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.

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.

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.

PML(NLS¯) protein: A novel marker for the early diagnosis of acute promyelocytic leukemia.

Promyelocyte leukemia­retinoic acid receptor α (PML­RARα) is known as a fusion gene of acute promyelocytic leukemia (APL). Previous studies have reported that neutrophil elastase (NE) cleaves PML­RARα in early myeloid cells, which leads to the removal of the nuclear localization signal (NLS) in PML and increases the incidence of APL. The resultant PML without the NLS is termed PML(NLS­). The aim of the present study was to verify the existence and location of the PML(NLS­) protein in NB4 cells. NB4 cells underwent electroporation with the pCMV­HA­NE plasmid to form NB4­HA­NE cells, which were then transplanted to produce tumors in nude mice and samples were collected from patients with APL. Western blot analysis, an immunofluorescence assay, confocal laser microscopy and immunohistochemistry were performed to detect the expression and localization of the PML(NLS­) protein. The findings demonstrated that PML(NLS­) was detectable in the cytoplasm of NB4­HA­NE cells, the tumors in nude mice and in neutrophils from patients with APL. This indicated that PML(NLS­) may be an effective and novel target for the diagnosis of APL.

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.