Involvement of angiotensin II type 2 receptor (AT2R) signaling in human pancreatic ductal adenocarcinoma (PDAC): a novel AT2R agonist effectively attenuates growth of PDAC grafts in mice

Abstract

We have recently discovered the potential involvement of angiotensin II type 2 receptor (AT2R) signaling in pancreatic cancer using AT2R deficient mice. To examine the involvement of AT2R expression in human PDAC, expressions of AT2R as well as the major angiotensin II receptor (type 1 receptor, AT1R) in human PDAC and adjacent normal tissue was evaluated by immunohistochemistry and real time PCR using surgically dissected human PDAC specimens. In immunohistochemical analysis, relatively strong AT1R expression was detected consistently in both normal pancreas and PDAC areas, whereas moderate AT2R expression was detected in 78.5% of PDAC specimens and 100% of normal area of the pancreas. AT1R, but not AT2R, mRNA levels were significantly higher in the PDAC area than in the normal pancreas. AT2R mRNA levels showed a negative correlation trend with overall survival. In cell cultures, treatment with a novel AT2R agonist significantly attenuated both murine and human PDAC cell growth with negligible cytotoxicity in normal epithelial cells. In a mouse study, administrations of the AT2R agonist in tumor surrounding connective tissue markedly attenuated growth of only AT2R expressing PAN02 murine PDAC grafts in syngeneic mice. The AT2R agonist treatment induced apoptosis primarily in tumor cells but not in stromal cells. Taken together, our findings offer clinical and preclinical evidence for the involvement of AT2R signaling in PDAC development and pinpoint that the novel AT2R agonist could serve as an effective therapeutic for PDAC treatment.

Abbreviations

PDAC

pancreatic ductal adenocarcinoma

Ang II

angiotensin II

AT1R

angiotensin II type 1 receptor

AT2R

angiotensin II type 2 receptor

PCR

polymerase chain reaction

cGMP

cyclic guanosine monophosphate

HIF-1

hypoxia inducible factor

VEGF

vascular endothelial growth factor

Ki

association constant

GFP

green fluorescent protein

BSA

bovine serum albumin

PLZF

promyelocytic leukemia zinc finger protein

PI3K

phosphatidylinositol-3 kinase

TUNEL

terminal deoxynucleotidyl transferase dUTP nick end labeling

FBS

fetal bovine serum

DMEM

Dulbecco`s modification of Eagle`s medium

Ad-

adenoviral vector

HBSS

Hanks’ balanced salt solution

Introduction

Pancreatic cancer is the most lethal cancer in the USA.¹ Pancreatic ductal adenocarcinoma (PDAC) is the most aggressive form of pancreatic cancer and constitutes approximately 90% of all primary malignant tumors arising from the pancreas.² This cancer is difficult to diagnose early and responds poorly to available therapies. Peritoneal dissemination and liver metastasis increase its mortality.^3,4 This cancer is one of a few cancers of which the death rate has not declined over the last 3 decades.¹ Therefore, development of an early diagnosis and an effective therapeutic strategy is urgently needed.

Angiotensin II (Ang II) is the key effecter of the renin-angiotensin system which plays important roles in maintaining blood pressure, body fluid and electrolyte homeostasis. Ang II receptors are composed of at least 2 well-defined receptor subtypes: Ang II type 1 receptor (AT1R) and Ang II type 2 receptor (AT2R), both of which are classified as G protein-coupled 7 transmembrane receptors.^5-8 The major isoform, AT1R, is expressed widely and is attributed to most Ang II-dependent actions in cardiovascular/renal tissues, such as vasoconstriction, cell growth, angiogenesis and collagen deposition.^9,10 In contrast, AT2R is expressed at significantly lower levels than AT1R in adults and AT2R-mediated Ang II actions are involved in vasodilation and anti-proliferation.^11,12 Multiple studies suggest that protein tyrosine and serine/threonine phosphatase activation, nitric oxide/cGMP production, and arachidonic acid/prostaglandins production are involved in the mechanism of AT2R-mediated biological reactions.^9,10 Both endogenous and over-expressed AT2R have been shown to mediate apoptosis.^9,10,12

