Combined targeting of histone deacetylases and hedgehog signaling ...

[Cancer Biology & Therapy 8:14, 1328-1339; 15 July 2009]; ©2009 Landes Targeting Bioscience HDACs and Hh in pancreatic cancer

Research Paper

Combined targeting of histone deacetylases and hedgehog signaling enhances cytoxicity in pancreatic cancer Stephen G. Chun,† Weiqiang Zhou† and Nelson S. Yee* Division of Hematology, Oncology and Blood & Marrow Transplantation; Department of Internal Medicine; University of Iowa; Carver College of Medicine; Program of Cell Signaling and Developmental Pharmacology; Holden Comprehensive Cancer Center; University of Iowa; Iowa City, IA USA †These

authors contributed equally to this work.

Abbreviations: ATCC, american type culture collection; CK7, cytokeratin 7; DAPI, 4',6 diamidino-2-phenylindole; DMEM, dulbecco’s modified essential medium; DMSO, dimethyl sulfoxide; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HDAC, histone deacetylases; Hh, hedgehog; HHIP, hedgehog interacting protein; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonylphenyl)-2H-tetrazolium, inner salt; PBS, phosphate buffered saline; PCR, polymerase chain reaction; Ptc-1, patched; SAHA, suberoylanilide hydroxamic acid; SANT-1, smoothened antagonist-1; SDS, sodium dodecyl sulfate; Shh, sonic hedgehog; Smo, smoothened; TBS, tris-buffered saline; Z-VAD-FMK, N-BenzyloxycarbonylVal-Ala-Asp(O-Me) fluoromethyl ketone Key words: hedgehog, histone deacetylases, pancreatic cancer, targeted therapy, SAHA, SANT-1, hedgehog interacting protein

Combined targeting of distinct cellular signaling mechanisms may improve the efficacy and reduce the toxicity of therapy in pancreatic cancer. Histone deacetylases (HDACs) control cellular functions through epigenetic modulation, and HDACs inhibitors suppress cell growth in pancreatic adenocarcinoma. The Hedgehog (Hh) pathway regulates the development of the pancreas, and aberrant Hh signaling promotes the initiation and progression of pancreatic neoplasia. We hypothesize that HDACs and the Hh pathway cooperatively interact to regulate cellular proliferation of the exocrine pancreas. A combination of the HDAC inhibitor SAHA and the Smoothened antagonist SANT-1 was evaluated for their ability to suppress growth of the Gemcitabine-resistant pancreatic adenocarcinoma cell lines Panc-1 and BxPC-3. The combination of SAHA and SANT-1 supra-additively suppressed cellular proliferation and colony formation. Flow cytometric and immunohistochemical analyses indicated that enhanced induction of apoptotic cell death, cell cycle arrest in G0/G1 phase, and ductal epithelial differentiation are involved. Cell death was associated with nuclear localization of survivin, increased bax expression, and activation of caspases 3 and 7. Consistent with the cell cycle arrest and cytodifferentiation, the cyclin-dependent kinase inhibitors p21waf and p27kip1 were upregulated, and cyclin D1 downregulated. The potentiated *Correspondence to: Nelson S. Yee; University of Iowa Carver College of Medicine; CBRB Room 3269B; 500 Newton Road; Iowa City, IA 52242 USA; Tel.: 319.356.1370; Fax: 319.353.8383; Email: [email protected] Submitted: 04/03/09; Accepted: 04/06/09 Previously published online as a Cancer Biology & Therapy E-publication: http://www.landesbioscience.com/journals/cbt/article/8633

1328

anti-proliferative effect by the combination of SAHA and SANT-1 may involve cooperative suppression of the Hh pathway activity, as shown by the upregulation of HHIP by SAHA, and enhanced repression of of Ptc-1 mRNA expression. In summary, we have developed a molecular target-based therapeutic approach that overcomes chemoresistance in pancreatic cancer cells by chemically inhibiting HDACs and Hh signaling in combination.

Introduction Pancreatic adenocarcinoma, the most common cancer of the exocrine pancreas, is the fourth leading cause of cancer mortality in the USA, with a 5-y survival rate of 4%.1 Despite recent advances in the understanding of the molecular genetics of pancreatic cancer and the development of mechanism-based targeted agents, prognosis remains dismal.2 The vast majority of patients develop metastatic disease that is refractory to the standard chemotherapeutic agent Gemcitabine, either alone or in combination with other cytotoxic or molecularly targeted agents. New therapeutic strategies based upon an improved understanding of the biology and signaling mechanisms of pancreatic tumorigenesis are urgently needed. The pathogenesis of pancreatic adenocarcinoma is a multiplestep process that involves the deregulation of developmental pathways, activation of oncogenes and silencing of tumor suppressor genes.3 The aberrantly activated Hedgehog (Hh) pathway plays a crucial role in the initiation and maintenance of pancreatic neoplasia, and contributes to chemotherapeutic resistance.4-6 Tumor suppressors such as p53, Smad4 and p16INK4A are often mutated or silenced by epigenetic modifiers such as histone deacetylases (HDACs).7,8 These lines of evidence suggest that the combination of agents that target non-overlapping

