Pancreatic tumors are resistant to conventional chemotherapies. The present study was aimed at evaluating the potential of a novel plant-derived product as a therapeutic agent for pancreatic cancer (PC). The effects of an extract from the tropical tree Annona Muricata, commonly known as Graviola, was evaluated for cytotoxicity, cell metabolism, cancer-associated protein/gene expression, tumorigenicity, and metastatic properties of PC cells. Our experiments revealed that Graviola induced necrosis of PC cells by inhibiting cellular metabolism. The expression of molecules related to hypoxia and glycolysis in PC cells (i.e. HIF-1α, NF-κB, GLUT1, GLUT4, HKII, and LDHA) were downregulated in the presence of the extract. In vitro functional assays further confirmed the inhibition of tumorigenic properties of PC cells. Overall, the compounds that are naturally present in a Graviola extract inhibited multiple signaling pathways that regulate metabolism, cell cycle, survival, and metastatic properties in PC cells. Collectively, alterations in these parameters led to a decrease in tumorigenicity and metastasis of orthotopically implanted pancreatic tumors, indicating promising characteristics of the natural product against this lethal disease.

2.1 Graviola Extract

Graviola supplement capsules were purchased from Raintree (Carson City, NV). The capsules consisted of 100% pure, finely milled Graviola leaf/stem powder with no binders or fillers. The capsule contents were suspended in DMSO (100mg/mL). After incubating for 5min, the suspension was centrifuged and the supernatant (i.e. extract) was filtered to remove any remaining particles. Subsequent dilutions were prepared in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 10% of fetal bovine serum (FBS). Stock solutions and respective dilutions were freshly prepared prior to treatment.

2.2 Cell Culture

The metastatic PC cell lines FG/COLO357 and CD18/HPAF were purchased from the American Type Culture Collection (ATCC). Before performing experiments, the PC cell lines were authenticated by short tandem repeat analysis. It was ensured that PC cells were used at fewer than 20 passages after purchase from ATCC. Cells were cultured in DMEM medium supplemented with 10% FBS and antibiotics (100μg/mL penicillin and 100μg/mL streptomycin). The cells were maintained at 37°C and 5% CO₂ in a humidified atmosphere.

2.3 Antibodies

The antibodies for phospho-ERK1/2, total ERK, phospho-Akt (Ser 473), total Akt, NF-κB, and caspase-3 were purchased from Cell Signaling Technology (Danvers, MA). The antibodies for Cyclin-D1, phospho-FAK (Tyr 925), and total FAK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The β-actin and β-Tubulin antibodies were obtained from Sigma Aldrich (St. Louis, MO), whereas the HIF-1α antibody was purchased from BD Biosciences (San Jose, CA). The MUC4 monoclonal antibody (8G7) used in these studies was developed by our group [22]. MMP9 antibody was obtained from a hybridoma cell supernatant kindly provided by Dr. Rakesh Singh at UNMC. The secondary antibodies used for western blot analyses were the ECL^™ anti-mouse and anti-rabbit IgG conjugated to horseradish peroxidase (GE healthcare, UK). Fluorescein isothiocyanate (FITC) conjugated-anti-mouse and Alexa Fluor conjugated anti-mouse antibodies were obtained from Invitrogen (Carlsbad, CA).

2.4 Cytotoxicity Assay

To determine the cytotoxicity of Graviola extract on PC cells, 1×10⁴ cells were seeded per well on a 96-well plate in DMEM supplemented with 10% FBS and antibiotics. After overnight incubation, different concentrations (10–200μg/mL) of the extract were added into triplicate wells. After 48hr, the media was replaced with fresh media containing thiazolyl blue tetrazolium bromide (MTT) reagent (Sigma Aldrich, St. Louis, MO). After 4hr incubation at 37°C in 5% CO₂ in humidified atmosphere, the media was replaced with 100μL of DMSO and the corresponding cytotoxicity values were calculated (λ=540nm). The experiment was repeated at least three times.