In relation to cancer in general, Ang II induces the expression of proto-oncogenes, such as c-fos and c-Myc, and promotes cell proliferation and growth through the AT1R.^13-15 Ang II-AT1R signaling also stimulates the expression of HIF-1α and VEGF,^16,17 signals that cause neovascularization and are required for solid tumor growth.^18,19 Ang II stimulates infiltration and activation of macrophages.^20,21 In addition, AT1R signaling is shown to be associated with cancer progressions and poor prognosis in various organ type cancers.^22-25 Inhibition of AT1R receptor function has been shown to attenuate solid tumor growth^26,27 and lung metastasis of renal cell carcinoma in mice.²⁸ On the contrary, over-expression of AT2R is shown to induce apoptosis in various cancer cells but not normal epithelial cells.^29,30 Our recent study demonstrated that the growth of PAN02 mouse PDAC grafts was significantly faster in AT2R knockout syngeneic mice.³⁰ This implies that the endogenously expressed AT2R plays a role in the attenuation of pancreatic cancer growth. These findings led to the hypotheses that the stimulation of AT2R in cancer cells induces death in cancer cells, thereby controlling tumor growth. Thus, AT2R could be a potential novel drug target for pancreatic cancer chemotherapy.

Recently, we developed a novel strategy to regulate selectivity for the AT2R/AT1R subtypes through electronic control of ligand aromatic-prolyl interactions.³¹ Through this strategy an AT2R high affinity (Ki = 3 nM) agonist analog that exerted 18.000-fold higher selectivity for AT2R versus AT1R was obtained.³¹ This novel AT2R agonist, bearing the peptide sequence DRVYIYPF, significantly stimulates neurite outgrowth in AT2R over-expressing PC12W rat pheochromocytoma cell line, in a dose-dependent manner.³¹ In the same study we pinpointed that this compound served as a negative regulator of AT1R signaling since it was able to inhibit MCF-7 breast carcinoma cellular proliferation in the low nanomolar range.³¹ These results suggested that this novel AT2R agonist binds selectively to the AT2R and its biological activity is equally strong as an endogenous ligand Ang II. Therefore, this novel AT2R agonist can serve as a valuable compound for AT2R-targeted stimulation and may control pathological conditions associated with Ang II-receptor signaling, such as hypertension, coronary artery disease, kidney disease and various cancers.

The pancreas is one of the few organs which express AT2R in normal adult animals.^32-35 The specific expression site appears to be throughout the pancreas including islets, acini and ductal epithelium.³² In the present study, we first evaluated the expression of AT1R and AT2R in human PDAC tissues and adjacent normal pancreatic tissues by immunohistochemistry and real time PCR. We then examined the effect of the newly synthesized AT2R agonist on the growth of PDAC cells in vitro using human and mouse PDAC cell lines and mouse allograft model. The results indicate that although the AT2R expression level in human PDAC ductal cells is slightly lower than that in adjacent normal ductal cells, the novel AT2R agonist has a strong growth attenuating effect on AT2R expressing PDAC grafts in mouse models. These results suggest that AT2R could potentially serve as a good drug target for human PDAC treatment and that our novel AT2R agonist is usable as a therapeutic agent for PDAC treatment.

Results

AT1R and AT2R expression in human PDAC specimens

In the first phase of the study, expression of the AT1R and the AT2R in the human PDAC specimens was investigated by immunohistochemical analysis. The AT1R expression was detected in all specimens including both cancerous (28/28) and adjacent normal pancreatic tissues (17/17). The strong expression of AT1R was observed in differentiated neoplastic ductal cells (Fig. 1A) and the stromal fibroblastic cells of PDAC and its expression in cancerous ductal cells was significantly higher than in normal ductal epithelium (Fig. 1A and E). The AT2R expression was also detected in both cancerous ductal cells (22/28) and adjacent normal pancreatic tissues (17/17) of specimens although the intensity of the expression was less than those of the AT1R expressions. The AT2R expression was mainly localized in differentiated ductal cells (Fig. 1B) as well as the mucin-filled ductal cells. The AT2R expression intensity in the stromal area was low-negligible and the expression did not show any anatomical characteristics. The AT2R expression level in cancerous ductal cells was similar to that in normal ductal epithelium (Fig. 1E). Although it was only observed in one specimen among all samples examined, a strong AT2R expression was localized in the nuclei of most adenocarcinoma cells (Fig. 1C). Survival length of 250 d for this particular patient is much shorter than the average length of all patients (658.8 days/28 patients).

Figure 1.

AT1R and AT2R expression in human PDAC. (A) AT1R immunoreactivity is strongly positive in the neoplastic ductal epithelial cells and fibroblasts in the stroma. (B) AT2R immunoreactivity is strongly positive in the neoplastic ductal epithelial cells. (C) Nuclear localization of AT2R in neoplastic ductal epithelial cells. (D) Negative control without primary antibody. These sections were counterstained by hematoxylin. (A–D; Original magnification 200×) (E) AT1R and AT2R protein expressions in cancerous (n = 28) and noncancerous ductal epithelium (n = 17) in the PDAC specimens were semi quantified by intensity of immunohistochemical staining (0 = negative; 1 = weak; 2 = intermediate; 3 = intense staining). (F) Expression of AT1R and AT2R mRNA in cancerous area (n = 8; black bar) and noncancerous area (n = 5; white bar) in the PDAC specimens was determined by real-time PCR using specific primers as described in the Methods. (G) Correlation between AT2R mRNA level and overall survival time (n = 8). (H) Average survival time (days) in AT2R positive and negative groups based on immunohistochemical observation. Average survival time was shown in diseased patients (n = 17).

