perjantai 15. huhtikuuta 2016

Groundbreaking new cancer study on graviola shows promise as a possible treatment

  • The overall downregulation of PC cell metabolism induced by Graviola extract resulted in PC cell death and necrosis.
  • The acetogenins recognize and selectively inhibit the cancer cells. 
  • The Graviola extract had a direct anti-tumorigenic effect on breast cancer cells.
     by downregulating the expression of the epidermal growth factor receptor (EGFR).
  • Pancreatic cancer cell metabolism is inhibited by Graviola extract.
  • Graviola extract inhibits tumor growth and metastasis of pancreatic cancer cells.
  • Acetogenins inhibit ATP transfer (complex I of mitochondria) into these cells, retarding their function in a process that eventually leads to cell death.

Groundbreaking new cancer study on graviola shows promise as a possible treatment

Thursday, January 10, 2013 by: Brad Chase

(NaturalNews) A new study on the herb graviola published in the October 2012 issue of Cancer Letterhas cancer researchers buzzing. Graviola, the tropical fruit with the unusual nickname "sour sop," stops cancer tumor cells from growing in pancreatic cancer.

Scientists prove the cancer-healing abilities of graviola in the lab and in living tissue
Oncologists agree that pancreatic cancer is one of the most difficult types of cancer to treat, and that being diagnosed with cancer of the pancreas is as good as a death sentence.

The new study, performed by a cancer research team at the Department of Biochemistry and Molecular Biology at the 
University of Nebraska Medical Center, shows that graviola kills pancreatic cancer cells by inhibiting cellular metabolism. This cancer tumor-fighting ability has been confirmed both in test tubes and in live subjects.
Graviola works by inhibiting numerous signaling pathways that manage how pancreatic cancer cells grow, how long they live, and how the cancer tumors spread within the host. By altering these parameters, the rate of new cancer cell growth and spread of the disease slowed significantly.
The team of researchers considers the characteristics possessed by graviola "promising."

Graviola stops breast cancer as well

Nutrition and Cancer confirms the cancer- inhibiting phytochemicals in graviola. The medical journal's June 2011 issue includes a study on graviola and breast cancer. In this study, scientists at Virginia Tech demonstrated that graviola fruit extract (juice) could reduce the growth of cancer on the skin of human breast cancer patients without damaging healthy breast tissue.

Mice who took 200 mg graviola fruit extract per kilogram of food in their diet for five weeks had a significant reduction in protein expression in breast cancer tumors. Overall, graviola extract was able to reduce tumor growth by 32 percent.

Graviola benefits do not stop at cancer

Memorial Sloan-Kettering Cancer Center has a new page on its website describing the health benefits of graviola. Besides slowing cancer, MSKCC gives clinical evidence that graviola also fights viruses, kills parasites, reduces inflammation, and reverses the glycemic load which leads to diabetes.

These medical studies suggest that graviola extract may be useful to treat herpes simplex virus-1 (Journal of Ethnopharmacology, 1998) and other viral infections.

Graviola is associated with neurotoxicity in one people group (This survey was faked by Big Pharma. See French Anses documents below.)

For most people, the graviola, or sour sop fruit, is considered a stand-alone dessert fruit with a flavor described as strawberry-pineapple mixed with coconut or banana cream. It is a popular flavor for juices and ice cream in Central American countries.

However, the fruit has been associated with a neurological disease similar to Parkinson's disease on the Caribbean Island of Guadeloupe, according to several studies, including one published in a 2006 article of the Journal of Neural Transmission.

