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Apoptosis induced by microwave radiation in pancreatic cancer JF305 cells Wenhe Zhu, Wei Zhang, Huiyan Wang, Junjie Xu, Yan Li, and Shijie Lv
Abstract: New therapeutic approaches are needed to improve the survival rate from pancreatic cancer, one of the most lethal human malignancies. In this study, JF305 cells were treated with microwaves at doses of 2.5, 5.0, 10.0, 15.0, and 20.0 mW/cm2 for 20 min. The inhibition of JF305 cell proliferation was tested using the MTT assay. Apoptotic cells were detected with Hoechst 33258 staining and a Nucleo-Counter NC-3000. The expression of apoptosis-related proteins was examined with Western blot. The results showed that microwaves inhibited the growth of JF305 cells in a dose–dependent manner, and caused morphological changes in apoptotic body formation. The percentages of apoptosis detected using annexin V–fluorescein isothiocyanate (FITC) were 4.0%, 10.0%, 12.0%, and 30.0% with the dosage of microwave (0, 5.0, 10.0, and 20.0 mW/cm2), respectively. Treatment with microwaves increased the activity of caspase-9 and caspase-3, down-regulated the expression of Bcl-2, and up-regulated the expression of Bax and CytoC. In addition, the expression level of p65 was increased whereas the level of IB␣ down-regulated. Those results suggest that microwaves inhibit cell growth and induce apoptosis in JF305 cells through an NF-B-regulated mitochondria-mediated pathway. Key words: anticancer, microwave, JF305 cells, apoptosis. Résumé : De nouvelles approches thérapeutiques sont requises afin d'améliorer le taux de survie des patients atteints du cancer du pancréas, un des cancers les plus létaux chez l'humain. Dans cette étude, les cellules JF305 ont été traitées par des microondes a` des doses de 2,5, 5,0, 10,0, 15,0 et 20,0 mW/cm2 pendant 20 minutes. L'inhibition de la prolifération des cellules JF305 a été testée a` l'aide d'un dosage au MTT. Les cellules en apoptose ont été détectées par une coloration au Hoechst 33258 sur un Nucleo-Counter NC-3000. L'expression de protéines reliées a` l'apoptose a été examinée par buvardage Western. Les résultats ont montré que les microondes peuvent inhiber la croissance des cellules JF305 en fonction de la dose et causer des changements morphologiques dans la formation de corps apoptotiques. Les pourcentages de cellules en apoptose détectés par l'Annexine V-FITC étaient respectivement de 4,0, 10,0, 12,0 et 30,0 % en fonction de la dose de microondes (0, 5,0, 10,0, et 20,0 mW/cm2). Le traitement aux microondes accroissait l'activité de la caspase-9 et de la caspase-3, régulait a` la baisse l'expression de Bcl-2 et régulait a` la hausse l'expression de Bax et CytoC. En plus, le niveau d'expression de p65 était accru alors que celui de IB␣ était diminué. Ces résultats suggèrent que les microondes inhibent la croissance cellulaire et induisent l'apoptose des cellules JF305 par l'intermédiaire d'une voie mitochondriale régulée par NF-B. [Traduit par la Rédaction] Mots-clés : anti-cancer, microonde, cellules JF305, apoptose.
Introduction Pancreatic cancer is one of the most lethal human malignancies, with a mortality rate of up to 96%. Pancreatic adenocarcinoma is locally invasive and is surrounded by a dense desmoplastic reaction, which can involve adjacent vital structures (Sener et al. 1999; Jemal et al. 2010). Most current pancreatic cancer therapies are based on the surgical removal of solid tumor masses, and chemotherapy (Neoptolemos et al. 2004). Although these 2 therapeutic strategies show improvements in disease-free survival and overall survival rates, new therapeutic approaches are still needed (Vonlaufen et al. 2008). Recently, microwave therapy has been clinically employed for the treatment of various types of cancer. Patients who receive proper doses of microwaves before surgery tend to have a better prognosis (Maier-Hauff et al. 2007; Ahmed et al. 2011). Previous studies have shown that microwaves selectively kill tumor cells through heat as well as non-heat-related effects, because tumor cells contain more water than normal cells and tumor cells have a
lower blood supply and are slower to eliminate heat. Microwave ablation techniques are applied clinically to treat benign and malignant tumors in the abdomen, chest, and musculoskeletal system, with other locations currently under investigation. Preclinical studies have shown that microwaves present an attractive therapy that is potentially superior compared with radio frequency in several therapeutic respects (Callstrom et al. 2006). Low-power microwave radiation can selectively inhibit tumor cell growth in culture (Beneduci et al. 2005). Many studies have attempted to elucidate the effects of low-power microwave radiation on cell growth, cell cycle progression, and induced cellular apoptosis; however, the mechanisms remain poorly characterized (McIntyre 2006). The technology is still in its infancy, and future in-depth future studies of the mechanisms and equipment requirements would facilitate clinical application in tumor therapy. Microwave radiation reportedly induces tumor cell apoptosis. However data on the effects of microwave radiation on pancreatic cancer cells is insufficient. In this study, we investigated the cytotoxic effect of microwave radiation on the pan-
Received 24 June 2013. Accepted 11 February 2014. W. Zhu, W. Zhang, J. Xu, Y. Li, and S. Lv. Department of Biochemistry, Ji Lin Medical College, Ji Lin 132013, China. H. Wang. Department of Laboratory, Ji Lin Medical College, Ji Lin 132013, China. Corresponding authors: Huiyan Wang (e-mail: [email protected]) and Shijie Lv (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 92: 324–329 (2014) dx.doi.org/10.1139/cjpp-2013-0220
