Deoxypodophyllotoxin induces G2/M cell cycle arrest and apoptosis in HeLa cells.

The natural flavolignan deoxypodophyllotoxin (DPPT) inhibits tubulin polymerization and induces cell cycle arrest at G(2)/M, followed by apoptosis. However, the precise mechanism of DPPT action is currently unknown. Here, we investigated the mechanism by which DPPT treatment of HeLa cervical carcinoma cells induces cell cycle arrest and apoptosis. We show that DPPT treatment inhibits cell viability in a dose-dependent manner and that this reduction in cell viability results from cell cycle arrest at G(2)/M phase, accompanied by an increase in apoptotic cell death. The induction of apoptosis by DPPT was confirmed by visualization of morphologic changes and internucleosomal DNA fragmentation. In addition, DPPT causes p53 and Bax to accumulate, accompanied by activation of DNA damage-sensing kinases, including ataxia-telangiectasia mutated (ATM) kinase and Chk2. Furthermore, DPPT activates caspase-3 and -7, suggesting that caspase-mediated pathways are involved in DPPT-induced apoptosis. Levels of the tumor suppressor PTEN were up-regulated during DPPT treatment, coincident with Akt inhibition. Together, these data suggest that DPPT induces G(2)/M cell-cycle arrest followed by apoptosis through multiple cellular processes, involving the activation of ATM, upregulation of p53 and Bax, activation of caspase-3 and -7, and accumulation of PTEN resulting in the inhibition of the Akt pathway.

[1]  C. Monneret,et al.  Hemi-synthesis and biological activity of new analogues of podophyllotoxin. , 2002, Bioorganic & medicinal chemistry.

[2]  J. Downward,et al.  Suppression of Egr‐1 transcription through targeting of the serum response factor by oncogenic H‐Ras , 2006, The EMBO journal.

[3]  K. Bhalla.  Microtubule-targeted anticancer agents and apoptosis , 2003, Oncogene.

[4]  P. Russell,et al.  Human Wee1 kinase inhibits cell division by phosphorylating p34cdc2 exclusively on Tyr15. , 1993, The EMBO journal.

[5]  R. Margolis,et al.  Microtubule treadmilling: what goes around comes around , 1998, BioEssays : news and reviews in molecular, cellular and developmental biology.

[6]  Mathias Schmidt,et al.  Mitotic drug targets and the development of novel anti-mitotic anticancer drugs. , 2007, Drug resistance updates : reviews and commentaries in antimicrobial and anticancer chemotherapy.

[7]  Scott W. Lowe,et al.  Apoptosis A Link between Cancer Genetics and Chemotherapy , 2002, Cell.

[8]  P. Cohen,et al.  Mechanism of activation of protein kinase B by insulin and IGF‐1. , 1996, The EMBO journal.

[9]  J. Loike,et al.  Structure-activity study of the inhibition of microtubule assembly in vitro by podophyllotoxin and its congeners. , 1978, Cancer research.

[10]  S. R. Datta,et al.  Akt Phosphorylation of BAD Couples Survival Signals to the Cell-Intrinsic Death Machinery , 1997, Cell.

[11]  T. Tursz,et al.  Overexpression of MDM2, due to enhanced translation, results in inactivation of wild-type p53 in Burkitt's lymphoma cells , 1998, Oncogene.

[12]  Y. Shen,et al.  p53-dependent apoptosis pathways. , 2001, Advances in cancer research.

[13]  M. Jordan,et al.  Microtubules as a target for anticancer drugs , 2004, Nature Reviews Cancer.

[14]  B. Gallagher.  Microtubule-stabilizing natural products as promising cancer therapeutics. , 2007, Current medicinal chemistry.

[15]  Se Hyun Kim,et al.  Clozapine, a neuroleptic agent, inhibits Akt by counteracting Ca2+/calmodulin in PTEN-negative U-87MG human glioblastoma cells. , 2006, Cellular signalling.

[16]  L. Schriml,et al.  Mutational spectra of PTEN/MMAC1 gene: a tumor suppressor with lipid phosphatase activity. , 1999, Journal of the National Cancer Institute.

[17]  V. Vandana,et al.  9-Deoxopodophyllotoxin derivatives as anti-cancer agents. , 1999, Bioorganic & medicinal chemistry letters.

[18]  D. Housman,et al.  p53-dependent apoptosis modulates the cytotoxicity of anticancer agents , 1993, Cell.

