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ORIGINAL ARTICLE
Year : 2018  |  Volume : 14  |  Issue : 8  |  Page : 190-196

Effect of bevacizumab combined with chemotherapy at different sequences in the gastric-cancer-bearing nude mice


1 Department of Medical Oncology, The Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, Hebei, China
2 Department of Epidemiology and Statistics, Hebei Medical University, Shijiazhuang 050011, Hebei, China
3 Palliative Care Center of Beijing Cancer Hospital, Beijing 100000, China

Date of Web Publication26-Mar-2018

Correspondence Address:
Wei Liu
Palliative Care Center of Beijing Cancer Hospital , Beijing 100000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0973-1482.171364

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 > Abstract 


Objective: To observe changes in the growth of fluorescence-labelled tumour cells in nude mice using small animal in vivo imaging technology and to compare the anti-tumour effects of the administration of bevacizumab monoclonal antibodies combined with chemotherapy at different time sequences.
Materials and Methods: Different time sequences of administration of bevacizumab monoclonal antibodies combined with the 5-fluorouracil and cisplatin (FP) chemotherapy regimen were used for intervention treatment of tumour growth in a subcutaneous xenograft model of human gastric cancer in nude mice. Tumour growth, that is, tumour volume, was evaluated with the changes in fluorescence signal strength and the inhibition rate.
Results: Compared with the control group (normal saline), experimental groups had a certain inhibition rate, while the tumour inhibition rate in the group with a bevacizumab treatment for 24 h followed by the FP chemotherapy regimen was the highest (68.42%). Moreover, the fluorescence signal strength changed significantly in all of the experimental groups. At the 3rd week of bevacizumab administration, the fluorescence signal value in the group with a bevacizumab treatment for 24 h followed by the FP chemotherapy regimen was the lowest, indicating this is the best treatment out of five groups.
Conclusion: Bevacizumab monoclonal antibodies combined with chemotherapy had synergistic effects. The small animal in vivo imaging system could dynamically obtain long and short diameters of tumours and their fluorescence signal values; compared with traditional methods that calculate tumour inhibition rates by weighing tumours, this method was more sensitive and more objective for drug evaluation.

Keywords: Angiogenesis inhibitors, drug evaluation, gastric cancer, small animal in vivo imaging


How to cite this article:
Lv Y, Song L, Chang L, Zhang X, Liu Y, Liu W. Effect of bevacizumab combined with chemotherapy at different sequences in the gastric-cancer-bearing nude mice. J Can Res Ther 2018;14, Suppl S1:190-6

How to cite this URL:
Lv Y, Song L, Chang L, Zhang X, Liu Y, Liu W. Effect of bevacizumab combined with chemotherapy at different sequences in the gastric-cancer-bearing nude mice. J Can Res Ther [serial online] 2018 [cited 2019 Jun 26];14:190-6. Available from: http://www.cancerjournal.net/text.asp?2018/14/8/190/171364




 > Introduction Top


Incidence of gastric cancer is high, and its treatment effect is not satisfied. Recent studies confirmed that angiogenesis inhibitors could inhibit gastric cancer growth through anti-angiogenesis therapy.[1],[2],[3],[4] How to evaluate the effect of targeted therapy combined with chemotherapy on gastric cancer treatment and the investigation of the best sequence for drug administration have become researchers' hotspots. The present study performed an intervention treatment of tumour growth in a subcutaneous xenograft model of human gastric cancer in nude mice using different time sequences of the vascular endothelial growth factor (VEGF) monoclonal antibody bevacizumab combined with the 5-fluorouracil (5-FU) and cisplatin (FP) chemotherapy regimen to investigate the best drug administration order. Tumour growth was also dynamically observed by a small animal in vivo imaging system.


 > Materials and Methods Top


Animals

Male BALB/c-nu nude mice aged 28–35 day with body weights of 20–22 g were provided by Beijing HFK Bioscience (permission number SCXK (Jing) 2009-0004). The nude mice used in this experiment were housed in a laminar air flow room at a specific pathogen free facility. In the laminar air flow room, everything that came into contact with the nude mice was sterilised, and staff entering the laminar air flow room had to wear sterile surgical masks, caps, gloves, and gowns. Room temperature of the laminar air flow room was controlled at 26–28°C, humidity was maintained at 40–60%, and the ventilation exchange was 10–15 times/h. Feed was made into a monolayer on a small plate and was autoclaved for 45 min at l20°C. Drinking water was filtered through a 0.2 μ microporous membrane. The pressure per square inch did not exceed 10 pounds. Water bottles were changed every other day. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal use protocol has been reviewed and approved by the Institutional Animal Care and Use Committee of Medical University.

