Hyperoside exerts potent anticancer activity in skin cancer
Quercetin-3-O-β-D-galactopyranoside, is a hyperoside flavonol glycoside with anti-cancer, anti-inflammatory, and anti-oxidant activities that is derived from Hypericum and Crataegus plants. To this end, we examined the effect of this hyperoside in skin cancer cells lines and in DMBA/TPA induced skin tumors in vivo. In vitro treatment of cancer cells with hyperoside significantly inhibited phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR)/p38 MAPK axis, concomitantly activated the 5’AMP-activated protein kinase (AMPK) signaling, inhibited proliferation, and induced apoptosis and autophagy. Hyperoside markedly inhibited diffuse epidermal hyperplasia and significantly reduced the changes in phosphorylated levels of PI3K, AKT, mTOR and AMPK while reducing p38 phosphorylation as well as of tumor burden in vivo in DMBA/TPA induced skin tumors. These data suggest that hyperoside might be of therapeutic value in chemoprevention of skin cancer.
Nonmelanoma skin cancers (NMSC) include squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) that account, respectively, for about 20% and 80% of all diagnosed NMSC cases in the world (1-2). However, some report a higher prevalence for SCC as compared to BCC (3). Apart from ultraviolet radiation (UVR), the common factors causing NMSC are occupational and environmental exposures to polycyclic aromatic hydrocarbons (PAHs), arsenic, as well as the ionizing radiation (4). PAHs, which are known for their toxic, carcinogenic and mutagenic effects, are produced during the incomplete combustion of organic materials, like petroleum, wood and coal (5). Among the PAHs, 7, 12 dimethylbenz(a)anthracene (DMBA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) are commonly used as a model for studying carcinogenesis of SCC, a multistep process that goes through initiation, promotion and formation of cancer (6-8). Impeding or reversal of such a process can be achieved by chemoprevention (9). One category of drugs considered for such chemoprevention is flavonoids, polyphenol compounds such as hyperoside (HS), that are derived from various plants (10-14) (Figure 1). SCCs undergo both apoptosis or programmed cell death (PCD) and autophagy (15). Thus, it follows that enhancement of these processes might stop or reverse the carcinogenesis. To this end, here, we examined the effect of hyperoside on cell proliferation, apoptosis and autophagy in vitro in a series of SCC cell lines and in vivo in SCCs that were induced by topical application of DMBA/TPA to skin.
3. Materials and methods
3.1. Cells and treatments
Squamous cell carcinoma cell lines (A431, A432, HS-4) were obtained from American Type Culture Collection (Manassas, VA). Normal human skin cells (HACAT, HFF) were obtained from KeyGENE Biotech (Nanjing, China). Cell lines were cultured and maintained at 37 °C in MEM containing 10% FBS (GIBCO, USA) and 1% penicillin/ streptomycin (GIBCO, USA) in a humidified atmosphere in presence of 5% CO2. Hyperoside (>98% purity) was purchased from Wako-Chem (Osaka, Japan), dissolved in DMSO and stored at –20oC until further use. Upon thawing, hyperoside was diluted in DMEM medium at the indicated concentrations, with a final concentration of no more than 0.1% (v/v) DMSO.
3.2. Animals and treatments
All experimental procedures were approved by the Research Ethical Committee of Huai'an First People's Hospital, Nanjing Medical University. All experiments were performed following the Guide for the Care and Use of Laboratory Animals. A total of 60, 6–7 week-old female mice were obtained from Institute of Cancer Research (Shanghai, China). Animals were kept, in germ-free conditions, in climate-controlled quarters with a 12 h light and dark cycle, and were fed ad libitum with food and water. Fifteen mice were randomly assigned into four groups as follows
1. Group I: This group that serves as negative control received only the solvent vehicle (DMSO).
2. Group II: This group was treated with indicated doses of DMBA/TPA (Sigma Aldrich, USA).
3. Group III: This group was treated with indicated dose of DMBA/TPA and 8 μM HS.
4. Group IV: This group was treated with indicated dose of DMBA/TPA and 16 μM HS.
SCCs were induced by intra-dermal injection of 60 μg of DMBA in skin of the back of the animals. Two weeks later, the animals received intra-dermal injection of 4 μg TPA twice a week for a total of 22 weeks. The effect of hyperoside was examined by 1 min topical skin application of either 8 or 16 μM HS dissolved in DMSO/acetone 1:9 (V/V), 30 min before DMBA/TPA mixture was introduced to skin. The control animals received the vehicle alone. The injections were carried out five days a week for 22 weeks, after which the size of tumors were measured with a caliper and the animals were sacrificed, the normal skin and tumors were removed and were immediately stored in liquid nitrogen, or fixed 10% Formalin.
