Strategies for surviving down-regulation : effects on tumour cell growth potential and chemosensitivity profile

Pennati, Marzia (2008). Strategies for surviving down-regulation : effects on tumour cell growth potential and chemosensitivity profile. PhD thesis The Open University.



Survivin is a bifunctional protein that acts as a suppressor of apoptosis and plays a central role in cell division. The protein is strongly expressed in the most common human neoplasms, has prognostic relevance for some of them, and appears to be involved in tumour cell resistance to anticancer agents and ionizing radiation. On the basis of these findings, survivin has been proposed as an attractive target for new anticancer interventions.

We generated a hammerhead ribozyme (Rz) targeting the CUA110 triplet in the survivin mRNA and transfected them into the JR8 human melanoma cell line. Cells endogenously expressing Rz were characterized by a lower survivin protein level than parental cells, and showed an increased caspase-9-dependent apoptotic response to treatment with the cytotoxic agents cisplatin and topotecan as well as with ɣ-irradiation. Moreover, an increased anti-tumour activity of oral topotecan was observed in Rz-expressing cells grown as xenograft tumours in athymic nude mice. In addition, we constructed a Moloney-based retroviral vector expressing Rz, encoded as a chimeric RNA within adenoviral VA1 RNA. Polyclonal cell populations, obtained by infection with the retroviral vector, of two androgen-independent human prostate cancer cell lines (DU145 and PC-3) were characterized by a significant reduction of survivin expression; the cells became polyploid and underwent caspase-9-dependent apoptosis. Survivin inhibition also enhanced their susceptibility to cisplatin-induced apoptosis and prevented tumour formation when cells were xenografted into athymic nude mice.

Again we used RNAi to specifically repress survivin in DU145 and PC-3 cell lines. RNAi-mediated survivin knock-down was able to significantly reduce cell proliferation and to enhance the rate of caspase-9-dependent apoptosis. Moreover, sequential treatment with survivin-specific siRNA followed by the Hsp90 inhibitor 17-allylamino-17-demethoxy-geldanamycin produced supra-additive anti-proliferative effects in both cell lines.

Finally, we investigated the effects of the novel cdk inhibitor NU6140, in term of ability to potentiate the response to paclitaxel in HeLa cells, in relation to its interference with survivin. Sequential administration of cdk inhibitors resulted in escape from the mitotic block imposed by paclitaxel and significantly increased the apoptotic rate, with inhibition of survivin expression/phosphorylation as the potential mechanism.

Overall, such results suggest that strategies aimed at interfering with survivin expression/activity can be adopted to improve the chemo/radio-sensitivity profile of treatment-refractory human malignancies.

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