Original Papers
Mechanism of Ascorbate-Induced Cell Death in Human Pancreatic Cancer Cells: Role of Bcl-2, Beclin 1 and Autophagy
Authors
Masayuki Fukui 1, 2, Noriko Yamabe 1, Hye-Joung Choi 1, Kishore Polireddy 1, Qi Chen 1, Bao Ting Zhu 1, 3
Affiliations
1
2 3
Key words " ascorbate l " pancreatic cancer l " cell death l " autophagy l
Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, KS, USA Department of Microbiology, Tropical Biosphere Research Center, University of the Ryukyus, Nishihara, Okinawa, Japan Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China
Abstract
some formation contributes to ascorbate-induced pancreatic cancer cell death.
!
The present study investigates the anticancer effect of ascorbate in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as important mechanistic elements in its anticancer actions. We showed that ascorbate suppresses the growth of human pancreatic cancer cells via the induction of oxidative stress and caspase-independent cell death. Ascorbate induces the formation of autophagosomes and the presence of autophagy inhibitors suppresses ascorbate-induced cell death. These data suggest that the induction of autophago-
Introduction !
received revised accepted
February 11, 2015 April 18, 2015 May 3, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1546132 Published online July 1, 2015 Planta Med 2015; 81: 838–846 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Bao Ting Zhu South University of Science and Technology of China Department of Biology 1088 Xueyuan Road, Xili, Nanshan District Shenzhen, Guangdong, 518055 China Phone: + 86 7 55 88 01 84 16 Fax: + 86 7 55 88 01 84 17 [email protected]
The use of dietary agents has been considered an alternative approach in the prevention and treatment of human cancers. Epidemiological studies have shown that a diet rich in fruits and vegetables is associated with a low incidence of many types of human malignancies, including pancreatic cancer [1, 2]. AA, commonly known as vitamin C and an essential nutrient in human diet, not only serves as a cofactor in many metabolic reactions, but it also serves as a strong antioxidant that protects cells against oxidative stress and damage [3–5]. However, AA may also serve as a prooxidant under certain conditions [4, 5]. Studies have shown that while the plasma and tissue levels of AA are usually maintained within a well-controlled range (under 0.2 mM) following oral administration, its effective anticancer concentrations (at 0.2–10 mM range) are achievable following parenteral (i. v. or i. p.) administrations [6–8]. Some previous studies showed that the production of hydrogen peroxide (H2O2) with pharmacological doses of AA is an important contributor to its anticancer activity both in vitro [5]
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Abbreviations !
AA: ERK: JNK: MAPK: ROS:
ascorbate extracellular signaling-regulated regulated protein kinase c-jun N-terminal kinase mitogen-activated protein kinase reactive oxygen species
Supporting information available online at http://www.thieme-connect.de/products
and in vivo [9]. Moreover, it was observed that the presence of other antioxidants could diminish the anticancer activity of AA in cancer cells, whereas the presence of prooxidants can enhance its anticancer activity [10, 11]. The cytotoxic activity of AA appears to be predominantly seen in cancer cells, but not in normal cells [9, 12]. The exact type of cell death seen in cultured cancer cells following exposure to high concentrations of AA remains controversial [13–25]. In this study, we sought to investigate the anticancer effect of AA in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as important mechanistic elements in its anticancer actions.
Results !
First, we confirmed the role of AA-mediated formation of extracellular hydrogen peroxide in its anticancer activity [5, 12]. We showed that AA induced cell death in a concentration-dependent manner in cultured MIA-PaCa-2 cells, and the co-
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Fig. 1 AA-induced cell death in MIA-PaCa-2 cells is mediated by hydrogen peroxide, but independent of MAPK activation. A MIA-PaCa-2 cells were treated with AA at indicated concentrations with or without 100 µg/mL catalase. After 24 h incubation, cell viability was analyzed using the MTT assay. Each value is the mean ± SD (N = 3). *P < 0.05. B MIA-PaCa-2 cells were treated with 1 mM AA in the presence or absence of 100 µg/mL catalase or 1 mM trolox for 8 h, and then analyzed for intracellular ROS accumulation using the H2-DCF‑DA staining method as described in Materials and Methods. The experiments were repeated three times and representative results are shown. The size bar in each image represents 50 µm in length. C MIA-PaCa-2 cells were pretreated with 10 µM SP600125 (SP is a JNK pathway inhibitor), 10 µM PD98059 (PD is an ERK pathway inhibitor), or 10 µM SB202190 (SB is a p38 pathway inhibitor) for 2 h and then treated with 2 mM AA for an additional 24 h. The cell viability was determined using the MTT assay. Each value is the mean ± SD (N = 3). In each panel, the filled columns are without an inhibitor, and the open columns are with an inhibitor.
presence of catalase or trolox (water-soluble derivatives of vitamin E) strongly protected the cells against AA-induced cell death " Fig. 1 A; Fig. S1, Supporting Information). In addition, we (l showed that while there was no appreciable accumulation of intracellular ROS in vehicle-treated cells, a significant accumulation was detected in AA-treated cells, and the presence of catalase or trolox almost completely abolished the intracellular accumula" Fig. 1 B). tion of ROS (l It is known that oxidative stress can cause the activation of the MAPKs, i.e., JNK, ERK, and p38-MAPK, and, subsequently, results in caspase activation and apoptotic cell death [13, 14]. Han et al. reported that p38-MAPK, but not JNK or ERK, mediates AA-induced cytotoxicity in human gastric cancer cells [15]. In this study, therefore, we examined whether MAPK signaling pathways are involved in AA-induced cell death in MIA-PaCa-2 cells. Cells were treated with AA in the presence or absence of the inhibitors of the MAPKs (i.e., SP600125 for JNK1/2, PD98059 for " Fig. 1 C, MEK1/2, and SB202190 for p38-MAPK). As shown in l these inhibitors (at 10 µM) did not have a significant effect on AA-induced cell death in cultured pancreatic cancer cells.
To determine whether the induction of apoptosis is an important contributor to AA-induced anticancer activity, we examined capsase-3 activation and nuclear morphological changes in cells treated with AA. Based on measuring the cleaved caspase-3 by Western blotting, we found that caspase-3 activation was not detected in AA-treated MIA-PaCa-2 cells, but the treatment of these cells with paclitaxel (PTX) clearly induced caspase-3 cleavage " Fig. 2 A). Further, as shown in l " Fig. 2 B, while typical apoptot(l ic nuclear morphological changes were readily observed in cells treated with PTX, these changes were essentially absent in cells treated with AA. Moreover, when cells were treated with AA in the presence or absence of z-VAD‑fmk (a pan-caspase inhibitor), changes in cell viability in AA-treated pancreatic cancer cells were not significantly altered (Fig. S2, Supporting Information). Together, these data indicate that AA-induced pancreatic cancer cell death is independent of caspase activation. Because caspase activation is known to be an ATP-dependent process [16–18], the lack of caspase activation in AA-treated cells is likely due to the depletion of the cellular ATP level resulting from rapid ROS accumulation. In support of this possibility, we
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Fig. 2 AA-induced cell death in MIA-PaCa-2 cells is independent of caspase activation. A MIA-PaCa-2 cells were treated with 1 mM AA for the indicated lengths of time or with 20 nM paclitaxel (PTX) for 24 h. Cell extracts were prepared and subjected to Western blotting of caspase-3. The experiments were repeated three times and representative results are shown. B MIA-PaCa-2 cells were treated with 1 mM AA or 20 nM PTX for 24 h and stained with Hoechst33342. Nuclear morphological changes were photographed under a fluorescence microscope. C MIA-PaCa-2 cells were treated with 2 mM AA for the indicated lengths of time, and then the cellular ATP levels were determined as described in Materials and Methods. Each value is the mean ± SD (N = 3). *P < 0.05 compared to the control (0 h).
