- Email: [email protected]
Aloin-induced cell growth arrest, cell apoptosis, and autophagy in human non-small cell lung cancer cells
Biomarkers and Genomic Medicine (2014) 6, 144e149
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.j-bgm.com
ORIGINAL ARTICLE
Aloin-induced cell growth arrest, cell apoptosis, and autophagy in human non-small cell lung cancer cells Ming-Chin Lee a, Ji-Der Liao b, Wan-Ling Huang b, Fang-Yi Jiang b, Yu-Zih Jheng b, Yuan-Yuan Jin b, Ya-Shih Tseng b,* a
Department of Surgery, Mau-Dou Sin-Lau Christian Hospital, Tainan, Taiwan Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, Tainan, Taiwan
b
Received 10 July 2014; received in revised form 17 August 2014; accepted 21 August 2014
Available online 28 September 2014
KEYWORDS aloin; apoptosis; autophagy; lung cancer
Abstract Aloin, the major anthraquinone in aloe exudates and gels, has been shown to be an effective antioxidant and anti-inflammatory agent. We studied the antitumor effects of aloin in human non-small cell lung cancer cells. The treatment of lung cancer cells with aloin suppressed cell growth in a time- and concentration-dependent manner. Aloin-induced apoptosis also showed a concentration-dependent behavior when studied by flow cytometry. In addition, autophagy occurred after treatment with aloin. Taken together, our data demonstrate that aloin induces apoptosis and autophagy in human lung cancer cells. Aloin is therefore a potential therapeutic agent for human lung cancer and is worthy of further investigation. Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction Lung cancer is the most common cause of cancer-related death worldwide and is classified as either small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). NSCLC * Corresponding author. Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No.89, Wenhwa 1st St., Rende District, Tainan 71703, Taiwan. E-mail address: [email protected] (Y.-S. Tseng).
accounts for 75e80% of patients with primary lung cancer. Compared with NSCLC, SCLC shows an aggressive behavior with rapid tumor growth and early spread to distant sites; its malignancy is the highest of all types of lung cancer.1e3 Surgery is the most effective therapeutic modality, but the postoperative prognosis remains poor. In addition, most patients present with advanced stage disease and chemotherapy with or without radiotherapy is recommended. Although NSCLC and SCLC have a relatively good initial response to clinical treatment, relapse or disease progression may occur quickly and the prognosis for these patients
http://dx.doi.org/10.1016/j.bgm.2014.08.005 2214-0247/Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Aloin suppresses growth of lung cancer cells remains poor.2,3 A novel and effective treatment for SCLC and NSCLC is therefore needed. Aloin, also known as barbaloin,4 is the major anthraquinone in aloe exudates and gels and has been characterized as the Cglycoside of aloe-emodin.5,6 Aloin is a potent inhibitor of tyrosinase and is an effective inhibitor of stimulated granulocyte matrix metalloproteinases.7,8 Aloin also protects against chemically induced colon toxicity and has been reported to act by increasing antioxidant levels and by having anti-inflammatory and antiproliferative effects.9 Aloin has been shown to cause a pronounced reduction in cell proliferation and has also been shown to induce cell apoptosis in HeLaS3 cells.10 At higher concentrations, it has caused a reduction in the proportion of cells undergoing mitosis by the induction of apoptosis, inhibition of topo II and protein expression, and the downregulation of cyclin B1 protein expression in MCF-7 cell lines.11 The treatment of breast and ovarian tumor cells with aloin showed a persistent aneuploid pattern and a significantly dose-dependent increase in the percentage of the S phase fraction and in the proportion of cells cycling at a higher ploidy level.12 Importantly, a loss of cell membrane integrity was preceded by a loss of mitochondrial membrane potential after treatment with aloin, suggesting a mitochondrial-dependent pathway for aloin-induced apoptosis.13 The combined treatment of cells with aloin and low doses of cisplatin increases the antiproliferative activity of aloin.14 Aloin may also inhibit tumor angiogenesis and growth via blocking STAT3 activation and has resulted in substantially reduced tumor volumes and weights in in vivo mouse xenografts without obvious toxicity.15 Aloin could therefore be a novel anticancer agent in a number of cancers. However, the antitumor behavior of aloin in lung cancer cells has not yet been clearly elucidated. This study showed that aloin can suppress the growth of human NSCLC cells. Apoptosis and autophagy were also observed after treatment with aloin. Our data therefore suggest that aloin can be used as an anticancer agent in human NSCLC.