Cancer Biology & Therapy

2009; Vol. 8 Issue 14

Targeting HDACs and Hh in pancreatic cancer

mechanisms such as HDACs and Hh signaling may be an effective strategy to overcome chemoresistance. HDACs are dynamic modulators of the acetylation status of nucleosomal histones as well as non-histone proteins, leading to selective alteration of gene expression and a variety of cellular processes.9 In various cancers, unbalanced acetylation of nucleo somal histones causes structural alteration of chromatin, resulting in the deregulation of gene transcription involved in controlling cell cycle progression, differentiation and cell death.9,10 A number of HDAC inhibitors such as suberoylanilide hydroxamic acid (SAHA) and valproic acid have been shown to exert antitumor effects, and they are generally well tolerated for clinical use.11,12 SAHA is an inhibitor of class I and II HDACs, and it induces cell cycle arrest, differentiation, and cell death in a variety of cancers.10,13 In pancreatic adenocarcinoma cell lines, the antitumor activity of SAHA can be enhanced by combination with either Gemcitabine or DNA methyltransferase inhibitors.14-16 These evidences suggest that SAHA is a good candidate for combination therapy in pancreatic cancer when used together with agents such as Hh pathway inhibitors that act through distinct molecular mechanisms. The Hh-induced signaling pathway regulates the embryonic development and tumorigenesis of a variety of organs including the pancreas.17-19 Binding of Hh to its receptor Patched (Ptc-1) dis-inhibits the transmembrane protein Smoothened (Smo), which subsequently activates the Gli family of transcription factors, leading to the transcription of Ptc-1, Gli, cyclin D1 and N-myc genes. Abnormal reactivation of Hh signaling in the adult pancreas contributes to pancreatic carcinogenesis,20,21 and protects pancreatic cancer cells from apoptotic stimuli by promoting the degradation of p53 and stabilization of bcl-2/bcl-xL.22,23 The Hh pathway has recently been implicated as a regulator of pancreatic cancer stem cell homeostasis,24 and is thought to contribute to resistance to chemotherapy and radiation therapy.25,26 The inhibition of the Hh pathway by the plant alkaloid cyclopamine prevents pancreatic tumor formation in vivo and induces apoptosis in pancreatic cancer cell lines.4,24 Hh pathway antagonists including cyclopamine and the SANT family of small molecules bind the heptahelical bundle of Smo, thereby repressing Gli-mediated transcription.27-29 SANT-1 has the potential for therapeutic use because it potently inhibits wild-type and oncogenic Smo.29 Considering the role that Hh plays in the pathogenesis and possible chemoresistance of pancreatic adenocarcinoma, SANT-1 is a therapeutic candidate especially in combination with antitumor agents such as HDAC inhibitors. Based upon their roles in pancreatic adenocarcinoma, we hypothesize that HDACs and the Hh pathway cooperatively interact to regulate pancreatic cellular proliferation. In this study, we determine if the combined inhibition of HDACs and Smo produce enhanced suppression of pancreatic cancer growth. We show that SANT-1 potentiates SAHA-induced apoptosis, cytodifferentiation, and cell cycle arrest in the gemcitabine-resistant pancreatic adenocarcinoma cell lines Panc-1 and BxPC-3. These effects were accompanied by elevation of the pro-apoptotic protein bax, nuclear localization of survivin, and activation of effector caspases 3 and 7. Additionally, the cyclin-dependent kinase inhibitors p21waf and p27kip1 were upregulated, and cyclin D1 www.landesbioscience.com

downregulated. We also demonstrate enhanced repression of Hh signaling as indicated by increase of the Hh antagonist Hedgehog interacting protein (HHIP) mRNA as well as repression of the Hh receptor Ptc-1 mRNA expression. This translational study suggests that the combination of SAHA and SANT-1 has potential for improving the clinical treatment of pancreatic adenocarcinoma, and provides insight into novel interactions between HDACs and Hh in the therapeutic resistance of pancreatic cancer.

Results Enhanced inhibition of proliferation by SAHA and SANT-1 in gemcitabine-resistant pancreatic adenocarcinoma cell lines. Both Panc-1 and BxPC-3 were derived from primary pancreatic adenocarcinoma,30,31 and they have been shown to express altered levels of HDACs, Hh signaling components, and HHIP.4,17,32,33 To define the anti-proliferative effects of SAHA, SANT-1 and Gemcitabine on each cell line, dose response curves were obtained using the MTS assay (Suppl. Fig. 1). Our data verified that Panc-1 and BxPC-3 were relatively resistant to Gemcitabine, such that at 1 μM (a clinically feasible concentration) Panc-1 showed 10% reduction of proliferation, and BxPC-3 did not respond. SAHA and SANT-1 inhibited proliferation of each cell line in various dose-dependent manners. For 50% reduction of proliferation, 150 μM and 8 μM SAHA were required for Panc-1 and BxPC-3, respectively; 100 μM SANT-1 was required for Panc-1 whereas BxPC-3 did not respond up to 200 μM SANT-1. Next, we determined the concentrations at which SAHA and SANT-1 would produce an anti-proliferative effect with a magnitude greater than the sum of the individual effects of SAHA and SANT-1, which we define as supra-additive. Various concentrations of SAHA and SANT-1 were used in combination, and the effects on cellular proliferation were analyzed using the MTS assay (Table 1). SAHA at 5 μM and SANT-1 50 μM reproducibly yielded a supraadditive inhibition of cellular proliferation in Panc-1 and BxPC-3. Therefore, both Gemcitabine-resistant Panc-1 and BxPC-3 cells exhibit differential sensitivities to SAHA and SANT-1, and the supra-additive anti-proliferative effects of this combination were further investigated. First, we determined the effect of the combination of SAHA and SANT-1 on colony formation in soft agar, which facilitates direct cell-cell interactions and thus simulates in vivo growth more closely than the monolayer conditions. The combination of SAHA and SANT-1 caused a supra-additive inhibition of colony formation by Panc-1 and BxPC-3 in soft agar (Fig. 1A). Neither SAHA nor SANT-1 affected the ability of Panc-1 to form colonies, but the combination significantly reduced the number of formed colonies by 53% (p = 0.007). In BxPC-3, SAHA diminished colony formation by 37% (p = 0.004), SANT-1 had no significant effect, and SAHA + SANT-1 suppressed colony formation by 64% (p = 0.001). DMSO did not significantly affect cellular proliferation either alone or in combination with SAHA. To estimate toxicity to normal cells, the anti-proliferative effects of SAHA + SANT-1 on pancreatic ductal epithelia were determined using MTS assay. In comparison to 1 μM Gemcitabine that reduced proliferation of normal ductal epithelia by 90%, SAHA + SANT-1 caused a 40% reduction in proliferation. By contrast,


1329


Table 1 SAHA and SANT-1 supra-additively suppress proliferation of Panc-1 and BxPC-3 Treatment Panc-1 BxPC-3 Mean % reduction P Mean % reduction +/- SEM +/- SEM