observed that the cellular ATP level in AA-treated cells was rap" Fig. 2 C), which is idly depleted in a time-dependent manner (l also consistent with earlier observations [5, 19]. Notably, the ATP level in CCD-34SK fibroblasts (a non-tumor cell line) was basically not changed following treatment with 5 mM of ascorbate for 1 and 4 h (Fig. S3, Supporting Information). To determine whether AA treatment induces autophagosome formation, we examined the changes in Beclin 1, LC3B–I, and LC3B-II protein levels following the treatment of cultured MIA" Fig. 3 A, there was no signifiPaCa-2 cells with AA. As shown in l cant change in the Beclin 1 level after AA treatment, but the expression of LC3B–I was decreased, and this decrease was accompanied by an increase in LC3B-II. To further investigate the effect of AA on autophagosome formation, we performed immunocytochemical staining of AA-treated cells using the same anti-LC3B antibody. After an 8-h AA treatment, intracellular LC3B accumu" Fig. 3 B). When lation and punctuated staining were observed (l the percentage of LC3B positive cells with a punctuated staining pattern was quantitated, we found that the percentages of positive cells treated with the vehicle and AA were 6.4 ± 0.3 and 27.6 ± 9.5, respectively (p < 0.05). The observation of an increased cell population with positive LC3B staining following AA treatment was further confirmed by detecting the formation of auto-
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phagosomes in AA-treated cells using transmission electron mi" Fig. 3 B). croscopy (l To determine the functional role of autophagosome formation in AA-induced cell death, we examined the effect of two commonly used autophagy inhibitors, namely, chloroquine (an inhibitor of endosomal acidication) and bafilomycin A1 (an inhibitor of the " Fig. 3 C, the formation of autophagic vacuoles). As shown in l AA-induced loss of cell viability was significantly suppressed by the presence of either inhibitor. In addition, we observed that the addition of catalase in the culture medium effectively sup" Fig. 3 D; pressed autophagosome formation in AA-treated cells (l Fig. S4, Supporting Information), suggesting that AA-induced autophagosome formation results from increased hydrogen peroxide formation and oxidative stress. It is known that Bcl-2 family proteins play an important role in regulating autophagosome formation [14, 20–22]. Next, we investigated the effect of AA on some of the Bcl-2 family proteins, Bax, Bcl-XL, and Bcl-2. The protein expression levels of Bax and Bcl-XL were not significantly changed by AA treatment (Fig. S5, Supporting Information), but Bcl-2 protein expression was strongly suppressed (Fig. S6 A, Supporting Information). It was reported that Bcl-2 can directly bind to Beclin 1 and maintains autophagy at a level that promotes cell survival but not cell death [20]. To investigate the mechanism by which AA triggers autophagy, we examined the binding between Bcl-2 and Beclin 1 in AA-treated cells using the immunocoprecipitation approach. After treatment with AA for 6 h, equal amounts of protein were prepared and immnocoprecipitated with the mouse anti-Beclin 1 antibody. The precipitated proteins were then subjected to Western blotting analysis using the rabbit anti-Beclin 1 and anti-Bcl-2 antibodies. As shown in Fig. S6 B (Supporting Information), Beclin 1 and Bcl-2 were found to interact with each other in the non-treated cells, but not in AA-treated cells. This observation is consistent with the very low levels of Bcl-2 protein detected in cells treated with AA for 12 h. Further, to confirm the role of Beclin 1 in AA-induced pancreatic cancer cell death, we used the Beclin 1-specific siRNAs (siBeclin 1) to knock down its expression. Beclin 1 protein expression was suppressed after transfection with siBeclin 1, but not with the control siRNA (siCon; Fig. S6 C, Supporting Information). Under these experimental conditions, we found that the AA-induced cell death was significantly suppressed in cells with Beclin 1 knockdown (Fig. S6 D, Supporting Information). These data indicate that Beclin 1 is involved in mediating AA-induced pancreatic cancer cell death via autophagosome formation. To shed light on the mechanism by which the treatment of cancer cells with AA results in a reduction in Bcl-2 protein levels, we hypothesized that the depletion of cellular ATP levels in AA-treated cells suppresses the mammalian target of the rapamycin (mTOR) pathway, which then leads to increased Bcl-2 degradation. To test this hypothesis, we determined the protein levels of the phosphorylated mTOR (the active form) in cells treated with AA (1 and 2 mM). The p-mTOR protein level was reduced by AA treat" Fig. 4 A). In addition, we found that co-treatment of cells ment (l with AA + rapamycin (an inhibitor of mTOR activity) enhanced " Fig. 4 B). Moreover, we also exthe anticancer activity of AA (l amined whether NF-κB was involved in regulating ascorbate-induced autophagy. No significant changes were found in the protein levels of either NF-κB or its inhibitor IKB (Fig. S7, Supporting Information), indicating that NF-κB most likely is not involved in this process. Together, these data suggest that the suppression of mTOR activity resulting from AA-induced ATP depletion is likely
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Fig. 3 AA induces autophagy in MIA-PaCa-2 cells. A MIA-PaCa-2 cells were treated with 1 mM AA for the indicated duration. Cell extracts were prepared and subjected to Western blotting of Beclin 1 and LC3B (I and II). Notably, the anti-LC3B antibody used in this study has a stronger reactivity with the LC3BII form than the LC3B–I form. The membrane was stripped and reprobed for GAPDH as a loading control. The left panel shows the representative Western blots for Beclin-1, LC3B, and β-actin, and the right panel shows the quantitative data from three experiments (mean ± SD) for the control and the 18-h treatment groups. *P < 0.05, **p < 0.01 compared to the vehicle treatment group. B MIA-PaCa2 cells were treated with 1 mM AA for 8 h. Then, cells were fixed and immunocytochemically stained with the anti-LC3B antibody. Formation of autophagosomes was examined under a fluorescence microscope. C MIA-PaCa-2 cells were pretreated with 5 µM chloroquine (CQ) or 25 nM bafiromycin (BF) for 2 h and then treated with 1 mM AA for an additional 24 h. Cell viability was analyzed using the MTT assay. Each value is the mean ± SD (N = 3); *p < 0.05. D MIA-PaCa-2 cells were treated with 1 mM AA for 8 h, and then the cells were fixed and analyzed for autophagosome formation using transmission electron microscopy (TEM). Experiments were repeated three times and representative results are shown.
an underlying cause for the reduction of cellular Bcl-2 protein levels in AA-treated cells. To evaluate the effects of AA in vivo, we used the growth of human pancreatic cancer cell xenografts in athymic nu/nu mice as a model. Each mouse was injected s. c. with the MIA-PaCa-2 cells (at 5 × 106 cells/100 µL PBS) in the left and right flanks. Two weeks later, the animals were randomly grouped, and received either the vehicle (i. p., 3 times per week) or AA (250 mg/kg BW, i. p., 3 times per week). No significant difference was observed in the animalsʼ body weight change between the control and AA treat" Fig. 5 A). Also, the amount of food intake between ment groups (l these two groups of mice was not significantly different through" Fig. 5 B). However, treatment with AA sigout the experiment (l nificantly suppressed tumor growth and tumor weight " Fig. 5 C, D). (l Histological examination (H/E staining) of the dissected tumor tissues revealed that the morphology and density of the tumor " Fig. 5 E; H/E staining). cells were not significantly different (l The cellular levels of 3-nitroso-cysteine, a commonly used marker for protein nitration and cellular oxidative stress, were determined in tumor xenografts as a parameter for intracellular ROS levels. We found that the 3-nitroso-cysteine level was signif-
icantly elevated in tumor tissues from mice treated with AA " Fig. 5 E; 3-nitroso-cysteine staining). Further, the number of (l LC3B positive cells was increased in tumor tissues from mice " Fig. 5 E; LC3B staining). These data indicate treated with AA (l that AA suppresses pancreatic cancer growth in vivo by promoting cancer cell death, likely resulting from increased oxidative stress and cell death plus the subsequent induction of autophagy.
Discussion !
