Antitumor effect of recombinant human endostatin combined with cisplatin on rats with transplanted Lewis lung cancer

Zhan-Wu Yu, Ying-Hua Ju, Cheng-Liang Yang, Han-Bing Yu, Quan Luo, Ye-Gang Ma,Yong-Yu Liu*

1Department of Thoracic Surgery, Liaoning Cancer Hospital and Institute, Dalian Medical University Affiliated Tumour Hospital, Shenyang City,Liaoning Province 110042, China

2Department of Biochemistry and Molecular Biology, China Medical University, Shenyang City, Liaoning Province 110122, China

1. Introduction

Lung cancer is a common malignancy in respiratory system,ranging the top of incidence of all the malignant cancers[1-3].According to the pathological characteristics, the lung cancer can be divided into small cell lung cancer and non-small-cell lung cancer;according to statistics, non-small-cell lung cancer in lung cancer patients accounts for about 75%-85%[4]. The conventional therapy for lung cancer is basically surgery, radiotherapy and chemotherapy,with toxic side effects, liability to relapse and metastasize, and low survival rate of 5 years for patients[5]. Therefore, how to improve the efficiency of non-small-cell lung cancer treatment and reduce the toxic side effects have been urgent problems in antitumor research field. It has been widely and clinically recognized that tumor grows depending on angiogenesis and that the tumor invasion and metastasis are closely connected to angiogenesis of tumor tissues; hence, anti-angiogenesis has become the research highlight of oncotherapy[6]. Angiostatin is a new angiogenesis inhibitor and can play a specific inhibitory role in vascular endothelial cells with heparin[5-7]. Researches confirm that vascular endostatin at normal level has the particular inhibitory effect in tumor growth. In the present research, the antitumor effect of Endostarinjection in tumor combined with intraperitoneal injection of cisplatin on subcutaneous transplanted Lewis lung cancer in rats was observed by taking C57 rats as models of subcutaneous transplanted Lewis lung cancer ready for the medication. The antitumor mechanism and effect were observed, aiming to provid a experimental basis for the clinical treatment.

2. Materials and methods

2.1. Experimental animals

A total of 30 clean C57/6J rats were purchased from Shanghai Lab,Animal Research Center, with age of 5-7 weeks, weight of (21±3)g,humidity for raising of (60±5)%, and temperature at (25±2)℃, and food and water were available ad libitum. The whole experimental process was conducted strictly sticking to Regulations for the Administration of Affairs Concerning Experimental Animals.

2.2. Equipments and reagents

Endostar injection was purchased from Shandong Simcere-Medgenn Bio-pharmaceutical Co.,Ltd., with batch number 20130404 and standard of 15 mg/piece. Cisplatin injection was provided by Mayne Pharma Pty. Ltd., with batch number U131881AA and standard of 50 mg/piece. The monoclonal antibodies of rabbit-antirat endothelial cell CD34 antigen and rabbit-anti-rat VEGF antigen were provided by Beijing Zhongshan Golden Bridge-Biotechnology Co., Ltd. Phosphate buffered saline (buffer, Strept Avidin-Biotin Complex kit, 3,3'-diaminobenzidine color-substrate solution and related reagents were purchased from Shanghai Senxiong Biotechnology Co., Ltd. Olympus BH-2 microscope was from Japan.

2.3. Model preparation and group treatments

Well-grown tumors in tumor-bearing rats were taken for the preparation of monoplast suspension 2×106/mL by homogenization.A total of 0.2 mL of the prepared suspension was taken to inoculate in the subcutaneous tissues of axilla beneath rats' right upper limb, after which the rats were divided randomly into Groups A,B, and C, with 10 in each. Medication was performed when the tumor volume reached 400 mm3. Group A was the control group without any medication. Group B was given Endostar 5 mg.kg-1.d injection directly in tumor for continuous 10 d while Group C was given Endostar injection 5 mg.kg-1.d combined with intraperitoneal injection of cisplatin 5 mg.kg-1.d for continuous 10 d.

2.4. Observation indicators

All the rats were executed the day after the 10-d medication. The volume and mass of tumor were then measured and the antitumor rate was calculated. The blood from rats' eyeballs was taken for the determination of vascular endothelial growth factor (VEGF)level in plasma by double antibody sandwich ELISA assay. After the measurement and weighing of tumor, conventional tissue biopsies were prepared. After hematoxylin-eosin staining was conducted,the necrosis and metastasis of tumor were observed under light microscope. VEGF and microvessel density (MVD)of tumor tissue were determined by immunohistochemical method.

2.5. Result determination

According to method of Rahman et al, the VEGF expression was scored and radio of positive cells were scored based on dyeing range and intensity. Dyeing intensity was ranged 0-3 levels, namely,level 0: negative; level 1: weakly positive; level 2: positive; level 3:strongly positive. Dyeing techniques were ranged from 0-4 levels,namely, level 0: negative; level 1: positive cells 1%-25%; level 2:positive cells 26%-50%; level 3: positive cells 51%-75%; level 4:positive 76%-100%. The densest dyeing area of blood vessels of tumor in biopsies at high magnification was for the MVD count and 5 random counts were taken for the average value of microvessel numbers.

2.6. Statistical processing

The data were processed by SPSS 13.0 and measurement data were expressed by mean±sd. One-way ANOVA was performed by Pairwise comparison and Q test was conducted. If P ＜ 0.05,statistical significance was considered to exist.

3. Results

3.1. Comparison of tumor volume, mass and antitumor rates in groups

Both tumor volume and mass in Groups B and C were significantly lower than Group A (P＜0.05).Tumor volume and mass in Group C were significantly lower than Group B (P＜0.05). The antitumor rate in Group C was significantly higher than Group B (P＜0.05). Specific results were shown in Table 1.