P

SAHA 1 µM

ND

ND

1 +/- 4

0.23

SAHA 5 µM

13 +/- 5

0.08

39 +/- 3

0.0001*

SAHA 10 µM

15 +/- 9

0.02*

45 +/- 12

0.0005*

SANT-1 10 µM

6 +/- 12

0.18

0 +/- 5

0.79

SANT-1 50 µM

8 +/- 3

0.07

1 +/- 12

0.89

16 +/- 9

0.01*

7 +/- 9

0.12

SAHA 1 µM + SANT-1 10 µM

ND

ND

-2 +/ -6

0.3


ND

ND

-14 +/- 2

0.13

SANT-1 100 µM


ND

ND

-2 +/- 13

0.86


23 +/- 6

0.006*

47 +/- 2

0.0001*


37 +/- 6

0.004*

46 +/- 3

0.0001*


51 +/- 7

0.0007*

52 +/- 2

0.0002*


20 +/- 7

0.003*

48 +/- 4

0.0001*


48 +/- 8

0.002*

46 +/- 7

0.0001*


49 +/- 9

0.0001*

53 +/- 4

0.0001*

Cellular proliferation was assessed by MTS assay. Each value represents the mean % reduction from controls (either no treatment or DMSO) +/- SEM of three independent experiment with each treatment performed in triplicate. ND, not determined. *P < 0.05.

neither Panc-1 nor BxPC-3 was significantly affected by 1 μM Gemcitabine. These results suggest that the combination of SAHA and SANT-1 suppresses the proliferation of Gemcitabine-resistant pancreatic adenocarcinoma cell lines to a greater extent than either agent alone at concentrations that are less toxic than Gemcitabine to pancreatic epithelial cells. SAHA and SANT-1 cooperatively induce cytodifferentiation, cell cycle arrest and apoptosis. To gain insight into the suppressive effects on cellular proliferation, cellular morphology was analyzed by phase contrast microscopy at 24 h (Fig. 1B). In Panc-1, consistent with their effects on proliferation, neither SAHA nor SANT-1 caused significant alteration of morphology, but caused pronounced morphologic changes indicative of cytodifferentiation and cell death when combined.14 SAHA + SANT-1 induced the formation of dendrite-like cytoplasmic projections, a decrease in the nucleus:cytoplasm ratio, cell rounding and decreased cell-cell adhesiveness. In BxPC-3, while SAHA as a single agent caused nuclear swelling, nuclear membrane blebbing, and a decrease in the nucleus:cytoplasm ratio, the addition of SANT-1 further enhanced these effects. The cooperative induction of morphologic changes by SAHA and SANT-1 suggested that suppression of cellular proliferation involved cytodifferentiation and apoptotic cell death. To verify the cytodifferentiation observed using phase contrast microscopy, BxPC-3 cells were examined for expression of cytokeratin 7 (CK7), a marker of pancreatic ductal differentiation (Fig. 2A). Consistent with the ability of SAHA and SAHA + SANT-1 to induce morphologic differentiation, these treatments increased CK7 expression at 48 h. Co-staining with DAPI revealed that the combination of SAHA + SANT-1 caused nuclear swelling and an increase in the number of fragmented nuclei. The ability of 1330

SAHA + SANT-1 to increase CK7 expression and induce nuclear fragmentation further implicated cytodifferentiation and apoptosis as the mechanisms by which cellular proliferation was suppressed. To assess whether cytodifferentiation was associated with cell cycle arrest, cellular DNA content was analyzed by FACS (Fig. 2B). In Panc-1 and BxPC-3, SAHA increased the proportion of cells in G0/G1 (by 28% and 20%, respectively), and decreased the proportion of cells in S phase (by 28% and 27%, respectively). SANT-1 produced small increase (by 6% in Panc-1) or no change (in BxPC-3) of proportion of cells in G0/G1. SAHA + SANT-1 caused an additional accumulation of cells in G0/G1 (by 5% in Panc-1 and 4% in BxPC-3) as compared to SAHA alone. These results show that cellular proliferation was suppressed in part by SANT-1 enhancement of SAHA-induced blocking of cell cycle progression at G0/G1. Panc-1 and BxPC-3 were incubated with Annexin-V and propidium iodide to quantify apoptosis by FACS. At 48 h, SAHA and SANT-1 supra-additively induced early and late apoptosis in both cell lines (Fig. 3A). In Panc-1, SAHA + SANT-1 increased the proportion of cell population in apoptosis by 20%, whereas SAHA increased it by 10% and SANT-1 by 3%. Similarly, in BxPC-3, SAHA + SANT-1 increased the proportion of cells in apoptosis by 57%, whereas SAHA increased it by 36% and SANT-1 by 1%. Using the Vybrant Apoptosis Assay, a similar supra-additive induction of apoptosis and cell death was also observed by quantification of YO-PRO-1 and propidium iodide uptake using FACS (data not shown). SAHA and SANT-1 induced apoptosis is associated with nuclear localization of survivin, activation of caspase 3/7, and elevation of bax. To further characterize apoptosis induced by


2009; Vol. 8 Issue 14


Figure 1. SAHA and SANT-1 inhibit anchorage-independent colony formation and induce cellular morphology consistent with ductal differentiation and apoptosis. (A) Soft agar colony assay. Panc-1 and BxPC-3 were treated with untreated medium (control), 5 μM SAHA, 50 μM SANT-1 and 5 μM SAHA + 50 μM SANT-1 and grown in a soft agar medium for 14 d. Each value is represented as the number of colonies as percent of control. Each column represents the mean of three independent experiments with each treatment in triplicate; bars represent SEM. *P = 0.007; **P = 0.004; ***P = 0.001. (B) cellular morphology at 24 h. Bright field images of the cells treated as described above were captured under phase contrast microscope. Morphologic features included dendrite-like cytoplasmic projections (light arrows), and nuclear membrane blebbing. Original magnification, 20x.

