALL samples, we noted the Ik-6 isoforms in 6 of 15 BCR-ABL1-, and 1 of 2 BCR-ABL1+ cases, with Ik-8 also expressed in one case. Cases with Ik-6 expression showed inferior survival as well as older age at presentation, lower expression of CD10 and more commonly a diploid karyotype.

LncRNA ANRIL promotes cell proliferation, migration and invasion during acute myeloid leukemia pathogenesis via negatively regulating miR-34a.

OBJECTIVE: LncRNA ANRIL (antisense non-coding RNA in the INK4 locus) was highly expressed in acute myeloid leukemia (AML) patients to promote AML pathogenesis. In this study, we aimed to investigate the roles and molecular events of ANRIL associated with AML progression. METHODS: Expression patterns of ANRIL and miR-34a in the bone marrow (BM) samples and cell lines were determined using qRT-PCR. Cell proliferation, apoptosis, migration and invasion of cells with ANRIL knockdown or miR-34a overexpression were assessed by CCK-8, EdU staining, flow cytometry and Transwell assays, respectively. The dual-luciferase reporter assay was employed to validate the relationship between miR-34a and Histone deacetylase 1 (HDAC1). The binding of E2 F1 (E2 F transcription factor 1) with gene promoter was analyzed by ChIP. Furthermore, the tumorigenicity of AML was determined by xenograft transplantation in nude mice. RESULTS: ANRIL was up-regulated both in the BM samples from AML patients and cell lines (HL-60 and THP-1), of which expression was negatively correlated with miR-34a expression. ANRIL knockdown inhibited cell proliferation, migration and invasion but promoted apoptosis of AML cells, while overexpression of miR-34a exerted opposite effects. miR-34a was verified as a downstream gene targeted by ANRIL. Moreover, HDAC1 was a direct target of miR-34a, and HDAC1 overexpression impaired the recruitment of E2 F1 to ASPP2 (apoptosis stimulating proteins of p53) gene promoter. ANRIL knockdown significantly inhibited the tumorigenesis of AML. CONCLUSION: ANRIL promotes AML development through HDAC1-mediated epigenetic suppression of ASPP2 via negatively regulating miR-34a, which might serve as a therapeutic target for AML treatment.

lncRNA CCAT1/miR-490-3p/MAPK1/c-Myc positive feedback loop drives progression of acute myeloid leukaemia.

Acute myeloid leukaemia (AML) is a frequently diagnosed malignancy in adults. Long non-coding RNA (lncRNA) colon cancer-associated transcript 1 (CCAT1) has been well known to play vital roles in multiple malignancies including AML. Unfortunately, the detailed mechanism of CCAT1 in AML progression remains obscure. In this study, we demonstrated that CCAT1 was up-regulated in AML samples while its target, miR-490-3p, was relatively down-regulated. CCAT1 markedly increased viability and metastasis of AML cells, while miR-490-3p had opposite effects. CCAT1 could specifically bind to miR-490-3p and reduce its expression and activity, and MAPK1 was a target gene of miR-490-3p. Overexpressed CCAT1 could induce MAPK1 expression and c-Myc reciprocally increased CCAT1 expression. Our data implied that miR-490-3p could be a novel therapeutic target for AML, and highlights the crucial role of CCAT1/miR-490-3p/MAPK1/c-Myc positive feedback loop in AML progression.

PARP inhibitor re­sensitizes Adriamycin resistant leukemia cells through DNA damage and apoptosis.

