Response of p53siRNA U2-OS cells to etoposide.

<p>(<b>A</b>) Inhibition of p53 expression in p53siRNA U2-OS. Actin was used as loading control. (<b>B</b>) p53siRNA U2-OS were less sensitive to etoposide when compared with parental and Ctrl U2-OS;). Student’s test from three independent experiments indicated significantly higher IC₅₀ mean values at 72 h of treatment in p53siRNA U2-OS than in Ctrl and parental U2-OS cells; p = 0.05 (<b>C</b>) Etoposide treatment did not induce mature miR-34a expression in p53siRNA U2-OS, as opposed to Ctrl U2-OS. (<b>D</b>) p53siRNA U2-OS cells presented CpG island methylation (M-MSP) of one of the two alleles of miR-34a. In Ctrl U2-OS both alleles were unmethylated. (<b>E</b>) p53siRNA transfection determined lengthening of G2/M phase after 48 h of etoposide treatment when compared to untreated cells. (<b>F</b>) Western blot of cyclin D1 and CDK4 in p53siRNA cell showed increased amount of CDK4 linked to cyclin D1 and total CDK4 after etoposide treatment when compared to control. No differences in cyclin D1 levels were seen. <i>Ctrl = siRNA negative control duplex; C = Untreated cells; T = Etoposide treated cells.</i></p

Western blot of total and D1-bound CDK4.

<p>In contrast to MG63 and Saos-2 cells, decreased levels of total CDK4 and CDK4 bound to cyclin D1 were observed in U2-OS and U2-OS175 cells after 48 h of etoposide treatment when compared to untreated cells. No differences in cyclin D1 levels were seen. Etoposide treatment did not affect CDK4 level in both MG63 and Saos-2 cell lines. A slight increase of D1-bound CDK4 was evident. <i>C = Untreated cells; T = Etoposide treated cells.</i></p

miR-34a gene genomic organization and methylation specific PCR.

<p>(<b>A</b>) The position of p53 binding site and primers for wild-type and methylation sequences on CpG region are indicated. (<b>B</b>) After bisulphite treatment, U2-OS, U2-OS/e and U2-OS175 showed complete unmethylation (U-MPS) of miR-34a promoter; in contrast MG63 and Saos-2 cells presented methylation (M-MPS) of both alleles.</p

p53 protein expression in OS cells.

<p>wt-p53 U2-OS, U2-OS transfected with empty vector (U2-OS/e) and p53-impaired U2-OS175 cells were positive to anti-p53 that binds the transactivation site of N-terminal domain (aa20-25), with increased expression in U2-OS175 cells. U2-OS and U2-OS/e also presented accumulation of p53 phosphorylated at Ser20 residue (p-p53). MG63 and Saos-2 were negative to both antibodies. Actina was used as loading control.</p

Chromatin Immunoprecipitation assay (ChIP).

<p>Interaction between p53 and miR-34a promoter was present in both U2-OS and U2-OS175. <i>INPUT = positive control; IgG = negative control.</i></p

Growth-inhibition assay.

<p>U2-OS and U2-OS175 cells showed a similar viability Trend with higher sensitivity to etoposide at 72 h than MG63 and Saos-2 cells. No differences between U2- OS and U2-OS/e were observed. Data were presented as mean ± SE from three independent experiments. Student’s test indicated significantly lower IC₅₀ mean values at 72 h of treatment in U2-OS, U2-OS/e and in U2-OS175 than in p53-deficient Saos-2 and MG63 ((p<0.05).</p

Cell cycle analysis and apoptosis.

<p>(<b>A</b>) After 48 h of etoposide treatment (T), BrDU incorporation showed cell accumulation in G1 phase in U2-OS and U2-OS175 cells and in G2/M phase in MG63 and Saos-2 when compared to untreated cells. (<b>B</b>) By Annexin V-FITC assay, no significant increase of apoptotic cells was observed in OS cell lines after 24 h and 48 h of treatment. Data were presented as mean ± SE from three independent experiments. <i>C = Untreated cells.</i></p

Synthesis and Biological Evaluation (in Vitro and in Vivo) of Cyclic Arginine–Glycine–Aspartate (RGD) Peptidomimetic–Paclitaxel Conjugates Targeting Integrin α_Vβ₃