[19]  J. Romashkova,et al.  NF-κB is a target of AKT in anti-apoptotic PDGF signalling , 1999, Nature.

[20]  John Calvin Reed,et al.  Regulation of cell death protease caspase-9 by phosphorylation. , 1998, Science.

[21]  T. Imbert.  Discovery of podophyllotoxins. , 1998, Biochimie.

[22]  T. Moon,et al.  Dual inhibition of cyclooxygenases-2 and 5-lipoxygenase by deoxypodophyllotoxin in mouse bone marrow-derived mast cells. , 2004, Biological & pharmaceutical bulletin.

[23]  T. Mak,et al.  Regulation of PTEN transcription by p53. , 2001, Molecular cell.

[24]  S. Elledge,et al.  DNA damage-induced activation of p53 by the checkpoint kinase Chk2. , 2000, Science.

[25]  T. Masuda,et al.  Flow cytometric estimation on cytotoxic activity of leaf extracts from seashore plants in subtropical Japan: isolation, quantification and cytotoxic action of (‐)‐deoxypodophyllotoxin , 2002, Phytotherapy research : PTR.

[26]  M. Gordaliza,et al.  Podophyllotoxin: distribution, sources, applications and new cytotoxic derivatives. , 2004, Toxicon : official journal of the International Society on Toxinology.

[27]  Chang Gun Kim,et al.  p21 Waf1/Cip1 expression by curcumin in U-87MG human glioma cells: role of early growth response-1 expression. , 2008, Cancer research.

[28]  Mari Haramoto,et al.  Isolation of Apoptosis- and Differentiation-Inducing Substances toward Human Promyelocytic Leukemia HL-60 Cells from Leaves of Juniperus taxifolia , 2008, Bioscience, biotechnology, and biochemistry.

[29]  P. Nurse.  Universal control mechanism regulating onset of M-phase , 1990, Nature.

[30]  Y. Lazebnik,et al.  Cleavage of poly(ADP-ribose) polymerase by a proteinase with properties like ICE , 1994, Nature.

[31]  Enrique Casado,et al.  PI3K/Akt signalling pathway and cancer. , 2004, Cancer treatment reviews.

[32]  Karl-Heinz Altmann,et al.  Anticancer drugs from nature--natural products as a unique source of new microtubule-stabilizing agents. , 2007, Natural product reports.

[33]  Marc W. Kirschner,et al.  cdc25 is a specific tyrosine phosphatase that directly activates p34cdc2 , 1991, Cell.

[34]  N. Banik,et al.  Differential sensitivity of human glioblastoma LN18 (PTEN-positive) and A172 (PTEN-negative) cells to Taxol for apoptosis , 2008, Brain Research.

[35]  S. Elledge,et al.  Linkage of ATM to cell cycle regulation by the Chk2 protein kinase. , 1998, Science.

[36]  H. Piwnica-Worms,et al.  The human Myt1 kinase preferentially phosphorylates Cdc2 on threonine 14 and localizes to the endoplasmic reticulum and Golgi complex , 1997, Molecular and cellular biology.

[37]  K. Hideg,et al.  EF24 Induces G2/M Arrest and Apoptosis in Cisplatin-resistant Human Ovarian Cancer Cells by Increasing PTEN Expression* , 2007, Journal of Biological Chemistry.

[38]  B. Ahn,et al.  Deoxypodophyllotoxin; the cytotoxic and antiangiogenic component from Pulsatilla koreana. , 2002, Planta medica.

[39]  G. Salvesen,et al.  The apoptosome: signalling platform of cell death , 2007, Nature Reviews Molecular Cell Biology.

[40]  J. Pezzuto,et al.  Natural products as inhibitors of carcinogenesis , 2008 .

[41]  K. Sudo,et al.  Inhibitory Effects of Podophyllotoxin Derivatives on Herpes Simplex Virus Replication , 1998, Antiviral chemistry & chemotherapy.

[42]  M. Barbacid,et al.  Mammalian cyclin-dependent kinases. , 2005, Trends in biochemical sciences.

[43]  Y. Nakano,et al.  Antiproliferative constituents in umbelliferae plants. III. Constituents in the root and the ground part of Anthriscus sylvestris Hoffm. , 1998, Chemical & pharmaceutical bulletin.

[44]  Y Taya,et al.  Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. , 1998, Science.