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Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death
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Kaohsiung Journal of Medical Sciences (2017) xx, 1e8
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.kjms-online.com
Original Article
Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death Hou-Long Long a, Feng-Feng Zhang a, Hong-Ling Wang b, Wen-Shi Yang a, Hai-Tao Hou a, Jing-Kui Yu a, Bin Liu a,* a
Department of Breast and Thyroid Surgery, Tengzhou Central People’s Hospital, Tengzhou City, Shangdong Province, PR China b Department of Ultrasound Tengzhou Central People’s Hospital, Tengzhou City, Shangdong Province, PR China Received 22 May 2017; accepted 8 November 2017
KEYWORDS Mulberry anthocyanins; Thyroid cancer; Apoptosis; Autophagic death
Abstract Dietary anthocyanin compounds have multiple biological effects, including antioxidant, anti-inflammatory, and anti-atherosclerotic characteristics. The present study evaluated the anti-tumor capacity of mulberry anthocyanins (MA) in thyroid cancer cells. Our data showed that MA suppressed SW1736 and HTh-7 cell proliferation in a time- and dosedependent manner. Meanwhile, flow cytometry results indicated that MA significantly increased SW1736 and HTh-7 cell apoptosis. We additionally observed that SW1736 and HTh7 cell autophagy was markedly enhanced after MA treatment. Importantly, anthocyanininduced cell death was largely abolished by 3-methyladenine (3-MA) or chloroquine diphosphate salt (CQ) treatment, suggesting that MA-induced SW1736 and HTh-7 cell death was partially dependent on autophagy. In addition, activation of protein kinase B (Akt), mammalian target of rapamycin (mTOR), and ribosomal protein S6 (S6) were significantly suppressed by anthocyanin exposure. In summary, MA may serve as an adjunctive therapy for thyroid cancer patients through induction of apoptosis and autophagy-dependent cell death. Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Conflicts of interest: All authors declare no conflicts of interests. * Corresponding author. Department of Breast and Thyroid Surgery, Tengzhou Central People’s Hospital, #181 Xingtan Road, Tengzhou City, Shangdong Province, PR China. E-mail address: [email protected] (B. Liu). https://doi.org/10.1016/j.kjms.2017.11.004 1607-551X/Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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Introduction In traditional Chinese medicine, mulberry serves as both a food and medicinal source [1]. Nutritional medical research indicates that mulberry ingestion can enhance immune function, promote hematopoietic cell growth, prevent atherosclerosis, and improve metabolism [2,3]. The pharmacologic activities of mulberry are due to its unique ingredients, including some bioactive substances [4]. Research shows that mature mulberry anthocyanins (MA) contains abundant dietary compounds and is a natural pigment widely present in vegetables, fruits and other foods [5]. Epidemiological and experimental studies have demonstrated that dietary anthocyanin compounds have multiple biological effects, including antioxidant, antiinflammatory, antitumor, and anti-atherosclerotic characteristics [6,7]. Thyroid cancer incidence is very high, and it is the most common malignant tumor in the endocrine system [8,9]. There are four main types of thyroid carcinoma: papillary thyroid carcinoma, medullary carcinoma, undifferentiated carcinoma and follicular thyroid carcinoma [10]. At present, the major therapeutic modality for thyroid papillary carcinoma is surgery combined with radioactive iodine cleaning or a chemical treatment program [11]. However, such treatment methods do not achieve satisfactory response in undifferentiated thyroid carcinoma and medullary thyroid carcinoma due to radiotherapy resistance. The major reason for this may be attributed to apoptosis escape [12,13]. Therefore, it is urgent to explore additional methods to induce the death of thyroid cancer cells. Autophagy refers to the process in which autophagosomes remove damaged or aging organelles to maintain intracellular stability [14]. Recent studies have shown that multiple anticancer treatments result in the disorders of excessive autophagy and further lead to cancer cell death [14,15]. The Akt/mTOR pathway is suggested to play a key role in nutrient-induced autophagy and is tightly correlated with oncogenesis in different cancer cells [16]. However, whether MA could induce autophagic death through this pathway in thyroid cancer cells has never been explored. In the present study, we explored the effects of MA on thyroid cancer cells. Our data showed that MA enhanced thyroid cancer cell death, primarily by inducing apoptosis and autophagic-induced cell death.
Materials and methods Cell lines and cultures SW1736 (BRAFV600E/wt) and HTh-7 (NRASQ61R) thyroid cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) F-12 culture medium (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% heatinactivated fetal calf serum (FBS), 100 U/ml penicillin, and streptomycin in 25 cm2 culture flasks at 37 C in a humidified atmosphere with 5% CO2.
