Cryptotanshinone induces G1 cell cycle arrest and autophagic cell death by activating the AMP-activated protein kinase signal pathway in HepG2 hepatoma

Abstract Abstract 4999 Natural killer (NK) cell neoplasms, including extranodal NK/T-cell lymphoma, nasal type (ENKL) and aggressive NK cell leukemia (ANKL), show a highly aggressive clinical course with poor response to chemotherapy, and new treatment approaches are urgently needed to improve cure rates. Patients with NK cell neoplasms cluster in Asia and Latin American countries, and the frequency of p53 mutations has been reported to be various by district. We have demonstrated that MDM2 protein was overexpressed in aggressive subclasses of NK cell neoplasms (Sugimoto et al. Jap J Cancer Res. 2002), which suggests that wild-type p53 expressing malignant NK cells may be a good candidate for biologic therapies that abrogate MDM2-p53 interactions and lead to cell death. Nutlin-3 is a small-molecule antagonist of MDM2 that efficiently blocks the MDM2-p53 interaction. In this study, we investigated the effects of nutlin-3 in 3 cell lines of ENKL and ANKL with known p53 mutation status (wt-p53: NK-YS, HANK-1; mt-p53: KHYG-1). Since aggressive NK-cell neoplasms arise in hypoxic environments and usually show an angiodestructive-infiltration pattern resulting in the tissue necrosis, we tried to assess the anti-proliferative effects and molecular mechanisms of nutlin-3 in the hypoxic condition. For hypoxia experiments, cells were cultured under 1.0% O2 for at least 14 days to assure their continuous proliferation and survival. Under hypoxia, more cells were positive to Annexin V than in normoxia, indicating that hypoxic conditions promote apoptosis in NK cell neoplasms. Nutlin-3 treatment in normoxia resulted in a reduction of cell proliferation with G0/G1 cell cycle arrest in a time and concentration-dependent manner in wt-p53 cells (IC50 at 48 hrs; 3.2 μM for NK-YS and 5.0 μM for HANK-1, MTT test). In hypoxia, nutlin-3 further enhanced cell growth inhibition and G0/G1 cell cycle arrest. An increase in the specific apoptosis (sub G1 and annexin V positivity) by nutlin-3 was observed with similar level between normoxia and hypoxia. The mt-p53 KHYG-1 cells demonstrated neither cell cycle arrest nor increase in the apoptotic cell fraction after nutlin-3 treatment. In the wt-p53 NK-YS and HANK-1 cells, nutlin-3 treatment increased the cellular levels of p53, and p53 dependent proteins including p21, MDM2 itself and the proapoptotic BH3-only proteins Noxa and Puma followed by the activation of caspase-9 and caspase-3 regardless of foxygen level. We observed no significant increase in the p53 targets in the mt-p53 overexpressing KHYG-1 cells. L-asparaginase has been demonstrated to induce apoptosis in aggressive NK cell neopplasms. To determine if inhibition of the TP53-MDM2 interaction by nutlin-3 in NK cell neoplasms might potentiate the effects of L-asparaginase, we assessed the effect of combining the two drugs. However, L-asparaginase induced apoptosis only in NK-YS cells, and no synergistic anti-proliferative effect was observed in any of the cell lines analyzed. These findings demonstrate that nutlin-3 successfully activates wt-p53 in NK cell neoplasms leading to the upregulation of traditional targets such as p21 and proapoptotic proteins including Noxa and Puma, and result in apoptotic cell death regardless of oxygen concentration. The data suggest that p53 activators such as nutlin-3 may be considerable for selected patients with wt-p53 NK cell neoplasms. Disclosures: No relevant conflicts of interest to declare.

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Apigenin induces autophagic cell death in human papillary thyroid carcinoma BCPAP cells

Food & Function ◽

10.1039/c5fo00671f ◽

2015 ◽

Vol 6 (11) ◽

pp. 3464-3472 ◽

Cited By ~ 43

Author(s):

Li Zhang ◽

Xian Cheng ◽

Yanyan Gao ◽

Jie Zheng ◽

Qiang Xu ◽

...

