Anticancer Effect of Ginger Extract against Pancreatic Cancer Cells Mainly through Reactive Oxygen Species-Mediated Autotic Cell Death

The extract of ginger (Zingiber officinale Roscoe) and its major pungent components, [6]-shogaol and [6]-gingerol, have been shown to have an anti-proliferative effect on several tumor cell lines. However, the anticancer activity of the ginger extract in pancreatic cancer is poorly understood. Here, we demonstrate that the ethanol-extracted materials of ginger suppressed cell cycle progression and consequently induced the death of human pancreatic cancer cell lines, including Panc-1 cells. The underlying mechanism entailed autosis, a recently characterized form of cell death, but not apoptosis or necroptosis. The extract markedly increased the LC3-II/LC3-I ratio, decreased SQSTM1/p62 protein, and enhanced vacuolization of the cytoplasm in Panc-1 cells. It activated AMPK, a positive regulator of autophagy, and inhibited mTOR, a negative autophagic regulator. The autophagy inhibitors 3-methyladenine and chloroquine partially prevented cell death. Morphologically, however, focal membrane rupture, nuclear shrinkage, focal swelling of the perinuclear space and electron dense mitochondria, which are unique morphological features of autosis, were observed. The extract enhanced reactive oxygen species (ROS) generation, and the antioxidant N-acetylcystein attenuated cell death. Our study revealed that daily intraperitoneal administration of the extract significantly prolonged survival (P = 0.0069) in a peritoneal dissemination model and suppressed tumor growth in an orthotopic model of pancreatic cancer (P < 0.01) without serious adverse effects. Although [6]-shogaol but not [6]-gingerol showed similar effects, chromatographic analyses suggested the presence of other constituent(s) as active substances. Together, these results show that ginger extract has potent anticancer activity against pancreatic cancer cells by inducing ROS-mediated autosis and warrants further investigation in order to develop an efficacious candidate drug.

section_content: Pancreatic cancer is a highly aggressive neoplasm with many chemotherapy-and radiotherapy-resistant phenotypes. The incidence of pancreatic cancer is increasing annually worldwide, becoming the fourth most common cause of cancer-related death in the world. Indeed, the estimated number of new pancreatic cancer cases was 277,000 in 2008 and 338,000 in 2012, and there were 266,000 pancreatic cancer deaths in 2008 and 331,000 pancreatic cancer deaths in 2012 [1, 2]. As the majority of pancreatic cancer patients are diagnosed at an inoperable stage [3, 4], the overall 5-year relative survival rate is low, and the median survival time is only 6 months even in patients receiving quality treatment. In order to obtain new leads for the development of preventive and therapeutic strategies, many research efforts have been focused on understanding the molecular mechanisms underlying pancreatic cancer progression [3, 4]. 

Ginger (Zingiber officinale Roscoe), a rhizomatous perennial plant, is widely used as a spice in foods and beverages and utilized primarily as a remedy for digestive disorders including dyspepsia, nausea, gastritis, vomiting, colic, and diarrhea [5, 6]. Ginger extract and its pungent components, such as [6] -gingerol and [6] -shogaol, are also known to exhibit many biological effects including anti-inflammation, antioxidation and anticancer activity [5, 6]. Because of its strong anti-inflammatory activity, ginger has recently drawn attention as a remedy for osteoarthritis and rheumatoid arthritis [7, 8]. With respect to anticancer activity, ginger and its constituents have been shown to inhibit the proliferation of and induce apoptosis of a variety of cancer cell types in vitro [9] [10] [11] [12] [13] [14] [15]. In addition, the use of ginger for the chemoprevention of colorectal cancer has attracted attention [16] [17] [18]. However, the anticancer activity of ginger extract and its constituents against pancreatic cancer has been poorly investigated. 