A small library of integrin ligand–paclitaxel conjugates <b>10</b>–<b>13</b> was synthesized with the aim of using the tumor-homing <i>cyclo</i>[DKP-RGD] peptidomimetics for site-directed delivery of the cytotoxic drug. All the paclitaxel–RGD constructs <b>10</b>–<b>13</b> inhibited biotinylated vitronectin binding to the purified α_Vβ₃ integrin receptor at low nanomolar concentration and showed in vitro cytotoxic activity against a panel of human tumor cell lines similar to that of paclitaxel. Among the cell lines, the cisplatin-resistant IGROV-1/Pt1 cells expressed high levels of integrin α_Vβ₃, making them attractive to be tested in in vivo models. <i>cyclo</i>[DKP-<i>f</i>3-RGD]-PTX <b>11</b> displayed sufficient stability in physiological solution and in both human and murine plasma to be a good candidate for in vivo testing. In tumor-targeting experiments against the IGROV-1/Pt1 human ovarian carcinoma xenotransplanted in nude mice, compound <b>11</b> exhibited a superior activity compared with paclitaxel, despite the lower (about half) molar dosage used

Design, Synthesis, and Biological Evaluation of Novel cRGD–Paclitaxel Conjugates for Integrin-Assisted Drug Delivery

The efficacy of taxane-based antitumor therapy is limited by several drawbacks which result in a poor therapeutic index. Thus, the development of approaches that favor selective delivery of taxane drugs (e.g., paclitaxel, PTX) to the disease area represents a truly challenging goal. On the basis of the strategic role of integrins in tumor cell survival and tumor progression, as well as on integrin expression in tumors, novel molecular conjugates were prepared where PTX is covalently attached to either cyclic AbaRGD (Azabicycloalkane-RGD) or AmproRGD (Aminoproline-RGD) integrin-recognizing matrices via structurally diverse connections. Receptor-binding assays indicated satisfactory-to-excellent α_Vβ₃ binding capabilities for most conjugates, while <i>in vitro</i> growth inhibition assays on a panel of human tumor cell lines revealed outstanding cell sensitivity values. Among the nine conjugate ensemble, derivative <b>21</b>, bearing a robust triazole ring connected to ethylene glycol units by an amide function and showing excellent cell sensitivity properties, was selected for <i>in vivo</i> studies in an ovarian carcinoma model xenografted in immunodeficient mice. Remarkable antitumor activity was attained, superior to that of PTX itself, which was associated with a marked induction of aberrant mitoses, consistent with the mechanism of action of spindle poisons. Overall, the novel cRGD-PTX conjugates disclosed here represent promising candidates for further advancement in the domain of targeted antitumor therapy

Antitumor Activity of a Novel Homodimeric SMAC Mimetic in Ovarian Carcinoma

Treatment of ovarian carcinoma often fails to be curative because of drug resistance, and many efforts are directed to overcome tumor cell resistance by increasing apoptosis induction. The potential of second mitochondria-derived activator of caspases (SMAC) mimetics (SMACm) has appeared in preclinical studies, but novel proapoptotic agents of this class with improved pharmacological profile are needed. To identify novel treatment options for ovarian carcinoma by interfering with antiapoptotic factors, in the present study a novel homodimeric SMACm (SM83) was employed in preclinical models both in vitro and in vivo. An investigation of the structural features of dimeric SM83 as compared to a closely related reference compound indicated slight differences, likely because of the interaction between one of the terminal phenyl groups and triazole rings of SM83 with the BIR2 domain. Although SM83 per se did not inhibit cell proliferation, it displayed a synergistic effect in combination with TNF-related apoptosis inducing ligand (TRAIL) in cell sensitivity assays. Because the tumor microenvironment is a reservoir of cytokines that may act in conjunction with SMACm to affect tumor growth, the activity of the novel compound was tested in vivo in ovarian carcinoma cells subcutaneously xenografted into immunodeficient mice. A significant tumor volume inhibition was observed together with activation of caspase 3 and apoptotic cell death. A biochemical analysis of tumor necrosis factor (TNF) and TRAIL content in specimens from xenografted mice indicated that SM83 downmodulated the levels of human TNF in plasma samples and tended to upmodulate human TRAIL levels in tumors. Thus, TRAIL appears to contribute to the antitumor activity of novel SMACm SM83 in subcutaneously grown ovarian carcinoma. Overall, our results indicate that SM83 is an attractive candidate for further development