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Extraction and determination of anthocyanins from mulberry Fresh mulberry (Morus alba L.) was purchased from a local fruit market in Tengzhou. Anthocyanins were isolated and purified as previously described [17]. Briefly, fruit was extracted with a threefold volume of 95% ethanol containing 1% HCl for 24 h at 4 C. Filtered fluid was evaporated at 38 C and then the concentrates were loaded onto an equilibrated macroporous resin column orderly eluted with 1% formic acid in methanol for further purification. MA was obtained by lyophilization and stored at 80 C before use. Composition and content of anthocyanins in MAE were determined by an HPLC instrument (Thermo UltiMate 3000, Waltham, MA, USA). A linear gradient from 5% A (acetonitrile) to 40% B (water containing 10% formic acid) in 30 min was used for HPLC assay. The column was operated at a temperature of 30 C and absorption spectrum was recorded at 520 nm.
MTT colorimetric assays To investigate the influence of MA on cancer cell proliferation, HTh-7 cells were seeded in 96-well tissue culture plates at a density of 5 104 cells per well. Then, SW1736 and HTh-7 cells were treated with 1, 10, and 30 mg/ml MA for 48 h. For time-dependent assay, SW1736 and HTh-7 cells were treated with 10 mg/ml MA at 24, 48, 72 h. Cell viability was determined with an MTT assay kit (Sigma, St. Louis, MO, USA). The blue formazan products in the SW1736 and HTh-7 cells were dissolved in DMSO and spectrophotometrically measured at a wavelength of 550 nm. All experiments were conducted in triplicate.
Cell migration and invasion assays Cell migration assays were performed using Boyden chambers with 8 mm-pore filters (Corning, New York, USA). For cell invasion assays, the upper chamber was pre-coated with matrigel (356234, BD, USA). SW1736 and HTh-7 cells treated with DMSO control or 10 mg/ml MA in (DMEM) F-12 medium without FBS were plated on the upper chamber for 48 h. 600 mL of medium with 20% FBS as chemoattractants were plated on the lower chamber of the 24-well plates. Cells were then incubated under standard culture conditions for 48 h. Non-invading cells in the upper chamber were completely removed using a cotton swab. Cells that invaded to the lower surface of the membrane were fixed in methanol for 30 min at 37 C and stained with 0.5% crystal violet for 1 h. Cell numbers were quantified by counting the number of stained nuclei in five random fields by fluorescence microscopy in triplicate.
Colony formation assay SW1736 or HTh-7 cells were seeded into 6-well plates at a density of 2000 cells per well. After incubation for 24 h, the non-adherent cells were removed, and the remaining cells
Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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MA improves TC by inducing apoptosis and autophagy continued to be cultured for 1 week. The colonies were fixed with 4% paraformaldehyde, stained with 1% crystal violet and countered under a microscope. Each colony should contain no less than 50 cells.
GFP-LC3 transient transfection SW1736 and HTh-7 cells were seeded at a density of 5 105 cells/well in 6-well plates for 24 h. At that time, a GFP-LC3 expressing plasmid was transfected into cells using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Then, GFPLC3-positive dots were counted under a confocal laser microscope, LSM700 (Carl Zeiss, Jena, Germany).
Flow cytometry assay Apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. After treatment, the cells were collected in a 5 ml culture tube and washed twice with cold PBS. Then, the cells were resuspended in 1 binding buffer and transferred to a 5 ml culture tube. FITCAnnexinV (5 ml) and propidium iodide (5 ml) were added and incubated with the cells at room temperature for 15 min in the dark. Then, 400 ml of 1 binding buffer was added, and the samples were analyzed within 1 h using flow cytometry.
Electron microscopy SW1736 and HTh-7 cells were fixed with 2% glutaraldehyde paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4 for 12 h at 4 C and washed three times for 30 min in 0.1 M PB. Then, the samples were postfixed with 1% OsO4 dissolved in 0.1 M PB for 2 h, dehydrated in an ascending gradual series (50e100%) of ethanol and infiltrated with propylene oxide. After pure fresh resin embedment and polymerization at 60 C in an electron microscope oven (TD700, DOSAKA, Kyoto, Japan) for 24 h, 70 nm thin sections were made with a LEICA Ultracut UCT Ultra-microtome (Leica Microsystems, Wetzlar, Germany). All thin sections were observed by TEM (JEM-1011, JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV.
Western blot Total protein samples were extracted from the cultured cells using RIPA buffer (Solarbio, Beijing, China) according to the instructions. The immunoblotting was carried out as previously described [12]. The primary antibodies were purchased from Cell Signaling: anti-p-AKT (Cell Signaling Technology, Inc., Boston, MA, USA), anti-Akt (Cell Signaling Technology, Inc., Boston, MA, USA), anti-p-mTOR (Cell Signaling Technology, Inc., Boston, MA, USA), anti-mTOR (Cell Signaling Technology, Inc., Boston, MA, USA), anti-pS6 (Cell Signaling Technology, Inc., Boston, MA, USA), anti-S6 (Cell Signaling Technology, Inc., Boston, MA, USA), anti-LC3 (SigmaeAldrich, St. Louis, MO, USA), and anti-bactin (Cell Signaling Technology, Inc., Boston, MA, USA). The protein of interest was further detected with goat antimouse or ant-rabbit IgG-HRP secondary antibody.
