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Hedgehog inhibitor decreases chemosensitivity to 5-fluorouracil and gemcitabine under hypoxic conditions in pancreatic cancer - PubMed

Hedgehog inhibitor decreases chemosensitivity to 5-fluorouracil and gemcitabine under hypoxic conditions in pancreatic cancer

Hideya Onishi et al. Cancer Sci. 2012 Jul.

Abstract

Pancreatic cancer is one of the deadliest types of cancer. Previously, we showed that hypoxia increases invasiveness through upregulation of Smoothened (Smo) transcription in pancreatic ductal adenocarcinoma (PDAC) cells. Here, we first evaluated whether hypoxia-induced increase in Smo contributes to the proliferation of PDAC cells. We showed that Smo, but not Gli1, inhibition decreases proliferation significantly under hypoxic conditions. To further investigate the effects of Smo on PDAC growth, cell cycle analysis was carried out. Inhibition of Smo under hypoxia led to G(0) /G(1) arrest and decreased S phase. As 5-fluorouracil (5-FU) and gemcitabine, which are first-line drugs for pancreatic cancer, are sensitive to S phase, we then evaluated whether cyclopamine-induced decreased S phase under hypoxia affected the chemosensitivity of 5-FU and gemcitabine in PDAC cells. Cyclopamine treatment under hypoxia significantly decreased chemosensitivity to 5-FU and gemcitabine under hypoxia in both in vitro and in vivo models. In contrast, cis-diamminedichloroplatinum, which is cell cycle-independent, showed significant synergistic effects. These results suggest that hypoxia-induced increase of Smo directly contributes to the proliferation of PDAC cells through a hedgehog/Gli1-independent pathway, and that decreased S phase due to the use of Smo inhibitor under hypoxia leads to chemoresistance in S phase-sensitive anticancer drugs. Our results could be very important clinically because a clinical trial using Smo inhibitors and chemotherapy drugs will begin in the near future.

© 2012 Japanese Cancer Association.

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Figures

Figure 1
Figure 1

Smooothened (

S

mo) is required for proliferation of pancreatic cancer cells. (a) Representative pictures of cell morphology in

ASPC

‐1 (

ASPC

) and

SUIT

‐2 (

SUIT

) pancreatic ductal adenocarcinoma (

PDAC

) cells 24 h after treatment with 10 μM cyclopamine (cyclo) under hypoxia. Bar = 20 μm. (b) Cyclopamine‐treated (10 μM)

PDAC

cells (cyc) and Smo si

RNA

‐transfected

PDAC

cells (7 × 104/well) (Smo‐si) were plated in 6‐well plates under normoxic (n) and hypoxic (h) conditions. After the indicated culture period, cells were harvested and cell numbers were counted by Coulter counter. (c) Proliferation rates under normoxic (normo) and hypoxic (hypo) conditions were examined after 64 h culture. Bar shows mean ±

SD

. *P < 0.05. (d) Gli1 si

RNA

‐transfected

PDAC

cells (7 × 104/well) (

G

li1‐si) were plated in 6‐well plates. After the indicated culture period, cells were harvested and counted by

C

oulter counter. Bar shows mean ±

SD

. Cont, control; c‐si, control si‐

RNA

‐transfected cells. *P < 0.05.

Figure 2
Figure 2

Cell cycle arrest at

G

0/

G

1 with the decrease of

S

phase is induced by

S

moothened (

S

mo) inhibition under hypoxic conditions in

ASPC

‐1 (

ASPC

) and

SUIT

‐2 (

SUIT

) pancreatic ductal adenocarcinoma (

PDAC

) cells. (a) Cell cycle analysis in cyclopamine‐treated (10 μM) (cyc)

PDAC

cells or Smo si

RNA

‐transfected

PDAC

cells (

S

mo‐si) was carried out under normoxia (n) and hypoxia (h) by

FACS

. cont, control. (b) Cell cycle analysis in Gli1 si

RNA

‐transfected

PDAC

cells (

G

li1‐si) was carried out under normoxia (n) and hypoxia (h) by

FACS

. The graph shows the mean ±

SD

. *P < 0.05. (c) Cyclin D1,

CDK

4, and p21 expressions in cyclopamine‐treated

PDAC

cells or Smo si

RNA

‐transfected

PDAC

cells under hypoxia were estimated by Western blot. α‐Tubulin was used as the loading control. c‐si, control si‐

RNA

‐transfected cells; ct, control.