Both AT1R and AT2R mRNA expressions in 8 cancerous areas and 5 normal areas of the pancreas were quantified by real-time PCR. Expression of the AT1R mRNA (4 fold increase), but not AT2R mRNA, was significantly up-regulated in cancerous areas compared to the normal pancreas adjacent to the carcinoma area (Fig. 1F). On the contrary, the AT2R mRNA level in cancerous areas was slightly lower than in normal areas (Fig. 1F). In addition, the AT2R mRNA (r = 0.663, p = 0.07) level, but not AT1R mRNA (r = 0.535, p = 0.17) showed a weak negative correlation with the length of the patient overall survival (Fig. 1G). This negative relationship between AT2R mRNA expression and patients overall survival length was also coincided with the AT2R protein expression in the ductal epithelium in the cancerous area which was detected by immunohistochemical analysis (Fig. 1H).

The novel AT2R agonist inhibited the growth of hAT2R-overexpressing PDAC cells

To explore the effect of AT2R signaling on pancreatic tumor cell growth, the effect of the novel AT2R-specific agonist on the growth of hAT2R over-expressing mouse (PAN02) and human (PANC-1) PDAC cell lines was evaluated in vitro. Basal expression of Ang II receptors in these cells was very low for AT1R and negligible for AT2R (Supplemental Table 1). However, adenoviral transfection of the hAT2R significantly increased AT2R expression in both cell lines one day after the transfection and their expression were sustained for 14 d although gradually declined. The AT2R expression in PAN02 and PANC-1 cell lines were 27 ± 5 and 35 ± 4 fmol/mg protein (triplicate determinations), respectively, on day 3 after the transfection. It was confirmed that AT2R over expression did not significantly influenced AT1R expression in both cell lines (Supplemental Table 1). With these cell lines, the growth of both PDAC cell lines treated with or without this AT2R agonist (10⁻⁸ M) was investigated by MTT assay. Overexpression of hAT2R alone significantly decreased the growth of both PDAC cell types as compared to those overexpressed with GFP (Fig. 2). AT2R agonist treatment further decreased the growth of both PDAC cell lines (15% decrease in PAN02 (P < 0.05) and 9% decrease in PANC-1 (P < 0.01)). These results suggest that AT2R signaling can significantly attenuate PDAC cell growth.

Figure 2.

AT2R agonist significantly decreased cell viability of hAT2R-overexpressing PAN02 cells (panel A) and PANC-1 cells (panel B). Cells transduced with Ad-GFP or Ad-hAT2R were seeded in a 96-well cell culture plate. After 24 hours, cells were treated with 10 nM Ang II, 10 nM AT2R agonist or 0.05% BSA (control vehicle solution). After 48 hours of treatment, cell viability was determined by MTT assay. The experiment was performed with triplicate determinations and repeated twice. Results are presented as the mean ± standard error of mean (n = 6). The statistical significance is indicated by *; P < 0.05, **; P < 0.01 and ***; P < 0.001.

AT2R agonist significantly attenuated the colony formation of PDAC cells

A two-layer 3-dimensional colony formation assay was carried out to evaluate the effect of the AT2R agonist on the colony growth of both hAT2R over-expressing PAN02 and PANC-1 PDAC cells. Number of colonies larger than 3,000 (PAN02) or 2,000 μm² (PANC-1) were counted 12 d after the agonist or the Ang II treatment by an automated colony counter. One-time treatment with 10 nM AT2R agonist, but not with 10 nM Ang II, significantly attenuated colony numbers both in hAT2R over-expressing PAN02 (P < 0.05) and PANC-1 (P < 0.01) cells as compared to 0.05% BSA containing vehicle solution (Fig. 3). Although 10 nM Ang II treatment slightly decrease the colony number in either GFP or hAT2R over-expressing PANC-1 human PDAC cells, this decrease was not statistically significant. In the PANC-1 cells, over-expression of AT2R itself significantly attenuated colony growth as compared to GFP over-expressing PANC-1 cells. These results corroborate with the results obtained in the 2-dimensional (2D) culture of these cells (Fig. 2) and suggest that the expression of the AT2R also attenuates anchorage-independent growth of the PDAC cells.

Figure 3.