The type of cell death seen in cultured cancer cells following exposure to high concentrations of AA remains controversial. While some of the studies reported that apoptotic cell death is induced by treatment with AA [15, 23–27], some other studies reported that the AA-induced cell death is a caspase-independent event [5, 28–33]. Recently, the role of autophagy in AA-induced cell death has also been suggested [5, 19, 34]. In this study, we sought to investigate the anticancer effect of AA in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as key mechanistic elements in its anticancer actions. A bet-
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Fig. 4 Role of mTOR in mediating AA-induced cell death. A MIA-PaCa-2 cells were treated with 1 or 2 mM AA for 6 h, and then whole-cell lysates were prepared for Western blotting of the p-mTOR protein level. β-Actin was determined as a loading control. The left panel shows the representative Western blots for p-mTOR and β-actin, and the right panel shows the quantitative data from three experiments (mean ± SD). *P < 0.05 compared to the vehicle treatment group. B MIA-PaCa-2 cells were treated for 48 h with 2 mM AA alone or in combination with 50 or 200 nM rapamycin, and cell viability was determined by the MTT assay. Each value is the mean ± SD (N = 3). *P < 0.05 compared to either AA or rapamycin treatment alone.
ter understanding of the mechanism of the anticancer actions of AA will aid in the development of new cancer chemotherapeutic strategies based on the use of this common nutritional compound. The results of this study showed that that AA can strongly suppress the growth of human pancreatic cancer cells both in vitro and in vivo, and that the growth suppression by AA is associated with the induction of oxidative stress and cell death. We also showed that the cell death induced by AA is abrogated by the presence of catalase, indicating that an increased formation of hydrogen peroxide from AA and the induction of oxidative stress play a critical role in cell death. These results are in accordance with earlier studies [5, 12]. Both caspase-dependent and caspase-independent mechanisms have been suggested in earlier studies for AA-induced cancer cell death. For example, it was reported that AA at pharmacological doses induces caspase-3 activation in gastric cancer cells [15]. On the other hand, it has been shown that AA can induce cell death in a caspase-independent mechanism in human breast cancer cells [24]. The results of our present study show that caspase-3 is not activated in AA-treated cells. Also, these cells lack characteristic apoptotic nuclear morphological changes. Consistent with these observations, the presence of the nonspecific caspase inhibitors does not have a significant effect on AA-induced cell death. In this study, we showed that the treatment of MIA-PaCa-2 cells induces autophagosome formation, based on both morphological
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and biochemical observations. The role of AA-induced autophagosome formation in modulating cell death is tested by selectively knocking down Beclin 1 expression or by using autophagy inhibitors. We found that either approach suppresses, to similar degrees, AA-induced cell death. These data suggest that the AAinduced autophagosome formation is part of the mechanism that causes death in treated cells. To understand the mechanism by which AA induces autophagosome formation in cancer cells, we found that treatment with AA results in the downregulation of Bcl-2 protein, which is an initiating event that subsequently leads to Beclin 1 upregulation and autophagy induction. The interaction between Beclin 1 and Bcl2 has been reported previously [20]. Bcl-2 can bind directly to Beclin 1 and maintain autophagy at a level that is favorable for cell survival rather than cell death. While Bcl-2 gene silencing increases starvation-induced autophagy, Bcl-2 transgenic expression reduces starvation-induced autophagy. In this study, data are presented to show that the AA-induced downregulation of the Bcl-2 protein level is related to the inhibition of the mTOR activity, resulting from the rapid depletion of cellular ATP following the induction of oxidative stress in AA-treated cells. Suppression of the mTOR activity is known to accelerate Bcl-2 degradation [35]. It is known that the induction of autophagy would lead to enhanced cell survival under many conditions, such as in response to nutrient deprivation, organelle damage, or other stresses. However, there are also reports suggesting that excessive or prolonged autophagy may cause cell death. For instance, embryonic fibroblasts from Bax/Bak double knockout mice are resistant to apoptosis, but they still undergo a non-apoptotic cell death after death stimulation. This form of cell death is thought to be associated with autophagic cell death [21, 35]. At present, the role of autophagy in mediating cell death remains controversial [36– 38]. Although more studies are needed, the observations made in this study provide an example for future study of the role of autophagy induction in mediating cell death under certain conditions. " Fig. 6), it is proposed that the exIn summary (as depicted in l tracellular AA generates hydrogen peroxide (H2O2), and subsequently it causes oxidative stress and rapid cellular ATP depletion. ATP depletion leads to the inhibition of the mTOR activity, which is known to increase Bcl-2 degradation. A reduction in cellular Bcl-2 levels would lead to Beclin 1 accumulation, which then mediates the induction of autophagosome formation. Increased autophagy observed in AA-treated cancer cells is part of the mechanism that contributes to AA-induced cell death. Selective knockdown of Beclin 1 protein levels or treatment with pharmacological autophagy inhibitors each inhibit AA-induced cell death in cultured MIA-PaCa-2 human pancreatic cancer cells.
Materials and Methods !
Chemicals Sodium ascorbate (≥ 98 % purity), trolox (97% purity), chloroquine diphosphate salt (abbreviated as chloroquine, ≥ 98 % purity), bafilomycin A1 (≥ 90% purity, based on HPLC analysis), paclitaxel (≥ 95 % purity), and fetal bovine serum (FBS) were obtained from Sigma-Aldrich Chemical Co. The antibiotics solution (containing 10 000 U/mL penicillin and 10 mg/mL streptomycin) was obtained from Invitrogen. All other reagents used in this study
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Fig. 5 Effect of AA on the growth of MIA-PaCa-2 cells in athymic nude mice. Panels A–D show the changes in body weight (A), food intake (B), tumor volume (C), and tumor weight (D), and panel E shows the H/E staining and the quantitative analysis of cells immunohistochemically stained positive for 3-nitroso-cysteine and LC3B. Nude mice were maintained for 5 weeks following subcutaneous inoculation of the MIA-PaCa-2 cells (5 × 106 cells in 100 µL PBS). Two weeks after inoculation, the mice started to receive ascorbate treatment: Group 1 received the vehicle treatment (i. p., 3 times a week, N = 6) and Group 2 received the sodium ascorbate treatment (at 250 mg/kg BW, i. p., 3 times a week, N = 6). Tumor size, body weight, and food intake amount were measured twice a week. Tumor volume was calculated assuming the tumor to be spherical according to the formula volume = π/6 × d3, where d is the mean diameter. At the end of the experiment, each tumor was removed, trimmed, and weighed. Tumor samples obtained from each animal were processed for regular H/E staining as well as for analysis of 3-nitroso-cysteine and LC3B positivity as described in the Materials and Methods section.
were obtained from standard suppliers and were of analytical grade or higher.
Cell culture conditions and assay of cell viability MIA-PaCa-2 human pancreatic cancer cells were obtained from the American Type Culture Collection (ATCC). They were maintained in DMEM supplemented with 10% FBS and 3.024 g/L NaHCO3, and incubated at 37 °C under 5 % CO2. Cells were subcultured every 3 to 4 days. The cells were seeded in 96-well plates at a density of 5000 cells per well. A stock solution of AA (at 1 M, dissolved in PBS) was diluted in the culture medium immediately before the addition to each well, and the treatment usually lasted for 24 h. For the initial assessment of gross cell viability, the MTT assay was used, as described in our earlier study [39].
Small interfering ribonucleic acid (siRNA) To study the role of Beclin 1 in AA-induced pancreatic cancer cell death, Beclin 1-siRNA (Santa Cruz) was used to selectively knock down the expression of Beclin 1 in MIA-PaCa-2 cells. Cells were seeded 24 h before transfection and reached a density of 30– 50 % confluence at the time of transfection. Then, Beclin 1-siRNA and the negative control-siRNA were used for transfection using Lipofectamine 2000, according to the manufactureʼs instruction. The transfected cells were maintained in culture for 24 h before harvesting and further analyses. The efficacy of the siRNA knockdown of target protein expression was determined by Western blot analysis with a specific antibody.
Immunoprecipitation The total cell lysates were incubated for 1 h at 4 °C with mouse anti-Beclin B1 antibody. The immunocomplex was collected on protein A-Sepharose beads (Sigma) for 1 h and washed five times with the TNN buffer (containing 40 mM Tris-HCl, 120 mM NaCl, 0.5 % NP-40, 0.1 mM sodium orthovanadate, 2 µg/mL aprotinin, 2 µg/mL leupeptin, and 100 µg/mL phenymethylsulfonyl fluoride, pH 8.0) prior to boiling in the SDS buffer. Immunoprecipitated proteins were separated on SDS-polyacrylamide gels, transferred to a polyvinylidene difluoride membrane (Bio-Rad), and analyzed using Western immunoblotting.