Materials and methods Lung cancer cell lines and cell culture The H1299 cells were maintained in RPMI1640 medium (Gibco BRL, Grand Island, NY, USA) and the A549 cells were grown in Dulbecco’s Modified Eagle Medium (Gibco) with 10% fetal calf serum (Gibco) and 1% penicillin/streptomycin (Gibco); they were incubated at 37 C in 5% carbon dioxide. A549 contains wild type p53. H1299 contains null deletion p53.
Cell proliferation assay Aloin was purchased from SigmaeAldrich (St Louis, MO, USA). A549 or H1299 cells (5 103/well) were plated into 96-well tissue culture plates and grown in the specified medium. The cells were treated with medium only (the negative control) or medium containing aloin (12.5e400mM). After incubation for 72 hours, metabolically active cells were determined by 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. Three
145 independent experiments were analyzed statistically to give the final result.
Measurement of multinucleated cells Cells were treated with or without aloin for 72 hours. The nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI) (Sigma) for 20 minutes at room temperature. The morphology of cells with multiple nuclei (2 or more nuclei in 1 cell) was confirmed under a fluorescent microscope and compared with the observations obtained with light microscopy.
Apoptosis analysis For the cells treated with or without aloin, Annexin-V staining (SigmaeAldrich) was performed to detect apoptotic cells. The cells were washed twice with phosphate-buffered saline and the cell pellets were resuspended in a staining solution of Annexin-V-fluorescein isothiocyanate and propidium iodide (PI) and incubated for 15 minutes at room temperature in darkness. The Annexin-V or PI fluorescent intensities were analyzed by guava easyCyte flow cytometers (Merk Millipore, Germany); 10,000 cells were evaluated in each sample.
Western blot analysis of LC3-II expression The induction of autophagy by aloin was evaluated by the expression of LC3-II using Western blot analysis (antibody purchased from Medical & Biological Laboratories Co., Nagoya, Japan). The expression of beta-actin (antibody purchased from Cell Signaling Technology, Inc., USA.) was used as an internal control.
Results Aloin inhibition of cell growth of human lung cancer cells Two human NSCLC cell lines, A549 and H1299, were used to evaluate the inhibition of cell growth after treatment with aloin. The cellular viability was determined by MTT assay. As shown in Fig. 1, the cell viability decreased significantly in a concentration-dependent manner in these two cell lines. The IC50 of aloin in A549 and H1299 cells was about 150mM and 200mM after 72 hours of treatment, respectively (Fig. 1). These data show that aloin can suppress the growth of NSCLC cells in vitro.
Aloin induction of multinucleated cells in human NSCLC cells The formation of giant multinucleated cells is a phenotype of mitotic catastrophe induced by ionizing radiation or certain anticancer drugs.16 Aloin has previously been reported to induce aneuploid and increased S phase populations in human ovarian and breast cancer cells.12 We therefore investigated whether aloin induced multinucleation in our two cell lines. As shown in Fig. 2A, cells
Cell viability
146
M.-C. Lee et al.
1.6
Aloin induction of apoptosis in human NSCLC cells
1.4
To confirm whether aloin induced apoptosis in human NSCLC cells, cells were incubated in medium or medium with aloin for 24 hours, 48 hours, and 72 hours, and the apoptotic cells were then determined by flow cytometry. As shown in Fig. 3A, after treatment with aloin (200mM), the percentages of apoptotic cells in human A549 cells at 24 hours, 48 hours, and 72 hours were 3.14%, 4.6%, and 27.04%, respectively. The percentage of apoptotic cells in human H1299 cells after treatment with aloin (200mM) at 24 hours, 48 hours, and 72 hours were 1.06%, 0.49%, and 8.58%, respectively (Fig. 3B). These data show that aloin-induced apoptosis in lung cancer cells occurred at concentrations of about 200mM with a 72 hour treatment. A549 cells were more susceptible to aloin-induced apoptosis than H1299 cells.
1.2 1 0.8 0.6
A549 48h
0.4
A549 72h H1299 48h
0.2 0
H1299 72h
0
12.5
25
50
100
200
400
Aloin (µM) Figure 1 Aloin inhibition of the growth of human non-small cell lung cancer cells. Cells were incubated with different concentrations of aloin for 48 hours or 72 hours and the cellular viability of A549 and H1299 cells was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay. All the data are expressed as mean SEM values for three independent experiments.
containing an enlarged phenotype could be observed by microscopy after treatment with aloin. This phenomenon was confirmed by DAPI staining, which showed the presence of multinucleated cells in the groups treated with aloin (Fig. 2B). Aloin therefore induces the formation of multinucleated cells in human NSCLC cell lines.
