NOSH-aspirin (NBS-1120) inhibits pancreatic cancer cell growth in a xenograft mouse model: Modulation of FoxM1, p53, NF-κB, iNOS, caspase-3 and ROS

Abstract

Pancreatic cancer has poor survival rates and largely ineffective therapies. Aspirin is the prototypical anti-cancer agent but its long-term use is associated with significant side effects. NOSH-aspirin belongs to a new class of anti-inflammatory agents that were designed to be safer alternatives by releasing nitric oxide and hydrogen sulfide. In this study we evaluated the effects of NOSH-aspirin against pancreatic cancer using cell lines and a xenograft mouse model. NOSH-aspirin inhibited growth of MIA PaCa-2 and BxPC-3 pancreatic cancer cells with IC₅₀s of 47 ± 5, and 57 ± 4 nM, respectively, while it did not inhibit growth of a normal pancreatic epithelial cell line at these concentrations. NOSH-aspirin inhibited cell proliferation, caused G₀/G₁ phase cycle arrest, leading to increased apoptosis. Treated cells displayed increases in reactive oxygen species (ROS) and caspase-3 activity. In MIA PaCa-2 cell xenografts, NOSH-aspirin significantly reduced tumor growth and tumor mass. Growth inhibition was due to reduced proliferation (decreased PCNA expression) and induction of apoptosis (increased TUNEL positive cells). Expressions of ROS, iNOS, and mutated p53 were increased; while that of NF-κB and FoxM1 that were high in vehicle-treated xenografts were significantly inhibited by NOSH-aspirin. Taken together, these molecular events and signaling pathways contribute to NOSH-aspirin mediated growth inhibition and apoptotic death of pancreatic cancer cells in vitro and in vivo.

Graphical Abstract

1. Introduction

Pancreatic cancer is one of the most aggressive human malignancies with poor survival rates of approximately 4% [1]. It is the fourth cause of death in cancer even though it constitutes 2–3% of all cancers [2]. Pancreatic cancer cells are infiltrating epithelium cells that are neoplastic and gland forming, and therefore, pancreatic cancers are ductal adenocarcinomas. Majority of pancreatic cancer patients present with advanced disease or distant metastatcies which is often non-operable. Monotherapies with gemcitabine, 5-fluorouracil (5-FU), cisplatin and capecitabine or combinations thereof have been used with modest improvements in survival, however, mutidrug resistance to chemotherapy is generally a significant problem. To control disease progression, it is believed that multiple modes of intervention are required. As more targeted investigational agents are developed, the major pathways of focus are those related to signaling pathways associated with induction of apoptosis or proliferation. Therefore, many of the current agents target growth factors mediated events, angiogenesis, and other activated signaling pathways in this malignant disease [3, 4]. Not surprizing therefore, newer therapies for pancreatic cancer are needed.

Inactivation of the TP53 gene is found in approximately 50%–70% of pancreatic cancers [5]. The p53 protein is needed for important functions in the cell, such as G1-S cell cycle checkpoint, arresting cells at G2-M, and inducing apoptosis of aberrant cells [5]. Mutated cells with loss of p53 function generally accumulate more genomic alterations and this allows abnormal pancreatic cells with damaged DNA to survive more and then continue to divide. The transcriptional regulator FoxM1 is also a player in the tumorigenesis process such as initiation, progression, and metastasis in many tumor types [6]. FoxM1 is activated in pancreatic cancer and other cancers such as lung cancer, glioblastomas, prostate cancer, basal cell and hepatocellular carcinoma [7, 8]. Considering the poor survival rates and burden of small window of time in pancreatic cancer, targeting FoxM1 constitutes a meaningful strategy. Pancreatic cancer chemotherapy is also associated with resistance. It is also reported that many primary pancreatic cancers and cell lines, but not normal pancreatic cells, show constitutive activity of NF-κB, which is an important transcription factor promoting survival of cells [9, 10].

Induction of reactive oxygen species (ROS) is also known to inhibit cell growth and induce apoptosis and cell death [11, 12]. In particular, in pancreatic adenocarcinoma cells with p53 deletions, cell growth is strongly inhibited by ROS-inducing compounds [13]. Further, it is reported that pancreatic cancer cells with lower levels of ROS are more resistant to chemotherapy [14, 15]. Collectively, there is an understanding that increases in ROS production presents a good strategy to induce apoptosis in those cancer cells with decreased antioxidant capacity) and also to improve upon chemotherapy-induced resistance during pancreatic cancer treatment [14]

Nonsteroidal anti-inflammatory drugs (NSAIDs) are prototypical anti-cancer agents whose long-term use is associated with adverse side effects mainly gastrointestinal, cardiovascular, and renal, reviewed in [16]. Nitric oxide (NO) and hydrogen sulfide (H₂S) are endogenous gaseous mediators that are associated with improved mucosal defense mechanisms [17, 18]. For these reasons, we recently developed NOSH-aspirin, a hybrid entity capable of releasing both NO and H₂S [19]. Our preliminary results indicated that NOSH-aspirin was a potent anti-inflammatory agent devoid of cellular toxicity, which was also active against a variety of cancer lines at very low concentrations [19]; was efficacious against established tumors in a xenograft mouse model of colon cancer [20]; was safe to the stomach yet it retained all the pharmacological hallmarks of its parent compound aspirin [21]. In this study we explored the effect of NOSH-aspirin on twopancreatic cancer cell lines kinetics and a xenograft mouse model evaluating redox sensitive proteins that are important in determining cellular mass and carcinogenesis.

