Cancer Lett. Author manuscript; available in PMC 2013 October 1.
Published in final edited form as:
Cancer Lett. 2012 October 1; 323(1): 29–40.
Published online 2012 April 1. doi: 10.1016/j.canlet.2012.03.031
María P. Torres,1,2 Satyanarayana Rachagani,1 Vinee Purohit,2 Poomy Pandey,3 Suhasini Joshi,1 Erik D. Moore,1 Sonny L. Johansson,2,4 Pankaj K. Singh,1,2 Apar K. Ganti,5 and Surinder K. Batra1,2,4
The publisher's final edited version of this article is available at Cancer Lett
See other articles in PMC that cite the published article.
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.
The overall five-year survival rate for pancreatic cancer (PC) patients was 5.5% for the period of 2001–2007, according to the National Cancer Institute (NCI), a statistic that has not varied significantly for over the last four decades . In 2012, it is estimated that 43,920 new PC cases will be diagnosed and approximately 85% of these (i.e. 37,390) will succumb to the disease . The main reason behind the poor prognosis of PC patients is the insidious and sporadic nature of the disease, which is often presented with no specific early clinical symptoms. By the time of diagnosis PC is already in advanced stages (i.e. III and IV) and is resistant to conventional chemotherapy and radiotherapy . Interestingly, even patients diagnosed with stage I PC that have the option to undergo surgery have a 5-year overall survival of approximately 20%, a clear indication of the general failure of current standard treatments for each stage of PC [4, 5]. What is even more alarming, are the statistics that predict possible 55% increase in the expected number of new PC cases by 2030 . Thus, immediate progress must be made in the prevention, early diagnosis, and systemic treatments against this lethal disease.
Gemcitabine has been the standard line of treatment for PC patients for over a decade and is associated with a median patient survival of 5.4 months . Over all these years, numerous clinical efforts have been devoted to improve PC chemotherapy outcomes, but unfortunately no significant improvements have been reported apart from a clinical trial reported in May of 2011 . This phase III clinical trial reported an improved overall survival of PC patients treated with a four-drug chemotherapy regimen comprising fluorouracil, leucovorin, irinotecan, and oxaliplatin (FOLFIRINOX). Nevertheless, a major disadvantage of this novel treatment was its related toxicity, which was noticeably high when compared to PC patients treated with gemcitabine alone. Therefore, novel, alternative PC therapeutics must not only improve the prognosis of PC patients but also minimize any possible toxicity-related side effects that will interfere with the quality of life of PC patients.
It is well known that an increased consumption of fruits and vegetables is associated with a reduced risk of most cancers, including PC . For this reason, the potential of natural products in PC therapies has been widely investigated . While some of these compounds have undergone clinical testing (i.e. curcumin, genistein) and have demonstrated some activity against PC, the poor bioavailability in patients minimizes their therapeutic efficacy. However, as compared with conventional chemotherapeutic drugs, the major benefit of these therapies is the apparent lack of toxicities to healthy tissues. This attracted our attention to find alternative, natural-derived chemotherapeutic drugs in order to improve the prognosis of PC patients. Traditionally, the leaves from the tropical tree Annona Muricata, also known as Graviola or Soursop, have been used for a wide range of human diseases including inflammatory conditions, rheumatism, neuralgia, diabetes, hypertension, insomnia, cystitis, parasitic infections, and cancer . The major bioactive components that have been extracted from different parts of the plant are known as Annonaceous acetogenins. These are derivatives of long chain (C35 or C37) fatty acids derived from the polyketide pathway  that is selectively toxic to cancer cells, including multidrug-resistant cancer cell lines [13–17]. Annonaceous acetogenins induce cytotoxicity by inhibiting the mitochondrial complex I, which is involved in ATP synthesis . As cancer cells have a higher demand for ATP than the normal cells, mitochondrial complex I inhibitors have potential in cancer therapeutics.
A few in vivo studies involving Annona Muricata have been reported. Among these, two reports have shown the ability of the leaf extract to regenerate pancreatic islet β cells in diabetic rats [18, 19]. These studies suggest an additional benefit of the natural product against PC given that diabetes has been classified as a risk factor of the malignant disease . More recently, one study analyzing the anti-tumor efficacy of Annona Muricata was published . The extract had a direct anti-tumorigenic effect on breast cancer cells by downregulating the expression of the epidermal growth factor receptor (EGFR). Although this study demonstrates the potential anti-tumorigenic properties of Graviola, the doses used in the experimental design were not properly controlled. The mice were fed with the extract mixed in the diet and the exact amount ingested by each animal could not be estimated accurately.
Although a few in vitro reports have shown the cytotoxic characteristics of Graviola against various cancer cell lines, including PC cells , the comprehensive in vivo effects and mechanistic scientific studies are still lacking. To our knowledge, the studies reported herein are the first to indicate that Graviola extract has promising characteristics for PC therapeutics. Comprehensive in vitro and in vivo studies in various PC cell lines revealed that the natural product inhibited multiple signaling pathways that regulate metabolism, cell cycle, survival, and metastatic properties of PC cells.
2. Materials and Methods
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.
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 . 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 .
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 . 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 . 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 . 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 .
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 . 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.
