Inhibitory Effects Of Survivin Antisense Oligonucleotide On Human Pancreatic Cancer Inoculated Into Nude Mice

[Objective]:To evaluate the inhibitory effect of Survivin antisense oligonucleotide (ASODN)on nude mice bearing human pancreatic carcinoma xenograft;the expression of Survivin PTEN、CEACAM-land Cox-2, and their correlation in pancreatic cancer.[Methods]:Twenty-eight nude mouse models bearing human pancreatic carcinoma xenograft were constructed and randomly assigned to control group, Lip group, SODN group and ASODN group. Each group has seven nude mice and PBS,lipofectin, ASODN/LiP, and SODN/LiP were injected in to the tumor or around the tumor, respectively. A total of ten injections were given every other day.Two days after the end of treatment,tumors were fully resected from each mouse and measured for tumor weight and inhibitory rate of tumor growth; Paraffin sections of each tumor were made, and respectively stained with HE, immunohistochemical and TUNEL method to evaluate the variations of pathological morphology, Survivin、Cox-2、PTEN and CEACAM-1protein expressions.The expression of Survivin mRNA、Cox-2mRNA、 PTEN mRNA and CEACAM-1mRNA by reverse transcription polymerase chain reaction(RT-PCR) respectively.[Results]:1.After union treated withSurvivin ASODN and lipofectin, the growth of transplanted tumor in nude mice was obviously inhibited. The tumor GI of group LiP, group control,group SODN, group ASODN were (6.81±2.79)%,(7.31±2.65)%,(6.7±2.06)%,(-0.58±4.27)%;and the weights were (1.39±0.09)g,(1.44±0.08)g,(1.33±0.11g),(0.84±0.07)g.There were significantly difference between group ASODN and other groups(P<0.05). The tumor suppres-sion effect of group ASODN was significantly higher than that of the other groups(P<0.05).There was no obvious difference in group SODN and the other two group.2.The observation of main organ in nude mouse after the Survivin-ASODN complex treatment.There were no obvious change in the nude mouse heart, lung, liver,spleen, and kidney observed through routine pathological check after the liposome-Survivin-ASODN treatment.3.The expression of Survivin mRNA in transplanted tumor of nude mouse.There were specific Surviving strap in346bp in control groups and ASODN group through the RT-PCR measurement.Survivin expression of ASODN group was(0.39±0.12),and control groups were respectively (0.57±0.14,0.62±0.15,0.68±0.09),There is significantly difference between ASODN group and control groups(P<0.05);no significantly statistical differences were found in the expressions of Survivin mRNA and protein between SODN group and the other groups.The expression levels of Cox-2、PTEN、CEACAM-1mRNA were detected by RT-PCR methods in ASODN group and control groups respectively.(1).The expression level of Cox-2mRNA was decreased in ASODN group up than in the control group, LiP group and SODN group(0.53±0.27VS O.76±.02,0.75±.21,0.72±.24,P<0.05),the expression of Cox-2mRNA was significantly difference in SODN group than that in LiP group and the control group(P<0.05).(2).The expression level of PTEN mRNA was increased in ASODN group up than in the control group,SODN group and LiP group(1.42±0.15VS0.97±0.63,1.13±0.15,1.06±0.12,P<0.05), no significantly statistical differences were found in the expressions of PTEN mRNA between SODN group and the other two groups.(3).The expression level of CEACAM-1mRNA was decreased in ASODN group than in the control group, LiP group and SODN group(0.50±0.22VS0.71±0.02,0.68±0.23,0.69±0.23, P<0.05), the expresson of CEACAM-1mRNA was significantly difference in SODN group than that in LiP group and the control group(P<0.05). Among the pancreatic cancer,there were positive significant correlations between the expression of Survivin mRNA and the expression of Cox-2、CEACAM-1(r was0.615,0.715,P<0.02), but there was negative significant correlations between the expression of PTEN mRNA and the expression of Survivin,Cox-2,CEACAM-1(r was-0.762,-0.561,-0.807, P<0.02)4.Typical histological changes of orthotopic tumors were observed in4groups.Morphologic observations.Gross:The transplanted tumor was ellipsoidal shape with intact membrane and smooth surface.Occasionally,the surface of tumor was broken.The cross section of tumor was solid with gray-white color.Light microscope(LM):The result revealed that the tumor cells were round or multisided.The cytoplasm of cells was stained red.The proportion of the nuclei and cytoplasm was imbalance.Nuclear division could be easily observed. There were many apoptotic cells with their cytoplasm shrank in group ASODN, and the chromatin condensed into block, and dispersed unevenly.Few apoptotic cells were seen in group SODN, group LiP and group control.5.The Survivin protein expression in the transplanted tumor in nude mouse.There expression of Survivin protein in the transplanted tumor in nude mouse was high. There were significant difference in ASODN group than that in the control group, LiP group and SODN group(P<0.05).The expression of Survivin protein was no significantly statistical differences were found in SODN group than that in LiP group and the control group (P<0.05).The expression levels of Cox-2、PTEN、CEACAM-1protein were detected by immunohistochemistry methods in ASODN group up and control groups respectively.(1).The expression level of Cox-2protein was decreased in ASODN group than in the control group, LiP group and SODN group(P<0.01),the expression of Cox-2protein was no significantly difference in SODN group than that in LiP group and the control group(P<0.05).(2)The expression level of PTEN protein was increased in ASODN group than in the control group and LiP group(P<0.01), no significantly statistical differences were found in the expressions of PTEN protein between SODN group and ASODN group.(3)The expression level of CEACAM-1protein was decreased in ASODN group up than in the control group, LiP group and SODN group(P<0.01), no significantly statistical differences were found in the expressions of CEACAM-1protein between SODN group and the other two groups(P<0.05).6.After treated with ASODN the percentage of apoptotic cells were increased to (20.2±.05)%by the TUNEL,and the value of group SODN was (7.71±0.61)%, the control group was (4.62±0.66)%,the LiP group was(4.98±0.064)%.[Conclusion]:Survivin ASODN can effectively suppress human pancreatic cancer cells growth rate in subcutaneous tumor of nude mice and accelerate the induction of apoptosis of tumor cells.Thus the result had shown that Survivin was a bifunctional protein capable both of suppressing apoptotic cell death and regulating cell proliferation,and it could promote the neoplastic progression by both inhibition of cell apoptosis and progress of cell proliferation.