2. Materials and Methods

2.1. Reagents and cell lines

NOSH-aspirin (NBS-1120) 4-(3-thioxo-3H-1,2-dithiol-5-yl)phenyl 2-((4-(nitrooxy)butanoyl)oxy), benzoate was synthesized as described previously [19] and was a gift from Avicenna Pharmaceuticals Inc, (New York, NY). The structural components of NOSH-aspirin are shown in Figure 1. Aspirin was obtained from Sigma (St Louis, MO). Stock solutions (10 mM or 100 mM) were made in DMSO; final DMSO concentration was adjusted in all media to 1%. The pancaspase inhibitor z-VAD-FMK (z-VAD) was obtained from Promega (Madison, WI) and used according to the manufacturer’s instructions. Human pancreatic cancer MIA PaCa-2 (CRL-1420) and BxPC-3 (CRL-1687) cells were purchased from American Type Culture Collection (ATCC, Manassas, VA) with verification of authenticity indicating them to be mycoplasma-free; human normal pancreatic epithelial ACBRI 515 was purchased from the European Collection of Cell Culture (ECACC, Salisbury, UK) and certified as such by the supplier. Cells were maintained in RPMI-1640 media (Invitrogen, Carlsbad, CA) that was supplemented with 10% fetal bovine serum and 100 μg/mL penicillin, and 100 μg/mL streptomycin (Invitrogen, Carlsbad, CA) and maintained at 37°C and in an atmosphere of 5% CO₂/95% air.

Figure 1.

Structural components of NOSH-aspirin. The parent compound aspirin is shown in the shaded box. The parts of the molecule that releases NO and H₂S are shown in the dotted ellipses.

2.2. Cell growth inhibition assay

The growth inhibitory effect of NOSH-aspirin was measured using a colorimetric MTT assay kit (Roche, Indianapolis, IN). Briefly, cells were plated in 96 well plates at a density of 10⁴ cells per well and, following overnight incubation, were washed and treated with vehicle, NOSH-aspirin or ASA. At indicated time periods of treatment, viable cells were incubated with MTT substrate (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide, 5 mg/mL in phosphate buffered saline), further for 2 hrs, and formazan formed by viable cells was dissolved in 100 μL of the solubilization solution (10% SDS in 0.01 M HCl) and absorbance was measured on a spectrophotometric plate reader at a wavelength of 570 nm. Each experiment was performed in triplicate, and the entire experiment was repeated three times. In some experiments pan-caspase inhibitor z-VAD-FMK, pretreatment was performed prior to NOSH-aspirin addition to cells, followed by MTT assay.

2.3. Cell proliferation

Proliferating cell nuclear antigen (PCNA) was determined using an ELISA Kit (Calbiochem, La Jolla, CA), in accordance with the manufacturers protocol and read on spectrophotometric plate reader as previously described [22].

2.4. Flow cytometry for phase distribution in the cell cycle and detection of apoptosis

Cell proliferation and cell cycle phases:

Cell proliferation and cell cycle analysis were performed as previously described [22, 23]. Cell cycle phase distributions were obtained using a Coulter Profile XL equipped with a single argon ion laser. For each subset, >10,000 events were analyzed. All parameters were collected in list mode files. Data were analyzed on a Coulter XL Elite Work station using the Software programs Multigraph™ and Multicycle™. The percentage of cells in G₀/G₁, G₂/M, and S phases was determined form DNA content histograms.

Apoptosis assay:

Cell death was determined by FITC-labeled Annexin V and propidium iodide (PI), using an Annexin V-FITC apoptosis detection kit, (BD Biosciences Pharmingen, San Jose, CA, USA), according to the manufacturer’s instructions, as reported previously [22]. Treated cells (floating and adherent cells) were collected, resuspended in an Annexin V binding buffer and transferred to test tubes containing FITC-labeled Annexin V and PI. The cells were then incubated for 15 minutes at room temperature in the dark, and analyzed by flow cytometry. Annexin V–FITC detects cells in early apoptosis by staining externalized phosphatidylserine, and the nuclear stain PI identifies cells that have lost their plasma membrane integrity such as necrotic or late apoptotic cells. Annexin V-FITC-positive/PI-negative cells were regarded as a good measure of early apoptosis. Percentage of apoptotic cells was obtained using a Becton Dickinson LSR II equipped with a single argon ion laser. For each subset, about 10,000 events were analyzed. All parameters were collected in list mode files. Data was analyzed by Flow Jo software.

