Page 13 - I. Chemical biology and drug discovery

The Natural alkaloid compound E inhibits both stem cell and non-stem-cell populations
in human cancer cells by targeting heat shock protein 70

Seung Yeob Hyun¹, Huong Thuy Le¹, Hye Young Min¹, Honglan Pei¹, Yijae Lim¹, Injae Song¹, Yen T. Nguyen¹,
Suckchang Hong¹, Byung Woo Han¹, Ho Young Lee¹ *
,
¹ College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea 151-742

Abstrac MATERIAL & METHODS
t
1. Effect of Compound E on CSCs and non – CSCs are examined
Cancer stem cells (CSCs) play critical roles in tumor recurrence and drug by ALDH assay, FACS analysis, sphere forming assay, MTT,
resistance. However, anticancer drugs targeting CSCs are limited due to the poor Anchorage dependent assay, FACS and Hoechst staining.
understanding of the underlying biology in CSCs. Here, we applied reporter
vectors carrying NANOG or POUSF1 promoters and selected ALDH high and 2. Effect of Compound E on HSPs interaction and Hsp70 ATP
sphere-forming subpopulations from non-small-cell lung cancer (NSCLC) and
patient-derived xenografts (PDXs) to obtain potential CSCs. Using these CSCs, Pocket binding are analyzed by Immunoprecipitation and
we demonstrated that activation of the heat shock protein (Hsp) system in CSCs ATP binding assay.
and pharmacological or genomic approaches that target the Hsp system eliminate
CSCs. From a screen with a large natural product chemical library, we identified
the natural alkaloid compound E as a novel antitumor drug that targets both the
CSC and bulk non-CSC populations in human lung, colon, and breast cancer cells Conclusion
by inducing apoptosis. Compound E administration decreased the multiplicity,
volume, and load of lung tumors in KrasG12D/+ transgenic mice and suppressed
the growth of cell line- and PDX-derived tumors by inducing apoptosis.
Mechanistically, Compound E disrupts the Hsp system by binding the N-terminal 1. A natural product derived compound E is a potential Hsp70 inhibitor.
ATP-binding pocket of Hsp70. The present study demonstrates that targeting
Hsp70 would be an effective anticancer strategy to eradicate CSCs. 2. Compound E inhibits both CSCs and non-CSCs populations in NSCLC.
Results

1. Compound E treatment decreases CSC population in NSCLC. 3. Compound E suppresses NSCLC cell (non CSC) viability by induce apoptosis
A.
A. 150 H1299 120 A549 150 H460 150 226B
100
H460 100 80 100 100
H1299 Oct4 promoter GFP O.D *** 60 ***
A549 50 40 *** 50 *** 50
226B 20
0 0 0 0
B. CT Comp E CT CompE μM 0 0.5 1 5 0 0.5 1 5 0 0.5 1 5 0 0.5 1 5
10.68% 7.76% 38.09% 26.42% B. 0uM 1uM C. 0uM 1uM
H460 Oct4 H1299 Oct4 A549 1.07% 46.74% A549

0uM 1uM
H460 3.61% 24.32% H460
17.13%
8.88%
A549 Oct4 33.16% 20.26% 226B Oct4 4. Compound E suppresses colony formation in both naïve and chemoresistant cells
H1299 120 A549 120 H460 150 226B
150 100 100
Colony Number (%) 50 40 40 *** 50 ***
C. CT CompE 100 80 60 80 100
60
+DEAB uM 0 0.5 *** 20 0 0 0.5 *** 20 0 0 0.5 1 0 0 0.5 1
0
1
1
0.3% 0.33% 150 H1299/R pe 200 H1299/R Cis 150 H460 R/PTX
Colony Number (%) 100 100 100
ALDH 50 *** 50 ***
2.67% 1.44% uM 0 0 0.5 1 0 0 0.5 *** 0 0 0.5 1
1
2. Compound E inhibits sphere forming ability and CSC markers 5. Compound E suppresses the growth 6. Compound E disrupts Hsp70 function by
expression. of xenograft tumor and PDX binding to the ATP binding site of Hsp70
CT Comp E 6000 Xenograft *** H460
H460 H1299 Tumor Volume(mm 3 ) 4000 IgG 0 1 10 (uM)
H460 120 150 2000 HSP90 IP : HSP70 ATP binding assay
100
Sphere (%) 80 60 40 *** 100 50 *** 0 0 4 8 PDX HSP70
0 1 10 uM
HSP70
12 16 20 (Days)
H1299 20 0 0 1 0 0 1 6000 *** HSP90 WCL
HSP70
4000
CT Comp E CT Comp E Tumor Volume(mm 3 ) 2000 Actin
0
0 3 7 11 15 19 (Days)