Combination of nadroparin with radiotherapy results in powerful synergistic antitumor effects in lung adenocarcinoma A549 cells

Zhuang,Xibing; Qiao,Tiankui; Xu,Guoxiong; Yuan,Sujuan; Zhang,Qi; Chen,Xue

doi:10.3892/or.2016.4990

Combination of nadroparin with radiotherapy results in powerful synergistic antitumor effects in lung adenocarcinoma A549 cells

Authors:
- Xibing Zhuang
- Tiankui Qiao
- Guoxiong Xu
- Sujuan Yuan
- Qi Zhang
- Xue Chen
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Affiliations: Department of Oncology, Jinshan Hospital, Fudan University, Shanghai 201508, P.R. China, Center Laboratory, Jinshan Hospital, Fudan University, Shanghai 201508, P.R. China
Published online on: August 1, 2016 https://doi.org/10.3892/or.2016.4990
Pages: 2200-2206

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Abstract

Low-molecular-weight heparins (LMWHs), which are commonly used in venous thromboprophylaxis and treatment, have recently been reported to have effects on cancer metastasis in pre-clinical research studies. This study was planned to define the synergistic antitumor effects of nadroparin (a kind of LMWH) combined with radiotherapy in A549 cells. Six experimental groups were set up in our study according to the different treatment: control group; irradiation (IR) group; low dose of nadroparin group (LMWH50, L50); high dose of nadroparin group (LMWH100, L100); LMWH50+IR group; LMWH100+IR group. The viability of A549 cells was assessed by Cell Counting Kit-8 (CCK-8) assay. The apoptosis of tumor cells was analyzed by flow cytometry (FCM) after treatment. The concentration of transforming growth factor-β1 (TGF-β1) in the culture supernatants was measured by enzyme-linked immunosorbent assay (ELISA). The migration and invasion of the A549 cells were tested by the Transwell chamber assay. The expression of survivin, CD147 and matrix metalloproteinase-2 (MMP-2) was analyzed by western blotting. CCK-8 assay showed that irradiation or nadroparin alone slightly inhibited the cell viability while the combined treatments significantly inhibited the cell viability in a dose- and time-dependent manner. The apoptosis rate showed greater improvement dose- and time‑dependently in the groups receiving combination therapy of nadroparin and irradiation than the control group or the group receiving nadroparin or irradiation alone by FCM. ELISA assay showed that the decreased TGF-β1 secretion was found after combined treatments with nadroparin and irradiation compared to either treatment alone. The Transwell chamber assay showed that nadroparin not only significantly suppressed the migration and invasion of A549 cells but also inhibited the enhanced ability of migration and invasion induced by X-ray irradiation. Western blotting showed that nadroparin inhibited the upregulated effects of survivin and MMP-2 expression induced by radiation in the combined treatment groups in a dose- and time-dependent manner. Moreover, the expression level of CD147 was the lowest in the combined treatment groups. This study identified that combination of nadroparin and irradiation had a strong synergistic antitumor effect in a dose- and time-related manner in vitro, which was reflected in the inhibition of cell viability, invasion and metastasis, promotion of apoptosis, inhibited secretion level of TGF-β1 and downregulation of CD147, MMP-2 and survivin expression.

