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The effect of earthworm extract on mice S180 tumor growth and apoptosis
Biomedicine & Pharmacotherapy 115 (2019) 108979
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
The effect of earthworm extract on mice S180 tumor growth and apoptosis a,b,1
Zhenhan Deng ⁎ Yusheng Lia,f,
c,1
d
e
e
g
, Shanshan Gao , Xiang Xiao , Ni Yin , Shiyang Ma , Wenping Li ,
T
a
Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha 410008, Hunan, China Department of Sports Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People’s Hospital, Shenzhen 518035, Guangdong, China Department of Cardiology, University of Colorado Anschutz Medical Campus, Aurora 800045, CO, USA d The Animal Health Inspection Institute of Yuelu District, Changsha 410000, Hunan, China e Department of Clinical Medicine (8-Year Program), Xiangya Medicine School, Central South University, Changsha 410013, Hunan, China f National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha 410008, Hunan, China g Department of Animal Science, College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, Hunan, China b c
ARTICLE INFO
ABSTRACT
Keywords: Earthworm extract Sarcoma Tumor growth Apoptosis Bcl-2 Bax
Great efforts have been made to explore the potential treatment for cancers, and the most common therapies include surgery, chemotherapy and radiotherapy. As an alternative medication, earthworms have drawn increased attention considering its abundance in resource, easy access and minor side effects compared to traditional therapies. However, few studies had focused on the antitumor effect of earthworm-derived components. The purpose of this study is to investigate whether earthworm extract has an effect on tumor cell apoptosis and growth. Earthworm extract (EE) was purified through multiple steps of centrifugation and chromatography. Mice were inoculated with ascitic fluid derived from mice inoculated with S180 sarcoma tumor cells and fed orally with different amounts of EE for 25 days. Tumor samples were analyzed for size and cell apoptosis. And we found that the weight and sizes of tumor decreased gradually as the amount of EE administered increased. More apoptotic cells and lowered level of lactate dehydrogenase (LDH), a biomarker of tumor invasiveness, was detected in EE-treated group than the untreated group. Our results suggested that EE could dramatically promote tumor apoptosis and reduce tumor size in vivo, suggesting a novel alternative therapy for cancer patients.
1. Introduction Cancer is among the leading causes of death world wide. In 2017, over one million new cases in the United States were diagnosed with cancer and over 600,000 individuals died of it [1]. Great efforts have been made for development of new anti-cancer drugs over time. However, the 5-year survival rates of several types cancers like lung cancer and pancreatic cancer are still below 20%. Additionally, patients who underwent chemotherapy and radiation therapy suffer from side effects such as muscle pain, hair loss, nausea and even the risk of developing a secondary cancer, due to the fact that besides cancer cells, healthy active cells were also damaged during the therapy [2]. Earthworms have long been used as a treatment for illness in the history of traditional medicine. People from Burma applied ashes from burnt earthworms to alleviate symptoms of fever; in Laos, earthworms
was used to treat small pox; ancient Chinese used earthworm to ease fever-associated convulsions, hemiplegia and blood clots [3]. In modern medicine, studies have revealed beneficial effects of earthworm in both in vitro and in vivo models, with a better understanding of the mechanism underlying earthworm’s disease-curing capability. Fu et al. found the earthworm extract (EE) in culture medium and the ablity of promoting cell proliferation and activating osteoblasts [4]. Chang et al. observed stimulated migration in Schwann cells by EE, through increased production of matrix-degrading proteolytic enzymes [5]. Intraperitoneal injection of EE could notably reduce pulmonary inflammation and fibrosis induced by silica inhalation in mice [6]. Isolated active ingredients from earthworms could protect mice from endoplasmic reticulum stress-induced liver injury [7]. Our previous study observed effect of EE on promoting wound healing in mice with excisional wounds [8].
Abbreviation: CAT, catalase; CTX, cyclophosphamide; EE, earthworm extract; H&E, hematoxylin and eosin; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; RBC, red blood cell; ROS, reactive oxygen species; SOD, superoxide dismutase; WBC, white blood cell ⁎ Corresponding author. E-mail address: [email protected] (Y. Li). 1 These two authors contributed equally. https://doi.org/10.1016/j.biopha.2019.108979 Received 25 March 2019; Received in revised form 20 April 2019; Accepted 8 May 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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In terms of cancer-related studies, Augustine et al. found earthworm coelomic fluid suppressed the proliferation in a squamous cancer cell line [9]. The earthworm fibrinolytic enzyme from Eiseniafoetida had shown to significantly inhibit the growth of human hepatoma tumor xenograft in nude mice [10]. Despite its rising role in development of alternative anti-cancer therapies and the advantages of low-cost, no reported side effects and therefore being possible to be consumed continuously, the potential outcome of treating cancer with EE still remains controversial. According to previous studies, activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were detected in live earthworms and EE [11,12]. Although antioxidant enzymes play a positive role in our body and protect our tissues against damage caused by excess reactive oxygen species (ROS), other studies advocated that antioxidant enzymes may promote tumor growth and invasiveness [13–15]. Hence, the effect of EE on cancer should be carefully examined case by case depending on the purification methods of earthworm components, types of cancers and degrees of cancer progression, the way of drug administration and other factors. S180 is a murine sarcoma cancer cell line and has been commonly used in cancer research because of its high proliferation rate in mice. Until today, there have been few studies on the influence of EE in S180 tumor-bearing mice [16]. Therefore, in the study we aim to investigate the effect of EE on S180 tumor growth and apoptosis.
saline. 200ul diluted S180 tumor cell solution was subsequently subcutaneously injected into mice used in our experiments for the establishment of a S180 tumor-bearing model. 2.5. Oral administration of EE The S180 tumor-bearing mice were divided randomly into five groups. Mice fed with PBS were used as control group. Cyclophosphamide (CTX) or EE was administered orally to mice for 25 days. CTX was applied at concentration of 30 mg/kg body weight/day, and EE was used at concentrations of 30, 60, 90 mg/kg body weight/ day, respectively. 2.6. Spleen/thymus index Immediately after mice were sacrificed, tumor, spleen and thymus were surgically collected and weighed. The spleen/thymus index was recorded using the following formula: Spleen/thymus index = the final weight of spleen/thymus (g)/the final average body weight (g) without tumor. 2.7. Histological analysis The collected tumor tissues were fixed with 10% formalin and embedded in paraffin, sectioned at 7 μm thickness. Hematoxylin and eosin (H&E) staining was performed to reveal morphology of the tumors. For immunohistochemical staining, Bcl-2 family antibody sampler kit (Boster Technology) was applied to detect Bax and Bcl-2 expression on tumor sections. The positive cell numbers were counted manually in 5 random fields from each single slide under a microscope.
2. Methods and materials 2.1. Extraction and purification of EE Indian blue earthworms (Perionyx excavates) were collected from an earthworm breeding farm, and the EE was isolated and purified as previously described [17]. Briefly, sexually mature earthworms were rinsed with tap water to remove attached mud, followed by homogenization with a tissue homogenizer. The homogenized sample was ground by a tissue grinder on ice, sonicated and centrifuged with 6000 rpm for 10 min at 4 °C. The supernatant was ultra-filtered with 50KD ultra-filter (Millipore) with 5000 g for 20 min, then the lower fraction left at the bottom of centrifuge tube was ultra-centrifuged with 10KD ultra-filter (Millipore) at 4 °C. Extract was purified from the ultrafiltered solution with Sephadex G-200 by chromatography. Desired protein sample was eluted with phosphate-buffered saline (PBS, 0.1 M, pH7.8) and stored at −20 °C.
2.8. DNA laddering DNA fragmentation, a key characteristic of apoptosis, was detected with DNA laddering kit (TianDZ, Inc) following user manual. DNA samples extracted from S180 tumor tissues were purified and separated on agarose gel and visualized with imaging system (YLN2000). 2.9. Hematological analysis Counting of red blood cells (RBCs) and white blood cells (WBCs) from mice whole blood was performed using a hematology analyzer. Enzymatic activities of lactate dehydrogenase(LDH) in mice serum were measured with assay kits (Jiancheng Bioengineering).
2.2. Cell culture S180 tumor cells were purchased from Cell Banks of the Chinese Academy of Sciences (Cat.TCM15) and maintained in RMPI 1640 medium (Gibco) containing 10% FBS at 37 °C in a CO2 incubator. Fresh medium was changed every 2–3 days.
2.10. Statistical analysis The values were expressed as means ± standard deviation (SD). All statistical analyses were performed using the SPSS 16.0 software (Chicago, IL, U.S.A.). Data were analyzed by the independent samples ttest compared between two groups and a one-way analysis of variance (ANOVA) was conducted to assess differences between multiple groups. P-value < 0.05 was considered statistically significant.
2.3. Animals Male Kunming mice (6–8 weeks old) purchased from Changsha Animal Center were used in the experiment. The mice were housed under normal laboratory conditions and were allowed to acclimate in the facility for 1 week prior to experiments. Animal protocol was approved by the Animal Care and Use Committee of Central South University and Hunan Agricultural University.