2.5 Western Blot Analysis

For protein analysis, 0.5×10⁶ of PC cells were seeded on each well of a six-well plate in DMEM supplemented with 10% FBS and antibiotics. After overnight incubation, fresh solutions of Graviola (0–200μg/mL) were prepared and added to the respective wells. Cells incubated with the corresponding amount of DMSO present in the highest concentrated solution of Graviola were used as a negative control (0μg/mL). After 48hr of incubation with the extract, protein lysates were isolated and prepared for western blot analysis, as previously described [23].

2.6 Real-time PCR

The transcripts levels of the glucose transporters GLUT1 and GLUT4, the glycolytic enzymes hexokinase II (HKII) and lactate dehydrogenase A (LDHA), and the mucin glycoprotein MUC4 in PC cells were determined after treatment with Graviola extract by real-time PCR. 0.5×10⁶ cells were seeded in each well of a six-well plate in complete media. After overnight incubation, fresh solutions of Graviola extract (50 and 100μg/mL) were prepared and cells were incubated for 48hr. Subsequently, cDNA was synthesized from purified RNA and real-time PCR was carried out as has been described by previous studies [23]. The sequences of the gene-specific primers used were: GLUT1: F 5′-GCCATGGAGCCCAGCAGCAA-3′; R 5′-CGGGGACTCTCGGGGCAGAA-3′ GLUT4: F 5′-GCCTGTGGCCACTGCTCCTG-3′; R 5′-GGGGTCTCTGGGCCGGGTAG-3′ HKII: F 5′-GTCATCCCCTTGTGTCAGAG-3′; R 5′-CTTCATTAGTGTCCCCATCCTG-3′ LDHA: F 5′-CCAGTGTGCCTGTATGGAGTG-3′; R 5′-GCACTCTCAACCACCTGCTTG-3′ MUC4: F 5′-GTGACCATGGAGGCCAGTG-3′; R 5′-TCATGCTCAGGTGTCCACAG-3′

2.7 Glucose Uptake

Glucose-uptake rate was assayed by utilizing [³H] 2-deoxyglucose ([³H] 2-DG). 5×10⁴ PC cells were seeded per well in a 24-well plate. 12hr later, the cells were treated with Graviola extract (10 and 50μg/mL) for 48hr. The cells were then starved for glucose for 2hr and incubated for 20min with 2 Ci [³H] 2-DG. Subsequently, cells were lysed with 1% SDS and the lysates were counted for [³H] by utilizing a scintillation counter. Cells treated with labeled and excess unlabeled 2-DG were used as controls to set a baseline for non-specific [³H] uptake. The results were normalized to the cell counts for treated and untreated groups. Glucose uptake was normalized with that of the control cells (0μg/mL) and it is presented as the mean values ± standard error from experiments performed in triplicate.

2.8 ATP Quantification

The CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Madison, WI) was used to measure the ATP content in the cells. Briefly, 1×10⁴ PC cells were seeded in each well of an opaque 96-well plate. Cells were seeded for both ATP quantification and protein concentration estimation. Starting the next day, the cells were incubated with Graviola extract-containing media for 48hr. Subsequently, the instructions of the manufacturer for ATP quantification were followed and luminescence was measured on a Synergy^™Mx Luminescent Plate Reader (BioTek, Winooski, VT). Data is presented as the mean value for samples in triplicates, normalized with the protein content for each treatment, as determined by utilizing micro-BCA protein estimation kit.

2.9 Detection and Quantification of Apoptosis and Necrosis

To quantify the number of PC cells undergoing apoptosis and necrosis after being incubated with Graviola extract, the Annexin-V-FLUOS staining kit (Roche Diagnostics, Indianapolis, IN) was used. PC cells were seeded and treated with Graviola extract as described above. After 48hr of treatment with Graviola extract, the instructions of the manufacturer were followed for staining cells for flow cytometry analysis. The experiment was repeated three times.