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1	Padera TP, Kadambi A, di Tomaso E, Carreira CM, Brown EB, Boucher Y, Choi NC, Mathisen D, Wain J, Mark EJ, et al: Lymphatic metastasis in the absence of functional intratumor lymphatics. Science. 296:1883–1886. 2002. View Article : Google Scholar : PubMed/NCBI
2	Mitrovska S and Jovanova S: Low-molecular weight heparin enoxaparin in the treatment of acute coronary syndromes without ST segment elevation. Bratisl Lek Listy. 110:45–48. 2009.PubMed/NCBI
3	Mousa SA and Petersen LJ: Anti-cancer properties of low-molecular-weight heparin: Preclinical evidence. Thromb Haemost. 102:258–267. 2009.PubMed/NCBI
4	Icli F, Akbulut H, Utkan G, Yalcin B, Dincol D, Isikdogan A, Demirkazik A, Onur H, Cay F and Büyükcelik A: Low molecular weight heparin (LMWH) increases the efficacy of cisplatinum plus gemcitabine combination in advanced pancreatic cancer. J Surg Oncol. 95:507–512. 2007. View Article : Google Scholar
5	Yu CJ, Ye SJ, Feng ZH, Ou WJ, Zhou XK, Li LD, Mao YQ, Zhu W and Wei YQ: Effect of Fraxiparine, a type of low molecular weight heparin, on the invasion and metastasis of lung adenocarcinoma A549 cells. Oncol Lett. 1:755–760. 2010.PubMed/NCBI
6	Carmazzi Y, Iorio M, Armani C, Cianchetti S, Raggi F, Neri T, Cordazzo C, Petrini S, Vanacore R, Bogazzi F, et al: The mechanisms of nadroparin-mediated inhibition of proliferation of two human lung cancer cell lines. Cell Prolif. 45:545–556. 2012. View Article : Google Scholar : PubMed/NCBI
7	Niu Q, Wang W, Li Y, Ruden DM, Wang F, Li Y, Wang F, Song J and Zheng K: Low molecular weight heparin ablates lung cancer cisplatin-resistance by inducing proteasome-mediated ABCG2 protein degradation. PLoS One. 7:e410352012. View Article : Google Scholar : PubMed/NCBI
8	Seidensticker M, Seidensticker R, Damm R, Mohnike K, Pech M, Sangro B, Hass P, Wust P, Kropf S, Gademann G, et al: Prospective randomized trial of enoxaparin, pentoxifylline and ursodeoxycholic acid for prevention of radiation-induced liver toxicity. PLoS One. 9:e1127312014. View Article : Google Scholar : PubMed/NCBI
9	Bae SM, Kim JH, Chung SW, Byun Y, Kim SY, Lee BH, Kim IS and Park RW: An apoptosis-homing peptide-conjugated low molecular weight heparin-taurocholate conjugate with antitumor properties. Biomaterials. 34:2077–2086. 2013. View Article : Google Scholar
10	Erduran E, Tekelioğlu Y, Gedik Y and Yildiran A: Apoptotic effects of heparin on lymphoblasts, neutrophils, and mononuclear cells: Results of a preliminary in vitro study. Am J Hematol. 61:90–93. 1999. View Article : Google Scholar : PubMed/NCBI
11	Jadhav U and Mohanam S: Response of neuroblastoma cells to ionizing radiation: Modulation of in vitro invasiveness and angiogenesis of human microvascular endothelial cells. Int J Oncol. 29:1525–1531. 2006.PubMed/NCBI
12	Kaliski A, Maggiorella L, Cengel KA, Mathe D, Rouffiac V, Opolon P, Lassau N, Bourhis J and Deutsch E: Angiogenesis and tumor growth inhibition by a matrix metalloproteinase inhibitor targeting radiation-induced invasion. Mol Cancer Ther. 4:1717–1728. 2005. View Article : Google Scholar : PubMed/NCBI
13	Park CM, Park MJ, Kwak HJ, Lee HC, Kim MS, Lee SH, Park IC, Rhee CH and Hong SI: Ionizing radiation enhances matrix metalloproteinase-2 secretion and invasion of glioma cells through Src/epidermal growth factor receptor-mediated p38/Akt and phosphatidylinositol 3-kinase/Akt signaling pathways. Cancer Res. 66:8511–8519. 2006. View Article : Google Scholar : PubMed/NCBI
14	Zhai GG, Malhotra R, Delaney M, Latham D, Nestler U, Zhang M, Mukherjee N, Song Q, Robe P and Chakravarti A: Radiation enhances the invasive potential of primary glioblastoma cells via activation of the Rho signaling pathway. J Neurooncol. 76:227–237. 2006. View Article : Google Scholar
15	Camphausen K, Moses MA, Beecken WD, Khan MK, Folkman J and O'Reilly MS: Radiation therapy to a primary tumor accelerates metastatic growth in mice. Cancer Res. 61:2207–2211. 2001.PubMed/NCBI
16	Norrby K: Low-molecular-weight heparins and angiogenesis. APMIS. 114:79–102. 2006. View Article : Google Scholar : PubMed/NCBI
17	Debergh I, Van Damme N, Pattyn P, Peeters M and Ceelen WP: The low-molecular-weight heparin, nadroparin, inhibits tumour angiogenesis in a rodent dorsal skinfold chamber model. Br J Cancer. 102:837–843. 2010. View Article : Google Scholar : PubMed/NCBI
18	Kakkar AK, Levine MN, Kadziola Z, Lemoine NR, Low V, Patel HK, Rustin G, Thomas M, Quigley M and Williamson RC: Low molecular weight heparin, therapy with dalteparin, and survival in advanced cancer: The fragmin advanced malignancy outcome study (FAMOUS). J Clin Oncol. 22:1944–1948. 2004. View Article : Google Scholar : PubMed/NCBI
19	Altinbas M, Coskun HS, Er O, Ozkan M, Eser B, Unal A, Cetin M and Soyuer S: A randomized clinical trial of combination chemotherapy with and without low-molecular-weight heparin in small cell lung cancer. J Thromb Haemost. 2:1266–1271. 2004. View Article : Google Scholar : PubMed/NCBI