SAHA and SANT-1, the expression of the anti-apoptotic protein survivin was analyzed under confocal microscope (Fig. 3B). In the untreated BxPC-3 cells, survivin was diffusely located in both the cytoplasm and nuclei, though it was less diffusely present in the SANT-1-treated cells. By contrast, either SAHA or SAHA + SANT-1 caused survivin expression to be located almost exclusively in the nucleus. The SAHA-induced nuclear localization of survivin was also observed under a compound microscope (data not shown). These results show that SAHA and SANT-1 induce apoptosis due in part to the ability of SAHA to restrict survivin expression to the nucleus. www.landesbioscience.com

Major effector caspase activity was then determined using caspase 3/7 assay to elucidate the mechanism by which cell death was induced. Proportionate to the amount of apoptosis quantified by FACS, SAHA and SANT-1 induced caspase 3/7 activities at 48 h in both cell lines (Fig. 4A). In order to determine whether the anti-proliferative effects of SAHA and SANT-1 were caspasedependent, both cell lines were treated with the non-selective caspase inhibitor Z-VAD-FMK. Although Z-VAD-FMK inhibited activation of caspase 3/7 (Fig. 4A), it failed to rescue cells from the anti-proliferative effects of SAHA and SANT-1. This was evident in the MTS proliferation assay in which SAHA and


1331

Targeting HDACs and Hh in pancreatic cancer Figure 2. Induction of cytodifferentiation and accumulation of cells in G0/G1 phases of cell cycle by SAHA and SANT-1. (A) immunofluorescent analysis of CK7. BxPC-3 was grown with untreated medium (control), 5 μM SAHA, 50 μM SANT-1 and 5 μM SAHA + 50 μM SANT-1 for 48 h. CK7 was detected with mouse anti-CK7 antibodies and then Alexa Fluor® 488 nm goat anti-mouse IgG (green); nuclei were co-stained with DAPI (blue). The pattern of CK7 expression is indicated with white arrows; DAPI-stained nuclear fragments, yellow arrows. Original magnification, 40x. (B) cell cycle analysis of Panc-1 and BxPC-3 at 48 h. The DNA content of the treated cells was analyzed by FACS quantification of propidium iodide staining. The proportion of cells in each phase of the cell cycle is indicated. The data shown is representative of three independent experiments with similar results each time.

SANT-1 suppressed the proliferation of Panc-1 and BxPC-3, but this effect was not abolished by the addition of Z-VAD-FMK (Fig. 4B). Z-VAD-FMK alone or in combination with either SAHA or SANT-1 did not affect proliferation of both Panc-1 and BxPC-3 (data not shown). These results indicate that the anti-proliferative effects of SAHA and SANT-1 could be mediated through caspaseindependent mechanisms. Expression of the pro-apoptotic protein bax was then analyzed by immunoblotting to further explore the mechanisms by which apoptosis was induced (Fig. 5A).In Panc-1, SAHA and SANT-1 slightly induced bax monomers expression (0.5 and 0.3 fold above control, respectively), but the combination upregulated Figure 3. SAHA and SANT-1 induce apoptosis and nuclear localization of survivin. (A) flow cytometric analysis of apoptosis in Panc-1 and BxPC-3. Cells were treated with 5 μM SAHA, 50 μM SANT-1, 5 μM SAHA + 50 μM SANT-1, or without treatment (control) for 48 h. Apoptosis was quantified by flow cytometric detection of FITC-conjugated Annexin-V binding (black border). The data shown is representative of three independent experiments with similar results each time. (B) immunofluorescent analysis of survivin in BxPC-3 at 48 h. Survivin was detected with mouse anti-survivin antibodies and then Alexa Fluor® 488 nm goat anti-mouse IgG (green); actin with phalloidin conjugated to Alexa Fluor® 546 nm (red) by confocal microscopy. Original magnification, 200x. 1332


2009; Vol. 8 Issue 14


bax monomers expression (increased by 13 fold). Similarly, in BxPC-3, bax monomers expression was induced by either SAHA or SANT-1 (0.7 and 0.2 fold above control, respectively), and this effect was most prominent with SAHA + SANT-1 (increased by 0.9 fold). Importantly, in BxPC-3, the expression of bax dimers was induced by either SAHA or SANT-1 (1.6 and 0.8 fold above control, respectively), and this effect was most with SAHA + SANT-1 (increased by 4.0 fold). No appreciable increase in the level of bax dimers by SAHA and/ or SANT-1 was noted in Panc-1. Taken together, the induction of bax implied that the intrinsic apoptosis pathway is an important mechanism by which SAHA and SANT-1 induced caspases 3/7. SAHA and SANT-1 arrest of cell cycle progression is associated with upregulation of p21waf and p27kip1, and downregulation of cyclin D1 mRNA. Next, to further characterize the impaired cell cycle progression from G1 to S phase, expression of the cyclin-dependent kinase inhibitor p21waf was assessed. SAHA induced p21waf protein expression in Panc-1 (3.7 fold of control) and to a less extent in BxPC-3 (1.3 fold of control), and this effect was enhanced by SANT-1 particularly in BxPC-3 (2.9 fold of control) (Fig. 5A). The induction of p21waf protein expression by SAHA and SANT-1 involved a supra-additive upregulation of p21waf mRNA levels (Fig. 5B). Consistent with this finding, SAHA and SANT-1 supra-additively upregulated p27kip1 mRNA levels. SAHA alone downregulated cyclin D1 mRNA, and this was further enhanced by the addition of SANT-1. These results imply that the upregulation of the cyclin-dependent kinase inhibitors p21waf and Figure 4. SAHA and SANT-1 induce caspase 3/7 activity that is not required for their p27kip1, and the downregulation of cyclin D1, anti-proliferative effects. (A) caspase 3/7 assay. Panc-1 and BxPC-3 were treated with 5 μM contribute to the mechanism by which SAHA and SAHA, 50 μM SANT-1, 5 μM SAHA + 50 μM SANT-1, 5 μM SAHA + 50 μM SANT-1 + 25 μM Z-VAD-FMK, or without treatment (control) for 48 h. Caspase 3/7 activity is indicated SANT-1 induce G0/G1 cell cycle arrest. by the units of fluorescence emission. Each column represents the mean of three independent SAHA and SANT-1 cooperatively repress experiments with each treatment in triplicate; bars represent SEM. (B) cellular proliferation the Hh pathway. In an attempt to explore the by MTS assay. Proliferation of the cells in each experimental group described above is molecular mechanisms underlying the enhanced represented as percent of control. Each column represents the mean of three independent suppression of cellular proliferation by SAHA experiments with each treatment in triplicate; bars represent SEM. *P < 0.05. and SANT-1, the Hh pathway was assessed by quantifying HHIP and Ptc-1 mRNA using real-time PCR, HHIP mRNA by 3.4 fold and 1.9 fold, respectively in BxPC-3 and the acetylation status of histones H3 and H4 by immu- (Fig. 6A). noblotting. HHIP is a competitive antagonist of Hh at the Consistent with SAHA-induced upregulation of HHIP and Ptc-1 receptor; Ptc-1 is a known target gene of Gli-mediated SANT-1 mediated repression of Smo,29,34 Ptc-1 mRNA levels in transcription controlled by the Hh pathway, and the expression Panc-1 and BxPC-3 were downregulated by SAHA by 45% and of Ptc-1 mRNA is therefore used as a marker of Hh pathway 46%, respectively, whereas Ptc-1 mRNA expression was reduced by activity.29,32,34 The expression of HHIP has been shown to be SANT-1 by 29% and 45%, respectively (Fig. 6B). The combinadiminished in both Panc-1 and BxPC-3, as compared to normal tion of SAHA and SANT-1 yielded enhanced repression of Ptc-1 pancreatic ductal epithelia.4,32 SAHA alone or in combination mRNA expression by 58% and 74%, respectively in Panc-1 and with SANT-1 upregulated HHIP mRNA expression by 17.4 fold BxPC-3 (Fig. 6B). and 20.6 fold, respectively in Panc-1 (Fig. 6A). Similarly but to To determine how the mRNA levels of HHIP and Ptc-1 correa lesser extent, SAHA or SAHA + SANT-1 increased the levels of lated with epigenetic modulation by SAHA, the protein levels of www.landesbioscience.com