2.5. Caspase-3 activity measurement

Cells were treated with NOSH-aspirin at its IC₅₀ (50 nM) for cell growth inhibition and caspase-3 activity was assayed as a function of time (0, 3, 6, 12 hr) using a colorimetric kit from Abcam bcam (Cambridge, MA) following the manufacturer’s instructions. The assay is based on spectrophotometric detection of the chromophore p-nitroaniline (p-NA) after cleavage from the labeled substrate DEVD-p-NA. The p-NA light emission was quantified using a plate reader at 400 nm. In some experiments the pan-caspase inhibitor z-VAD-FMK was used following the manufacturer’s instructions.

2.6. Determination of reactive oxygen species (ROS)

Cells (0.3 × 10⁶ cells/well) were plated in 6-well plates for 24 h after which they were treated with various concentrations of NOSH-aspirin for 6 h. Cells were then trypsinized, washed once in PBS resuspended and then incubated for 30 min at 37°C in the dark with the oxidation-sensitive fluorescent probes 2’,7’-dichlorodihydrofluorescein diacetate (H2DCFDA, 10 μM) or dihydroethidium (DHE, 5 μM) (Molecular Probes, Life Technologies, NY). Fluorescence intensity was then measured by flow cytometry using a FACS Calibur (BD Bioscience). H2DCFDA is a probe for H₂O₂, peroxyl radical, including both alkylperoxyl and hydroperoxyl, while DHE is an intracellular probe that preferentially measures superoxide anion [24, 25]. A minimum of 10,000 events were analyzed and expressed as fluorescent intensity versus events.

2.7. Mouse xenograft study

Our institutional animal care and research committee approved all experimental procedures described here. Male athymic SCID mice, age 5 weeks, were purchased from Charles River Laboratories, Inc., (Wilmington, MA) and were housed according to institutional and National Institute’s of Health (NIH) guidelines. In this post-initiation study, tumors were initiated and followed with NOSH-aspirin treatment. MIA PaCa-2 cells (2×10⁶) were suspended in Matrigel (BD Biosciences, San Jose, CA) 50% v/v and were inoculated subcutaneously in the right flanks of each mouse (N=10) using a 1-mL syringe and 22-gauge needles. After 10 days (average tumor sizes of ~70 mm³) the mice were randomly divided into 2 groups (N=5/gp) and gavaged daily with NOSH-aspirin (100 mg/kg/d) or vehicle (1% methylcelloluse). The mice were closely monitored for signs of toxicity. Tumor volume and animal weight were recorded every 3 days. Tumor volumes were calculated using the following formula: length × width²/2. After 30 days of treatment, mice were sacrificed, tumors excised, weighed, photographed and fixed in 10% buffered formalin for immunohistochemistry studies.

2.8. Immunohistochemistry

Paraffin embedded sections were dewaxed, rehydrated and microwave heated for 15 min in 0.01 M citrate acid buffer (pH 6.0) for antigen retrieval. This was followed by 3% hydrogen peroxide for 15 min to block endogenous peroxidase activity and normal horse serum for 15 min as a blocking solution. The primary antibody or isotype IgG control were applied and incubated overnight at 4° C. Slides were washed 3 times with PBS, each for 5 min. The biotinylated secondary antibody and the streptavidin-biotin complex (Zymed Laboratories, San Francisco, CA) were applied, each for 30 min at room temperature with an interval washing. After rinsing with PBS, the slides were immersed for 5 min in the substrate 3,3’-diaminobenzidine (DAB, Sigma-Aldrich Corp. St. Louis, MO) 0.4 mg/mL with 0.003% hydrogen peroxide, then rinsed with distilled water, counterstained with hematoxylin, dehydrated and cover-slipped. Antibodies: PCNA, p53 and FoxM1 were from Santa Cruz Biotechnology (Santa Cruz, CA); NF-ĸB p65 (which recognizes activated NF-ĸB), was from Chemicon International (Temecula, CA).

2.9. TUNEL and ROS staining in tissues

TUNEL staining was performed using the In Situ Cell Death Detection kit (Roche Applied Science, Indianapolis, IN) following the manufacturer’s instructions. Briefly, 4 μm thick formalin-fixed, paraffin-embedded tissue sections were deparaffinized and rehydrated. Endogenous peroxidase activity was quenched by hydrogen peroxide and tissue protein was hydrolyzed with proteinase K (Roche Applied Science, Indianapolis, IN). Negative control: sections incubated with Label Solution (without TdT enzyme). All other sections were incubated with TUNEL reaction mixture (fluorescein-labeled nucleotides) at 37°C for 1 h in a humid chamber, incubated with Converter-POD solution (anti-fluorescein antibody conjugated with POD) for 30 min at 37°C, treated with DAB and counterstained with hematoxylin. For ROS detection by immunofluorescence in tumor tissues, paraffin embedded sections were stained with 5 μmol/L CM-H2DCFDA (Invitrogen, Carlsbad, CA) for 1.5 hours at 37°C. Images were taken using a fluorescent microscope (BX41, Olympus) controlled by IPlab (Scanalytics Inc., Fairfax, VA). Randomly selected areas were photographed with the same exposure time. The images were processed using the same fixed threshold in all samples by Imagej software; representative images are shown.