Väitteet tutkimuksesta Guadeloupella, jossa olisi todettu Graviolan aiheuttavan "epätyypillistä Parkinsonin tautia", eivät pidä paikkaansa:

Ranskan terveysviranomaiset
 (Anses - Agence nationale de sécurité sanitaire de l’alimentation, de l’environnement et du travail) ovat nimittäin osoittaneet, että tuo neurotoksisuus/Parkinsonin tauti -väite, ts. lääketeollisuuden masinoitu hyökkäys, toteutettiiin hätäisesti Guadeloupella.
Ihmisiä haastateltiin opiskelijoiden toimesta ja rotillle painettiin pumpuilla Graviola -extractia suoraan suoneen (reisivaltimoon) 28 päivän ajan, niin paljon että ne menivät shokkiin. 
Ranskalaisten tarkastuksessa todetaan, että: "
ei voida katsoa, että Parkinsonin taudin ja hedelmän syönnin välillä olisi osoitettu yhteys!

Tämä hätäinen "tutkimus" -operaatio julkaistiin virallisena tutkimuksena.

Ranskan viranomaisten (Anses) ovat myöhemmin, "Avis signé le 28/04/2010", tarkastaneet tämän "tutkimuksen" jossa todettiin tietyllä ihmisryhmällä Guadoloupella etätyypilisen Parkinsonin taudin oireita.

Tuon Ranskan viranomaisten julkistama, lääketeollissuuden masinoiman tutkimus-huijauksen paljastavaRanskan Elintarviketurvallisuusviraston, ANSES, perus-dokumentit linkissä alla 

Fr: "En l’absence de données précises sur les habitudes de consommation des patients atteints de syndromes parkinsoniens, l’Afssa considère que la responsabilité d’autres causes, aussi bien environnementales que génétiques, ne peut être exclue (Angibaud et al. 2004, Caparros et al. 2006, Aubeneau et al. 2008)."

En: "
In the absence of precise data on the consumption habits of patients with Parkinson's syndromes, Afssa considers that the responsibility for other causes, both environmental and genetic, can not be ruled out (Angibaud et al., 2004, Caparros et al. 2006, Aubeneau et al., 2008)."

Fi: "Koska Parkinsonin oireyhtymästä kärsivien potilaiden kulutustottumuksista ei ole tarkkoja tietoja, Afssa katsoo, että vastuuta muista syistä, sekä ympäristöllisistä että geneettisistä, ei voida sulkea pois (Angibaud et al., 2004, Caparros et al. 2006, Aubeneau ym., 2008).


Memorial- Sloane Kettering Cancer, "Graviola", Nutrition and Cancer. 2011; 63(5):795-801. "Selective growth inhibition of human breast cancer cells by graviola fruit extract in vitro and in vivo involving downregulation of EGFR expression." Dai and Hogan, et al., Cancer Letters. 2012 Oct 1; 323(1):29-40. "Graviola: a novel promising natural-derived drug that inhibits tumorigenicity and metastasis of pancreatic cancer cells in vitro and in vivo through altering cell metabolism." Torres, et al., Journal of Ethnopharmacology. 1998 May; 61(1):81-3. "Effect of the extract of Annona muricata and Petunia nyctaginiflora on Herpes simplex virus."
Padma P, et al., "Antinociceptive and Anti-Inflammatory Activities of the Ethanol Extract of Annona muricata L. Leaves in Animal Models." International Journal of Molecular Science. 2010 May 6; 11(5):2067-78. De Sousa OV, et al.

About the author:
Brad Chase is the President of His website provides articles and natural remedies to help people solve their health concerns.



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.

2.2 Cell Culture
2.3 Antibodies
2.4 Cytotoxicity Assay
2.5 Western Blot Analysis
2.6 Real-time PCR
2.7 Glucose Uptake
2.8 ATP Quantification
2.10 Cell Cycle Analysis
2.11 Confocal Microscopy
2.12 Wound Healing Assay
2.13 Motility Assay

1. Introduction

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 [1]. 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 [2]. 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 [3]. 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 [6]. 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 [7]. 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 [8]. 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 [9]. For this reason, the potential of natural products in PC therapies has been widely investigated [10]. 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 [11].