Nude mice mode

The establishment of a subcutaneous xenograft nude mice model with fluorescence-labelled MGC803 human gastric cancer cells. The fluorescence-labelled logarithmic phase human gastric cancer MGC803 cells (provided by Shanghai Sciencelight Biology Science and Technology) were collected and adjusted to a concentration of 2.5 × 107/L. Each nude mouse was inoculated subcutaneously on the back with 0.2 ml of the cell suspension. Xenograft modules with hard properties <1 cm were considered to be successfully established.

Experimental grouping and time sequences of drug administration

Twenty-five nude mice of the xenograft model were randomly divided equally into five groups, named A, B, C, D, E. The treatment program was a 5 mg/kg/day bevacizumab treatment twice each week on Tuesday and Friday. The FP chemotherapy regimen was intraperitoneal injection of 5-FU at 40 mg/kg/day + DDP 4 mg/kg/day 4 times each week on Monday, Tuesday, Thursday, and Friday. Group A was the combined bevacizumab + FP chemotherapy treatment group. Group B was the FP chemotherapy for 24 h followed by bevacizumab treatment group. Group C was the bevacizumab treatment for 24 h followed by FP treatment group. Group D was the bevacizumab treatment group. Group E was the control group with intraperitoneal injection of 1 ml normal saline on Monday, Tuesday, Thursday, and Friday each week. After drug treatment for 28 day, all tumour-bearing nude mice were sacrificed.

Detection indicators and methods

Tumour growth and distribution in the body was observed every 7 day, and tumour volume was measured (V = ½ × a × b 2; a and b were length and width, respectively). Tumour inhibition rate was calculated as follows: Tumour inhibition rate = 1 − (tumour volume in the control group–tumour volume in the experimental group)/tumour volume in the control group × l00%. Detection of fluorescence signals of tumour in tumour-bearing nude mice. During observation, each nude mouse was subcutaneously injected with 0.4 g/kg chloral hydrate (4%) and 150 mg/kg luciferase substrate. Ten minutes after the injection, in vivo imaging was performed by the in vivo imaging system and the fluorescence signal strength was recorded.

Statistical analysis

Datas following a normal distribution were presented as x ± s. Comparisons of homogeneity of body weight, tumour volume, and fluorescence signal strength between different drug treatment groups before the experiment were performed using one-way analysis of variance (ANOVA). Comparison of each indicator between different time points was performed with repeated-measures ANOVA as well. P < 0.05 indicated that the differences were statistically significant. All data were analysed using SPSS 16.0 software (SPSS Inc., Chicago, IL, USA).


 > Results Top


The comparison of homogeneity of body weight, tumour volume, and fluorescence signal strength between nude mice in the five groups before the treatment. Results of the one-way ANOVA for the homogeneity of body weight, tumour volume, and fluorescence signal strength between nude mice in the five groups before treatment did not show statistical significance, indicating that body weight, tumour volume, and fluorescence signal strength of the nude mice in five groups were homogeneous before treatment [Table 1].
Table 1: The body weight, tumour volume, and fluorescence signal strength of the nude mice in each group were homogeneous before treatment

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The effects of different time sequences of administration of combined bevacizumab with 5-fluorouracil and cisplatin on the body weight of nude mice

Changes in the body weight of nude mice in different groups did not show a significant difference (F = 1.578, P= 0.219), whereas, changes in body weight at different time points after the treatment had statistical significance (F = 26.995, P < 0.001), and time points and groups had interactive effects (F = 2.547, P= 0.031). Further group-wise comparison showed that the changes in body weight at different time points of the nude mice in Group A did not exhibit a significant difference (F = 0.375, P= 0.773); whereas the body weight of the nude mice in the B, C, D, and E Groups showed an increasing trend over time (P < 0.05) [Figure 1].
Figure 1: The changes in the body weight of nude mice in different groups

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The effects of different time sequences of administration of bevacizumab combined with 5-fluorouracil and cisplatin on the xenograft tumour volume of nude mice