3. Results 3.1. In vivo antitumor activity of EE To investigate whether EE has antitumor effect, mice injected with S180 tumor cells were sacrificed on day 25 post-implantation. No tumor formation was observed in control group. The collected tumors from S180 tumor-bearing mice were photographed and weighed. It was found that tumors in CTX treated group and all the EE treated group were significantly smaller in size (Fig. 1A) and lower in weight (Fig. 1B) compared to control group. There was an inverse correlation between the amount of EE orally administrated and the weight of tumors,
2.4. Ascites tumor-bearing mouse model To establish a S180-bearing mouse model, we followed a protocol previously published by other groups [18,19]. Briefly, 20 mice were injected intraperitoneally with 0.8–1.2 × 106 S180 cells resuspended in 0.4 mL saline solution. One week later, enlarged abdomen was observed in these mice and ascitic fluid was collected, diluted 3-fold with sterile 2
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Fig. 1. Tumor size and weight in S180 tumor-bearing mice with different treatment. (A) Gross view of tumors from inoculated mice in different treated groups. (B) Quantification of tumor weight in different treated groups (N = 8/group). *p < 0.05. vs Ctrl, #p < 0.05. vs CTX.
Fig. 2. Spleen and thymus index of S180 tumor-bearing mice with different treatment (N = 8/group). *p < 0.05. vs Ctrl, #p < 0.05. vs Uninoculated. Ψ < 0.05 vs CTX.
Fig. 3. Serum levels of LDH in S180 tumor-bearing mice with different treatment (N = 5/group).*p < 0.05, **p < 0.01. vs Uninoculated; #p < 0.05, ##p < 0.01 vs Ctrl; Ψp < 0.05, ΨΨp < 0.01 vs CTX.
indicating the antitumor activity of EE is dose-dependent. However, tumor weight in 30 mg/kg EE treated group was significantly higher than that in CTX (30 mg/kg) treated group, suggesting that CTX acts more efficiently in inhibiting tumor growth than EE at the same dosage. Spleen and thymus are the two primary immune organs involved in antitumor activity [20,21], and the spleen and thymus indexes were measured to evaluate the impact of tumor formation and subsequent drug treatment on these immune organs (Fig. 2). Compared to control group, a significant enhancement of both spleen and thymus index was observed in CTX and EE treated groups, but more prominent in CTX treated group. However, it has been reported that CTX treatment leads to splenomegaly in mice [22]. Interestingly, while S180 cell inoculation reduced the thymus index, no difference was found in spleen index between uninoculated mice and control group, indicating S180 cellinduced tumor formation didn’t exert much influence on spleen size.
3.3. Oral administration of EE induces S180 tumor cell death in vivo In H&E staining, the tumor tissues in EE and CTX treated groups showed cells with pyknotic nuclei and hypereosinophilic cytoplasm, which served as an indicator of cell apoptosis. More apoptotic cells were observed in 90 mg/kg EE treated mice than those treated with 30 or 60 mg/kg EE (Fig. 4). Bax is an essential promoter for apoptosis and Bcl is considered as an apoptosis inhibitor. Immunohistochemical analysis of Bax and Bcl-2 revealed increased Bax-positive cells in mice treated with CTX or EE, while cells stained with Bcl-2 antibody decreased in tumor tissues from these drug-treated groups (Fig. 5A). Statistical analysis of Bax- and Bcl-2- positive cells from tumor sections showed a significantly higher ratio of Bax-positve cells to Bcl-2 positive cells in CTX and EE treated mice than control group (Fig. 5B). Analysis of DNA fragmentation, a feature of early stage apoptosis, was performed by DNA laddering kit with agarose gel electrophoresis. Sequential degraded DNA fragments were detected in tumor tissues from CTX and EE treated mice, but not in control group (Fig. 5C). These results suggested that EE be able to induce apoptosis in S180 tumors.
3.2. The effect of EE on activities of serum LDH Previous studies had detected enhanced expression of LDH in several types of human cancers compared to normal tissues [23]. LDH has been used as a prognostic biomarker for evaluation of tumor progression, invasiveness and patient survival rate [24]. Serum LDH level is served as a useful test to determine outcome of antitumor treatments [25,26]. The S180 tumor-bearing mice without drug treatments showed significantly increased LDH level compared to uninoculated group (Fig. 3). LDH level in the CTX treated group was significantly lower than the control group, indicating that CTX treatment could efficiently inhibit tumor growth. This result was consistent with the results in Fig. 1, showing reduced size and weight of tumors in CTX treated group. All groups of mice that fed with EE exhibited a suppression of S180 tumor-induced LDH level in a dose-dependent manner.
3.4. Blood cell counts Blood cell number counts were performed in normal and S180 cellsimplanted mice (Fig. 6). No significant difference in RBC count or WBC count was found between uninoculated mice and control mice, suggesting tumor formation has little effect on the numbers of these two types of blood cells. RBCs were dramatically increased in CTX-fed mice compared to control mice or S180 tumor-bearing mice treated with 3
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Fig. 4. H&E staining of the tumor tissues in S180 tumor-bearing mice with different treatment (N = 5/group, 200×). Ctrl stands for mice without any drug treatment.
incubation with LDH inhibitor in culture medium inhibits the growth of cancer cells [36]. Additionally, inactivation of LDH gene in mice models of lung cancer suppresses tumor initiation and progression [37]. In our studies, we discovered that oral administration of EE deceased the serum LDH level in S180-inoculated mice in a dosage-dependent manner, but as dramatically as the CTX treated group (Fig. 3). More experiments should be performed to investigate whether EE can sensitize cancer cells or animal models of cancer for responses to radio- or chemotherapy in the future. Apoptosis in cancer has been extensively studied for many years and cytotoxic drugs that target certain apoptotic pathways have been developed for clinical cancer therapies [38,39]. So far there are mainly two principle types of apoptotic pathways identified: the intrinsic pathway and the extrinsic pathway [40], both involved in tumorigenesis [39]. The intrinsic pathway is mitochondria-dependent, in which the Bcl-2 protein family members, including pro-apoptotic Bax and anti-apoptotic Bcl-2, interact with each other to trigger or inhibit apoptosis [41]. Sabah et al [42] detected Bcl-2 expression in soft tissue sarcoma samples, and the protein expression of Bcl-2 is more prevalent in high-grade sarcoma tissues. Liu et al [43] have shown that direct activation of Bax protein induces apoptosis of cancer cells, which may provide clues for development of new anti-cancer drugs. Nevertheless, whether and how treatment with EE affects the apoptosis of tumor cells in vitro and in vivo remains elusive. One group has found that earthworm coelomic fluid leads to apoptosis of Hela cells [44], a cervical cancer cell line; another group has also detected apoptosis-like cell death in tumor cells incubated with coelomocyte lysates from earthworm [45]. On the other hand, EE exhibit an inhibitory effect on cardiomyoblast apoptosis induced by lipopolysaccharides [45]. It will be interesting to investigate why components derived from earthworms show different impacts on distinct types of cells. In this study we examined the histology of tumor samples from S180-inoculated mice treated with or without EE, and found that EE leads to apoptosis in tumors, partially via regulation of Bax and Bcl-2 proteins. We noticed that although in the tumor samples the ratio of Bax-positive cells to Bcl2-positive cells was the highest in CTX treated animals compared to all the EE treated groups, mice fed with 90 mg/kg EE had the most obvious DNA fragmentation among all the groups, a hallmark of apoptosis
30 mg/kg EE. Oral administration of EE raised the RBC counts in a dosage-dependent manner, but none of the EE treated group showed statistic difference compared to uninoculated group, indicating EE has weaker effect on RBC number than CTX at the same dosage. WBCs were significantly increased in CTX treated mice compared to uninoculated group and 30 mg/kg EE group. In mice treated with 90 mg/kg EE, WBC count was much higher than the uninoculated group, suggesting EE treatment stimulates the production of WBCs in vivo. 4. Discussion In this study, we investigated the antitumor potential of EE and compare its effect on tumor growth and progression with CTX, a medication to treat several types of cancers including leukemia, lymphoma, breast cancer and sarcoma. We used a sarcoma mouse model inoculated with S180 tumor cells and administrated CTX or EE orally for a defined period of time to observe the outcome of drug treatment. When the tumor tissues were collected at the endpoint of the experiment, it was found that at the same dosage, CTX treated mice had smaller tumors size compared to EE treated group (Fig. 1), but lower thymus and spleen index (Fig. 2), which could be due to the thymus atrophyor splenomegaly caused by CTX 27,28]. It was shown that CTX has an inhibitory effect on the growth of all the lymphocyte subsets [29]. It also needs to be taken into consideration that expansion and activation of effector T cells induced by CTX could overcome the decrease in exact T cell counts [29,30] and therefore, exhibit a better antitumor effect in CTX treated animals. It is interesting to notice that all the EE treated mice had higher thymus and spleen indexes than the uninoculated and control groups, although how EE led to the increased indexes of both immune organs remains unknown and needs to be further explored. Upregulated in many types of cancers, LDH has been used as a biomarker [31] and potential therapeutic target for cancer treatment [32,33]. LDH catalyzes the conversion of pyruvate to lactate during glycolysis, a process critical for providing energy supply in cancer cells due to their increased cellular proliferation metabolism [34]. In sarcoma, high serum LDH level has been associated with poor outcome of radiotherapy and chemotherapy [35]. Previous studies have found that 4
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Fig. 5. Evaluation of apoptosis in tumor tissues from S180 tumor-bearing mice with different treatment (N = 5/group). (A) Immunohistochemical analysis of Bax and Bcl-2 expression in tumor tissues (400×). (B) Quantification of Bax and Bcl-2 positive cells in tumor tissues. (C) DNA fragmentation from tumor tissues demonstrated by agarose gel electrophoresis. *p < 0.05., **p < 0.01., ***p < 0.001.vs Ctrl, #p < 0.05., ##p < 0.01.vs CTX.
