In brief, ESCC cells were pretreated with increasing concentrations of SNS-032 for 24?h; cells were then harvested and incubated with a m assay kit with JC-1 (Beyotime, Shanghai, China) at 37?C for 30?min

In brief, ESCC cells were pretreated with increasing concentrations of SNS-032 for 24?h; cells were then harvested and incubated with a m assay kit with JC-1 (Beyotime, Shanghai, China) at 37?C for 30?min. SNS-032 on ESCC growth in vitro. Tumor xenograft in nude mice was used to assess the antitumor activity of SNS-032 in vivo. The roles of SNS-032 in ESCC metastasis were conducted by wound healing and transwell assays in vitro, and by a lung and a popliteal lymph node metastasis model in vivo. The results showed that CDK7 and CDK9 were highly expressed in ESCC cells; AXIN1 SNS-032 effectively inhibited cellular viability, abrogated anchorage-independent growth, and potentiated the sensitivity to cisplatin in ESCC cells in vitro and in vivo. In addition, SNS-032 induced a mitochondrial-dependent apoptosis of ESCC cells by reducing Mcl-1 transcription. SNS-032 also potently abrogated the abilities of ESCC cell migration and invasion through transcriptional downregulation of MMP-1. Importantly, SNS-032 remarkably inhibited the growth of ESCC xenograft, increased the overall survival, as well as diminished the lung and lymph node metastasis in nude mice. Taken together, our findings highlight that the CDK7/9 inhibitor SNS-032 is a promising therapeutic agent, and warrants a clinical trial for its efficacy in ESCC patients, even those with metastasis. test. D Western blot SB-222200 analysis of MMP-1 and MMP-2 in KYSE30 and KYSE150 cells treated with increasing concentrations of SNS-032 for 48?h. E SNS-032 dose-dependently decreased mRNA levels of MMP-1 in ESCC cells. *test. To further determine the inhibitory effects of SNS-032 on ESCC metastasis, an in vivo popliteal lymph node metastasis model was employed in nude mice by inoculating the foot pads with KYSE30 cells stably expressing green fluorescent protein (GFP; Fig. ?Fig.6C).6C). We found that SNS-032 greatly attenuated lymph node metastasis, as evidenced by smaller volumes of lymph nodes and decreased numbers of GFP-positive tumor cells in tumors formed from SNS-032-treated mice, compared with those of the vehicle-treated mice (Fig. 6D, E). Strikingly, the ratios of metastatic to total dissected popliteal lymph nodes were markedly lower in mice from the SNS-032-treated group (100% (6/6)) than in the vehicle controls (16.7% (1/6); Fig. ?Fig.6F6F). Taken together, these results demonstrate that SNS-032 effectively abrogates the metastasis of ESCC cells in nude mice. Discussion In this study, we discovered that both CDK7 and CDK9 are highly elevated in all five tested human ESCC cells, suggesting existence of elevated transcription activity. Transcription inhibition by SNS-032 significantly suppressed the cellular proliferation, anchorage-independent growth, and the growth of xenografted ESCC cells in nude mice. Moreover, SNS-032 drastically inhibited the migration and invasion of ESCC cells by reducing the transcription of MMP-1. Most importantly, SB-222200 SNS-032 profoundly diminished the lung and lymph node metastasis of ESCC cells in vivo. Our studies provided a new strategy for the treatment of ESCC by targeting transcription of the oncogene. As SB-222200 a specific CDK inhibitor against CDK7 and CDK9, SNS-032 has displayed potent anticancer effects in various human malignant cells, such as uveal melanoma [5], acute myeloid leukemia [24], and cervical cancer cells [25], and exerted chemopreventive effects in both NOD-SCID mouse xenograft model and NOG mouse model of uveal melanoma [5]. In this study, our results showed that SNS-032 significantly inhibited tumor cell growth and induced apoptosis in all the investigated cell lines of ESCC at nanomolar concentrations. Consistently, a phase I clinical trial in patients with metastatic SB-222200 refractory solid tumors showed that the therapeutic SNS-032 plasma concentrations were 754?nM [22], which can be extrapolated to kill ESCC cells. Intriguingly, our results also showed a notable synergistic antiproliferative effect between SNS-032 and CDDP, the first-line chemotherapy for patients with locally advanced ESCC [26]. In agreement with our findings, a few reports have showed that there is a synergistic effect to inhibit malignant cell growth when SNS-032 combined with radiation in cervical cancer and non-small cell lung cancer [25, 27]. Altogether, these findings suggest that SNS-032 in combination with the traditional chemotherapeutic agents is a promising therapeutic strategy for cancer patients. Thus, future investigations are needed to further evaluate the efficiency of SNS-032 together with CDDP in mouse models and clinical trials of ESCC. In this study, we also identified that Mcl-1 played an important role in SNS-032-induced apoptosis. Mcl-1 belongs to an antiapoptotic.