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 [12] 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 [14]. 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 [20]. More recently, one study analyzing the anti-tumor efficacy of Annona Muricata was published [21].
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 [12], 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.

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 [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).

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.

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].

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′

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.

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.

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.

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. Results (myös lähdelinkissä alla)

3.1 Graviola extract induces cytotoxicity of pancreatic cancer cells

3.2 Pancreatic cancer cell metabolism is inhibited by Graviola extract
3.3 Graviola extract induces necrosis of pancreatic cancer cells
3.4 Motility of pancreatic cancer cells decreases after treatment with Graviola extract 

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

4. Discussion
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 [46], a pathway that can be targeted and explored in PC therapies [47].
Natural products have been investigated in PC therapeutics over several decades, but to date none has been incorporated in routine chemotherapies [10].

Traditionally, the leaves from Graviola (Annona Muricata) have been used for a wide range of human diseases including cancer [11]. 

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 [47]. 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 [51], 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 [52]. 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 [57], 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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Graviola, aka Soursop: What You Need to Know

Graviola: What You Need to Know

Graviola is the ­Portuguese name for a plant that is widely grown and consumed in Latin America. In Spanish-speaking countries, the fruit is called guanábana. Common names for it are soursop, custard apple, cherimoya, and Brazilian paw paw. By whatever name, this tropical evergreen tree produces a fruit with white flesh, many large seeds and an extremely sweet, slightly acidic flavor. Because it is difficult to eat, its pulp is commonly made into juice. In fact, your local grocery store probably sells the popular guanábana nectar.

Not only the fruit but also other parts of this plant -- the leaves, stem, bark, roots, and seeds -- have a long history of medicinal use in the Americas. Graviola is used as a natural remedy for infections, fever, digestive problems and high blood pressure [source: Cassileth]. Researchers have documented many other traditional uses among the indigenous people of the Andes, the Amazon and the Caribbean [source: Taylor].

Recently, scientists have begun to explore the potential of the bioactive chemicals in graviola leaves, stems and seeds, called annonaceous acetogenins. These acetogenins appear to have powerful anti-tumor and anti-cancer qualities. Some test-tube studies have concluded that graviola compounds may be able to target and kill cancer cells, even drug-resistant ones, without interfering with healthy cells. These results, circulated through alternative medicine networks and on the Internet, have created considerable excitement and a measure of hype. Natural health guru Andrew Weil is among those who are skeptical of the claims made for graviola and recommends against its use [source: Weil]. It may take years before clinical trials are conducted to legitimate or disprove the claims made by graviola proponents. In the meantime, the plant has hit the herbal market and many cancer patients are taking it.
This article will attempt to cut through the controversy regarding this form of alternative medicine, its known uses and current research.

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Graviola Benefits
Graviola is a rainforest plant that has been part of the natural and traditional medicine of Central and South America and the Caribbean for centuries. It has an extremely wide range of medicinal properties, which are distributed through the different parts of the plant. The fruit or juice is taken to reduce fever, counteract diarrhea and dysentery, and kill worms and other parasites. The seeds are also a potent antiparasitic and are used traditionally as a remedy for lice. The bark, leaves and roots can be made into a soothing medicinal tea, taken as a sedative or an antispasmodic. Research also bears out the traditional use of graviola tea as a hypotensive -- that is, a remedy for high blood pressure [source: Taylor]. The bark can also be used to treat fever, and the leaves are used topically to speed the healing of wounds. The unripe fruit is especially prized as a digestive aid [source: Weil].