In this study, we examined the anticancer activity of ginger extract against pancreatic cancer cells both in vitro and in vivo and investigated its potential mechanism. Here, we report that ginger extract leads to the reduction of cell viability and tumor growth of Panc-1 cells mainly through ROS-mediated autosis, a recently characterized form of cell death.
section_name: Introduction

section_name: Materials and Methods

section_content: Human pancreatic cancer cells, Panc-1, AsPC-1, BxPC-3, CAPAN-2, CFPAC-1, MIAPaCa-2 and SW1990, and mouse pancreatic cancer cells, Panc02, were used in this study. Panc-1 and MIAPaCa-2 cells were obtained from the RIKEN BRC Cell Bank (Tsukuba, Japan), and other human pancreatic cell lines were purchased from ATCC (Manassas, VA). Panc02 cells were kindly provided by Dr. T. Hollingsworth, University of Nebraska Medical Center [19, 20]. Panc-1-Luc-ZsGreen cells and Panc02-Luc-ZsGreen cells expressing firefly luciferase and ZsGreen were established by lentiviral transduction of the plasmid pHIV-Luc-ZsGreen, which has been deposited with Addgene (http://www. addgene. org/Bryan_Welm/), and subsequent cloning. Human pulmonary alveolar epithelial cells (HPAEpiC) were purchased from ScienCell (Carlsbad, CA, USA) and were maintained in alveolar epithelial cell medium (AEpiCM) supplemented with 2% fetal bovine serum (FBS), epithelial cell growth supplement (EpiCGS) and penicillin/streptomycin. Human umbilical vein endothelial cells (HUVEC) were obtained from Lonza Walersville, Inc. (Walkersville, MD, USA) and were grown in EBM-2 supplemented with EGM SingleQuats (Lonza). Mitochondria DNA-less P29 (ρ 0 P29) cells and the cybrid P29mtP29 cells that were reintroduced with P29 mtDNA into ρ 0 P29 cells were established from Lewis lung carcinoma P29 cells [21]. Colon cancer cells (Colo320DM, HT29, LoVo, LS174T, SW480, SW620) [22], gastric cancer cells (MKN1, MKN45) [23], lung cancer cells (A549, QG56, PC-10, PC-1) [24], breast cancer cells (MCF7, BT549, MDA-MB-231, MDA-MB-468) [25, 26], leukemia cells (THP-1, K562) [27], osteosarcoma cells (Saos-2) [28], cervical cancer cells (HeLa) [29], hepatoma cells (HepG2) [30], fibrosarcoma cells (HT1080) [29], and mouse colon carcinoma LuM1 cells derived from colon 26 tumor [31] were also used in this study. HT29, LS174T, SW480, SW620 and MDA-MB-468 were purchased from ATCC. MCF7 and HT1080 cells were obtained from the JCRB Cell Bank. THP-1 and K562 cells were kindly provided by Dr. Y. Honma, Shimane University Faculty of Medicine. Gastric cancer cell lines were supplied by the Department of Pathology (Dr. S. Morikawa), Shimane University Faculty of Medicine. Other cell lines were supplied by Dr. A. Nakagawara, Chiba Cancer Center Research Institute. Characteristics of the cell lines used in this study are described elsewhere [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31]. Leukemia cell lines were cultured in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS) and 40 μg/ml gentamicin. Other cell lines were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS and 40 μg/ml gentamicin in a humidified atmosphere with 21% O 2 /5% CO 2 (normoxia) or 1% O 2 /5% CO 2 (hypoxia). Hypoxic culture conditions were achieved in a humidified automatic O 2 /CO 2 incubator (Wakenyaku, Kyoto, Japan).
section_name: Cells and cell culture

section_content: [6]-Shogaol and [6] -gingerol were purchased from TOKIWA PHYTOCHEMICAL CO., Ltd. (Chiba, Japan).
section_name: Reagents

section_content: Dry powder of the root parts of Syussai Shoga (ginger in Japanese), which was cultivated in the Hikawa area in Izumo, Shimane prefecture, was extracted with ethanol (10:1; volume for weight) for 20 min in a sonication water bath. Ethanol was evaporated at 80°C to yield a crude ethanol extract of the ginger (referred to as SSHE). The extract was weighed and dissolved in ethanol or dimethylsulfoxide (DMSO) at the desired concentration.
section_name: Preparation of ginger extract (SSHE)

section_content: Cell growth and viability was measured by using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Briefly, cells (2x10 4 cells/well) were cultured in 96-well tissue culture plates and treated in triplicate in 100 μl DMEM/10% FBS containing different concentrations of SSHE or solvent alone for the indicated period. At the end of the incubation, 10 μl of MTT (2. 5 mg/ml) (Sigma-Aldrich Japan, Tokyo, Japan) was added to the wells to allow formation of MTT formazan crystals for 4 h. After the medium was removed, the crystals were solubilized in 100 μl of DMSO. Absorbance was recorded at 550 nm. Cell viability was also assayed by a trypan blue dye exclusion test.
section_name: Cell growth and viability assay

