Figure 2. Cytotoxicity Assay of PECE Copolymer. Different amounts of PECE copolymer were added from 5 to 400 mg each well, and after 24, 48 and 72 hours, survival rate of HOK16E6E7 and HSC-3 cells was determined by MTT assay. Data were obtained from three independent triplicate experiments and were presented as mean 6 S.D (*P,0.01). doi:10.1371/journal.pone.0033860.g002 have detected the expression of acetyl-Histone H3 in order to reflect the controlled releases ability of SAHA-DDP/PECE indirectly. As shown Fig. 3C, the level of acetyl-Histone H3 was observably reduced with the lapse of time in SAHA-DDP group while high expression level of acetyl-Histone H3 was maintained in the SAHA-DDP/PECE group in an extended period. Systematic Toxicity in the SAHA-DDP/PECE Treated Mice Since earlier researches confirmed the severe irreversible side effects of DDP and SAHA treatment, we compared the body weight and other side effects on mice treated with free SAHA, DDP or SAHA-DDP/PECE. No difference of body weight was observed among the SAHA-DDP/PECE group compared with the other groups at the end of the study (Fig. 4A). After mice were sacrificed, their liver, lung, kidney, spleen and heart were harvested and H&E histological staining was performed. As observed by two pathologists in a blinded manner, we found that mice treated with SAHA-DDP/PECE showed no slight changes of toxicity to the organ tissues, compared with the normal organ tissues from mice receiving NS (Fig. 4B). In addition, no conspicuous adverse effects in gross measures were observed, such as appetite, feeding, ruffling of fur, behavior change, etc. SAHA-DDP/PECE Inhibited Cell Proliferation and Intratumoral Angiogenesis In Vivo To address possible mechanisms responsible for the in vivo antitumor activity of combination-therapy groups, we made an inquiry into phenotypic changes of tumor tissues in cell pro- liferation and intratumoral angiogenesis. We first performed PCNA staining to assess the effect of combination-therapy groups, suppression on tumor cell proliferation. Tumor tissues of two combination-therapy groups, especially the group treated with Anti-Tumor Effect of Controlled Drug Delivery SAHA-DDP/PECE were exhibit much less staining for PCNA compared with that in tumor tissues of mice treated with NS, PECE, SAHA, DDP (Fig. 5A, p , 0.05). Some studies have shown that MVD and perimeter are significantly different between normal mucosa and OSCC [29–31]. Therefore, MVD was evaluated in the tumors harvested from mice. We examined the tumor tissues with anti-CD34 and the most highly vascularized area of each tumor was identified and five high-powered fields were counted in this area for MVD. As shown in Fig. 5B, the most significant reduction of MVD was observed in the group treated with SAHA-DDP/PECE (19.764.8) compared with the other groups. Thus, it may be hypothesized that the anti-tumor effects of SAHA-DDP/PECE could be contributed to its ability of inhibits cell proliferation and intratumoral angiogenesis. However, no significant difference was found among SAHA, DDP group and SAHA-DDP group. SAHA-DDP/PECE Induced Apoptosis In Vivo The cytotoxic effects mediated by combination-therapy groups were also analyzed to evaluate whether the cytotoxic mechanisms of the constructs observed included the element of apoptosis in the HSC-3 tumor model. We performed the in situ TUNEL assay to evaluate the influence of combination-therapy groups on apoptosis in tumor cells. We observed an apparently elevated apoptosis rate in tumors of mice with combination-therapy, especially the group treated with SAHA-DDP/PECE compared with that in tumors of mice injected with NS, PECE, SAHA, DDP and (Fig. 6A, p , 0.05). Therefore, SAHA-DDP, especially SAHA-DDP/PECE could led to reduced cell proliferation and increased apoptosis in vivo, which associated with inhibition of tumor growth. To further determine the apoptotic mechanism in the protein level in vivo, western blot assay was performed. The result showed that the protein level of p53, cleaved Caspase-8, cleaved caspase-9 and cleaved caspase-3 in the tumor tissue of the SAHA-DDP/ PECE treated mice much higher than the other groups (Fig. 6B). These data indicated that the observed cytotoxic effects of SAHA- DDP/PECE on HSC-3 tumor xenografts in nude mice also seemed to be mediated by the efficient induction of apoptotic mechanism just like our results in vitro [32]. Discussion In this study, we provided a novel chemotherapy protocol for oral squamous cell carcinoma. The PECE thermosensitive hydrogel have showed promising application as a chemotherapeu- tics controlled drug delivery [24]. We combined SAHA with DDP and utilized the SAHA-DDP/PECE complexes to induce an antitumor activity against the oral squamous cell carcinoma xenografts in a therapeutic setting. We found that the PECE hydrogel could be a novel delivery system and PECE hydrogel delivering DDP and SAHA had promising application in cancer chemotherapy. DDP has a powerful therapeutic effect against oral carcinoma, but the resistance of tumor cells to DDP and dose-related toxicity remain two of the most important problems in the chemotherapy of clinical OSCC [32]. When the DDP dose was too high, the side- toxic effect was enhanced; when the DDP dose was low, the anticancer effect was too low. Researchers have been seeking a combinative treatment regimen to improve the effect of chemotherapy, so combination therapy using two or more chemotherapeutic agents was one of the most common strategies used in current oncology. SAHA was a newly developed HDAC inhibitor which could alter gene transcription and expression profile involved in cell-cycle regulation, tumor suppression, PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e33860