Additional utilization of graviola has been documented within specific native healing traditions. In the Andean mountain ranges of Peru, graviola leaves are brewed to discharge mucus and soothe inflamed mucous membranes.
To the east, in the Amazon region, the bark, leaves and roots are used by diabetics to stabilize blood sugar. The leaf tea is taken as a heart tonic in Guyana, a liver remedy in Brazil, and a treatment for asthma, coughs and flu in the West Indies. It is also used for arthritis and rheumatism, and some mothers eat and drink the graviola fruit to increase lactation [source: Taylor].
New York's Memorial Sloan-Kettering Cancer Center affirms a number of the plant's beneficial properties, including antiviral, antiparasitic, antirheumatic and emetic effects on its Web site [source: Memorial Sloan-Kettering]. In view of this extensive list of benefits, the claims for graviola's cytotoxic effects on tumors and cancer cells have acquired a certain credibility for many people, despite the absence of scientific evidence on human subjects.
Like any potent medicine, albeit natural in origin, graviola has certain contra-indications and side effects. Continue reading to discover what they are.
Results of a neurological study, published in 1998, found that graviola has the capability to stimulate the brain's receptors for serotonin and may have an antidepressant effect [source: Cassileth]. Traditional usage supports this conclusion. To treat anxiety, one herbal manufacturer markets a tincture of graviola combined with the bark of mulungu, another rainforest tree [source:Amazon Botanicals].

Graviola Side Effects
Some side effects follow from graviola's areas of bioactivity. Studies on animal subjects have demonstrated that the plant can dilate blood vessels and lower blood pressure, so those whose blood pressure is already low, or are already on medication to reduce hypertension, should consult their physician before taking graviola [source: Wright]. Also, a large dose taken at one time can cause nausea and vomiting [source: Taylor].
Graviola's purported anti-cancer potency comes largely from its ability to reduce the supply of adenosine triphosphate (ATP) to cancer cells.
ATP often provides metabolic energy to healthy cells as well, and some nutritional supplements, notably Coenzyme Q10, are known for increasing ATP. For this reason, CoQ10 may neutralize the effect of graviola and they should not be taken together [source: Taylor].
Some side effects follow from graviola's areas of bioactivity.
Studies on animal subjects have demonstrated that the plant can dilate blood vessels and lower blood pressure, so those whose blood pressure is already low, or are already on medication to reduce hypertension, should consult their physician before taking graviola [source: Wright]. 
Also, a large dose taken at one time can cause nausea and vomiting [source: Taylor].


I entsyymi on NADH-dehydrogenaasi, II on sukkinaattidehydrogenaasi, III on sytokromi b-ckompleksi ja IV entsyymi on sytokromi-c-oksidaasi

Elektroninsiirtoketju on mitokondrion sisäkalvolla (eukaryootit) tai solukalvon kalvoproteiineissa (bakteerit) tapahtuva energiaa tuottava reaktiosarja, jossa sitruunahappokierrossa ja sitä edeltäneissä reaktioissa koentsyymeille NADH ja FADHsiirtyneitä elektroneja siirrellään elektroninsiirtoketjun entsyymiltä toiselle, jolloin elektronit menettävät potentiaalienergiaansa vähitellen, vapauttaen samalla energiaa. 
Vapautuvan energian avulla mitokondrion matriksista pumpataan protoneja mitokondrion kalvojen välitilaan, mikä aiheuttaa elektrokemiallisen gradientin eli potentiaali- ja protonikonsentraatioeron matriksin ja välitilan välille. Muodostunut gradientti purkautuu ATP-syntaasientsyymin kautta, jolloin muodostuu suurenergiaista fosfaattiyhdistettä, 

Electron transport chain
Electron transfer chain in the inner-membrane of mitochondrion. The electron transfer chain contains five complexes designated as complex I, II, III, IV, and V (F 1 F 0 -ATP synthase). The electrochemical H + gradient provided by these membrane-bound complexes serve as energy source for ATP synthesis from ADP and inorganic phosphate by an F 1 F 0 -ATP synthase

The Annonaceous acetogenins are promising new antitumor and pesticidal agents that are found only in the plant family Annonaceae. 
Chemically, they are derivatives of long-chain fatty acids. Biologically, they exhibit their potent bioactivities through depletion of ATP levels via inhibiting complex I of mitochondria and inhibiting the NADH oxidase of plasma membranes of tumor cells. Thus, they thwart ATP-driven resistance mechanisms. This review presents the progress made in the chemistry, biology, and development of these compounds since December 1995

