Experimental Study On Variation Of Radiosensitivity Of B16 Cells Transfected By STAT3 Antisense Oligodeoxynucleotide

Radiation therapy and chemotherapy in conjunction with surgical operation have been commonly used for the treatment of many tumors. However a significant number of tumors fail respond to radiation therapy and/or chemotherapy because many forms of tumors appear less sensitive to radiation and anticancer drugs. Over the last few years, studies manipulating the expression of certain genes have led to a better understanding of the genetic basis of sensitivity to ionizing radiation. Such studies are important not only to gain fundamental insights into the response of cells to radiation, but also to provide clues as to how and where one could intervene to make tumor cells more radiosensitive. Examples of genetic manipulations affecting radiosensitivity have proliferated in the literature over the last few years, probing the many processes and pathways that influence whether a cell lives or dies after radiation. There are many manipulation strategies and also lots of genes to be selected or targeted to intervene tumor radiosensitivity. Here we use STAT3 antisense oligodeoxynucleotides to prohibit the expression and activity of STAT3 protein so as to enhance the radiosensitivity of mouse melanoma B16 cell lines.Signal transducers and activators of transcription (STATs) were identified originally as key components of cytokine signaling pathways, involved in mediating responses to IFNs, IL-6 and other cytokines. On phosphorylation of STAT monomers by tyrosine kinases, the monomers dimerized, translocate to the nucleus, and bind to specific promoter sequences, thereby inducing expression of multiple genes associated with cellular proliferation and survival. As a point of convergence for many tyrosine kinase signaling pathways, such as EGFR, IL-6/JAK and Src, STAT3, one of the most important members of the seven STATs family, has been found to be constitutively activated in a wide variety of human tumor specimens and tumor cell lines, including breast cancer, head and neck aquamous cell carcinoma, prostatecancer, various leukemias, multiple myeloma and melanoma cells. Furthermore, accumulating evidence is defining a critical role for STAT3 in oncogenesis. In particular, STAT3 signaling has been shown to prevent apoptosis and enhance cell proliferation through regulating genes involved in cell growth and programmed cell death, including Bcl-xL, Mcl-1, c-Myc, cyclin Dl, and P53. In addition to its role in promoting tumor cell growth and survival, constitutive STAT3 activation has also been shown to up-regulate VEGF expression and to promote tumor angiogenesis. Numerous studies have also demonstrated that abrogation of STAT3 signaling blocks oncogenesis in vitro and in vivo. Interrupting STAT3 signaling has been shown to induce tumor apoptosis and /or cell cycle arrest in a wide range of cancer cell lines which provided a rationale for designing new drugs for therapy of these cancers.In this study we use antisense oligodeoxynucleotide of STAT3 (ASO-STAT3) as an inhibitor to block the STAT3 signal pathway, observe the variation of the radiosensitivity of mouse melanoma B16 cell lines after transfected with AS0-STAT3 and probe into the mechanism of the observed phenomena. First we detected the expression and phosphorylated levels of STAT3 protein using WESTERN BLOT with special antibody targeting total STAT3 or 705-tyrosine phosphorylated STAT3 respectively in B16, 7721, HepG-2, A549 and Hela tumor cells, both of which were highly expressed in all of the detected cell lines.After B16 cells were transfected with ASO-STAT3, synthesized using phosphorothioate chemistry according to reference articles to improve its stabilization in cells, obvious decreasing of total STAT3 protein was observed in B16 cells, the decreasing extent elevated with the rising concentration of ASO-STAT3. The level of phosphorylated STAT3 diminished either, showing the same variational trend with total STAT3 protein, which implied the decline activity of STAT3 protein. Proliferation inhibition of B16 cells was evaluated by MTT assay. Forty-eight hours later after transfected by Oligofectamine method, MTT was practiced, and the result showed that the higher the concentration of the AS0-STAT3 was, the heavier the inhibition was displayed in a certain scope varied from 0 to 200 nM (P<0.01), while no statistic inhibitive effects were found in blank or senseoligodeoxynucleotide-STAT3 (SO- STAT3) control. When we tried to transfect more