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

Abstract

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.

Conflict of interest statement

Conflicts of Interest Statement

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

Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Figures

Figure 1. Effect of Graviola extract on pancreatic cancer cell viability

(A) MTT cytotoxicity assay for Graviola extract-treated PC cells. Cells were incubated with different concentrations of Graviola extract and corresponding DMSO controls for 48hr. Data represents the mean value of experiments performed in triplicate ± standard error of mean. (*p-value<0.0001, **0.0001<p-value<0.001, ***p-value=0.007, compared to untreated cells); (B) Inverted microscope images (40X) of PC cells after treatment with Graviola extract for 48hr; (C) Western blot analysis of proteins involved in PC cell proliferation after 48hr treatment with Graviola extract. Protein lysates (30μg) were resolved by 10% SDS-PAGE. β-actin was used as the loading control. Each experiment was performed three times in triplicate.

Figure 2. Effect of Graviola extract on the metabolism of pancreatic cancer cells

(A) Western blot analysis of HIF-1α and NF-κB expression in PC cells after treatment with Graviola extract. Protein lysates (30μg) were resolved on 10% SDS-PAGE gels. β-actin was used as a loading control; (B) Real-time PCR-based measurement of transcript levels of glucose transporters 1 and 4 (GLUT1, GLUT4), hexokinase II (HKII), and lactate dehydrogenase A (LDHA) in PC cells after incubation with Graviola extract. Data is presented as the average fold difference in gene expression for the gene of interest 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. (*0.01<p-value<0.05, **0.005<p-value<0.001, ***p-value<0.005); (C) Measurement of glucose uptake in PC cells after treatment with Graviola extract. Radioactive counts of cells labeled with [3H]-2-deoxyglucose were normalized with controls (***p-value<0.0001); (D) ATP quantification of PC cells after treatment with Graviola extract. A Luminescent Cell Viability assay was used to measure the ATP content in the cells. Data is presented as mean value from experiments performed in triplicates normalized with the protein content ± standard error of mean. (*p-value=0.003, **p-value=0.002, ***p-value=0.0002) Data in the left panel is from FG/COLO357 cells, whereas data in the right panel is from CD18/HPAF cells.

Figure 3. Analysis of cytotoxic mechanism of Graviola extract in pancreatic cancer cells

(A) Quantification of apoptotic and necrotic PC cells after treatment with Graviola extract (Apoptotic cells Annexin V+/PI− staining; Necrotic cells Annexin V+/PI+staining). Data is presented as the mean value of the corresponding % cell population in duplicate samples ± standard error of mean; (B) The production of reactive oxygen species (ROS) in PC cells after treatment with Graviola extract was determined after incubating Graviola extract-treated PC cells with 2′,7′-Dichlorofluorescein diacetate (DCFH-DA). Cells were then analyzed by confocal microscopy. Scale bar represents 20μm; (C) Western blot analysis of Caspase-3 expression in PC cells after treatment with Graviola extract. Protein lysates (30μg) were resolved on 10% SDS-PAGE gels. β-actin was used as a loading control; (D) Cell cycle analysis of FG/COLO357 PC cells after treatment with Graviola extract. Cells were synchronized in the G1/S phase by thymidine block before adding Graviola extract. The effect of Graviola extract on the distribution of cells in different phases of the cell cycle was analyzed by flow cytometry. The data is presented as the mean value of the corresponding % cell population in duplicate samples ± standard error of mean. Representative flow cytometry histograms of cells treated with different concentrations of Graviola extract are shown. (*p-value=0.0001; **p-value<0.0001); (E) Western blot analysis of the expression of the cell cycle-related protein CyclinD1 in PC cells after being incubated with Graviola extract. Protein lysates (30μg) were resolved in 10% SDS-PAGE gels. β-actin was used as the loading control. In (A), (B), and (C), data in the left panel is from FG/COLO357 cells, whereas data in the right panel is from CD18/HPAF cells.

Figure 4. Effect of Graviola extract in the motility, migration, and cytoskeleton of pancreatic cancer cells

(A) Wound healing assay of FG/COLO357 PC cells after treatment with Graviola extract. Microscope images (40X) of the artificially created wound in PC cells monolayer were taken before (0hr) and after adding Graviola extract (24hr); (B) Migration of FG/COLO357 PC cells after treatment with Graviola extract. The number of cells that migrated through the 8μm pores of a polyethylene terephtalate (PET) membrane was quantified in 10 random fields. Data represent the mean value of migrating cells ± standard error of mean (*p-value = 0.0009; **p-value < 0.0001, compared to untreated control cells); (C) Actin filaments were analyzed by confocal microscopy by Rhodamine-anti-Phalloidin staining of FG/COLO357 cells after treatment with Graviola extract. Nucleus was stained with DAPI. Scale bars represent 20μm; (D) Microtubules were analyzed by confocal microscopy after FITC-anti-β Tubulin staining of FG/COLO357 cells after treatment with Graviola extract. Nucleus was stained with DAPI. Scale bars represent 20μm; (E) Expression of proteins related to migration/motility of PC cells after treatment with Graviola extract. Protein lysates (30μg) were resolved by 10% SDS-PAGE. β-actin was used as a loading control.

Figure 5. Evaluation of Graviola Extract in pancreatic cancer orthotopic xenograft model

(A) Pancreatic tumor weight results after treatment with Graviola extract. CD18/HPAF-Luciferase cells were injected orthotopically in the pancreas of athymic nude mice. After 1 week of tumor growth, oral gavage treatment of PBS-suspended Graviola extract was given daily for 35 days (N=8). Data is presented as box plots of the mean tumor weight of mice in each treatment group. (*p-value = 0.006; **p-value=0.0008, compared to tumors of PBS-treated mice); (B) Major sites of metastasis in each treatment group. Results are presented as number of animals having metastasis out of total number of animals per group. Statistical analysis was done comparing Graviola extract-treated mice with untreated mice (0mg/kg Graviola extract); (C)In vivo biophotonic imaging of pancreatic tumors during the course of treatment with Graviola extract. Representative IVIS images of mice from different treatment groups are shown (D) Hematoxylin and Eosin (H&E) staining of paraffin embedded pancreatic tumors. Images on the right (20X) are magnified areas from the images located at the left (10X). Yellow arrows in H&E sections represent necrotic areas in tumors from mice treated with Graviola extract.

Figure 6. Immunohistochemical analyses of pancreatic tumors after treatment with Graviola extract

(A) Immunohistochemical staining of MMP9 in paraffin-embedded pancreatic tumors. Representative images (20X) of tumors from different treatment groups are shown with the average composite score shown at the right. Data from experiments performed in triplicates is presented as the mean value of the composite score of tumors ± standard error of mean. MMP9 expression in pancreatic tumors was also assessed by western blot analysis. Homogenized protein tumor lysates (30μg) were resolved by 10% SDS-PAGE. β-actin was used as a loading control. (B) Immunohistochemistry staining of MUC4 in paraffin-embedded pancreatic tumors. Representative images (200X) of tumors from different treatment groups are shown with the average composite score shown at the right. Data from experiments performed in triplicate is presented as the mean value of the composite score of tumors ± standard error of mean. MUC4 expression in pancreatic tumors was also assessed by western blot analysis. Homogenized protein tumor lysates (30μg) were resolved by 2% agarose gels. β-actin was used as the loading control.