2.10. Scoring the expression of biomarkers

For each animal and treatment group, 5 slides were prepared and scored by a pathologist blind to the identity of the specimens (x400). For PCNA, and TUNEL staining, cells with a brown nucleus were considered labeled and those with a blue nucleus unlabeled. For each, the percentage of positive cells over the total cells counted was calculated. For NF-κB, FoxM1, and p53 the following semi-quantitative scoring system was used. The extent of staining was graded as follows: 0 = no staining; 1+ = ≤25% of cells positive; 2+ = 26% – 50% of cells positive; 3+ = ≥51% of cells positive. The intensity of staining was scored as follows: 0 = no staining; 1+ = faint; 2+ = moderate; 3+ = strong. 1+, 2+ and 3+ were recorded as 1, 2 and 3 points, respectively. To compare differences in staining, an Expression index (EI) was calculated by the following formula: EI = Extent of staining × Intensity of staining, as described previously [26].

2.11. Statistical analysis

In vitro data are presented as means ± SEM for at least 3 different sets of plates and treatment groups done in duplicate or triplicate. Xenograft data, tumor volume and mass are presented as means ± SEM for 5 animals in each group. Comparisons between groups were performed using a one-way analysis of variance followed by the Student-t test.

3. Results

3.1. NOSH-aspirin inhibits pancreatic cancer cell growth

The effects of NOSH-aspirin and ASA were examined on cell growth of two pancreatic cancer cell lines MIA PaCa-2 (Cox-1 and Cox-2 negative, p53 mutated, K-ras mutated, p16 deleted) and BxPC-3 (Cox-1 and Cox-2 positive, p53 mutated, K-ras wild type) [27], and a normal pancreatic ductile epithelial ACBRI 515 cell line. Cells were exposed to various concentrations of these compounds for 24 hrs and MTT assays were performed. From their growth inhibition curves, IC₅₀ values were calculated (Table 1). NOSH-aspirin inhibited the growth of MIA PaCa-2 and BxPC-3 cells at nanomolar levels with calculated IC₅₀ values 47 ± 5 nM and 57± 4 nM at 24h, respectively, while it did not inhibit growth of normal ACBRI 515 cells at these low concentrations. These results strongly suggest that NOSH-aspirin’s effect on cancer cell growth maybe COX independent, as MIA PaCa-2 cells are COX null while BxPC-3 cells express both COX-1 and COX-2. The IC₅₀ for the normal cell line was determined to be 28,000 ± 2000 nM (Table 1), that is ~500-fold higher, demonstrating the differential behavior of this novel compound towards cancer cells. The IC₅₀ of the parent compound aspirin was more than 5mM in these cells at 24 hr. Overall, NOSH-aspirin demonstrated an increase in potency as determined by the ratio of the IC₅₀s (ASA / NOSH-aspirin) which was calculated to be ~80,000-fold more than ASA. Finally, as K-ras wild type and K-ras mutated cell lines responded to NOSH-aspirin treatment much the same way, it appears that NOSH-aspirin growth inhibition are independent of the RAS/RAF/MAPK pathway.

Table 1.

IC₅₀s for cell growth inhibition at 24 hr.

	IC50 nM at 24 hr
	MIA PaCa-2	BxPc-3	ACBRI 515
COX-1, −2 status	−	+	NK
NOSH-aspirin	47± 5	57 ± 4	28,000 ± 2000
ASA	> 5,000,000		ND
Enhanced potency	> 80,000*		~500†

Pancreatic cancer cell lines MIA PaCa-2 and BxPC-3 and a normal pancreatic ductile epithelial cell line ACBRI 515 were treated with different concentrations of NOSH-aspirin or ASA for 24 hr and IC₅₀s for cell growth inhibition determined. Results are mean ± SEM for at least 3–5 different experiments performed in triplicate.

ND = not determined. NK = not known

^*= Fold-increase for NOSH-ASA vs ASA determined as IC₅₀ ASA / IC₅₀ NOSH-aspirin.

^†= Fold-increase in potency towards cancer cells determined as IC₅₀ normal / IC₅₀ cancer cells.

3.2. NOSH-aspirin modulates cellular mechanisms

MIA PaCa-2 and BxPC-3 cells were analyzed for effects of NOSH-aspirin on their cell proliferation, cell cycle phase transitions and apoptosis by flow cytometry. The concentration of 50 nM was selected for these studies representing a dose very close to their respective IC₅₀ values. Proliferation status, as measured by proportion of cells expressing PCNA when treated with the vehicle DMSO or 50 nM NOSH-aspirin for 0, 3, 6 and 12 hr showed a time-dependent anti-proliferative effect observed by reductions in PCNA expression for both cell types (Fig 2A, MIA PaCa-2 and 2D, BxPC-3). This was associated with block of cell cycle transitions by NOSH-aspirin at G₀/G₁ phase, based on the increase of population of cells in the G₀/G₁ phase from 42% to 59% for MIA PaCa-2 cells (Fig 2B), and from 39% to 58% for BxPC-3 cells (Fig 2E) at 12 hrs. Early apoptosis as quantified by Annexin V FITC-positive and PI-negative stained cell populations at these time points produced similar increases to approximately 65% to 70% early apoptotic cells for both cell types at 12 hrs (Figs 2C and 2F). Overall, it appears that NOSH-aspirin produces similar effects on these two cell lines and in general, inhibits cell growth via inhibition of proliferation, G₀/G₁ cell cycle arrest and apoptosis. We have reported earlier that effects of NOSH-aspirin are independent of cell type [19].