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Materials and methods Materials The 2450-MHz microwave radiation source (MY8C-1) was manufactured by Huiyan Systems Engineering (Nanjing, China).The antibodies against Bcl-2, Bax, and caspase-3 were purchased from Santa Cruz Biotechnology. NF-B p65 and -actin were purchased from Sigma–Aldrich (St. Louis, Missouri, USA). All other chemicals were of analytical grade. Cell culture The human pancreatic cancer cell line JF305 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 U/mL of streptomycin, at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells from the stock flask were suspended by treatment with trypsin, buffered with phosphate buffered saline (PBS), and counted using a hemocytometer. After about 3 days from seeding, active growth of cells began, and it was only after this period was the microwave irradiation started. Cell viability assay The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) cytotoxicity assay was used to measure cell viability as described. Cells (5 × 104 cells/well) were seeded in 96-well flatbottom tissue culture clusters and incubated overnight. Then, cells were treated with microwave radiation at doses of 2.5, 5.0, 10.0, 15.0, or 20.0 mW/cm2 for 20 min. Non-treated cells were used as the control. Each treatment condition was tested in 5 replicate wells. At 24 h after irradiation, 20 L MTT (5 mg/mL MTT dissolved in PBS) solution was added to each well and incubated for another 4 h. Then we added 100 L DMSO per well. Absorbance in each well was detected at 450 nm using a microplate reader (model 550; Bio-Rad, Richmond, California, USA). The relative cell viability was expressed as the ratio of the absorbance of microwave-treated cells to that of the control cells. All experiments were performed in triplicate. Apoptosis JF305 cells treated with different doses of microwaves were harvested by centrifugation at 1000g for 5 min, and washed with ice-cold PBS. The cell suspension (100 L) was centrifuged at 1000g for 5 min. After that, the supernatant was discarded and the pellet was gently resuspended in 195 L annexinV–FITC binding buffer, and incubated with 10 L propidium iodide (PI) solution on an ice bath in the dark. After filtration (300 m), the suspension from each group was analyzed using a Nucleo-Counter NC-3000 (Chemometec). Morphological examination for apoptosis Cells were seeded on slides at a density of 5 × 104/mL in 6-well plates. After treatment as mentioned above, cells from all 5 groups were washed twice with PBS, fixed in 4% paraformaldehyde for 10 min, and then stained with Hoechst 33258 for 5 min. Then the cells were observed under a fluorescence microscope. The nuclei of the living cells were a homogeneous blue; those of apoptotic cells were compact, condensed, and whitish-blue. Effects of microwaves on the MDA content, as well as the activity of SOD and GSH-Px in JF305 cells The enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as the content of malondialdehyde (MDA), were measured using Biotech SOD-525 and MDA-586 assay kits (OXIS International, Portland, Orregon, USA)
Fig. 1. Cytotoxicity of microwaves to JF305 cells. JF305 cells were seeded in 96-well plates and treated with different doses of microwaves for 20 min. After culturing for 24 h, cell proliferation was determined with an MTT assay. Data are the means ± SD, n = 5; *, P < 0.05 compared with the control cells. 100
The inhibition ratio (%)
creatic cancer cell line JF305. The possible modes of action were also explored.
325
*
80
* *
60
*
40
*
20 0 Control
2.5
5
10
15
20 mW/cm2
and a GSH-Px assay kit (Jiancheng Institute of Biomedical Technology, Nanjing) respectively. Western blot After treatment as mentioned above, 5 × 105 cells were harvested and sonicated in radioimmunoprecipitation assay (RIPA) buffer. After centrifugation at 12 000g for 10 min at 4 °C, the protein content was estimated as for the Bio-Rad protein assay, and 50 g protein/lane were loaded onto 12% polyacrylamide SDS gel (SDS–PAGE). The separated proteins were then transferred electrophoretically to nitrocellulose paper and soaked in transfer buffer (25 mmol/L Tris, 192 mmol/L glycine) and 20% (v/v) methanol. Nonspecific binding was blocked by incubation of the blots in 5% no-fat dry milk in TBS–0.1% Tween (25 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween v/v) for 60 min. After washing, the blots were incubated overnight at 4 °C with the primary antibody. After incubation with the primary antibodies and washing in TBS–0.1% Tween, the appropriate secondary antibody was added and left for 1 h at room temperature. Immunoreactive protein bands were detected by chemiluminescence using enhanced chemilumunescence reagents (ECL). Blots were also stained with anti -actin antibody as the internal control for the amounts of target proteins. The films were then subjected to densitometric analysis using a Gel Doc 2000 system (Bio-Rad). Statistical analysis Results are the mean ± SEM. All data were analyzed by one-way ANOVA using SPSS version 13 and are expressed as the mean ± SD. Values for P < 0.05 were considered statistically significant.
Results Cytotoxicity of microwave radiation on pancreatic cancer JF305 cells To evaluate the cytotoxic effects of microwave radiation on JF305 cells, the MTT assay was conducted. JF305 cells were treated with microwaves at the doses of microwave 2.5, 5.0, 10.0, 15.0, or 20.0 mW/cm2 for 20 min. The cytotoxic and growth-inhibitory effects were examined at 24 h after irradiation. As shown in Fig. 1, the microwave radiation caused dramatic cell growth inhibition in JF305 cells in a dose-dependent manner. Further experiments are recommended to investigate the cytotoxic mechanisms of microwave radiation. Effect of microwave on apoptosis in irradiated JF305 cells To determine how microwaves induce apoptosis, we stained the cells with annexin V, a marker for early apoptosis, and PI for detection of late apoptosis. A Nucleo-Counter NC-3000 was used to Published by NRC Research Press
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Fig. 2. The rate of apoptosis in JF305 cells treated with microwaves, as determined using a Nucleo-Counter NC-3000 with annexinV–FITC and PI double labeling. (A) Nucleo-Counter NC-3000 analysis of annexin-V stained cells. (B) Quantitative analysis of data from the lower right quadrant in (A). Data are the mean ± SD, n = 3; *, P < 0.05 compared with the control cells.
quantify fluorescent cells. The number of cells showing early apoptosis increased with increasing doses of microwave radiation (Fig. 2). The percentages of apoptosis detected by annexin V–FITC were 4.0%, 10.0%, 12.0%, and 30.0% with microwave doses of 0, 5.0, 10.0, and 20.0 mW/cm2, respectively. In general, the number of apoptotic JF305 cells significantly increased in a dose-dependent manner.