Measurement of reactive oxygen species Reactive oxygen species (ROS) were detected using the 2′,7′-dichlorofluorescin diacetate (H2-DCF-DA) method. Cells were first cultured in a 96-well plate and treated with 1 mM AA for 8 h. After the cells were washed with PBS five times, 10 µM H2-DCFDA were added to each well. The intracellular ROS accumulation was observed and photographed with a fluorescence microscope (AXIO, Carl Zeiss Corporation). Here it is of note that since H2DCF-DA basically does not react with hydrogen peroxide, these observations were mainly used to reflect the formation of other intracellular ROS species.
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Immunocytochemical analysis Immunofluorescence staining was done in cells cultured in chamber slides. Briefly, after AA treatment, cells were fixed in 2 % formaldehyde and incubated in ice-cold 100% methanol. Then, the cells were blocked with 5 % normal goat serum and incubated with rabbit anti-LC3B antibody (Cell Signaling). Then, the cells were incubated with FITC-conjugated goat anti-rabbit antibody (Vector Laboratories, Inc.). Subsequently, nuclei were stained with Hoechst-33342 and examined under a fluorescence microscope.
Western blotting For Western blotting, cells were washed first, and then were suspended in 100 µL lysis buffer (containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 v/v, 10 mM NaF, 2 mM Na3VO4, and a protease inhibitor cocktail, pH 7.5). The amount of proteins was determined using the Bio-Rad protein assay (Bio-Rad). After an equal amount of protein was loaded in each lane, they were separated by 10% SDS w/v-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrically transferred to a polyvinylidene difluoride membrane (Bio-Rad). After blocking the membrane with 5 % skim milk w/v, target proteins were immunodetected using specific antibodies (from Cell Signaling Technology). Thereafter, the horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Invitrogen) was applied as the secondary antibody, and the positive bands were detected using Amersham ECL Plus Western blotting detection reagents (GE Healthcare).
Analysis of cells by transmission electron microscopy Fig. 6 Schematic illustration of the mechanism of AA-induced formation of autophagosomes and their role in AA-induced cell death in pancreatic cancer.
Cellular adenosine triphosphate detection by high-performance liquid chromatography As high concentrations of ascorbate may interfere with the commonly used luciferase-based ATP assays, high-performance liquid chromatography (HPLC) was utilized for the detection of ATP. Briefly, cells in a six-well plate (2 × 105 cells/well) were treated with ascorbate for the indicated length, and then the cells were harvested using a rubber scraper, washed, and resuspended in PBS. ATP was extracted by quickly lysing the cells in a 0.05 mM potassium hydroxide solution, and the lysate was immediately neutralized to pH 6 with 0.1 mM KH2PO4. After centrifugation, the supernatant was analyzed using a gradient method on a Waters E2695 HPLC with ultraviolet detection set at 254 and 340 nm (Waters 2489 diode array UV detector; Waters). Reversed-phase chromatography was performed with an XBridgeTM C18 column 3.5 µm (Waters). The mobile phase (pH 6) contained acetonitrile (2% for solvent A and 30% for solvent B), 0.1 M KH2PO4, and 8 mM tetrabutylammonium hydrogen sulfate. The fractions of solvent A to solvent B at 0, 4, 7, 12, 15, and 22 min were 100/0, 90/10, 80/20, 60/40, 0/100, and 100/0, respectively. Waters Empower II software was used for instrument control and data analysis. All values were normalized to the protein content of the whole-cell lyate using the bicinchoninic acid method (Pierce Biotechnology) [19].
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Cells were harvested using trypsin-EDTA and fixed in 2 % glutaraldehyde for 4 h, and centrifuged to form pellets. A sample preparation was carried out according to the previous method [39– 41]. Briefly, the pellets were rinsed in 0.1 M cacodylate buffer [purchased from Electron Microscopy Sciences (EMS) and postfixed in 1% osmium tetroxide (EMS)]. Cell pellets were dehydrated through a graded series of ethanol and then passed through a propylene oxide twice and, lastly, placed in propylene oxide/Embed 812 resin (EMS) overnight for infiltration, and then polymerized in a 60 °C oven overnight. Then sections were cut on a Leica UCT ultra microtome at 80 nm using a Diatome diamond knife. Sections were contrasted with uranyl acetate and Satoʼs lead citrate (EMS), and viewed and photographed on a JEOL 100CXII TEM at 60 KV (J. E. O. L. Ltd.).
Growth of human pancreatic cancer cell xenografts in athymic nude mice All animal use procedures were approved on January 25, 2010 (protocol # 2009–1841) by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center, and the investigators followed the NIH guidelines for the humane treatment of animals. Female athymic nu/nu mice, 4–5 weeks of age, were obtained from Harlan Laboratories. They were exposed to a 12-h light/12-h dark cycle, and had free access to Harlan Teklad Rodent Diet 8604 (Harlan Teklad) and water. Mice were housed under aseptic conditions (positive air pressure in a designated mouse room, with microisolator tops) and all mouse-handling procedures were carried out under a laminar flow hood. MIA-PaCa-2 cells (5 × 106 cells/100 µL PBS) were injected s. c. into the right and left flanks of the mice. Two weeks after inoculation, the mice were randomly grouped according to body weight and tumor size, and started to receive vehicle or ascorbate treatment: Group 1 received the vehicle treatment (i. p., 3 times a week,
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Morphological and histopathological analyses Tumor samples from each mouse were fixed in 10 % buffered formalin phosphate (Fisher Scientific), dehydrated, embedded in paraffin, sectioned in 5-µm thickness, and stained with hematoxylin and eosin (H/E). For immunohistochemical staining of the 3nitroso-cysteine and LC3B, the antigen retrieval procedure was performed by placing the slides in 10 mM citrate buffer (pH 3.0), heating in a microwave oven for 20 min, and then allowing the slides to cool to room temperature for 20 min. Slides were rinsed once with PBS, and endogenous peroxidase activity was blocked by incubating the samples for 30 min with 3% H2O2 in PBS, followed by rinsing threee times with PBS. Nonspecific binding was blocked by incubating the slides for 30 min in 2% normal goat serum (Vector Laboratories) in 1% Triton X-100 containing PBS, followed by incubation with the specific antibodies against 3-nitroso-cysteine (1 : 200 dilution, Sigma) and LC3B (1 : 400 dilution, Cell Signaling Technology, Inc.) in the blocking solution as described above. Then, slides were incubated with biotinylated goat anti-rabbit IgG (dilution, 1 : 500, Vector Laboratories) in the blocking solution as described above. Next, the slides were incubated for 2 h with the avidin-biotin peroxidase complex (Vector Laboratories) according to the manufacturerʼs instructions, followed by a 5-min incubation with a DAB substrate kit (Vector Laboratories). Counterstaining was performed using Mayerʼs hematoxylin. Negative controls lacking the primary antibody were used for each staining.
Reproducibility of experiments and statistical analysis For the in vitro cell culture study, each experiment was repeated at least three times. The data were presented as the mean ± SD of multiple independent experiments. For the in vivo animal study, we have obtained similar results from two independent experiments, and only one set of the representative data was shown. Statistical significance was analyzed using one-way ANOVA and Dunnettʼs test (SPSS software). A p value of less than 0.05 was considered statistically significant.
Supporting information Because of space constraint, some of the results are shown in Figs. S1, S2, S3, S4, S5, S6, and S7.
Acknowledgements !
This work was supported, in part, by a grant from the National Institutes of Health (Grant No. CA97109).
Conflict of Interest !
The authors declare no conflict of interest.