Figure 2.

Effect of NOSH-aspirin on MIA PaCa-2 and BxPC-3 pancreatic cancer cell kinetics. NOSH-aspirin inhibits proliferation by altering cell cycle progression and inducing apoptosis. Cells were treated with vehicle or 50 nM NOSH-aspirin (close to IC₅₀ for cell growth inhibition in both cell lines) and analyzed at the indicated times for proliferation by PCNA antigen expression (Panels A and D, MIA PaCa-2 and BxPC-3 cells respectively); cell cycle phases by PI staining and flow cytometry (Panels B and E, MIA PaCa-2 and BxPC-3 cells respectively); and apoptosis by Annexin V staining and flow cytometry (Panels C and F, MIA PaCa-2 and BxPC-3 cells respectively). In (A), (C), (D), and (F), results are mean ± SEM for 3 different experiments performed in duplicate, *P < 0.05 at the indicated time points. In (B) and (E), results are representative of two different experiments. This study was repeated twice generating results within 10% of those presented here.

3.3. NOSH-aspirin induces caspase-3 and reactive oxygen species

Caspase-3 activation, which is important for mediating the induction of apoptosis, was measured in both cell lines treated with 50 nM NOSH-aspirin at time intervals up to 12hrs. NOSH-aspirin increased the activity of caspase-3 in a time dependent manner to a statistically significant four- to five-fold increase above control levels at 12 hrs (P < 0.01) (Fig 3A, BxPC-3 and Fig 3B, MIA PaCa-2). Overall, both cell lines behaved similarly in their responses. Pretreatment for 1 hr with z-VAD-FMK, a cell permeable pan-caspase inhibitor abolished the increases in caspase-3 activity at all time intervals, demonstrating that NOSH-aspirin induces cell death through caspase activation in both cell lines.

Figure 3.

NOSH-aspirin treatment increases caspase-3 activity. MIA PaCa-2 (Pane A) and BxPC-3 (Panel B) pancreatic cancer cells were treated with vehicle or 50 nM NOSH-aspirin. Caspase-3 activity was increased in both cell lines at all time points, 3, 6, and 12 hr. Results are mean ± SEM for 4 different experiments performed in duplicates. *P < 0.05 and ^†P < 0.01 and the indicated times. The pan-caspase inhibitor z-VAD-FMK inhibited caspase-3 activity at all time points.

Very high ROS levels are known to push cells towards apoptosis [12]. We examined whether NOSH-aspirin induced ROS in these cells. A molecular probe that detects several individual reactive oxygen species including peroxides is H2DCFDA, also abbreviated as DCFDA [28, 29]. DHE is a selective probe for detection of superoxide anions (•O₂-) [30]. Cells were treated with 50 nM NOSH-aspirin for 6 hr and analyzed for levels of intracellular peroxides by flow cytometry. At 6 hr, 50 nM NOSH-aspirin increased production of intracellular peroxide and •O₂- levels in MIA PaCa2 cells by approximately two orders of magnitude (Fig 4A and 4B, respectively). Similar results were also obtained with the BxPC-3 cells (data not shown). We also determined the levels of ROS in MIA PaCa2 cells following treatment with z-VAD-FMK and NOSH-aspirin. ROS that was produced at 6 hr with NOSH-aspirin was blocked when caspase-3 was inhibited with z-VAD-FMK pretreatment in MIA PaCa2 (Fig. 4C) and BxPC-3 M cells (Fig. 4D). This strongly suggests that caspase-3 activation precedes and is necessary for ROS generation.

Figure 4.

NOSH-aspirin induces ROS levels. MIA PaCA-2 cells were treated with 50 nM NOSH-aspirin for 6 h followed by staining with a general ROS probe DCFDA (A) or DHE which detects superoxide anions in cells (B). These experiments were repeated twice and representative histograms are shown. ROS in MIA PaCA-2 cells was also probed with DCFDA following treatment with z-VAD and NOSH-aspirin (C) and similarly examined in BxPC-3 cells (D). The coloring of the backgrounds in the histograms and cleaning of the axis was done by using Adobe Photoshop CS5.

3.4. Caspases are necessary for cell growth inhibition by NOSH-aspirin

Since caspase-3 activation and ROS production appear to be key features of NOSH-aspirin’s mechanism of action, we examined whether blocking caspase-3 activation could indeed block the inhibition of growth that we observed at the IC₅₀ concentrations of NOSH-aspirin. We did not include ASA in these studies because ASA’s IC₅₀ for cell growth inhibition at 24 hr was greater than 5mM. MIA PaCa-2 and BxPC-3 cells were pretreated with z-VAD (2 μM) for 4 hr followed by NOSH-aspirin (50 nM) or vehicle (control) for 24 h. Growth inhibition was measured by the MTT assay. Whereas cells were approximately 50% growth inhibited by NOSH-aspirin, this inhibition was markedly and partially reversed by z-VAD suggesting that caspases are necessary for the effects of NOSH-aspirin. (Fig 5).

