Anti-hepatocellular carcinoma effect of low dose cyclophosphamide combined with CIK cells in mice

Abstract

Objective To explore the influence of low dose cyclophosphamide (CTX) acting on regulatory T cells (Tregs) of hepatocellular carcinoma-bearing mice, and the anti-tumor effect of low dose CTX combined with cytokine induced killer cells (CIKs). Methods Models of tumor-bearing mice were established by subcutaneous inoculation with hepatocellular carcinoma cells. The mice were randomly divided into 4 groups (n=35), there were normal control group, single tumor group, single CTX group (1 time, 100 mg/kg, intraperitoneal) and cyclical CTX group (once every 6 days, 3 times, 100 mg/kg, intraperitoneal). The changes of the Treg/CD4+ in spleens were detected by flow cytometry. We isolated mononulear cells from spleen of mice to culture CIKs. To study the effect of anti-tumor in vitro, tumor-bearing mice were randomly divided into 6 treatment groups, there were single tumor group, single CTX group, single CIK group, cyclical CTX group, single CTX+ CIK group and cyclical CTX+ CIK group. The growth curves of tumor were drawn. At the end of the experiment, tumor was separated and weighted. The growth inhibition ratio of tumor was calculated. Results With time prolonged after tumor inoculation, the proportion of Treg/CD4+ increased gradually in spleen of mice in single tumor group. On the 10th day, this proportion of single CTX group was (8.95±1.90)% and the single tumor group was (9.25±1.74)%, and there was no difference between two groups (t=0.374, P=0.714). The proportion in the cyclical CTX group (8.99±2.11)% was still lower than that in the single tumor group (16.76±2.02)% on the 19th day, and the difference was significant (t=8.544, P=0.000). Treatment for 21 days, the volume and weight of the single tumor group, single CTX group, single CIK group, cyclical CTX group, single CTX+ CIK group and cyclical CTX+ CIK group were (3 800.4±607.5), (3 764.8±537.7), (3 352.2±485.4), (2 076.6±620.2), (1 867.1±533.6), (970.7±135.3)mm3 and (4.01±0.66), (3.86±0.74), (3.80±0.42), (2.08±0.27), (1.83±0.93), (0.86±0.25)g. The tumor volume and weight were minimum in the cyclical CTX+ CIK group. The tumor inhibition effects were weaker in the cyclical CTX and single CTX+ CIK groups. But tumor in single CIK and CTX groups were unsuppressed. The differences among the 6 groups had statistically significance (F=213.750, P=0.000; F=27.142, P=0.000). Conclusion Cyclical administration of low-dose CTX combined with CIKs has an obvious therapeutic effect on hepatocellular carcinoma bearing mice and can provide a new idea for anti-tumor therapy. Key words: Cyclophosphamide; Liver neoplasms; T-lymphocytes, regulatory; Immunotherapy