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Mechanism of Ascorbate-Induced Cell Death in Human Pancreatic Cancer Cells: Role of Bcl-2, Beclin 1 and Autophagy
Authors
Masayuki Fukui 1, 2, Noriko Yamabe 1, Hye-Joung Choi 1, Kishore Polireddy 1, Qi Chen 1, Bao Ting Zhu 1, 3
Affiliations
1
2 3
Key words " ascorbate l " pancreatic cancer l " cell death l " autophagy l
Department of Pharmacology, Toxicology and Therapeutics, School of Medicine, University of Kansas Medical Center, Kansas City, KS, USA Department of Microbiology, Tropical Biosphere Research Center, University of the Ryukyus, Nishihara, Okinawa, Japan Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, China
Abstract
some formation contributes to ascorbate-induced pancreatic cancer cell death.
!
The present study investigates the anticancer effect of ascorbate in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as important mechanistic elements in its anticancer actions. We showed that ascorbate suppresses the growth of human pancreatic cancer cells via the induction of oxidative stress and caspase-independent cell death. Ascorbate induces the formation of autophagosomes and the presence of autophagy inhibitors suppresses ascorbate-induced cell death. These data suggest that the induction of autophago-
Introduction !
received revised accepted
February 11, 2015 April 18, 2015 May 3, 2015
Bibliography DOI http://dx.doi.org/ 10.1055/s-0035-1546132 Published online July 1, 2015 Planta Med 2015; 81: 838–846 © Georg Thieme Verlag KG Stuttgart · New York · ISSN 0032‑0943 Correspondence Bao Ting Zhu South University of Science and Technology of China Department of Biology 1088 Xueyuan Road, Xili, Nanshan District Shenzhen, Guangdong, 518055 China Phone: + 86 7 55 88 01 84 16 Fax: + 86 7 55 88 01 84 17 [email protected]
The use of dietary agents has been considered an alternative approach in the prevention and treatment of human cancers. Epidemiological studies have shown that a diet rich in fruits and vegetables is associated with a low incidence of many types of human malignancies, including pancreatic cancer [1, 2]. AA, commonly known as vitamin C and an essential nutrient in human diet, not only serves as a cofactor in many metabolic reactions, but it also serves as a strong antioxidant that protects cells against oxidative stress and damage [3–5]. However, AA may also serve as a prooxidant under certain conditions [4, 5]. Studies have shown that while the plasma and tissue levels of AA are usually maintained within a well-controlled range (under 0.2 mM) following oral administration, its effective anticancer concentrations (at 0.2–10 mM range) are achievable following parenteral (i. v. or i. p.) administrations [6–8]. Some previous studies showed that the production of hydrogen peroxide (H2O2) with pharmacological doses of AA is an important contributor to its anticancer activity both in vitro [5]
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Abbreviations !
AA: ERK: JNK: MAPK: ROS:
ascorbate extracellular signaling-regulated regulated protein kinase c-jun N-terminal kinase mitogen-activated protein kinase reactive oxygen species
Supporting information available online at http://www.thieme-connect.de/products
and in vivo [9]. Moreover, it was observed that the presence of other antioxidants could diminish the anticancer activity of AA in cancer cells, whereas the presence of prooxidants can enhance its anticancer activity [10, 11]. The cytotoxic activity of AA appears to be predominantly seen in cancer cells, but not in normal cells [9, 12]. The exact type of cell death seen in cultured cancer cells following exposure to high concentrations of AA remains controversial [13–25]. In this study, we sought to investigate the anticancer effect of AA in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as important mechanistic elements in its anticancer actions.
Results !
First, we confirmed the role of AA-mediated formation of extracellular hydrogen peroxide in its anticancer activity [5, 12]. We showed that AA induced cell death in a concentration-dependent manner in cultured MIA-PaCa-2 cells, and the co-
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Fig. 1 AA-induced cell death in MIA-PaCa-2 cells is mediated by hydrogen peroxide, but independent of MAPK activation. A MIA-PaCa-2 cells were treated with AA at indicated concentrations with or without 100 µg/mL catalase. After 24 h incubation, cell viability was analyzed using the MTT assay. Each value is the mean ± SD (N = 3). *P < 0.05. B MIA-PaCa-2 cells were treated with 1 mM AA in the presence or absence of 100 µg/mL catalase or 1 mM trolox for 8 h, and then analyzed for intracellular ROS accumulation using the H2-DCF‑DA staining method as described in Materials and Methods. The experiments were repeated three times and representative results are shown. The size bar in each image represents 50 µm in length. C MIA-PaCa-2 cells were pretreated with 10 µM SP600125 (SP is a JNK pathway inhibitor), 10 µM PD98059 (PD is an ERK pathway inhibitor), or 10 µM SB202190 (SB is a p38 pathway inhibitor) for 2 h and then treated with 2 mM AA for an additional 24 h. The cell viability was determined using the MTT assay. Each value is the mean ± SD (N = 3). In each panel, the filled columns are without an inhibitor, and the open columns are with an inhibitor.
presence of catalase or trolox (water-soluble derivatives of vitamin E) strongly protected the cells against AA-induced cell death " Fig. 1 A; Fig. S1, Supporting Information). In addition, we (l showed that while there was no appreciable accumulation of intracellular ROS in vehicle-treated cells, a significant accumulation was detected in AA-treated cells, and the presence of catalase or trolox almost completely abolished the intracellular accumula" Fig. 1 B). tion of ROS (l It is known that oxidative stress can cause the activation of the MAPKs, i.e., JNK, ERK, and p38-MAPK, and, subsequently, results in caspase activation and apoptotic cell death [13, 14]. Han et al. reported that p38-MAPK, but not JNK or ERK, mediates AA-induced cytotoxicity in human gastric cancer cells [15]. In this study, therefore, we examined whether MAPK signaling pathways are involved in AA-induced cell death in MIA-PaCa-2 cells. Cells were treated with AA in the presence or absence of the inhibitors of the MAPKs (i.e., SP600125 for JNK1/2, PD98059 for " Fig. 1 C, MEK1/2, and SB202190 for p38-MAPK). As shown in l these inhibitors (at 10 µM) did not have a significant effect on AA-induced cell death in cultured pancreatic cancer cells.
To determine whether the induction of apoptosis is an important contributor to AA-induced anticancer activity, we examined capsase-3 activation and nuclear morphological changes in cells treated with AA. Based on measuring the cleaved caspase-3 by Western blotting, we found that caspase-3 activation was not detected in AA-treated MIA-PaCa-2 cells, but the treatment of these cells with paclitaxel (PTX) clearly induced caspase-3 cleavage " Fig. 2 A). Further, as shown in l " Fig. 2 B, while typical apoptot(l ic nuclear morphological changes were readily observed in cells treated with PTX, these changes were essentially absent in cells treated with AA. Moreover, when cells were treated with AA in the presence or absence of z-VAD‑fmk (a pan-caspase inhibitor), changes in cell viability in AA-treated pancreatic cancer cells were not significantly altered (Fig. S2, Supporting Information). Together, these data indicate that AA-induced pancreatic cancer cell death is independent of caspase activation. Because caspase activation is known to be an ATP-dependent process [16–18], the lack of caspase activation in AA-treated cells is likely due to the depletion of the cellular ATP level resulting from rapid ROS accumulation. In support of this possibility, we
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Fig. 2 AA-induced cell death in MIA-PaCa-2 cells is independent of caspase activation. A MIA-PaCa-2 cells were treated with 1 mM AA for the indicated lengths of time or with 20 nM paclitaxel (PTX) for 24 h. Cell extracts were prepared and subjected to Western blotting of caspase-3. The experiments were repeated three times and representative results are shown. B MIA-PaCa-2 cells were treated with 1 mM AA or 20 nM PTX for 24 h and stained with Hoechst33342. Nuclear morphological changes were photographed under a fluorescence microscope. C MIA-PaCa-2 cells were treated with 2 mM AA for the indicated lengths of time, and then the cellular ATP levels were determined as described in Materials and Methods. Each value is the mean ± SD (N = 3). *P < 0.05 compared to the control (0 h).
