In vivo study of anticancer activity of ginsenoside Rh2-containing arginine-reduced graphene in a mouse model of breast cancer

Document Type : Original Article

Authors

1 Department of Biology, Science and Arts University, Yazd, Iran

2 Reproductive Immunology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

3 Hematology and Oncology Research Center, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

4 Department of Biomedical Engineering, Meybod University, Meybod, Iran

5 Department of Pathology, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

6 Faculty of Interdisciplinary Science and Technology, Tarbiat Modares University, Tehran, Iran

Abstract

Objective(s): This study aims to evaluate the in vivo anticancer activity of arginine-reduced graphene (Gr-Arg) and ginsenoside Rh2-containing arginine-reduced graphene (Gr-Arg-Rh2). 
Materials and Methods: Thirty-two mice with breast cancer were divided into four groups and treated every three days for 32 days: Group 1, PBS, Group 2, Rh2, Group 3, Gr-Arg, and Group 4, Gr-Arg-Rh2. The tumor size and weight, gene expression (IL10, INF-γ, TGFβ, and FOXP3), and pathological properties of the tumor and normal tissues were assessed. 
Results: Results showed a significant decrease in TGFβ expression for all drug treatment groups compared with the controls (P=0.04). There was no significant difference among the groups regarding IL10 and FOXP3 gene expression profiles (P>0.05). Gr-Arg-Rh2 significantly inhibited tumor growth (size and weight) compared with Rh2 and control groups. The highest survival rate and the highest percentage of tumor necrosis (87.5%) belonged to the Gr-Arg-Rh2 group. Lungs showed metastasis in the control group. No metastasis was observed in the Gr-Arg-Rh2 group. Gr-Arg-Rh2 showed partial degeneration of hepatocytes and acute cell infiltration in the portal spaces and around the central vein. The Gr-Arg group experienced a moderate infiltration of acute cells into the port spaces and around the central vein. The Rh2 group also showed a mild infiltration of acute and chronic cells in portal spaces. 
Conclusion: Based on the results, Gr-Arg-Rh2 can reduce tumor size, weight, and growth, TGF-β gene expression, and increase tumor necrosis and survival time in mice with cancer. 