Morphological changes in microwave-radiation-induced apoptosis in JF305 cells Hoechst 33258, a DNA-sensitive fluorochrome, was used to assess changes in the nuclear morphology following treatment with microwaves. The nuclei in normal cells exhibited diffused staining of the chromatin. However, after microwave radiation, the cells underwent typical morphological changes (chromatin Published by NRC Research Press
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Fig. 3. JF305 cells treated with different doses of microwaves, as observed by fluorescence microscopy (magnification ×200) after nuclei staining with Hoechst 33258. (A) Morphological apoptosis was determined by staining with Hoechst 33258. (B) The percentage of nuclear condensation in the cultured cells was counted in response to control. Data are the mean ± SD, n = 3; *, P < 0.05 compared with the control cells.
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Table 1. The effect of microwaves on the level of malondialdehyde (MDA), the activities of the enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Group (mW/cm2)
SOD (U/mL)
MDA (mol/L)
GSH-Px (mol/L)
Control 5.0 10.0 20.0
48.00±0.2 40.96±0.78* 35.73±1.43* 31.00±4.68*
2.48±0.13 2.52±0.09 2.88±0.22* 3.02±0.43*
42.21±0.03 40.27±0.42* 36.70±0.22* 28.38±0.13*
Note: Data are the mean ± SD, n = 5; *, P < 0.05 compared with the control cells.
increase in the cytosolic CytoC levels following treatment with microwaves (Figs. 4A and 4D). Studies have shown that Bcl-2 and its dominant inhibitor, Bax, are key regulators of cell proliferation and apoptosis. Overexpression of Bcl-2 enhances cell survival by suppressing apoptosis, but the overexpression of Bax accelerates cell death. The ratio of Bax/Bcl-2 is crucial for the activation of the mitochondrial apoptotic pathway. Microwave treatment caused an increase in the ratio of Bax/Bcl-2 protein (Figs. 4A and 4C). Since caspases are known to play a central role in mediating various apoptotic responses, we checked their activation status and observed that cleaved caspase-9 and caspase-3 were activated after microwave treatment (Figs. 4A and 4D). Then we further examined p65 protein, a subunit of NF-B, and IB␣, an inhibitor of NF-B. We found that the level of p65 was increased whereas the level of IB␣ was down-regulated (Figs. 4B and 4E). These results indicate that microwave-induced JF305 cell apoptosis may occur through an NF-B-regulated mitochondriamediated pathway. However, the mechanisms behind this process need further study.
Discussion
condensation, margination, and shrunken nucleus) indicative of apoptosis (Fig. 3). Effect of microwaves on the content of MDA, and the enzyme activity of GSH-Px and SOD MDA is regarded as a major marker of lipid peroxidation in tissue, whereas SOD and GSH-Px are 2 important enzymes in the antioxidant defense system. After exposure to microwave radiation, the MDA content and SOD and GSH-Px activities in JF305 cells were measured (Table 1). As shown in the table, microwave radiation decreased the activity of SOD and GSH-Px and increased MDA content. Microwave radiation modulates the expression of apoptosis-related proteins in JF305 cells The release of cytochrome c (CytoC) from the mitochondria is a critical step in the apoptotic cascade, since this activates downstream caspases. The results demonstrated a concentration-dependent
Pancreatic cancer is among the most aggressive human malignancies worldwide, but all clinical management procedures remain rather palliative in nature, and have been without significant improvement over the last decade (Lockhart et al. 2005). Monotherapy with chemotherapeutic agents or in combination with other agents has become the standard treatment for advanced pancreatic cancer. However, the overall efficacy to date is not satisfactory. Given that chemotherapeutic agents have systemic toxicity, new therapeutic approaches are still needed. Apoptosis is ubiquitous in most tumor cells; it is important in the genesis and progression of tumors. Previous studies have demonstrated that antitumor drugs typically inhibit tumors by inducing apoptosis of the sensitive tumor cells (Li and Yuan 2008; Li and Sheng 2012). Therefore, using apoptosis as an intervention to treat tumors has become a new target in the search for antitumor drugs, and a new direction for development in tumor pharmacology. Studies have revealed that microwave radiation inhibits cell growth, induces cell cycle arrest, and promotes apoptosis in tumor cells. However, microwave-radiation-induced apoptosis in JF305 cells has not yet been reported. In this study, JF305 cell proliferation was inhibited after microwave treatment for 20 min, and the rate of apoptosis increased in a dose-dependent manner. This finding indicates that microwave radiation can induce apoptosis in JF305 cells. SOD and GSH-Px, 2 of the major components of the oxidase defense system, are natural scavengers of active oxygen and superoxide anion radicals (Li et al 2012). SOD in animal blood is stored in relatively high amounts and is in the first line of defense against free radicals. SOD can specifically bind to superoxide anions in vivo and can act synergistically with GSH-Px to prevent lipid peroxidation and its metabolites from damaging the body. It can also directly capture and remove free radicals, such as the superoxide anion. MDA is widely used to determine the extent of lipid peroxidation in vivo, which can cause changes in cell function, gePublished by NRC Research Press
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Fig. 4. Western blot analysis of protein extracts obtained from JF305 cells treated with microwaves. (A) Total protein extracts were prepared after treatment with microwaves, and analyzed with antibodies to Bcl-2, Bax, CytoC, caspase-9, and caspase-3. (B) Analysis of NF-B p65 and IB␣. (C) Quantitation of Bax/Bcl-2. (D) Quantitation of CytoC, caspase-9, and caspase-3 protein levels. (E) Quantitation of NF-B p65 and IB␣ protein levels. Data are the mean ± SD, n = 3; *, P < 0.05 compared with the control.
