Effects of Ifosfamide and Curcumin Combination on Apoptotic Biomarkers and Cell Migration in Non-Small Cell Lung Cancer Cell

Abstract

Lung cancer is one of the most common malignancies and aggressive tumour, which causes the highest cancer-related death in the world. Ifosfamide is an alkylating antineoplastic agent used in the treatment of cancer including, lung cancer. Curcumin, a flavonoid from the rhizome of Curcuma longa has been shown to chemotherapeutic activity through inhibition of cell growth, cell cycle arrest, and pro-apoptotic activity in human cancer. However, the effects of Ifosfamide and Curcumin combination on non-small cell lung cancer (NSCLC) remain unclear. In this study, the influences of Ifosfamide, cell proliferation, ROS accumulation, apoptosis, migration, and its combined effects with curcumin were investigated in NSCLC cell line A549 by RT-Cell analyse, DCFDA assay, western blot and wound healing-migration assays. A549 cells were treated with the Ifosfamide-Curcumin combination and the results showed that the combination was effective in ROS accumulation, which contributed to inducing apoptosis via increasing pro-apoptotic Bax and decreasing anti-apoptotic TIGAR, and depression of cell migration. The treatment of combination significantly induced apoptosis, together with the downregulation of TIGAR and upregulation of pro-apoptotic Bax protein. Moreover, Curcumin and Ifosfamide combination suppressed cell migration into the scratched area as compared to untreated cells. Our findings suggest that Curcumin enhanced the efficacy of Ifosfamide in human non-small cell lung cancer cell line.

Keywords

• Ifosfamide
• Curcumin
• A549
• Apoptosis
• Cell migration

İfosfamid ve Kurkumin Kombinasyonun Küçük Hücreli Dışı Akciğer Kanseri Hücresinde Apoptotik Biyobelirteçler ve Hücre Göçü Üzerine Etkileri

Abstract

Akciğer kanseri, dünyada kansere bağlı en yüksek ölüme neden olan kötü huylu ve saldırgan tümörlerden biridir. İfosfamid, akciğer kanserinin de dâhil olduğu birçok kanser tedavisinde kullanılan alkilleyici bir antineoplastik ajandır. Curcuma longa rizomundan elde edilen bir flavonoid olan kurkumin'in, insan kanserlerinde hücre büyümesinin inhibisyonu, hücre döngüsü dudurulması ve pro-apoptotik etki yoluyla kemoterapötik aktiviteye sahip olduğu gösterilmiştir. Bununla birlikte, İfosfamid ve Kurkumin kombinasyonunun küçük hücreli olmayan akciğer kanseri (NSCLC) üzerindeki etkileri henüz doğrudan ele alınmamıştır. Bu çalışmada akciğer kanseri hücre hattı A549'da, İfosfamid ve Kurkumin kombinasyonunun, hücre proliferasyonu, hücre içi ROS birikimi, apoptozis ve hücre göçü üzerine olan etkileri gerçek-zamanlı hücre analizi, DCFDA floresan işaretleme, western blot ve yara iyileşme-hücre göçü testleri ile araştırılmıştır. A549 hücrelerinde, İfosfamid-kurkumin kombinasyonu ile tedavi sonrasında, hücre içi ROS birikiminde ve pro-apoptotik Bax proteinin düzeyinde bir artış olduğu belirlendi. Ayrıca, kombinasyonun, tedavi edilmeyen ve tek ajan ile tedavi edilen hücrelere kıyasla, anti-apoptotik TIGAR protein düzeyinin azalmasında ve çizilen alana hücre göçünün bastırılmasında etkili olduğu belirlendi. Bulgularımız, Kurkuminin, küçük hücreli olmayan akciğer kanseri hücre hattın A549’da İfosfamidin etkinliğini artırdığını göstermektedir.

Keywords

• İfosfamid
• Kurkumin
• A549
• Apoptozis
• Hücre Göçü

References

1. [1] Bray F, Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin. 2018;68(6):394–424.
2. [2] Siegel RL, Miller KD, Jemal A. Cancer statistics, 2017. CA Cancer J Clin. 2017;67(1):7–30.
3. [3] Pirker R. Chemotherapy remains a cornerstone in the treatment of nonsmall cell lung cancer. Current opinion in oncology. 2020;32(1):63-7.
4. [4] Belani CP. Paclitaxel/carboplatin in the treatment of non-small-cell lung cancer. ONCOLOGY-WILLISTON PARK THEN HUNTINGTON-. 1998; 12:74-9.
5. [5] Emadi A, Jones RJ, Brodsky RA. Cyclophosphamide and cancer: golden anniversary. Nature reviews Clinical oncology. 2009 Nov;6(11):638.
6. [6] Agca CA, Kırıcı M, Nedzvetsky VS, Gundogdu R, Tykhomyrov AA. The Effect of TIGAR Knockdown on Apoptotic and Epithelial‐Mesenchymal Markers Expression in Doxorubicin‐Resistant Non‐Small Cell Lung Cancer A549 Cell Lines. Chemistry & Biodiversity. 2020;17(9): 1-14.
7. [7] Dilruba S, Kalayda GV. Platinum-based drugs: past, present and future. Cancer chemotherapy and pharmacology. 2016;77(6):1103-24.
8. [8] Liu RM, Xu P, Chen Q, Feng SL, Xie Y. A multiple-targets alkaloid nuciferine overcomes paclitaxel-induced drug resistance in vitro and in vivo. Phytomedicine. 2020;79: 1-12.

