Alyamkina Ekaterina A, Nikolin Valeriy P, Popova Nelly A, Dolgova Evgenia V, Proskurina Anastasia S, Orishchenko Konstantin E, Efremov Yaroslav R, Chernykh Elena R, Ostanin Alexandr A, Sidorov Sergey V, Ponomarenko Dmitriy M, Zagrebelniy Stanislav N, Bogachev Sergey S, Shurdov Mikhail A
Institute of Cytology and Genetics, Siberian Branch, Russian Academy of Sciences, Novosibirsk, Russia.
Genet Vaccines Ther. 2010 Nov 1;8:7. doi: 10.1186/1479-0556-8-7.
Immunization of mice with tumor homogenate after combined treatment with cyclophosphamide (CP) and double-stranded DNA (dsDNA) preparation is effective at inhibition of growth of tumor challenged after the treatment. It was assumed that this inhibition might be due to activation of the antigen-presenting cells. The purpose was to develop improved antitumor strategy using mice. We studied the combined action of cytostatics doxorubicin (Dox) plus CP with subsequent dsDNA preparation on tumor growth.
Three-month old CBA/Lac mice were used in the experiments. Mice were injected with CP and human dsDNA preparation. The percentage of mature dendritic cells (DCs) was estimated by staining of mononuclear cells isolated from spleen and bone marrow 3, 6, and 9 days later with monoclonal antibodies CD34, CD80, and CD86. In the next set of experiments, mice were given intramuscularly injections of 1-3 × 105 tumor cells. Four days later, they were injected intravenously with 6-6.7 mg/kg Dox and intraperitoneally with 100-200 mg/kg CP; 200 mkg human DNA was injected intraperitoneally after CP administration. Differences in tumor size between groups were analyzed for statistical significance by Student's t-test. The MTT-test was done to determine the cytotoxic index of mouse leucocytes from treated groups.
The conducted experiments showed that combined treatment with CP and dsDNA preparation produce an increase in the total amount of mature DCs in vivo. Treatment of tumor bearers with preparation of fragmented dsDNA on the background of pretreatment with Dox plus CP demonstrated a strong suppression of tumor growth in two models. RLS, a weakly immunogenic, resistant to alkalyting cytostatics tumor, grew 3.4-fold slower when compared with the control (p < 0.001). In experiment with Krebs-2 tumor, only 2 of the 10 mice in the Dox+CP+DNA group had a palpable tumor on day 16. The cytotoxic index of leucocytes was 86.5% in the Dox+CP+DNA group, but it was 0% in the Dox+CP group.
Thus, the set of experiments we performed showed that exogenous dsDNA, when administered on the background of pretreatment with Dox plus CP, has an antitumor effect possibly due to DC activation.
用环磷酰胺（CP）和双链DNA（dsDNA）制剂联合处理后，用肿瘤匀浆对小鼠进行免疫接种，可有效抑制处理后接种的肿瘤生长。据推测，这种抑制可能是由于抗原呈递细胞的激活。目的是开发一种改进的小鼠抗肿瘤策略。我们研究了细胞抑制剂阿霉素（Dox）加CP与随后的dsDNA制剂对肿瘤生长的联合作用。
实验使用3个月大的CBA/Lac小鼠。给小鼠注射CP和人dsDNA制剂。在3、6和9天后，用单克隆抗体CD34、CD80和CD86对从脾脏和骨髓中分离的单核细胞进行染色，估计成熟树突状细胞（DC）的百分比。在接下来的一组实验中，给小鼠肌肉注射1 - 3×105个肿瘤细胞。4天后，给它们静脉注射6 - 6.7 mg/kg的Dox，腹腔注射100 - 200 mg/kg的CP；在给予CP后腹腔注射200 μg人DNA。通过学生t检验分析各组肿瘤大小的差异有无统计学意义。进行MTT试验以确定处理组小鼠白细胞的细胞毒性指数。
进行的实验表明，CP和dsDNA制剂联合处理可使体内成熟DC的总量增加。在Dox加CP预处理的背景下，用片段化dsDNA制剂处理荷瘤小鼠，在两种模型中均显示出对肿瘤生长的强烈抑制。RLS是一种弱免疫原性、对烷化细胞抑制剂耐药的肿瘤，与对照组相比生长速度慢3.4倍（p < 0.001）。在Krebs - 2肿瘤实验中，Dox + CP + DNA组的10只小鼠中只有2只在第16天可触及肿瘤。Dox + CP + DNA组白细胞的细胞毒性指数为86.5%，而Dox + CP组为0%。
因此，我们进行的这组实验表明，在Dox加CP预处理的背景下给予外源性dsDNA具有抗肿瘤作用，可能是由于DC的激活。