netic toxicity, DNA damage, and carcinogenesis (Thorne et al 2009). In this study, microwave radiation decreased SOD and GSH-Px activities and increased MDA content in JF305 cells. These data suggest that the change in activities of antioxidative stress enzymes are related to DNA damage or cell apoptosis in JF305 cells after microwave irradiation. Cell apoptosis or programmed cell death is significant in the maintenance of the intrinsic stability of multicellular organisms. Various signaling pathways for apoptosis exist within an organism, with the mitochondrial pathway being one of the most important. CytoC is an important mitochondrial protein that induces apoptosis when accumulated in the cytosol in response to diverse stress stimuli. CytoC binds caspase-9 to form a complex that activates other caspase family members, including caspase-3, to induce apoptosis (Hyman and Yuan 2012; Martin et al. 2012). This study shows that treatment with microwaves increased caspase-9 activity in JF 305 cells. This is consistent with the release of CytoC into the cytosol from mitochondria, potentially activating caspase-9. The activated upstream caspase-9 acts on the downstream target of caspase-3 enzymes, subsequently the activated caspase-3 acts on the target cells as an effector molecule, damaging the cell structure and causing functional disorder by proteolysis, ultimately inducing apoptosis. To further understand the mechanisms for microwave-radiationinduced apoptosis in JF305 cells, we investigated the expression
levels of Bcl-2 and Bax. Bcl-2 inhibits apoptosis by negatively regulating the apoptotic activity of Bax and forming Bcl-2/Bax heterodimers. The Bcl-2/Bax ratio, a measure of the cell death switch, determines whether a cell will live or die upon being exposed to an apoptotic stimulus (Oltval et al. 1993). In this study, exposure to increasing levels of microwaves gradually down-regulated and up-regulated the expression levels of Bcl-2 and Bax proteins in JF305 cells, respectively. Taken together, these findings indicate that a caspase-dependent mitochondrial pathway is involved in microwave-induced apoptosis in JF305 cells. Most tumors activate NF-B, whereas natural chemopreventive agents suppress it, indicating a strong link between the tumor biology and the anticancer properties of various natural compounds (Hecker et al. 1996). Pro-inflammatory cytokines, chemotherapeutic agents, and radiation therapy that induce apoptosis also activate NF-B and may thus mediate the chemoresistance and radio-resistance of tumor cells. NF-B exists in the cytoplasm and is inhibited by complexing with IB. Phosphorylation of IB by IB kinase (IKK) causes ubiquitination and degradation of IB. This study shows that microwave stimulation of JF305 tumor cells induces the activation of NF-B p65, possibly by inhibiting IB␣ expression and thereby causing apoptosis. In conclusion, the cytotoxic effect of microwave-induced cell death is through the induction of apoptosis, as indicated by the current data. Treatment with microwaves led to a decrease in the Published by NRC Research Press
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activity of SOD and GSH-Px, and increased MDA levels. Subsequently, the Bax/Bcl-2 ratio was elevated, CytoC, caspase-9, and caspase-3 were up-regulated, and NF-B p65 protein expression was activated. A NF-B-regulated mitochondria-mediated pathway was activated, which may contribute to the proapoptotic effects of microwaves in JF305 tumor cells. These observations provide information on a potentially useful tumor therapeutic approach for the treatment of human pancreatic cancer. However, further study of the exact mechanisms is still needed to determine the therapeutic effects.
Acknowledgements This work was supported by Department of Education of Jilin Province (No. 2013 [479]), Project Agreement for Science & Technology Development, Jilin Province (No. 20120941 and 20140204002YY), National Natural Science Foundation of China (No. 81273421), and National Science and Technology Major Projects (2012ZX09103301003).
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Jemal, A., Siegel, R., Xu, J., and Ward, E. 2010. Cancer statistics, 2010. CA Cancer J. Clin. 60(5): 277–300. doi:10.3322/caac.20073. PMID:20610543. Li, Z., and Sheng, M. 2012. Caspases in synaptic plasticity. Mol. Brain, 5(1): 15–21. doi:10.1186/1756-6606-5-15. PMID:22583788. Li, J., and Yuan, J. 2008. Caspases in apoptosis and beyond. Oncogene, 27(48): 6194–6206. doi:10.1038/onc.2008.297. PMID:18931687. Li, W.F., Cui, Z.W., Rajput, I.R., Li, Y.L., Wu, H.Z., and Yu, D.Y. 2012. Effect of Enterococcus faecium 1 (EF1) on antioxidant functioning activity of Caco-2 cells under oxidative stress. J. Anim. Vet. Adv. 11(13): 2307–2312. doi:10.3923/javaa. 2012.2307.2312. Lockhart, A.C., Rothenberg, M.L., and Berlin, J.D. 2005. Treatment for pancreatic cancer: current therapy and continued progress. Gastroenterology, 128(6): 1642–1654. doi:10.1053/j.gastro.2005.03.039. PMID:15887156. Maier-Hauff, K., Rothe, R., Scholz, R., Gneveckow, U., Wust, P., Thiesen, B., et al. 2007. Intracranial thermotherapy using magnetic nanoparticles combined with external beam radiotherapy: results of a feasibility study on patients with glioblastoma multiforme. J. Neurooncol. 