3.3. Cell viability analysis
1×103 cells/well were seeded in 96-well plates (Corning, USA) containing complete growth medium. On the following day, all cells were treated with different concentrations of hyperoside (0, 1, 5, 10, 25, 50 and 100 μM) and incubated at 37 °C for 0, 12, 24 and 48 h. Then, the cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) Cell Viability Assay Kit (Abnova, USA). Briefly, post hyperoside treatment, 15 µL of MTT reagent was added to each well and incubated for 4 hours at 37°C. Later, 100 µL of the solubilizer was added to each well and mixed gently on an orbital shaker for 1 hour at room temperature. Then, optical density was measured at 570 nm on an absorbance plate reader (Elx800TM absorbance microplate reader, BioTek Instrument, Inc., Vermont). Each experiment was repeated three times.
3.4. Flow cytometric assay
Apoptosis was analyzed by the Annexin V-FITC/ propidium iodide (PI) apoptosis detection kit from KeyGENE (Nanjing, China). Cells were harvested and washed with cold PBS twice and processed according to manufacturer’s instructions. In brief, cells were treated with 100 µl binding buffer containing annexin V-FITC and PI and incubated on ice for 15 min in the dark. Then, 400 µL ice-cold binding buffer was added to cells and these were analyzed by a flow cytometer (BD Biosciences, USA) (16).
3.5. Migration analysis
1×105 skin cancer cells/well were seeded in the top chamber of 24-well Transwell micropore polycarbonate membrane filter, with a pore size of 8-μm (Millipore, USA).After 24 h, cells on the top surface of membrane were gently removed, and cells that migrated onto the bottom surface of wells stained and then counted in five random fields, under a light microscope.
3.6. Scratch wound-healing assay
Human skin cancer cells were seeded in a 6-well plate and once reaching confluence, a thin “wound” was introduced by scratching the monolayer with a pipette tip. Cells then were washed with PBS and images were captured under an inverted scope along the length of the scratch at 0 h and 24 h after introduction of the wound.
3.7. Western blot analysis
Western blot analysis was performed as previously described (17). Briefly, cells were rinsed with ice-cold PBS three times and lysed in ice-cold lysis buffer in the presence of a freshly prepared protease inhibitor cocktail. For carrying Western blotting on skin and skin tumors, 100 mg of frozen tissue were also lysed in 1ml lysis buffer (pH 7.4, 50 mM Tris-HCl, 150 mM NaCl, 1 mM NaF, 1 mM ethyleneglycol-bis(aminoethylether)-tetraacetic acid, 1% NP-40, 1 mM phenylmethane-sulfonyl fluoride, and 10 μg/ml leupeptin). Following centrifugation of the lysates at x12,000 g for 20 min at 4°C, the total protein concentration in the lysates were quantified by BSA protein assay kit (Thermo, USA). Then, 40 ng of protein extracts were resolved in 10%-12% SDS-PAGE, and then blotted to polyvinylidene fluoride membrane (PVDF) (Millipore, USA). The membranes were blocked for 2 hr with 5% skim fat dry milk dissolved in 0.1% Tween-20 and Tris-Buffered Saline (TBS). Membranes were incubated, overnight at 4°C, with primary antibodies (Table 1) dissolved in blocking buffer. Bands were revealed by chemiluminescence using Pierce ECL Western Blotting Substrate reagents (Thermo Scientific). All experiments were performed in triplicates and validated three times.
|Primary antibodies||Dilution ratio||Corporation|
|Rabbit anti-Bcl-2||1:1000||Cell Signaling Technology|
|Rabbit anti-Bcl-xl||1:1000||Cell Signaling Technology|
|Mouse anti- Caspase-9||1:1000||Abcam|
|Rabbit anti-PARP||1:1000||Cell Signaling Technology|
|Rabbit anti-Beclin-1||1:1000||Cell Signaling Technology|
|Rabbit anti-LC3-I/II||1:1000||Cell Signaling Technology|
|Rabbit anti-p-PI3K||1:1000||Cell Signaling Technology|
|Rabbit anti-PI3K||1:1000||Cell Signaling Technology|
|Rabbit anti-AKT||1:500||Cell Signaling Technology|
|Rabbit anti-p-AKT||1:1000||Cell Signaling Technology|
|Rabbit anti- mTOR||1:1000||Abcam|
|Rabbit anti- p38||1:1000||Cell Signaling Technology|
|Rabbit anti-p-p38||1:1000||Cell Signaling Technology|
3.8. Hematoxylin and eosin staining
Formalin fixed skin and tumor tissues of experimental mice were embedded in paraffin blocks and sectioned at 3 μm thickness. Sections were de-paraffinized, rehydrated in descending series of alcohol and stained with hematoxylin and eosin (H&E) stain. The thickness of epidermis was measured by using Magnuspro software and in H&E staining sections by Image-J software (USA).