1333


induction of cell death in Gemcitabineresistant pancreatic adenocarcinoma cell lines. Our data provides new insight into the mechanism of the antitumor activity of SAHA, including repression of Hh signaling, and nuclear localization of the anti-apoptotic protein survivin. Taken together, the findings of this study suggest that the combination of SAHA and SANT-1 has therapeutic potential in pancreatic cancer, and help elucidate the molecular mechanisms to overcome chemoresistance in these cancer cells. This study revealed a novel action of SAHA that acts to repress Hh pathway activity. Our data showed that SANT-1 reduced the mRNA level of Ptc-1 as previously reported.29,34 SAHA induced the upregulation of HHIP, that coincided with the downregulation of Ptc-1 mRNA level. The latter effect was further enhanced when combined with SANT-1. This is consistent with the finding that hypermethylation of the HHIP promoter represses HHIP expression in Panc-1 and BxPC-3, and this effect can be partially overcome by the HDAC inhibitor trichostatin A.32 However, trichostatin A has been previFigure 5. SAHA and SANT-1 induce bax, p21waf and p27kip1, and downregulate cyclin D1 expresously shown not to have an effect on HHIP sion. (A) immunoblot analysis of bax and p21. Panc-1 and BxPC-3 cells were incubated for 48 h expression in the pancreatic cancer cell line with 5 μM SAHA, 50 μM SANT-1, 5 μM SAHA + 50 μM SANT-1, 0.01% DMSO, or with no treatMIA PaCa-2, unless it is combined with the ment (control). The protein levels of bax and p21waf were assessed by immunoblotting using anti-bax and anti-p21waf antibodies. Anti-actin antibodies were used to indicate equivalent amount of protein DNA methyltransferase inhibitor 5-aza-2'loaded in each lane. (B) quantification of p21waf, p27kip1 and cyclin D1 mRNA. The mRNA levels of deoxycytidine.32 We therefore suspect that p21waf, p27kip1 and cyclin D1 in the cells of each experimental group described above were deterSAHA likewise, does not upregulate HHIP mined by real-time PCR and standard curves. Each column represents the mean of three independent in MIA PaCa-2, offering an explanation experiments with each treatment in triplicate as a ratio of that in control. for our observed absence of cooperative anti-proliferative effects by SAHA and acetylated histones H3 and H4 were examined by immunoblotting. SANT-1 in MIA PaCa-2 (data not shown). In agreement with the In Panc-1, SAHA increased the level of acetylated histone H3 and effect of SAHA in Panc-1 and BxPC-3, transcriptional analyses of H4 by 2.8 fold and 0.7 fold, respectively (Fig. 6C). In BxPC-3, the zebrafish germ-line mutation with loss-of-function in hdac1 SAHA upregulated acetylated histones H3 and H4 by 0.4 and and human pancreatic adenocarcinoma cells with targeted knock5.9 fold, respectively. In both Panc-1 and BxPC-3, SANT-1 had down of HDAC1 reveal upregulation of HHIP mRNA (Yee NS and no significant effect on the levels of acetylated histones H3 and Zhou WQ, unpublished data). Moreover, the differential effect of H4, and it did not influence the effect of SAHA when combined SAHA on the upregulation of HHIP and other antitumor effects (Fig. 6C). Therefore, the enhanced suppression of cellular prolif- in Panc-1 and BxPC-3 may be explained by the differential induceration by the combination of SAHA and SANT-1 is associated tion of histones H3 and H4 acetylation in the respective cell lines. with cooperative inhibition of Hh signaling, and SAHA-induced The precise mechanisms responsible for mediating the interaction hyperacetylation of nucleosomal histones. between HDACs and Hh remain to be determined. However, our results demonstrating enhanced suppression of the Hh pathway Discussion by SAHA and SANT-1 suggest a benefit of combining HDACs This study represents a novel translational approach to inhibitors and Smo antagonists in overcoming Hh-induced theraimprove treatment response of pancreatic adenocarcinoma by peutic resistance of pancreatic cancer stem cells.22,24 The supra-additive induction of apoptosis and the associated targeting epigenetic modulators and developmental regulators. In combination with an Hh pathway inhibitor, we have achieved enhanced expression of bax produced by the combination of enhanced SAHA-induced suppression of cellular proliferation and SAHA and SANT-1 are likely the primary cellular events that 1334