20	Klerk CP, Smorenburg SM, Otten HM, Lensing AW, Prins MH, Piovella F, Prandoni P, Bos MM, Richel DJ, van Tienhoven G, et al: The effect of low molecular weight heparin on survival in patients with advanced malignancy. J Clin Oncol. 23:2130–2135. 2005. View Article : Google Scholar : PubMed/NCBI
21	Tuxhorn JA, McAlhany SJ, Yang F, Dang TD and Rowley DR: Inhibition of transforming growth factor-beta activity decreases angiogenesis in a human prostate cancer-reactive stroma xenograft model. Cancer Res. 62:6021–6025. 2002.PubMed/NCBI
22	Li C, Guo B, Bernabeu C and Kumar S: Angiogenesis in breast cancer: The role of transforming growth factor beta and CD105. Microsc Res Tech. 52:437–449. 2001. View Article : Google Scholar : PubMed/NCBI
23	Kanekura T and Chen X: CD147/basigin promotes progression of malignant melanoma and other cancers. J Dermatol Sci. 57:149–154. 2010. View Article : Google Scholar : PubMed/NCBI
24	Weidle UH, Scheuer W, Eggle D, Klostermann S and Stockinger H: Cancer-related issues of CD147. Cancer Genomics Proteomics. 7:157–169. 2010.PubMed/NCBI
25	Wu J, Li Y, Dang YZ, Gao HX, Jiang JL and Chen ZN: HAb18G/CD147 promotes radioresistance in hepatocellular carcinoma cells: A potential role for integrin β1 signaling. Mol Cancer Ther. 14:553–563. 2015. View Article : Google Scholar
26	Ambrosini G, Adida C and Altieri DC: A novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 3:917–921. 1997. View Article : Google Scholar : PubMed/NCBI
27	Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T and Reed JC: IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res. 58:5315–5320. 1998.PubMed/NCBI
28	Suzuki A, Ito T, Kawano H, Hayashida M, Hayasaki Y, Tsutomi Y, Akahane K, Nakano T, Miura M and Shiraki K: Survivin initiates procaspase 3/p21 complex formation as a result of interaction with Cdk4 to resist Fas-mediated cell death. Oncogene. 19:1346–1353. 2000. View Article : Google Scholar : PubMed/NCBI
29	Li F, Ambrosini G, Chu EY, Plescia J, Tognin S, Marchisio PC and Altieri DC: Control of apoptosis and mitotic spindle checkpoint by survivin. Nature. 396:580–584. 1998. View Article : Google Scholar : PubMed/NCBI
30	Skoufias DA, Mollinari C, Lacroix FB and Margolis RL: Human survivin is a kinetochore-associated passenger protein. J Cell Biol. 151:1575–1582. 2000. View Article : Google Scholar
31	Suzuki A, Hayashida M, Ito T, Kawano H, Nakano T, Miura M, Akahane K and Shiraki K: Survivin initiates cell cycle entry by the competitive interaction with Cdk4/p16(INK4a) and Cdk2/cyclin E complex activation. Oncogene. 19:3225–3234. 2000. View Article : Google Scholar : PubMed/NCBI
32	Nassar A, Lawson D, Cotsonis G and Cohen C: Survivin and caspase-3 expression in breast cancer: Correlation with prognostic parameters, proliferation, angiogenesis, and outcome. Appl Immunohistochem Mol Morphol. 16:113–120. 2008. View Article : Google Scholar : PubMed/NCBI
33	Monzó M, Rosell R, Felip E, Astudillo J, Sánchez JJ, Maestre J, Martín C, Font A, Barnadas A and Abad A: A novel anti-apoptosis gene: Re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol. 17:2100–2104. 1999.PubMed/NCBI
34	Kren L, Brazdil J, Hermanova M, Goncharuk VN, Kallakury BV, Kaur P and Ross JS: Prognostic significance of anti-apoptosis proteins survivin and bcl-2 in non-small cell lung carcinomas: A clinicopathologic study of 102 cases. Appl Immunohistochem Mol Morphol. 12:44–49. 2004. View Article : Google Scholar : PubMed/NCBI
35	Yoshida H, Sumi T, Hyun Y, Nakagawa E, Hattori K, Yasui T, Morimura M, Honda K, Nakatani T and Ishiko O: Expression of survivin and matrix metalloproteinases in adenocarcinoma and squamous cell carcinoma of the uterine cervix. Oncol Rep. 10:45–49. 2003.
36	Ikeguchi M, Liu J and Kaibara N: Expression of survivin mRNA and protein in gastric cancer cell line (MKN-45) during cisplatin treatment. Apoptosis. 7:23–29. 2002. View Article : Google Scholar : PubMed/NCBI
37	Jin XD, Gong L, Guo CL, Hao JF, Wei W, Dai ZY and Li Q: Survivin expressions in human hepatoma HepG2 cells exposed to ionizing radiation of different LET. Radiat Environ Biophys. 47:399–404. 2008. View Article : Google Scholar : PubMed/NCBI
38	Rödel F, Reichert S, Sprenger T, Gaipl US, Mirsch J, Liersch T, Fulda S and Rödel C: The role of survivin for radiation oncology: Moving beyond apoptosis inhibition. Curr Med Chem. 18:191–199. 2011. View Article : Google Scholar
39	Rödel C, Haas J, Groth A, Grabenbauer GG, Sauer R and Rödel F: Spontaneous and radiation-induced apoptosis in colorectal carcinoma cells with different intrinsic radiosensitivities: Survivin as a radioresistance factor. Int J Radiat Oncol Biol Phys. 55:1341–1347. 2003. View Article : Google Scholar : PubMed/NCBI