section_content: Panc-1 cells treated with SSHE for 20 h were fixed in 70% ethanol and stored at -20°C until use. The fixed cells were washed with Dulbecco's PBS (DPBS) and incubated with 100 μg/ml RNase A and 50 μg/ml PI (Sigma-Aldrich Japan). The cells were then subjected to flow cytometric analysis using a FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ).
section_name: Cell cycle analysis

section_content: Mitochondria membrane potential was monitored by the JC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemical, Ann Arbor, MI). For this assay, 100 μl/ml of the JC-1 Staining Solution was added to Panc-1 cells treated with SSHE for 20 h in 6-well plates, and the cells were incubated for 15 min. Afterwards, the cells were detached by trypsinization, washed twice with the assay buffer, and then subjected to flow cytometry.
section_name: Measurement of mitochondria membrane potential

section_content: The Annexin V-FITC Apoptosis Detection Kit (BECKMAN COULTER Inc., Pasadena, CA) was used to detect annexin V and/or PI-positive cells. Briefly, Panc-1 cells were washed with ice-cold DPBS and then stained with Annexin V-FITC for 15 min at room temperature in the dark and PI in ice-cold Binding Buffer. Annexin V and/or PI-positive cells were counted using a FACS Calibur flow cytometer.
section_name: Annexin V/Propidium iodide (PI) staining

section_content: The production of ROS was monitored by flow cytometry with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) (Molecular Probe-Life Technologies, Carlsbad, CA) and mitochondrial superoxide indicator MitoSOX Red (Invitrogen) as probes. Briefly, Panc-1 cells treated with SSHE were incubated with 10 μM of H2DCFDA or 5 μM of MitoSOX Red in serum-free DMEM for 10 min. The medium was removed, and the cells were detached with a brief treatment of 0. 25% trypsin in Hank's balanced salt solution. After addition of fresh culture medium, the cells were collected by centrifugation, washed once with DPBS and suspended in DPBS. The fluorescence was monitored using the FACSCalibur flow cytometer or under a laser confocal microscope (Fluoview FV1000, Olympus, Tokyo).
section_name: Measurement of ROS generation

section_content: Cell extracts were prepared by lysing cells with RIPA buffer (50 mM Tris-HCl, pH 7. 4, 150 mM NaCl, 1% NP-40, 0. 5% deoxycholate, 0. 1% sodium dodecyl sulfate, 2 mM EDTA, protease inhibitor cocktail and phosphatase inhibitor cocktail) on ice for 20 min. The lysates were centrifuged at 12,000 ×g for 10 min at 4°C,and the supernatants were used for subsequent analyses. SDS-polyacrylamide gel electrophoresis and immunoblot analyses were performed as described previously [32]. The primary antibodies used were rabbit monoclonal anti-SQSTM1/ p62 (D5E2, 1:1,000 dilution, Cell Signaling Technology, Danvers, MA), rabbit polyclonal anti-LC3B (1:1,000 dilution, Cell Signaling), rabbit polyclonal anti-phospho-mTOR (S2481) (1:1,000 dilution, Cell Signaling), rabbit monoclonal anti-mTOR (7C10, 1:1,000 dilution, Cell Signaling), rabbit monoclonal anti-phospho-AMPKα (T172) (40H9, 1:1,000 dilution, Cell Signaling), and rabbit monoclonal anti-AMPKα antibody (23A3, 1:1,000 dilution, Cell Signaling). The secondary antibodies were HRP-conjugated rabbit or anti-mouse IgG (1:3,000 dilution, Cell Signaling). For loading controls, anti-β-actin antibody (sc-47778, 1:3,000 dilution, Santa Cruz Biotechnology) was used. Signals were visualized using ECL plus (GE Healthcare, Little Chalfont, UK). The membranes were scanned with a Luminoimaging Analyzer LAS4000 (GE Healthcare).
section_name: Western blotting