The colony growth of hAT2R-overexpressing PAN02 (panel A) and PANC-1 cells (panel B) was attenuated significantly by the AT2R agonist treatment in soft agar. Cells transduced with Ad-GFP or hAT2R were individually suspended in agar layer in the 12-well culture plate. After 24 hours, cells were treated with 10 nM Ang II, 10 nM AT2R agonist or 0.05% BSA (control vehicle solution). Colony growth was evaluated 12 d after treatment using an automated colony counter. The pictures in the upper panels show the colony growth of PAN02 (A) or PANC-1 cells (B) transduced with Ad-GFP or Ad-hAT2R. The bar graphs in panel B represent the total colony numbers of PAN02 cells (C) and PANC-1 cells (D) that received the various treatments indicated in the legends. The experiment was performed with sextuplicate determinations and repeated twice. Results are presented as the mean ± standard error of mean (n = 12). The statistical significance is indicated by *, P < 0.05 and **, P < 0.01.

AT2R agonist attenuated the growth of PAN02 tumor graft in syngeneic mouse

To evaluate the effect of the AT2R agonist on pancreatic tumor growth, PAN02 cells transduced with either the hAT2R or GFP were inoculated in the right flank of syngeneic C57BL/6 mice (n = 6). As shown in Figure 4, the growth of the hAT2R expressing tumor grafts was slightly faster than that of the GFP-transfected grafts. Treatment with the AT2R agonist (5 mg/Kg/day) starting on day 10 (every 3 d for 10 d) effectively attenuated AT2R transduced PAN02 grafts, but this effect was not detected in the control GFP-transduced grafts. Statistical significance (P < 0.05) between the AT2R agonist and saline treatments became clear on day 18 and thereafter. Moreover, every day treatment with the AT2R agonist (5 mg/Kg/day) stating 23 d after the tumor cell inoculation caused a decrease in the tumor volume 5 d after every day treatment began and sustained low tumor volume thereafter (P < 0.001, Fig. 4A). Average tumor weight of the hAT2R-transduced and the agonist treated group (0.076 g) was also significantly smaller than that of the hAT2R-transduced and saline treated group (0.156 g) at the end of this in vivo study (P < 0.05). However, AT2R agonist did not show any effect on the growth of GFP transduced PAN02 grafts in average tumor weight measurements (Fig. 4B).

Figure 4.

The effect of the AT2R agonist on the growth of hAT2R-overexpressing PAN02 grafts was evaluated in syngeneic mice. Five million GFP-transduced (GFP-) or hAT2R-transduced (hAT2R-) PAN02 cells were inoculated into the right flank of C57BL/6 mice (n = 6). Administrations of the AT2R agonist (5mg/kg/day) were started 10 d after tumor cell inoculation (red arrows) and continued every 3 d until day 19, and then every other day until day 23, and finally every day until the mice were sacrificed. Panel A, tumor volume was evaluated by measuring tumor diameter using a caliper. Arrows indicate administration of the AT2R agonist. Panel B, the tumor weight was measured and are presented as the mean ± standard error of mean of 6 tumors from 6 mice. Panel C, mouse AT1Ra (A) and AT2R (B) expression in tumor tissues was determined by real-time PCR. The values of mouse AT1Ra and AT2R show relative quantity (RQ) which was compared to the value of the saline-treated GFP-PAN02 group. Panel D, Cell proliferation (A) and apoptotic analysis (B) was conducted using Ki-67 expression and TUNEL assay in PAN02 tumors, respectively. The statistical significances in this figure are indicated by *, P < 0.05, **, P < 0.01 and ***, P < 0.001.

The real-time PCR analysis of the AT1R and the AT2R expression in the PAN02 grafts indicated that the overexpression of hAT2R increased an endogenous mouse AT1R, but not the AT2R expression. Although the AT2R mRNA expression in naïve PAN02 cells was negligible (Supplemental Table 1), AT2R agonist treatment also increased endogenous mouse AT2R expression at the end of this study (Fig. 4C). Taken together, these mouse studies suggest that the AT2R agonist is able to attenuate the growth of AT2R expressing PDAC grafts in immunocompetent mice through the stimulation of AT2R signaling.

Histological analysis of cell proliferation and apoptosis in PAN02 grafts

Cell proliferation and apoptosis was histologically investigated. Cell proliferation index measured by Ki-67 positive cells did not show statistical difference among 4 groups. However, the treatment with the AT2R agonist significantly increased apoptotic cell number in either GFP or hAT2R-transduced PAN02 grafts (Fig. 4D). The percentage of apoptotic cells in AT2R agonist-treated hAT2R-PAN02 was increased 4 fold as compared to those in saline-treated GFP-PAN02 (P < 0.01) or hAT2R-PAN02 (P < 0.05) grafts. These results suggest that the primary mechanism by which the AT2R agonist attenuates tumor growth may be associated with AT2R signaling-induced apoptosis.

