METTL1 overexpression is correlated with poor prognosis and promotes hepatocellular carcinoma via PTEN.

RNA methylation is emerging as an important regulator of gene expression. Dysregulation of methyltransferase that is essential for RNA modification contributes to the development and progression of human cancers. Here we show that methyltransferase-like 1 (METTL1) is upregulated in hepatocellular carcinoma (HCC) and exhibits oncogenic activities via PTEN/AKT signaling pathway. High expression of METTL1 is correlated with larger tumor size, higher serum AFP level, tumor vascular invasion, and poor prognosis in two independent cohorts containing 892 patients with HCC. Multivariate analyses suggest METTL1 as an independent factor for unfavorable overall survival. In vitro studies demonstrate that METTL1 overexpression promotes cell proliferation and migration, whereas its knockdown results in opposite phenotypes. Gene set enrichment analysis (GSEA) indicates PTEN pathway is activated in patients with low METTL1 expression. Ectopic expression of PTEN or inhibition of AKT activity significantly attenuates the METTL1-mediated malignant phenotypes. In clinical samples, METTL1 expression is reversely associated with PTEN expression. Combination of low METTL1 expression and high PTEN expression is significantly correlated with overall survival, more so than either METTL1 or PTEN expression alone. Collectively, our data suggest that METTL1 serves as a promising prognostic biomarker and that targeting METTL1/PTEN axis may provide therapeutic potential in HCC intervention. KEY MESSAGES: METTL1 is upregulated in HCC and correlated with poor outcomes. METTL1 promotes cell proliferation and migration in HCC. METTL1 exerts oncogenic activities via suppression of PTEN signaling.

Salinomycin induces apoptosis and differentiation in human acute promyelocytic leukemia cells.

At present, acute promyelocytic leukemia (APL) is the most curable form of acute myeloid leukemia and can be treated using all-trans retinoic acid and arsenic trioxide. However, the current treatment of APL is associated with some issues such as drug toxicity, resistance and relapse. Therefore, other strategies are necessary for APL treatment. In the present study, we investigated the effects of salinomycin (SAL) on APL cell lines NB4 and HL-60 and determined its possible mechanisms. We observed that SAL inhibited cell proliferation, as determined by performing Cell Counting Kit-8 (CCK-8) assay, promoted cell apoptosis, as determined based on morphological changes, and increased Annexin V/propidium iodide (PI)-positive apoptotic cell percentage. Treatment with SAL increased Bax/Bcl-2 and cytochrome c expression and activated caspase-3 and -9, thus leading to poly(ADP-ribose) polymerase (PARP) cleavage and resulting in cell apoptosis. These results revealed that SAL induced cell apoptosis through activation of the intrinsic apoptosis pathway. The present study is the first to show that SAL induced the differentiation of APL cells, as determined based on mature morphological changes, increased NBT-positive cell and CD11b-positive cell percentages and increased CD11b and C/EBPß levels. Furthermore, SAL decreased the expression of ß-catenin and its targets cyclin D1 and C-myc. Results of immunofluorescence analysis revealed that SAL markedly decreased the ß-catenin level in both the nucleus and cytoplasm. Combination treatment with SAL and IWR-1, an inhibitor of Wnt signaling, synergistically triggered SAL-induced differentiation of APL cells. These findings demonstrated that SAL effectively inhibited cell proliferation accompanied by induction of apoptosis and promotion of cell differentiation by inhibiting Wnt/ß-catenin signaling. Collectively, these data revealed that SAL is a potential drug for treatment of APL.

Effects of lapatinib on cell proliferation and apoptosis in NB4 cells.