Keywords


1. Siegel RL, Miller KD. Cancer statistics, 2020.  2020; 70:7-30.
2. Maughan KL, Lutterbie MA, Ham PS. Treatment of breast cancer. Am Fam Physician 2010; 81:1339-1346.
3. Moo T-A, Sanford R, Dang C, Morrow M. Overview of Breast Cancer Therapy. PET Clinics 2018; 13:339-354.
4. Nounou MI, ElAmrawy F, Ahmed N, Abdelraouf K, Goda S, Syed-Sha-Qhattal H. Breast Cancer: Conventional Diagnosis and Treatment Modalities and Recent Patents and Technologies. Breast Cancer Basic Clin Res 2015; 9:17-34.
5. Pearce A, Haas M, Viney R, Pearson S-A, Haywood P, Brown C, et al. Incidence and severity of self-reported chemotherapy side effects in routine care: A prospective cohort study. PLoS One 2017; 12:e0184360-e0184360.
6. Shewach DS, Kuchta RD. Introduction to cancer chemotherapeutics. Chem Rev 2009; 109:2859-2861.
7.Desai AG, Qazi GN, Ganju RK, El-Tamer M, Singh J, Saxena AK, et al. Medicinal plants and cancer chemoprevention. Curr Drug Metab 2008; 9:581-591.
8. Greenwell M, Rahman PKSM. Medicinal Plants: Their Use in Anticancer Treatment. Int J Pharm Sci Res 2015; 6:4103-4112.
9. Wan Y, Wang J, Xu JF, Tang F, Chen L, Tan YZ, Rao CL, Ao H, Peng C. Panax ginseng and its ginsenosides: Potential candidates for the prevention and treatment of chemotherapy-induced side effects. J Ginseng Res 2021 45(6):617-630. 
10. Oh M, Choi YH, Choi S, Chung H, Kim K, Kim SI, et al. Anti-proliferating effects of ginsenoside Rh2 on MCF-7 human breast cancer cells. Int J Oncol 1999; 14:869-875.
11. Nakata H, Kikuchi Y, Tode T, Hirata J, Kita T, Ishii K, et al. Inhibitory effects of ginsenoside Rh2 on tumor growth in nude mice bearing human ovarian cancer cells. Jpn J Cancer Res 1998; 89:733-740.
12. Gu Y, Wang GJ, Sun JG, Jia YW, Wang W, Xu MJ, et al. Pharmacokinetic characterization of ginsenoside Rh2, an anticancer nutrient from ginseng, in rats and dogs. Food Chem Toxicol 2009; 47:2257-2268.
13. Patra JK, Das G, Fraceto LF, Campos EVR, Rodriguez-Torres MdP, Acosta-Torres LS, et al. Nano based drug delivery systems: recent developments and future prospects. J Nanobiotechnology 2018; 16:1-33.
14. Naghibzadeh M, Firoozi S, Nodoushan FS, Adabi M, Khoradmehr A, Fesahat F, et al. Application of electrospun gelatin nanofibers in tissue engineering. Biointerface Res Appl Chem 2018; 8:3048-3052.
15. Compton OC, Nguyen ST. Graphene oxide, highly reduced graphene oxide, and graphene: versatile building blocks for carbon‐based materials. Small 2010; 6:711-723.
16. Zare-Zardini H, Taheri-Kafrani A, Ordooei M, Amiri A, Karimi-Zarchi M. Evaluation of toxicity of functionalized graphene oxide with ginsenoside Rh2, lysine and arginine on blood cancer cells (K562), red blood cells, blood coagulation and cardiovascular tissue: In vitro and in vivo studies. J Taiwan Inst Chem Eng 2018; 93:70-78.
17. Zare-Zardini H, Taheri-Kafrani A, Amiri A, Bordbar A-K. New generation of drug delivery systems based on ginsenoside Rh2-, Lysine-and Arginine-treated highly porous graphene for improving anticancer activity. Sci Rep 2018; 8:1-15.
18.Foroutan T, Nazemi N, Tavana M, Kassaee MZ, Motamedi E, Sonieshargh S, et al. Suspended graphene oxide nanoparticle for accelerated multilayer osteoblast attachment. J Biomed Mater Res A 2018; 106:293-303.
19.Moon IK, Lee J, Ruoff RS, Lee H. Reduced graphene oxide by chemical graphitization. Nat Commun 2010; 1:1-6.
20.Zare-Zardini H, Taheri-Kafrani A, Amiri A, Bordbar A-K. New generation of drug delivery systems based on ginsenoside Rh2-, Lysine- and Arginine-treated highly porous graphene for improving anticancer activity. Sci Rep 2018; 8:1-5.