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Statistical analysis Data are expressed as the means SE. Multiple comparisons were evaluated by ANOVA followed by Turkey’s multiple-comparison procedure with p < 0.05 being considered significant.
Results MA suppressed thyroid cancer cell proliferation in a dose- and time-dependent manner We first evaluated the effect of MA on SW1736 and HTh-7 cell viability using MTT assay. Our data showed that 1, 10, and 30 mg/ml MA treatment for 48 h markedly reduced SW1736 and HTh-7 cell viability (Fig. 1A and B). In addition, SW1736 and HTh-7 cell viability was significantly inhibited by treatment with 10 mg/ml MA at 24, 48, 72 h, respectively (Fig. 1C and D).
MA decreased SW1736 and HTh-7 cell invastion and increased apoptosis Next, we explored the role of MA on SW1736 and HTh-7 cell migration and apoptosis. As shown in Fig. 2A and B, treatment with 10 mg/ml MA for 48 h decreased SW1736 and HTh7 colony formation and invasion, respectively. Moreover, we also found that the levels of apoptosis were significantly increased in SW1736 and HTh-7 cells in the presence of anthocyanins for 48 h (Fig. 2C), indicating a tumor suppressor role for anthocyanins in thyroid cancer cells.
MA enhanced SW1736 and HTh-7 cell autophagy To further explore the effects of MA on thyroid cancer cells, we evaluated the autophagy pathway in MA-treated SW1736 and HTh-7 cells. Western blot analysis demonstrated that MA significantly increased the LC3II/LC3I ratio in a pattern comparable to rapamycin after 48 h (Fig. 3A and B). Combination use of MA and rapamycin significantly increased autophagic activity in these cells after 48 h (Fig. 3A and B). Transmission electron microscopy also demonstrated an enhanced presence of autophagic vesicles in cells treated with 10 mg/ml MA for 48 h (Fig. 3C). Additionally, GFP-LC3 transfection was used to detect autophagic activity, and our data showed that treatment with MA increased GFP puncta, indicating autophagic vacuoles, in SW1736 and HTh-7 cells. These effects could be suppressed by addition of 3-MA, indicating that MA induced autophagic vacuole formation, as evidenced by LC3II accumulation (Fig. 3D).
MA-induced SW1736 and HTh-7 cell death correlated with severe autophagy To explore whether the MA-induced cell death observed in SW1736 and HTh-7 cells was partially attributed to MA (10 mM)-induced autophagy, 3-MA (5 mM) or chloroquine (CQ, 40 mM), the autophagolysosome fusion inhibitor, was utilized. As shown in Fig. 4A and C, MA-induced cell death
Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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Figure 1. MA suppressed thyroid cancer cell proliferation in a dose- and time-dependent manner. MA markedly reduced SW1736 (A) and HTh-7 (B) cell viability at doses of 1, 10, and 30 mg/ml for 48 h. SW1736 (C) and HTh-7 (D) cell viability was significantly inhibited by treatment with 10 mg/ml MA at 24, 48, and 72 h, respectively. )p < 0.05, ))p < 0.01, )))p < 0.001 vs. control.
was largely abolished by 3-MA incubation for 48 h in SW1736 and HTh-7 cells. Likewise, after treatment with CQ for 48 h, cell death of SW1736 and HTh-7 caused by MA was largely repressed (Fig. 4B and D). These results demonstrated that MA-induced SW1736 and HTh-7 cell death was dependent on autophagy in these thyroid cancer lines.
MA suppressed Akt/mTOR signaling in SW1736 and HTh-7 cells The Akt/mTOR signaling pathway is an important regulator of both autophagy and cancer cell proliferation. Western blot analysis illustrated that MA markedly reduced Akt, mTOR, and S6 activation in SW1736 and HTh-7 cells, indicating that MA acted as a tumor suppressor in thyroid cancer cells (Fig. 5).