section_content: Panc-1 cells treated with solvent alone or SSHE were fixed with 4% formaldehyde/5% sucrose in DPBS for 20 min, rinsed with DPBS, and permeabilized with 0. 5% Triton X-100 in DPBS for 4 min. In some experiments, cells were stained with 100 nM of MitoTracker Red CMXRos (Invitrogen) for 10 min before fixation. The cells were blocked with 3% BSA/0. 1% glycine in DPBS for 1 h, rinsed, and then incubated with rabbit monoclonal anti-AIF (D39D2, 1:200 dilution, Cell Signaling) or rabbit polyclonal anti-LC3B antibody (1:200 dilution) for 1 h. After extensive washing with DPBS, the cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit IgG (1:300 dilution, Invitrogen) for 1 h. The cells were counterstained with DAPI and observed under a laser scanning confocal microscope.
section_name: Immunofluorescent staining

section_content: Untreated Panc-1 cells and the cells treated with 200 μg/ml SSHE for 28 h were placed in 2% paraformaldehyde and 2% glutaraldehyde in 30 mM HEPES buffer (30 mM HEPES, 100 mM NaCl, 2 mM CaCl 2, pH 7. 4) for 2 h at 4°C and post-fixed with 1% OsO 4 for 1 h at 4°C. The samples were dehydrated with graded alcohol and embedded in TAAB812 resin (TAAB Laboratories Equipment Ltd., Berkshire, UK). Ultrathin sections were stained for 1 h in 3% aqueous uranyl acetate, washed, and counterstained with 0. 3% lead citrate, and they were examined on a transmission electron microscope (EM-002B, JEOL Ltd., Tokyo, Japan).
section_name: Transmission electron microscopy (TEM) analysis

section_content: EGFP-LC3B fusion plasmid (pCMX-SAH/Y145F-LC3B-GFP) was constructed by cloning LC3B cDNA, which was amplified by PCR, into a pCMX-SAH/Y145F-GFP vector [33]. The construct was verified by DNA sequencing. Panc-1 ells were transiently transfected with the plasmid using Lipofectamine 2000 (Invitrogen-Life Technologies, Carlsbad, CA) according to the manufacturer's instructions. After 24 h, the cells were exposed to SSHE and examined under a laser scanning confocal microscope.
section_name: GFP-LC3 plasmid and transfection

section_content: All animal experiments were performed in compliance with the institutional guidelines for the care and use of animal research. The protocol was approved by the IZUMO Campus Animal Care and Use Committee of Shimane University (Permission Number: IZ26-7). All the mice were housed in the animal center of Shimane University Faculty of Medicine under specific pathogen-free conditions at a controlled temperature of 23±2°C, relative humidity 55±10%, and with 12 h light/12 h dark cycles. They were given food and water ad libitum. Mice were checked for their health during the entire experimental period at least once a day after tumor injection. All surgery was performed under medetomidine (0. 3 mg/kg)/midazolam (4. 0 mg/ kg)/butorphanol (5. 0 mg/kg) anesthesia. Mice were normally euthanized by CO 2 inhalation at the end of a study. For the peritoneal dissemination model, Panc02-Luc-ZsGreen cells (5x10 5 cells/mouse) were injected intraperitoneally as a cell suspension into 7-week-old male C57BL/6 mice (Crea Japan, Tokyo, Japan), and the mice were randomized and grouped into the control (n = 8) and the SSHE groups (n = 8). The treatment regimens were started the day after tumor inoculation. Mice were euthanised in a CO 2 chamber when they were moribund, measured by a lack of sustained purposeful response to gentle stimuli. All efforts including subcutaneous administration of meloxicam (5 mg/kg) were made to minimize suffering. The experiment was performed twice, and the combined data were subjected to analyses. For the orthotopic model of pancreatic cancer, Panc-1-Luc-ZsGreen cells (1x10 6 cells/mouse) were implanted with 50% Matrigel into the pancreas of 6-week-old female nude mice (BALB/c nu/nu, Japan SLC, Shizuoka, Japan) [32]. One week after the injection, they were randomized and grouped into the control (n = 6) and the SSHE groups (n = 6). The treatment regimens were started one week after tumor injection. For mouse colon carcinoma experiments, LuM1 cells (3 x 10 5 cells) were subcutaneously implanted in male Balb/c mice (n = 6 for the control and the SSHE group). The volumes of LuM1 tumors were evaluated by measuring two perpendicular diameters with calipers. Tumor volume (V) was calculated using the following equation: V = (a 2 x b)/2, where a is the small diameter and b the large diameter. In the SSHE group, mice were intraperitoneally administered 80 mg/kg SSHE once daily. In the control group, mice were administered solvent alone in DPBS.
section_name: Animal experiments