81(1): 53–60. doi:10.1007/s11060006-9195-0. PMID:16773216. Martin, S.J., Henry, C.M., and Cullen, S.P. 2012. A perspective on mammalian caspases as positive and negative regulators of inflammation. Mol. Cell, 46(4): 387–397. doi:10.1016/j.molcel.2012.04.026. PMID:22633484. McIntyre, G.I. 2006. Cell hydration as the primary factor in carcinogenesis: A unifying concept. Med. Hypotheses, 66(3): 518–526. doi:10.1016/j.mehy.2005. 09.022. PMID:16271440. Neoptolemos, J.P., Stocken, D.D., Friess, H., Bassi, C., Dunn, J.A., Hickey, H., et al. 2004. A randomized trial of chemoradiotherapy and chemotherapy after resection of pancreatic cancer. N. Engl. J. Med. 350(12): 1200–1210. doi:10.1056/ NEJMoa032295. PMID:15028824. Oltval, Z.N., Milliman, C.L., and Korsmeyer, S.J. 1993. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programed cell death. Cell, 74(4): 609–619. doi:10.1016/0092-8674(93)90509-O. PMID:8358790. Sener, S.F., Fremgen, A., Menck, H.R., and Winchester, D.P. 1999. Pancreatic cancer: a report of treatment and survival trends for 100,313 patients diagnosed from 1985–1995, using the National Cancer Database. J. Am. Coll. Surg. 189(1): 1–7. doi:10.1016/S1072-7515(99)00075-7. PMID:10401733. Thorne, D., Wilson, J., Kumaravel, T.S., Massey, E.D., and McEwan, M. 2009. Measurement of oxidative DNA damage induced by mainstream cigarette smoke in cultured NCI-H292 human pulmonary carcinoma cells. Mutat. Res. 673(1): 3–8. doi:10.1016/j.mrgentox.2008.11.008. PMID:19100859. Vonlaufen, A., Phillips, P.A., Xu, Z., Goldstein, D., Pirola, R.C., Wilson, J.S., and Apte, M.V. 2008. Pancreatic stellate cells and pancreatic cancer cells: an unholy alliance. Cancer Res. 68(19): 7707–7710. doi:10.1158/0008-5472.CAN-081132. PMID:18829522.
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Apoptosis induced by microwave radiation in pancreatic cancer JF305 cells Wenhe Zhu, Wei Zhang, Huiyan Wang, Junjie Xu, Yan Li, and Shijie Lv
Abstract: New therapeutic approaches are needed to improve the survival rate from pancreatic cancer, one of the most lethal human malignancies. In this study, JF305 cells were treated with microwaves at doses of 2.5, 5.0, 10.0, 15.0, and 20.0 mW/cm2 for 20 min. The inhibition of JF305 cell proliferation was tested using the MTT assay. Apoptotic cells were detected with Hoechst 33258 staining and a Nucleo-Counter NC-3000. The expression of apoptosis-related proteins was examined with Western blot. The results showed that microwaves inhibited the growth of JF305 cells in a dose–dependent manner, and caused morphological changes in apoptotic body formation. The percentages of apoptosis detected using annexin V–fluorescein isothiocyanate (FITC) were 4.0%, 10.0%, 12.0%, and 30.0% with the dosage of microwave (0, 5.0, 10.0, and 20.0 mW/cm2), respectively. Treatment with microwaves increased the activity of caspase-9 and caspase-3, down-regulated the expression of Bcl-2, and up-regulated the expression of Bax and CytoC. In addition, the expression level of p65 was increased whereas the level of IB␣ down-regulated. Those results suggest that microwaves inhibit cell growth and induce apoptosis in JF305 cells through an NF-B-regulated mitochondria-mediated pathway. Key words: anticancer, microwave, JF305 cells, apoptosis. Résumé : De nouvelles approches thérapeutiques sont requises afin d'améliorer le taux de survie des patients atteints du cancer du pancréas, un des cancers les plus létaux chez l'humain. Dans cette étude, les cellules JF305 ont été traitées par des microondes a` des doses de 2,5, 5,0, 10,0, 15,0 et 20,0 mW/cm2 pendant 20 minutes. L'inhibition de la prolifération des cellules JF305 a été testée a` l'aide d'un dosage au MTT. Les cellules en apoptose ont été détectées par une coloration au Hoechst 33258 sur un Nucleo-Counter NC-3000. L'expression de protéines reliées a` l'apoptose a été examinée par buvardage Western. Les résultats ont montré que les microondes peuvent inhiber la croissance des cellules JF305 en fonction de la dose et causer des changements morphologiques dans la formation de corps apoptotiques. Les pourcentages de cellules en apoptose détectés par l'Annexine V-FITC étaient respectivement de 4,0, 10,0, 12,0 et 30,0 % en fonction de la dose de microondes (0, 5,0, 10,0, et 20,0 mW/cm2). Le traitement aux microondes accroissait l'activité de la caspase-9 et de la caspase-3, régulait a` la baisse l'expression de Bcl-2 et régulait a` la hausse l'expression de Bax et CytoC. En plus, le niveau d'expression de p65 était accru alors que celui de IB␣ était diminué. Ces résultats suggèrent que les microondes inhibent la croissance cellulaire et induisent l'apoptose des cellules JF305 par l'intermédiaire d'une voie mitochondriale régulée par NF-B. [Traduit par la Rédaction] Mots-clés : anti-cancer, microonde, cellules JF305, apoptose.