(Fig. 5). This indicates there may be other apoptotic pathways EE is involved with and more studies need to be done in order to further explore the mechanism underlying the apoptosis-inducible effect of EE in cancer. Strong chemotherapies come with unpleasant side effects including inhibition of blood cell production [46,47]. Lowered white blood cell
count can weaken the immune system of cancer patients, rendering them susceptible to infections [48]. In this study lowering of white blood cells or red blood cells was not detected in EE treated animals (Fig. 6), suggesting EE does not exert immune suppression effect when it’s orally administered. In conclusion, our results demonstrated that EE is able to inhibit
Fig. 6. RBC counts (A) and WBC counts (B) in whole blood obtained fromS180 tumor-bearing mice with different treatment (N = 5/group). *p < 0.05 vs CTX, #p < 0.05 vs Uninoculated. 5
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tumor growth, as well as induce apoptosis of tumor cells via Bax/Bcl-2 regulation, without significantly reducing blood cells in S180-inoculated mice. This study sheds new insights into the potential of EE as an alternative antitumor drug; however, thorough investigations are required in the future to identify and purify the active antitumor component(s) from earthworm due to the complex composition of EE.
[14] K. Le Gal, M.X. Ibrahim, C. Wiel, V.I. Sayin, M.K. Akula, C. Karlsson, M.G. Dalin, L.M. Akyurek, P. Lindahl, J. Nilsson, M.O. Bergo, Antioxidants can increase melanoma metastasis in mice, Sci. Transl. Med. 7 (308) (2015) 308re308, https://doi. org/10.1126/scitranslmed.aad3740. [15] A.R. Mendelsohn, J.W. Larrick, Paradoxical effects of antioxidants on cancer, Rejuvenation Res. 17 (3) (2014) 306–311, https://doi.org/10.1089/rej.2014.1577. [16] P.M. Ferreira, P.M. Da Costa, M. Costa Ade, D.J. Lima, R.R. Drumond, N. Silva Jdo, D.R. Moreira, G.B. De Oliveira Filho, J.M. Ferreira, M.G. De Queiroz, A.C. Leite, C. Pessoa, Cytotoxic and toxicological effects of phthalimide derivatives on tumor and normal murine cells, An. Acad. Bras. Cienc. 87 (1) (2015) 313–330, https://doi. org/10.1590/0001-3765201520130345. [17] W. Luo, Z.H. Deng, R. Li, G. Cheng, R.N. Kotian, Y.S. Li, W.P. Li, Study of analgesic effect of earthworm extract, Biosci. Rep. 38 (1) (2018), https://doi.org/10.1042/ BSR20171554. [18] X. Wang, H. Wang, C. Zhang, K. Zhang, Experimental study on inhibition of S180 tumor cells by Agrimonia pilosa extract, Afr. J. Tradit. Complement. Altern. Med. 10 (3) (2013) 475–479. [19] C. Wang, C. Lu, Y. Hsueh, W. Liu, C. Chen, Activation of antitumor immune responses by Ganoderma formosanum polysaccharides in tumor-bearing mice, Appl. Microbiol. Biotechnol. 98 (22) (2014) 9389–9998, https://doi.org/10.1007/ s00253-014-6027-6. [20] S.W. Wen, S.J. Everitt, J. Bedo, M. Chabrot, D.L. Ball, B. Solomon, M. MacManus, R.J. Hicks, A. Moller, A. Leimgruber, Spleen volume variation in patients with locally advanced non-small cell lung cancer receiving platinum-based chemo-radiotherapy, PLoS One 10 (11) (2015) e0142608, https://doi.org/10.1371/journal. pone.0142608. [21] D. Metcalf, R. Moulds, B. Pike, Influence of the spleen and thymus on immune responses in ageing mice, Clin. Exp. Immunol. 2 (1) (1967) 109–120. [22] W. Lu, D. Jia, S. An, M. Mu, X. Qiao, Y. Liu, X. Li, D. Wang, Calf Spleen Extractive Injection protects mice against cyclophosphamide-induced hematopoietic injury through G-CSF-mediated JAK2/STAT3 signaling, Sci. Rep. 7 (1) (2017) 8402, https://doi.org/10.1038/s41598-017-08970-3. [23] R.D. Goldman, N.O. Kaplan, T.C. Hall, Lactic dehydrogenase in human neoplastic tissues, Cancer Res. 24 (1964) 389–399. [24] S.Y. Suh, H.Y. Ahn, Lactate dehydrogenase as a prognostic factor for survival time of terminally ill cancer patients: a preliminary study, Eur. J. Cancer 43 (6) (2007) 1051–1059, https://doi.org/10.1016/j.ejca.2007.01.031. [25] L. Faloppi, M. Scartozzi, M. Bianconi, G. Svegliati Baroni, P. Toniutto, R. Giampieri, M. Del Prete, S. De Minicis, D. Bitetto, C. Loretelli, M. D’Anzeo, A. Benedetti, S. Cascinu, The role of LDH serum levels in predicting global outcome in HCC patients treated with sorafenib: implications for clinical management, BMC Cancer 14 (2014) 110, https://doi.org/10.1186/1471-2407-14-110. [26] S.L. Yu, L.T. Xu, Q. Qi, Y.W. Geng, H. Chen, Z.Q. Meng, P. Wang, Z. Chen, Serum lactate dehydrogenase predicts prognosis and correlates with systemic inflammatory response in patients with advanced pancreatic cancer after gemcitabine-based chemotherapy, Sci. Rep. 7 (2017) 45194, https://doi.org/10.1038/ srep45194. [27] H.R. Smith, T.M. Chused, A.D. Steinberg, Cyclophosphamide-induced changes in the MRL-lpr/lpr mouse: effects upon cellular composition, immune function, and disease, Clin. Immunol. Immunopathol. 30 (1) (1984) 51–61. [28] R. Brelinska-Peczalska, U. Mackiewicz, Effect of cyclophosphamide on the thymus in mice, Arch. Immunol. Ther. Exp. (Warsz) 29 (6) (1981) 773–777. [29] M. Scurr, T. Pembroke, A. Bloom, D. Roberts, A. Thomson, K. Smart, H. Bridgeman, R. Adams, A. Brewster, R. Jones, S. Gwynne, D. Blount, R. Harrop, R. Hills, A. Gallimore, A. Godkin, Low-dose cyclophosphamide induces antitumor T-cell responses, which associate with survival in metastatic colorectal cancer, Clin. Cancer Res. 23 (22) (2017) 6771–6780, https://doi.org/10.1158/1078-0432.CCR17-0895. [30] M.T. Madondo, M. Quinn, M. Plebanski, Low dose cyclophosphamide: mechanisms of T cell modulation, Cancer Treat. Rev. 42 (2016) 3–9, https://doi.org/10.1016/j. ctrv.2015.11.005. [31] V. Jurisic, S. Radenkovic, G. Konjevic, The actual role of LDH as tumor marker, biochemical and clinical aspects, Adv. Exp. Med. Biol. 867 (2015) 115–124, https:// doi.org/10.1007/978-94-017-7215-0_8. [32] J.R. Doherty, J.L. Cleveland, Targeting lactate metabolism for cancer therapeutics, J. Clin. Invest. 123 (9) (2013) 3685–3692, https://doi.org/10.1172/JCI69741. [33] P. Miao, S. Sheng, X. Sun, J. Liu, G. Huang, Lactate dehydrogenase A in cancer: a promising target for diagnosis and therapy, IUBMB Life 65 (11) (2013) 904–910, https://doi.org/10.1002/iub.1216. [34] M.G. Vander Heiden, L.C. Cantley, C.B. Thompson, Understanding the Warburg effect: the metabolic requirements of cell proliferation, Science 324 (5930) (2009) 1029–1033, https://doi.org/10.1126/science.1160809. [35] S. Li, Q. Yang, H. Wang, Z. Wang, D. Zuo, Z. Cai, Y. Hua, Prognostic significance of serum lactate dehydrogenase levels in Ewing’s sarcoma: a meta-analysis, Mol. Clin. Oncol. 5 (6) (2016) 832–838, https://doi.org/10.3892/mco.2016.1066. [36] M.A. Lea, Y. Guzman, C. Desbordes, Inhibition of growth by combined treatment with inhibitors of lactate dehydrogenase and either phenformin or inhibitors of 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3, Anticancer Res. 36 (4) (2016) 1479–1488. [37] K.J. Song, X.N. Yu, T. Lv, Y.L. Chen, Y.C. Diao, S.L. Liu, Y.K. Wang, Q. Yao, Expression and prognostic value of lactate dehydrogenase-A and -D subunits in human uterine myoma and uterine sarcoma, Medicine (Baltimore) 97 (14) (2018) e0268, https://doi.org/10.1097/MD.0000000000010268. [38] S. Fulda, K.M. Debatin, Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy, Oncogene 25 (34) (2006) 4798–4811, https://doi.org/10.1038/sj. onc.1209608.