Resistance to Adriamycin (ADR) is an increasing problem in the treatment of leukemia and the development of novel therapeutic strategies is becoming increasingly important. Olaparib is a poly (adenosine diphosphate­ribose) polymerase (PARP) 1 inhibitor, which has promising antitumor activity in patients with metastatic breast cancer and germline BRCA mutations. Previously published studies have indicated that Olaparib is able to overcome drug resistance in cancer; however, its underlying mechanism of action is yet to be elucidated. The aim of the present study was to explore the mechanism underlying re­sensitization. Annexin V­propidium iodide staining indicated that the percentage of apoptotic ADR resistant cells was markedly increased and the cell cycle was blocked at the G2/M­phase following treatment with ADR combined with Olaparib, when compared with the control group. The alkaline comet assay demonstrated that ADR combined with Olaparib significantly upregulated the induction of the DNA damage response in ADR­resistant cells. Western blot analysis revealed that the protein expression of γ­H2A histone family member X, cleaved PARP, caspase 3 and cleaved caspase 3 was markedly enhanced, while the cell cycle­associated protein cyclin B1 was downregulated in K562/ADR cells following treatment with a combination of ADR and Olaparib. Similar synergistic cytotoxicity was observed in blood mononuclear cells, which were isolated from patients with chemotherapy­resistant leukemia. As Olaparib is available for clinical use, the results of the present study provide a rationale for the development of Olaparib combinational therapies for cases of ADR resistant leukemia.

Potent induction of apoptosis by givinostat in BCR-ABL1-positive and BCR-ABL1-negative precursor B-cell acute lymphoblastic leukemia cell lines.

We have previously shown that givinostat can induce potent apoptosis in the BCR-ABL1-positive, TP53-wild type B-cell acute lymphoblastic leukemia (B-ALL) cell line SUP-B15. We extend our studies here to two additional B-ALL cell lines, BCR-ABL1-negative CCRF-SB and p210 BCR-ABL1-positive NAML1. Givinostat induced significant cell growth inhibition in both cell lines, with an IC50 of 0.65±0.052µM and 0.25±0.028µM in CCRF-SB and NAML1, respectively. The key signal protein of the BCR-ABL1, Crk-L1, was significantly reduced by givinostat treatment in NAML1. As in SUP-B15, givinostat induced apoptosis in both cell lines but showed different levels of cleavage of the procaspase proteins Casp-3, Casp-7 and PARP. Levels of cell cycle-DNA repair regulator p21, CHK1 and FANCD2 levels were markedly affected by givinostat treatment. These data further enrich our understanding of the mechanisms of the antineoplastic effects of givinostat in B-ALL and provide a preclinical rationale for the inclusion of givinostat or similar agent in leukemia therapy.

Givinostat, a type II histone deacetylase inhibitor, induces potent caspase-dependent apoptosis in human lymphoblastic leukemia.

Unlike chronic myeloid leukemia, patients with acute lymphoblastic leukemia (ALL) with Philadelphia chromosome (Ph+) do not respond well to Imatinib or tyrosine kinase inhibitors (TKI). In addition, TKI might induce resistant mutations in kinase domain (KD) of ABL in patients with relapsed diseases. Of the histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic acid (SAHA) has shown to induce potent cytotoxicity on acute myeloid leukemia cell lines but Givinostat effect on acute lymphoblastic leukemia (ALL) has not been reported. We investigated if Givinostat could exert similar inhibitory effect on SUP-B15, an established B-cell ALL with Philadelphia chromosome (Ph+). Two Ph+ leukemia cell lines, SUP-B15 and an AML cell line K562 were studied in parallel for their responses to Givinostat. Mutation status of TP53 genes was also examined to correlate cellular proliferation and apoptosis. Givinostat significantly inhibited cell proliferation of SUP-B15 (IC50:0.18±0.03µM) and simultaneously inhibited BCR-ABL signal pathway. A remarkable apoptosis was induced by 0.25µM Givinostat in SUP-B15 along with the activation of caspase cascades and increased expression of p21. These inhibitory and proapoptotic effects were not observed in K562 simultaneously treated with Givinostat. Finally our studies showed that TP53 mutation status might determine responder or non-responder to Givinostat in these two Ph+ leukemia cell lines.

Involvement of Fanconi anemia genes FANCD2 and FANCF in the molecular basis of drug resistance in leukemia.

The Fanconi anemia (FA)­associated proteins FANCF and FANCD2 are important components of the FA pathway of DNA crosslink repair. FANCF and FANCD2 have been found to be involved in drug­resistant multiple myeloma, ovarian cancer, non­small­cell lung cancer, and head and neck cancer. However, it is unclear whether these two genes participate in adriamycin (ADR)­resistant leukemia. Therefore, the aim of the current study was to investigate FANCF and FANCD2 expression in drug­resistant and drug­sensitive leukemia cells. Western blot analysis revealed enhanced FANCF expression and monoubiquitination of FANCD2 in ADR­resistant cells. Additionally, it was observed that drug­resistant cells had reduced DNA damage compared with drug­sensitive cells. The results of this study indicate that the FA pathway may confer leukemia resistance to ADR via enhanced DNA interstrand crosslink repair.