3.9. Immunohistochemical staining
The tissue sections were subjected to a pH based antigen retrieval, by exposing the sections to HCl (3.5 M) for 20 min at room temperature. Sections were then washed in PBS for 3 times. Following quenching of endogenous peroxidase by immersion of sections in peroxidase (0.3%), the tissue sections were incubated with normal goat serum (5%) for 30 min followed by incubation with the anti-Ki67 primary antibody (1:100 dilution; ab15580, Abcam, USA) for 2 h at room temperature. The sections were then incubated with HRP-conjugated secondary antibody, and after the sections were washed in PBS, they were developed using diaminobenzidine (DAB, ChemService, USA).
3.10. Statistical analysis
Data were expressed as the means ± standard errors of the mean (S.E.M.). Statistical analysis was carried out using GraphPad PRISM (version 6.0; Graph Pad Software) and ANOVA with Dunnet’s least significant difference. A p-value of less than 0.05 was considered to be statistically significant.
4.1. Hyperoside reduces viability, migration, colony formation, apoptosis and autophagy of skin cancer cells in vitro
Hyperoside treatment did not impact the viability of HACAT and HFF normal human skin cell lines (Figure 2 A-B) whereas the viability was significantly reduced in A431, A432 and HS-4 cancer ce lls that were treated for 0, 12, 24 and 48 h with escalating doses of hyperoside (0, 1, 5, 10, 25, 50 and 100 μM) (Figure 3 A-D). This treatment increased apoptosis and reduced the migration, and colony formation in cancer cells (Figure 4-5). Moreover, consistent with such results, there was down-regulation of Bcl-2 and Bcl-xl that protect cells from apoptosis whereas the pro-apoptotic signals, Bax and Bad were upregulated (Figure 6). In addition, there was an increase in the executors of apoptosis including cytochrome C, caspase-9, caspase-3 as well as evidence for PARP cleavage (Figure 6). Examining the PTEN, Beclin-1 and LC3I/II that participate in the autophagic cell death showed that these were all increased in cells that were treated with hyperoside in a dose dependent manner while those that inhibit autophagic cell death, namely, phosphorylated levels of PI3K/AKT/mTOR were reduced (20-21, 27)(Figure 7 A-B). The phosphorylated AMPK and MAPK that maintain energy homeostasis and survival were increased in cancer cells treated with hyperoside (Figure 7 C). Consistent with reduced proliferation, phosphorylated AURKA that is increased in cells undergoing mitosis (18), was reduced in skin cancer cells treated with hyperoside (Figure 8).
4.2. Hyperoside reduces DMBA/TPA induced skin cancer In vivo
Fifteenth weeks after treatment with DMBA/TPA, animals developed skin tumors. The body weight of animals that harbored DMBA/TPA induced cancers and were treated without or with hyperoside did not change indicating safety of hyperoside for animal use (Figure 9). Treatment with DMBA/TPA increased the epidermal thickness, while this effect was suppressed by hyperoside (Figure 10 A-B). Consistent with such a hyperproliferative state in skin, there was an increase in the phosphorylated AURKA as well as the number of Ki-67 positive cells in DMBA/TPA-treated mice was higher as compared to the control group whereas treatment with hyperoside reduced such an increase (Figure 10 C).
The size of the tumors was significantly reduced with 8 μM or 16 μM hypreoside in the DMBA/TPA treated mice indicating that hyperoside suppresses tumorigenesis (Figure 11). The tumors showed increased apoptosis, down-regulation of Bcl-2 and Bcl-xl and up-ergulation of Bax, Bad, Cyto-c and Apaf-1, activation of caspase-9, caspase-3 and PARP cleavage and these effects were reduced by treatment with hyperoside (Figure 12 A). Hyperoside treatment also increased autophagy-associated proteins including PTEN, Beclin-1 and LC3II (Figure 12 B). Furthermore hypreoside reversed the DMBA/TPA-induced changes in phosphorylated levels of PI3K, AKT, mTOR and AMPK while reducing p38 phosphorylation (Figure 12 C-D).
