Abstract The aim of the present study was observation E838 antitumor effect on mice transplanted lymphoma and related biological indicators to explore co-137Cs γ-ray with tumor growth synergies. Methods 2 ~ 3mm3 lymphoma tumor block IRM  2 mice were subcutaneously inoculated in the 24h after Dutch-bearing mice were randomly divided into a control group, radiotherapy alone group, E838 low, medium, high drug group and drug combination irradiated group , cyclophosphamide group. Drug group and drug combination corresponding to the irradiated group intraperitoneal injection of the same dose E838, twice daily, for seven days in a row, cyclophosphamide every other day 1 x 4. The combined irradiation group in the first four days of the administration the systemic 1Gy irradiation, 1 day, 5 days in a row. Observed that the mice in each group of bone marrow nucleated cells and tumor inhibition rate. Results, respectively, for the the E838 3 doses in mice transplanted tumors by lymphocytes (44.14 ± 15.96)%, (70.74 ± 11.17)% and (50.00 ± 18.09)%, compared with the control group difference was statistically significant ( P <0.001), bone marrow nucleated cells and compared to the control group was significantly improved. E838 share 137Csγ ray can enhance the anti-tumor effect, inhibition rates were (65.43 ± 2.13)%, (77.13 ± 6.38)% and (67.55 ± 11.17)% (P <0.001), higher than the single-killing effect on tumor radiotherapy group and the monotherapy group. Conclusion E838 has good inhibition of tumor cells in mice, E838 combined gamma ray has a synergistic inhibitory effect, can promote bone marrow damage and repair in tumor-bearing mice after radiotherapy in the appropriate dose range.

Key words E838; antitumor; IRM  2 mice; gamma rays; synergies

Antitumor Effects of Combination Treatment of E838 with γ  ray for Mouse Lymphoma

    WANG Yue  ying, LI De  guan, LIU Qiang, WU Hong  ying, WANG Yong, LU Lu, MENG Ai  min, WANG Ru  qin, ZHANG Liang  an

    Institute of Radiation Medicine, Chinese Academy of Medical Sciences, Tianjin Key laboratory of Molecular Nuclear Medicine, Tianjin 300192, China

    Corresponding Author: MENG Ai  min, E  mail: ai_min_meng@126.comAbstract: Objective To investigate the tumor inhibitory effects of E838 and the combined antitumor effects of E838 and 137Cs γ  ray irradiation. Methods IRM  2 mice transplanted with 2 ~ 3mm3 lymphoma (LM) tissue for 24h, were randomized into nine groups: control group, radiation group, high, middle, low E838 dose group, different dose E838 combined with radiation group and cyclophosphamide group. E838 group and combined group administered with same dose E838 daily for 7 days, cyclophosphamide group administered every other day 4 times. Combined group radiated with 1 Gy / day for 5 days after the mice wereadministered with E838 for 4 days.The size of LM tumor and the bone marrow cells of different group were measured. Results The mouse LM tumor inhibitory ratios of three E838 doses groups were (44.14 ± 15.96)%, (70.74 ± 11.17)% and (50.00 ± 18.09)% respectively. There was a significant difference between E838 groups and control group (P <0.001). The level of bone marrow cells counting was markedly elevated. E838 combined with 137Csγ  ray could enhance tumor inhibitory effect, the tumor inhibitory ratios of three combined groups were (65.43 ± 2.13)%, (77.13 ± 6.38)% and (67.55 ± 11.17)%, there were obviously increased compare with control and radiation group respectively (P <0.001). Conclusion E838 has good tumor inhibitory effects on lymphoma transplanted mice tumor. The synergistic antitumor effects was found after the mice were treated with E838 and γ  ray irradiation, the bone marrow function recovery after irradiation was speed up when the mice were administrate with E838 of some definite doses.

    Key words: E838; Tumor inhibitory effects; IRM  2 mouse; γ  ray; Synergistic effects
Malignant lymphoma is one of the common malignant tumors. Treatment measures include surgery, radiation therapy, and chemotherapy, but the treatment, bone marrow suppression, immune function and injury, and will also give the body side effects. E838 is a new type of radiation protectants, the the pharmacodynamic experiment confirmed: E838 with radiological protection and high titer [1], the characteristics of the toxic side effects (estrogen ethinyl estradiol activity was only 1/10000) [2] can significantly improve survival and protection index of irradiated mice 30 days. The trials of a new class of drugs in accordance with national approval requirements [3] using IRM  2 mouse tumor models, the antitumor effect observed E838 is whether we can enhance the inhibitory effect of lymphoma and joint 137Cs γ-ray for the clinical treatment of lymphoma provide a reference.

    1 Materials and methods

    1.1 Materials

    Animals and tumor strain, by my own cultivated IRM  2 inbred mice of either sex, weight 26 ~ 27g. The cell lines IRM  2 mice spontaneously lymphoma, feed for the full price of clean grade mouse material, provided by the Beijing the Keao third feed Limited.

    1.2 reagent preparation

    Injection tea oil E838 formulated into 5,7.5,10 mg / kg dose group. Cyclophosphamide (CTX) Shanghai Hualian Pharmaceutical Co., Ltd., batch number 031018, with saline preparation of 25mg/kg dose group.

    1.3 irradiation conditions

    The irradiation source our 137Csγ, the radiation dose to 1Gy, dose rate 0.853Gy/min.

    1.4 Method

    1.4.1 animal grouping selected IRM  2 mice 99, were randomly divided into nine groups, the control group, the radiotherapy alone group, CTX group, E838 (low, medium, high) dose group, E838 (low, medium, high) + irradiation group. Take 2 ~ 3mm3 tumor inoculation subcutaneous mice groin.