observed that the cellular ATP level in AA-treated cells was rap" Fig. 2 C), which is idly depleted in a time-dependent manner (l also consistent with earlier observations [5, 19]. Notably, the ATP level in CCD-34SK fibroblasts (a non-tumor cell line) was basically not changed following treatment with 5 mM of ascorbate for 1 and 4 h (Fig. S3, Supporting Information). To determine whether AA treatment induces autophagosome formation, we examined the changes in Beclin 1, LC3B–I, and LC3B-II protein levels following the treatment of cultured MIA" Fig. 3 A, there was no signifiPaCa-2 cells with AA. As shown in l cant change in the Beclin 1 level after AA treatment, but the expression of LC3B–I was decreased, and this decrease was accompanied by an increase in LC3B-II. To further investigate the effect of AA on autophagosome formation, we performed immunocytochemical staining of AA-treated cells using the same anti-LC3B antibody. After an 8-h AA treatment, intracellular LC3B accumu" Fig. 3 B). When lation and punctuated staining were observed (l the percentage of LC3B positive cells with a punctuated staining pattern was quantitated, we found that the percentages of positive cells treated with the vehicle and AA were 6.4 ± 0.3 and 27.6 ± 9.5, respectively (p < 0.05). The observation of an increased cell population with positive LC3B staining following AA treatment was further confirmed by detecting the formation of auto-
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phagosomes in AA-treated cells using transmission electron mi" Fig. 3 B). croscopy (l To determine the functional role of autophagosome formation in AA-induced cell death, we examined the effect of two commonly used autophagy inhibitors, namely, chloroquine (an inhibitor of endosomal acidication) and bafilomycin A1 (an inhibitor of the " Fig. 3 C, the formation of autophagic vacuoles). As shown in l AA-induced loss of cell viability was significantly suppressed by the presence of either inhibitor. In addition, we observed that the addition of catalase in the culture medium effectively sup" Fig. 3 D; pressed autophagosome formation in AA-treated cells (l Fig. S4, Supporting Information), suggesting that AA-induced autophagosome formation results from increased hydrogen peroxide formation and oxidative stress. It is known that Bcl-2 family proteins play an important role in regulating autophagosome formation [14, 20–22]. Next, we investigated the effect of AA on some of the Bcl-2 family proteins, Bax, Bcl-XL, and Bcl-2. The protein expression levels of Bax and Bcl-XL were not significantly changed by AA treatment (Fig. S5, Supporting Information), but Bcl-2 protein expression was strongly suppressed (Fig. S6 A, Supporting Information). It was reported that Bcl-2 can directly bind to Beclin 1 and maintains autophagy at a level that promotes cell survival but not cell death [20]. To investigate the mechanism by which AA triggers autophagy, we examined the binding between Bcl-2 and Beclin 1 in AA-treated cells using the immunocoprecipitation approach. After treatment with AA for 6 h, equal amounts of protein were prepared and immnocoprecipitated with the mouse anti-Beclin 1 antibody. The precipitated proteins were then subjected to Western blotting analysis using the rabbit anti-Beclin 1 and anti-Bcl-2 antibodies. As shown in Fig. S6 B (Supporting Information), Beclin 1 and Bcl-2 were found to interact with each other in the non-treated cells, but not in AA-treated cells. This observation is consistent with the very low levels of Bcl-2 protein detected in cells treated with AA for 12 h. Further, to confirm the role of Beclin 1 in AA-induced pancreatic cancer cell death, we used the Beclin 1-specific siRNAs (siBeclin 1) to knock down its expression. Beclin 1 protein expression was suppressed after transfection with siBeclin 1, but not with the control siRNA (siCon; Fig. S6 C, Supporting Information). Under these experimental conditions, we found that the AA-induced cell death was significantly suppressed in cells with Beclin 1 knockdown (Fig. S6 D, Supporting Information). These data indicate that Beclin 1 is involved in mediating AA-induced pancreatic cancer cell death via autophagosome formation. To shed light on the mechanism by which the treatment of cancer cells with AA results in a reduction in Bcl-2 protein levels, we hypothesized that the depletion of cellular ATP levels in AA-treated cells suppresses the mammalian target of the rapamycin (mTOR) pathway, which then leads to increased Bcl-2 degradation. To test this hypothesis, we determined the protein levels of the phosphorylated mTOR (the active form) in cells treated with AA (1 and 2 mM). The p-mTOR protein level was reduced by AA treat" Fig. 4 A). In addition, we found that co-treatment of cells ment (l with AA + rapamycin (an inhibitor of mTOR activity) enhanced " Fig. 4 B). Moreover, we also exthe anticancer activity of AA (l amined whether NF-κB was involved in regulating ascorbate-induced autophagy. No significant changes were found in the protein levels of either NF-κB or its inhibitor IKB (Fig. S7, Supporting Information), indicating that NF-κB most likely is not involved in this process. Together, these data suggest that the suppression of mTOR activity resulting from AA-induced ATP depletion is likely
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Fig. 3 AA induces autophagy in MIA-PaCa-2 cells. A MIA-PaCa-2 cells were treated with 1 mM AA for the indicated duration. Cell extracts were prepared and subjected to Western blotting of Beclin 1 and LC3B (I and II). Notably, the anti-LC3B antibody used in this study has a stronger reactivity with the LC3BII form than the LC3B–I form. The membrane was stripped and reprobed for GAPDH as a loading control. The left panel shows the representative Western blots for Beclin-1, LC3B, and β-actin, and the right panel shows the quantitative data from three experiments (mean ± SD) for the control and the 18-h treatment groups. *P < 0.05, **p < 0.01 compared to the vehicle treatment group. B MIA-PaCa2 cells were treated with 1 mM AA for 8 h. Then, cells were fixed and immunocytochemically stained with the anti-LC3B antibody. Formation of autophagosomes was examined under a fluorescence microscope. C MIA-PaCa-2 cells were pretreated with 5 µM chloroquine (CQ) or 25 nM bafiromycin (BF) for 2 h and then treated with 1 mM AA for an additional 24 h. Cell viability was analyzed using the MTT assay. Each value is the mean ± SD (N = 3); *p < 0.05. D MIA-PaCa-2 cells were treated with 1 mM AA for 8 h, and then the cells were fixed and analyzed for autophagosome formation using transmission electron microscopy (TEM). Experiments were repeated three times and representative results are shown.
an underlying cause for the reduction of cellular Bcl-2 protein levels in AA-treated cells. To evaluate the effects of AA in vivo, we used the growth of human pancreatic cancer cell xenografts in athymic nu/nu mice as a model. Each mouse was injected s. c. with the MIA-PaCa-2 cells (at 5 × 106 cells/100 µL PBS) in the left and right flanks. Two weeks later, the animals were randomly grouped, and received either the vehicle (i. p., 3 times per week) or AA (250 mg/kg BW, i. p., 3 times per week). No significant difference was observed in the animalsʼ body weight change between the control and AA treat" Fig. 5 A). Also, the amount of food intake between ment groups (l these two groups of mice was not significantly different through" Fig. 5 B). However, treatment with AA sigout the experiment (l nificantly suppressed tumor growth and tumor weight " Fig. 5 C, D). (l Histological examination (H/E staining) of the dissected tumor tissues revealed that the morphology and density of the tumor " Fig. 5 E; H/E staining). cells were not significantly different (l The cellular levels of 3-nitroso-cysteine, a commonly used marker for protein nitration and cellular oxidative stress, were determined in tumor xenografts as a parameter for intracellular ROS levels. We found that the 3-nitroso-cysteine level was signif-
icantly elevated in tumor tissues from mice treated with AA " Fig. 5 E; 3-nitroso-cysteine staining). Further, the number of (l LC3B positive cells was increased in tumor tissues from mice " Fig. 5 E; LC3B staining). These data indicate treated with AA (l that AA suppresses pancreatic cancer growth in vivo by promoting cancer cell death, likely resulting from increased oxidative stress and cell death plus the subsequent induction of autophagy.
Discussion !