Discussion

Although AT1R and AT2R-mediated signaling are diverse, it is known that they are involved in cell growth in pathophysiological conditions, i.e., AT1R signaling is mainly involved in cell proliferation,^9,10,36 whereas AT2R signaling is involved in anti-proliferation through various mechanisms and they often counteract each other.^11,12,37 Since an involvement of the AT1R-mediated signaling in PDAC has been reported,^38,39 the primary objectives of this study were to clarify a potential involvement of AT2R signaling in human PDAC and to determine how AT2R signaling controls PDAC cell growth. In addition, whether the stimulation of the AT2R signaling by the novel AT2R agonist effectively controls PDAC grafts was evaluated in syngeneic immunocompetent mice by utilizing human AT2R over-expressing PDAC cells.

In the first study, expression levels of AT1R and AT2R in human PDAC specimens and normal pancreas specimens adjacent to the cancerous area were evaluated by immunohistochemistry and real-time PCR. This study clearly indicated that the AT1R expression was uniformly detectable in all neoplastic and normal ductal epithelial cells in the pancreas and its expression level in neoplastic ductal epithelium was significantly higher than that in the normal pancreas (2.1 fold in immunohistochemistry and 3.1 fold in PCR). The AT2R protein expression was mainly localized in neoplastic ductal epithelium and its expression level in neoplastic ductal epithelium was slightly lower than that in normal pancreas (0.99 fold in immunohistochemistry and 0.89 fold in PCR, Fig. 1). These expression pattern of 2 Ang II receptors in human PDAC specimens, high AT1R and moderate AT2R expressions, coincide with the observation described in other reports.^38,40,41 Analysis of the interrelationship between Ang II receptor expressions in PDAC specimens and patient overall survival length indicated that AT1R expression is unrelated with patient overall survival. However, both AT2R mRNA and protein expressions were weakly correlated with a shorter survival length in deceased patients (p = 0.07, Fig. 1G and H). Accordingly, it is suggested that AT2R expression in neoplastic ductal epithelium of PDAC is potentially associated with the progression of PDAC and is worthy of further study.

In a detailed analysis of AT2R expression in human PDAC tissues, strong nuclear localization of AT2R was observed in one patient (Fig. 1C). Nuclear localization of AT2R has been observed in the cardiac myocytes in mice bearing pressure-overloaded cardiac hypertrophy.⁴² Since interaction of the AT2R with the cytosolic transcription factor, promyelocytic leukemia zinc finger protein (PLZF) has been shown to stimulates the phosphatidylinositol-3 kinase (PI3K)/Akt pathway,⁴³ AT2R nuclear localization may be involved in cell survival of PDAC cells. Therefore, AT2R nuclear localization in PDAC tissues may indicate sustained tumor growth. Indeed, this patient survival length (250 days) was much shorter than the average overall survival length (658.8 d/28 patients). However, due to the small number of human PDAC specimens, confirmation of this issue must await for additional study.

In the second phase of this study, the effect of the novel AT2R agonist was examined to determine whether this agonist controls the growth of AT2R expressing PDAC cells using PANC-1 human and PAN02 murine PDAC cells transduced with human AT2R gene in ordinary 2D cell culture and 3-dimensional (3D) anchorage-independent cell culture (colony formation assay). These in vitro cell culture studies clarified that treatment with low concentrations of the AT2R agonist and natural ligand Ang II significantly attenuated the growth of the PDAC cells transduced with human AT2R but not GFP (Fig. 2) in 2D cell culture. Although Ang II showed a significant cell growth attenuation in the 2D culture, its effect was not statistically significant in the 3D colony formation assay (Fig. 3). This discrepancy of Ang II effects in 2 cell culture conditions may be explained by the stimulation of Ang II-AT1R signaling axis. First, as shown in the mouse study (Fig. 4C), AT1R expression in PDAC cells is significantly increased by the AT2R over-expression. It is presumed that this increased AT1R expression in PDAC cells has evoked Ang II-AT1R-dependent cell proliferation. Second, it took only 2 d to complete the 2D culture study, whereas it was required for a total of 12 d for the 3D culture study. This long term culture appears to be a sufficient period to induce AT1R expression in PDAC cells. Third, since the affinities of Ang II to AT1R and AT2R are in nanomolar range,⁴⁴ added Ang II easily stimulates the growth of hAT2R expressing PDAC cells. This speculation is supported by several reports that describe Ang II–AT1R-dependent growth of PDAC cells.^38,40,45 Although the crosstalk between AT1R and AT2R has also been described in various pathological conditions in cardiovascular tissues,³⁷ this is the first to describe that AT2R overexpression upregulates AT1R expression in PDAC cells. The present study clearly indicated that the cell growth attenuation effect of AT2R agonist is consistent in both 2D and 3D cultures when the PDAC cells are expressing AT2R, suggesting that the AT2R agonist is potentially an effective treatment for controlling PDAC tumor growth.

