A Potent Chemotherapeutic Strategy with Eg5 Inhibitor against Gemcitabine Resistant Bladder Cancer.

Development of resistance to gemcitabine is a major concern in bladder cancer therapy, and the mechanism remains unclear. Eg5 has been recently identified as an attractive target in cancer chemotherapy, so novel targeted chemotherapy with Eg5 inhibitor is expected to improve the anticancer effect in gemcitabine-resistant bladder cancer. In this research, RT112-Gr cells were 350-fold less sensitive to gemcitabine than the parental cell lines, while KU7-Gr cells were 15-fold less sensitive to gemcitabine than the parental cell lines. Human OneArray Microarray analysis was performed to obtain broad spectrum information about the genes differentially expressed in RT112 and RT112-Gr cells. The anti-proliferative activity of S(MeO)TLC, an Eg5 inhibitor, was analyzed in RT112-Gr cell lines using a cell viability assay. Furthermore, the inhibitory effect was evaluated in vivo using subcutaneous xenograft tumor model. According to the result of Human OneArray GeneChip, RRM1 and RRM2 were up-regulated, while there was no significant change in Eg5. Trypan blue staining confirmed that in S(MeO)TLC and Gemcitabine combining S(MeO)TLC group cell viability were significantly decreased in RT112-Gr cells as compared with other groups. S(MeO)TLC and S(MeO)TLC+gemcitabine groups prominently suppressed tumor growth in comparison with other groups' in vivo. There were no significant differences in S(MeO)TLC and gemcitabine+S(MeO)TLC group in the effect of inhibition of bladder cancer in vivo and in vitro. Our data collectively demonstrated that S(MeO)TLC represents a novel strategy for the treatment of gemcitabine resistant bladder cancer

S(MeO)TLC induces Gemcitabine resistance bladder cancer cells apoptosis.

<p>A: RT112-Gr cells were left untreated; B: RT112-Gr cells were treated with 45nM Gemcitabine; C: RT112-Gr cells were treated with 0.5μM S(MeO)TLC; D: RT112-Gr cells were treated with both together (45nM Gemcitabine + 0.5μM S(MeO)TLC) for the indicated times and then nuclear morphology was examined with Hoechst staining and visualized by fluorescent microscopy.</p

RNAi-mediated Knockdown of RRM1/2 in RT112-Gr bladder cancer cells.

<p>RT112-Gr cells were left untreated or were treated with 45nM Gemcitabine (GEM), RNAi-mediated Knockdown of RRM1 (A) and RRM2 (B) in RT112-Gr bladder cancer cells were left untreated or were treated with 45nM Gemcitabine (GEM). Seventy-two hours later viability was analyzed by Trypan blue staining. RNAi-mediated Knockdown of RRM1 (A) and RRM2 (B) +GEM, inhibiting tumor effect is the best among the four groups. Quantification of each value is from triplicate independent experiments. (<i>p</i> < 0.01).</p

Expression of RRM1 and RRM2 in clinical bladder samples.

<p>A:Immunohistochemical staining of RRM1 and RRM2 in clinical tissues. RRM1 and RRM2 immunostaining was considered positive if >10% of the cytoplasm of cancer cells showed weak or greater intensity. (I) High expression of RRM1 in clinical bladder cancer. (II) Low expression of RRM1 in clinical bladder cancer (III) High expression of RRM2 in clinical bladder cancer. (Ⅳ) Low expression of RRM2 in clinical bladder cancer (all figures were captured at 400× magnification). B: (I) Bar graph illustrates combined immunostaining score for RRM1 expression according to tumor grade. (II)Bar graph illustrates combined immunostaining score for RRM2 expression according to tumor stage (The color of black and gray represent weakly and strongly positive, respectively).</p

S(MeO)TLC overcomes Gemcitabine resistance.

<p>A: The IC50s of Gemcitabine in KU7、KU7-Gr、RT112 and RT112-Gr in 72 hours. B: The IC50s of S(MeO)TLC of KU7、KU7-Gr、RT112 and RT112-Gr in 72 hours. C: KU7-Gr and RT112-Gr cells were left untreated or were treated with 45nM Gemcitabine (GEM), 0.5μM S(MeO)TLC or both together (GEM+ S(MeO)TLC). Seventy-two hours later viability was analyzed by Trypan blue staining. S(MeO)TLC and GEM+ S(MeO)TLC, inhibiting tumor effect is the best among the four group. Quantification of each value is from triplicate independent experiments. (<i>p</i> < 0.01).</p

Anticancer activity of S(MeO)TLC in subcutaneous xenograft tumors.

<p>A. After successful establishment of subcutaneous xenograft tumors, DMSO (vehicle control), 20 mg/kg S(MeO)TLC, 50 mg/kg Gemcitabine or both(S(MeO)TLC + GEM) were administered intraperitoneally daily for 5 days (arrows). Tumor volumes were measured every other day. B. The mean body weights of mice were assessed every other day. There is no significant difference among three groups (<i>P</i>>0.05).</p

Clustering analysis of microarray.

<p>Clustering was performed to visualize the correlations between the replicates and varying sample conditions. Up- and down-regulated genes are represented in red and green colors, respectively. A subset of differential genes was selected for clustering analysis. An intensity filter was used to select genes where the difference between the maximum and minimum intensity values exceeds 1000 among all microarrays. For this microarray project, the number of genes clustered was 227.</p

Expression of RRM in relation to clinicopathological characteristics of patients with Bladder Cancer.

<p>RRM1 and RRM2 staining was localized in the cytoplasm. Tumor grade and stage were evaluated using standard hematoxylin and eosin (H&E) staining.</p><p>Expression of RRM in relation to clinicopathological characteristics of patients with Bladder Cancer.</p

Expression of RRM1 and RRM2 in bladder cancer cell lines.

<p>RT-PCR for RRM1 and RRM2 mRNA expression in bladder cancer cell lines. GAPDH served as the loading control. Bar graph illustrates RRM1 and RRM2 mRNA expression in RT112、RT112-Gr and siRNA-RT112-Gr bladder cancer cell lines. (<i>p</i> < 0.001).</p