Acute promyelocytic leukemia (APL), characterized by the presence of the promyelocytic leukemia (PML)-retinoic acid α receptor (RARα) fusion protein, responds to treatment with all-trans retinoic acid (ATRA) and arsenic trioxide (ATO). However, drug resistance and side effects restrict the application of these reagents. Hence, the development of novel therapeutic drugs for APL treatment is critical. Lapatinib, a small-molecule tyrosine kinase inhibitor, has been used in the treatment of different tumors. However, it is unclear whether lapatinib exerts antitumor effects on APL. The present study investigated the antitumor effects and potential mechanisms of lapatinib on NB4 cells derived from APL. Cell Counting Kit-8 assay and colony forming analysis indicated that lapatinib inhibited NB4 cell proliferation in a dose-dependent manner. Flow cytometry analysis revealed that lapatinib induced cell cycle arrest at the S phase and promoted cell apoptosis. Furthermore, Liu's staining and Hoechst 33258 staining revelaed that lapatinib treatment induced an apoptotic nuclear phenomenon. Furthermore, lapatinib induced apoptosis by decreasing Bcl-2 and PML-RARα levels, and by increasing the levels of Bax, cleaved PARP, cleaved caspase-3 and cleaved caspase-9. In addition, lapatinib increased the levels of phospho-p38 MAPK and phospho-JNK, and decreased the levels of phospho-Akt. The p38 inhibitor PD169316 partially blocked lapatinib-induced proliferation inhibition and apoptosis, whereas the JNK inhibitor SP600125 had no such effects. Therefore, treatment with lapatinib may be a promising strategy for APL therapy.

[Mechanism underlying inhibition of proliferation and promotion of apoptosis by lapatinib in HL60 cells].

Objective To investigate the effect of lapatinib on cell proliferation and apoptosis in acute myeloid leukemia HL60 cells in vitro and the related molecular mechanisms. Methods The HL60 cells were treated with 5, 10, 15 µmol/L lapatinib for 24 hours, and then morphological changes of the cells were observed under optical microscope. CCK-8 assay was used to assess the cell viability. Colony formation assay was performed to detect the cell proliferation ability. Cell apoptosis labeled by annexinV-FITC/PI were analyzed by flow cytometry. Wright modified LIU's staining and Hoechst33342 fluorescent staining were used to observe the morphology of the nucleus. Western blotting was utilized to detect the expressions of Bax, Bcl2, caspase-3, caspase-9, cleaved caspase-3, cleaved caspase-9, cleaved poly-ADP-ribose polymerase (cleaved PARP), cell proliferation regulating inhibitor of protein phosphatase 2A (CIP2A), c-MYC, AKT and p-AKT. Results Compared with the control group, lapatinib inhibited cell proliferation and promoted apoptosis, induced nuclear fragmentation, chromatin condensation of HL60 cells in a dose-dependent manner. Meanwhile, it down-regulated the expression of Bcl2, up-regulated the levels of Bax, cleaved caspase-3, cleaved caspase-9 and cleaved PARP, and decreased the levels of CIP2A, p-AKT and c-MYC. Conclusion Lapatinib could inhibit cell proliferation and promote apoptosis in HL60 cells by inhibiting the CIP2A/AKT/c-MYC signal pathway.

Epigallocatechin-3-gallate induces apoptosis in acute promyelocytic leukemia cells via a SHP-1-p38α MAPK-Bax cascade.

Acute promyelocytic leukemia (APL) is characterized by a specific chromosomal translation, resulting in a fusion gene that affects the differentiation, proliferation and apoptosis of APL cells. Epigallocatechin-3-gallate (EGCG), a catechin, exhibits numerous biological functions, including antitumor activities. Previous studies have reported that EGCG induces apoptosis in NB4 cells. However, the molecular mechanism underlying EGCG-induced apoptosis remains unclear. The present study aimed to determine the molecular basis of EGCG-induced apoptosis in NB4 cells. EGCG treatment significantly inhibited the viability of NB4 cells in a dose-dependent manner. In addition, EGCG treatment induced apoptosis and increased the levels of (Bcl-2-like protein 4) Bax protein expression. Moreover, EGCG treatment was able to increase phosphorylated (p)-p38α mitogen-activated protein kinase (MAPK) and Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1) expression. Pretreatment with PD169316 (a p38 MAPK inhibitor) partially blocked EGCG-induced apoptosis and inhibited EGCG-mediated Bax expression. Similarly, pretreatment with NSC87877, an inhibitor of SHP-1, partially blocked EGCG-induced apoptosis and inhibited EGCG-mediated increases in p-p38α MAPK and Bax expression. Therefore, the results of the present study indicate that EGCG is able to induce apoptosis in NB4 cells via the SHP-1-p38αMAPK-Bax cascade.