2.10 Cell Cycle Analysis

PC cells were synchronized at the G1/S phase using a double thymidine block. After seeding cells in 100cm² Petri dishes, thymidine (2mM) was added for 12hr. After washing cells with serum-free media, the cells were released from thymidine block by culturing in fresh medium containing 24mM 2-deoxycytidine for 9hr. Then, cells were washed and incubated once more with thymidine (2mM) for 14hr. Subsequently, the cells were released from the second thymidine block and the respective treatment prepared in complete media was added for 48hr. For cell cycle analysis, cells were trypsinized and washed with PBS after the duration of the treatment. Cells were then fixed in 70% ethanol at 4°C for 1hr. After washing, cells were incubated with Telford reagent (EDTA, RNAse A, propidium iodide, Triton X-100 in PBS) at 4°C and analyzed by flow cytometry on the next day.

2.11 Confocal Microscopy

For confocal analysis, 2×10⁵ PC cells were seeded on sterilized round glass cover slips. After overnight incubation, Graviola extract (0, 50 and 100μg/mL) was added to the cells, followed by a 48hr incubation. For the detection of reactive oxygen species (ROS), Graviola extract-treated PC cells were incubated with 1μM 2′-7′-Dichlorofluorescein diacetate (DCFH-DA) (Sigma Aldrich, St. Louis, MO) for 15 min. After three washes with PBS, glass cover slips were mounted on glass slides and visualized by confocal microscopy. For β-tubulin and MUC4 confocal analysis, details of the procedure are published elsewhere [23]. Finally, to visualize the arrangement of actin filaments in Graviola extract-treated cells, the cells were stained with fluorescent phallotoxins (Invitrogen, Carlsbad, CA). The instructions of the manufacturer were followed for formaldehyde-fixed cells. Post-staining, the glass cover slips were mounted with Vectashield medium (Vector Laboratories, Burlingame, CA). LSM 510 microscope, a laser scanning confocal microscope (Carl Zeiss GmbH, Thornwood, NY) was utilized to image the cells in the respective channels at a magnification of 630X.

2.12 Wound Healing Assay

For wound healing assays, 3×10⁶ of PC cells were seeded in 60mm petri dishes in DMEM media supplemented with 10% FBS and antibiotics. After overnight incubation, an artificial wound was induced on 100% confluent PC cell monolayers using a sterile pipette tip. Graviola extract-containing (0, 50, 100μg/mL) media solutions were then added to the respective treatment plate. Images (40X) were captured immediately after adding Graviola extract (0hr) and after 24hr of treatment, by a light microscope. The motility of the cells across the wound was visualized in each treatment group.

2.13 Motility Assay

The effect of Graviola extract on the migration of PC cells was also analyzed by a transwell migration assay. FG/COLO357 cells (0.5×10⁶) were suspended in Graviola extract-containing (0–100μg/mL) 1% FBS-DMEM media and seeded for 48hr in 8μm pore size polyethylene terephthalate (PET) membranes (Becton Dickinson, San Jose, CA). DMEM supplemented with 10% FBS was added at the bottom of each well and after 48hr of incubation, the cells that migrated to the bottom of the PET membrane were stained with Diff-Quick cell staining kit (Dade Behring Inc., Newark, DE). The number of cells migrated was quantified by performing cell counts of 10 random fields at 100X magnification. The results are presented as the average number of cells in one field.

2.14 In vivo tumorigenicity studies

The effect of Graviola extract on pancreatic tumor growth was evaluated on orthotopic tumor xenografts. 6–8 week old female athymic immunodeficient mice were purchased from the Animal Production Area of the NCI/Frederick Cancer Research and Development Center (Frederick, MD). The mice were treated in accordance with the Institutional Animal Care and Use Committee (IACUC) guidelines at UNMC and were housed in pathogen-free environment and were fed sterile water and food ad libitum.