Understanding mitochondrial complex I assembly in health and disease

Complex I (NADH:ubiquinone oxidoreductase) is the largest multimeric enzyme complex of the mitochondrial respiratory chain, which is responsible for electron transport and the generation of a proton gradient across the mitochondrial inner membrane to drive ATP production. Eukaryotic complex I consists of 14 conserved subunits, which are homologous to the bacterial subunits, and more than 26 accessory subunits. In mammals, complex I consists of 45 subunits, which must be assembled correctly to form the properly functioning mature complex.

Complex I dysfunction is the most common oxidative phosphorylation (OXPHOS) disorder in humans and defects in the complex I assembly process are often observed.

Researchers exploring the mechanisms that graviola uses claim that the acetogenins in the plant can distinguish cancerous cells from healthy cells because cancer cells have a consistently higher level of cellular activity. 
The acetogenins recognize and selectively inhibit the cancer cells. Pregnant women are advised to avoid graviola because the high energy in the cells of the developing fetus may trigger the botanical's toxic activity [source: Wright]. The plant was also found to stimulate the uterus in an animal study [source: Taylor].
The most detrimental effect attributed to graviola is that it "may cause neural dysfunction and degeneration leading to symptoms reminiscent of Parkinson's Disease" [source: Memorial Sloan-Kettering]. The first study to make this assertion was conducted by French researchers in Guadeloupe, who found an abnormally high presence of atypical Parkinson's amongst a poor population that used graviola for both food and medicine. However, the outbreak of neurological disorders was relatively confined, whereas the popularity of graviola is widespread in the region [source: Wright]. In her book "The Healing Power of Rainforest Herbs," botanist Leslie Taylor acknowledges that graviola seeds and roots contain alkaloids that have shown neurotoxic effects in tests. For this reason, she recommends using the leaves instead [source: Taylor].

Graviola -hedelmän ja -lehtien aineosat, annonaceus acetogenins, etsivät
nopeasti kasvavat, paljon energiaa tuhlaavat syöpäsolut ja estävät niiden ravinnon
ja hapen saannin.
- Graviolan aineosat pysäyttävät syöpäsolujen kasvun jo niiden energian-
tuotannossa, mitokondrioissa, Complex 1: n elektronisiirto -ketjussa.
Electron transport chain COMPLEX 1 (NADH ubiquinone oxidoreductase)

The Warburg hypothesis, sometimes known as the Warburg theory of cancer,
postulates that the driver of tumorigenesis is an insufficient cellular respiration caused by insult to mitochondria.[1] The term Warburg effect describes the observation that

cancer cells, and many cells grown in-vitro, exhibit glucose fermentation even when
enough oxygen is present to properly respire. In other words, instead of fully respiring
in the presence of adequate oxygen, cancer cells ferment.

He hypothesized that cancer, malignant growth, and tumor growth are caused by the
fact that tumor cells mainly generate energy (as e.g., adenosine triphosphate / ATP)
by non-oxidative breakdown of glucose (a process called glycolysis).
This is in contrast to "healthy" cells which mainly generate energy from oxidative
breakdown of pyruvate.
Pyruvate is an end-product of glycolysis, and is oxidized within the mitochondria.

4.2. Annonaceous Acetogenins
AGEs are a unique class of C-35/C37 secondary metabolites derived from long chain
(C-32/C34) fatty acids in the polyketide pathway. They are usually characterized by a combination of fatty acids with a 2-propanol unit at C-2 that forms a methyl-substituted α,β-unsaturated γ-lactone [72]. Since the discovery of uvaricin from Uvaria accuminata
in 1982, more than 500 AGEs have been identified from different parts of the plants in the Annonaceae family [73,74]. Due to the special structures and extensive biological activities, AGEs have attracted significant scientific interest in recent years.
Various biological activities have been reported for AGEs, including antimalarial, antiparasitic and pesticidal activities [72,75].