2009; Vol. 8 Issue 14


contribute to their antitumor effect. Hh overexpression has been shown to upregulate the endogenous anti-apoptotic proteins in pancreatic cancer cell lines,4,5,35 and inhibit p53-dependent apoptosis.23 We speculate that the downregulation of these Hh-induced anti-apoptotic proteins by SANT-1 likely accounts for enhanced bax-mediated apoptosis. In Panc-1 that expresses relatively high levels of Shh, Ptc-1, Smo and Gli-1 mRNA,4,17,36 both SAHA and SANT-1 were required to induce bax monomers expression. In contrast to Panc-1, expression of bax dimers was induced in BxPC-3 that expresses relatively high level of Shh and Ptc-1 mRNA, but relatively low levels of Smo and Gli-1 mRNA.4,17 As the dimerization of bax has been shown to enhance its mitochondrial translocation,37 we speculate that this finding may explain the relative sensitivity of BxPC-3 to SAHA and SANT-1 in comparison to Panc-1. Moreover, expression levels of Hh-mediated downstream apoptotic effectors may partially explain the differential sensitivities of Panc-1 and BxPC-3 to SAHA, especially as HDACs expression level does not correlate with response to HDAC inhibitors in pancreatic cancer cell lines.33 The observed potentiation of caspase activity was also likely mediated in part by the sequestration of survivin to the nucleus by SAHA. In summary, the activation of caspases provides compelling evidence that SAHA and SANT-1 induce apoptosis by enhancing bax expression. SAHA-induced apoptosis was accompanied by the localization of survivin to the nucleus. Although HDAC inhibitors have previously been shown to reduce survivin expression in a dose-dependent fashion in pancreatic cancer cell lines,38 survivin’s pattern of cellular localization in response to HDAC inhibition has not been described. Figure 6. SAHA and SANT-1 upregulate HHIP and reduce Ptc-1 mRNA levels and Ubiquitination of survivin at lysine residues has been induce histone acetylation. (A and B) quantification of HHIP and Ptc-1 mRNA, shown to target it to the mitotic spindle,39 and to the respectively. The levels of HHIP and Ptc-1 mRNA in Panc-1 and BxPC-3 treated with proteasome.40 Thus, it is tempting to speculate that 5 μM SAHA, 50 μM SANT-1, 5 μM SAHA + 50 μM SANT, or control (0.01% SAHA induces acetylation of lysine residues of survivin, DMSO) at 48 h were determined using real-time PCR. Each column represents the mean of three independent experiments with each treatment in triplicate as a causing the acetylated survivin to become ubiquitinated ratio of that in control. (C) analysis of histone acetylation. The levels of acetylated and targeted for translocation from the cytoplasm into the histones H3 and H4 were assessed by immunoblotting using total protein extracted nucleus. Further study of this phenomenon may reveal a from the cells in each of the experimental group as described above. Anti-total novel mechanism of counteracting the action of survivin histones H3 and H4 antibodies were used to indicate equivalent amount of protein loaded in each lane. The data shown is representative of three independent experiand overcoming therapeutic resistance in cancer cells. ments with similar results each time. Although SANT-1 potentiated the induction of caspases by SAHA, the anti-proliferative effects of SAHA and SANT-1 were not caspase-dependent. This finding is in agree- has not been evaluated directly, Z-VAD-FMK’s inability to abolish ment with previous studies showing that SAHA-induced apoptosis the anti-proliferative effects in MTS assays suggests a potential in pancreatic cancer cell lines is caspase-independent.13 This benefit of this combination in treating pancreatic cancer with effect may be mediated by the upregulation of bax by SAHA defects in caspase-dependent apoptotic pathways. and SANT-1, as bax has also been shown to induce cell death The suppression of cell proliferation by SAHA and SANT-1 by triggering autophagy through release of free radicals in the also involved the induction of epithelial differentiation and cell mitochondria.41 Moreover, we observed that SAHA and SANT-1 cycle arrest in G0/G1 phases. These events were associated with cooperatively upregulated the endogenous caspase inhibitor bcl-2 upregulation of the cyclin-dependent kinase inhibitors p21waf protein level (data not shown), lending further support to our and p27kip1 and downregulation of cyclin D1. Although HDAC observation that the suppression of cellular proliferation was inhibition is frequently associated with p21waf upregulation,42 caspase-independent. Although the effect of the pan-caspases Panc-1 and BxPC-3 have been shown to be resistant to SAHAinhibitor Z-VAD-FMK on SAHA and SANT-1-induced cell death induced upregulation of p21waf.15,16 The induction of p21waf www.landesbioscience.com


1335


by SAHA and SANT-1 in Panc-1 and BxPC-3 suggests that the overexpression of the Hh pathway contributes to the mechanism of resistance to p21waf upregulation by HDAC inhibitors. As the overexpression of Hh has been shown to downregulate p21waf in pancreatic ductal epithelium,5 we speculate that SANT-1 relieves an unidentified Hh-induced repressor of p21waf. Another potential explanation for the upregulation of p21waf and p27kip1 is modulation of SAHA-induced lysine acetylation by SANT-1. Although immunoblot analysis showed that SAHA alone or in combination with SANT-1 induced histone acetylation to similar extent, we cannot rule out the possibility that SANT-1 alters patterns of lysine acetylation leading to the upregulation of p21waf and p27kip1. The enhanced accumulation of cells in G0/G1 phases by the upregulation of p21waf and p27kip1 may suggest an important clinical benefit as cells in the G0/G1 phases are known to be radiation-sensitive. While the molecular mechanisms underlying the enhanced antitumor activity of SAHA and SANT-1 remain to be determined, we have evidence that argues against a mechanism that involves a non-specific interaction between the inhibitors. This is supported by our unpublished observation that combining SANT-1 and the HDAC inhibitor valproic acid, also produced a supra-additive suppression of proliferation in Panc-1 and BxPC-3 cells in MTS assays. However, such an effect was not observed when SANT-1 was combined with Gemcitabine, in agreement with Feldman et al. (2008) who similarly failed to demonstrate synergy or potentiation when combining a novel Hh inhibitor with Gemcitabine in pancreatic cancer cell lines.24 In conclusion, combined inhibition of HDACs and the Hh pathway using SAHA and SANT-1, respectively produces a supraadditive anti-proliferative effect in pancreatic adenocarcinoma cells through induction of apoptotic cell death and cell cycle arrest. We propose that the enhanced antitumor effects of SAHA and SANT-1 involve cooperative repression of the Ptc-1-mediated Hh pathway by restoring expression of HHIP by SAHA and Smo antagonism by SANT-1. Ongoing studies aim to evaluate the in vivo efficacy of SAHA and SANT-1 in mouse tumor models, and to further elucidate the interaction between HDACs and the Hh pathway, with the goals of determining their clinical potential to overcome chemotherapeutic resistance.