section_content: In vivo bioluminescent imaging was performed using the IVIS imaging system (Caliper Life Sciences, Hopkinton, MA). All mice were injected intraperitoneally with 150 mg/kg d-luciferin (Promega, Fitchburg, WI) and anesthetized with 2. 5% isoflurane. Ten minutes later, photons from animals' whole bodies were imaged using the IVIS imaging system (Caliper Life Sciences) according to the manufacturer's instructions. Data were analyzed by living image 2. 50 software (Caliper Life Sciences).
section_name: Bioluminescent imaging

section_content: Mice were anesthetized, and blood was collected from the heart. Peripheral blood profiles were analyzed by the Sysmex KX-21NV automated hematology analyzer (Sysmex, Kobe, Japan). The levels of white blood cells (WBC), red blood cells (RBC), platelets (PLT), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were examined. Glucose (Glu) levels, total cholesterol (T-Cho) levels, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, and blood urea nitrogen (BUN) levels were analyzed with an automated analyzer for clinical chemistry, SPOTCHEM EZ SP-4430 (ARKRAY, Inc., Kyoto), using SPOTCHEM II test strips.
section_name: Blood hematology and biochemistry test

section_content: Liquid chromatographic separations were achieved using a reversed-phase C-18, 3 μm, 2. 4 × 250 mm column (COSMOSIL. NAKARAI TESQUE, Inc., Kyoto, Japan) at a flow rate of 1 ml/min. The mobile phase was 70% methanol. The elution profile was monitored by UV spectrophotometry at 228 nm.
section_name: Reversed-phase high-performance liquid chromatography (HPLC)

section_content: All data are presented as the mean ± SD. Statistical significance between data sets was tested by unpaired Student's t test. Survival of mice was analyzed using the log-rank test. P < 0. 05 was considered to be statistically significant. [35]. Thus, we concluded that SSHE-induced cell death was caspase-independent and resembled autophagy cell death. This form of cell death coincides well with autosis, a recently characterized Na +,K + -ATPase-regulated form of cell death [36]. Unfortunately, because the cardiac glycoside digoxin, an antagonist of Na +,K + -ATPase that is reported to inhibit autosis [36], was too cytotoxic to Panc-1 cells, we could not evaluate its effect on SSHE-induced cell death. Mitochondria are reported to be the primary source of ROS required for autophagy induction [37]. To examine whether mitochondrial ROS play a role in SSHE-induced autotic cell
section_name: Statistical analysis

section_name: Results

section_name: SSHE inhibits cell cycle progression and induces the death of pancreatic cancer cell lines

section_name: Mitochondrial ROS generation is involved in SSHE-induced cell death

section_content: When Panc02-Luc-ZsGreen cells (5 x 10 5 cells) were intraperitoneally transplanted into C57BL/6 mice, they tended to form disseminated nodules preferentially around the pancreas and on the peritoneal surface, and the mice developed ascites (approximately 2. To examine any toxicity of SSHE in more detail, hematological and biochemical blood tests were performed on Day 15. In a routine peripheral blood analysis, there were no differences in the levels of WBC, RBC, PLT, HGB, HCT, MCV, MCH, or MCHC between the control and the SSHE-treated groups (S7 Fig). The Glu levels were slightly higher in the SSHE-treated group than in the control group (P < 0. 01). The T-Cho levels were not affected by SSHE. In terms of renal function, BUN levels in the SSHE-treated group were only slightly higher than those in the control group (P < 0. 05). With respect to liver function, there were no significant differences in T-Bil, AST and ALT levels (S7 Fig). Overall, we did not observe serious abnormalities in the SSHE-treated mice. 