However, the biological activities of AGEs are primarily characterized with toxicity against cancer cells and inhibitory effects against the mitochondrial complex I (mitochondrial NADH: ubiquinone oxidoreductase) [76,77].

Phytochemical investigations and biological studies on different parts of the A. muricata plant resulted in the identification of a wide array of AGE compounds, as summarized in Table 1.
The chemical structures of the major acetogenins are shown in Figure 2.
To the best of our knowledge, at the time of preparation (January 2015) of the present review over 100 AGEs have been identified in A. muricata.

Electron Transport Chain | 8.12.2016
The Electron Transport Chain & complexes I-IV that pump protons out of the Mitochondria by the transfer of the electrons carried on NADH & FADH2 to maintain the concentration gradient of the protons "high in the intermembrane space & low in the matrix of the Mitochondria"

Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1

(Scientific Reports volume 6, Article number: 36462 (2016) doi:10.1038/srep36462)

In this study, we evaluated a novel PAI-1 inhibitor, annonacinone, a natural product from the Annonaceous acetogenins group
- High plasma levels of PAI-1 are related to the development of thrombosis as well as several other pathologies such as cardiovascular diseases and metabolic disturbances 1,2,3.
- Moreover PAI-1 is able to promote tumor angiogenesis and high PAI-1 level in solid tumors are associated with a poor prognosis 4,5.


 In conclusion, our work showed that, as well as their other biological properties, natural Annonaceous acetogenins, and particularly annonacinone, have an effect on fibrinolysis.
Indeed, annonacinone is a potent inhibitor of PAI-1 in vitro, ex vivo and in vivo.
Annonacinone mechanism of action and binding site on PAI-1 were also enlightened.
Altogether, annonacinone appears to be a very promising antithrombotic agent and should be further studied.

Fibrinolysis is a process that prevents blood clots from growing and becoming problematic.[1] The fibrinolytic system is closely linked to control of inflammation, and plays a role in disease states associated with inflammation.Plasmin is produced in an inactive form, plasminogen, in the liver.

PAI-1 is present in increased levels in various disease states, such as a number of forms of cancer, as well as in obesity and the metabolic syndrome. It has been linked to the increased occurrence of thrombosis in patients with these conditions.

Figure 5: Depiction of a putative binding mode of annonacinone against the active form of PAI-1


Chemical structures of the major compounds isolated from Annona muricata.
Published online 2015 Jul 10. doi:  10.3390/ijms160715625

Graviola and Cancer
The National Cancer Institute first noted the anticancer activity of graviola leaves in 1976, in an internal study not publicly released. Much of the subsequent research has been conducted at Purdue University in Indiana [source: Bluestein].
The studies concentrate on the antitumor properties and selective toxicity of annonaceous acetogenins. In 1997, the Purdue team announced that these phytochemicals, in studies, appeared especially effective at destroying cells that had survived chemotherapy. Such cells can develop resistance to several anti-cancer agents, earning the name multi-drug resistant (MDR). Typically, less than two percent of cancer cells have MDR properties, but this small set can quickly multiply after initial chemotherapy, rendering subsequent rounds of chemo useless. Expelling the anti-cancer agents requires large amounts of cellular energy, which MDR cells acquire from the chemical ATP. 

Acetogenins inhibit ATP transfer (complex I of mitochondria) into these cells, retarding their function in a process that eventually leads to cell death.
This process bypasses the healthy cells, which do not require infusions of ATP [source: Taylor].