The tumour inhibition rate was calculated based on the tumour volume. The tumour inhibition rates were 40.95% in Group A, 37.57% in Group B, 68.42% in Group C, and 19.84% in Group D. Changes in the tumour volume of nude mice in different groups did not imply statistical significance (F = 1.812, P= 0.166), while changes in tumour volume at different time points after the treatment had statistical significance (F = 4.566, P= 0.006), and there were interactive effects between time points and group (F = 6.688, P < 0.001). Further group-wise comparison showed that the differences between before treatment and 1 week after treatment in different groups were not statistically significant (P > 0.05). The differences between the 2nd week and the 3rd week after treatment in different groups all had statistical significance (P < 0.05). The mean tumour volume of different groups from small to large was listed as follows: C, A, B, D, and E. The volume in Group C was significantly smaller than those in Groups D and E (P < 0.05). The changes in the tumour volume of nude mice in Groups A, B, and D at different time points did not have statistical significance (P > 0.05). The tumour volume of nude mice in Group C exhibited a dramatically decreasing trend over time (P < 0.05). The tumour volume of nude mice in Group E exhibited a significantly increasing trend over time (P < 0.05) [Figure 2].
Figure 2: The effects of different time sequences of administration of bevacizumab combined with 5-fluorouracil and cisplatin on the xenograft tumour volume of nude mice

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The effects of different time sequences of administration of bevacizumab combined with 5-fluorouracil and cisplatin on fluorescence signals in the xenograft tumours of nude mic

Changes in the fluorescence signals of nude mouse tumours were significantly different among groups (F = 18.219, P < 0.001). Moreover, the changes in the fluorescence signals were also significantly different among different time points after treatment (F = 36.229, P < 0.001), and time point and group had interactive effects (F = 8.721, P < 0.001). Further group-wise comparisons indicated that the changes in different groups before treatment were not significantly different (F = 2.979, P= 0.074). The differences at the 1st, 2nd, and 3rd week after drug treatment among the different groups all had statistical significance (P < 0.05). At the 1st and 2nd week, Group E had higher signals than Groups A, B, C, and D. At the 3rd week, the fluorescence signals ordered from low to high were Groups C, A, B, D, and E. The fluorescence signals of the nude mouse tumours in Groups A and C at the 3rd week were lower than those before drug treatment (P < 0.05). The fluorescence signals of the nude mouse tumours in Group E showed a dramatically increasing trend over time (P < 0.05) [Figure 3] and [Figure 4].
Figure 3: The effects of different time sequences of administration of bevacizumab combined with 5-fluorouracil and cisplatin on fluorescence signals in the xenograft tumours of nude mice

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Figure 4: The effects of different time sequences of administration of bevacizumab combined with 5-fluorouracil and cisplatin on the fluorescence signals of the nude mice. Group A: Bevacizumab + 5-fluorouracil and cisplatin chemotherapy contemporary, Group B: 5-fluorouracil and cisplatin chemotherapy for 24 h followed by bevacizumab, Group C: The bevacizumab treatment for 24 h followed by 5-fluorouracil and cisplatin chemotherapy, Group D: The bevacizumab treatment group, Group E: The control group with intraperitoneal injection of 1 ml normal saline. Tumour growth and distribution in the body were observed every 7 day. The tumour inhibition rates of five groups were calculated based on the tumour volume and reduction in the fluorescence signals of nude mouse tumours. Differences between the 2nd and the 3rd week after treatment in five groups all had statistical significance (P < 0.05). At the 1st and 2nd week, Group E had the highest signals, while the fluorescence signals ordered from low to high were Groups C, A, B, D, and E at the 3rd week. The fluorescence signals of the nude mouse tumours in Groups A and C at the 3rd week were dramatically lower than the rest before drug treatment (P < 0.05). The fluorescence signals of the nude mouse tumours in Group E showed a significant increasing trend over time (P < 0.05)

