| Objective1. To observe the different anti-tumor effect of5-fluorouracil (5-FU)、bevacizumab (Bev) and the combination of the two drugs,so that to determine if the combination of5-FU with Bev could synergisticly inhibit the growth of subcutaneous xenograft model of colorectal carcinoma.2. To examine whether the intratumoral (i.t.) injection of5-FU Bev and the combination of the two drugs could inhibit the growth of distant tumors.3. To observe the side effects of5-FU、Bev、5-FU and Bev.MethordsBALB/c nude mice subcutaneous xenograft models of colorectal cancer were established,both flanks were injected subcutaneously(s.c) with1×107HT-29tumor cells. Only the tumors on one side(the intratumoral injected side) were intratumoral(i.t.) injected with drugs, once every four days, a total of3times.The other side(un-i.t. injected side) tumor was a self-control for each nude mouse. Based on the diameter of the i.t. injected tumor, the mice were divided into5mm and10mm block.Each block contained16mice which was divided to four different groups:group A (control group, i.t. injection of Normal Saline,NS), group B (5-FU group, i.t. injection of5-FU,20mg/Kg dissolved in NS), group C (Bev group, i.t. injection of Bev,4mg/Kg, dissolved in NS), group D (5-FU+Bev combined treated group, i.t.20mg/Kg of5-FU and4mg/Kg Bev, dissolved in NS),each group contains4nude mice.The volume of tumors were observed every tow days,and Tumor Grouth mhibition(T/C value)、Tumor Grouth Delay(T-C value)、Log cell kill(LCK value) were compared amang the four groups。Tumors’orgenic and lymph node metastasis were examined.The side effects of drugs were evaluated from the change of blood cell count、skin ulceration and bleeding、loss of appetite、fatigue and loss of weight of mice.Results1. The influence to tumor growth of each treatment groupFrom the tumor growth curves we drawn, we observed that the growth speed of i.t. injected tumor for each treatment group, from fast to slow was as follow:the control group, the Bev group, the5-FU group and the5-FU+Bev combined treated group. In5mm block and10mm block, the i.t. injected tumor of each corresponding treatment group had the same growth trend. There was no significant difference of the growth speed of the un-i.t. injected tumors among all the treatment groups (data not shown)2. Tumor growth inhibition effectT/C value(Tumor Growth Inhibition)=(Median tumor weight of the treated group/Median tumor weight of the control group)×100%。T/C value≤42%is the minimum level for antitumor activity, T/C value<10%is the high antitumor level for drugs. In5mm block, T/C value of the treatment groups were as follow:5-FU:42%, Bev:85%,5-FU+Bev:20%.in10mm block, the T/C value of the treatment groups were as follow:5-FU:66%, Bev:98%,5-FU+Bev:32%.3. Tumor growth delay effectThe T-C value(Tumor Growth Delay) based on the median time(in days),required for the treatment group(T) and the control group(C) tumors, to reach a predetermined size. Td(Tumor-doubling Time)=The days to reach the predetermined size of the treated group/The days to reach the size of the control group. In our experiment, this predetermined size was:the diameter of the i.t. injected tumor reached15millimeter.The T-C values of5-FU group, Bev group,5-FU+Bev combined treated group were5.5,-2.5,14.5respectively in the5mm block,and10.5,2,20.5respectively in the10mm block.The LCK value (Log Cell Kill)=T-C value in days/(Td×3.32) LCK>1, means the tumor is sensitive to drug, LCK<1, stands for the drug is inactive to tumor,tumor models deemed resistant.The LCK values of5-FU group, Bev group,5-FU+Bev combined treated group were1.24,-0.89,2.32respectively in5mm block,and1.09,0.44,1.31respectively in10mm block.Both the T-C value and the LCK value of the combined treated group were higher than5-FU group and Bev group,it indicated that the5-FU and Bev had a synergistic anti-tumor effection.4. Tumor volume regression effectIn our experiment, we observed that the5-FU+Bev combined treated group showed a tumor growth regression effect. In5mm block, from d6-d14,the i.t. injected tumor volume regressed from213.191mm3to170.483mm3,with a regression ratio of20.03%.while in10mm block, there were two period,from d4-d6,d12-d14,the i.t. injected tumor volume was regressed,from389.512mm3to373.114mm3,and422.830mm3to349.573mm,with a tumor volume regression ratio of4.21%and17.33%respectively.5. The influence of i.t. injection therapy to distant tumorsWhen the diameter of the i.t. injected tumor reached15millimeter,the mice were sacrificed by cerviral dislocation. The time(days) for the i.t. injected tumor reach15mm diameter was as follow, in the5mm block,the control group24,5-FU group32, Bev group20,5-FU+Bev group40. In the10mm block, the days for i.t. injected tumor diameter growed to15mm of the control group,5-FU group, Bev group,5-FU+Bev group were18,24,20,36respectively.We examined the wet weight and volume of the tumors in each group. The wet weight of un-injected tumor in5mm block were as follow, control group0.85,5-FU group1.52, Bev group1.04,5-FU+Bev group2.26, In the10mm block, the wet weight of un-injected tumor of the control group,5-FU group Bev group,5-FU+Bev group were0.84,1.10,0.97,2.12respectively. The volume(mm3) of un-injected tumor in5mm block were as follow, control group88052,5-FU group1525.88, Bev group1007.06,5-FU+Bev group2283.10. In the10mm block, the volume(mm3) of un-injected tumor of the control group,5-FU group, Bev group,5-FU+Bev group were868.14,1099.13,971.35,2105.39respectively.In addition to the control group, either the wet weight or the volume of the i.t. injected tumors were significantly reduced than the un-injected tumors in5-FU group,Bev group and the combined treated group (P<0.05).6. Influence of tumor metastasis of liver, spleen, lung and lymph node by i.t. injection therapyBy anatomical examination, no tumor metastasis was found in liver, spleen, lung of the nude mice. The number of metastatic lymph node of the control group,5-FU group, Bev group,5-FU+Bev combined treated group were4.75,4.75,5.00,5.25respectively, which had no significant difference among all of the treated groups(P>0.05).7.Evaluation of drug side effectsThe skin ulceration and bleeding of nude mice was observed in each group. The weight loss ratio showed no significant difference among the control group,5-FU group and Bev group (P>0.05),while the incidence of this side effect of the combined treated group was significantly lower(P<0.05). Compared with other groups, the white blood cell count of nude mice in the5-FU group was significantly lower (P<0.05); while the white blood cell count of nude mice in the control group,Bev group,5-FU+Bev combined treated group had no difference(P>0.05).Conclusion1. i.t. injection of5-FU or Bev could inhibit the growth of colorectal carcinoma, and the anti-tumor effect of5-FU was more effective than Bev. I.t. injection of5-FU and Bev could synergisticly inhibit the growth of nude mice subcutaneous xenograft model of colorectal carcinoma.2.1ntratumoral injection of5-FU、Bev and the combination of the two drugs has no significant influence to the growth of distant un-i.t. injected tumors.3.5-FU and Bev combination therapy can reduce the side effects such as weight loss and white blood cell reduce of each drug. |
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