Changes in LC3-II expression level after treatment with aloin in human NSCLC cells We also investigated whether autophagosome was formed in human NSCLC cells during treatment with aloin. Cells were treated with either medium or medium with aloin and the expression of LC3-II, a marker of autophagosome,17e22 was determined by Western blotting. As shown in Fig. 4, after treatment with aloin (100mM or 200mM) for 72 hours, the expression of LC3-II in A549 cells was increased on treatment with 100mM aloin, but reduced on treatment with 200mM aloin. However, LC3-II expression in H1299 cells was reduced in a concentration-dependent manner. These results suggest that the formation of autophagosome by aloin induction may be an anti-apoptosis effect in A549 cells at lower concentrations (100mM). However, 72 hours of treatment at the higher concentration (200mM) induced
Figure 2 Aloin-induced formation of multinuclear cells in human non-small cell lung cancer cells. (A) Cells were treated with 100mM or 200mM aloin for 72 hours and the cellular morphology was observed with microscopy. (B) Cells were treated with 100mM or 200mM aloin for 72 hours and the cellular nuclei were stained by 40 ,6-diamidino-2-phenylindole and observed under fluorescence microscopy. The multinuclear cells are indicated by arrows.
Aloin suppresses growth of lung cancer cells
147
Figure 3 Detection of aloin-mediated apoptosis in human non-small cell cancer cells. Cells were treated with 100mM or 200mM aloin and the apoptotic cells of (A) A549 and (B) H1299 cells were assessed by flow cytometry with Annexin-V/propidium iodide double staining.
148
M.-C. Lee et al. A549 0
100
H1299 200
0
100
200
(Aloin, μM)
susceptible to the cytotoxic effects of aloin than H1299 cells. Aloin may be a potential therapeutic agent against human NSCLCs and is worthy of further investigation.
LC3-I LC3-II
Conflicts of interest All contributing authors declare no conflicts of interest.
Figure 4 Autophagy induced by aloin in human non-smallcell lung cancer cells. Cells were treated with 100mM or 200mM aloin for 72 hours and the expression of LC3 was detected by Western blotting. Beta-actin was used as a loading control. Two independent experiments were carried, but only one is shown here.
apoptosis and suppressed autophagy. The decreased expression of LC3-II in H1299 cell lines requires further investigation.
Discussion We have shown the cytotoxic effects of aloin on human NSCLC cells (Fig. 1). The aloin-induced phenomenon of multinucleated cells is similar to previous observations in breast and ovarian tumor cells12 and was also observed in human NSCLC cells (Fig. 2). Compared with the H1299 cells, A549 cells are more susceptible to growth inhibition by aloin. Aloin-induced apoptosis has been reported in several human cancer cell lines.10e13 In this study, aloin was shown to induce a high percentage of apoptotic cells in the A549 cell line. Interestingly, the aloin-induced apoptosis of A549 cells is higher than that of H1299 cells (Figs. 3A and 3B). A549 contains wild type p53 and H1299 contains null deletion p53. The involvement of p53 in aloin-induced apoptosis in human NSCLC cells therefore needs further investigation. Recent reports have suggested that stress stimulates the autophagy process to adapt to changes in the surroundings. When the stress is above the survival threshold, cells will shift to an apoptotic process. If the apoptotic process is initiated, the autophagy program will stop.23 In the A549 cell line, the lower concentration of aloin (100mM) treatment consistently induced a higher level of expression of LC3-II and the apoptotic cells did not significantly increase. Furthermore, the IC50 concentration of aloin (200mM) promoted apoptosis and suppressed the expression of LC3-II. In addition, the cellular location of the p53 protein regulates autophagy.23 The cytosolic portion of p53 restrains autophagosome formation; however, the nuclear portion of p53 activates many genes to adapt to environmental stress.23 Under lethal conditions, p53 will translocate to the mitochondria to influence the permeability transition of the mitochondria and to eliminate any dysfunctional mitochondria.23 In the H1299 cell line, which contains the null mutation p53 gene, the expression of LC3-II was reduced in a concentration-dependent manner. Therefore the role played by p53 in aloin-induced autophagy in human NSCLC cells should be investigated further. We have shown the in vitro antitumor effects of aloin on human NSCLC cells through the formation of multinuclear cells, apoptosis, and/or autophagy. A549 cells are more
Acknowledgments This study was supported by the cooperative research grants (Grant No.10101009) of Madou Sin-Lau Hospital, Tainan, Taiwan and Chung Hwa University of Medical Technology, Tainan, Taiwan.