The type of cell death seen in cultured cancer cells following exposure to high concentrations of AA remains controversial. While some of the studies reported that apoptotic cell death is induced by treatment with AA [15, 23–27], some other studies reported that the AA-induced cell death is a caspase-independent event [5, 28–33]. Recently, the role of autophagy in AA-induced cell death has also been suggested [5, 19, 34]. In this study, we sought to investigate the anticancer effect of AA in MIA-PaCa-2 human pancreatic cancer cells using both in vitro and in vivo models, with a focus on assessing the role of oxidative stress and autophagy as key mechanistic elements in its anticancer actions. A bet-
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Fig. 4 Role of mTOR in mediating AA-induced cell death. A MIA-PaCa-2 cells were treated with 1 or 2 mM AA for 6 h, and then whole-cell lysates were prepared for Western blotting of the p-mTOR protein level. β-Actin was determined as a loading control. The left panel shows the representative Western blots for p-mTOR and β-actin, and the right panel shows the quantitative data from three experiments (mean ± SD). *P < 0.05 compared to the vehicle treatment group. B MIA-PaCa-2 cells were treated for 48 h with 2 mM AA alone or in combination with 50 or 200 nM rapamycin, and cell viability was determined by the MTT assay. Each value is the mean ± SD (N = 3). *P < 0.05 compared to either AA or rapamycin treatment alone.
ter understanding of the mechanism of the anticancer actions of AA will aid in the development of new cancer chemotherapeutic strategies based on the use of this common nutritional compound. The results of this study showed that that AA can strongly suppress the growth of human pancreatic cancer cells both in vitro and in vivo, and that the growth suppression by AA is associated with the induction of oxidative stress and cell death. We also showed that the cell death induced by AA is abrogated by the presence of catalase, indicating that an increased formation of hydrogen peroxide from AA and the induction of oxidative stress play a critical role in cell death. These results are in accordance with earlier studies [5, 12]. Both caspase-dependent and caspase-independent mechanisms have been suggested in earlier studies for AA-induced cancer cell death. For example, it was reported that AA at pharmacological doses induces caspase-3 activation in gastric cancer cells [15]. On the other hand, it has been shown that AA can induce cell death in a caspase-independent mechanism in human breast cancer cells [24]. The results of our present study show that caspase-3 is not activated in AA-treated cells. Also, these cells lack characteristic apoptotic nuclear morphological changes. Consistent with these observations, the presence of the nonspecific caspase inhibitors does not have a significant effect on AA-induced cell death. In this study, we showed that the treatment of MIA-PaCa-2 cells induces autophagosome formation, based on both morphological
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and biochemical observations. The role of AA-induced autophagosome formation in modulating cell death is tested by selectively knocking down Beclin 1 expression or by using autophagy inhibitors. We found that either approach suppresses, to similar degrees, AA-induced cell death. These data suggest that the AAinduced autophagosome formation is part of the mechanism that causes death in treated cells. To understand the mechanism by which AA induces autophagosome formation in cancer cells, we found that treatment with AA results in the downregulation of Bcl-2 protein, which is an initiating event that subsequently leads to Beclin 1 upregulation and autophagy induction. The interaction between Beclin 1 and Bcl2 has been reported previously [20]. Bcl-2 can bind directly to Beclin 1 and maintain autophagy at a level that is favorable for cell survival rather than cell death. While Bcl-2 gene silencing increases starvation-induced autophagy, Bcl-2 transgenic expression reduces starvation-induced autophagy. In this study, data are presented to show that the AA-induced downregulation of the Bcl-2 protein level is related to the inhibition of the mTOR activity, resulting from the rapid depletion of cellular ATP following the induction of oxidative stress in AA-treated cells. Suppression of the mTOR activity is known to accelerate Bcl-2 degradation [35]. It is known that the induction of autophagy would lead to enhanced cell survival under many conditions, such as in response to nutrient deprivation, organelle damage, or other stresses. However, there are also reports suggesting that excessive or prolonged autophagy may cause cell death. For instance, embryonic fibroblasts from Bax/Bak double knockout mice are resistant to apoptosis, but they still undergo a non-apoptotic cell death after death stimulation. This form of cell death is thought to be associated with autophagic cell death [21, 35]. At present, the role of autophagy in mediating cell death remains controversial [36– 38]. Although more studies are needed, the observations made in this study provide an example for future study of the role of autophagy induction in mediating cell death under certain conditions. " Fig. 6), it is proposed that the exIn summary (as depicted in l tracellular AA generates hydrogen peroxide (H2O2), and subsequently it causes oxidative stress and rapid cellular ATP depletion. ATP depletion leads to the inhibition of the mTOR activity, which is known to increase Bcl-2 degradation. A reduction in cellular Bcl-2 levels would lead to Beclin 1 accumulation, which then mediates the induction of autophagosome formation. Increased autophagy observed in AA-treated cancer cells is part of the mechanism that contributes to AA-induced cell death. Selective knockdown of Beclin 1 protein levels or treatment with pharmacological autophagy inhibitors each inhibit AA-induced cell death in cultured MIA-PaCa-2 human pancreatic cancer cells.
Materials and Methods !
Chemicals Sodium ascorbate (≥ 98 % purity), trolox (97% purity), chloroquine diphosphate salt (abbreviated as chloroquine, ≥ 98 % purity), bafilomycin A1 (≥ 90% purity, based on HPLC analysis), paclitaxel (≥ 95 % purity), and fetal bovine serum (FBS) were obtained from Sigma-Aldrich Chemical Co. The antibiotics solution (containing 10 000 U/mL penicillin and 10 mg/mL streptomycin) was obtained from Invitrogen. All other reagents used in this study
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Fig. 5 Effect of AA on the growth of MIA-PaCa-2 cells in athymic nude mice. Panels A–D show the changes in body weight (A), food intake (B), tumor volume (C), and tumor weight (D), and panel E shows the H/E staining and the quantitative analysis of cells immunohistochemically stained positive for 3-nitroso-cysteine and LC3B. Nude mice were maintained for 5 weeks following subcutaneous inoculation of the MIA-PaCa-2 cells (5 × 106 cells in 100 µL PBS). Two weeks after inoculation, the mice started to receive ascorbate treatment: Group 1 received the vehicle treatment (i. p., 3 times a week, N = 6) and Group 2 received the sodium ascorbate treatment (at 250 mg/kg BW, i. p., 3 times a week, N = 6). Tumor size, body weight, and food intake amount were measured twice a week. Tumor volume was calculated assuming the tumor to be spherical according to the formula volume = π/6 × d3, where d is the mean diameter. At the end of the experiment, each tumor was removed, trimmed, and weighed. Tumor samples obtained from each animal were processed for regular H/E staining as well as for analysis of 3-nitroso-cysteine and LC3B positivity as described in the Materials and Methods section.
were obtained from standard suppliers and were of analytical grade or higher.
Cell culture conditions and assay of cell viability MIA-PaCa-2 human pancreatic cancer cells were obtained from the American Type Culture Collection (ATCC). They were maintained in DMEM supplemented with 10% FBS and 3.024 g/L NaHCO3, and incubated at 37 °C under 5 % CO2. Cells were subcultured every 3 to 4 days. The cells were seeded in 96-well plates at a density of 5000 cells per well. A stock solution of AA (at 1 M, dissolved in PBS) was diluted in the culture medium immediately before the addition to each well, and the treatment usually lasted for 24 h. For the initial assessment of gross cell viability, the MTT assay was used, as described in our earlier study [39].
Small interfering ribonucleic acid (siRNA) To study the role of Beclin 1 in AA-induced pancreatic cancer cell death, Beclin 1-siRNA (Santa Cruz) was used to selectively knock down the expression of Beclin 1 in MIA-PaCa-2 cells. Cells were seeded 24 h before transfection and reached a density of 30– 50 % confluence at the time of transfection. Then, Beclin 1-siRNA and the negative control-siRNA were used for transfection using Lipofectamine 2000, according to the manufactureʼs instruction. The transfected cells were maintained in culture for 24 h before harvesting and further analyses. The efficacy of the siRNA knockdown of target protein expression was determined by Western blot analysis with a specific antibody.