Table 1Comparison of tumor volume, mass and antitumor rates in groups.

3.2. Comparison of tumor VEGF, MVD and plasma VEGF in groups

The tumor VEGF score, MVD and plasma VEGF level in Group C were significantly lower than Groups A and B (P ＜ 0.05). The tumor VEGF score, MVD and plasma VEGF level in Group B were significantly lower than Groups A (P ＜ 0.05). Specific results were shown in Table 2.

Table 2Comparison of tumor VEGF, MVD and plasma VEGF in groups.

3.3. Histological observation

In Group A, there was clear demarcation between tumor and the surrounding tissues. The tumor was with hilic growth, complete but easy-to-peel capsule. The cut of tumor was grey white. The tumor cells grew vigorously and the tumor was surrounded by abundant blood capillary. In Group B, several caseous necrosis and large area of focal necrosis were seen in the central part of tumor. The number of tumor cells and blood capillary surrounding tumor was obviously smaller than Group A. In Group C, the active tumor cells were obviously fewer than Groups A and B. Atrophy and liquefaction necrosis were easily seen in parts of tumor, and the blood capillary surrounding tumor was the fewest in Group C. Specific results were shown in Figure 1.

4. Discussion

Lung cancer is the common malignant disease in respiratory system, with increasing incidence in recent years, and has topped the morbidity and mortality of malignancy. At present, the surgery,radiotherapy and chemotherapy are still the major therapeutic regimens, but radiotherapy and chemotherapy are of heavy toxic side effects which are detrimental to the survival rate of patients[10-13]. The growth and metastasis of malignant tumor are dependent on the vasifaction of new blood vessels which can ensure the nutrient supply of tumor cells; hence, anti-angiogenesis has been the new focus in therapy for malignant tumor[14]. Anti-angiogenesis targeted drugs that are not prone to show drug resistance and of low toxicity have become the research focus in tumor therapeutic area. The present research observed the effect of anti-angiogenesis drug, endostatin, combined with cisplatin, on growth and metastasis of tumor in rats with transplanted Lewis lung cancer and aimed to provide the experimental basis for clinical treatment.

Experiments have confirmed that angiostatin carries significant inhibitory effect on growth of malignant tumors of all kinds and can reduce the proliferation and metastasis of tumors[15-18]. Other experiments also confirm that endostatin at normal level can significantly inhibit tumor growth but when it is lower than normal level, the tumor would grow 2-3 times faster. Many researchers believe that the process is the result of multiple target points and multiple steps[19]. Endostar is the new recombinant human endostatin in recent years, whose functional mechanism is to inhibit the new tumor angiogenesis by inhibiting the metastasis of endothelial cells in blood vessels, so that the nutrient supply of tumor can be blocked and therefore the proliferation and metastasis of tumor can be inhibited[20]. In-vitro experiment results showed that Endostar had inhibitory effect in the metastasis of human microvascular endothelial cells and formation of tubes, and meanwhile could significantly inhibit angiogenesis of chick chorioallantoic membrane which implied that Endostar carries in-vitro anti-angiogenesis effect; in addition, Endostar plays an inhibitory effect in human lung adenocarcinoma cell SPC-A4 and gastric carcinoma cell SGC7901[21,22]. cis-Dichlorodiamineplatinum(Ⅱ)or DDP is the first line chemotherapy for treatment in kinds of solid tumors, with broad anticancer spectrum, strong function, no cross-resistance and other advantages, and it can be used with many antineoplastic drugs,producing a synergistic effect which makes it the most common drug in current combined chemotherapy; VP-16 plus EP regimen is the first line therapy for treatment in small cell lung cancer or nonsmall-cell lung cancer, with significant antitumor effect[23,24]. The present research treated the rats with subcutaneous transplanted Lewis lung cancer, having Endostar injection in tumor combined with intraperitoneal injection of cisplatin. After the treatment,in Group C, the volume and quality of tumor were significantly lower than Groups A and B (P ＜ 0.05)and the antitumor rate was significantly higher than Group B (P ＜ 0.05), which showed that Endostar injection in tumor can significantly inhibit the growth and proliferation of tumor, and that injection combined with intraperitoneal injection of cisplatin can play a synergetic inhibitory effect with better efficacy than single medication. The present research showed that tumor VEGF, MVD and plasma VEGF levels in Group C were significantly lower than Groups A and B (P ＜0.05)and tumor VEGF, MVD and plasma VEGF levels in Group B were significantly lower than Group A (P ＜ 0.05), suggesting that Endostar injection combined with cisplatin can effectively reduce the tumor VEGF expression and plasma VEGF level of rats with subcutaneous transplanted Lewis lung cancer so that the tumor angiogenesis can be reduced and antitumor goal can be achieved.Histopathological observation showed that the positive tumor cells in tumor tissue of rats in Group C were obviously fewer than Groups A and B, with atrophy and liquefaction necrosis in parts of tumor and the fewest blood capillary around tumor; that also confirmed that Endostar can effectively inhibit the tumor angiogenesis so that the nutrient supply of tumor cells can be blocked and tumor cell apoptosis can be accelerated. Furthermore, cisplatin can directly kill tumor cells and cause atrophy and liquefaction necrosis in tumor tissue. The combination of two medications makes a better antitumor effect.

The research confirmed that Endostar injection in tumor combined with intraperitoneal injection of cisplatin can significantly inhibit the VEGF and MVD of rats with subcutaneous transplanted Lewis lung cancer so that the nutrient supply of tumor cells can be blocked and tumor cells can be killed directly, so the proliferation and metastasis of tumor cells can be inhibited.

Conflict of interest statement

We declare that we have no conflict of interest.

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