Skin cancer is caused by a variety of carcinogens including chemicals, radiation, and UV light. Given that the incidence of skin cancer has been rising, there is a need for evaluating strategies that can offer a safe chemopreventive effect. Here, we tested Quercetin-3-O-β-D-galactopyranoside, as a candidate with such an effect. In vitro treatment of cancer cells with hyperoside inhibited proliferation, and induced apoptosis and reduced autophagy. In vivo, hyperoside markedly inhibited diffuse epidermal hyperplasia that is often seen in skin cancers as well as of tumor burden in vivo in DMBA/TPA induced skin tumors. Consistent with such an impact on proliferation, phosphorylated AURKA that is increased in cells undergoing mitosis, was reduced in skin cancer cells treated with hyperoside (18). These findings show that hyperoside stops cancer progression through various mechanisms, including regulation of proliferation, and by inducing apoptosis and autophagy. The effect of hyperoside is likely mediated by increasing Bcl-2 and Bcl-xl that protect cells from apoptosis as well as by reducing the pro-apoptotic signals, Bax and Bad. Hyperoside also significantly halted activation of apoptotic cascade induced by executors of apoptosis including cytochrome C, caspase-9, caspase-3 and markedly reduced PARP cleavage which occurs in cells that undergo apoptosis. These data suggest that hyperoside might be of therapeutic value in chemoprevention of skin cancer.
The mammalian target of rapamycin (mTOR) is considered as a pro-survival signal transduction pathway that prevents apoptosis (19). Following stress, mTOR depletion inhibits cell growth and proliferation and increases autophagy and apoptosis (20). The PI3K/AKT are also involved in regulation of cell cycle, protein synthesis, apoptosis and proliferation (19, 21-22). We show here, that hyperoside normalizes phosphorylated levels of PI3K/AKT/mTOR (23-24, 25) and effectively reduces p38 phosphorylation, a MAPK known to play a crucial role in several cellular processes, including cell proliferation (26-27).
AMPK was highly phosphorylated, while p38 was de-phosphorylated by hyperoside administration, further confirming the suppressive effects of hyperoside on skin cancer, which was directly proved by reduced tumor incidence and the number of tumor lesions formed in DMBA/TPA treated mice, in vivo. Additionally, hyperoside induced reduction in epidermal thickness and the number of Ki-67 positive cells further showing that it has the ability to restrain tumor cell growth or skin hyperplasia. The treatment of cancer cells with hyperoside also increased phosphorylated AMPK, an evolutionarily conserved serine/threonine protein kinase, that serves as an energy sensor in all eukaryotic cells, maintains energy homeostasis and regulates both proliferation and apoptosis (28). Thus, treatment with hyperoside might confer resistance to the tumor development and halts further progression of tumors.
Hyperoside also alters autophagy, a conserved cellular process involved in cell survival and death whereby cellular organelles and proteins are engulfed by autophagosomes, digested within lysosomes, and recycled to sustain cellular homeostasis (29-32). Regulators of autophagy include microtubule-associated protein II light chain 3 (LC3-II) that regulates autophagy by binding to the membranes of the autophagosomes (30, 33), Beclin 1 which acts as a tumor suppressor (34) and PTEN that, by virtue of inducing autophagy, causes inhibition of tumor growth (35). The effect of hyperoside treatment included preventing cancer induced changes of LC3I/II, Beclin-1 and PTEN.
In conclusion, this study demonstrates that hyperoside shows potential in reducing tumor burden by increasing apoptosis and autophagy and by activating the signaling required in such processes.
1. Rogers HW, Weinstock MA, Harris AR, Hinckley MR, Feldman SR, Fleischer AB and Coldiron BM: Incidence estimate of nonmelanoma skin cancer in the United States, 2006. Arch Dermatol. 146:283-287 (2010)
2. National Comprehensive Cancer Network Guidelines of Cancers by Site Available online. http://www.nccn.org/professionals/physicianAccessed (2014)
3. Tanvetyanon T, Padhya T, McCaffrey J, Kish JA, Deconti RC, Trotti A and Rao NG: Postoperative concurrent chemotherapy and radiotherapy for high-risk cutaneous squamous cell carcinoma of the head and neck. Head Neck, 37:840-845 (2015).