Discussion Previous studies have shown that MA includes abundant compounds that are the material basis of mulberry’s dark brown color [3,18,19]. Anthocyanin is a kind of natural plant pigment, which is widely found in plants and a variety of fruits and vegetables [18]. Anthocyanins are present in the form of glycosides or aglycone, which are
characterized by antioxidant, anti-inflammatory, antitumor, anti-atherosclerotic biological effects [20]. Malignant tumors are among the world’s most common disease and a cause of significant morbidity and mortality in human health [21]. Recently, isolating dietary compounds from natural food has become an important research avenue with respect to the prevention and treatment of various tumors [18,22,23]. Thyroid cancer is the most common endocrine system malignancy with an overall good prognosis. However, due to the development of radiation resistance, traditional antitumor treatments do not achieve satisfactory therapeutic efficacy in papillary thyroid carcinoma, undifferentiated carcinoma and medullary carcinoma [24,25]. In the present study, we mainly evaluated the anti-carcinogenic and antiproliferative effects of MA on thyroid cancer cells. Our data indicated that MA significantly suppressed SW1736 and HTh7 cell viability in a time- and dose-dependent manner. Further study revealed that MA significantly reduced SW1736 and HTh-7 cell colony formation and invasion capacity. These data indicated an important tumor suppressor role of MA in thyroid cancer cells. Meanwhile, we also investigated the induction of cell death by MA treatment. Firstly, we focused on apoptosis, which plays a critical role in many biological activities, including the evolution of organisms and the stability of the
Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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Figure 2. MA decreased SW1736 and HTh-7 cell migration while increasing apoptosis. Treatment with 10 mg/ml MA for 48 h decreased SW1736 (A) and HTh-7 (B) cell migration and invasion capacity. Apoptosis in SW1736 (C) and HTh-7 (D) cells was significantly increased in response to 10 mg/ml MA for 48 h. )p < 0.05, ))p < 0.01 vs. control.
microenvironment [21,26]. Apoptosis evasion is closely related to the occurrence and development of tumors, and activation of apoptosis could be a helpful strategy to eliminate tumor cells [24]. The ability to induce apoptosis
in tumor cells is a powerful anti-tumor mechanism [27]. Here, our data showed that MA enhanced apoptosis in two thyroid cancer cell lines, suggesting the anti-carcinogenesis role of MA in thyroid cancer cells.
Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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Figure 3. MA enhanced autophagy in SW1736 and HTh-7 cells. Western blot analysis of SW1736 (A) and HTh-7 (B) cells showed the ratio between LC3II/LC3I was increased by 10 mg/ml MA for 48 h. (C) Transmission electron microscopy demonstrated enhanced autophagic vesicles in SW1736 and HTh-7 cells treated with 10 mg/ml MA for 48 h. (D) GFP-LC3 transfection indicated 10 mg/ml MA for 48 h increased SW1736 and HTh-7 cell autophagy. )p < 0.05 vs. control.
Furthermore, researchers have also begun to focus on the autophagy pathway with respect to the pathogenesis and development of thyroid cancer, particularly in tumors that are not sensitive to conventional therapy. Autophagy responds quickly to myriad internal and external environmental pressures that exist in eukaryotic cells and that happen during the aging process [28,29]. Autophagy not only is a cell survival mechanism but also can induce cell death. Thus, the potential role of MA in thyroid cancer cell autophagy was further explored. In line with rapamycin, MA
treatment could enhance the LC3II/LC3I ratio, indicating the activation of autophagy in thyroid cancer cells. And such effects were further determined by transmission electron microscopy and GFP-LC3 transfection. The above findings motivated us to further explore the potential mechanism in which MA induced autophagy. The Akt/mTOR pathway plays an important role in regulating cell autophagy [30]. Therefore, we next investigated whether the effect of MA on SW1736 and HTh-7 cell autophagy is achieved through this pathway. Here, we observed
Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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Figure 4. MA-induced SW1736 and HTh-7 cell death correlated with heightened autophagic activity. MA-induced cell death was largely abolished by addition of 3-MA for 48 h in a dose-dependent manner in SW1736 (A) and HTh-7 cells (C). After inhibition of autophagy by CQ for 48 h, cell death of SW1736 (B) and HTh-7 (D) caused by MA was greatly reduced. )p < 0.05 vs. control.
Figure 5. 10 mg/ml MA treatment for 48 h suppressed the activation of Akt/mTOR signaling in SW1736 and HTh-7 cells. )p < 0.05, ))p < 0.01 vs. control.
that MA suppressed the activation of Akt/mTOR signaling in the thyroid cancer cells, further supporting the tumor suppressor role of MA. In conclusion, MA may serve as a novel therapy method for thyroid cancer mainly by inducing apoptosis and autophagic-induced cell death. Increasing research is investigating the relationship between autophagy and thyroid cancer, and it will be important to explore novel methods in which autophagy can be applied to the clinical treatment of thyroid cancer.
