Quantitative RT-PCR confirmed enhanced expression of s-CLU strictly correlated to

mRNA expression in both KF-TX and SKOV-3-TX cells when compared with their parental cell lines (Figure 2C). Table 4 List of ovarian cancer cells and their IC50 for TX in a three days treatment experiment. Histological type Cell line IC50 (TX; nM) Serous KF 100 Serous KF-TX 500 Serous SKOV-3 20 Serous SKOV-3-TX 100 Serous OVK18 50 Clear cell TU-OC-1 6900 Clear cell KOC-7c 6700 Figure 2 S-CLU is up-regulated in the chemo-resistant cells: A. Western blotting analysis of CLU in a panel of ovarian cancer cells. Equal amount of proteins were loaded, resolved by SDS-PAGE Hedgehog inhibitor and immunoprobed with anti-CLU mAb. S-CLU was found in the cells and media. Some ovarian cancer cells

express relatively high levels of CLU in comparison to immortalized non tumorigenic ovarian epithelial OSE cells. B. Chemo-resistant KF-TX cells shows higher expression levels of CLU compared to parental KF cells. A similar result is found in SKOV-3 compared to SKOV-3-TX cells. C. Quantitative PCR showing the difference in CLU transcript level between the TX-sensitive and TX-resistant clones in both KF (left) and Skov-3 (right) systems. To investigate whether upregulation of s-CLU expression is a cause or a result of TX-induced resistance, both parental KF and KF-TX cells were treated with TX in a dose dependent fashion for 6 h. Sensitive KF cells p53 inhibitor rapidly responded by increasing s-CLU expression level under low doses of TX. In this experiment cellular viability mainly decreased Ricolinostat molecular weight when TX dose surpassed IC50. KF-TX cells already selleck chemicals llc expressing higher CLU levels, did not further express CLU following TX treatment (Figure

3A). When we treated cells with TX up to 48 h, KF parental cells progressively increased CLU expression levels up to IC50 doses (100 nM) then CLU was down-regulated at higher doses. On the other hand, CLU expression level (already high) did not change in KF-TX cells. Again, only at doses higher than IC50 (500 nM), CLU was down-regulated (Figure 3B). S-CLU detected in cells’ medium progressively decreased up to IC50 doses in the sensitive cells suggesting its retention inside cells. However, secretion of CLU into the media by resistant cells clearly extended up to higher concentration of TX if compared with parental cells. Considering that changes in CLU expression seem independent of CLU mRNA, which did not change significantly as indicated by real-time PCR (data not shown), these results suggest that post-translational modification of CLU, including maturation and secretion, may be altered in response to TX treatment. Figure 3 Induction of CLU in a time and dose dependent fashion by TX. A. Western analysis showing s-CLU expression after 6 h treatment with TX. Induction of s-CLU is evident in chemo-sensitive KF cells when treated with high doses of TX but not in KF-TX, in which the high levels of s-CLU remained unchanged.