Authors’ contribution LY, WL, and SG designed and directed the study. ZD and XX performed the experiments. ZD, NY, and SM analyzed the data. ZD and SG prepared the manuscript. YL was responsible for funding acquisition. Conflicts of interest None. Acknowledgments This research was funded by National Natural Science Foundation of China [grant numbers 81874030, 81402224]; the Provincial Science Foundation of Hunan [grant number 2015JJ3139]; the Health and Family Planning Commission of Hunan Province [grant number B2016105]; the Administration of Traditional Chinese Medicine of Hunan Province [grant number 2015116]; and the China Scholarship Council [grant number 201606375101]. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2017, CA Cancer J. Clin. 67 (1) (2017) 7–30, https://doi.org/10.3322/caac.21387. [2] M. Popovic, T.M. Hrcenjak, T. Babic, J. Kos, M. Grdisa, Effect of earthworm (G-90) extract on formation and lysis of clots originated from venous blood of dogs with cardiopathies and with malignant tumors, Pathol. Oncol. Res. 7 (3) (2001) 197–202. [3] Y.T. Fu, K.Y. Chen, Y.S. Chen, C.H. Yao, Earthworm (Pheretima aspergillum) extract stimulates osteoblast activity and inhibits osteoclast differentiation, BMC Complement. Altern. Med. 14 (2014) 440, https://doi.org/10.1186/1472-6882-14440. [4] E.L. Cooper, K. Hirabayashi, M. Balamurugan, Dilong: food for thought and medicine, J. Tradit. Complement. Med. 2 (4) (2012) 242–248. [5] Y.M. Chang, Y.T. Shih, Y.S. Chen, C.L. Liu, W.K. Fang, C.H. Tsai, F.J. Tsai, W.W. Kuo, T.Y. Lai, C.Y. Huang, Schwann cell migration induced by earthworm extract via activation of PAs and MMP2/9 mediated through ERK1/2 and p38, Evid. Complement. Alternat. Med. 2011 (2011) 395458, https://doi.org/10.1093/ecam/ nep131. [6] J. Yang, T. Wang, Y. Li, W. Yao, X. Ji, Q. Wu, L. Han, R. Han, W. Yan, J. Yuan, C. Ni, Earthworm extract attenuates silica-induced pulmonary fibrosis through Nrf2-dependent mechanisms, Lab. Invest. 96 (12) (2016) 1279–1300, https://doi.org/10. 1038/labinvest.2016.101. [7] Y.F. Zhao, Y. Gao, X.F. Wu, J.G. Wang, L.X. Duan, Protective effect of earthworm active ingredients against endoplasmic reticulum stress-induced acute liver injury in mice, Zhongguo Zhong Yao Za Zhi 42 (6) (2017) 1183–1188, https://doi.org/10. 19540/j.cnki.cjcmm.20170121.028. [8] Z.H. Deng, J.J. Yin, W. Luo, R.N. Kotian, S.S. Gao, Z.Q. Yi, W.F. Xiao, W.P. Li, Y.S. Li, The effect of earthworm extract on promoting skin wound healing, Biosci. Rep. 38 (2) (2018), https://doi.org/10.1042/BSR20171366. [9] D. Augustine, R.S. Rao, J. Anbu, K.N. Chidambara Murthy, In vitro antiproliferative effect of earthworm coelomic fluid of Eudrilus eugeniae, Eisenia foetida, and Perionyx excavatus on squamous cell Carcinoma-9 cell line: a pilot study, Pharmacognosy Res. 9 (Suppl. 1) (2017) S61–S66, https://doi.org/10.4103/pr.pr_ 52_17. [10] H. Chen, S. Takahashi, M. Imamura, E. Okutani, Z.G. Zhang, K. Chayama, B.A. Chen, Earthworm fibrinolytic enzyme: anti-tumor activity on human hepatoma cells in vitro and in vivo, Chin. Med. J. (Engl.) 120 (10) (2007) 898–904. [11] S. Zhao, L. He, Y. Lu, L. Duo, The impact of modified nano-carbon black on the earthworm Eisenia fetida under turfgrass growing conditions: assessment of survival, biomass, and antioxidant enzymatic activities, J. Hazard. Mater. 338 (2017) 218–223, https://doi.org/10.1016/j.jhazmat.2017.05.035. [12] Y.J. Shi, X.B. Xu, X.Q. Zheng, Y.L. Lu, Responses of growth inhibition and antioxidant gene expression in earthworms (Eisenia fetida) exposed to tetrabromobisphenol A, hexabromocyclododecane and decabromodiphenyl ether, Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 174–175 (2015) 32–38, https://doi.org/ 10.1016/j.cbpc.2015.06.005. [13] M.A. Hawk, C. McCallister, Z.T. Schafer, Antioxidant activity during tumor progression: a necessity for the survival of cancer cells? Cancers (Basel) 8 (10) (2016), https://doi.org/10.3390/cancers8100092.
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Z. Deng, et al. [39] K.M. Debatin, Apoptosis pathways in cancer and cancer therapy, Cancer Immunol. Immunother. 53 (3) (2004) 153–159, https://doi.org/10.1007/s00262-003-0474-8. [40] S. Elmore, Apoptosis: a review of programmed cell death, Toxicol. Pathol. 35 (4) (2007) 495–516, https://doi.org/10.1080/01926230701320337. [41] J.M. Adams, S. Cory, Bcl-2-regulated apoptosis: mechanism and therapeutic potential, Curr. Opin. Immunol. 19 (5) (2007) 488–496, https://doi.org/10.1016/j. coi.2007.05.004. [42] M. Sabah, R. Cummins, M. Leader, E. Kay, Immunoreactivity of p53, Mdm2, p21(WAF1/CIP1) Bcl-2, and Bax in soft tissue sarcomas: correlation with histologic grade, Appl. Immunohistochem. Mol. Morphol. 15 (1) (2007) 64–69. [43] P. Pluta, P. Smolewski, A. Pluta, B. Cebula-Obrzut, A. Wierzbowska, D. Nejc, T. Robak, R. Kordek, L. Gottwald, J. Piekarski, A. Jeziorski, Significance of Bax expression in breast cancer patients, Pol. Przegl. Chir. 83 (10) (2011) 549–553, https://doi.org/10.2478/v10035-011-0087-4. [44] L. Yanqin, S. Yan, S. Zhenjun, L. Shijie, W. Chong, L. Yan, G. Yuhong, Coelomic fluid of the earthworm Eisenia fetida induces apoptosis of HeLa cells in vitro, Eur. J. Soil
Biol. 43 (2007) S143–S148, https://doi.org/10.1016/j.ejsobi.2007.08.049. [45] P.C. Li, Y.C. Tien, C.H. Day, P. Pai, W.W. Kuo, T.S. Chen, C.H. Kuo, C.H. Tsai, D.T. Ju, C.Y. Huang, Impact of LPS-induced cardiomyoblast cell apoptosis inhibited by earthworm extracts, Cardiovasc. Toxicol. 15 (2) (2015) 172–179, https://doi. org/10.1007/s12012-014-9281-z. [46] A. Akinbami, A. Popoola, A. Adediran, A. Dosunmu, O. Oshinaike, P. Adebola, S. Ajibola, Full blood count pattern of pre-chemotherapy breast cancer patients in Lagos, Nigeria, Caspian J. Intern. Med. 4 (1) (2013) 574–579. [47] J.R. Germa Lluch, R.B. Piulats, Correlation between white blood cell count and neutrophil count after chemotherapy administration at a day hospital, Rev. Esp. Oncol. 32 (4) (1985) 627–632. [48] N.I. Abdou, M. Richter, Cells involved in the immune response. VI. The immune response to red blood cells in irradiated rabbits after administration of normal, primed, or immune allogeneic rabbit bone marrow cells, J. Exp. Med. 129 (4) (1969) 757–774.