References 1. World Health Organization. Cancer. Geneva: World Health Organization; 2006. 2. Hansen HH. Treatment of advanced non-small cell lung cancer. BMJ. 2002;325:452e453. 3. Chen YT, Feng BG, Chen LB. Update of research on drug resistance in small cell lung cancer chemotherapy. Asian Pacific J Cancer Prev. 2012;13:3577e3581. 4. Gru ¨n M, Franz G. In vitro biosynthesis of the C-glycosidic bond in aloin. Planta. 1981;152:562e564. 5. Groon QJ, Reynols T. Barbaloin in aloe species. Planta Med. 1987;53:345e348. 6. Reynold T. The compounds in aloe leaf exudates: a review. Bot J Lin Soc. 1985;90:157e177. 7. Tan C, Zhu W, Lu Y. Aloin, cinnamic acid and sophorcarpidine are potent inhibitors of tyrosinase. Chin Med J (Engl). 2002; 115:1859e1862. 8. Barrantes E, Guinea M. Inhibition of collagenase and metalloproteinases by aloins and aloe gel. Life Sci. 2003;72: 843e850. 9. Hamiza O, Rehman M, Khan R, et al. Chemopreventive effects of aloin against 1,2-dimethylhydrazine-induced preneoplastic lesions in the colon of Wistar rats. Hum Exp Toxicol. 2014;33: 148e163. 10. Niciforovic A, Adzic M, Spasic SD, et al. Antitumor effects of a natural anthracycline analog (aloin) involve altered activity of antioxidant enzymes in HeLaS3 cells. Cancer Biol Ther. 2007;6: 1e6. 11. Esmat AY, Tomasetto C, Rio MC. Cytotoxicity of a natural anthraquinone (aloin) against human breast cancer cell lines with and without ErbB-2-topoisomerase IIa coamplification. Cancer Biol Ther. 2006;5:97e103. 12. Esmat AY, El-Gerzawy SM, Rafaat A. DNA ploidy and S phase fraction of breast and ovarian tumor cells treated with a natural anthracycline analog (aloin). Cancer Biol Ther. 2005;4: 108e112. 13. Buenz EJ. Aloin induces apoptosis in Jurkat cells. Toxicol in Vitro. 2008;22:422e429. 14. Tabolacci C, Rossi S, Lentini A, et al. Aloin enhances cisplatin antineoplastic activity in B16-F10 melanoma cells by transglutaminase-induced differentiation. Amino Acids. 2013; 44:293e300. 15. Pan Q, Pan H, Lou H, et al. Inhibition of the angiogenesis and growth of aloin in human colorectal cancer in vitro and in vivo. Cancer Cell Int. 2013;13:1e9. 16. Zhu J, Beattie EC, Yang Y, et al. Centrosome impairments and consequent cytokinesis defects are possible mechanisms of Taxane drugs. Anticancer Res. 2005;25:1919e1992.
Aloin suppresses growth of lung cancer cells 17. Mizushima N. Physiological functions of autophagy. Curr Top Microbiol Immunol. 2009;335:71e84. 18. Eskelinen EL, Saftig P. Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta. 2009;1793:664e673. 19. Mizushima N, Yamamoto A, Hatano M, et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol. 2001;152:657e668. 20. Sou YS, Waguri S, Iwata J, et al. The Atg8 conjugation system is indispensable for proper development of autophagic
149 isolation membranes in mice. Mol Biol Cell. 2008;19: 4762e4775. 21. Eskelinen EL. Fine structure of autophagosome. Methods Mol Biol. 2008;445:11e28. 22. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000;19:5720e5728. 23. Marin ˜o G, Niso-Santano M, Baehrecke EH, et al. Self-consumption: the interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol. 2014;15:81e94.
Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.j-bgm.com
ORIGINAL ARTICLE
Aloin-induced cell growth arrest, cell apoptosis, and autophagy in human non-small cell lung cancer cells Ming-Chin Lee a, Ji-Der Liao b, Wan-Ling Huang b, Fang-Yi Jiang b, Yu-Zih Jheng b, Yuan-Yuan Jin b, Ya-Shih Tseng b,* a
Department of Surgery, Mau-Dou Sin-Lau Christian Hospital, Tainan, Taiwan Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, Tainan, Taiwan
b
Received 10 July 2014; received in revised form 17 August 2014; accepted 21 August 2014
Available online 28 September 2014
KEYWORDS aloin; apoptosis; autophagy; lung cancer
Abstract Aloin, the major anthraquinone in aloe exudates and gels, has been shown to be an effective antioxidant and anti-inflammatory agent. We studied the antitumor effects of aloin in human non-small cell lung cancer cells. The treatment of lung cancer cells with aloin suppressed cell growth in a time- and concentration-dependent manner. Aloin-induced apoptosis also showed a concentration-dependent behavior when studied by flow cytometry. In addition, autophagy occurred after treatment with aloin. Taken together, our data demonstrate that aloin induces apoptosis and autophagy in human lung cancer cells. Aloin is therefore a potential therapeutic agent for human lung cancer and is worthy of further investigation. Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Introduction Lung cancer is the most common cause of cancer-related death worldwide and is classified as either small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). NSCLC * Corresponding author. Department of Medical Laboratory Science and Biotechnology, College of Medicine and Life Science, Chung Hwa University of Medical Technology, No.89, Wenhwa 1st St., Rende District, Tainan 71703, Taiwan. E-mail address: [email protected] (Y.-S. Tseng).
accounts for 75e80% of patients with primary lung cancer. Compared with NSCLC, SCLC shows an aggressive behavior with rapid tumor growth and early spread to distant sites; its malignancy is the highest of all types of lung cancer.1e3 Surgery is the most effective therapeutic modality, but the postoperative prognosis remains poor. In addition, most patients present with advanced stage disease and chemotherapy with or without radiotherapy is recommended. Although NSCLC and SCLC have a relatively good initial response to clinical treatment, relapse or disease progression may occur quickly and the prognosis for these patients
http://dx.doi.org/10.1016/j.bgm.2014.08.005 2214-0247/Copyright ª 2014, Taiwan Genomic Medicine and Biomarker Society. Published by Elsevier Taiwan LLC. All rights reserved.
Aloin suppresses growth of lung cancer cells remains poor.2,3 A novel and effective treatment for SCLC and NSCLC is therefore needed. Aloin, also known as barbaloin,4 is the major anthraquinone in aloe exudates and gels and has been characterized as the Cglycoside of aloe-emodin.5,6 Aloin is a potent inhibitor of tyrosinase and is an effective inhibitor of stimulated granulocyte matrix metalloproteinases.7,8 Aloin also protects against chemically induced colon toxicity and has been reported to act by increasing antioxidant levels and by having anti-inflammatory and antiproliferative effects.9 Aloin has been shown to cause a pronounced reduction in cell proliferation and has also been shown to induce cell apoptosis in HeLaS3 cells.10 At higher concentrations, it has caused a reduction in the proportion of cells undergoing mitosis by the induction of apoptosis, inhibition of topo II and protein expression, and the downregulation of cyclin B1 protein expression in MCF-7 cell lines.11 The treatment of breast and ovarian tumor cells with aloin showed a persistent aneuploid pattern and a significantly dose-dependent increase in the percentage of the S phase fraction and in the proportion of cells cycling at a higher ploidy level.12 Importantly, a loss of cell membrane integrity was preceded by a loss of mitochondrial membrane potential after treatment with aloin, suggesting a mitochondrial-dependent pathway for aloin-induced apoptosis.13 The combined treatment of cells with aloin and low doses of cisplatin increases the antiproliferative activity of aloin.14 Aloin may also inhibit tumor angiogenesis and growth via blocking STAT3 activation and has resulted in substantially reduced tumor volumes and weights in in vivo mouse xenografts without obvious toxicity.15 Aloin could therefore be a novel anticancer agent in a number of cancers. However, the antitumor behavior of aloin in lung cancer cells has not yet been clearly elucidated. This study showed that aloin can suppress the growth of human NSCLC cells. Apoptosis and autophagy were also observed after treatment with aloin. Our data therefore suggest that aloin can be used as an anticancer agent in human NSCLC.