We also investigated the effect of intraperitoneal administration of SSHE on the tumor growth of orthotopically implanted Panc-1 cells. Panc-1-Luc-ZsGreen cells (1 x10 6 cells) were injected into the pancreas of nude mice with Matrigel. One week after the injection, the mice were randomized (6 mice/group) and then treated with 80 mg/kg SSHE. Two 2-week periods of once-daily administration of SSHE were performed, with a 5-day resting phase in between treatment periods (Fig 7A ). Unfortunately, a mouse in the treatment group showed splenomegaly of unknown cause during the first treatment period, so we excluded that mouse from the experiment. Bioluminescence imaging on Day 35 clearly showed that SSHE significantly suppressed the growth of Panc-1 cells (P < 0. 01) without loss of body weight (Fig 7B and 7C ). 

Additionally, SSHE also suppressed tumor growth of subcutaneously transplanted mouse colon carcinoma LuM1 cells without loss of body weight (S8 Fig).
section_name: SSHE retards tumor growth of pancreatic cancer

section_content: To investigate which constituents of SSHE are active in retarding cell growth and inducing the death of Panc-1 cells, we examined the effect of [6] -shogaol and [6] -gingerol [14, [38] [39] [40] [41] [42]. The results showed that [6] -shogaol reduced the viability of Panc-1 cells with an IC50 value of ~18. 8 μM (~5. 2 μg/ml). It also caused cell growth retardation and cell death in other pancreatic Presence of potent cell death-inducing component(s) other than [6] shogaol in SSHE To investigate whether [6] -shogaol is the major cell death-inducing component in SSHE, we estimated the concentration of [6] -shogaol in SSHE. To this end, we carried out reversed-phase HPLC followed by the measurement of area corresponding to [6] -shogaol in SSHE. By extrapolating the area to the standard curve, the concentration of [6] -shogaol in SSHE was determined to be approximately 4. 6 μg in 200 μg SSHE (S12 Fig). This concentration was not sufficient to achieve full activity of SSHE (Fig 1B ), which suggested the presence of other active constituent (s). We then fractionated the constituents in SSHE and examined the cell death-inducing activity of each fraction. The results showed that potent activity was eluted in a few fractions, all of which were ahead of the [6] -gingerol fraction (S12 Fig), although the chemical composition of these fractions is presently unknown. [6] -Gingerol fractions showed moderate activity, most likely due to its high content in the extract. [6] -Shogaol fractions showed weak activity.
section_name: [6]-Shogaol causes cell growth retardation and death of pancreatic cancer cells

section_content: The present study demonstrated that the of Syussai ginger (SSHE) had potent growthinhibitory and cell death-inducing activity against pancreatic cancer cells including Panc-1 cells. Normal cells such as HUVEC and HPAEpiC were relatively resistant to SSHE compared to Panc-1 cells. The extract was also effective under hypoxic conditions, which inevitably develop in all solid tumors to varying degrees and influence the resistance of tumor cells to radiotherapy and conventional chemotherapy [43, 44]. Among various cancers, pancreatic cancer is notorious for its unusually hypoxic microenvironments [45]. Our results showed that SSHE could induce the death of hypoxic Panc-1 cells, which may be a notable characteristic of SSHE for treating pancreatic cancers. In addition to pancreatic cancer, SSHE also induced a marked cell growth retardation and cell death in a variety of tumor cells, which supports the possibility of applying ginger extract and its active constituents for treating cancers. 

After the treatment of Panc-1 cells with SSHE, the mitochondrial membrane potential was dramatically decreased and the number of annexin V-positive cells increased by 24 h after administration of SSHE. However, little increase in subG1 phase cells, fragmented nuclei and caspase-3 activation, all of which are features of apoptosis, were observed. Moreover, zVAD-fmk failed to rescue SSHE-induced cell death. Because annexin V stains both primary necroptotic and apoptotic cells [46] and mitochondria dysfunction can also be brought about by mitophagy, the autophagy-dependent elimination of mitochondria [47], we considered the possibility that SSHE-induced cell death was not due to apoptosis. Additionally, necroptosis was excluded because necrostatin-1 could not ameliorate the cell death induced by SSHE. We then explored the possibility of autophagic cell death and noticed many cytoplasmic vacuoles and LC3 puncta in the SSHE-treated Panc-1 cells at early stages. SSHE treatment dramatically increased the ratio of LC3-II/LC3-I and caused loss of SQSTM1/p62. SSHE activated AMPK and inhibited mTOR. 3-Methyladenine and chloroquine partially rescued SSHE-induced cell death. All of these data suggested that autophagy was occurring in the SSHE-treated cells. However, a close look at the SSHE-treated cells revealed several features that are not observed in classical type autophagy, including nuclear shrinkage, focal membrane rupture, electrondense mitochondria, empty vacuoles and focal perinuclear swelling. Eventually, it appeared that these morphological features coincided well with the recently discovered form of cell death, "autosis" [36]. Thus, we presently consider that the SSHE-induced cell death of Panc-1 cells is mainly due to caspase-independent, autotic cell death rather than due to apoptosis and necroptosis. 