Over 90% viable luciferase-labeled CD18/HPAF cells transduced with retroviral particles (Addgene, Cambridge, MA) were orthotopically injected into the head of the pancreas of immunodeficient mice. Details of the orthotopic implantation procedure are described elsewhere [22, 24]. After 1 week of tumor growth, oral gavage treatment of PBS-suspended Graviola extract was given daily for 35 days. The doses of Graviola extract for these studies were based on previous in vivo studies [18, 19, 25] and on the recommended dose for human consumption [11]. Treatment groups (N=8) included: PBS only (0 mg/kg), 50mg/kg, and 100mg/kg Graviola extract. Graviola extract was not dissolved in DMSO for these studies in order to demonstrate the benefit of the aqueous natural oral supplement in PC therapy. Nevertheless, the cytotoxic properties of the Graviola extract suspended in PBS were corroborated beforehand (Supplementary Fig. 1). In vivo IVIS 200 biophotonic imaging system was used to capture images (Caliper Life Sciences, Hopkinton, MA) of pancreatic tumors within every two weeks during the course of treatment with Graviola extract. Mice were sacrificed after 42 days of tumor growth and 35 days of treatment with Graviola extract. Changes in tumor growth and sites of metastasis were evaluated in each treatment group. Body weights of mice were measured before the treatment.

2.15 Analysis of pancreatic tumor tissues

On the necropsy day, pancreatic tumors from the different treatment groups were divided for protein and immunohistochemistry (IHC) analyses. The tumors were immediately frozen under liquid nitrogen for protein analysis. To prepare tumor lysates, the tumors were then suspended on radioimmunoprecipitation (RIPA) buffer and sonicated for three cycles with a Branson digital sonifier^® (60% amplitude, 10s). After centrifuging the homogeneous suspension, the protein concentration in each sample was estimated and respective solutions for western blot analyses were prepared as previously described [23].

For histopathological and IHC analyses, the tumor tissues were fixed in 10% Formalin for 48hr. The tumors were embedded in paraffin and 5μm sections were cut and stained with hematoxylin and eosin stains (H&E) and various antibodies (i.e. MMP9 and MUC4). Details of the procedure for IHC staining is described elsewhere [24]. The IHC and H&E stained slides were evaluated by pathologist at University of Nebraska Medical Centre.

2.16 Statistical Analysis

The JMP^® Statistical Discovery Software (Cary, NC) was used to determine the statistical significance within the treatment replicates in each experiment. A Student’s t-test was used to calculate the corresponding p-value. All p values < 0.05 were considered statistically significant.

3.1 Graviola extract induces cytotoxicity of pancreatic cancer cells

The PC cells FG/COLO357 and CD18/HPAF were incubated for 48hr with different concentrations of Graviola extract. The results from the MTT cytotoxicity assay indicated a progressive decrease in cell viability with the successive increase in the concentrations of the extract (Fig. 1A). After 48hr of treatment, the resulting IC50 of Graviola extract on FG/COLO357 and CD18/HPAF cells was 200 and 73μg/mL, respectively (Fig. 1B) and the results indicated that CD18/HPAF cell line is more sensitive to the Graviola extract than the FG/COLO357 cell line.

It is well known that the activation of the extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3′kinase (PI3K/Akt) pathways play a crucial role in the proliferation and survival of PC [26] and inhibition of these pathways leads to the inhibition of pancreatic tumor growth [27, 28]. The present study revealed that treatment of PC cells with Graviola extract resulted in decreased activation of both ERK and Akt pathways in PC cells (Fig. 1C). Thus, the inhibition of these pathways is in agreement with the decreased viability of PC cells treated with Graviola extract.

3.2 Pancreatic cancer cell metabolism is inhibited by Graviola extract

Previous studies have shown that major bioactive components present in Graviola extract that inhibit mitochondrial complex I [13–17], suggesting their direct involvement in cell metabolism. It has already been well-documented that cancer cells undergo a metabolic shift to adapt and survive under harsh environments by enhancing aerobic glycolysis [29, 30]. Also, Akt activation leads to glycolytic ATP generation in tumor cells [31]. Hence, the effect of Graviola extract on several stages of the glycolytic pathway in PC cells was analyzed.

The expression of HIF-1α, a critical regulator of aerobic glycolysis in cancer cells [32], was analyzed in PC cells after incubation with Graviola extract (Fig. 2A). We observed reduced HIF-1α expression in both PC cell lines, suggesting a direct effect of this natural product on the metabolism of PC cells. Likewise, it has been previously reported that the NF-κB upregulates the expression of HIF-1α [33, 34]. Not surprisingly, the expression levels of NF-κB were also reduced in PC cells after being incubated with Graviola extract (Fig. 2A).