4. Jarkowski A III, Hare R, Loud P, Skitzki JJ, Kane JM III, May KS, Zeitouni NC, Nestico J, Vona KL, Groman A and Khushalani NI: Systemic therapy in advanced cutaneous squamous cell carcinoma (CSCC): The Roswell Park experience and a review of the literature. Am J Clin Oncol, 39(6), 545-548 (2016)
5. Banik BK and Becker FF: Synthesis, electrophilic substitution and structure-activity relationship studies of polycyclic aromatic compounds towards the development of anticancer agents. Curr Med Chem, 8, 1513-1533 (2001)
6. Zhaorigetu S, Yanaka N, Sasaki M, Watanabe H and Kato N: Silk protein, sericin, suppresses DMBA-TPA-induced mouse skin tumorigenesis by reducing oxidative stress, inflammatory responses and endogenous tumor promoter TNF-α. Oncology Reports, 10, 537-543 (2003)
8. Budunova IV, Perez P, Vaden VR, Spiegelman VS, Slaga TJ and Jorcano JL: Increased expression of p50-NF-kappaB and constitutive activation of NF-kappaB transcription factors during mouse skin carcinogenesis. Oncogene, 18, 7423-7431 (1999)
11. Wang ZD, Huang C, Li ZF, Yang J, Li BH, Liang RR, Dai ZJ, Liu ZW: Chrysanthemum indicum ethanolic extract inhibits invasion of hepatocellular carcinoma via regulation of MMP/TIMP balance as therapeutic target. Oncol Rep, 23, 413-421 ( 2010)
12. Li W, Liu M, Xu YF, Feng Y, Che JP, Wang GC, Zheng JH: Combination of quercetin and hyperoside has anticancer effects on renal cancer cells through inhibition of oncogenic microRNA-27a. Oncol Rep, 31, 117-124 (2014)
13. Li ZL, Hu J, Li YL, Xue F, Zhang L, Xie JQ, Liu ZH, Li H, Yi DH, Liu JC, Wang SW: The effect of hyperoside on the functional recovery of the ischemic/reperfused isolated rat heart: potential involvement of the extracellular signal-regulated kinase 1/2 signaling pathway. Free Radic Biol Med, 57, 132-140 (2013)
14. Liu Y, Liu GH, Mei JJ, Wang J: The preventive effects of hyperoside on lung cancer in vitro by inducing apoptosis and inhibiting proliferation through Caspase-3 and P53 signaling pathway. Biomedicine & Pharmacotherapy, 83, 381-391 (2016)
16. Robles-Escajeda E, Lerma D, Nyakeriga AM, Ross JA, Kirken RA, Aguilera RJ, Varela-Ramirez A: Searching in mother nature for anti-cancer activity: anti-proliferative and pro-apoptotic effect elicited by green barley on leukemia/lymphoma cells. PLoS One, 8, e73508 (2013)
18. Liang CZ, Zhang X, Li H, Tao YQ, Tao LJ, Yang ZR, Zhou XP, Shi ZL, Tao HM: Gallic acid induces the apoptosis of human osteosarcoma cells in vitro and in vivo via the regulation of mitogen-activated protein kinase pathways. Cancer Biother Radiopharm, 27, 701-710 (2012)
22. Lee ER, Kim JY, Kang YJ, Ahn JY, Kim JH, Kim BW, Choi HY, Jeong MY and Cho SG: Interplay between PI3K/Akt and MAPK signaling pathways in DNA-damaging drug-induced apoptosis. Biochim Biophys Acta, 1763, :958-968 (2006)
23. Yue Z, Jin S, Yang C, Levine AJ and Heintz N: Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci USA, 100, 15077-15082 (2003)
27. Sui XB, Kong N, Ye L, Han W, Zhou J, Zhang Q, He C, Pan H: p38 and JNK MAPK pathways control the balance of apoptosis and autophagy in response to chemotherapeutic agents. Cancer Lett, 344174-179 ( 2014)
29. Huang X, Bai HM, Chen L, Li B and Lu YC: Reduced expression of LC3B-II and Beclin 1 in glioblastoma multiforme indicates a down-regulated autophagic capacity that relates to the progression of astrocytic tumors. J Clin Neurosci, 17, 1515-1519, (2010)
30. Xu MX, Zhu YF, Chang HF, Liang Y: Nanoceria restrains PM2. 5-induced metabolic disorder and hypothalamus inflammation by inhibition of astrocytes activation related NF-κB pathway in Nrf2 deficient mice. Free Radical Biology and Medicine, 99, 259-272, (2016)
31. Katayama H, Sasai K, Kawai H, Yuan ZM, Bondaruk J, Suzuki F, Fujii S, Arlinghaus RB, Czerniak BA, Sen S: Phosphorylation by aurora kinase A induces Mdm2-mediated destabilization and inhibition of p53. Nat Genet 36, 55-62 (2004)
32. Gong K, Chen C, Zhan Y, Chen Y, Huang Z and Li W: Autophagy-related gene 7 (ATG7) and reactive oxygen species/extracellular signal-regulated kinase regulate tetrandrine-induced autophagy in human hepatocellular carcinoma. J Biol Chem, 287, 35576-35588 (2012)