Atorvastatin prevents Plasmodium falciparum cytoadherence and endothelial damage

<p>Abstract</p> <p>Background</p> <p>The adhesion of <it>Plasmodium falciparum </it>parasitized red blood cell (PRBC) to human endothelial cells (EC) induces inflammatory processes, coagulation cascades, oxidative stress and apoptosis. These pathological processes are suspected to be responsible for the blood-brain-barrier and other organs' endothelial dysfunctions observed in fatal cases of malaria. Atorvastatin, a drug that belongs to the lowering cholesterol molecule family of statins, has been shown to ameliorate endothelial functions and is widely used in patients with cardiovascular disorders.</p> <p>Methods</p> <p>The effect of this compound on PRBC induced endothelial impairments was assessed using endothelial co-culture models.</p> <p>Results</p> <p>Atorvastatin pre-treatment of EC was found to reduce the expression of adhesion molecules and <it>P. falciparum </it>cytoadherence, to protect cells against PRBC-induced apoptosis and to enhance endothelial monolayer integrity during co-incubation with parasites.</p> <p>Conclusions</p> <p>These results might suggest a potential interest use of atorvastatin as a protective treatment to interfere with the pathophysiological cascades leading to severe malaria.</p

Evaluation d'une nouvelle stratégie antitumorale en utilisant des peptides pénétrants qui ciblent l'interaction Caspase-9/PP2A

PARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

Specific targeting of caspase-9/PP2A interaction as potential new anti-cancer therapy.

PURPOSE: PP2A is a serine/threonine phosphatase critical to physiological processes, including apoptosis. Cell penetrating peptides are molecules that can translocate into cells without causing membrane damage. Our goal was to develop cell-penetrating fusion peptides specifically designed to disrupt the caspase-9/PP2A interaction and evaluate their therapeutic potential in vitro and in vivo. EXPERIMENTAL DESIGN: We generated a peptide containing a penetrating sequence associated to the interaction motif between human caspase-9 and PP2A (DPT-C9h), in order to target their association. Using tumour cell lines, primary human cells and primary human breast cancer (BC) xenografts, we investigated the capacity of DPT-C9h to provoke apoptosis in vitro and inhibition of tumour growth (TGI) in vivo. DPT-C9h was intraperitoneally administered at doses from 1 to 25 mg/kg/day for 5 weeks. Relative Tumour Volume (RTV) was calculated. RESULTS: We demonstrated that DPT-C9h specifically target caspase-9/PP2A interaction in vitro and in vivo and induced caspase-9-dependent apoptosis in cancer cell lines. DPT-C9h also induced significant TGI in BC xenografts models. The mouse-specific peptide DPT-C9 also induced TGI in lung (K-Ras model) and breast cancer (PyMT) models. DPT-C9h has a specific effect on transformed B cells isolated from chronic lymphocytic leukemia patients without any effect on primary healthy cells. Finally, neither toxicity nor immunogenic responses were observed. CONCLUSION: Using the cell-penetrating peptides blocking caspase-9/PP2A interactions, we have demonstrated that DPT-C9h had a strong therapeutic effect in vitro and in vivo in mouse models of tumour progression

Effect of DPT-C9h on caspase-9 activation, mitochondrial membrane depolarization, cytochrome <i>c</i> release and cell cycle.

<p>A) HBCx-3 cells were cultured for 3 or 6 h with medium (control), 100 µM of DPT-C9h or 10 µM of the caspase inhibitor Z-VAD (pre incubation of 1h) and 100 µM of DPT-C9h. Caspase-9 activity was estimated using a luminogenic substrate. Results are represented relative to control non-treated cells as arbitrary units. P values are shown. B) HBCx-3 cells were cultured for 24 h with medium (control), DPT-Sh1 (100 µM), DPT-C9h (100 µM) or Z-VAD (10 µM, pre incubation of 1h) and DPT-C9h (100 µM). Apoptosis was estimated by Annexin-V-FITC binding. C) HBCx-3 cells were treated for different periods of time with DPT-C9h (100 µM) and then incubated for 30 min at 37°C protected from the light with the fluorescent probe JC-10. Green and red fluorescence were measured. Data are represented relative to the control non-treated cells. P values are shown. D) HBCx-12A and HBCx-3 cell lines were treated for 24 h with 100 µM of DPT-C9 h. Mitochondrial fraction was separated from whole cell lysates and immunoblotted for cytochrome <i>c</i>. The WB was also hybridized with the mitochondrial marker Tim23 as internal control of protein loading. E) HBCx-3 cells were non-treated (control) or treated with 10 or 25 µM of DPT-C9h for 24 or 48 h and the cell cycle was analyzed by FACS.</p

Apoptotic effect of DPT-C9h peptide on primary and tumour cells.

<p><b>A</b>) Peripheral blood mononuclear cells (PBMC) from healthy donors or CLL patients were cultured in the presence of DPT-C9h (150 µM) for 3 h, then washed, transferred to complete medium and apoptosis was estimated 6h later. Selection of B cells was done by anti-CD19 antibody before Annexin V-FITC staining. Non-treated cells were used as control. <b>B</b>) Cells isolated from bone marrow of CLL patients and healthy donors were treated as in A and analyzed for apoptosis. P values are shown.</p

<i>In vivo</i> antibody responses and toxicity induced by DPT-C9h.

<p><b>A</b><b> </b>) Serum antibodies taken from nude mice treated for different periods of time were detected by ELISA at two different concentrations of DPT-C9h peptide (10 and 50 µM). <b>B</b>) Serum antibodies from wild type mice treated for different periods for time were tested by ELISA against DPT-C9h and DPT-Sh1 (50 µM). <b>C</b>) DPT-C9h was intraperitoneally administered in mice bearing tumors HBCx-12A at 1, 5, or 25 mg/kg once daily for 5 weeks; the median weight of mice for each experimental group is represented at different times. A total of 10 mice were included per group. Similarly, DPT-C9h was intraperitoneally administered in mice bearing tumours HBCx-8 at 10 mg/kg twice daily for 4 weeks. DPT-C9 was IP administrated at mice model PyMT model at dose of 5 mg/kg. The median weight of mice for each experimental group is represented at different times. Ten mice were included per group.</p

DPT-C9h induces apoptosis in human cell lines.

<p><b>A</b>) Daudi, Jurkat, and HeLa cell lines were cultured in the presence of DPT-C9h, DPT-Sh1, C9h, or C9 peptides for 20 h at 100 µM and apoptosis was estimated by Annexin-V staining. <b>B</b>) Mouse lung cancer cell lines LKR10 and LKR13 were cultured in the presence of DPT-C9h, DPT-C9, or DPT-Sh1 at 100 µM. After 24 h of incubation, apoptosis was estimated by Annexin staining. The basal level of apoptosis of control non-treated cells is shown. P values are also shown (*<0.05; **<0.001; ***<0.0001). <b>C</b>) Breast, uveal melanoma and lung cancer cell lines isolated from primary human xenografs were cultured in the presence or absence of DPT-C9h peptide (100 µM) for 24h and apoptosis was estimated by Annexin V-FITC. Basal level of apoptosis without peptide addition is shown (grey colour) p values are shown. <b>D</b>). Breast cancer cell lines derived from the primary human xenografts, were incubated with C9h in culture medium at 150 µM and apoptosis induction was estimated at different times. <b>E</b>) Breast cancer cell lines isolated from primary human xenografts BCx-3 and Bcx-12 were cultured in the presence or absence (control) of the peptide DPT-C9h for 24 h and apoptosis was estimated.</p