The Fanconi anemia/BRCA pathway is involved in DNA interstrand cross-link repair of adriamycin-resistant leukemia cells.

The Fanconi anemia/BRCA (FA/BRCA) pathway plays a vital role in DNA damage repair induced by DNA cross-linking agents and is closely related to drug response in cancer treatment. Here we demonstrate that the FA/BRCA pathway contributes to acquired drug resistance in adriamycin (ADR)-resistant leukemia cell lines, and disruption of this pathway partially reverses the drug resistance. We observed that ADR-resistant cells have reduced DNA interstrand cross-links (ICL) compared with ADR-sensitive cells. Western blot studies demonstrated enhanced FA protein expression in ADR-resistant cells. Using siRNA to knock down FANCF in K562/R drug-resistant cells showed increases in sensitivity to ADR and ADR-induced DNA damage, and demonstrated a direct relationship between the FA/BRCA pathway and drug sensitivity. Overexpression of FANCF in K562 drug-sensitive cells partially reproduced the drug-resistant phenotype. These results show that the FA/BRCA pathway is involved in acquired ADR resistance of leukemia cells. The FA/BRCA pathway may be a new target to reverse ADR resistance in leukemia treatment.

Associations of IL-10 gene polymorphisms with acute myeloid leukemia in Hunan, China.

We investigated the possible association of interleukin-10 (IL-10) single nucleotide polymorphisms (SNPs) and susceptibility to acute myeloid leukemia (AML) in 115 patients and 137 healthy controls. Genetic analysis of IL-10 SNPs at -819 and -592 was carried out with the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) approach. The IL-10 mRNA expression of AML patients and controls with different genotype was detected by real-time quantitative polymerase chain reaction (RT-PCR). Genetic analysis of IL-10 revealed that the -819AA genotype frequencies and the -819A allele frequencies in the AML group were higher than in the controls (59.1% vs 40.9%; 75.6% vs 63.9%, respectively); there were remarkable differences in -819T/C and -592A/C gene distribution (P<0.05) and the TA haploid frequencies were higher in the AML group (75.6% vs 63.9%, P<0.05). IL-10 mRNA expression in incipient AML patients was obvious higher than the non- tumor group and the remission group (7.78?10-3 vs 2.43?10-3, 3.64?10-3, P<0.05).The study suggested that the haploid TA and genotype TA/TA may be associated with AML in Han people in Hunan province.The IL-10 SNPs at -819 and -592 sites were associated with AML and may affect IL-10 mRNA expression in AML patients.

Potentiation of arsenic trioxide-induced apoptosis by 8-bromo-7-methoxychrysin in human leukemia cells involves depletion of intracellular reduced glutathione.

The novel chrysin analog 8-bromo-7-methoxychrysin (BrMC) has been reported to induce apoptosis of various cancer cell lines. Arsenic trioxide (ATO) treatment induces clinical remission in acute promyelocytic leukemia patients. The combination of ATO with other agents has been shown to improve therapeutic effectiveness in vitro and in vivo. In this report, the mechanism of apoptosis induced by treatment with ATO alone or in combination with BrMC was studied in U937, HL-60, and Jurkat cells. Our results demonstrated that BrMC cooperated with ATO to induce apoptosis in human leukemia cells. This co-treatment caused mitochondrial transmembrane potential dissipation and stimulated the mitochondrial apoptotic pathway, as evidenced by cytochrome c release, down-regulation of X-linked inhibitor of apoptosis (XIAP) and Bcl-XL, and up-regulation of Bax. BrMC alone or in combination with ATO, decreased Akt phosphorylation as well as intracellular reduced glutathione (GSH) content. The thiol antioxidant N-acetylcysteine and exogenous GSH restored GSH content and attenuated apoptosis induced by co-treatment with ATO plus BrMC. In contrast, the non-thiol antioxidant butylated hydroxyanisole and mannitol failed to do so. These findings suggest that GSH depletion explains at least in part the potentiation of ATO-induced apoptosis by BrMC.