Materials and methods Lung cancer cell lines and cell culture The H1299 cells were maintained in RPMI1640 medium (Gibco BRL, Grand Island, NY, USA) and the A549 cells were grown in Dulbecco’s Modified Eagle Medium (Gibco) with 10% fetal calf serum (Gibco) and 1% penicillin/streptomycin (Gibco); they were incubated at 37 C in 5% carbon dioxide. A549 contains wild type p53. H1299 contains null deletion p53.
Cell proliferation assay Aloin was purchased from SigmaeAldrich (St Louis, MO, USA). A549 or H1299 cells (5 103/well) were plated into 96-well tissue culture plates and grown in the specified medium. The cells were treated with medium only (the negative control) or medium containing aloin (12.5e400mM). After incubation for 72 hours, metabolically active cells were determined by 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyl tetrazolium bromide (MTT) assay. Three
145 independent experiments were analyzed statistically to give the final result.
Measurement of multinucleated cells Cells were treated with or without aloin for 72 hours. The nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI) (Sigma) for 20 minutes at room temperature. The morphology of cells with multiple nuclei (2 or more nuclei in 1 cell) was confirmed under a fluorescent microscope and compared with the observations obtained with light microscopy.
Apoptosis analysis For the cells treated with or without aloin, Annexin-V staining (SigmaeAldrich) was performed to detect apoptotic cells. The cells were washed twice with phosphate-buffered saline and the cell pellets were resuspended in a staining solution of Annexin-V-fluorescein isothiocyanate and propidium iodide (PI) and incubated for 15 minutes at room temperature in darkness. The Annexin-V or PI fluorescent intensities were analyzed by guava easyCyte flow cytometers (Merk Millipore, Germany); 10,000 cells were evaluated in each sample.
Western blot analysis of LC3-II expression The induction of autophagy by aloin was evaluated by the expression of LC3-II using Western blot analysis (antibody purchased from Medical & Biological Laboratories Co., Nagoya, Japan). The expression of beta-actin (antibody purchased from Cell Signaling Technology, Inc., USA.) was used as an internal control.
Results Aloin inhibition of cell growth of human lung cancer cells Two human NSCLC cell lines, A549 and H1299, were used to evaluate the inhibition of cell growth after treatment with aloin. The cellular viability was determined by MTT assay. As shown in Fig. 1, the cell viability decreased significantly in a concentration-dependent manner in these two cell lines. The IC50 of aloin in A549 and H1299 cells was about 150mM and 200mM after 72 hours of treatment, respectively (Fig. 1). These data show that aloin can suppress the growth of NSCLC cells in vitro.
Aloin induction of multinucleated cells in human NSCLC cells The formation of giant multinucleated cells is a phenotype of mitotic catastrophe induced by ionizing radiation or certain anticancer drugs.16 Aloin has previously been reported to induce aneuploid and increased S phase populations in human ovarian and breast cancer cells.12 We therefore investigated whether aloin induced multinucleation in our two cell lines. As shown in Fig. 2A, cells
Cell viability
146
M.-C. Lee et al.
1.6
Aloin induction of apoptosis in human NSCLC cells
1.4
To confirm whether aloin induced apoptosis in human NSCLC cells, cells were incubated in medium or medium with aloin for 24 hours, 48 hours, and 72 hours, and the apoptotic cells were then determined by flow cytometry. As shown in Fig. 3A, after treatment with aloin (200mM), the percentages of apoptotic cells in human A549 cells at 24 hours, 48 hours, and 72 hours were 3.14%, 4.6%, and 27.04%, respectively. The percentage of apoptotic cells in human H1299 cells after treatment with aloin (200mM) at 24 hours, 48 hours, and 72 hours were 1.06%, 0.49%, and 8.58%, respectively (Fig. 3B). These data show that aloin-induced apoptosis in lung cancer cells occurred at concentrations of about 200mM with a 72 hour treatment. A549 cells were more susceptible to aloin-induced apoptosis than H1299 cells.
1.2 1 0.8 0.6
A549 48h
0.4
A549 72h H1299 48h
0.2 0
H1299 72h
0
12.5
25
50
100
200
400
Aloin (µM) Figure 1 Aloin inhibition of the growth of human non-small cell lung cancer cells. Cells were incubated with different concentrations of aloin for 48 hours or 72 hours and the cellular viability of A549 and H1299 cells was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide assay. All the data are expressed as mean SEM values for three independent experiments.
containing an enlarged phenotype could be observed by microscopy after treatment with aloin. This phenomenon was confirmed by DAPI staining, which showed the presence of multinucleated cells in the groups treated with aloin (Fig. 2B). Aloin therefore induces the formation of multinucleated cells in human NSCLC cell lines.