21. Sun Q, Wang X, Cui C, Li J, Wang Y. Doxorubicin and anti-VEGF siRNA co-delivery via nano-graphene oxide for enhanced cancer therapy in vitro and in vivo. Int J Nanomedicine 2018; 13: 3713-3728.
22. Dehghan-Manshadi M, Nikpoor AR, Hadinedoushan H, Zare F, Sankian M, Fesahat F, et al. Protective immune response against P32 oncogenic peptide-pulsed PBMCs in mouse models of breast cancer. Int Immunopharmacol 2021; 93:107414.
23. Nikpoor AR, Tavakkol-Afshari J, Sadri K, Jalali SA, Jaafari MR. Improved tumor accumulation and therapeutic efficacy of CTLA-4-blocking antibody using liposome-encapsulated antibody: In vitro and in vivo studies. Nanomedicine: NBM. 2017;13:2671-2682.
24.Economopoulos SP, Rotas G, Miyata Y, Shinohara H, Tagmatarchis N. Exfoliation and chemical modification using microwave irradiation affording highly functionalized graphene. ACS Nano 2010; 4:7499-7507.
25. SreeHarsha N, Maheshwari R, Al-Dhubiab BE, Tekade M, Sharma MC, Venugopala KN, et al. Graphene-based hybrid nanoparticle of doxorubicin for cancer chemotherapy. Int J Nanomed 2019; 14:7419.
26. Yang XX, Li CM, Li YF, Wang J, Huang CZ. Synergistic antiviral effect of curcumin functionalized graphene oxide against respiratory syncytial virus infection. Nanoscale 2017; 9:16086-16092.
27. Javanbakht S, Namazi H. Doxorubicin loaded carboxymethyl cellulose/graphene quantum dot nanocomposite hydrogel films as a potential anticancer drug delivery system. Mater Sci Eng C Mater Biol Appl 2018; 87:50-59.
28. Dillekås H, Rogers MS, Straume O. Are 90% of deaths from cancer caused by metastases? Cancer Med 2019; 8:5574-5576.
29. Gennari A, Conte P, Rosso R, Orlandini C, Bruzzi P. Survival of metastatic breast carcinoma patients over a 20-year period: a retrospective analysis based on individual patient data from six consecutive studies. Cancer 2005; 104:1742-1750.
30. Jin L, Han B, Siegel E, Cui Y, Giuliano A, Cui X. Breast cancer lung metastasis: Molecular biology and therapeutic implications. Cancer Biol Ther 2018; 19:858-868.
31. Zheng H, Yuan C, Cai J, Pu W, Wu P, Li C, et al. Early diagnosis of breast cancer lung metastasis by nanoprobe-based luminescence imaging of the pre-metastatic niche. J Nanobiotechnology 2022; 20:1-7.
32. Patil MD, Bhaumik J, Babykutty S, Banerjee UC, Fukumura D. Arginine dependence of tumor cells: targeting a chink in cancer’s armor. Oncogene 2016; 35:4957-4972.
33. Albaugh VL, Pinzon-Guzman C, Barbul A. Arginine-Dual roles as an onconutrient and immunonutrient. J Surg Oncol 2017; 115:273-280.
34. Lubec B, Hoeger H, Kremser K, Amann G, Koller DY, Gialamas J. Decreased tumor incidence and increased survival by one year oral low dose arginine supplementation in the mouse. Life Sci 1996; 58:2317-2325.
35. Al-Koussa H, El Mais N, Maalouf H, Abi-Habib R, El-Sibai M. Arginine deprivation: a potential therapeutic for cancer cell metastasis? A review. Cancer Cell Int 2020; 20:150-157.
36. Patel SC, Lee S, Lalwani G, Suhrland C, Chowdhury SM, Sitharaman B. Graphene-based platforms for cancer therapeutics. Ther Deliv 2016; 7:101-116.
37. Sharma H, Mondal S. Functionalized Graphene Oxide for Chemotherapeutic Drug Delivery and Cancer Treatment: A Promising Material in Nanomedicine. Int J Mol Sci. 2020; 21:6280.
38. Ou L, Lin S, Song B, Liu J, Lai R, Shao L. The mechanisms of graphene-based materials-induced programmed cell death: A review of apoptosis, autophagy, and programmed necrosis. Int J Nanomedicine 2017; 12: 6633–6646.
39. Tao K, Fang M, Alroy J, Sahagian GG. Imagable 4T1 model for the study of late stage breast cancer. BMC Cancer 2008; 8:1-9.