[Apoptosis-inducing Effect of 8-Bromo-7-Methoxychrysin on K562 cells].

This study was purposed to investigate the apoptosis-inducing effect of 8-bromo-7-methoxychrysin (BrMChR) on leukemia K562 cells as well as the variation of caspase-3 activity and phosphorylated Akt (p-Akt) expression of K562 cells during the process of apoptosis. MTT assay was used to determine the inhibitory effect of BrMChR on proliferation of K562 cells. Cell apoptosis was assayed by AO/EB staining under fluorescent microscope and flow cytometry with Annexin V-FITC/PI staining. The expression level of p-Akt was measured by Western blot. The results showed that BrMChR had the inhibitory effect on proliferation of K562 cells and could induce apoptosis of these cells in dose-dependent manner, and these effects were significantly stronger than ChR. After treatment of K562 cells with 3 µmol/L ChR for 12 hours, the apoptosis rate was only 3.68%, but the apoptosis rate of K562 cells treated with 3 µmol/L BrMChR was 21.8%. In the same time, the caspase-3 activity significantly increased (p < 0.05), but the expression of p-Akt was down-regulated (p < 0.01). It is concluded that BrMChR can induce apoptosis of K562 cells and with effect stronger than chR. P-Akt may participate in the apoptosis process of K562 cells induced by BrMChR.

[Expression and SNP analysis of BRD7 gene in acute leukemia cells].

OBJECTIVE: To evaluate the expression level of BRD7 gene in bone marrow mononuclear cells (BMNCs) in patients with acute leukemia (AL) and to analyze BRD7 single nucleotide polymorphism(SNP). METHODS: RT-PCR was used to detect BRD7 expression in patients with AL and normal bone marrow subjects. Single-strand conformation polymorphism and DNA sequence analysis were also used to identify BRD7 mutation or SNP to investigate the relation between BRD7 and AL. RESULTS: BRD7 mRNA in BMNCs from 52 patients with AL and 30 control subjects was expressed. The mRNA relative expression of BRD7 in patients with AL was higher than that of the control group (P=0.001). Three SNPs (C657A,C495T and A737G) in BRD7 gene coding region (447 approximately 844 bp) were found, and A737G was coupled with C495T . The allele frequencies of SNP C657A were not significantly different between AL and the control group. The genotype and the allele frequencies of the 2 coupled SNPs were significantly different (P<0.01). But there was no significant discrepancy among the mRNA expression levels of AA, AG, and GG genotypes in the leukemia group (P>0.05). CONCLUSION: Expression of BRD7 gene is up-regulated in AL cells. The 2 coupled SNPs (C495T and A737G ) in BRD7 gene coding region (447 approximately 844 bp) are correlated with AL, indicating that SNPs may be one of the genetic susceptibility factors of AL.

[Effect of diallyl disulfide on expression and secretion of VEGF in HL-60 leukemic cells].

The study was aimed to investigate the expression of VEGF mRNA and VEGF protein in HL-60 cells treated with diallyl disulfide (DADS), and to explore the antileukemic mechanism of DADS in respect of VEGF production. Semi-quantitative RT-PCR and ELISA were used to detect the expression of VEGF mRNA and secretion of VEGF protein in HL-60 cell lines treated by DADS respectively. The results showed that the expression of VEGF mRNA and secretion of VEGF protein were found in HL-60 cells. The expression of VEGF mRNA and secretion of VEGF protein in HL-60 cells could be down regulated by treatment with 0.625, 1.25, and 2.5 microg/mL DADS for 48 and 72 hours and the effects had a dose dependent relationship (r > 0.9, P < 0.01). The differences between DADS treated HL-60 cell groups and the control group were statistically significant (P < 0.01), there were also statistically significant differences among three DADS-treated HL-60 cell groups (P < 0.05). It is concluded that DADS effectively inhibits the proliferation of human leukemia cell line HL-60 cells; DADS exerts its antileukemic effects by reduction of the expression of VEGF mRNA and VEGF protein secretion.