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Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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Kaohsiung Journal of Medical Sciences (2017) xx, 1e8
Available online at www.sciencedirect.com
ScienceDirect journal homepage: http://www.kjms-online.com
Original Article
Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death Hou-Long Long a, Feng-Feng Zhang a, Hong-Ling Wang b, Wen-Shi Yang a, Hai-Tao Hou a, Jing-Kui Yu a, Bin Liu a,* a
Department of Breast and Thyroid Surgery, Tengzhou Central People’s Hospital, Tengzhou City, Shangdong Province, PR China b Department of Ultrasound Tengzhou Central People’s Hospital, Tengzhou City, Shangdong Province, PR China Received 22 May 2017; accepted 8 November 2017
KEYWORDS Mulberry anthocyanins; Thyroid cancer; Apoptosis; Autophagic death
Abstract Dietary anthocyanin compounds have multiple biological effects, including antioxidant, anti-inflammatory, and anti-atherosclerotic characteristics. The present study evaluated the anti-tumor capacity of mulberry anthocyanins (MA) in thyroid cancer cells. Our data showed that MA suppressed SW1736 and HTh-7 cell proliferation in a time- and dosedependent manner. Meanwhile, flow cytometry results indicated that MA significantly increased SW1736 and HTh-7 cell apoptosis. We additionally observed that SW1736 and HTh7 cell autophagy was markedly enhanced after MA treatment. Importantly, anthocyanininduced cell death was largely abolished by 3-methyladenine (3-MA) or chloroquine diphosphate salt (CQ) treatment, suggesting that MA-induced SW1736 and HTh-7 cell death was partially dependent on autophagy. In addition, activation of protein kinase B (Akt), mammalian target of rapamycin (mTOR), and ribosomal protein S6 (S6) were significantly suppressed by anthocyanin exposure. In summary, MA may serve as an adjunctive therapy for thyroid cancer patients through induction of apoptosis and autophagy-dependent cell death. Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/ by-nc-nd/4.0/).
Conflicts of interest: All authors declare no conflicts of interests. * Corresponding author. Department of Breast and Thyroid Surgery, Tengzhou Central People’s Hospital, #181 Xingtan Road, Tengzhou City, Shangdong Province, PR China. E-mail address: [email protected] (B. Liu). https://doi.org/10.1016/j.kjms.2017.11.004 1607-551X/Copyright ª 2017, Kaohsiung Medical University. Published by Elsevier Taiwan LLC. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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Introduction In traditional Chinese medicine, mulberry serves as both a food and medicinal source [1]. Nutritional medical research indicates that mulberry ingestion can enhance immune function, promote hematopoietic cell growth, prevent atherosclerosis, and improve metabolism [2,3]. The pharmacologic activities of mulberry are due to its unique ingredients, including some bioactive substances [4]. Research shows that mature mulberry anthocyanins (MA) contains abundant dietary compounds and is a natural pigment widely present in vegetables, fruits and other foods [5]. Epidemiological and experimental studies have demonstrated that dietary anthocyanin compounds have multiple biological effects, including antioxidant, antiinflammatory, antitumor, and anti-atherosclerotic characteristics [6,7]. Thyroid cancer incidence is very high, and it is the most common malignant tumor in the endocrine system [8,9]. There are four main types of thyroid carcinoma: papillary thyroid carcinoma, medullary carcinoma, undifferentiated carcinoma and follicular thyroid carcinoma [10]. At present, the major therapeutic modality for thyroid papillary carcinoma is surgery combined with radioactive iodine cleaning or a chemical treatment program [11]. However, such treatment methods do not achieve satisfactory response in undifferentiated thyroid carcinoma and medullary thyroid carcinoma due to radiotherapy resistance. The major reason for this may be attributed to apoptosis escape [12,13]. Therefore, it is urgent to explore additional methods to induce the death of thyroid cancer cells. Autophagy refers to the process in which autophagosomes remove damaged or aging organelles to maintain intracellular stability [14]. Recent studies have shown that multiple anticancer treatments result in the disorders of excessive autophagy and further lead to cancer cell death [14,15]. The Akt/mTOR pathway is suggested to play a key role in nutrient-induced autophagy and is tightly correlated with oncogenesis in different cancer cells [16]. However, whether MA could induce autophagic death through this pathway in thyroid cancer cells has never been explored. In the present study, we explored the effects of MA on thyroid cancer cells. Our data showed that MA enhanced thyroid cancer cell death, primarily by inducing apoptosis and autophagic-induced cell death.
Materials and methods Cell lines and cultures SW1736 (BRAFV600E/wt) and HTh-7 (NRASQ61R) thyroid cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) F-12 culture medium (GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% heatinactivated fetal calf serum (FBS), 100 U/ml penicillin, and streptomycin in 25 cm2 culture flasks at 37 C in a humidified atmosphere with 5% CO2.