Verteporfin Inhibits Cell Proliferation and Induces Apoptosis in Human Leukemia NB4 Cells without Light Activation.

Background and Aims: Verteporfin (VP), clinically used in photodynamic therapy for neovascular macular degeneration, has recently been proven a suppressor of yes-associated protein (YAP) and has shown potential in anticancer treatment. However, its anti-human leukemia effects in NB4 cells remain unclear. In this study, we investigated the effects of VP on proliferation and apoptosis in human leukemia NB4 cells. Methods: NB4 cells were treated with VP for 24 h. The effects of VP on cell proliferation were determined using a Cell-Counting Kit-8 assay (CCK-8) assay and colony forming assay. Apoptosis and cell cycle were evaluated by flow cytometry (FCM). The protein levels were detected by western blot. Results: We found that VP inhibited the proliferation of NB4 cells in a concentration and time-dependent manner. FCM analysis showed that VP induced apoptosis in a concentration dependent manner and that VP treatment led to cell cycle arrest at G0/G1 phase. Moreover, VP significantly decreased the protein expression of YAP, p-YAP, Survivin, c-Myc, cyclinD1, p-ERK, and p-AKT. In addition, VP increased the protein expression of cleaved caspase3, cleaved PARP, Bax, and p-p38 MAPK. Conclusions: VP inhibited the proliferation and induced apoptosis in NB4 cells.

Effect of YAP Inhibition on Human Leukemia HL-60 Cells.

Background: Yes-associated protein (YAP), the nuclear effector of the Hippo pathway, is a candidate oncoprotein and participates in the progression of various malignancies. However, few reports have examined the effect of YAP inhibition in human leukemia HL-60 cells. Methods: We examined the effects of YAP knockdown or inhibition using short hairpin RNA (shRNA) or verteporfin (VP), respectively. Western blot assays were used to determine the expression levels of YAP, Survivin, cyclinD1, PARP, Bcl-2, and Bax. Cell proliferation was assessed using the cell counting kit (CCK-8) assay. Cell cycle progression and apoptosis were evaluated by flow cytometry, and apoptotic cell morphology was observed by Hoechst 33342 staining. Results: Knockdown or inhibition of YAP led to cell cycle arrest at the G0/G1 phase and increased apoptosis, inhibited cell proliferation, increased levels of Bax and cleaved PARP, and decreased levels of PARP, Bcl-2, Survivin, and cyclinD1. Moreover, Hoechst 33342 staining revealed increased cell nuclear fragmentation. Conclusion: Collectively, these results show that inhibition of YAP inhibits proliferation and induces apoptosis in HL-60 cells. Therefore, a novel treatment regime involving genetic or pharmacological inhibition of YAP could be established for acute promyelocytic leukemia.

Epigallocatechin-3-gallate promotes all-trans retinoic acid-induced maturation of acute promyelocytic leukemia cells via PTEN.

Acute promyelocytic leukemia (APL) is a distinctive subtype of acute myeloid leukemia (AML) in which the hybrid protein promyelocytic leukemia protein/retinoic acid receptor α (PML/RARα) acts as a transcriptional repressor impairing the expression of genes that are critical to myeloid cell mutation. We aimed at explaining the molecular mechanism of green tea polyphenol epigallocatechin-3-gallate (EGCG) enhancement of ATRA-induced APL cell line differentiation. Tumor suppressor phosphatase and tensin homolog (PTEN) was found downregulated in NB4 cells and rescued by proteases inhibitor MG132. A significant increase of PTEN levels was found in NB4, HL-60 and THP-1 cells upon ATRA combined with EGCG treatment, paralleled by increased myeloid differentiation marker CD11b. EGCG in synergy with ATRA promote degradation of PML/RARα and restores PML expression, and increase the level of nuclear PTEN. Pretreatment of PTEN inhibitor SF1670 enhances the PI3K signaling pathway and represses NB4 cell differentiation. Moreover, the induction of PTEN attenuated the Akt phosphorylation levels, pretreatment of PI3K inhibitor LY294002 in NB4 cells, significantly augmented the cell differentiation and increased the expression of PTEN. These results therefore indicate that EGCG targets PML/RARα oncoprotein for degradation and potentiates differentiation of promyelocytic leukemia cells in combination with ATRA via PTEN.

