Survivin overexpression is associated with poor prognosis of human gastric cancer

Survivin overexpression is associated with poor prognosis of human gastric cancer and is a target for gastric cancer therapy. cancer cells. YM155 infusion NHS-Biotin at 5 mg/kg/day for 7 days markedly inhibited growth of gastric cancer xenograft in a nude mouse model. Immunohistochemistry staining and Western Blot showed that YM155 treatment inhibited expression of survivin and CD44 induced apoptosis and reduced CD44+ CSCs in xenograft tumor tissues [22]. While those approaches are effective it is still difficult to use in clinic. Recent studies showed that YM155 a novel small imidazolium-based compound can specifically inhibit survivin expression and induce apoptosis in human cancer cells [23]. Preclinical studies demonstrated that three to seven-day continuous infusion of YM155 (1-10mg/kg.d) significantly inhibited tumor growth in hormone-refractory prostate cancer melanoma and non-small-cell lung cancer [24]. Moreover recent results from completed phase I/II clinical studies show that YM155 was safe at a dose of 4.8 mg/m2/day for 168 hours every 3 weeks and exhibited encouraging anti-cancer effect in advanced cancer patients [25-29]. These results suggest that YM155 is a promising agent for cancer therapy. However there are no studies to show that YM155 inhibit gastric tumor growth gastric cancer SGC-7901 and MKN-28 cells were treated with YM155 for 48 hours; cell proliferation was measured by MTT. The results showed NHS-Biotin that YM155 significantly inhibited cell proliferation. The mean IC50 of SGC-7901 and MKN-28 cells were 13. 2 nM and 11.6 nM (Figure ?(Figure1A) 1 respectively. YM155 has also shown a great activity against other gastric cancer cell lines such as AGS and Hs 764T cell lines with IC50 values 0.8 nM and 7.3 nM [24]. Figure 1 YM155 inhibits anchored-dependent and anchored-independent growth in gastric cancer cells To further investigated the effect of YM155 NHS-Biotin Rabbit Polyclonal to ABHD12. on cell transformation. Soft agar assay was conducted to determine cell transformation method “spheroid colony formation” that candidate CSCs were cultured in serum-free medium containing only EGF and bFGF (stem cell medium SCM) using a ultra-low-attachment plates. We first investigated the effect of YM155 on sphere formation. SGC-7901 cells and AGS cells were cultured in SCM with and without YM155 for 2 weeks. Quantification analysis of spheres showed that YM155 reatment significantly reduced spheres formation in SGC-7901 and AGS cells in dose dependent patterns (Figure ?(Figure4A).4A). These results indicate that YM155 can inhibit formation of spheres in gastric cancer cells. To determine whether YM155 also reduces expansion of gastric CSCs SGC-7901 cells were first cultured in SCM for one week to form sphere. One week after culturing SGC-7901 cells formed small spheroid colonies (spheres) (Figure ?(Figure4B 4 upper panel). These formed spheres of SGC-7901 cells were then treated with YM155 at the doses of 1 1 nM 10 nM and 20 nM in SCM for one more week. We observed that vehicle-treated spheres greatly expanded and formed large size colonies and that YM155-treated spheres grew slowly and formed smaller size colonies compared to vehicle-treated spheres (Number ?(Number4B 4 bottom panel). The related inhibition of sphere growth was also observed in AGS cells treated with YM155 (data not shown). The results suggest that YM155 inhibits development of gastric CSCs. Number 4 YM155 inhibits formation and development NHS-Biotin of gastric malignancy spheres YM155 inhibits manifestation of CSC molecules We then explored the underlying mechanisms by which YM155 inhibits the development of gastric CSCs. Since Wnt/1.07 ± 0.15 % < 0.01) (Number ?(Figure6D).6D). These results indicate that YM155 induces apoptosis of gastric malignancy we determined manifestation of CD44 in xenograft cells by Western Blot and IHC staining. Western blot showed the protein levels (densities of band) of CD44 in the tumors from YM155-treated mice were lower than those in the mice from NHS-Biotin vehicle-treated mice (Number ?(Number6A6A remaining and right panel). Although IHC staining showed that the number of CD44+ staining cells was not significantly different between NHS-Biotin the groups the intensity of CD44+ staining were markedly weaker in tumor cells from YM155-treated mice compared to those in tumor cells from vehicle-treated mice (Number ?(Figure6B).6B). Quantification analysis showed the densities.