Materials and Methods Cell culture. The pancreatic adenocarcinoma cell lines Panc-1 and BxPC-3 were obtained from ATCC. Panc-1 was maintained in DMEM (ATCC), and 10% FBS (Hyclone), and BxPC-3 maintained in RPMI-1640 (ATCC) and 10% FBS. All cell culture media were supplemented with 100 U/ml penicillin (Gibco) and 100 μg/ml streptomycin (Gibco). Cells were grown at 37°C in an atmosphere of 95% air/5% CO2. All experiments were performed using culture medium unless otherwise specified. The cells were used within 20 passages of the frozen stocks from which the cells were periodically recovered. Small molecules and drugs. Stock solutions were prepared as follows: SAHA (Biomol) 50 mM in DMSO (Sigma), SANT-1 (Calbiochem) 5 mg/ml in DMSO, Z-VAD-FMK (Sigma-Aldrich) 1336

5 mM in DMSO, and Gemcitabine-HCl (Toronto Research Chemicals) 10 mM in PBS, pH 7.4 (Sigma). Each of the stock solutions was divided into aliquots and stored at -20°C. Proliferation assay. The effects of SAHA and SANT-1 on cellular proliferation were quantified using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega). This is a colorimetric method for determining the number of viable cells by utilizing the MTS as substrate; the quantity of formazan product as measured by the amount of absorbance at 490 nm is directly proportional to the number of living cells. In 96-well microtiter plates (Costar), 4 x 104 cells were seeded per well containing culture medium and allowed to adhere overnight. For 48 h, cells were incubated with 100 μl of medium containing the chemicals at the indicated concentrations. As a control, cells were incubated with the amount of DMSO used to dissolve small molecules (0.01%), especially as DMSO has been shown to weakly inhibit HDACs. According to the manufacturer’s instructions, 20 μl of MTS was added per well and allowed to incubate with the cells for 1 h. Absorbance of the samples at 490 nm was measured using a VersaMax Microplate Reader (Molecular Devices) and analyzed with SoftMax Pro software (Molecular Devices). For each data value, the absorbance of medium with 20 μl of MTS was subtracted from absorbance values of the cells to adjust for background absorbance. All experiments were performed with each sample in triplicate. Soft agar colony assay. A two-layer soft agar gel was used to assess the ability of SAHA and SANT-1 to inhibit anchorageindependent proliferation in Panc-1 and BxPC-3, as previously described.15 Briefly, in six-well tissue culture dishes (Costar), a 2.3-ml bottom layer of 1.6% agarose (Amresco) in cell medium was allowed to solidify overnight. A 3-ml top layer of 1.6% agarose in cell medium containing 5 x 103 cells treated as indicated was added to each well. The plates were then incubated for 14 d at 37°C in 95% air/5% CO2. Cells were stained with 5 x 10-3% crystal violet (Sigma), and colonies consisting of at least 50 cells were counted under an inverted light microscope (CKX31, Olympus). The experiment was repeated three times, with each treatment in triplicate. Morphologic analysis. Cells were plated in six-well tissue culture dishes (Costar) at a density of 5 x 105 per well and allowed to adhere overnight. Cells were then incubated with the indicated treatment for 24 h. Images of 20x magnification fields were captured under an inverted light microscope with phase contrast (IX81, Olympus). Immunohistochemistry of cytokeratin 7 (CK7) and survivin. BxPC-3 cells were grown on glass-chambered slides (Nunc) to approximately 60% confluency. For 48 h, BxPC-3 cells were incubated with the indicated treatment. Cells were then fixed with 4% paraformaldehyde (Fisher) in PBS for 20 min, and rinsed with PBS. Immunohistochemistry for CK7 and survivin was performed using an automated immunostainer (DakoCytomation) according to the manufacturer’s instructions. The cells were permeabilized by treating with proteinase K for 5 min. After rinsing with Trisbuffered saline (TBS), pH 7.5, cells were blocked for 30 min with Dako Serum-Free Blocking Solution. Immediately after blocking, a 1:50 dilution of monoclonal mouse anti-CK7 (DakoCytomation)


2009; Vol. 8 Issue 14


or a 1:100 dilution of monoclonal mouse anti-survivin (Cell Signaling) was applied for 1 h. Cells were then rinsed with TBS, and a 1:500 dilution of Alexa Fluor® 488 nm goat anti-mouse IgG (Invitrogen) was applied for 30 min. The slides stained for CK7 were rinsed with TBS and mounted with Vectashield® containing 4',6 diamidino-2-phenylindole (DAPI, Vector). The slides stained for survivin were rinsed with TBS, incubated for 20 min with a 1:250 dilution of phalloidin-Alexa Fluor® 546 nm (Invitrogen), and mounted with VectaShield® (Vector). The pattern of CK7 expression was visualized under a compound microscope with fluorescence (Olympus BX51); the images captured using a digital camera (DP71, Olympus), analyzed using DP Manager software (Olympus), and constructed using Adobe® Photoshop® 7.0. The cellular localization of survivin was examined under a laser scanning confocal microscope (MRC-1024, BioRad). In all experiments, negative controls were incubated without primary antibody to rule out non-specific binding by the secondary antibody, and to confirm the efficiency of blocking. Each experiment was repeated at least three times. Flow cytometric analysis of cell cycle. Panc-1 was incubated in DMEM without the addition of FBS for 48 h, and BxPC-3 in serum-free RPMI-1640 for 24 h to synchronize cells in the G0/G1 phase. Panc-1 and BxPC-3 were then incubated with the indicated treatment cell culture media supplemented with 10% FBS for 48 h. Both adherent and non-adherent cells were collected. Cells were washed in PBS and fixed with 1 ml 70% ethanol for at least 1 h at 4°C. Prior to flow cytometry, cells were centrifuged for 5 min at 103 rpm, and the cell pellet re-suspended in 1 ml PBS containing 100 μg/ml RNase A (Fermentas) and 50 μg/ml propidium iodide (Sigma). Within 1 h, cells were filtered through a 70-micron nylon mesh filter (Falcon) and analyzed by fluorescence activated cell sorting (FACS) on a FACScan (Becton Dickinson) with 104 events per histogram. The data was then analyzed using CellQuest Pro (Becton Dickinson) and ModFit (Verity Software House) software. As previously described in these cell lines,16 non-specific sub-G0/ G1 events were gated out of analyses, allowing for more accurate analysis of cell cycle distribution curves. All experiments were repeated at least three times. FACS analysis of cell survival and apoptosis. Panc-1 and BxPC-3 were seeded in six-well tissue culture plates at a density of 5 x 105 cells per well and allowed to adhere overnight. For 48 h, cells were treated as indicated. Adherent and floating cells were collected and washed with PBS. Cells were re-suspended in 0.4 ml Annexin-V binding Buffer (BioSource). Aliquots of 100 μl of each cell suspension were incubated with 5 μl Annexin-V (BioSource) and 5 μg/ml propidium iodide (Sigma) in the dark for 15 min. After the incubation period, 400 μl Annexin-V binding buffer was added to each tube. The cell suspensions were filtered through a 70-micron mesh, and analyzed by FACS within 1 h. FACS was performed on a FACScan, obtaining 104 events per histogram. The data was analyzed using CellQuest Pro software. All experiments were repeated at least three times. Caspase 3/7 assay. The activity of caspases 3 and 7 were quantified using the Apo-ONE® Homogeneous Caspase-3/7 Assay (Promega). In 96-well microtiter plates, 4 x 104 cells were www.landesbioscience.com