Subsequently, the expression of the glucose transporters 1 and 4 (GLUT1 and GLUT4), and the expression of the glycolytic enzymes hexokinase II (HKII) and lactate dehydrogenase A (LDHA), all of which are upregulated by HIF1-α in cancer cells [32, 35], were analyzed in Graviola extract-treated PC cells by real-time PCR analysis (Fig 2B). Overall, the transcript levels of GLUT1, HKII, and LDHA were significantly reduced in both PC cell lines when compared to untreated cells (i.e. 60–87% downregulation).

Cancer cells have an increased expression of glucose transporters to enhance glucose uptake, which in turn increases the glycolytic rate for an enhanced ATP production that will ultimately lead to an enhanced tumor growth [10]. Thus, based on the results discussed above, it was not surprising that PC cells treated with Graviola extract doses over 10 μg/mL had a decreased rate of glucose uptake when compared to untreated cells (0μg/mL) (Fig. 2C). Finally, to evaluate the energy outcome of the glycolytic pathway of PC cells, we measured ATP production in Graviola extract-treated PC cells (Fig. 2D), and observed significant inhibition by 42–47% and by 31–43% doses in FG/COLO357 and CD18/HPAF PC cells, respectively. Altogether, these results indicate that Graviola extract impairs the metabolism of PC cells that will ultimately lead to decreased cell viability.

3.3 Graviola extract induces necrosis of pancreatic cancer cells

In order to evaluate the cytotoxic pathways induced by Graviola extract. PC cells were stained with annexin-V and propidium iodide (PI) staining to measure the necrotic and apoptotic cell populations by performing flow cytometry. While the necrotic cell population in both PC cell lines increased significantly after incubation with Graviola extract, the apoptotic cell population remained unchanged (Fig. 3A). Subsequently, the production of Graviola extract-induced reactive oxygen species (ROS) in FG/COLO357 and CD18/HPAF PC cells was confirmed by confocal microscopy (Fig. 3B). Additionally, it was also observed that cells incubated with Graviola extract have a gain in cell volume, a characteristic of necrotic cell death.

In order to confirm that Graviola extract was not inducing apoptosis of PC cells, the levels of caspase-3 expression were analyzed by western blot analysis. The Caspase-3 expression values remained statisticallyunaltered by treatment with the extract, suggesting that apoptotic pathways are not involved (Fig. 3C). Furthermore, the apoptotic cells population in Graviola extract-treated cells was also analyzed by Telford staining, and the results corroborated the findings from AnnexinV/PI staining studies, where the number of apoptotic cell population did not vary after being incubated with the natural compound (data not shown).

An analysis of the different phases of the cell cycle after treatment with Graviola extract demonstrated cell cycle arrest at G1 phase (Fig. 3D). While the G1 cell population increased from 43 to 65%, the S phase decreased from 56 to 32% with increasing concentrations of Graviola extract (0, 5, 100μg/mL). To support these results, the expression of CyclinD1 in Graviola extract-treated PC cells was analyzed (Fig. 3E). In agreement with previous studies indicating that a decreased CyclinD1 expression induces G0/G1 cell cycle arrest [36], Graviola extract-treated PC cells had also reduced expression of the cell cycle regulatory protein.

3.4 Motility of pancreatic cancer cells decreases after treatment with Graviola extract

The effect of Graviola extract on the functional properties of PC cells was analyzed in vitro wound healing and migration assays (Fig. 4A, B). As it can be observed in the images from the wound healing assays, PC cells treated with Graviola extract did not close the wound even after 24hr, as opposed to untreated cells (0μg/mL), indicating reduced motility of PC cells after treatment with Graviola extract (Fig. 4A). Similarly, the migratory capacity of PC cells was also reduced after treatment with Graviola extract, as evaluated by a transwell assay (Fig. 4B), suggesting that the natural extract reduces the motility of PC cells.