Shikonin suppresses proliferation and induces apoptosis in human leukemia NB4 cells through modulation of MAPKs and c­Myc.

Acute promyelocytic leukemia (APL) is a special subtype of acute myeloid leukemia that responds to treatment with all­trans retinoic acid and arsenic trioxide. However, severe side effects and drug resistance limit the effectiveness of these treatments. Hence, new drugs for APL are required urgently. Shikonin, an active naphthoquinone derived from the Chinese medical herb Zi Cao exerts antitumor activity in several cancers. In the present study, the effects of shikonin on proliferation and apoptosis in NB4 cells, as well as related mechanisms were assessed. Treatment of NB4 cells with shikonin inhibited proliferation in a concentration­ and time­dependent manner. The cell cycle was arrested in the G1 phase. NB4 cells treated with shikonin exhibited more apoptosis and higher levels of cleaved caspase­3 and poly ADP­ribose polymerase than control cells. Western blotting results demonstrated that the expression of p­p38 mitogen­activated protein kinase (p­p38MAPK) and p­c­Jun N­terminal kinase (p­JNK) was increased significantly by shikonin treatment, while the expression of p­ERK and c­Myc was decreased. In summary, these findings indicated that shikonin inhibited cell proliferation and induced apoptosis partly through modulation of the MAPKs and downregulation of c­Myc.

NLS-RARα Inhibits the Effects of All-trans Retinoic Acid on NB4 Cells by Interacting with P38α MAPK.

Nuclear localization signal retinoic acid receptor alpha(NLS-RARα), which forms from the cleavage of promyelocytic leukemia-retinoic acid receptor alpha(PML-RARα) protein by neutrophil elastase(NE), possesses an important role in the occurrence and development of acute promyelocytic leukemia(APL). However, the potential mechanism underlying the effects of NLS-RARα on APL is still not entirely clear. Here, we investigated the effects of NLS-RARα on APL NB4 cells and its mechanism. We found that all-trans retinoic acid(ATRA) could promote differentiation while inhibit proliferation of APL NB4 cells via upregulating the expression of phosphorylated p38α mitogen-activated protein kinase(p-p38α MAPK). We also found that NLS-RARα could inhibit differentiation while accelerate proliferation of NB4 cells via downregulating the expression of p-p38α protein in the presence of ATRA. Furthermore, immunofluorescence and co-immunoprecipitation assays confirmed NLS-RARα interacted with p38α protein directly. Finally, application of PD169316, an inhibitor of p38α protein, suggested that recruitment p38α-combinded NLS-RARα by ATRA eventually caused activation of p38α protein. In summary, our study demonstrated that ATRA cound promote differentiation while inhibit proliferation of APL NB4 cells via activating p38α protein after recruiting p38α-combinded NLS-RARα, while NLS-RARα could inhibit the effects of ATRA in the process.

Effects of LG268 on Cell Proliferation and Apoptosis of NB4 Cells.

AIMS: To investigate the effect of LG100268 (LG268) on cell proliferation and apoptosis in NB4 cells. METHODS: NB4 cells were treated with LG268 for 24 h or 48 h. The effect of LG268 on cell proliferation was assessed by the CCK-8 assay and colony-forming assay. Apoptosis and cell cycle were evaluated by flow cytometry. The protein expression levels of Survivin, PARP, c-Myc, cyclin D1, ERK, p-ERK, p38 MAPK, and p- p38 MAPK were detected by western blot. RESULTS: We found that LG268 inhibited the proliferation of NB4 cells in a dose-dependent manner. Flow cytometry analysis showed that LG268 accelerated apoptosis in NB4 cells in a time- dependent manner and that LG268 treatment led to cell cycle arrest at G0/G1 phase. Moreover, LG268 significantly decreased the protein levels of Survivin, c-Myc, and cyclinD1. Cleaved PARP was observed in the LG268 treatment group but not in the control group. In addition, LG268 increased the phosphorylation level of p38 MAPK and decreased the phosphorylation level of ERK. CONCLUSIONS: LG268 inhibited cell proliferation and promoted cell apoptosis in NB4 cells.