Figure 5.

NOSH-aspirin mediated cell growth inhibition is reversed by zVAD. MIA PaCa-2 and BxPC-3 cells, pretreated with 2 μM z-VAD-FMK for 4 hr or untreated, were followed by 50 nM NOSH-aspirin (~IC₅₀ concentration) and subsequently analyzed for growth as described in Section 2.2. Values represent means ± SEM of three representative experiments performed in triplicate. *P < 0.05 compared to absence of z-VAD.

3.5. NOSH-aspirin inhibits tumor growth in a xenograft model with no evidence of gross toxicity

In this treatment model, tumor xenografts were transplanted by human pancreatic cancer cells to evaluate the antitumor effects of NOSH-aspirin in vivo. Male athymic nude mice were used for subcutaneous implantation of MIA PaCa-2 cells (2 × 10⁶) in the right flank. The mice were divided into two groups after 10 days when tumors were palpable and had a volume of about 70 mm³. The test group received daily administration of NOSH-aspirin (100 mg/kg, gavage), while the control group was gavaged with the vehicle (1% methylcellulose) daily till the end of the experiment, the protocol followed is shown in Fig 6A. During the 30 days of this study, mice did not die as a result of the received treatment; the body weights of the NOSH-aspirin-treated and vehicle-treated mice throughout the duration of the study did not vary (Fig 6B). After 30 days and post sacrifice, NOSH-aspirin-treated mice showed a significant reduction in tumor volume compared with the control mice (Fig 6C and inset). The control- and NOSH-aspirin- mean tumor volumes at the end of the study were 3265 ± 520 mm³ and 330 ± 95 mm³, respectively (Fig 6C), representing a mean reduction of about 90% (P=0.001). Tumor mass for control and NOSH-aspirin-treated mice at the end of the protocol were 2.47 ± 0.24 g and 0.62 ± 0.25 g respectively (Fig 6D), with 75% reduction (P=0.003). Figure 6E shows the vehicle and NOSH-aspirin-treated mice with the xenografts shown with arrows and Fig 6F shows the excised tumors in the two groups. After sacrifice, close inspection of the main organs (heart, liver, kidney, spleen) did not show any gross signs of toxicity (deformity, edema or bleeding) in the NOSH-aspirin treated mice.

Figure 6.

NOSH-aspirin inhibits growth of established tumor xenografts. Athymic SCID male mice were injected subcutaneously with MIA PaCa-2 cells for the development of subcutaneous tumors as described in Section 2.7. Following tumor formation, the mice were randomly divided into 2 groups (N = 5) and treated daily with vehicle or 100 mg/kg NOSH-aspirin for 30 days according to the protocol in panel A. Body weight of both groups over 30 days (panel B) and average tumor volume as function of time and tumor mass at sacrifice are shown in panels C and D. The inset shows an expanded scale for the NOSH-aspirin-treated mice. Representative mice and tumors for untreated and treated mice are shown in panels E and F, respectively. NOSH-aspirin significantly reduced tumor volume from day 15 of treatment to sacrifice, *P < 0.01. Tumor mass was also significantly reduced by NOSH-aspirin treatment, P = 0.003.

The tumor sections were examined for proliferating cells by the expression of PCNA stained cells, for apoptotic cells by TUNEL staining, and for promoters of cell survival by the expression by NF-ĸB p65. Compared to control tumors, the NOSH-aspirin group had reduced expression of PCNA (from 78.4 ± % to 25.2 ± 3 %, P < 0.01) (Fig 7A), increase in number of apoptotic cells (from 3.4 ± 0.3 % to 83.9 ± 3 %, P < 0.01) (Fig 7B), and reduced expression of NF-ĸB cells (from 63.6 ± 7 % to 14.7 ± 3 %, P < 0.01), (Fig 7C). These corroborated the in vitro observations from cell culture studies and demonstrate that in vivo tumor growth inhibition occurs by a combination of apoptosis induction and proliferation reduction, and reduced NF-ĸB expression. Since ROS induction was observed in vitro and may contribute to apoptosis, tumor sections were stained for ROS and observed by fluorescence. The NOSH-aspirin group showed marked increases in peroxide- (7 ± 1% control to 90 ± 2% treated) (Fig 8A) and superoxide (9 ± 1% control to 84 ± 3% treated) anion compared to vehicle treated controls (Fig 8B). Another target of interest that may be associated with induction of apoptosis is the inducible nitric oxide synthase (iNOS), which was visualized by staining in the NOSH-aspirin-treated group, and determined to be induced by NOSH-aspirin (1.6 ± 0.1% control versus 33 ± 2% treated), (Fig 8C).

Figure 7.