Little or no progress has been accomplished in PC treatment over the last 40 years.
Novel therapeutics against this lethal malignancy must inhibit several pathways that promote survival, progression, and metastasis of PC cells.
Based on the fact that cancer cells are mainly dependent on the glycolytic pathway for ATP production, glucose deprivation by anti-glycolytic drugs can induce cancer cell death , a pathway that can be targeted and explored in PC therapies .
Natural products have been investigated in PC therapeutics over several decades, but to date none has been incorporated in routine chemotherapies .
Traditionally, the leaves from Graviola (Annona Muricata) have been used for a wide range of human diseases including cancer .
The present study is the first to demonstrate that Graviola extract reduces the viability of PC cells and tumors by inducing necrosis and cell cycle arrest, and by inhibiting PC cell motility (i.e. cytoskeleton rearrangement), migration, and metabolism. Overall, in vitro experiments revealed that the compounds present in the natural extract inhibited several pathways involved in PC cell proliferation and metabolism, simultaneously. Such inhibitions ultimately led to a decrease in tumor growth and metastasis in orthotopically transplanted pancreatic tumor-bearing mice.
In PC patients, an increased metabolic activity and glucose concentration of malignant tumors has been linked to pancreatic tumor aggressiveness . Additionally, the presence of hypoxia in PC has been associated with tumor growth and metastasis [48, 49]. Indeed, the presence of hypoxic environment has been linked to the oncogenic and metabolic transformation (i.e. glycolysis) of PC cells that results in resistance to conventional cancer therapeutics [48, 50]. More specifically, it has been suggested that hypoxia can induce resistance to gemcitabine through the activation of PI3K/Akt/NF-κB and MAPK/ERK pathways , which are also related to PC progression and survival. The activation of both of these signaling pathways was evaluated in PC cells after treatment with Graviola extract and it was found that the extract suppressed phosphorylation of the key molecules involved in these pathways, which correlated with reduced viability of PC cells. Subsequently, the expression of HIF-1α, the major transcription factor activated under hypoxic conditions, and its ensuing downstream effects on PC cell metabolism were analyzed in Graviola extract-treated cells. The results indicated the natural product inhibited PC cell metabolism by inhibiting the expression of HIF-1α, NF-κB, glucose transporters (i.e. GLUT1, GLUT4), and glycolytic enzymes (i.e. HKII, LDHA), all of which lead to the reduction of glucose uptake and ATP production by PC cells.
The overall downregulation of PC cell metabolism induced by Graviola extract resulted in PC cell death and necrosis. In agreement with previous studies of ATP reduction, the metabolic and therapeutic stress induced by Graviola extract led to an acute ATP depletion, which is accompanied by increased intracellular ROS, ultimately leading to necrosis [52–54].
While necrotic agents have not been considered beneficial in cancer therapies due to induction of local inflammation, the process itself can lead to the activation of the innate immune system capable of initiating anti-tumor immunity . It makes it imperative to evaluate the effect of a necrosis-inducing product such as Graviola extract in an immune competent host. In this regard, we plan to evaluate the effect of the natural product on the progression of pancreatic adenocarcinoma in the KrasG12DPdx1-Cre spontaneous animal model, where the effect on the immune system can be evaluated.[55, 56]. In order to evaluate the potential of Graviola extract in preventing PC progression, we plan to supplement the diet of KrasG12DPdx1-Cre mice with Graviola extract after the mice start developing pancreatic intraepithelial neoplastic (PanIN) lesions. The effective concentrations of Graviola metabolites after oral absorption and effects on the immune system will be measured as well. Additional experiments will be carried out to evaluate the potential of a combination therapy of Graviola extract with the standard chemotherapeutic drug Gemcitabine. With the results discussed in the present study, it is expected that minimum doses of the chemotherapeutic drug will be needed to eradicate the malignant disease.
The major bioactive compounds identified in Annona Muricata have been classified as Annonaceous acetogenins, which inhibit mitochondrial complex I that leads to a decreased ATP production [13–17]. Although the natural extract capsules used in these studies contained numerous compounds, the presence of Annonaceous acetogenins was evident by the depletion of ATP production in PC cells after being incubated with Graviola extract.
Bioactivity-guided fractionation for the identification of potent bioactive (i.e. anti-tumorigenic) compounds that are present in the Graviola extract is currently being investigated. We are also ensuring that cytotoxic effects are specific to tumorigenic cells only, by including the non-transformed immortalized pancreatic epithelial cell line HPNE, which is derived from pancreatic duct (data not shown).
Pancreatic tumors develop from a complex interplay of numerous signaling pathways and Graviola extract has shown promising anti-tumorigenic characteristics by targeting some of these pathways all at once. Although novel glycolytic inhibitors, such as Graviola extract, may have broad therapeutics applications , inhibition of glycolysis alone may not be sufficient to eradicate tumor cells completely. Perhaps the use of alternative medicine, like taking Graviola capsules on a regular basis, should still be considered a supplement, not a replacement for standard therapies. Currently, in vitro studies evaluating the potential of the natural product in combination with chemotherapeutic drugs are being conducted.
Conflicts of Interest Statement
There are no potential conflicts of interest involved with this work.
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