1. 山竹子素調控miR-200c與調降Notch1路徑來抑制PANC-1胰臟癌類癌幹細胞的癌化
Garcinol downregulates Notch1 signaling via modulating miR-200c and suppresses oncogenic properties of PANC-1 cancer stem-like cells
山竹子素調控miR-200c與調降Notch1路徑來抑制PANC-1胰臟癌類癌幹細胞的癌化
黃其晟1 李偉華2 吳駿翃3
1國泰綜合醫院 外科部 一般外科
2台北醫學大學雙和醫院 病理部
3台北醫學大學 轉譯醫學博士學位學程

2. Purpose
Pancreatic cancer represents one of the most aggressive types of malignancy due to its high resistance towards most clinically available treatments.
The presence of pancreatic cancer stem-like cells (CSCs) have been attributed to the intrinsically high resistance and highly metastatic potential of this disease.
CD44, CD133, Oct4, Notch
A traditional plant-derived anti-oxidant, garcinol, have been implicated for its anti-cancer properties.
Phytochemicals: Polyisoprenylated benzophenone isolated from Garcinia indica.
Anti-inflammatory and antioxidant properties.

3. Methods
We identified and isolated pancreatic cancer stem-like cells using side-population (SP) method from human pancreatic cancer cell line, PANC-1 and then compared the SP and non-SP PANC-1 cells genetically.
Stem/progenitor cells: enhancing drug transporters such as ABCG2.
Approximately 2% of the entire cell population as SP cells with CSC properties.
Isolation of pancreatic SP cells by fluorescence-activated cell sorting (FACS) with FACSAria TM III sorter (BD Biosciences, Taipei, Taiwan).
SP cells express ATP-binding cassette ABCG2 and Hoechst efflux activity.

4. PANC-1 SP cells exhibited enhanced self-renewal ability and expressed higher stemness markers
Phase contrast photomicrographs:
self-renewal
RT-PCR

5. Results
PANC-1 SP cells exhibited cancer stem-like cell properties including enhanced self-renewal ability, increased metastatic potential and resistance towards gemcitabine treatment.
These cancer stem-like phenotypes were supported by their enhanced expression of ABCG2, Oct4 and CD44.
Here, we found that garcinol treatment to PANC-1 SP cells significantly suppressed the stem-like properties of PANC-1 SP cells and metastatic potential by down-regulating the expression of Mcl-1(prosurvival
signal), EZH2(oncogenic/stemness), ABCG2(drug resistance), Gli-1 and Notch1(stemness).
More importantly, garcinol treatment led to the up-regulation of several tumor suppressor miRNAs and miR-200c increased by garcinol treatment was found to target and down-regulate Notch1.

6. PANC-1 SP cells possessed increased tumorigenic properties
PANC-1 SP cells also exhibited enhanced migratory and invasive abilities.
Clone formation
efficiency
Wound healing (migration) assay
Matrigel invasive assay

7. PANC-1 SP cells exhibited chemotherapeutic resistance
PANC-1 SP cells exhibited approximately sevenfold higher gemcitabine tolerance as compared with the non-SP counterparts.
Gemcitabine sensitivity in SP and non-SP cells isolated from the PANC-1 cell line. The cells were treated with different doses of gemcitabine for 48 H.

8. Garcinol treatment suppressed CSC properties in PANC-1 SP cells
Cell viability (SRB)
assay.
Garcinol increased the miRNA
expression of miR-29b, miR-101, miR-181, and
miR-200c.

9. Mir-200c suppressed Notch1 and PANC-1 self-renewal ability
TarqetScan database prediction indicates that Has-miR-20Oc binds to the 3’ UTR of Notch.
Garcinol treatment led to the increase in several antioncogenic miR molecules.

10. Conclusions
PANC-1 SP cells may serve as a model for studying drug-resistant pancreatic cancer stem-like cells and garcinol has the potential as an antagonist against pancreatic CSCs.
PANC-1 SP cells: elevated stem and oncogenic expression profiles resemble stem cells with similar properties including increased colony-forming ability, drug resistance, and enhanced metastatic potential.
Garcinol-treated PANC-1 SP cells exhibited a significantly downregulated oncogenic and stem cell properties.
Garcinol targets several established tumor suppressor miRNAs, specifically miR-200c that targets and downregulate Notch1 expression.

11. Thank You
This study is funded by Cathay General Hospital-Taipei Medical University joint research fund (101CGH-TMU-06).