NOSH-aspirin inhibits proliferation, induces apoptosis and decreases NF-κB p65 in vivo. Stored tumors were sectioned, probed, and scored as described in Section 2.8–10. Average mitotic index at sacrifice was determined by PCNA staining (P < 0.05), TUNEL staining (P < 0.01) and p65 staining (P < 0.02). Representative fields used for quantification of the staining are shown. The scale bar represents 200 μm.

Figure 8.

NOSH-aspirin induces ROS, iNOS, and p53 and inhibits FoxM1 in vivo. Stored tumors were sectioned, probed, and scored as described in Section 2.8–10. NOSH-aspirin-treatment increased peroxide and super oxide anions, as well as iNOS and p53 expression while it reduced FoxM1 levels. Five different representative fields were used for quantification from each tumor (N=5), in all cases P < 0.01 compared to vehicle treated mice. The scale bar represents 200 μm.

3.6. FoxM1 is modulated by NOSH-aspirin

FoxM1 (Forkhead box M1) is a transcription factor that regulates a network of proliferation-associated genes critical to mitosis [31, 32]. Its expression increases at G₁ to S phase transition, and reaches maximal levels in G₂ to M phase, promoting M phase entry [33] with depletion leading to cell cycle arrest. Importantly, aberrant upregulation of FoxM1 has been shown to be a key driver of cancer progression and has been proposed as an initiating factor of oncogenesis [34–36]. Since NOSH-aspirin accumulated cells in the G₁ phase, we examined the expression of FoxM1 by immunohistochemical staining. The tumors from the NOSH-aspirin group had approximately 4-fold reduced levels of FoxM1 expression, compared to control (80 ± 3% control to 21 ± 1% treated, Fig 8D). It is known that p53 negatively regulates the expression of FoxM1 [8]. We therefore, evaluated the expression of p53 in our xenograft tumors. Staining demonstrated that in NOSH-aspirin-treated tumors, there was a large increase (about 40-fold) in p53 protein expression, 1.8 ± 0.1% to 79 ± 5% in control and treated tumors, respectively (Fig 8E).

4. Discussion

This study demonstrates that NOSH-aspirin inhibits tumor growth of pancreatic cell xenografts as well as pancreatic cancer cell lines with the advantages of strong potency and differential activity in cancer- versus normal- cell. Nanomolar concentrations inhibited pancreatic cancer cell growth, arrested cells in G₀/G₁ phase transition and induced apoptosis in vitro, while normal pancreatic epithelial cells were fairly unaffected. Mutations in the proto-oncogene Kras and tumor suppressor p53 are known to be major genetic alterations associated with cell cycle deregulation and apoptosis inhibition [37]. As MIA PaCa-2 cells have mutations in K-Ras and p53 [38], while BxPC-3 cells exhibit wild type K-Ras and mutated p53, it is promising that NOSH-aspirin inhibited both of these cell lines with low IC₅₀ values. Apoptosis and cancer cell growth inhibition were also corroborated in the NOSH-aspirin xenograft model. Progression of MIA PaCa-2 xenograft tumors was strongly reduced and a combination of mutiple mechanisms may contribute to this process. Caspase-dependent apoptosis induction occurred in tumors and in cancer cells in vitro. This conclusion is based on our current observation that the pan-caspase inhibitor z-VAD-FMK inhibited caspase-3 activation and on our previous studies with NO-releasing aspirin [39] and H₂S-releasing aspirin [40] where we had shown that the IC₅₀ for cell growth inhibition in Jurkat T-leukemia cells increased by almost a factor of 5 in the presence z-VAD-FMK. Increases in ROS such as peroxide and superoxide anion production and an increase in iNOS expression by NOSH-aspirin were also associated with reduced tumor progression. Very high ROS levels are known to push cells to apoptosis by oxidative stress through both p53 and iNOS pathways [41].

As it is accepted that iNOS-mediated NO has the capacity to either promote or prevent tumorogenesis, therefore, the role of iNOS/NO in pancreatic cancer also remains controversial. For example, iNOS/NO promotes development and progression of pancreatic ductal adenocarcinoma [42] and increased iNOS expression is associated with poor survival in pancreatic ductal adenocarcinoma patients [43]. On the contrary, the aggressive pancreatic cancer cell line Panc02-H7 was associated with a low iNOS expression [44] and exogenous NO addition was able to reduce the proliferation and invasion [44]. Studies also note that iNOS affects the tumor microenvironment such that tumors escape the immune response [45], However, ectopic or orthotopic xenograft mouse models injected with iNOS transfectedpancreatic cancer cells did not form tumors or metastases likely due to NO-mediated apoptosis [46]. In our study, considering that the source of ROS production is not known, tumor microenvironment may be suspected to contribute to the tumor growth inhibition observed as iNOS expression is increased in the tumors. Desmoplastic stroma makes up 80% of tumor mass in pancreatic ductal adenocarcinoma [47], therefore, the interplay of tumor and its microenvironment components by NOSH-aspirin would be of interest for future investigation.