Several lines of evidence indicate that ROS, more specifically mitochondria-derived ROS, are the inducer of autophagy upon various stresses such as nutrient deprivation [48] [49] [50] [51] [52] [53] [54]. Our data demonstrated that ROS production was suppressed in SSHE-treated Panc-1 cell at early stages. This may be owed to the antioxidant properties of ginger extract [5, 6]. However, prolonged treatment of the cells with SSHE caused a marked increase in ROS production. The ROS production was most likely the cause of SSHE-induced autotic cell death because NAC partially rescued the cell death induced by SSHE. This was further corroborated by the fact that ρ 0 P29 cells that produced little ROS were refractory to SSHE compared with P29mtP29 cells. 

Our study in mice demonstrated that SSHE showed potent anticancer activity in both the peritoneal dissemination model and the orthotopic model. It also suppressed the tumor growth of subcutaneously implanted mouse colon carcinoma cells. Notably, apparent severe adverse effects, such as loss of body weight and low blood cell counts, were not evident in the SSHEtreated mice, although relatively minor abnormalities in the blood Glu and BUN levels were observed. Other side effects, such as diarrhea, constipation, or hair loss, also did not occur. Therefore, even at such a high tested dose, SSHE seemed to be safe and tolerable. Although it is difficult to extrapolate our observations to humans, recent pilot clinical study demonstrated that oral intake of 2. 0 g/day whole ginger extract (the materials extracted with 50% ethanol) for 28 days was tolerable and safe in humans [18]. Additionally, it has recently been reported that oral administration of whole ginger extract (100 mg/kg) was effective in suppressing xenografted PC-3 prostate cancer growth without any toxicity [15]. Thus, treatment with whole ginger extract may be beneficial for cancer patients. 

[6]-Shogaol and [6]-gingerol are the major components of ginger extract, and both have been shown to exhibit anti-proliferative and apoptosis-inducing activities in tumor cells [55, 56]. Our data showed that, although [6] -gingerol had cell growth-inhibitory activity at very high concentrations, [6] -shogaol exerted potent activity at relatively low concentrations. However, the content of [6] -shogaol in SSHE was not sufficient enough to exhibit cell death-inducing activity. To search for other active component(s), reversed-phase HPLC followed by fractionation revealed three major peaks showing cell death-inducing activity; in the order of elution, the first peak had the most potent activity, and the second and the third peaks corresponded to the elution position of [6] -gingerol and [6] -shogaol, respectively. Identification of the component(s) in the first peak is currently under way. 

In conclusion, the present study demonstrated that ginger extract inhibited cell proliferation and subsequently induced the autotic death of pancreatic cancer Panc-1 cells. The extract suppressed tumor growth without serious adverse effects in a Panc02 peritoneal dissemination mouse model and Panc-1 xenografted mice when administered intraperitoneally. Our results suggest that whole ginger extract or its constituents may have clinical implications for therapeutic intervention against pancreatic cancer.
section_name: Discussion

section_name: Acknowledgments

section_content: We would like to thank Tsunao Yoneyama and Yuko Okui for the TEM work and Chika Imaoka, Momoko Ogawa, Reona Kishimoto and Yuna Kimura, Izumo High School, who collaborated with us.
section_name: Acknowledgments

section_content: This work was supported in part by Grantsin-Aid from: Shimane University "SUIGANN" Project (http://www. shimane-u. ac. jp) (to KT); Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (http://www. mext. go. jp) (no. 25430110 to KT); and Japan Arteriosclerosis Research Foundation (to KT). This work was also supported by the Support for Super Science High Schools from Japan Science and Technology Agency to Izumo High School (http://rikai. jst. go. jp) (to KT). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

section_content: All relevant data are within the paper and its Supporting Information files.

section_name: Supporting Information