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Extraction and determination of anthocyanins from mulberry Fresh mulberry (Morus alba L.) was purchased from a local fruit market in Tengzhou. Anthocyanins were isolated and purified as previously described [17]. Briefly, fruit was extracted with a threefold volume of 95% ethanol containing 1% HCl for 24 h at 4 C. Filtered fluid was evaporated at 38 C and then the concentrates were loaded onto an equilibrated macroporous resin column orderly eluted with 1% formic acid in methanol for further purification. MA was obtained by lyophilization and stored at 80 C before use. Composition and content of anthocyanins in MAE were determined by an HPLC instrument (Thermo UltiMate 3000, Waltham, MA, USA). A linear gradient from 5% A (acetonitrile) to 40% B (water containing 10% formic acid) in 30 min was used for HPLC assay. The column was operated at a temperature of 30 C and absorption spectrum was recorded at 520 nm.
MTT colorimetric assays To investigate the influence of MA on cancer cell proliferation, HTh-7 cells were seeded in 96-well tissue culture plates at a density of 5 104 cells per well. Then, SW1736 and HTh-7 cells were treated with 1, 10, and 30 mg/ml MA for 48 h. For time-dependent assay, SW1736 and HTh-7 cells were treated with 10 mg/ml MA at 24, 48, 72 h. Cell viability was determined with an MTT assay kit (Sigma, St. Louis, MO, USA). The blue formazan products in the SW1736 and HTh-7 cells were dissolved in DMSO and spectrophotometrically measured at a wavelength of 550 nm. All experiments were conducted in triplicate.
Cell migration and invasion assays Cell migration assays were performed using Boyden chambers with 8 mm-pore filters (Corning, New York, USA). For cell invasion assays, the upper chamber was pre-coated with matrigel (356234, BD, USA). SW1736 and HTh-7 cells treated with DMSO control or 10 mg/ml MA in (DMEM) F-12 medium without FBS were plated on the upper chamber for 48 h. 600 mL of medium with 20% FBS as chemoattractants were plated on the lower chamber of the 24-well plates. Cells were then incubated under standard culture conditions for 48 h. Non-invading cells in the upper chamber were completely removed using a cotton swab. Cells that invaded to the lower surface of the membrane were fixed in methanol for 30 min at 37 C and stained with 0.5% crystal violet for 1 h. Cell numbers were quantified by counting the number of stained nuclei in five random fields by fluorescence microscopy in triplicate.
Colony formation assay SW1736 or HTh-7 cells were seeded into 6-well plates at a density of 2000 cells per well. After incubation for 24 h, the non-adherent cells were removed, and the remaining cells
Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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MA improves TC by inducing apoptosis and autophagy continued to be cultured for 1 week. The colonies were fixed with 4% paraformaldehyde, stained with 1% crystal violet and countered under a microscope. Each colony should contain no less than 50 cells.
GFP-LC3 transient transfection SW1736 and HTh-7 cells were seeded at a density of 5 105 cells/well in 6-well plates for 24 h. At that time, a GFP-LC3 expressing plasmid was transfected into cells using Lipofectamine 2000 Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Then, GFPLC3-positive dots were counted under a confocal laser microscope, LSM700 (Carl Zeiss, Jena, Germany).
Flow cytometry assay Apoptosis was measured using an Annexin V-FITC Apoptosis Detection kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s protocol. After treatment, the cells were collected in a 5 ml culture tube and washed twice with cold PBS. Then, the cells were resuspended in 1 binding buffer and transferred to a 5 ml culture tube. FITCAnnexinV (5 ml) and propidium iodide (5 ml) were added and incubated with the cells at room temperature for 15 min in the dark. Then, 400 ml of 1 binding buffer was added, and the samples were analyzed within 1 h using flow cytometry.
Electron microscopy SW1736 and HTh-7 cells were fixed with 2% glutaraldehyde paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4 for 12 h at 4 C and washed three times for 30 min in 0.1 M PB. Then, the samples were postfixed with 1% OsO4 dissolved in 0.1 M PB for 2 h, dehydrated in an ascending gradual series (50e100%) of ethanol and infiltrated with propylene oxide. After pure fresh resin embedment and polymerization at 60 C in an electron microscope oven (TD700, DOSAKA, Kyoto, Japan) for 24 h, 70 nm thin sections were made with a LEICA Ultracut UCT Ultra-microtome (Leica Microsystems, Wetzlar, Germany). All thin sections were observed by TEM (JEM-1011, JEOL, Tokyo, Japan) at an acceleration voltage of 80 kV.
Western blot Total protein samples were extracted from the cultured cells using RIPA buffer (Solarbio, Beijing, China) according to the instructions. The immunoblotting was carried out as previously described [12]. The primary antibodies were purchased from Cell Signaling: anti-p-AKT (Cell Signaling Technology, Inc., Boston, MA, USA), anti-Akt (Cell Signaling Technology, Inc., Boston, MA, USA), anti-p-mTOR (Cell Signaling Technology, Inc., Boston, MA, USA), anti-mTOR (Cell Signaling Technology, Inc., Boston, MA, USA), anti-pS6 (Cell Signaling Technology, Inc., Boston, MA, USA), anti-S6 (Cell Signaling Technology, Inc., Boston, MA, USA), anti-LC3 (SigmaeAldrich, St. Louis, MO, USA), and anti-bactin (Cell Signaling Technology, Inc., Boston, MA, USA). The protein of interest was further detected with goat antimouse or ant-rabbit IgG-HRP secondary antibody.
