Zhuang Xibing, Qiao Tiankui, Xu Guoxiong, Yuan Sujuan, Zhang Qi, Chen Xue
Department of Oncology, Jinshan Hospital, Fudan University, Shanghai 201508, P.R. China.
Center Laboratory, Jinshan Hospital, Fudan University, Shanghai 201508, P.R. China.
Oncol Rep. 2016 Oct;36(4):2200-6. doi: 10.3892/or.2016.4990. Epub 2016 Aug 1.
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.
低分子量肝素（LMWHs）常用于静脉血栓预防和治疗，最近在临床前研究中报道其对癌症转移有影响。本研究旨在确定那屈肝素（一种LMWH）与放疗联合对A549细胞的协同抗肿瘤作用。根据不同治疗方法，本研究设立了六个实验组：对照组；照射（IR）组；低剂量那屈肝素组（LMWH50，L50）；高剂量那屈肝素组（LMWH100，L100）；LMWH50 + IR组；LMWH100 + IR组。采用细胞计数试剂盒-8（CCK-8）法评估A549细胞的活力。处理后通过流式细胞术（FCM）分析肿瘤细胞的凋亡情况。采用酶联免疫吸附测定（ELISA）法检测培养上清液中转化生长因子-β1（TGF-β1）的浓度。通过Transwell小室试验检测A549细胞的迁移和侵袭能力。采用蛋白质印迹法分析生存素、CD147和基质金属蛋白酶-2（MMP-2）的表达。CCK-8试验表明，单独照射或那屈肝素均可轻微抑制细胞活力，而联合治疗则以剂量和时间依赖性方式显著抑制细胞活力。通过FCM检测，与对照组或单独接受那屈肝素或照射的组相比，那屈肝素与照射联合治疗组的凋亡率在剂量和时间依赖性方面有更大改善。ELISA试验表明，与单独任何一种治疗相比，那屈肝素与照射联合治疗后TGF-β1分泌减少。Transwell小室试验表明，那屈肝素不仅显著抑制A549细胞的迁移和侵袭，还抑制X射线照射诱导的迁移和侵袭能力增强。蛋白质印迹法表明，在联合治疗组中，那屈肝素以剂量和时间依赖性方式抑制辐射诱导的生存素和MMP-2表达上调。此外，联合治疗组中CD147的表达水平最低。本研究确定，那屈肝素与照射联合在体外以剂量和时间相关方式具有强大的协同抗肿瘤作用，这体现在对细胞活力、侵袭和转移的抑制、凋亡的促进、TGF-β1分泌水平的抑制以及CD147、MMP-2和生存素表达的下调。