Having data showing that the AT2R-specific agonist can significantly attenuate growth of AT2R-expressing PDAC cells, the effect of the AT2R agonist on tumor growth was examined using murine PDAC grafts in syngeneic immunocompetent mice. As expected, AT2R agonist treatment attenuated growth of tumors in volume and weight by increasing apoptosis in PDAC cells (Fig. 4A, B and D), indicating that AT2R agonist was effective. In the tumor tissues from hAT2R expressing PAN02 cells, the expression of the AT2R was detected primarily in the tumor cells, which apparently sensitively responded to the agonist treatment thereby leading to the attenuation of tumor growth. However, AT2R expression was negligible in tumor tissues grown from naïve PAN02 cells and the growth of the tumors was unaltered by the agonist treatment (Fig. 4). The degree of cell proliferation and apoptosis in the tumors measured by Ki-67 staining and TUNEL assay, respectively, suggested that injections of the AT2R agonist in connective tissues around tumors induced apoptosis of tumor cells rather than the attenuation of cell proliferation (Fig. 4D). These results are consistent with previous reports that stimulation of AT2R induces apoptosis in various types of cells.^11,12,29,30 Accordingly, specific agonist-induced AT2R stimulation is a potential treatment scheme for AT2R expressing PDAC. Since AT2R expression in PDAC cells has been confirmed in the human PDAC specimens by this study and other studies,⁴¹ the AT2R agonist therapy may be applicable to human patients. This implies that the safety studies of the AT2R agonist and its therapy by formal multi-species toxicity and pharmacokinetic studies should be of high priority.

Recent studies suggest that AT2R over-expression alone also exerts biological functions such as apoptosis^29,30 by receptor dimerization without ligand binding.⁴⁶ The present study also exhibited such results in cell cultures (Figs. 2 and 3) in which the growth rate of hAT2R transfected cells was significantly slower than that of untransfected control cells. Since the AT2R agonist treatment tended to upregulate the endogenous mouse AT2R expression in PDAC cells transfected hAT2R and PDAC tumor growth was inhibited by the agonist treatment (Fig. 4A–C), AT2R upregulation in tumor cells may be the key to the success in this AT2R agonist therapy. However, on the contrary to cell culture studies (Figs. 2 and 3), tumor growth of AT2R transfected PDAC cells was faster in mice (Fig. 4A). This result may be explained by the AT1R-mediated tumor growth since AT1R expression in PDAC tumor tissue was significantly increased (Fig. 4C) and since AT1R-dependent tumor growth was previously observed.^26,40 Nonetheless, the present study suggests that the AT2R agonist therapy is effective in attenuating AT2R expressing PDAC tumors. Whether a longer period or higher dose of the AT2R agonist treatment is effective in attenuation of PDAC tumors should be the priority subjects to study. In addition, efficacy of the co-treatment of the AT2R agonist and an AT1R antagonist for PDAC may be of great interest as a future study.

In conclusion, this study strongly suggests that the AT2R expression in human PDAC cells is involved in tumor growth and potentially in overall patient survival. Although further studies are required to substantiate the in vivo safety of the AT2R agonist by formal multi-species toxicity and pharmacokinetic studies, our data indicate that our novel AT2R selective agonist, [Y]⁶-Ang II analog, could be a safe and effective therapeutic for PDAC. The present study provides clear evidence that local administration of the AT2R agonist causes AT2R gene expression in tumor cells and significantly attenuates the growth of fast growing murine PDAC tumors, suggesting that AT2R agonist therapy is an effective and useful procedure for PDAC treatment.

Materials and Methods

Human PDAC specimen

Human PDAC specimens were obtained from patients (12 males and 16 females for a total of 28 specimens), ranging between 42–83 y of age, who underwent surgical treatment with pancreatectomy during 2005 and 2012 at Yamaguchi University Hospital, Yamaguchi, Japan and Kagoshima University Medical and Dental Hospital, Kagoshima, Japan. All patients signed a written consent form on tissue acquisition and use for study. This study is approved by the Institutional Review Board of Yamaguchi and Kagoshima Universities.