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Statistical analysis Data are expressed as the means SE. Multiple comparisons were evaluated by ANOVA followed by Turkey’s multiple-comparison procedure with p < 0.05 being considered significant.
Results MA suppressed thyroid cancer cell proliferation in a dose- and time-dependent manner We first evaluated the effect of MA on SW1736 and HTh-7 cell viability using MTT assay. Our data showed that 1, 10, and 30 mg/ml MA treatment for 48 h markedly reduced SW1736 and HTh-7 cell viability (Fig. 1A and B). In addition, SW1736 and HTh-7 cell viability was significantly inhibited by treatment with 10 mg/ml MA at 24, 48, 72 h, respectively (Fig. 1C and D).
MA decreased SW1736 and HTh-7 cell invastion and increased apoptosis Next, we explored the role of MA on SW1736 and HTh-7 cell migration and apoptosis. As shown in Fig. 2A and B, treatment with 10 mg/ml MA for 48 h decreased SW1736 and HTh7 colony formation and invasion, respectively. Moreover, we also found that the levels of apoptosis were significantly increased in SW1736 and HTh-7 cells in the presence of anthocyanins for 48 h (Fig. 2C), indicating a tumor suppressor role for anthocyanins in thyroid cancer cells.
MA enhanced SW1736 and HTh-7 cell autophagy To further explore the effects of MA on thyroid cancer cells, we evaluated the autophagy pathway in MA-treated SW1736 and HTh-7 cells. Western blot analysis demonstrated that MA significantly increased the LC3II/LC3I ratio in a pattern comparable to rapamycin after 48 h (Fig. 3A and B). Combination use of MA and rapamycin significantly increased autophagic activity in these cells after 48 h (Fig. 3A and B). Transmission electron microscopy also demonstrated an enhanced presence of autophagic vesicles in cells treated with 10 mg/ml MA for 48 h (Fig. 3C). Additionally, GFP-LC3 transfection was used to detect autophagic activity, and our data showed that treatment with MA increased GFP puncta, indicating autophagic vacuoles, in SW1736 and HTh-7 cells. These effects could be suppressed by addition of 3-MA, indicating that MA induced autophagic vacuole formation, as evidenced by LC3II accumulation (Fig. 3D).
MA-induced SW1736 and HTh-7 cell death correlated with severe autophagy To explore whether the MA-induced cell death observed in SW1736 and HTh-7 cells was partially attributed to MA (10 mM)-induced autophagy, 3-MA (5 mM) or chloroquine (CQ, 40 mM), the autophagolysosome fusion inhibitor, was utilized. As shown in Fig. 4A and C, MA-induced cell death
Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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Figure 1. MA suppressed thyroid cancer cell proliferation in a dose- and time-dependent manner. MA markedly reduced SW1736 (A) and HTh-7 (B) cell viability at doses of 1, 10, and 30 mg/ml for 48 h. SW1736 (C) and HTh-7 (D) cell viability was significantly inhibited by treatment with 10 mg/ml MA at 24, 48, and 72 h, respectively. )p < 0.05, ))p < 0.01, )))p < 0.001 vs. control.
was largely abolished by 3-MA incubation for 48 h in SW1736 and HTh-7 cells. Likewise, after treatment with CQ for 48 h, cell death of SW1736 and HTh-7 caused by MA was largely repressed (Fig. 4B and D). These results demonstrated that MA-induced SW1736 and HTh-7 cell death was dependent on autophagy in these thyroid cancer lines.
MA suppressed Akt/mTOR signaling in SW1736 and HTh-7 cells The Akt/mTOR signaling pathway is an important regulator of both autophagy and cancer cell proliferation. Western blot analysis illustrated that MA markedly reduced Akt, mTOR, and S6 activation in SW1736 and HTh-7 cells, indicating that MA acted as a tumor suppressor in thyroid cancer cells (Fig. 5).