Clinical Summary

Graviola, a tree prevalent in the rain forests of Africa, South America, and Southeast Asia, has been used in traditional medicine in many countries.
Extracts of graviola show antiviral (1), antiparasitic, antirheumatic, astringent, emetic (2), antileishmanial and cytotoxic (3) (4), antinociceptive, anti-inflammatory (9), antihyperglycemic (10) and anticancer effects (5)(12) (13) in vitro and in vivo.

Purported Uses
Cancer treatment, Herpes, Infections, Parasitic infections

These research findings have generated tremendous excitement, as well as an effort to market graviola supplements. Skeptical analysts point out that test-tube experiments are only a preliminary stage in cancer research, and it is therefore premature to ascribe a potent anticancer effect to graviola. Nevertheless, one study claimed that graviola was 10,000 times more effective against cancer than the well-known chemotherapy drug Adriamycin, and this dubious assertion has found its way to numerous promotional sites [source:]. Ralph Moss, a respected cancer writer who has been critical of mainstream oncology, comments that "astounding claims concerning cancer cures spread like a virus from Web site to Web site." However, Moss admits that graviola is "of potential importance to the future of medicine" [source: Moss]. Its increasing popularity indicates that some individuals are not content to wait for the blessing of the scientific establishment.
To learn more about graviola, visit the sites on the following page.

Pharmaceutical companies have succeeded in reproducing several annonaceous acetogenins in the laboratory. They are presently tinkering with chemical structures, with the goal of creating a synthetic acetogenin unique enough to patent and effective enough to market. They cannot patent the natural phytochemical, and therefore cannot assure a profit from it. This may explain the conundrum of why no clinical studies have been done on such a promising medicinal plant [source: Taylor].


  • Amazon Botanicals. "Anti-Anxiety Herbs." (Accessed March 8, 2009)
  • Bluestein, Chuck. "Cancer Cure: The Story About Graviola and Cancer." (Accessed March 7, 2009)
  • Cassileth, Barrie. "Integrative Oncology: Complementary Therapies, Herbs, and Other OTC Agents." Oncology, September 2008. (Accessed March 8, 2009)
  • "The Graviola Information Site." (Accessed March 8, 2009)
  • Memorial Sloan-Kettering Cancer Center. "About Herbs: Graviola." (Accessed March 7, 2009)
  • Moss, Ralph W. "The War on Cancer: A Friendly Skeptic Looks at Graviola." (Accessed March 7, 2009)
  • Taylor, Leslie. "Graviola." From The Healing Power of Rainforest Herbs (Square One Publishers, 2005). (Accessed March 7, 2009)
  • Weil, Andrew. "Graviola: A Worthwhile Botanical Against Cancer?" (Accessed March 7, 2009)
  • Wright, Kathryn Mays. "Groundbreaking Plant From the Amazon Takes on Cancer, Skeptics, and Controversy." Health Sciences Institute newsletter, October 2005. (Accessed March 8, 2009)
Characterization of the Annonaceous acetogenin, annonacinone, a natural product inhibitor of plasminogen activator inhibitor-1
High plasma levels of PAI-1 are related to the development of thrombosis as well as several other pathologies such as cardiovascular diseases and metabolic disturbances1,2,3. Moreover PAI-1 is able to promote tumor angiogenesis and high PAI-1 level in solid tumors are associated with a poor prognosis4,5. Therefore, development of small molecule PAI-1 inhibitors should prove useful not only in the treatment of thrombotic disorders but also in diverse disease states.

Database File for: GRAVIOLA (Annona muricata)

Family: Annonaceae 
Genus: Annona 
Species: muricata 
Synonyms: Annona macrocarpa, A. bonplandiana, A. cearensis, Guanabanus muricatus 
Common names: Graviola, soursop, Brazilian paw paw, guanábana, guanábano, guanavana, guanaba, corossol épineux, huanaba, toge-banreisi, durian benggala, nangka blanda, cachiman épineux 
Part Used: Leaves, fruit, seeds, bark, roots


The Truth About Cancer: A Global Quest - Episode 1

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