The motility and migration of cancer cells is associated with the rearrangements of the cortical actin and microtubules network [37, 38]. Additionally, cellular ATP depletion has been associated with reorganization of the actin cytoskeleton [39] and suppression of the dynamics of microtubules is known to induce mitotic arrest [40]. Taking this into consideration, the cytoskeleton of Graviola extract-treated PC cells was analyzed by confocal microscopy (Fig. 4C, D). The image results of phallotoxins (i.e. phalloidin) staining indicate a disruption of the cortical actin network and dissolution of stress fibers in Graviola extract-treated PC cells (Fig. 4C). Similarly, a disruption of microtubules dynamics was evident after β-tubulin staining of PC cells incubated with Graviola extract (Fig. 4D). To further analyze the effect of Graviola extract on motility and migration of PC cells, the expression levels of the phosphorylated focal adhesion kinase (pFAK), which is involved in mitogenic signaling and motility [41], and matrix metalloproteinase 9 (MMP9), which targets many extracellular proteins including adhesion molecules [42], were analyzed by western blot analysis (Fig. 4E). In agreement with the experiments discussed above, we observed that the expression levels of both pFAK and MMP9 were downregulated in Graviola extract-treated cells.

3.5 Graviola extract inhibits tumor growth and metastasis of pancreatic cancer cells

Based on the results obtained from in vitro experiments, Graviola extract has promising properties to be incorporated in PC therapeutics. Nevertheless, these anti-tumorigenic properties require further validation through in vivo experiments. In order to evaluate the therapeutic potential of Graviola extract, a more realistic situation for administering the extract was mimicked. It is recommended that Graviola extract supplement must be taken on a regular basis [11], and therefore, it was decided that the extract must be administered by oral gavage after suspending contents of the capsule in aqueous solution instead of dissolving it in DMSO. Prior to evaluating the anti-tumorigenic properties of aqueous Graviola extract suspension by in vivo experiments, pertinent in vitro experiments corroborating the cytotoxic potential of the aqueous suspensions on PC cells were completed beforehand (Supplementary Fig. 1).

For tumorigenic studies, CD18/HPAF cells expressing luciferase were orthotopically injected into the pancreas of athymic mice. After 1 week, in vivo biophotonic imaging confirmed tumor growth in all animals and the treatment regimen was initiated. The tumor growth during the treatment was monitored by imaging every two weeks. After 35 days of treatment, the animals were euthanized and the pancreatic tumors were removed and weighed. Although pancreatic tumors were not completely eradicated, the results indicate that tumor growth decreased significantly in Graviola extract-treated mice in comparison to the control group (Fig. 5A). Specifically, the tumor growth inhibition in mice treated with a dose of 50mg/kg Graviola extract was 59.8% (p-value=0.0008) whereas in mice treated with 100 mg/kg Graviola extract the inhibition was 50.3% (p-value = 0.006), indicating the efficacy of the natural product in PC regression. The metastatic lesions in each mouse were evaluated in various vital organs including the liver, spleen, mesenteric lymph nodes (LN), small and large intestines, peritoneum, diaphragm, and ovaries (Fig. 5B). Although all the metastatic lesions were reduced in Graviola extract-treated mice in comparison to the untreated control mice, the incidence of metastasis in the liver, mesenteric LN, and ovaries was significantly reduced (p-values ≤ 0.02). Representative biophotonic tumor images illustrate the tumor growth across the different groups during the course of the treatment (Fig. 5C).

Further, tumors were evaluated by H&E (Fig. 5D) and IHC staining (Fig. 6). The H&E stained tumor sections showed necrotic cells in 20–50% of the pancreatic tumor tissues from Graviola extract-treated mice as compared with tumors from the control mice. These results further strengthen the results from in vitro experiments, which demonstrate that Graviola extract-mediated reduction in PC cell viability was through the induction of necrosis.

The tumor lysates and paraffin embedded pancreatic tumors were also evaluated by IHC for the expression of MMP9 (Fig. 6A) and MUC4 (Fig. 6B). In agreement with in vitro data, the levels of MMP9 were reduced in tumors from Graviola extract-treated mice compared to the untreated controls. As the expression of MMP9 has been related to invasion and metastasis, the reduced levels of the protein in Graviola extract-treated tumors substantiate our findings of reduced metastatic sites in these mice.