Introduction Pancreatic cancer is one of the most lethal human malignancies, with a mortality rate of up to 96%. Pancreatic adenocarcinoma is locally invasive and is surrounded by a dense desmoplastic reaction, which can involve adjacent vital structures (Sener et al. 1999; Jemal et al. 2010). Most current pancreatic cancer therapies are based on the surgical removal of solid tumor masses, and chemotherapy (Neoptolemos et al. 2004). Although these 2 therapeutic strategies show improvements in disease-free survival and overall survival rates, new therapeutic approaches are still needed (Vonlaufen et al. 2008). Recently, microwave therapy has been clinically employed for the treatment of various types of cancer. Patients who receive proper doses of microwaves before surgery tend to have a better prognosis (Maier-Hauff et al. 2007; Ahmed et al. 2011). Previous studies have shown that microwaves selectively kill tumor cells through heat as well as non-heat-related effects, because tumor cells contain more water than normal cells and tumor cells have a
lower blood supply and are slower to eliminate heat. Microwave ablation techniques are applied clinically to treat benign and malignant tumors in the abdomen, chest, and musculoskeletal system, with other locations currently under investigation. Preclinical studies have shown that microwaves present an attractive therapy that is potentially superior compared with radio frequency in several therapeutic respects (Callstrom et al. 2006). Low-power microwave radiation can selectively inhibit tumor cell growth in culture (Beneduci et al. 2005). Many studies have attempted to elucidate the effects of low-power microwave radiation on cell growth, cell cycle progression, and induced cellular apoptosis; however, the mechanisms remain poorly characterized (McIntyre 2006). The technology is still in its infancy, and future in-depth future studies of the mechanisms and equipment requirements would facilitate clinical application in tumor therapy. Microwave radiation reportedly induces tumor cell apoptosis. However data on the effects of microwave radiation on pancreatic cancer cells is insufficient. In this study, we investigated the cytotoxic effect of microwave radiation on the pan-
Received 24 June 2013. Accepted 11 February 2014. W. Zhu, W. Zhang, J. Xu, Y. Li, and S. Lv. Department of Biochemistry, Ji Lin Medical College, Ji Lin 132013, China. H. Wang. Department of Laboratory, Ji Lin Medical College, Ji Lin 132013, China. Corresponding authors: Huiyan Wang (e-mail: [email protected]) and Shijie Lv (e-mail: [email protected]). Can. J. Physiol. Pharmacol. 92: 324–329 (2014) dx.doi.org/10.1139/cjpp-2013-0220
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Materials and methods Materials The 2450-MHz microwave radiation source (MY8C-1) was manufactured by Huiyan Systems Engineering (Nanjing, China).The antibodies against Bcl-2, Bax, and caspase-3 were purchased from Santa Cruz Biotechnology. NF-B p65 and -actin were purchased from Sigma–Aldrich (St. Louis, Missouri, USA). All other chemicals were of analytical grade. Cell culture The human pancreatic cancer cell line JF305 was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and cultured in Dulbecco's modified Eagle's medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U/mL of penicillin, and 100 U/mL of streptomycin, at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. Cells from the stock flask were suspended by treatment with trypsin, buffered with phosphate buffered saline (PBS), and counted using a hemocytometer. After about 3 days from seeding, active growth of cells began, and it was only after this period was the microwave irradiation started. Cell viability assay The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) cytotoxicity assay was used to measure cell viability as described. Cells (5 × 104 cells/well) were seeded in 96-well flatbottom tissue culture clusters and incubated overnight. Then, cells were treated with microwave radiation at doses of 2.5, 5.0, 10.0, 15.0, or 20.0 mW/cm2 for 20 min. Non-treated cells were used as the control. Each treatment condition was tested in 5 replicate wells. At 24 h after irradiation, 20 L MTT (5 mg/mL MTT dissolved in PBS) solution was added to each well and incubated for another 4 h. Then we added 100 L DMSO per well. Absorbance in each well was detected at 450 nm using a microplate reader (model 550; Bio-Rad, Richmond, California, USA). The relative cell viability was expressed as the ratio of the absorbance of microwave-treated cells to that of the control cells. All experiments were performed in triplicate. Apoptosis JF305 cells treated with different doses of microwaves were harvested by centrifugation at 1000g for 5 min, and washed with ice-cold PBS. The cell suspension (100 L) was centrifuged at 1000g for 5 min. After that, the supernatant was discarded and the pellet was gently resuspended in 195 L annexinV–FITC binding buffer, and incubated with 10 L propidium iodide (PI) solution on an ice bath in the dark. After filtration (300 m), the suspension from each group was analyzed using a Nucleo-Counter NC-3000 (Chemometec). Morphological examination for apoptosis Cells were seeded on slides at a density of 5 × 104/mL in 6-well plates. After treatment as mentioned above, cells from all 5 groups were washed twice with PBS, fixed in 4% paraformaldehyde for 10 min, and then stained with Hoechst 33258 for 5 min. Then the cells were observed under a fluorescence microscope. The nuclei of the living cells were a homogeneous blue; those of apoptotic cells were compact, condensed, and whitish-blue. Effects of microwaves on the MDA content, as well as the activity of SOD and GSH-Px in JF305 cells The enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), as well as the content of malondialdehyde (MDA), were measured using Biotech SOD-525 and MDA-586 assay kits (OXIS International, Portland, Orregon, USA)
Fig. 1. Cytotoxicity of microwaves to JF305 cells. JF305 cells were seeded in 96-well plates and treated with different doses of microwaves for 20 min. After culturing for 24 h, cell proliferation was determined with an MTT assay. Data are the means ± SD, n = 5; *, P < 0.05 compared with the control cells. 100
The inhibition ratio (%)
creatic cancer cell line JF305. The possible modes of action were also explored.
325
*
80
* *
60
*
40
*
20 0 Control
2.5
5
10
15
20 mW/cm2
and a GSH-Px assay kit (Jiancheng Institute of Biomedical Technology, Nanjing) respectively. Western blot After treatment as mentioned above, 5 × 105 cells were harvested and sonicated in radioimmunoprecipitation assay (RIPA) buffer. After centrifugation at 12 000g for 10 min at 4 °C, the protein content was estimated as for the Bio-Rad protein assay, and 50 g protein/lane were loaded onto 12% polyacrylamide SDS gel (SDS–PAGE). The separated proteins were then transferred electrophoretically to nitrocellulose paper and soaked in transfer buffer (25 mmol/L Tris, 192 mmol/L glycine) and 20% (v/v) methanol. Nonspecific binding was blocked by incubation of the blots in 5% no-fat dry milk in TBS–0.1% Tween (25 mmol/L Tris, 150 mmol/L NaCl, 0.1% Tween v/v) for 60 min. After washing, the blots were incubated overnight at 4 °C with the primary antibody. After incubation with the primary antibodies and washing in TBS–0.1% Tween, the appropriate secondary antibody was added and left for 1 h at room temperature. Immunoreactive protein bands were detected by chemiluminescence using enhanced chemilumunescence reagents (ECL). Blots were also stained with anti -actin antibody as the internal control for the amounts of target proteins. The films were then subjected to densitometric analysis using a Gel Doc 2000 system (Bio-Rad). Statistical analysis Results are the mean ± SEM. All data were analyzed by one-way ANOVA using SPSS version 13 and are expressed as the mean ± SD. Values for P < 0.05 were considered statistically significant.