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The effect of earthworm extract on mice S180 tumor growth and apoptosis a,b,1
Zhenhan Deng ⁎ Yusheng Lia,f,
c,1
d
e
e
g
, Shanshan Gao , Xiang Xiao , Ni Yin , Shiyang Ma , Wenping Li ,
T
a
Department of Orthopaedics, Xiangya Hospital, Central South University, Changsha 410008, Hunan, China Department of Sports Medicine, The First Affiliated Hospital of Shenzhen University, Shenzhen Second People’s Hospital, Shenzhen 518035, Guangdong, China Department of Cardiology, University of Colorado Anschutz Medical Campus, Aurora 800045, CO, USA d The Animal Health Inspection Institute of Yuelu District, Changsha 410000, Hunan, China e Department of Clinical Medicine (8-Year Program), Xiangya Medicine School, Central South University, Changsha 410013, Hunan, China f National Clinical Research Center for Geriatric Disorders, Xiangya Hospital, Central South University, Changsha 410008, Hunan, China g Department of Animal Science, College of Animal Science and Technology, Hunan Agricultural University, Changsha 410128, Hunan, China b c
ARTICLE INFO
ABSTRACT
Keywords: Earthworm extract Sarcoma Tumor growth Apoptosis Bcl-2 Bax
Great efforts have been made to explore the potential treatment for cancers, and the most common therapies include surgery, chemotherapy and radiotherapy. As an alternative medication, earthworms have drawn increased attention considering its abundance in resource, easy access and minor side effects compared to traditional therapies. However, few studies had focused on the antitumor effect of earthworm-derived components. The purpose of this study is to investigate whether earthworm extract has an effect on tumor cell apoptosis and growth. Earthworm extract (EE) was purified through multiple steps of centrifugation and chromatography. Mice were inoculated with ascitic fluid derived from mice inoculated with S180 sarcoma tumor cells and fed orally with different amounts of EE for 25 days. Tumor samples were analyzed for size and cell apoptosis. And we found that the weight and sizes of tumor decreased gradually as the amount of EE administered increased. More apoptotic cells and lowered level of lactate dehydrogenase (LDH), a biomarker of tumor invasiveness, was detected in EE-treated group than the untreated group. Our results suggested that EE could dramatically promote tumor apoptosis and reduce tumor size in vivo, suggesting a novel alternative therapy for cancer patients.
1. Introduction Cancer is among the leading causes of death world wide. In 2017, over one million new cases in the United States were diagnosed with cancer and over 600,000 individuals died of it [1]. Great efforts have been made for development of new anti-cancer drugs over time. However, the 5-year survival rates of several types cancers like lung cancer and pancreatic cancer are still below 20%. Additionally, patients who underwent chemotherapy and radiation therapy suffer from side effects such as muscle pain, hair loss, nausea and even the risk of developing a secondary cancer, due to the fact that besides cancer cells, healthy active cells were also damaged during the therapy [2]. Earthworms have long been used as a treatment for illness in the history of traditional medicine. People from Burma applied ashes from burnt earthworms to alleviate symptoms of fever; in Laos, earthworms
was used to treat small pox; ancient Chinese used earthworm to ease fever-associated convulsions, hemiplegia and blood clots [3]. In modern medicine, studies have revealed beneficial effects of earthworm in both in vitro and in vivo models, with a better understanding of the mechanism underlying earthworm’s disease-curing capability. Fu et al. found the earthworm extract (EE) in culture medium and the ablity of promoting cell proliferation and activating osteoblasts [4]. Chang et al. observed stimulated migration in Schwann cells by EE, through increased production of matrix-degrading proteolytic enzymes [5]. Intraperitoneal injection of EE could notably reduce pulmonary inflammation and fibrosis induced by silica inhalation in mice [6]. Isolated active ingredients from earthworms could protect mice from endoplasmic reticulum stress-induced liver injury [7]. Our previous study observed effect of EE on promoting wound healing in mice with excisional wounds [8].
Abbreviation: CAT, catalase; CTX, cyclophosphamide; EE, earthworm extract; H&E, hematoxylin and eosin; LDH, lactate dehydrogenase; PBS, phosphate-buffered saline; RBC, red blood cell; ROS, reactive oxygen species; SOD, superoxide dismutase; WBC, white blood cell ⁎ Corresponding author. E-mail address: [email protected] (Y. Li). 1 These two authors contributed equally. https://doi.org/10.1016/j.biopha.2019.108979 Received 25 March 2019; Received in revised form 20 April 2019; Accepted 8 May 2019 0753-3322/ © 2019 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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In terms of cancer-related studies, Augustine et al. found earthworm coelomic fluid suppressed the proliferation in a squamous cancer cell line [9]. The earthworm fibrinolytic enzyme from Eiseniafoetida had shown to significantly inhibit the growth of human hepatoma tumor xenograft in nude mice [10]. Despite its rising role in development of alternative anti-cancer therapies and the advantages of low-cost, no reported side effects and therefore being possible to be consumed continuously, the potential outcome of treating cancer with EE still remains controversial. According to previous studies, activities of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) were detected in live earthworms and EE [11,12]. Although antioxidant enzymes play a positive role in our body and protect our tissues against damage caused by excess reactive oxygen species (ROS), other studies advocated that antioxidant enzymes may promote tumor growth and invasiveness [13–15]. Hence, the effect of EE on cancer should be carefully examined case by case depending on the purification methods of earthworm components, types of cancers and degrees of cancer progression, the way of drug administration and other factors. S180 is a murine sarcoma cancer cell line and has been commonly used in cancer research because of its high proliferation rate in mice. Until today, there have been few studies on the influence of EE in S180 tumor-bearing mice [16]. Therefore, in the study we aim to investigate the effect of EE on S180 tumor growth and apoptosis.
saline. 200ul diluted S180 tumor cell solution was subsequently subcutaneously injected into mice used in our experiments for the establishment of a S180 tumor-bearing model. 2.5. Oral administration of EE The S180 tumor-bearing mice were divided randomly into five groups. Mice fed with PBS were used as control group. Cyclophosphamide (CTX) or EE was administered orally to mice for 25 days. CTX was applied at concentration of 30 mg/kg body weight/day, and EE was used at concentrations of 30, 60, 90 mg/kg body weight/ day, respectively. 2.6. Spleen/thymus index Immediately after mice were sacrificed, tumor, spleen and thymus were surgically collected and weighed. The spleen/thymus index was recorded using the following formula: Spleen/thymus index = the final weight of spleen/thymus (g)/the final average body weight (g) without tumor. 2.7. Histological analysis The collected tumor tissues were fixed with 10% formalin and embedded in paraffin, sectioned at 7 μm thickness. Hematoxylin and eosin (H&E) staining was performed to reveal morphology of the tumors. For immunohistochemical staining, Bcl-2 family antibody sampler kit (Boster Technology) was applied to detect Bax and Bcl-2 expression on tumor sections. The positive cell numbers were counted manually in 5 random fields from each single slide under a microscope.
2. Methods and materials 2.1. Extraction and purification of EE Indian blue earthworms (Perionyx excavates) were collected from an earthworm breeding farm, and the EE was isolated and purified as previously described [17]. Briefly, sexually mature earthworms were rinsed with tap water to remove attached mud, followed by homogenization with a tissue homogenizer. The homogenized sample was ground by a tissue grinder on ice, sonicated and centrifuged with 6000 rpm for 10 min at 4 °C. The supernatant was ultra-filtered with 50KD ultra-filter (Millipore) with 5000 g for 20 min, then the lower fraction left at the bottom of centrifuge tube was ultra-centrifuged with 10KD ultra-filter (Millipore) at 4 °C. Extract was purified from the ultrafiltered solution with Sephadex G-200 by chromatography. Desired protein sample was eluted with phosphate-buffered saline (PBS, 0.1 M, pH7.8) and stored at −20 °C.
2.8. DNA laddering DNA fragmentation, a key characteristic of apoptosis, was detected with DNA laddering kit (TianDZ, Inc) following user manual. DNA samples extracted from S180 tumor tissues were purified and separated on agarose gel and visualized with imaging system (YLN2000). 2.9. Hematological analysis Counting of red blood cells (RBCs) and white blood cells (WBCs) from mice whole blood was performed using a hematology analyzer. Enzymatic activities of lactate dehydrogenase(LDH) in mice serum were measured with assay kits (Jiancheng Bioengineering).
2.2. Cell culture S180 tumor cells were purchased from Cell Banks of the Chinese Academy of Sciences (Cat.TCM15) and maintained in RMPI 1640 medium (Gibco) containing 10% FBS at 37 °C in a CO2 incubator. Fresh medium was changed every 2–3 days.
2.10. Statistical analysis The values were expressed as means ± standard deviation (SD). All statistical analyses were performed using the SPSS 16.0 software (Chicago, IL, U.S.A.). Data were analyzed by the independent samples ttest compared between two groups and a one-way analysis of variance (ANOVA) was conducted to assess differences between multiple groups. P-value < 0.05 was considered statistically significant.