Previous studies performed by our group have established the correlation of the expression of mucin4 (MUC4) glycoprotein with progression and metastasis of PC [24, 43–45]. Therefore, we were particularly interested in evaluating the effect of Graviola extract on the expression of MUC4 in PC cells and pancreatic tumors. In vitro experiments demonstrated a significant downregulation in MUC4 expression, both at the translational (Supplementary Figs. 1C, 2A, B) and transcriptional levels (Supplementary Fig. 2C) in Graviola extract treated PC cells. Similarly, the expression of the MUC4 was reduced in pancreatic tumors from mice treated with Graviola extract as compared to the untreated mice (Fig. 6B), Further supporting our findings of reduced tumor growth and metastasis after treatment with Graviola extract.

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Supplementary Figure 1 In vitro evaluation of aqueous solutions of Graviola extract in pancreatic cancer cells:

(A) Cytotoxicity of Graviola extract suspended in PBS. The PC cells CD18/HPAF were incubated with different doses of Graviola extract suspended in PBS for 48hr. Cytotoxicity was analyzed by MTT cytotoxic assay. Data from experiments performed in triplicate is presented as the mean value ± standard error of mean. (*p-value<0.0001, **p-value=0.0017, compared to untreated cells); (B) ATP quantification of CD18/HPAF PC cells after treatment with Graviola extract suspended in PBS. Data from experiments performed in triplicate is presented as the mean value normalized with total protein content ± standard error of mean; (C) Western blot analysis of the expression of proteins related to the proliferation, invasion, and metastasis of PC in CD18/HPAF cells after incubation with Graviola extract suspended in PBS. Protein lysates (30μg) were resolved on 2% SDS agarose gels (MUC4) and 10% SDS-PAGE (HIF-1α, Caspase3, MMP9, and β-actin). β-actin was used as a loading control.

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Supplementary Figure 2 Expression of MUC4 glycoprotein in pancreatic cancer cells after treatment with Graviola extract:

(A) Western blot analysis of MUC4 and HER2 in PC cells after treatment with Graviola extract. Protein lysates (30μg) were resolved on 2% agarose gels for MUC4 and 10% SDS-PAGE for HER2. β-actin was used as a loading control; (B) Confocal microscope images of PC cells stained against MUC4 (anti-Alexa Fluor 594) after treatment with Graviola extract. Cell nuclei were stained with DAPI. Scale bars represent 10μm; (C) Measurement of MUC4 transcripts in PC cells incubated with Graviola extract by real-time PCR. Data is presented as the average fold difference of MUC4 levels in Graviola extract-treated cells versus untreated cells (0μg/mL) ± standard error of mean. The housekeeping gene β-actin was used as an internal control. (*p-value<0.005; **p-value<0.005, compared to untreated PC cells)

The invaluable technical support from Kavita Mallya is greatly appreciated. We would like to give special thanks to UNMC professors: Dr. Michel Ouellette for kindly providing CD18/HPAF-Luciferase and HPNE cells, Dr. Shilpa Buch for allowing us to use the Luminescence plate reader, Dr. Vimla Band for allowing us to use the microscope to image tumor H&E and IHC sections, and Dr. Steve Caplan for assisting with the analysis of confocal images and providing us the β-Tubulin antibody. We also thank Janice A. Tayor and James R. Talaska of the Confocal Laser Scanning Microscope Core Facility at UNMC, Victoria B. Smith and Megan Michalak of the UNMC Cell Analysis Core Facility, and Ms. Kristi Berger, the Eppley Cancer Center for editing this manuscript. We are also very grateful for the expertise and involvement of Drs. Amarnath Natarajan and Abijah Nyong from the Chemistry department at UNMC in the bioactivity-guided fractionation of the Graviola extract. The authors of this work are supported by grants from the National Institutes of Health: NIH-NCI Cancer Biology Training Grant UNMC T32CA009479, R01 CA78590, U01EDRN CA111294, R01 CA131944, R01 CA133774, R01 CA 138791, P50 SPORE CA127297 and U54 CA160163.

Conflicts of Interest Statement

There are no potential conflicts of interest involved with this work.

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