Results Cytotoxicity of microwave radiation on pancreatic cancer JF305 cells To evaluate the cytotoxic effects of microwave radiation on JF305 cells, the MTT assay was conducted. JF305 cells were treated with microwaves at the doses of microwave 2.5, 5.0, 10.0, 15.0, or 20.0 mW/cm2 for 20 min. The cytotoxic and growth-inhibitory effects were examined at 24 h after irradiation. As shown in Fig. 1, the microwave radiation caused dramatic cell growth inhibition in JF305 cells in a dose-dependent manner. Further experiments are recommended to investigate the cytotoxic mechanisms of microwave radiation. Effect of microwave on apoptosis in irradiated JF305 cells To determine how microwaves induce apoptosis, we stained the cells with annexin V, a marker for early apoptosis, and PI for detection of late apoptosis. A Nucleo-Counter NC-3000 was used to Published by NRC Research Press
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Fig. 2. The rate of apoptosis in JF305 cells treated with microwaves, as determined using a Nucleo-Counter NC-3000 with annexinV–FITC and PI double labeling. (A) Nucleo-Counter NC-3000 analysis of annexin-V stained cells. (B) Quantitative analysis of data from the lower right quadrant in (A). Data are the mean ± SD, n = 3; *, P < 0.05 compared with the control cells.
quantify fluorescent cells. The number of cells showing early apoptosis increased with increasing doses of microwave radiation (Fig. 2). The percentages of apoptosis detected by annexin V–FITC were 4.0%, 10.0%, 12.0%, and 30.0% with microwave doses of 0, 5.0, 10.0, and 20.0 mW/cm2, respectively. In general, the number of apoptotic JF305 cells significantly increased in a dose-dependent manner.
Morphological changes in microwave-radiation-induced apoptosis in JF305 cells Hoechst 33258, a DNA-sensitive fluorochrome, was used to assess changes in the nuclear morphology following treatment with microwaves. The nuclei in normal cells exhibited diffused staining of the chromatin. However, after microwave radiation, the cells underwent typical morphological changes (chromatin Published by NRC Research Press
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Fig. 3. JF305 cells treated with different doses of microwaves, as observed by fluorescence microscopy (magnification ×200) after nuclei staining with Hoechst 33258. (A) Morphological apoptosis was determined by staining with Hoechst 33258. (B) The percentage of nuclear condensation in the cultured cells was counted in response to control. Data are the mean ± SD, n = 3; *, P < 0.05 compared with the control cells.
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Table 1. The effect of microwaves on the level of malondialdehyde (MDA), the activities of the enzymes glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD). Group (mW/cm2)
SOD (U/mL)
MDA (mol/L)
GSH-Px (mol/L)
Control 5.0 10.0 20.0
48.00±0.2 40.96±0.78* 35.73±1.43* 31.00±4.68*
2.48±0.13 2.52±0.09 2.88±0.22* 3.02±0.43*
42.21±0.03 40.27±0.42* 36.70±0.22* 28.38±0.13*
Note: Data are the mean ± SD, n = 5; *, P < 0.05 compared with the control cells.
increase in the cytosolic CytoC levels following treatment with microwaves (Figs. 4A and 4D). Studies have shown that Bcl-2 and its dominant inhibitor, Bax, are key regulators of cell proliferation and apoptosis. Overexpression of Bcl-2 enhances cell survival by suppressing apoptosis, but the overexpression of Bax accelerates cell death. The ratio of Bax/Bcl-2 is crucial for the activation of the mitochondrial apoptotic pathway. Microwave treatment caused an increase in the ratio of Bax/Bcl-2 protein (Figs. 4A and 4C). Since caspases are known to play a central role in mediating various apoptotic responses, we checked their activation status and observed that cleaved caspase-9 and caspase-3 were activated after microwave treatment (Figs. 4A and 4D). Then we further examined p65 protein, a subunit of NF-B, and IB␣, an inhibitor of NF-B. We found that the level of p65 was increased whereas the level of IB␣ was down-regulated (Figs. 4B and 4E). These results indicate that microwave-induced JF305 cell apoptosis may occur through an NF-B-regulated mitochondriamediated pathway. However, the mechanisms behind this process need further study.