40. Liaw C-C, Chang H, Yang T-S, Wen M-S. Pulmonary Venous Obstruction in Cancer Patients. J Oncol 2015; 2015:1-10.
41. Sangisetty SL, Miner TJ. Malignant ascites: A review of prognostic factors, pathophysiology and therapeutic measures. World J Gastrointest Surg 2012; 4:87-95.
42. Gupta A, Sedhom R, Beg MS. Ascites, or Fluid in the Belly, in Patients With Cancer. JAMA Oncology 2020; 6:308-308.
43. Méndez-García LA, Nava-Castro KE, Ochoa-Mercado TL, Palacios-Arreola MI, Ruiz-Manzano RA, Segovia-Mendoza M, et al. Breast cancer metastasis: Are cytokines important players during its development and progression? J Interferon Cytokine Res 2019; 39:39-55.
44. Esquivel-Velázquez M, Ostoa-Saloma P, Palacios-Arreola MI, Nava-Castro KE, Castro JI, Morales-Montor J. The role of cytokines in breast cancer development and progression. J Interferon Cytokine Res 2015; 35:1-16.
45. Kretzschmar M. Transforming growth factor-beta and breast cancer: Transforming growth factor-beta/SMAD signaling defects and cancer. Breast Cancer Res 2000; 2:107-115.
46. Fang J, Xu H, Yang C, Kayarthodi S, Matthews R, Rao VN, et al. Molecular Mechanism of Activation of Transforming Growth Factor Beta/Smads Signaling Pathway in Ets Related Gene-Positive Prostate Cancers. J Pharm Sci Pharmacol 2014; 1:82-85.
47. Tian M, Neil JR, Schiemann WP. Transforming growth factor-β and the hallmarks of cancer. Cell Signal 2011; 23:951-962.
48. Kocic J, Bugarski D, Santibanez JF. SMAD3 is essential for transforming growth factor-β1-induced urokinase type plasminogen activator expression and migration in transformed keratinocytes. Eur J Cancer 2012; 48:1550-1557.
49. Hamidullah, Changkija B, Konwar R. Role of interleukin-10 in breast cancer. Breast Cancer Res Treat 2012; 133:11-21.
50. Changkija B, Konwar R. Role of interleukin-10 in breast cancer. Breast Cancer Res Treat  2012;133: 11-21.
51. Chang CM, Lam HYP, Hsu HJ, Jiang SJ. Interleukin-10: A double-edged sword in breast cancer. Tzu Chi Med J 2021; 33:203-211.
52. Jiang X. Macrophage-produced IL-10 limits the chemotherapy efficacy in breast cancer. J Zhejiang Univ Sci B 2015; 16:44-45.
53. Martin F, Ladoire S, Mignot G, Apetoh L, Ghiringhelli F. Human FOXP3 and cancer. Oncogene 2010; 29:4121-4129.
54. Yang S, Liu Y, Li M-Y, Ng CSH, Yang S-l, Wang S, et al. FOXP3 promotes tumor growth and metastasis by activating Wnt/β-catenin signaling pathway and EMT in non-small cell lung cancer. Mol Cancer 2017; 16:1-12.
55. Takenaka M, Seki N, Toh U, Hattori S, Kawahara A, Yamaguchi T, et al. FOXP3 expression in tumor cells and tumor-infiltrating lymphocytes is associated with breast cancer prognosis. Mol Clin Oncol 2013; 1:625-632.
56. Ni L, Lu J. Interferon gamma in cancer immunotherapy. Cancer medicine 2018; 7:4509-4516.
57. Kotredes KP, Gamero AM. Interferons as inducers of apoptosis in malignant cells. J Interferon Cytokine Res 2013; 33:162-170.
58. Pusztai L, Mendoza TR, Reuben JM, Martinez MM, Willey JS, Lara J, et al. Changes in plasma levels of inflammatory cytokines in response to paclitaxel chemotherapy. Cytokine 2004; 25:94-102.
59. Reers S, Pfannerstill A-C, Rades D, Maushagen R, Andratschke M, Pries R, et al. Cytokine changes in response to radio-/chemotherapeutic treatment in head and neck cancer. Anticancer Res 2013; 33:2481-2489.
60. Jorgovanovic D, Song M, Wang L, Zhang Y. Roles of IFN-γ in tumor progression and regression: A review. Biomark Res 2020; 8:49.
61. Connolly EC, Freimuth J, Akhurst RJ. Complexities of TGF-β targeted cancer therapy. Int J Biol Sci 2012; 8:964-978.