2.3. Animals Male Kunming mice (6–8 weeks old) purchased from Changsha Animal Center were used in the experiment. The mice were housed under normal laboratory conditions and were allowed to acclimate in the facility for 1 week prior to experiments. Animal protocol was approved by the Animal Care and Use Committee of Central South University and Hunan Agricultural University.
3. Results 3.1. In vivo antitumor activity of EE To investigate whether EE has antitumor effect, mice injected with S180 tumor cells were sacrificed on day 25 post-implantation. No tumor formation was observed in control group. The collected tumors from S180 tumor-bearing mice were photographed and weighed. It was found that tumors in CTX treated group and all the EE treated group were significantly smaller in size (Fig. 1A) and lower in weight (Fig. 1B) compared to control group. There was an inverse correlation between the amount of EE orally administrated and the weight of tumors,
2.4. Ascites tumor-bearing mouse model To establish a S180-bearing mouse model, we followed a protocol previously published by other groups [18,19]. Briefly, 20 mice were injected intraperitoneally with 0.8–1.2 × 106 S180 cells resuspended in 0.4 mL saline solution. One week later, enlarged abdomen was observed in these mice and ascitic fluid was collected, diluted 3-fold with sterile 2
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Fig. 1. Tumor size and weight in S180 tumor-bearing mice with different treatment. (A) Gross view of tumors from inoculated mice in different treated groups. (B) Quantification of tumor weight in different treated groups (N = 8/group). *p < 0.05. vs Ctrl, #p < 0.05. vs CTX.
Fig. 2. Spleen and thymus index of S180 tumor-bearing mice with different treatment (N = 8/group). *p < 0.05. vs Ctrl, #p < 0.05. vs Uninoculated. Ψ < 0.05 vs CTX.
Fig. 3. Serum levels of LDH in S180 tumor-bearing mice with different treatment (N = 5/group).*p < 0.05, **p < 0.01. vs Uninoculated; #p < 0.05, ##p < 0.01 vs Ctrl; Ψp < 0.05, ΨΨp < 0.01 vs CTX.
indicating the antitumor activity of EE is dose-dependent. However, tumor weight in 30 mg/kg EE treated group was significantly higher than that in CTX (30 mg/kg) treated group, suggesting that CTX acts more efficiently in inhibiting tumor growth than EE at the same dosage. Spleen and thymus are the two primary immune organs involved in antitumor activity [20,21], and the spleen and thymus indexes were measured to evaluate the impact of tumor formation and subsequent drug treatment on these immune organs (Fig. 2). Compared to control group, a significant enhancement of both spleen and thymus index was observed in CTX and EE treated groups, but more prominent in CTX treated group. However, it has been reported that CTX treatment leads to splenomegaly in mice [22]. Interestingly, while S180 cell inoculation reduced the thymus index, no difference was found in spleen index between uninoculated mice and control group, indicating S180 cellinduced tumor formation didn’t exert much influence on spleen size.
3.3. Oral administration of EE induces S180 tumor cell death in vivo In H&E staining, the tumor tissues in EE and CTX treated groups showed cells with pyknotic nuclei and hypereosinophilic cytoplasm, which served as an indicator of cell apoptosis. More apoptotic cells were observed in 90 mg/kg EE treated mice than those treated with 30 or 60 mg/kg EE (Fig. 4). Bax is an essential promoter for apoptosis and Bcl is considered as an apoptosis inhibitor. Immunohistochemical analysis of Bax and Bcl-2 revealed increased Bax-positive cells in mice treated with CTX or EE, while cells stained with Bcl-2 antibody decreased in tumor tissues from these drug-treated groups (Fig. 5A). Statistical analysis of Bax- and Bcl-2- positive cells from tumor sections showed a significantly higher ratio of Bax-positve cells to Bcl-2 positive cells in CTX and EE treated mice than control group (Fig. 5B). Analysis of DNA fragmentation, a feature of early stage apoptosis, was performed by DNA laddering kit with agarose gel electrophoresis. Sequential degraded DNA fragments were detected in tumor tissues from CTX and EE treated mice, but not in control group (Fig. 5C). These results suggested that EE be able to induce apoptosis in S180 tumors.
3.2. The effect of EE on activities of serum LDH Previous studies had detected enhanced expression of LDH in several types of human cancers compared to normal tissues [23]. LDH has been used as a prognostic biomarker for evaluation of tumor progression, invasiveness and patient survival rate [24]. Serum LDH level is served as a useful test to determine outcome of antitumor treatments [25,26]. The S180 tumor-bearing mice without drug treatments showed significantly increased LDH level compared to uninoculated group (Fig. 3). LDH level in the CTX treated group was significantly lower than the control group, indicating that CTX treatment could efficiently inhibit tumor growth. This result was consistent with the results in Fig. 1, showing reduced size and weight of tumors in CTX treated group. All groups of mice that fed with EE exhibited a suppression of S180 tumor-induced LDH level in a dose-dependent manner.
3.4. Blood cell counts Blood cell number counts were performed in normal and S180 cellsimplanted mice (Fig. 6). No significant difference in RBC count or WBC count was found between uninoculated mice and control mice, suggesting tumor formation has little effect on the numbers of these two types of blood cells. RBCs were dramatically increased in CTX-fed mice compared to control mice or S180 tumor-bearing mice treated with 3
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Fig. 4. H&E staining of the tumor tissues in S180 tumor-bearing mice with different treatment (N = 5/group, 200×). Ctrl stands for mice without any drug treatment.
incubation with LDH inhibitor in culture medium inhibits the growth of cancer cells [36]. Additionally, inactivation of LDH gene in mice models of lung cancer suppresses tumor initiation and progression [37]. In our studies, we discovered that oral administration of EE deceased the serum LDH level in S180-inoculated mice in a dosage-dependent manner, but as dramatically as the CTX treated group (Fig. 3). More experiments should be performed to investigate whether EE can sensitize cancer cells or animal models of cancer for responses to radio- or chemotherapy in the future. Apoptosis in cancer has been extensively studied for many years and cytotoxic drugs that target certain apoptotic pathways have been developed for clinical cancer therapies [38,39]. So far there are mainly two principle types of apoptotic pathways identified: the intrinsic pathway and the extrinsic pathway [40], both involved in tumorigenesis [39]. The intrinsic pathway is mitochondria-dependent, in which the Bcl-2 protein family members, including pro-apoptotic Bax and anti-apoptotic Bcl-2, interact with each other to trigger or inhibit apoptosis [41]. Sabah et al [42] detected Bcl-2 expression in soft tissue sarcoma samples, and the protein expression of Bcl-2 is more prevalent in high-grade sarcoma tissues. Liu et al [43] have shown that direct activation of Bax protein induces apoptosis of cancer cells, which may provide clues for development of new anti-cancer drugs. Nevertheless, whether and how treatment with EE affects the apoptosis of tumor cells in vitro and in vivo remains elusive. One group has found that earthworm coelomic fluid leads to apoptosis of Hela cells [44], a cervical cancer cell line; another group has also detected apoptosis-like cell death in tumor cells incubated with coelomocyte lysates from earthworm [45]. On the other hand, EE exhibit an inhibitory effect on cardiomyoblast apoptosis induced by lipopolysaccharides [45]. It will be interesting to investigate why components derived from earthworms show different impacts on distinct types of cells. In this study we examined the histology of tumor samples from S180-inoculated mice treated with or without EE, and found that EE leads to apoptosis in tumors, partially via regulation of Bax and Bcl-2 proteins. We noticed that although in the tumor samples the ratio of Bax-positive cells to Bcl2-positive cells was the highest in CTX treated animals compared to all the EE treated groups, mice fed with 90 mg/kg EE had the most obvious DNA fragmentation among all the groups, a hallmark of apoptosis
30 mg/kg EE. Oral administration of EE raised the RBC counts in a dosage-dependent manner, but none of the EE treated group showed statistic difference compared to uninoculated group, indicating EE has weaker effect on RBC number than CTX at the same dosage. WBCs were significantly increased in CTX treated mice compared to uninoculated group and 30 mg/kg EE group. In mice treated with 90 mg/kg EE, WBC count was much higher than the uninoculated group, suggesting EE treatment stimulates the production of WBCs in vivo. 4. Discussion In this study, we investigated the antitumor potential of EE and compare its effect on tumor growth and progression with CTX, a medication to treat several types of cancers including leukemia, lymphoma, breast cancer and sarcoma. We used a sarcoma mouse model inoculated with S180 tumor cells and administrated CTX or EE orally for a defined period of time to observe the outcome of drug treatment. When the tumor tissues were collected at the endpoint of the experiment, it was found that at the same dosage, CTX treated mice had smaller tumors size compared to EE treated group (Fig. 1), but lower thymus and spleen index (Fig. 2), which could be due to the thymus atrophyor splenomegaly caused by CTX 27,28]. It was shown that CTX has an inhibitory effect on the growth of all the lymphocyte subsets [29]. It also needs to be taken into consideration that expansion and activation of effector T cells induced by CTX could overcome the decrease in exact T cell counts [29,30] and therefore, exhibit a better antitumor effect in CTX treated animals. It is interesting to notice that all the EE treated mice had higher thymus and spleen indexes than the uninoculated and control groups, although how EE led to the increased indexes of both immune organs remains unknown and needs to be further explored. Upregulated in many types of cancers, LDH has been used as a biomarker [31] and potential therapeutic target for cancer treatment [32,33]. LDH catalyzes the conversion of pyruvate to lactate during glycolysis, a process critical for providing energy supply in cancer cells due to their increased cellular proliferation metabolism [34]. In sarcoma, high serum LDH level has been associated with poor outcome of radiotherapy and chemotherapy [35]. Previous studies have found that 4
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Fig. 5. Evaluation of apoptosis in tumor tissues from S180 tumor-bearing mice with different treatment (N = 5/group). (A) Immunohistochemical analysis of Bax and Bcl-2 expression in tumor tissues (400×). (B) Quantification of Bax and Bcl-2 positive cells in tumor tissues. (C) DNA fragmentation from tumor tissues demonstrated by agarose gel electrophoresis. *p < 0.05., **p < 0.01., ***p < 0.001.vs Ctrl, #p < 0.05., ##p < 0.01.vs CTX.