9. [9] Huang RY, Pei L, Liu Q, Chen S, Dou H, Shu G, Yuan ZX, Lin J, Peng G, Zhang W, Fu H. Isobologram analysis: a comprehensive review of methodology and current research. Frontiers in pharmacology. 2019 29;10:1222.
10. [10] Martín AJM, Alfonso PG, Rupérez AB, Jiménez MM. Nab-paclitaxel plus gemcitabine as first-line palliative chemotherapy in a patient with metastatic pancreatic cancer with Eastern Cooperative Oncology Group performance status of 2. Oncol Lett. 2016;12(1):727–30.
11. [11] Ebara S, Kobayashi Y, Sasaki K, Araki M, Sugimoto M, Wada K, et al. A case of metastatic urachal cancer including a neuroendocrine component treated with gemcitabine, cisplatin and paclitaxel combination chemotherapy. Acta Med Okayama. 2016;70(3):223–7.
12. [12] Wagner T. Ifosfamide clinical pharmacokinetics. Clinical pharmacokinetics. 1994 Jun;26(6):439-56.
13. [13] Binotto G, Trentin L, Semenzato G. Ifosfamide and cyclophosphamide: effects on immunosurveillance. Oncology. 2003;65(2):17-20.
14. [14] Aziz K, Nowsheen S, Pantelias G, Iliakis G, Gorgoulis VG, Georgakilas AG. Targeting DNA damage and repair: embracing the pharmacological era for successful cancer therapy. Pharmacology & therapeutics. 2012 Mar;133(3):334-50.
15. [15] Jacot W, Pujol JL, Chakra M, Molinier O, Bozonnat MC, Gervais R, et al. Epirubicin and ifosfamide in relapsed or refractory small cell lung cancer patients. Lung Cancer. 2012;75(2):213–6.
16. [16] Furlanut M, Franceschi L. Pharmacology of ifosfamide. Oncology. 2003;65(2):2-6.
17. [17] Wang HT, Chen TY, Weng CW, Yang CH, Tang M. Acrolein preferentially damages nucleolus eliciting ribosomal stress and apoptosis in human cancer cells. Oncotarget. 2016;7(49):80450–64.
18. [18] Lee L-K, Chen P-M, Tzeng C-H, Liu J-H, Yen C-C. Ifosfamide-Induced Fanconi’s Syndrome. J Cancer Res Pract. 2014;1(1):46–9.
19. [19] Issels RD, Meier TH, Müller E, Multhoff G, Wilmanns W. Ifosfamide induced stress response in human lymphocytes. Mol Aspects Med. 1993;14(3):281–6.
20. [20] Meng B, Li J, Cao H. Antioxidant and Antiinflammatory Activities of Curcumin on Diabetes Mellitus and its Complications. Curr Pharm Des. 2013;19(11):2101–13.
21. [21] Trujillo J, Chirino YI, Molina-Jijón E, Andérica-Romero AC, Tapia E, Pedraza-Chaverrí J. Renoprotective effect of the antioxidant curcumin: Recent findings. Redox biology. 2013;1(1):448-56.
22. [22] Agrawal DK, Mishra PK. Curcumin and its analogues: potential anticancer agents. Medicinal research reviews. 2010;30(5):818-60.
23. [23] Salehi M, Movahedpour A, Tayarani A, Shabaninejad Z, Pourhanifeh MH, Mortezapour E, et al. Therapeutic potentials of curcumin in the treatment of non‐small‐cell lung carcinoma. Phyther Res. 2020;34(10):2557–76.
24. [24] Hewlings S, Kalman D. Curcumin: A Review of Its Effects on Human Health. Foods. 2017;6(10):92.
25. [25] Chen L, Zhan C-Z, Wang T, You H, Yao R. Curcumin Inhibits the Proliferation, Migration, Invasion, and Apoptosis of Diffuse Large B-Cell Lymphoma Cell Line by Regulating MiR-21/VHL Axis. Yonsei Med J. 2020;61(1):20.
26. [26] Sak K. Radiosensitizing potential of curcumin in different cancer models. Nutrition and cancer. 2020;72(8):1276-89.
27. [27] Zhu Y, Bu S. Curcumin induces autophagy, apoptosis, and cell cycle arrest in human pancreatic cancer cells. Evidence-Based Complementary and Alternative Medicine. 2017; 4:1-13.
28. [28] Chen QY, Lu GH, Wu YQ, Zheng Y, Xu K, Wu LJ, et al. Curcumin induces mitochondria pathway mediated cell apoptosis in A549 lung adenocarcinoma cells. Oncol Rep. 2010;23(5):1285–92.
29. [29] Devassy JG, Nwachukwu ID, Jones PJH. Curcumin and cancer: Barriers to obtaining a health claim. Nutr Rev. 2015;73(3):155–65.