Changes in LC3-II expression level after treatment with aloin in human NSCLC cells We also investigated whether autophagosome was formed in human NSCLC cells during treatment with aloin. Cells were treated with either medium or medium with aloin and the expression of LC3-II, a marker of autophagosome,17e22 was determined by Western blotting. As shown in Fig. 4, after treatment with aloin (100mM or 200mM) for 72 hours, the expression of LC3-II in A549 cells was increased on treatment with 100mM aloin, but reduced on treatment with 200mM aloin. However, LC3-II expression in H1299 cells was reduced in a concentration-dependent manner. These results suggest that the formation of autophagosome by aloin induction may be an anti-apoptosis effect in A549 cells at lower concentrations (100mM). However, 72 hours of treatment at the higher concentration (200mM) induced
Figure 2 Aloin-induced formation of multinuclear cells in human non-small cell lung cancer cells. (A) Cells were treated with 100mM or 200mM aloin for 72 hours and the cellular morphology was observed with microscopy. (B) Cells were treated with 100mM or 200mM aloin for 72 hours and the cellular nuclei were stained by 40 ,6-diamidino-2-phenylindole and observed under fluorescence microscopy. The multinuclear cells are indicated by arrows.
Aloin suppresses growth of lung cancer cells
147
Figure 3 Detection of aloin-mediated apoptosis in human non-small cell cancer cells. Cells were treated with 100mM or 200mM aloin and the apoptotic cells of (A) A549 and (B) H1299 cells were assessed by flow cytometry with Annexin-V/propidium iodide double staining.
148
M.-C. Lee et al. A549 0
100
H1299 200
0
100
200
(Aloin, μM)
susceptible to the cytotoxic effects of aloin than H1299 cells. Aloin may be a potential therapeutic agent against human NSCLCs and is worthy of further investigation.
LC3-I LC3-II
Conflicts of interest All contributing authors declare no conflicts of interest.
Figure 4 Autophagy induced by aloin in human non-smallcell lung cancer cells. Cells were treated with 100mM or 200mM aloin for 72 hours and the expression of LC3 was detected by Western blotting. Beta-actin was used as a loading control. Two independent experiments were carried, but only one is shown here.
apoptosis and suppressed autophagy. The decreased expression of LC3-II in H1299 cell lines requires further investigation.
Discussion We have shown the cytotoxic effects of aloin on human NSCLC cells (Fig. 1). The aloin-induced phenomenon of multinucleated cells is similar to previous observations in breast and ovarian tumor cells12 and was also observed in human NSCLC cells (Fig. 2). Compared with the H1299 cells, A549 cells are more susceptible to growth inhibition by aloin. Aloin-induced apoptosis has been reported in several human cancer cell lines.10e13 In this study, aloin was shown to induce a high percentage of apoptotic cells in the A549 cell line. Interestingly, the aloin-induced apoptosis of A549 cells is higher than that of H1299 cells (Figs. 3A and 3B). A549 contains wild type p53 and H1299 contains null deletion p53. The involvement of p53 in aloin-induced apoptosis in human NSCLC cells therefore needs further investigation. Recent reports have suggested that stress stimulates the autophagy process to adapt to changes in the surroundings. When the stress is above the survival threshold, cells will shift to an apoptotic process. If the apoptotic process is initiated, the autophagy program will stop.23 In the A549 cell line, the lower concentration of aloin (100mM) treatment consistently induced a higher level of expression of LC3-II and the apoptotic cells did not significantly increase. Furthermore, the IC50 concentration of aloin (200mM) promoted apoptosis and suppressed the expression of LC3-II. In addition, the cellular location of the p53 protein regulates autophagy.23 The cytosolic portion of p53 restrains autophagosome formation; however, the nuclear portion of p53 activates many genes to adapt to environmental stress.23 Under lethal conditions, p53 will translocate to the mitochondria to influence the permeability transition of the mitochondria and to eliminate any dysfunctional mitochondria.23 In the H1299 cell line, which contains the null mutation p53 gene, the expression of LC3-II was reduced in a concentration-dependent manner. Therefore the role played by p53 in aloin-induced autophagy in human NSCLC cells should be investigated further. We have shown the in vitro antitumor effects of aloin on human NSCLC cells through the formation of multinuclear cells, apoptosis, and/or autophagy. A549 cells are more
Acknowledgments This study was supported by the cooperative research grants (Grant No.10101009) of Madou Sin-Lau Hospital, Tainan, Taiwan and Chung Hwa University of Medical Technology, Tainan, Taiwan.
