Investigational New Drugs

, Volume 29, Issue 5, pp 786–799 | Cite as

5k, a novel β-O-demethyl-epipodophyllotoxin analogue, inhibits the proliferation of cancer cells in vitro and in vivo via the induction of G2 arrest and apoptosis

  • Danqing Xu
  • Ji Cao
  • Shijing Qian
  • Lin Li
  • Chunqi Hu
  • Qinjie Weng
  • Jianshu Lou
  • Difeng Zhu
  • Hong Zhu
  • Yongzhou Hu
  • Qiaojun He
  • Bo Yang


Etoposide (VP-16), a topoisomerase II (Topo II) inhibitor, has been widely used to treat malignancies. Its clinical application, however, has been hindered by the rise of acquired multidrug resistance (MDR). Here, we report that 4β-{[4-(pyrrolidin-1-ylmethyl)phenyl]amino}-4′-O-Demethyl-4-Epipodophyllotoxin (5k), a novel β-O-demethyl-epipodophyllotoxin analogue, possesses higher antitumor activity than its parent compound (VP-16) in a panel of various human tumor cell lines. More importantly, it was also effective against MDR cells both in vitro and in vivo. Using a KB/VCR MDR tumor xenograft model that overexpresses P-gp, 5k (2.5 mg/kg) exhibited a 2.4-fold higher growth inhibition rate versus VP-16 (5 mg/kg). In contrast, 5k and VP-16 displayed similar antitumor activities in a KB tumor xenograft model. Molecular and cellular mechanism studies revealed that 5k targeted Topo II by trapping DNA-Topo II cleavage complexes that could directly cause DNA damage. There were two distinct cellular responses to DNA damage elicited by the treatment with 5k: at low concentrations (20–80 nM), mitotic entry was arrested through the suppression of the activity of Cyclin B1/Cdc 2 complexes via the ATM/ATR signaling pathway; at high concentrations (1.25–5.00 μM), 5k-induced apoptotic signaling was mediated by the mitochondrial death pathways. Collectively, these data demonstrate the potential value of 5k as an antitumor drug candidate that should be further developed.


Topo II inhibitor MDR DNA damage G2 arrest Apoptosis 







5,5′6,6′-tetrachloro-1,1′3,3′-tetraethylbenzimidazol-carbocyanine iodide


mitochondrial membrane potential


multidrug resistance


DNA damage response

Topo II

Topoisomerase II




relative tumor volume


Ataxia-Telangiectasia Mutated


Ataxia-Telangiectasia and Rad3 related



Supplementary material

10637_2010_9423_Fig8_ESM.gif (109 kb)
Figure S1

The chromatogram of 5k. To validate the purity, liquid chromatography–mass spectrum analysis was performed. UV absorbance at 254 nm, the y-axis (Au) means the absorbance, while the x-axis represents elution time (min). Area percent was calculated, the purity of 5k was 99.86%. (GIF 109 kb)

10637_2010_9423_Fig1_ESM.tif (9.1 mb)
High Resolution Image (TIFF 9273 kb)
10637_2010_9423_Fig9_ESM.gif (80 kb)
Figure S2

Effects of 5k and VP-16 on P-gp ATPase activity. The decrease in luminescence of untreated samples compared to samples plus Na3VO4 represents basal P-gp ATPase activity. The P-gp ATPase activity in response to the treatment of verapamil (200 μM), 5k (100 μM, 25 μM), VP-16(100 μM, 25 μM) were expressed as the percentage of basal activity. (GIF 79 kb)

10637_2010_9423_Fig2_ESM.tif (25 kb)
High Resolution Image (TIFF 24 kb)
10637_2010_9423_Fig10_ESM.gif (144 kb)
Figure S3

Morphological alterations of KB cells after 5.0 μM 5k exposition in the presence or absence of 100 μM Boc-D-FMK pretreatment for 30 min, detected by microscope at the same magnification. (GIF 144 kb)

10637_2010_9423_Fig3_ESM.tif (6.4 mb)
High Resolution Image (TIFF 6516 kb)


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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Danqing Xu
    • 1
  • Ji Cao
    • 1
  • Shijing Qian
    • 1
  • Lin Li
    • 1
  • Chunqi Hu
    • 2
  • Qinjie Weng
    • 1
  • Jianshu Lou
    • 1
  • Difeng Zhu
    • 1
  • Hong Zhu
    • 1
  • Yongzhou Hu
    • 2
  • Qiaojun He
    • 1
  • Bo Yang
    • 1
  1. 1.Institute of Pharmacology & Toxicology, College of Pharmaceutical SciencesZhejiang UniversityHangzhouPeople’s Republic of China
  2. 2.Zhejiang University-Ecole Normole Superienre Joint Laboratory Medicinal ChemistryZhejiang UniversityHangzhouPeople’s Republic of China

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