30. [30] Hu A, Huang J-J, Zhang J-F, Dai W-J, Li R-L, Lu Z-Y, et al. Curcumin induces G2/M cell cycle arrest and apoptosis of head and neck squamous cell carcinoma in vitro and in vivo through ATM/Chk2/p53-dependent pathway. Oncotarget. 2017;8(31):50747–60.
31. [31] Guan F, Ding Y, Zhang Y, Zhou Y, Li M, Wang C. Curcumin suppresses proliferation and migration of MDA-MB-231 breast cancer cells through autophagy-dependent Akt degradation. PLoS One. 2016;11(1):1-18.
32. [32] Tan BL, Norhaizan ME. Curcumin combination chemotherapy: The implication and efficacy in cancer. Molecules. 2019;24(14):1–21.
33. [33] Chen Q, Wang Y, Xu K, Lu G, Ying Z, Wu L, et al. Curcumin induces apoptosis in human lung adenocarcinoma A549 cells through a reactive oxygen species-dependent mitochondrial signaling pathway. Oncol Rep. 2010;23(2):397–403.
34. [34] Yanagawa H, Haku T, Takeuchi E, Suzuki Y, Nokihara H, Sone S. Intrapleural therapy with MDP-Lys (L18), a synthetic derivative of muramyl dipeptide, against malignant pleurisy associated with lung cancer. Lung Cancer. 2000;27(2):67–73.
35. [35] Wu SH, Hang LW, Yang JS, Chen HY, Lin HY, Chiang JH, et al. Curcumin induces apoptosis in human non-small cell lung cancer NCI-H460 cells through ER stress and caspase cascade- and mitochondria-dependent pathways. Anticancer Res. 2010;30(6):2125–33.
36. [36] Yue Q, Gao G, Zou G, Yu H, Zheng X. Natural products as adjunctive treatment for pancreatic cancer: recent trends and advancements. BioMed research international. 2017 23;4:1-13.
37. [37] Aggarwal V, Tuli HS, Varol A, Thakral F, Yerer MB, Sak K,et al. Role of reactive oxygen species in cancer progression: molecular mechanisms and recent advancements. Biomolecules. 2019;9(11):735.
38. [38] Chanvorachote P, Pongrakhananon V, Wannachaiyasit S, Luanpitpong S, Rojanasakul Y, Nimmannit U. Curcumin sensitizes lung cancer cells to cisplatin-induced apoptosis through superoxide anion-mediated Bcl-2 degradation. Cancer Invest. 2009;27(6):624–35.
39. [39] Papież MA, Krzyściak W, Szade K, Bukowska-Straková K, Kozakowska M, Hajduk K, et al. Curcumin enhances the cytogenotoxic effect of etoposide in leukemia cells through induction of reactive oxygen species. Drug Des Devel Ther. 2016; 10:557–70.
40. [40] Jayakiran M. Apoptosis-Biochemistry: A Mini Review. J Clin Exp Pathol. 2015;05(01):1–4.
41. [41] Tiwari P, Khan MJ. Molecular and computational studies on apoptotic pathway regulator, Bcl-2 gene from breast cancer cell line MCF-7. Indian J Pharm Sci. 2016;78(1):87–93.
42. [42] Nakazawa M, Matsubara H, Matsushita Y, Watanabe M, Vo N, Yoshida H, et al. The human Bcl-2 family member Bcl-rambo localizes to mitochondria and induces apoptosis and morphological aberrations in drosophila. PLoS One. 2016 ;11(6) 1-23.
43. [43] Liu G, Pei F, Yang F, Li L, Amin AD, Liu S,et al. Role of autophagy and apoptosis in non-small-cell lung cancer. International journal of molecular sciences. 2017;18(2):367.
44. [44] Bensaad K, Cheung EC, Vousden KH. Modulation of intracellular ROS levels by TIGAR controls autophagy. EMBO J. 2009;28(19):3015–26.
45. [45] Bensaad K, Tsuruta A, Selak MA, Vidal MNC, Nakano K, Bartrons R, et al. TIGAR, a p53-Inducible Regulator of Glycolysis and Apoptosis. Cell. 2006;126(1):107–20.
46. [46] Wan Mohd Tajuddin WNB, Lajis NH, Abas F, Othman I, Naidu R. Mechanistic Understanding of Curcumin’s Therapeutic Effects in Lung Cancer. Nutrients. 2019;11(12):2989.
47. [47] Chen Q yong, Zheng Y, Jiao D min, Chen F yuan, Hu H zhen, Wu Y quan, et al. Curcumin inhibits lung cancer cell migration and invasion through Rac1-dependent signaling pathway. J Nutr Biochem. 2014;25(2):177–85.
48. [48] Tsai JR, Liu PL, Chen YH, Chou SH, Cheng YJ, Hwang JJ, et al. Curcumin inhibits non-Small cell lung cancer cells metastasis through the adiponectin/nf-κb/mmps signaling pathway. PLoS One. 2015 Dec 1;10(12).