References 1. World Health Organization. Cancer. Geneva: World Health Organization; 2006. 2. Hansen HH. Treatment of advanced non-small cell lung cancer. BMJ. 2002;325:452e453. 3. Chen YT, Feng BG, Chen LB. Update of research on drug resistance in small cell lung cancer chemotherapy. Asian Pacific J Cancer Prev. 2012;13:3577e3581. 4. Gru ¨n M, Franz G. In vitro biosynthesis of the C-glycosidic bond in aloin. Planta. 1981;152:562e564. 5. Groon QJ, Reynols T. Barbaloin in aloe species. Planta Med. 1987;53:345e348. 6. Reynold T. The compounds in aloe leaf exudates: a review. Bot J Lin Soc. 1985;90:157e177. 7. Tan C, Zhu W, Lu Y. Aloin, cinnamic acid and sophorcarpidine are potent inhibitors of tyrosinase. Chin Med J (Engl). 2002; 115:1859e1862. 8. Barrantes E, Guinea M. Inhibition of collagenase and metalloproteinases by aloins and aloe gel. Life Sci. 2003;72: 843e850. 9. Hamiza O, Rehman M, Khan R, et al. Chemopreventive effects of aloin against 1,2-dimethylhydrazine-induced preneoplastic lesions in the colon of Wistar rats. Hum Exp Toxicol. 2014;33: 148e163. 10. Niciforovic A, Adzic M, Spasic SD, et al. Antitumor effects of a natural anthracycline analog (aloin) involve altered activity of antioxidant enzymes in HeLaS3 cells. Cancer Biol Ther. 2007;6: 1e6. 11. Esmat AY, Tomasetto C, Rio MC. Cytotoxicity of a natural anthraquinone (aloin) against human breast cancer cell lines with and without ErbB-2-topoisomerase IIa coamplification. Cancer Biol Ther. 2006;5:97e103. 12. Esmat AY, El-Gerzawy SM, Rafaat A. DNA ploidy and S phase fraction of breast and ovarian tumor cells treated with a natural anthracycline analog (aloin). Cancer Biol Ther. 2005;4: 108e112. 13. Buenz EJ. Aloin induces apoptosis in Jurkat cells. Toxicol in Vitro. 2008;22:422e429. 14. Tabolacci C, Rossi S, Lentini A, et al. Aloin enhances cisplatin antineoplastic activity in B16-F10 melanoma cells by transglutaminase-induced differentiation. Amino Acids. 2013; 44:293e300. 15. Pan Q, Pan H, Lou H, et al. Inhibition of the angiogenesis and growth of aloin in human colorectal cancer in vitro and in vivo. Cancer Cell Int. 2013;13:1e9. 16. Zhu J, Beattie EC, Yang Y, et al. Centrosome impairments and consequent cytokinesis defects are possible mechanisms of Taxane drugs. Anticancer Res. 2005;25:1919e1992.
Aloin suppresses growth of lung cancer cells 17. Mizushima N. Physiological functions of autophagy. Curr Top Microbiol Immunol. 2009;335:71e84. 18. Eskelinen EL, Saftig P. Autophagy: a lysosomal degradation pathway with a central role in health and disease. Biochim Biophys Acta. 2009;1793:664e673. 19. Mizushima N, Yamamoto A, Hatano M, et al. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells. J Cell Biol. 2001;152:657e668. 20. Sou YS, Waguri S, Iwata J, et al. The Atg8 conjugation system is indispensable for proper development of autophagic
149 isolation membranes in mice. Mol Biol Cell. 2008;19: 4762e4775. 21. Eskelinen EL. Fine structure of autophagosome. Methods Mol Biol. 2008;445:11e28. 22. Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J. 2000;19:5720e5728. 23. Marin ˜o G, Niso-Santano M, Baehrecke EH, et al. Self-consumption: the interplay of autophagy and apoptosis. Nat Rev Mol Cell Biol. 2014;15:81e94.