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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

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Summary

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.

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Abbreviations

5k :

4β-{[4-(pyrrolidin-1-ylmethyl)phenyl]amino}-4′-O-Demethyl-4-Epipodophyllotoxin

DAPI:

4′6-diamidino-2-phenylindole

JC-1:

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

ΔΨ:

mitochondrial membrane potential

MDR:

multidrug resistance

DDR:

DNA damage response

Topo II:

Topoisomerase II

VCR:

vincristine

RTV:

relative tumor volume

ATM:

Ataxia-Telangiectasia Mutated

ATR:

Ataxia-Telangiectasia and Rad3 related

p-:

phosphorylated

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Authors and Affiliations

  1. Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Zhejiang University, Room 113, Building of College of Pharmaceutical Sciences, Zijin’gang campus, Hangzhou, People’s Republic of China, 310058

    Danqing Xu, Ji Cao, Shijing Qian, Lin Li, Qinjie Weng, Jianshu Lou, Difeng Zhu, Hong Zhu, Qiaojun He & Bo Yang

  2. Zhejiang University-Ecole Normole Superienre Joint Laboratory Medicinal Chemistry, Zhejiang University, Hangzhou, People’s Republic of China

    Chunqi Hu & Yongzhou Hu

Authors
  1. Danqing Xu
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  2. Ji Cao
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  3. Shijing Qian
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  4. Lin Li
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  5. Chunqi Hu
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  6. Qinjie Weng
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  7. Jianshu Lou
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  8. Difeng Zhu
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  9. Hong Zhu
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  10. Yongzhou Hu
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  11. Qiaojun He
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  12. Bo Yang
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Corresponding authors

Correspondence to Qiaojun He or Bo Yang.

Additional information

This work was supported by the Scientific Research Foundation of Zhejiang Provincial Health Bureau (No. 2008A043), Natural Science Foundation of Zhejiang Province (Z2090053).

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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)

High Resolution Image (TIFF 9273 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)

High Resolution Image (TIFF 24 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)

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Xu, D., Cao, J., Qian, S. et al. 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. Invest New Drugs 29, 786–799 (2011). https://doi.org/10.1007/s10637-010-9423-5

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  • DOI: https://doi.org/10.1007/s10637-010-9423-5

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