    1.4.2 administered drug group in the first two days after inoculation, intraperitoneal injection of 0.2 ml / only seven days in a row, the control group received an equal volume of tea oil, CTX every other day, a total of 4 times, drug + irradiation The group vaccinated four days the systemic 1Gy irradiation, 1 times / day for 5 consecutive days.

    1.5 OUTCOME MEASURES

    1.5.1 daily known as tumor-bearing mouse body weight, and tumor volume was measured, according to the formula V = AB2 / 2 calculating the volume [4]. Tumor growth and draw the change curve.

    1.5.2 experimental mice were sacrificed on day 12, said the tumor inhibitory rate was calculated. Tumor inhibition rate% = (control group, the average tumor weight – the experimental group, the average tumor weight) / control group, the average tumor weight × 100%.

    1.5.3 bone marrow nucleated cells from mice one femur, flushing the marrow cavity, collection made of bone marrow cells in suspension by trypan blue identification of cell activity, counting more than 95% of bone marrow nucleated cells.

    1.5.4 histological paraffin sections of the tumor was observed after HE staining, histological observation.

    1.5.5 synergistic effect was evaluated according to the Guinness Book of formula [5]. Q = EA + B (Found combined effect) / EA + EB-EA series · EB (anticipated combined effect), EA, EB-A, the effect produced in the the B two drugs when used alone; EA + EB-A, B two drugs effects of combination q value from 0.85 to 1.15, the role simple sum of the two drugs; q values ​​greater than 1.15, the enhanced role of the two drugs; q value of less than 0.85, the role of the two drugs in combination against each other.

    1.5.6 Statistical Methods All data results ± s, using group t test.

    2 Results

    2.1 E838 transplanted tumor growth curve

    E838 three-dose mice tumor volume was significantly lower than that of the control group were the most obvious, the middle dose group, as shown in Figure 1.

    Figure 1 E838 LM transplanted tumor growth curve

    Fig 1 The effects of E838 on the LM tumor sizes

    2.2 E838 with tumor growth curves after irradiation combined effect of change

    E838 combined with radiotherapy, tumor growth was slower in the control group and the radiotherapy alone group, the relative growth rate slows down, the middle dose group, tumor growth was significantly slower than the control group and the radiotherapy alone group, as shown in Figure 2.

    2.3 E838 drugs on tumor-bearing mice bearing

    The results are shown in Table 1. E838 drug groups tumor were lower than the control group (P <0.001); E838 low, the high dose group inhibition rates were 44.14%, 70.74% and 50.00%, respectively, compared with the control group difference was statistically significance (P <0.001), each group BMNC are higher than that in control group and CTX group, their difference was statistically significant (P <0.001).

    Γ-ray 2.4 E838 combined synergies of tumor-bearing mice

    Seen from Table 2, E838 small dose of tumor inhibition rate rose to 65.43% from 44.14%, the middle dose group rose to 77.13% from 70.74%, 50.00% to 67.55% of inhibitory rate of the high-dose group, and to the Guinness Book of formula , q values ​​were 1.03,0.95 and 1.00, additive effect, combined with γ-ray with the control group and the radiotherapy alone group showed significant table the Inhibition table 1 E838 on IRM  2 mouse lymphoma 2 E838 joint gamma-ray of IRM  2 mouse lymphoma Inhibition P <0.001 significant differences, the group BMNC were higher radiotherapy alone group (P <0.001).

    2.5 xenograft tumor histopathological observation

    Endoscopic transplantation tumor cell irregular shapes, sizes, within each group are visible organization of the visible part of the necrotic tissue necrosis, control group, middle dose group showed multifocal necrosis, but the distribution is not very uniform. Middle dose + radiation group, showing significant areas of necrosis, necrotic tissue area increases.

    3 Discussion

    The cause of malignant lymphoma is related with the patient’s immune function [6]. While radiotherapy and chemotherapy to further suppress the immune function has been reduced. Chemotherapy drugs can damage the patient’s immune system and lead to immune dysfunction or decline caused by bone marrow suppression, also confirmed this positive control of the experimental drug CTX. The main side effects of radiotherapy is bone marrow suppression, local injury, fatigue, loss of appetite. Tumor chemoradiotherapy one of the difficulties is how to avoid the rays and chemotherapy drugs damage normal tissue to reduce severe inhibition of the immune system after chemotherapy [7].

    Our results show that the E838 in a certain dose range, can significantly inhibit the growth of the the IRM  2 tumor-bearing mouse lymphoma, tumor growth has slowed significantly, significantly reduced tumor dose from the administration point of view, in the dose suppression tumor of the best, the inhibition rate of 70.74%. In antitumor while, each dose group BMNC of number were higher and CTX group. An increase in body weight of the mice in each group, while the the CTX group of mice significant weight loss. Description E838 addition to tumor cell growth inhibition, as well as the role of the strong body. This also reflects one of the advantages of the E838 treatment tumor toxicity is less than the chemotherapy drug. E838 in combination with γ-ray can enhance the anti-tumor effect, the inhibition rate in the radiotherapy alone group and individual drug group and CTX group. Inhibition rates were 65.43%, 77.13% and 67.55%, respectively, the inhibitory effect was an additive effect, each group BMNC number were higher and radiotherapy alone group, E838 the antagonistic radiation injury on the body and hematopoietic function. Prompted the E838 with radiotherapy has important guiding significance to further improve the cure rate and quality of life of the tumor.