seeded per well containing culture medium and allowed to adhere overnight. At 48 h, cells were incubated with 100 μl of medium containing the indicated treatment. According to the manufacturer’s instructions, the Caspase-3/7 Substrate was diluted in the Apo-ONE® Homogeneous Buffer 1:100 to make the Apo-ONE® Caspase-3/7 Reagent. In each well, 100 μl of Apo-ONE® Caspase3/7 Reagent was added to the cell medium and allowed to incubate for 1 h. For every experiment, the absorbance of medium with 100 μl of Apo-ONE® Caspase-3/7 Reagent was subtracted from the values of the cells to adjust for background. Fluorescence emission at 485 nm was measured using a FL600 Microplate Fluorescence Reader (BioTek). Each experiment was performed three times, with each sample in triplicate. Immunoblot analysis. Panc-1 and BxPC-3 were grown to approximately 60% confluence in 100-mm tissue culture dishes (Falcon) and then treated for 48 h as indicated. Total protein was extracted using a lysis buffer (pH 6.8) containing 63 mM Tris-HCl (Sigma), 10% glycerol (EMD BioSciences), 5% β-mercaptoethanol (Sigma), 3.5% SDS (Research Products International), and protease inhibitors (Roche Diagnostics) at 4°C for 1 h. Cell lysates were then sonicated on ice for 30 s, centrifuged at 2 x 104 g for 10 min at 4°C, and the protein concentration of the supernatant was quantified using the BCA Protein Assay Kit (BioRad). For SDS-PAGE, 20 μg protein was loaded in 4–12% Bis-Tris gels (NuPage, Invitrogen), transferred to a polyvinylidene difluoride membrane (Bio-Rad), and blocked with a buffer of 5% skim milk and 0.1% Tween for 1 h. Membranes were incubated for 1 h with primary antibodies including rabbit anti-human acetylated histone H3, rabbit anti-human acetylated histone H4, rabbit anti-human histone H3, rabbit anti-human histone H4 (Millipore/Upstate Biotechnology); goat anti-human bax (Santa Cruz Biotechnology), rabbit anti-human p21 (Abcam), and rabbit anti-human actin (Sigma) antibodies according to the manufacturers’ instructions. After washing with PBS-Tween, membranes were incubated with the horseradish peroxidase-conjugated goat anti-rabbit IgG (Pierce) or donkey anti-goat IgG (Santa Cruz Biotechnology) for 1 h. Protein antigens were detected using SuperSignal West Pico Chemiluminescent Substrate (Pierce) and exposed on HyBlot CL film (Denville). The intensity of the protein bands was quantified using Adobe® Photoshop® 7. The protein levels of bax and p21waf were compared to actin; those of acetylated histones H3 and H4 were relative to total histones H3 and H4, respectively. Quantification of mRNA using real-time polymerase chain reaction (PCR). After 48 h of the indicated treatment, total RNA was prepared using RNeasy mini kits (Qiagen Sciences). RNA (2 μg) was reverse-transcribed with SuperScript® reverse transcriptase and random primers (Invitrogen) according to the manufacturer’s instructions. Gene expression was quantified using real-time PCR (TaqMan® and SYBR® Green, Applied Biosystems), and primers were generated by International DNA Technology (IDT). The real-time PCR conditions were 2 min at 50°C, and 10 min at 95°C, followed by 40 cycles of 15 s at 95°C and 1 min at 60°C, followed by a final step of 10 min at 72°C. The sequences of the primers used were designed using


1337


the NCBI accession number (p21waf: NM_000389; p27kip1: BC001971; cyclin D1: NM_053056; HHIP: NM_022475; Ptc-1: NM_000264; GAPDH: NM_002046) as follows: p21waf: 5'-ATG CAG CTC CAG ACA GAT GA-3', 5'-CGC AAA CAG ACC AAC ATC AC-3'; p27kip1: 5'-TGA AGC CTG GAA CTT CGA CT-3', 5'-TGT GAA TAT CGG AGC CCT TC-3'; cyclin D1: 5'-CTG TGC GAC AGA CGT CAA CT-3', 5'-GGT GAG GTT CTG GGA TGA GA-3'; HHIP: 5'-GCA GAG GAG ACC TCA GCA TC-3', 5'-GCA GTT GTG CCA GTG TCA GT-3'; Ptc-1: 5'-AAA TTC AAG AGT GGA TGT GG-3', 5'-ATC ATT TCT GGG AGA CTG TG-3' GAPDH: 5'-GAG TCA ACG GAT TTG GTC GT-3'; 5'-TTG ATT TTG GAG GGA TCT CG-3'. The mRNA levels of each gene were determined with a standard curve using known concentrations of each mRNA, or relative to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using the Comparative CT Method (Applied Biosystems). Real-time PCR was performed using the ABI Prism 7500 (Applied Biosystems). Statistics. The significance of the difference between the experimental group and control was analyzed using the Student’s t-test. Statistical significance was considered at a P value