Immunoprecipitation The total cell lysates were incubated for 1 h at 4 °C with mouse anti-Beclin B1 antibody. The immunocomplex was collected on protein A-Sepharose beads (Sigma) for 1 h and washed five times with the TNN buffer (containing 40 mM Tris-HCl, 120 mM NaCl, 0.5 % NP-40, 0.1 mM sodium orthovanadate, 2 µg/mL aprotinin, 2 µg/mL leupeptin, and 100 µg/mL phenymethylsulfonyl fluoride, pH 8.0) prior to boiling in the SDS buffer. Immunoprecipitated proteins were separated on SDS-polyacrylamide gels, transferred to a polyvinylidene difluoride membrane (Bio-Rad), and analyzed using Western immunoblotting.
Measurement of reactive oxygen species Reactive oxygen species (ROS) were detected using the 2′,7′-dichlorofluorescin diacetate (H2-DCF-DA) method. Cells were first cultured in a 96-well plate and treated with 1 mM AA for 8 h. After the cells were washed with PBS five times, 10 µM H2-DCFDA were added to each well. The intracellular ROS accumulation was observed and photographed with a fluorescence microscope (AXIO, Carl Zeiss Corporation). Here it is of note that since H2DCF-DA basically does not react with hydrogen peroxide, these observations were mainly used to reflect the formation of other intracellular ROS species.
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Immunocytochemical analysis Immunofluorescence staining was done in cells cultured in chamber slides. Briefly, after AA treatment, cells were fixed in 2 % formaldehyde and incubated in ice-cold 100% methanol. Then, the cells were blocked with 5 % normal goat serum and incubated with rabbit anti-LC3B antibody (Cell Signaling). Then, the cells were incubated with FITC-conjugated goat anti-rabbit antibody (Vector Laboratories, Inc.). Subsequently, nuclei were stained with Hoechst-33342 and examined under a fluorescence microscope.
Western blotting For Western blotting, cells were washed first, and then were suspended in 100 µL lysis buffer (containing 20 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 v/v, 10 mM NaF, 2 mM Na3VO4, and a protease inhibitor cocktail, pH 7.5). The amount of proteins was determined using the Bio-Rad protein assay (Bio-Rad). After an equal amount of protein was loaded in each lane, they were separated by 10% SDS w/v-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrically transferred to a polyvinylidene difluoride membrane (Bio-Rad). After blocking the membrane with 5 % skim milk w/v, target proteins were immunodetected using specific antibodies (from Cell Signaling Technology). Thereafter, the horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Invitrogen) was applied as the secondary antibody, and the positive bands were detected using Amersham ECL Plus Western blotting detection reagents (GE Healthcare).
Analysis of cells by transmission electron microscopy Fig. 6 Schematic illustration of the mechanism of AA-induced formation of autophagosomes and their role in AA-induced cell death in pancreatic cancer.
Cellular adenosine triphosphate detection by high-performance liquid chromatography As high concentrations of ascorbate may interfere with the commonly used luciferase-based ATP assays, high-performance liquid chromatography (HPLC) was utilized for the detection of ATP. Briefly, cells in a six-well plate (2 × 105 cells/well) were treated with ascorbate for the indicated length, and then the cells were harvested using a rubber scraper, washed, and resuspended in PBS. ATP was extracted by quickly lysing the cells in a 0.05 mM potassium hydroxide solution, and the lysate was immediately neutralized to pH 6 with 0.1 mM KH2PO4. After centrifugation, the supernatant was analyzed using a gradient method on a Waters E2695 HPLC with ultraviolet detection set at 254 and 340 nm (Waters 2489 diode array UV detector; Waters). Reversed-phase chromatography was performed with an XBridgeTM C18 column 3.5 µm (Waters). The mobile phase (pH 6) contained acetonitrile (2% for solvent A and 30% for solvent B), 0.1 M KH2PO4, and 8 mM tetrabutylammonium hydrogen sulfate. The fractions of solvent A to solvent B at 0, 4, 7, 12, 15, and 22 min were 100/0, 90/10, 80/20, 60/40, 0/100, and 100/0, respectively. Waters Empower II software was used for instrument control and data analysis. All values were normalized to the protein content of the whole-cell lyate using the bicinchoninic acid method (Pierce Biotechnology) [19].
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Cells were harvested using trypsin-EDTA and fixed in 2 % glutaraldehyde for 4 h, and centrifuged to form pellets. A sample preparation was carried out according to the previous method [39– 41]. Briefly, the pellets were rinsed in 0.1 M cacodylate buffer [purchased from Electron Microscopy Sciences (EMS) and postfixed in 1% osmium tetroxide (EMS)]. Cell pellets were dehydrated through a graded series of ethanol and then passed through a propylene oxide twice and, lastly, placed in propylene oxide/Embed 812 resin (EMS) overnight for infiltration, and then polymerized in a 60 °C oven overnight. Then sections were cut on a Leica UCT ultra microtome at 80 nm using a Diatome diamond knife. Sections were contrasted with uranyl acetate and Satoʼs lead citrate (EMS), and viewed and photographed on a JEOL 100CXII TEM at 60 KV (J. E. O. L. Ltd.).
Growth of human pancreatic cancer cell xenografts in athymic nude mice All animal use procedures were approved on January 25, 2010 (protocol # 2009–1841) by the Institutional Animal Care and Use Committee of the University of Kansas Medical Center, and the investigators followed the NIH guidelines for the humane treatment of animals. Female athymic nu/nu mice, 4–5 weeks of age, were obtained from Harlan Laboratories. They were exposed to a 12-h light/12-h dark cycle, and had free access to Harlan Teklad Rodent Diet 8604 (Harlan Teklad) and water. Mice were housed under aseptic conditions (positive air pressure in a designated mouse room, with microisolator tops) and all mouse-handling procedures were carried out under a laminar flow hood. MIA-PaCa-2 cells (5 × 106 cells/100 µL PBS) were injected s. c. into the right and left flanks of the mice. Two weeks after inoculation, the mice were randomly grouped according to body weight and tumor size, and started to receive vehicle or ascorbate treatment: Group 1 received the vehicle treatment (i. p., 3 times a week,
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Morphological and histopathological analyses Tumor samples from each mouse were fixed in 10 % buffered formalin phosphate (Fisher Scientific), dehydrated, embedded in paraffin, sectioned in 5-µm thickness, and stained with hematoxylin and eosin (H/E). For immunohistochemical staining of the 3nitroso-cysteine and LC3B, the antigen retrieval procedure was performed by placing the slides in 10 mM citrate buffer (pH 3.0), heating in a microwave oven for 20 min, and then allowing the slides to cool to room temperature for 20 min. Slides were rinsed once with PBS, and endogenous peroxidase activity was blocked by incubating the samples for 30 min with 3% H2O2 in PBS, followed by rinsing threee times with PBS. Nonspecific binding was blocked by incubating the slides for 30 min in 2% normal goat serum (Vector Laboratories) in 1% Triton X-100 containing PBS, followed by incubation with the specific antibodies against 3-nitroso-cysteine (1 : 200 dilution, Sigma) and LC3B (1 : 400 dilution, Cell Signaling Technology, Inc.) in the blocking solution as described above. Then, slides were incubated with biotinylated goat anti-rabbit IgG (dilution, 1 : 500, Vector Laboratories) in the blocking solution as described above. Next, the slides were incubated for 2 h with the avidin-biotin peroxidase complex (Vector Laboratories) according to the manufacturerʼs instructions, followed by a 5-min incubation with a DAB substrate kit (Vector Laboratories). Counterstaining was performed using Mayerʼs hematoxylin. Negative controls lacking the primary antibody were used for each staining.
Reproducibility of experiments and statistical analysis For the in vitro cell culture study, each experiment was repeated at least three times. The data were presented as the mean ± SD of multiple independent experiments. For the in vivo animal study, we have obtained similar results from two independent experiments, and only one set of the representative data was shown. Statistical significance was analyzed using one-way ANOVA and Dunnettʼs test (SPSS software). A p value of less than 0.05 was considered statistically significant.
Supporting information Because of space constraint, some of the results are shown in Figs. S1, S2, S3, S4, S5, S6, and S7.
Acknowledgements !
This work was supported, in part, by a grant from the National Institutes of Health (Grant No. CA97109).
Conflict of Interest !
The authors declare no conflict of interest.
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