HSP70 shows strong expression (scored as 3+, a), moderate expression

(scored as 2+, b), weak expression (scored as 1+, c), and negative expression (scored as 0, d) in cytoplasm(×400). Screening the antisense oligodeoxynucleotides (ODNs) which could downregulate HSP70 expression in Hep-2 cells effectively Based on the mRNA complete sequence of human HSP70 mTOR activity (GeneBank accession NO. BC002453), we designed three antisense oligos (ASODNs) at the different sites in human HSP70 sequence (AS-1, AS-2 and AS-3). After being transfected with HSP70 ASODN for 48 h, the total proteins were isolated and the expression level of HSP70 was determined by western blot. The results showed that AS-1 significantly inhibited the expression of HSP70. Both AS-2 and AS-3, however, did not show any effect (Fig 2a). All the following experiments were thus carried out by using AS-1. And the corresponding sense and random oligos selleck were designed based on AS-1 sequence. Western blot showed that the random and sense oligos had no repressive effect on the expression of HSP70 (Fig 2b). Figure 2 Knock-down effect of HSP70 antisense oligos. Hep-2 cells were transfected with HSP70 antisense oligos (AS) or control oligos (sense oligos and random oligos) as described under materials and methods. After incubation for 48 h, cells were harvested,

lysed. Western-blotted (WB) with the corresponding antibodies was carried out to show the knock-down effect of AS. AS-1 has the best knock-down effect of all 3 oligos. The radiation sensitizing effect of HSP70 antisense oligos on laryngeal carcinoma To investigate whether the HSP70 antisense oligos have radiation sensitizing effect on laryngeal carcinoma xenografts in vivo, the PFKL antisense and random oligos were injected into tumor through intratumoral injection. The mice were treated with radiation (5Gy). The treatment effect was measured.

The results showed that there was no significant difference in the tumor growth between group antisense (368 ± 129 mm3) and group random(384 ± 179 mm3) before selleck kinase inhibitor radiotherapy (P > 0.05, Fig. 3a). However, eight days after radiotherapy, the volumes and weights of implantation tumor in group antisense (229 ± 28 mm3 and 0.18 ± 0.04 g) were significantly smaller than that of group random (417 ± 103 mm3 and 0.27 ± 0.05 g) (P < 0.05; Fig. 3b, c, d). To determine the efficiency of intratumoral injection, we used oligos with green fluorescent marker and observed the tumor under a fluorescence microscope after the first injection. Fig. 3e shows obvious infection efficiency. Figure 3 Effect of HSP70 antisense oligos on radiotherapy of laryngeal carcinoma xenografts. (a) shows the tumor growth curve before radiation, no difference between the 2 groups were found, the tumor volum were for antisense and random group, respectively (P > 0.05, 368 ± 129 mm3vs 384 ± 179 mm3).