Keyword(s):

Cell Cycle ◽

Dna Damage ◽

Cell Death ◽

Papillary Thyroid Carcinoma ◽

Thyroid Carcinoma ◽

Cell Cycle Arrest ◽

Autophagic Cell Death ◽

Ros Generation ◽

Papillary Thyroid ◽

Cycle Arrest

Apigenin-induced autophagic cell death in human papillary thyroid carcinoma BCPAP cells is associated with ROS generation, DNA damage and cell cycle arrest.

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Alisol B 23-acetate-induced HepG2 hepatoma cell death through mTOR signaling-initiated G1 cell cycle arrest and apoptosis: A quantitative proteomic study

Chinese Journal of Cancer Research ◽

10.21147/j.issn.1000-9604.2019.02.12 ◽

2019 ◽

Vol 31 (2) ◽

pp. 375-388

Author(s):

Ji Xia ◽

◽

Qiang Luo ◽

Shengbin Huang ◽

Fuquan Jiang ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Cell Cycle Arrest ◽

Hepatoma Cell ◽

Mtor Signaling ◽

Cycle Arrest ◽

Proteomic Study ◽

G1 Cell Cycle Arrest ◽

Hepg2 Hepatoma Cell

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Both G0/G1 Cell Cycle Arrest and Continuous Activation of MAPK Induce Autophagic Cell Death in bcr-abl-Positive Leukemic Cells.

Blood ◽

10.1182/blood.v106.11.4301.4301 ◽

2005 ◽

Vol 106 (11) ◽

pp. 4301-4301

Author(s):

Yuko Mishima ◽

Yasuhito Terui ◽

Yuji Mishima ◽

Toshihiro Takizawa ◽

Shinya Kimura ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Programmed Cell Death ◽

Cell Cycle Arrest ◽

Cell Line ◽

Immunohistochemical Analysis ◽

K562 Cells ◽

Leukemic Cells ◽

Cycle Arrest ◽

G1 Cell Cycle Arrest

Abstract Back ground; Accumulating evidence suggests that programmed cell death is not defined as apoptosis but cells use different pathways for active self-destruction as reflected by different morphology. Autophagic cell death (APCD), which also be designated type II programmed cell death and appears to be a phylogenetically old phenomenon, is observed in physiological and disease state. Indeed, APCD is observed in several neurodegenerative diseases and cancer cells, however, the APCD in leukemic cells has not been reported yet. Here, we found APCD was occurred in bcr-abl-expressing CML cell lines. Methods; Bcr-abl-positive cell line, K562, were cultured with 2nM of TPA up to 48hours. Subsequently, we examined the morphological change by light microscopy and electronmicroscopy. To evaluate the APCD, TPA-treated K562 cells were labeled with autofluorescent agent Monodansylcadaverin(MDC), which specifically accumulates in autophagosomes and we measured accumulation of autophagosmes in cytoplasm by fluorescent microplate reader. TPA continuous activated MAPK of K562 cells and induced cell cycle arrest in G0/G1 phase. In order to elucidate the role of activation of MAPK in APCD, MAPK in K562 cells were inactivated by MAPK inhibitor, U0126 or dominant negative MAPK-transfection, and then, the cells were treated with TPA. Furthermore, to evaluate the involvement of cell cycle arrest in APCD, bcr-abl-transfected murine leukemic cell line, BAF3, was treated with cell cycle arrest inducer, mimosine. In addition, we investigated the expression of an autophagy-related molecule, Beclin 1 by immunohistochemical analysis. Result; The 48 hours’ treatment with TPA induced cell death in K562 cells, which accumulated plenty of autophagosomes in cytoplasm. MDC labeling assay revealed that accumulation of autophagosomes were increased in time- and dose-dependent manner. On the other hand, MAPK-inactivated K562 cells exhibited resistance to TPA-induced APCD. Furthermore, the bcr-abl-transfected BAF3 cells exhibited continuous activation of MAPK and underwent APCD by cell cycle arrest by mimosine. Immunohistochemical analysis revealed that Beclin 1 shuttled between nuclear and cytoplasms and co-localized with MAPK during APCD progression. Conclusion: APCD in bcr-abl-positive leukemia cells were closely involved in G0/G1 cell cycle arrest and continuous activation of MAPK. These findings are useful in developing the novel strategy to treatment of Ph1 positive leukemia via alternative pathways and another type of cell death.