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Then tumor xenografts were used for histopathology assay by the hematoxylin and eosin (H and E) staining. Compared to the control group, the test group had a significantly lower microvessel density (MVD) (8.0 ± 1.0, 6.8 ± 1.2, 6.5 ± 1.2 and 4.0 ± 1.2 vs. 13.8 ± 1.6, P < 0.05) [Figure 5.1]. The expression of the apoptosis factor Bax was increased in tumor xenografts of the combined treatment of bevacizumab with FP 24 h later group compared with the other groups (P < 0.05) [Figure 5.2]. The expression of the marker VEGF-A was significantly lower in tumor xenografts of the bevacizumab with FP 24 h later group compared with the other groups [Figure 5.3].
Figure 5.1: The histopathology and immunohistochemistry assay for tumor xenograft. Magnification, ×400. The expression of MVD in tumor tissues in Groups

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Figure 5.2: The histopathology and immunohistochemistry assay for tumor xenograft. Magnification, ×400. The expression of BAX in tumor tissues in Groups

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Figure 5.3: The histopathology and immunohistochemistry assay for tumor xenograft. Magnification, ×400.The expression of VEGF-A in tumor tissues in Groups

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 > Discussion Top


The synergistic effect of the combination of bevacizumab and the 5-fluorouracil and cisplatin chemotherapy regimen

In addition to killing tumour cells, traditional chemotherapy drugs also have a certain cytotoxic effect on cells in normal tissues and have a strong toxic side effect; therefore, their efficacy in treating gastric cancer is limited. With the appearance of anti-tumour targeted drugs, targeted therapy has more choices. The treatment effect of bevacizumab alone, a macromolecular anti-angiogenic drug, is minimal; however, when combined with chemotherapy, bevacizumab treatment has shown some successes in clinical studies, suggesting that these two have synergistic effects.[5],[6],[7],[8],[9]

The present study suggests that the tumour inhibition rates of combined targeted drugs and double-drug chemotherapy were significantly higher than the bevacizumab-alone group; the tumour inhibition rates were 40.95% in Group A, 37.57% in Group B, 68.42% in Group C, and 19.84% in Group D. The treatment with bevacizumab for 24 h followed by the FP chemotherapy regimen, that is, Group C, had the highest rate of inhibition of the transplanted tumour in tumour-bearing nude mice.

This phenomenon has already been confirmed in a study of the other anti-angiogenic drug, endostar. The administration of targeted drugs combined with different chemotherapy drugs with different time sequences may have opposite results,[10],[11],[12],[13],[14],[15] which may be associated with the induction of the cell cycle. This mechanism will be discussed in a follow-up study.

Changes in fluorescence strength could intuitively reflect treatment effects

Current detection method for the evaluation of anti-tumour drug efficacy is generally to sacrifice the animal after drug administration, weigh the tumour, and obtain experimental data through calculation of tumour inhibition rates using formulas; which has a certain human manipulation error. However, a small animal in vivo imaging system can trace the changes in the same subjects (labelled cells) by recording the experimental subjects in different groups at different time points. Because it can perform continuous detection on the same animal to dynamically obtain the long and short diameters of tumours and their fluorescence signal strength, the effects of errors caused by the traditional detection method on experimental results can be minimized to the maximum extent.[16],[17],[18],[19],[20]

The results of the present study confirmed that this imaging system could evaluate whether established cell line had tumorigenicity in animals, dynamically obtain long and short diameters of tumours to calculate tumour volume, and obtain the dynamics of the fluorescence signal strength of tumours to reflect tumour cell activities. The tumour volume in Group C (the bevacizumab treatment for 24 h followed by FP treatment group) no longer changed after the 3rd week of drug administration, but the fluorescence signal strength continuously decreased, indicating that using tumour cell activity to determine treatment effects is more sensitive and more objective.

We can see from this experiment, bevacizumab medication after 24 h FP scheme, tumor size, fluorescence value, VEGF and MVD and Bax than other groups have statistically significant difference between, beacizumab bead sheet resistance to chemotherapy after 24 h compared to other treatment group and the blank control group, the tumor inhibition effect is better.

In summary, different time sequences of targeted drugs combined with chemotherapy had different anti-tumour activities. The results showed that the application of anti-angiogenesis targeted drugs before chemotherapy had stronger anti-tumour activities. Monitoring anti-tumour activities with a small animal in vivo imaging system could perform continuous and dynamic observation and could accurately obtain the long and short diameters of tumours, providing a new intuitive and sensitive method for the study of anti-tumour drugs.

Acknowledgements

This study was supported by the Laboratory Animal Centre of The Fourth Hospital of Hebei Medical University, Shijiazhuang, Hebei, China.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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