(Fig. 5). This indicates there may be other apoptotic pathways EE is involved with and more studies need to be done in order to further explore the mechanism underlying the apoptosis-inducible effect of EE in cancer. Strong chemotherapies come with unpleasant side effects including inhibition of blood cell production [46,47]. Lowered white blood cell
count can weaken the immune system of cancer patients, rendering them susceptible to infections [48]. In this study lowering of white blood cells or red blood cells was not detected in EE treated animals (Fig. 6), suggesting EE does not exert immune suppression effect when it’s orally administered. In conclusion, our results demonstrated that EE is able to inhibit
Fig. 6. RBC counts (A) and WBC counts (B) in whole blood obtained fromS180 tumor-bearing mice with different treatment (N = 5/group). *p < 0.05 vs CTX, #p < 0.05 vs Uninoculated. 5
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tumor growth, as well as induce apoptosis of tumor cells via Bax/Bcl-2 regulation, without significantly reducing blood cells in S180-inoculated mice. This study sheds new insights into the potential of EE as an alternative antitumor drug; however, thorough investigations are required in the future to identify and purify the active antitumor component(s) from earthworm due to the complex composition of EE.
[14] K. Le Gal, M.X. Ibrahim, C. Wiel, V.I. Sayin, M.K. Akula, C. Karlsson, M.G. Dalin, L.M. Akyurek, P. Lindahl, J. Nilsson, M.O. Bergo, Antioxidants can increase melanoma metastasis in mice, Sci. Transl. Med. 7 (308) (2015) 308re308, https://doi. org/10.1126/scitranslmed.aad3740. [15] A.R. Mendelsohn, J.W. Larrick, Paradoxical effects of antioxidants on cancer, Rejuvenation Res. 17 (3) (2014) 306–311, https://doi.org/10.1089/rej.2014.1577. [16] P.M. Ferreira, P.M. Da Costa, M. Costa Ade, D.J. Lima, R.R. Drumond, N. Silva Jdo, D.R. Moreira, G.B. De Oliveira Filho, J.M. Ferreira, M.G. De Queiroz, A.C. Leite, C. Pessoa, Cytotoxic and toxicological effects of phthalimide derivatives on tumor and normal murine cells, An. Acad. Bras. Cienc. 87 (1) (2015) 313–330, https://doi. org/10.1590/0001-3765201520130345. [17] W. Luo, Z.H. Deng, R. Li, G. Cheng, R.N. Kotian, Y.S. Li, W.P. Li, Study of analgesic effect of earthworm extract, Biosci. Rep. 38 (1) (2018), https://doi.org/10.1042/ BSR20171554. [18] X. Wang, H. Wang, C. Zhang, K. Zhang, Experimental study on inhibition of S180 tumor cells by Agrimonia pilosa extract, Afr. J. Tradit. Complement. Altern. Med. 10 (3) (2013) 475–479. [19] C. Wang, C. Lu, Y. Hsueh, W. Liu, C. Chen, Activation of antitumor immune responses by Ganoderma formosanum polysaccharides in tumor-bearing mice, Appl. Microbiol. Biotechnol. 98 (22) (2014) 9389–9998, https://doi.org/10.1007/ s00253-014-6027-6. [20] S.W. Wen, S.J. Everitt, J. Bedo, M. Chabrot, D.L. Ball, B. Solomon, M. MacManus, R.J. Hicks, A. Moller, A. Leimgruber, Spleen volume variation in patients with locally advanced non-small cell lung cancer receiving platinum-based chemo-radiotherapy, PLoS One 10 (11) (2015) e0142608, https://doi.org/10.1371/journal. pone.0142608. [21] D. Metcalf, R. Moulds, B. Pike, Influence of the spleen and thymus on immune responses in ageing mice, Clin. Exp. Immunol. 2 (1) (1967) 109–120. [22] W. Lu, D. Jia, S. An, M. Mu, X. Qiao, Y. Liu, X. Li, D. Wang, Calf Spleen Extractive Injection protects mice against cyclophosphamide-induced hematopoietic injury through G-CSF-mediated JAK2/STAT3 signaling, Sci. Rep. 7 (1) (2017) 8402, https://doi.org/10.1038/s41598-017-08970-3. [23] R.D. Goldman, N.O. Kaplan, T.C. Hall, Lactic dehydrogenase in human neoplastic tissues, Cancer Res. 24 (1964) 389–399. [24] S.Y. Suh, H.Y. Ahn, Lactate dehydrogenase as a prognostic factor for survival time of terminally ill cancer patients: a preliminary study, Eur. J. Cancer 43 (6) (2007) 1051–1059, https://doi.org/10.1016/j.ejca.2007.01.031. [25] L. Faloppi, M. Scartozzi, M. Bianconi, G. Svegliati Baroni, P. Toniutto, R. Giampieri, M. Del Prete, S. De Minicis, D. Bitetto, C. Loretelli, M. D’Anzeo, A. Benedetti, S. Cascinu, The role of LDH serum levels in predicting global outcome in HCC patients treated with sorafenib: implications for clinical management, BMC Cancer 14 (2014) 110, https://doi.org/10.1186/1471-2407-14-110. [26] S.L. Yu, L.T. Xu, Q. Qi, Y.W. Geng, H. Chen, Z.Q. Meng, P. Wang, Z. Chen, Serum lactate dehydrogenase predicts prognosis and correlates with systemic inflammatory response in patients with advanced pancreatic cancer after gemcitabine-based chemotherapy, Sci. Rep. 7 (2017) 45194, https://doi.org/10.1038/ srep45194. [27] H.R. Smith, T.M. Chused, A.D. Steinberg, Cyclophosphamide-induced changes in the MRL-lpr/lpr mouse: effects upon cellular composition, immune function, and disease, Clin. Immunol. Immunopathol. 30 (1) (1984) 51–61. [28] R. Brelinska-Peczalska, U. Mackiewicz, Effect of cyclophosphamide on the thymus in mice, Arch. Immunol. Ther. Exp. (Warsz) 29 (6) (1981) 773–777. [29] M. Scurr, T. Pembroke, A. Bloom, D. Roberts, A. Thomson, K. Smart, H. Bridgeman, R. Adams, A. Brewster, R. Jones, S. Gwynne, D. Blount, R. Harrop, R. Hills, A. Gallimore, A. Godkin, Low-dose cyclophosphamide induces antitumor T-cell responses, which associate with survival in metastatic colorectal cancer, Clin. Cancer Res. 23 (22) (2017) 6771–6780, https://doi.org/10.1158/1078-0432.CCR17-0895. [30] M.T. Madondo, M. Quinn, M. Plebanski, Low dose cyclophosphamide: mechanisms of T cell modulation, Cancer Treat. Rev. 42 (2016) 3–9, https://doi.org/10.1016/j. ctrv.2015.11.005. [31] V. Jurisic, S. Radenkovic, G. Konjevic, The actual role of LDH as tumor marker, biochemical and clinical aspects, Adv. Exp. Med. Biol. 867 (2015) 115–124, https:// doi.org/10.1007/978-94-017-7215-0_8. [32] J.R. Doherty, J.L. Cleveland, Targeting lactate metabolism for cancer therapeutics, J. Clin. Invest. 123 (9) (2013) 3685–3692, https://doi.org/10.1172/JCI69741. [33] P. Miao, S. Sheng, X. Sun, J. Liu, G. Huang, Lactate dehydrogenase A in cancer: a promising target for diagnosis and therapy, IUBMB Life 65 (11) (2013) 904–910, https://doi.org/10.1002/iub.1216. [34] M.G. Vander Heiden, L.C. Cantley, C.B. Thompson, Understanding the Warburg effect: the metabolic requirements of cell proliferation, Science 324 (5930) (2009) 1029–1033, https://doi.org/10.1126/science.1160809. [35] S. Li, Q. Yang, H. Wang, Z. Wang, D. Zuo, Z. Cai, Y. Hua, Prognostic significance of serum lactate dehydrogenase levels in Ewing’s sarcoma: a meta-analysis, Mol. Clin. Oncol. 5 (6) (2016) 832–838, https://doi.org/10.3892/mco.2016.1066. [36] M.A. Lea, Y. Guzman, C. Desbordes, Inhibition of growth by combined treatment with inhibitors of lactate dehydrogenase and either phenformin or inhibitors of 6phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3, Anticancer Res. 36 (4) (2016) 1479–1488. [37] K.J. Song, X.N. Yu, T. Lv, Y.L. Chen, Y.C. Diao, S.L. Liu, Y.K. Wang, Q. Yao, Expression and prognostic value of lactate dehydrogenase-A and -D subunits in human uterine myoma and uterine sarcoma, Medicine (Baltimore) 97 (14) (2018) e0268, https://doi.org/10.1097/MD.0000000000010268. [38] S. Fulda, K.M. Debatin, Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy, Oncogene 25 (34) (2006) 4798–4811, https://doi.org/10.1038/sj. onc.1209608.