Discussion Previous studies have shown that MA includes abundant compounds that are the material basis of mulberry’s dark brown color [3,18,19]. Anthocyanin is a kind of natural plant pigment, which is widely found in plants and a variety of fruits and vegetables [18]. Anthocyanins are present in the form of glycosides or aglycone, which are
characterized by antioxidant, anti-inflammatory, antitumor, anti-atherosclerotic biological effects [20]. Malignant tumors are among the world’s most common disease and a cause of significant morbidity and mortality in human health [21]. Recently, isolating dietary compounds from natural food has become an important research avenue with respect to the prevention and treatment of various tumors [18,22,23]. Thyroid cancer is the most common endocrine system malignancy with an overall good prognosis. However, due to the development of radiation resistance, traditional antitumor treatments do not achieve satisfactory therapeutic efficacy in papillary thyroid carcinoma, undifferentiated carcinoma and medullary carcinoma [24,25]. In the present study, we mainly evaluated the anti-carcinogenic and antiproliferative effects of MA on thyroid cancer cells. Our data indicated that MA significantly suppressed SW1736 and HTh7 cell viability in a time- and dose-dependent manner. Further study revealed that MA significantly reduced SW1736 and HTh-7 cell colony formation and invasion capacity. These data indicated an important tumor suppressor role of MA in thyroid cancer cells. Meanwhile, we also investigated the induction of cell death by MA treatment. Firstly, we focused on apoptosis, which plays a critical role in many biological activities, including the evolution of organisms and the stability of the
Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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MA improves TC by inducing apoptosis and autophagy
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Figure 2. MA decreased SW1736 and HTh-7 cell migration while increasing apoptosis. Treatment with 10 mg/ml MA for 48 h decreased SW1736 (A) and HTh-7 (B) cell migration and invasion capacity. Apoptosis in SW1736 (C) and HTh-7 (D) cells was significantly increased in response to 10 mg/ml MA for 48 h. )p < 0.05, ))p < 0.01 vs. control.
microenvironment [21,26]. Apoptosis evasion is closely related to the occurrence and development of tumors, and activation of apoptosis could be a helpful strategy to eliminate tumor cells [24]. The ability to induce apoptosis
in tumor cells is a powerful anti-tumor mechanism [27]. Here, our data showed that MA enhanced apoptosis in two thyroid cancer cell lines, suggesting the anti-carcinogenesis role of MA in thyroid cancer cells.
Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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Figure 3. MA enhanced autophagy in SW1736 and HTh-7 cells. Western blot analysis of SW1736 (A) and HTh-7 (B) cells showed the ratio between LC3II/LC3I was increased by 10 mg/ml MA for 48 h. (C) Transmission electron microscopy demonstrated enhanced autophagic vesicles in SW1736 and HTh-7 cells treated with 10 mg/ml MA for 48 h. (D) GFP-LC3 transfection indicated 10 mg/ml MA for 48 h increased SW1736 and HTh-7 cell autophagy. )p < 0.05 vs. control.
Furthermore, researchers have also begun to focus on the autophagy pathway with respect to the pathogenesis and development of thyroid cancer, particularly in tumors that are not sensitive to conventional therapy. Autophagy responds quickly to myriad internal and external environmental pressures that exist in eukaryotic cells and that happen during the aging process [28,29]. Autophagy not only is a cell survival mechanism but also can induce cell death. Thus, the potential role of MA in thyroid cancer cell autophagy was further explored. In line with rapamycin, MA
treatment could enhance the LC3II/LC3I ratio, indicating the activation of autophagy in thyroid cancer cells. And such effects were further determined by transmission electron microscopy and GFP-LC3 transfection. The above findings motivated us to further explore the potential mechanism in which MA induced autophagy. The Akt/mTOR pathway plays an important role in regulating cell autophagy [30]. Therefore, we next investigated whether the effect of MA on SW1736 and HTh-7 cell autophagy is achieved through this pathway. Here, we observed
Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004
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Figure 4. MA-induced SW1736 and HTh-7 cell death correlated with heightened autophagic activity. MA-induced cell death was largely abolished by addition of 3-MA for 48 h in a dose-dependent manner in SW1736 (A) and HTh-7 cells (C). After inhibition of autophagy by CQ for 48 h, cell death of SW1736 (B) and HTh-7 (D) caused by MA was greatly reduced. )p < 0.05 vs. control.
Figure 5. 10 mg/ml MA treatment for 48 h suppressed the activation of Akt/mTOR signaling in SW1736 and HTh-7 cells. )p < 0.05, ))p < 0.01 vs. control.
that MA suppressed the activation of Akt/mTOR signaling in the thyroid cancer cells, further supporting the tumor suppressor role of MA. In conclusion, MA may serve as a novel therapy method for thyroid cancer mainly by inducing apoptosis and autophagic-induced cell death. Increasing research is investigating the relationship between autophagy and thyroid cancer, and it will be important to explore novel methods in which autophagy can be applied to the clinical treatment of thyroid cancer.
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Please cite this article in press as: Long H-L, et al., Mulberry anthocyanins improves thyroid cancer progression mainly by inducing apoptosis and autophagy cell death, Kaohsiung Journal of Medical Sciences (2017), https://doi.org/10.1016/j.kjms.2017.11.004