Figure 3
Figure 3

Cyclopamine treatment under hypoxic conditions inhibits chemosensitivity in pancreatic ductal adenocarcinoma cell lines

ASPC

‐1 (

ASPC

) and

SUIT

‐2 (

SUIT

). Twenty‐four hours after treatment with 10 μM cyclopamine (cyclo), 100 μg/

mL

5‐fluorouracil (5‐

FU

), 100 μg/

mL

gemcitabine (

G

em), and 500 μM cis‐diamminedichloroplatinum (

CDDP

) was added to the culture. After an additional 48 h, cells were harvested and the sub‐G1 population was estimated by FACS. (a) Histogram shows representative results in cells treated with 5‐FU, Gem, and CDDP under hypoxic conditions. The value in each upper left corner shows the mean percentage of sub‐G1 population. (b) Summary of the experiment under normoxic (n) and hypoxic (h) conditions showing the mean ± SD. cont, control. *P < 0.05.

Figure 4
Figure 4

Hypoxia inducible factor (

HIF

)‐1α does not improve cyclopamine‐induced chemoresistance in

ASPC

‐1 (

ASPC

) and

SUIT

‐2 (

SUIT

) pancreatic ductal adenocarcinoma (

PDAC

) cell lines under hypoxic conditions. (a) Cell cycle analysis of

HIF

‐1α si

RNA

‐transfected

PDAC

cells (

HIF

‐si) in the absence or presence of 10 μM cyclopamine (+cyc) was carried out under hypoxic condition by

FACS

. (b) Twenty‐four hours after the treatment with 10 μM cyclopamine in

HIF

‐1α siRNA‐transfected PDAC cells, 100 μg/

mL

5‐fluorouracil (5‐

FU

) and 100 μg/

mL

gemcitabine (

G

em) was added to the culture. After an additional 48 h, cells were harvested and the sub‐G1 population was estimated by

FACS

. The graph shows the mean ± SD. *P < 0.05.

Figure 5
Figure 5

Cyclopamine treatment inhibited chemosensitivity to 5‐fluorouracil (5‐

FU

) in vivo. Mice bearing tumors were injected s.c. with 0.1

mL

vehicle (triolein : ethanol, 4:1) or cyclopamine (triolein : cyclopamine, 1 mg/body) once a week (on days 0 and 8). From 2 days after the first injection of cyclopamine (day 0), injection of 5‐

FU

(30 mg/kg) or equivalent normal saline by i.p. injection was given three times a week. (a) Representative photographs of immunofluorescent staining with

CA

‐9 (green) and cyclin

D

1 (red) in cyclopamine‐treated or non‐treated

ASPC

‐1 pancreatic tumor obtained from mouse xenograft model. Bars = 40 μm.

CA

‐9/cyclin

D

1 double positive cells turned yellow. (b) Tumor volume was estimated at the indicated days. cyc, cyclopamine‐treated pancreatic tumor cells; cont, non‐treated pancreatic tumor cells. *P < 0.05, comparing 5‐

FU

group with 5‐

FU

/cyclopamine group. (c) Apoptotic cells in tumor from xenograft model were examined by the

TUNEL

method. Apoptotic cells were labeled by

FITC

. Representative pictures are shown. Bar = 30 μm.

Figure 6
Figure 6

Schematic figure of findings. New findings in the present study (bold line) overlapped with our previous findings.

HIF

, hypoxia inducible factor;

S

mo,

s

moothened.

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