Authors’ contribution LY, WL, and SG designed and directed the study. ZD and XX performed the experiments. ZD, NY, and SM analyzed the data. ZD and SG prepared the manuscript. YL was responsible for funding acquisition. Conflicts of interest None. Acknowledgments This research was funded by National Natural Science Foundation of China [grant numbers 81874030, 81402224]; the Provincial Science Foundation of Hunan [grant number 2015JJ3139]; the Health and Family Planning Commission of Hunan Province [grant number B2016105]; the Administration of Traditional Chinese Medicine of Hunan Province [grant number 2015116]; and the China Scholarship Council [grant number 201606375101]. References [1] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2017, CA Cancer J. Clin. 67 (1) (2017) 7–30, https://doi.org/10.3322/caac.21387. [2] M. Popovic, T.M. Hrcenjak, T. Babic, J. Kos, M. Grdisa, Effect of earthworm (G-90) extract on formation and lysis of clots originated from venous blood of dogs with cardiopathies and with malignant tumors, Pathol. Oncol. Res. 7 (3) (2001) 197–202. [3] Y.T. Fu, K.Y. Chen, Y.S. Chen, C.H. Yao, Earthworm (Pheretima aspergillum) extract stimulates osteoblast activity and inhibits osteoclast differentiation, BMC Complement. Altern. Med. 14 (2014) 440, https://doi.org/10.1186/1472-6882-14440. [4] E.L. Cooper, K. Hirabayashi, M. Balamurugan, Dilong: food for thought and medicine, J. Tradit. Complement. Med. 2 (4) (2012) 242–248. [5] Y.M. Chang, Y.T. Shih, Y.S. Chen, C.L. Liu, W.K. Fang, C.H. Tsai, F.J. Tsai, W.W. Kuo, T.Y. Lai, C.Y. Huang, Schwann cell migration induced by earthworm extract via activation of PAs and MMP2/9 mediated through ERK1/2 and p38, Evid. Complement. Alternat. Med. 2011 (2011) 395458, https://doi.org/10.1093/ecam/ nep131. [6] J. Yang, T. Wang, Y. Li, W. Yao, X. Ji, Q. Wu, L. Han, R. Han, W. Yan, J. Yuan, C. Ni, Earthworm extract attenuates silica-induced pulmonary fibrosis through Nrf2-dependent mechanisms, Lab. Invest. 96 (12) (2016) 1279–1300, https://doi.org/10. 1038/labinvest.2016.101. [7] Y.F. Zhao, Y. Gao, X.F. Wu, J.G. Wang, L.X. Duan, Protective effect of earthworm active ingredients against endoplasmic reticulum stress-induced acute liver injury in mice, Zhongguo Zhong Yao Za Zhi 42 (6) (2017) 1183–1188, https://doi.org/10. 19540/j.cnki.cjcmm.20170121.028. [8] Z.H. Deng, J.J. Yin, W. Luo, R.N. Kotian, S.S. Gao, Z.Q. Yi, W.F. Xiao, W.P. Li, Y.S. Li, The effect of earthworm extract on promoting skin wound healing, Biosci. Rep. 38 (2) (2018), https://doi.org/10.1042/BSR20171366. [9] D. Augustine, R.S. Rao, J. Anbu, K.N. Chidambara Murthy, In vitro antiproliferative effect of earthworm coelomic fluid of Eudrilus eugeniae, Eisenia foetida, and Perionyx excavatus on squamous cell Carcinoma-9 cell line: a pilot study, Pharmacognosy Res. 9 (Suppl. 1) (2017) S61–S66, https://doi.org/10.4103/pr.pr_ 52_17. [10] H. Chen, S. Takahashi, M. Imamura, E. Okutani, Z.G. Zhang, K. Chayama, B.A. Chen, Earthworm fibrinolytic enzyme: anti-tumor activity on human hepatoma cells in vitro and in vivo, Chin. Med. J. (Engl.) 120 (10) (2007) 898–904. [11] S. Zhao, L. He, Y. Lu, L. Duo, The impact of modified nano-carbon black on the earthworm Eisenia fetida under turfgrass growing conditions: assessment of survival, biomass, and antioxidant enzymatic activities, J. Hazard. Mater. 338 (2017) 218–223, https://doi.org/10.1016/j.jhazmat.2017.05.035. [12] Y.J. Shi, X.B. Xu, X.Q. Zheng, Y.L. Lu, Responses of growth inhibition and antioxidant gene expression in earthworms (Eisenia fetida) exposed to tetrabromobisphenol A, hexabromocyclododecane and decabromodiphenyl ether, Comp. Biochem. Physiol. C: Toxicol. Pharmacol. 174–175 (2015) 32–38, https://doi.org/ 10.1016/j.cbpc.2015.06.005. [13] M.A. Hawk, C. McCallister, Z.T. Schafer, Antioxidant activity during tumor progression: a necessity for the survival of cancer cells? Cancers (Basel) 8 (10) (2016), https://doi.org/10.3390/cancers8100092.
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Z. Deng, et al. [39] K.M. Debatin, Apoptosis pathways in cancer and cancer therapy, Cancer Immunol. Immunother. 53 (3) (2004) 153–159, https://doi.org/10.1007/s00262-003-0474-8. [40] S. Elmore, Apoptosis: a review of programmed cell death, Toxicol. Pathol. 35 (4) (2007) 495–516, https://doi.org/10.1080/01926230701320337. [41] J.M. Adams, S. Cory, Bcl-2-regulated apoptosis: mechanism and therapeutic potential, Curr. Opin. Immunol. 19 (5) (2007) 488–496, https://doi.org/10.1016/j. coi.2007.05.004. [42] M. Sabah, R. Cummins, M. Leader, E. Kay, Immunoreactivity of p53, Mdm2, p21(WAF1/CIP1) Bcl-2, and Bax in soft tissue sarcomas: correlation with histologic grade, Appl. Immunohistochem. Mol. Morphol. 15 (1) (2007) 64–69. [43] P. Pluta, P. Smolewski, A. Pluta, B. Cebula-Obrzut, A. Wierzbowska, D. Nejc, T. Robak, R. Kordek, L. Gottwald, J. Piekarski, A. Jeziorski, Significance of Bax expression in breast cancer patients, Pol. Przegl. Chir. 83 (10) (2011) 549–553, https://doi.org/10.2478/v10035-011-0087-4. [44] L. Yanqin, S. Yan, S. Zhenjun, L. Shijie, W. Chong, L. Yan, G. Yuhong, Coelomic fluid of the earthworm Eisenia fetida induces apoptosis of HeLa cells in vitro, Eur. J. Soil
Biol. 43 (2007) S143–S148, https://doi.org/10.1016/j.ejsobi.2007.08.049. [45] P.C. Li, Y.C. Tien, C.H. Day, P. Pai, W.W. Kuo, T.S. Chen, C.H. Kuo, C.H. Tsai, D.T. Ju, C.Y. Huang, Impact of LPS-induced cardiomyoblast cell apoptosis inhibited by earthworm extracts, Cardiovasc. Toxicol. 15 (2) (2015) 172–179, https://doi. org/10.1007/s12012-014-9281-z. [46] A. Akinbami, A. Popoola, A. Adediran, A. Dosunmu, O. Oshinaike, P. Adebola, S. Ajibola, Full blood count pattern of pre-chemotherapy breast cancer patients in Lagos, Nigeria, Caspian J. Intern. Med. 4 (1) (2013) 574–579. [47] J.R. Germa Lluch, R.B. Piulats, Correlation between white blood cell count and neutrophil count after chemotherapy administration at a day hospital, Rev. Esp. Oncol. 32 (4) (1985) 627–632. [48] N.I. Abdou, M. Richter, Cells involved in the immune response. VI. The immune response to red blood cells in irradiated rabbits after administration of normal, primed, or immune allogeneic rabbit bone marrow cells, J. Exp. Med. 129 (4) (1969) 757–774.
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