than 250nM of oligodeoxynucleotides, proliferation inhibition was found in both groups of the cells transfected by antisense and sense oligodeoxynucleotide STAT3 (P<0.05). We suggest it as a nonspecific toxic reaction of oligodeoxynucleotides causes by too high relative concentration of the short DNA sequences. We also found that the inhibitive effects early appeared 24 hours after transfected with ASO-STAT3 and more obvious differences could be found after transfected 48 hours (P<0.01). All of these data indicate that there are some effects for the loss of cell viability and enhancement apoptosis with B16 cells transfected with AS0-STAT3, which supports a role for STAT3 as a potential molecular target for tumor therapies as has been testified by many researchers.Cells were transfected with 200nM AS0-STAT3 or control S0-STAT3 in 96 wells cultures (or 400 nM in 6 wells), exposed to different dose of irradiation 36 hours later, then compared for cell viability and apoptotic response. Effects on cells activity were confirmed using the MTT assay and CCK-8 assay. Apoptotic assays were performed using Hoechst33258 and Annexin V/PI with FACS analysis. Obvious enhancement of inhibition were observed when ASO-STAT3 transfected combining with Y irradiation within low dose scope characterized from OGy to 8Gy, yet no obvious changes in radiosensitivity could be detected when the radiation dose increased to 8Gy higher, which could be interpreted as that high dose of radiation itself could kill so much that little damage effect of ASO-STAT3 could be detected.Similar results were obtained when refered to levels of apoptosis of different experimental groups. In the visible light, apoptotic cells were characterized as shrinkage of cell membrane, loss cell volume, condensation of cytoplasm, some cells falling off from the wall of the culture, et al. After Hoechst 33258 staining was performed, cells were viewed with a fluorescence microscope. Chromatin condensation, nuclear shrinkage or nucleosomal fragmentations were considered morphologic markers of apoptosis. We found that part of the cells underwent apoptosis 48 hours after transfected with ASO-STAT3, more apoptotic cells could be detected following the rising of concentration of the antisense sequence, but nostatistic difference could be found between the SO-STAT3 and blank control group. No typical apoptotic bodies were found in all these groups. The ratio of condensed or fragmented nuclei to normal nuclei increased following the improving of radiation dose, round or irregular apoptotic bodies could also be found in some high dose irradiation groups. In the groups of ASO-STAT3 transfection with subsequent exposure to 16Gy irradiation, increasing number of apoptotic cells and apoptotic bodies were detected in both cells transfected with ASO-STAT3 alone and cells treated with irradiation only.Annexin V/PI assay was then performed using FACs for detecting early apoptosis of B16 cells after different treatment of each group in 6 wells. Statistic difference was found between the blank control and different concentrations groups of ASO-STAT3 and SO-STAT3 (400nM or 800nM or 2000nM) (P<0.01), and no obvious difference could be found in groups between blank control and 400nM or 800nM of SO-STAT3 groups. When refered to 2000nM SO-STAT3 transfection groups and blank control, evidently difference were discovered (P<0.01), which maybe a nonspecific toxic effect of oligodeoxynucleotides as described above. When treated with irradiation combined, although not so high differences were found in our data as has been reported, statistic contrast could be obtained among different groups (P<0.01). In groups irradiated with different dose after transfected with 400nM ASO-STAT3, ratio of early apoptosis was found increasing in different extent contrast to blank control, irradiated only, SO-STAT3 transfection with subsequent irradiation. All these data support that AS0-STAT3 itself can induce apoptosis of B16 cells, and it could produce more apoptosis cells together with Y irradiation than SO-STAT3 could.In summary, AS0-STAT3 can abrogate the activity of STAT3 protein, inducing subsequent proliferation inhibition and apoptosis of B16 cells. When practiced together with Y irradiation, AS0-STAT3 can not only kill the tumor cells itself but also partly enhance the cells radiosensitivity which improves the therapeutic effects of radiotherapy to radioresistant tumors or cancers. These results support roles for STAT3 as a potential molecular target for tumor therapies and also as a potential...