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JAZ “Targeting” Compounds Induce G1 Cell Cycle Arrest Followed by Cell Death in Human Leukemia Cells

Blood ◽

10.1182/blood.v112.11.2658.2658 ◽

2008 ◽

Vol 112 (11) ◽

pp. 2658-2658

Author(s):

Mingli Yang ◽

George Q. Yang ◽

Jinghua Jia ◽

David Ostrov ◽

W. Stratford May

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Cell Cycle Arrest ◽

Cell Lines ◽

Lymphoblastic Leukemia ◽

Leukemia Cell ◽

Leukemia Cells ◽

Human Leukemia ◽

Cycle Arrest ◽

G1 Cell Cycle Arrest

Abstract JAZ (just another zinc finger protein) was previously identified in our laboratory as a unique ZFP that preferentially binds to double-stranded (ds) RNA rather than dsDNA. We found that interleukin-3 growth factor withdrawal upregulates JAZ expression in factor-dependent hematopoietic cells in association with p53 activation and induction of apoptotic cell death. We recently discovered JAZ as a novel direct, positive regulator of p53 transcriptional activity. The mechanism involves direct binding to p53’s C-terminal (negative) regulatory domain to activate “latent” p53 in response to non-genotoxic stress signals. Our preliminary data indicate that JAZ is differentially expressed in murine and human bone marrow cells and in normal and malignant hematopoietic tissues and cell lines. Thus, we have explored JAZ as a potentially novel molecular target in human leukemia by identifying small molecules that bind and activate JAZ. Using a high-throughput, “molecular docking” strategy, we have screened approximately 240,000 small molecules for their ability to interact with JAZ. Based on the Lipinski Rules for Drug Likeness (molecular characteristics favorable for absorption and permeability), we identified ~70 putative “drug-like” binding molecules with high scores and obtained ~40 of them from the NCI Developmental Therapeutics Program. We first tested their cytotoxic effect on various human leukemia cell lines including wt p53 expressing Reh pre-B lymphoblastic leukemia and Molt-3 T-cell lymphoblastic leukemia cells, and p53-deficient U937 leukemic monocyte lymphoma and KU812 and K562 chronic myelogenous leukemia cells. We have selected four “candidate” JAZ-targeting (J1-J4) compounds for further investigation because they are potent (IC50 = <1 to ~50 μM) in killing leukemia cells in association with upregulation of JAZ protein expression and p53 activation. Since we previously demonstrated that JAZ can induce G1 cell cycle arrest prior to apoptosis in NIH3T3 mouse fibrablast cells in association with upregulation of p21, dephosphorylation of Rb and repression of cyclin A, we have tested these J-compounds for their potential effect on cell cycle progression. Drug treatment followed by flow cytometry analysis was carried out in human leukemia cell lines. Results reveal that the J2, J3 and J4 but not J1 compounds induce significant G1 cell cycle arrest followed by cell death in a dose- and time-dependent manner (e.g. an increase in the G1 population by up to 35 % at 24 hr following the treatment at doses of 0.1 to 50 μM). These data indicate that the J2-J4 compounds can not only induce leukemia cell killing but also mediate growth arrest. Interestingly, J3 and J4 are FDA-approved drugs (for the treatment of non-cancer diseases), suggesting a potentially novel role for these clinically available drugs as therapy for hematologic malignancies. Therefore, while further in vitro and in vivo characterization remains to be carried out, the JAZ-“targeting” compound(s) points the way to develop a potentially novel therapeutic strategy targeting JAZ to treat human leukemia.

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