Discussion
condensation, margination, and shrunken nucleus) indicative of apoptosis (Fig. 3). Effect of microwaves on the content of MDA, and the enzyme activity of GSH-Px and SOD MDA is regarded as a major marker of lipid peroxidation in tissue, whereas SOD and GSH-Px are 2 important enzymes in the antioxidant defense system. After exposure to microwave radiation, the MDA content and SOD and GSH-Px activities in JF305 cells were measured (Table 1). As shown in the table, microwave radiation decreased the activity of SOD and GSH-Px and increased MDA content. Microwave radiation modulates the expression of apoptosis-related proteins in JF305 cells The release of cytochrome c (CytoC) from the mitochondria is a critical step in the apoptotic cascade, since this activates downstream caspases. The results demonstrated a concentration-dependent
Pancreatic cancer is among the most aggressive human malignancies worldwide, but all clinical management procedures remain rather palliative in nature, and have been without significant improvement over the last decade (Lockhart et al. 2005). Monotherapy with chemotherapeutic agents or in combination with other agents has become the standard treatment for advanced pancreatic cancer. However, the overall efficacy to date is not satisfactory. Given that chemotherapeutic agents have systemic toxicity, new therapeutic approaches are still needed. Apoptosis is ubiquitous in most tumor cells; it is important in the genesis and progression of tumors. Previous studies have demonstrated that antitumor drugs typically inhibit tumors by inducing apoptosis of the sensitive tumor cells (Li and Yuan 2008; Li and Sheng 2012). Therefore, using apoptosis as an intervention to treat tumors has become a new target in the search for antitumor drugs, and a new direction for development in tumor pharmacology. Studies have revealed that microwave radiation inhibits cell growth, induces cell cycle arrest, and promotes apoptosis in tumor cells. However, microwave-radiation-induced apoptosis in JF305 cells has not yet been reported. In this study, JF305 cell proliferation was inhibited after microwave treatment for 20 min, and the rate of apoptosis increased in a dose-dependent manner. This finding indicates that microwave radiation can induce apoptosis in JF305 cells. SOD and GSH-Px, 2 of the major components of the oxidase defense system, are natural scavengers of active oxygen and superoxide anion radicals (Li et al 2012). SOD in animal blood is stored in relatively high amounts and is in the first line of defense against free radicals. SOD can specifically bind to superoxide anions in vivo and can act synergistically with GSH-Px to prevent lipid peroxidation and its metabolites from damaging the body. It can also directly capture and remove free radicals, such as the superoxide anion. MDA is widely used to determine the extent of lipid peroxidation in vivo, which can cause changes in cell function, gePublished by NRC Research Press
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Fig. 4. Western blot analysis of protein extracts obtained from JF305 cells treated with microwaves. (A) Total protein extracts were prepared after treatment with microwaves, and analyzed with antibodies to Bcl-2, Bax, CytoC, caspase-9, and caspase-3. (B) Analysis of NF-B p65 and IB␣. (C) Quantitation of Bax/Bcl-2. (D) Quantitation of CytoC, caspase-9, and caspase-3 protein levels. (E) Quantitation of NF-B p65 and IB␣ protein levels. Data are the mean ± SD, n = 3; *, P < 0.05 compared with the control.
netic toxicity, DNA damage, and carcinogenesis (Thorne et al 2009). In this study, microwave radiation decreased SOD and GSH-Px activities and increased MDA content in JF305 cells. These data suggest that the change in activities of antioxidative stress enzymes are related to DNA damage or cell apoptosis in JF305 cells after microwave irradiation. Cell apoptosis or programmed cell death is significant in the maintenance of the intrinsic stability of multicellular organisms. Various signaling pathways for apoptosis exist within an organism, with the mitochondrial pathway being one of the most important. CytoC is an important mitochondrial protein that induces apoptosis when accumulated in the cytosol in response to diverse stress stimuli. CytoC binds caspase-9 to form a complex that activates other caspase family members, including caspase-3, to induce apoptosis (Hyman and Yuan 2012; Martin et al. 2012). This study shows that treatment with microwaves increased caspase-9 activity in JF 305 cells. This is consistent with the release of CytoC into the cytosol from mitochondria, potentially activating caspase-9. The activated upstream caspase-9 acts on the downstream target of caspase-3 enzymes, subsequently the activated caspase-3 acts on the target cells as an effector molecule, damaging the cell structure and causing functional disorder by proteolysis, ultimately inducing apoptosis. To further understand the mechanisms for microwave-radiationinduced apoptosis in JF305 cells, we investigated the expression
levels of Bcl-2 and Bax. Bcl-2 inhibits apoptosis by negatively regulating the apoptotic activity of Bax and forming Bcl-2/Bax heterodimers. The Bcl-2/Bax ratio, a measure of the cell death switch, determines whether a cell will live or die upon being exposed to an apoptotic stimulus (Oltval et al. 1993). In this study, exposure to increasing levels of microwaves gradually down-regulated and up-regulated the expression levels of Bcl-2 and Bax proteins in JF305 cells, respectively. Taken together, these findings indicate that a caspase-dependent mitochondrial pathway is involved in microwave-induced apoptosis in JF305 cells. Most tumors activate NF-B, whereas natural chemopreventive agents suppress it, indicating a strong link between the tumor biology and the anticancer properties of various natural compounds (Hecker et al. 1996). Pro-inflammatory cytokines, chemotherapeutic agents, and radiation therapy that induce apoptosis also activate NF-B and may thus mediate the chemoresistance and radio-resistance of tumor cells. NF-B exists in the cytoplasm and is inhibited by complexing with IB. Phosphorylation of IB by IB kinase (IKK) causes ubiquitination and degradation of IB. This study shows that microwave stimulation of JF305 tumor cells induces the activation of NF-B p65, possibly by inhibiting IB␣ expression and thereby causing apoptosis. In conclusion, the cytotoxic effect of microwave-induced cell death is through the induction of apoptosis, as indicated by the current data. Treatment with microwaves led to a decrease in the Published by NRC Research Press
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activity of SOD and GSH-Px, and increased MDA levels. Subsequently, the Bax/Bcl-2 ratio was elevated, CytoC, caspase-9, and caspase-3 were up-regulated, and NF-B p65 protein expression was activated. A NF-B-regulated mitochondria-mediated pathway was activated, which may contribute to the proapoptotic effects of microwaves in JF305 tumor cells. These observations provide information on a potentially useful tumor therapeutic approach for the treatment of human pancreatic cancer. However, further study of the exact mechanisms is still needed to determine the therapeutic effects.
Acknowledgements This work was supported by Department of Education of Jilin Province (No. 2013 [479]), Project Agreement for Science & Technology Development, Jilin Province (No. 20120941 and 20140204002YY), National Natural Science Foundation of China (No. 81273421), and National Science and Technology Major Projects (2012ZX09103301003).
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