Immunohistochemical analysis of Ang II receptors in human PDAC specimens

To analyze the relationship among the expression of AT1R, AT2R and histological tumor types in human PDAC specimens, 20 8 PDAC and 17 normal pancreatic tissues adjacent to the cancerous area were obtained. These tissues were fixed by 10% buffered formalin, paraffin embedded and thin sectioned. After deparaffinization of sections, heat-induced epitope unmasking was performed in citrate buffer followed by incubation with 3% H₂O₂/methanol for 3 minutes to block endogenous peroxidase activity. Sections were incubated with polyclonal anti-AT1R (1:50 dilution, for 1 hour at 37°C, Novous Biological), anti-AT2R (1:100 dilution, for 18 hours at 4°C, Abcam) antibodies. After the incubation with primary antibodies, sections were reacted with a biotin-conjugated anti-rabbit IgG antibody (Vector Laboratories) at a 1:100 dilution for 1 hour at 37°C, followed by reaction with the avidin-biotin-peroxidase complex reagent (Vector Laboratories) for 40 minutes at 37°C. Reactions were developed with 3,3′-diaminobenzodine tetrahydrochloride (Sigma) and counterstained lightly with Mayer`s hematoxylin.

Cell culture

The PAN02 murine pancreatic ductal adenocarcinoma cell line was obtained from the National Cancer Institute and maintained in RPMI-1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; EQUITECH-BIO Inc.), 100 U/ml penicillin, and 100 μg/ml streptomycin (Lonza Rockland, Inc.). The PANC-1 human pancreatic ductal adenocarcinoma cell line was purchased from the American Type Culture Collection and maintained in DMEM (Mediatech) supplemented with 10% FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin. These cells were incubated at 37°C with 5% CO₂. Cell line was authenticated by short-tandem repeat (STR) DNA profiling. Both the cells were maintained in low passage (<15) for this study.

Preparation of AT2R agonist

The novel AT2R agonist, [Y]⁶-Ang II analog (Asp-Arg-Val-Tyr-Ile-Tyr-Pro-Phe) discovered in our previous study,³¹ synthesized as before,³¹ was diluted in 0.1 N hydrochloric acid. After being neutralized by an equal volume of 0.1 N sodium hydroxide, the solution was diluted at the optimal concentration by 0.05% fatty acid-free bovine serum albumin (BSA; Sigma) and used for each experiment.

Gene transduction by adenoviral vectors

PAN02 and PANC-1 cells (1.0 × 10⁵ cells) were seeded in a 6-well plate. After 24 hours, gene transfection was carried out as described previously using adenoviral vectors coding human AT2R (Ad-hAT2R) or GFP (Ad-GFP) gene.³⁰

[¹²⁵I]Ang II Binding Assay

To evaluate binding characteristics of the AT2R agonist, [Y]⁶-Ang II, the radioligand receptor binding assay was performed as described previously with slight modifications.⁴⁷ Briefly, [¹²⁵I]-Ang II was purchased from PerkinElmer. Subconfluent hAT2R over-expressing PAN02 and PANC-1 cells in 24-well plates were washed twice with Hanks’ balanced salt solution (HBSS) and incubated with 0.5 nM [¹²⁵I]-Ang II with or without 1 μM unlabeled Ang II or [Y]⁶-Ang II for 3 hours at 4°C in the presence of 1 μM PD123319 or vehicle solution (0.5 mg/mL BSA). Unbound ligand was thoroughly washed out with HBSS at 4°C. Cells were solubilized with 0.5 M NaOH, and the remaining radioactivity was counted. Specific binding was estimated by subtracting the nonspecific binding obtained in the presence of 1 μM unlabeled ligand from the total binding. An aliquot of the solubilized cells was subjected to protein assay (BCA protein assay method, Pierce Chemical Co). Specific binding was normalized by protein quantity per well. Saturation isotherm data were analyzed according to the Scatchard method.⁴⁸

MTT cell proliferation assay

The MTT cell proliferation assay was performed to evaluate the effect of the AT2R agonist on PAN02 and PANC-1 cell proliferation as described previously.³⁰

Colony formation assay

A two-layer 3-dimensional colony formation assay was carried out as follows: 0.5 mL 0.8% agarose (SeaPlaque Agarose; 50101, Lonza Rockland, Inc.) in RPMI-1640 medium for PAN02 cells or defined medium for PANC-1 cells containing 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin was poured into wells of a 12-well tissue culture plate (bottom layer). PAN02 (5.0 × 10⁴ cells) and PANC-1 (0.5 × 10⁴ cells) transduced with Ad-AT2 R and GFP were suspended in 0.5 mL RPMI-1640 medium for PAN02 cells or defined medium for PANC-1 cells containing 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.4% agarose, and plated on top of the bottom agar layer (top layer). The cells were incubated at 37°C with 5% CO₂ for growth of colonies. After 24 hours, 10⁻⁸ M of Ang II and Ago was administrated to the top layer. 0.05% BSA was added as a control. On day 12, colony growth was evaluated with an automated phase-contrast microscope equipped with the Micro Suite Five (CKX41; Olympus). Colonies greater than 3,000 μm² (PAN02) or 2,000 μm² (PANC-1) in area were counted by using Micro Suite Five software.