A molecular target that is relevant to inflammatory processes and is inhibited by NSAIDs is cyclo oxygenase (COX). When we compared the GI toxicities of NOSH-aspirin to that of aspirin [21], we showed that NOSH-aspirin was significantly less toxic than aspirin and that in the gastric mucosa, both aspirin and NOSH-aspirin reduced PGE₂ content indicating significant COX inhibition. This is inline with another report from us indicating preferential dose-dependent inhibition of pure ovine COX-1 vs COX-2 enzymatic activity by NOSH-aspirin [20]. We also showed that NOSH-aspirin inhibits platelet aggregation, a COX-1 mediated effect; effectively reduces LPS-induced fever, a COX-2 mediated effect, and has excellent anti-inflammatory properties using the carrageenan paw oedema model representing a COX-2 mediated effect [21]. Using this model, we also showed that NOSH-aspirin dose dependently inhibited COX-1 and COX-2 expression [19]. These results underscore the importance of being cautious when applying in vitro data to any in vivo settings. In the present study the actions of NOSH-aspirin appear to be COX independent since the cell lines that we used, BxPc-3 that expresses both COX-1 and COX-2 and MIA PaCa-2, which does not express COX at all [48], responded similarly to the actions of NOSH-aspirin.

Pancreatic cancers are associated with activated FoxM1 signaling pathway and its downregulation produces anti-cancer effects in pancreatic cancer cell lines [49]. Our study demonstrated that NOSH-aspirin treatement lead to FoxM1 downregulation. FoxM1 has been implicated in regulation of proliferation [35] and in cell cycle control. For example it was found that G2/M arrest occurred if FoxM1 was repressed [50].

The current understanding is that p53 executes its pancreatic tumor-suppressive activity at least in part via downregulation of FoxM1 expression [51]. In tumors where p53 is inactivated, it is believed that this in part, may lead to FoxM1 overexpression [8, 50]. In other studies, although FoxM1 repression and its reduced protein levels has been associated with G2 arrest [50], however, here we found that NOSH-aspirin caused a large accumulation of the cells in the G1 phase and G2 arrest was not strongly evident.

Examining other pre-clinical studies on pancreatic cancer models and our current study, it is not possible to make a straight comparison. In this study, p53 defective xenograft pancreatic tumors showed strong reductions in tumor growth and mass. Yet it is important to note that in mice with p53-defective tumors, a targeted approach of EGFR downregulation did not inhibit tumor development [52]. NOSH-aspirin reduces tumors and inhibits cancer cells through mechanisms that converge on caspases and apoptosis, and which maybe in part ROS- mediated and NF-κB inhibition-mediated. ROS production appears to be caspase-dependent and a downstream event since inhibition of caspases completely blocked NOSH-aspirin-mediated ROS generation to basal levels of the cancer cells. It is known that caspase-induced ROS generation can contribute to the process of apoptosis [53]. In this regard, our overarching hypothesis, supported by in vitro and in vivo studies has been that NOSH-aspirin increases ROS within the cancer cells, shifting the balance towards apoptosis; of course these events may or may not be caspase-dependent. In our previous preliminary studies with breast cancer cells, we have shown that treatment of the cells with N-acetyl cysteine (NAC) partially reverses the effects of NOSH-aspirin on apoptosis [54].

Another aspect of our studies that leads to the conclusion that these effects/observations are COX-independent is the fact that our IC₅₀s for cell growth inhibition in the two cell lines studied were very similar. MIA PaCa-2 cells are COX-null and BxPC-3 cells express both COX-1 and COX-2 [27]. COX-independent effects of NSAIDs are now well accepted [55].

Regarding the potency of NOSH-aspirin in terms of growth inhibition observed here, comparison of the IC₅₀ values (aspirin/NOSH-aspirin) suggests that NOSH-aspirin was at least 80,000-fold more potent than aspirin. Such fold increases imply that the NO and H₂S-related structural modifications of the aspirin molecule imparts a differential enhancement in potency. Currently we cannot explain the underlying mechanism(s) for the enhanced potency of NOSH-aspirin observed in these studies; however, our very preliminary data suggests that NO and H₂S that are released from NOSH-aspirin react to form highly reactive polysulfides. Our earlier studies had indicated that both NO and H₂S contribute towards the potency of the intact NOSH-aspirin molecule. We had shown that the biological activity of aspirin plus SNAP (S-Nitroso-N-acetylpenicillamine, which releases NO) plus ADT-OH (5-(4-hydroxyphenyl)-3H-1, 2-dithiole-3-thione, which releases H₂S) was not the same as the biological activity of the intact NOSH-aspirin molecule [20]. Thus the sum of parts did not equal the whole. Other reports indicate that NO can react with H₂S to produce HSNO (the smallest S-nitrosothiol) which is a highly reactive intermediate [56, 57]. Furthermore, NO and H₂S signaling pathways appear to be intimately intertwined with mutual potentiation of biological responses [58].

Overall, these studies demonstrate that NOSH-aspirin is inhibitory to human pancreatic tumor xenograft via combined effects of increases in ROS, iNOS, caspases, mutated p53 and downregulation of FoxM1. Collectively this study and our previous reports with H₂S-releasing aspirin support our hypothesis and proposed working model that NOSH-aspirin induces ROS levels, induces p53, down-regulates FoxM1, which leads to growth arrest and apoptosis.