Animals

Seven week-old wild-type female C57BL/6 mice were obtained from Charles River Laboratories International, Inc. All mice were housed in a clean facility and held for 10 d to acclimatize. All animal experiments were done under strict adherence to the Institutional Animal Care and Use Committee (IACUC) protocols set by Kansas State University (Manhattan, KS). All experiments were carried out under the approvals of Kansas State University institutional IACUC and Institutional Biosafety Committee.

Pancreatic cancer graft in syngeneic mouse and treatment of AT2R agonist

Nine week-old C57BL/6 were anesthetized with isoflurane. PAN02 cells transduced with or without 50MOI of Ad-AT2R (hAT2R-PAN02) or Ad-GFP (GFP-PAN02) were trypsinized and washed with PBS. Five million PAN02 cells in 100 μl of PBS were subcutaneously inoculated into the flank using a 1 ml syringe with a 27 G needle.⁴⁹ The tumor size was measured by caliper every 3 d and the volume was calculated using the formula (short diameter)² × (long diameter) × 0.5.⁵⁰ Administration of AT2R agonist (5 mg/kg/day, 50 μl 2 mg/ml PBS) adjacent to tumor was started 10 d after PAN02 cell inoculation. Administration was carried out every 3 d until day 19, and then every other day until day 23, and finally every day until the mice were sacrificed. At the end of the experiments, the mice were sacrificed by decapitation under anesthesia. The tumors were dissected and weighed.

Gene expression analysis using real-time PCR

Total RNA was extracted from cells using TRIzol reagent (Invitrogen). Real-time PCR was carried out using an iScript One-Step RT-PCR Kit with SYBR Green (Bio-Rad), and the reactions were conducted on a StepOnePlus real-time PCR system (Applied Biosystems). The results were quantified relatively by a delta-delta Ct method.⁵¹ The mouse AT1Ra primers were 5′- GGC AGC ATC GGA CTA AAT GG -3′ (forward) and 5′- CCA GCT CCT GAC TTG TCC TTG -3′ (reverse), the mouse AT2R primers were 5′- GCC TGC ATT TTA AGG AGT GC -3′ (forward) and 5′- GTG CCA GTT GCG TTG AGA TT -3′ (reverse) and the 18S ribosome RNA primers were 5′-TCG CTC CAC CAA CTA AGA AC-3′ (forward) and 5′-GAG GTT CGA AGA CGA TCA GA-3′ (reverse).

Immunohistochemical analysis for AT1R, AT2R, cell proliferation and apoptosis in PAN02 tumor

Immunohistochemical analysis on AT1R, AT2R, cell proliferation and anti-Ki-67 (1:100, for 1 hour at 37°C, Abcam) in PAN02 tumors was carried out as described above for human PDAC. Apoptotic cells in PAN02 tumors were detected by a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay by using the DeadEnd Colorimetric TUNEL System (Promega), according to the manufacturer's instructions with a slight modification.⁵² The cell proliferation index was assessed as a percentage of Ki-67 positive cells per tumor, while the apoptotic index was assessed as the average number of positive cells in 5 randomly selected fields.

Statistical analysis

All values are expressed as the mean ± standard error of mean. For all in vitro and in vivo experiments, statistical significance was assessed by unpaired t-test or ANOVA followed by Tukey's Honestly Significant Difference test or Kruskal-Wallis test. All experiments were conducted with multiple sample determinations. Actual sample numbers/experiments are described in the figure legends. Statistical significance was set at *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are grateful to Ms. Katie Turner (Department of Anatomy and Physiology, Kansas State University) for critical reading and constructive comments during the preparation of the manuscript.

Funding

This work was supported in part by Kansas State University (KSU) Johnson Cancer Research Center, KSU College of Veterinary Medicine Dean's fund, NIH grants P20 RR017686, P20 RR016475, 5P20RR015563 and Kansas Bioscience Authority Collaborative Cancer Research grant. In addition, this work was supported by the Grant-in-Aid for Scientific Research (B) 25293288 from the Japanese Ministry of Education, Culture, Sports, Science and Technology (ST), and the European Union (European Social Fund-ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF)-Research Funding Program: ARISTEIA II. Investing in knowledge society through the European Social Fund (AGT).