Μελέτη συγχορήγησης μετρονομικής βινορελμπίνης και αντιαγγειογενετικών φαρμάκων μοριακής στόχευσης σε καλλιέργεια ενδοθηλιακών κυττάρων ανθρώπινου πλακούντα: διερεύνηση συνεργίας σε κυτταρικό και μοριακό επίπεδο

Abstract

Metronomic chemotherapy is the protracted dose dense administration of low sub-toxic doses of | 6chemotherapy. Vinorelbine is a semisynthetic vinca alkaloid with the additional advantage of the oralformulation which favors its use in the chronic administration protocol of metronomic chemotherapy.Clinical studies have demonstrated that the metronomic administration of vinorelbine createssustainable anti-tumor efficacy. Considering the low nanomolar concentrations of the drug, thenegligible toxicity and the profile of circulating angiogenic biomarkers we speculated that the antitumoractivity is most likely attributed to anti-angiogenic activity.We sought to determine whether the clinically relevant concentration is anti-angiogenic in vitro. Wefound that 10 nM vinorelbine inhibited critical functions of the angiogenic process. In particular, itinhibited human umbilical vein endothelial cell (HUVEC) migration and tube formation withoutsimultaneous induction of cell death as opposed to the conventional concentration of 1 κΜ which wastoxic. Moreover metronomic vinorelbine inhibited HUVEC proliferation and angiogenic sprouting.We next investigated the mechanism of this inhibitory effect. Apart from the direct anti-tubular activitythat was observed we also examined Notch signaling. Notch pathway is a critical coordinator of tip/stalkcell specification and is known to suppress angiogenic sprouting by downregulating VEGFR2. Wefound that 10nM vinorelbine increased the mRNA of the Notch target gene Hey1 without concomitantincrease of the Notch Intracellular Domain (NICD). NFθB and JNK have been shown to cross talk withNotch and upregulate its target genes. We found that the metronomic concentration of vinorelbineinduced the translocation of the NFθB subunits in the nucleus and JNK phosphorylation. However theIKK inhibitor iii and JNK inhibitor ii did not block the effect on Hey1 mRNA suggesting that is notattributable to NFθB and JNK pathway activation. Moreover the upregulation of Hey1 mRNA wasunexpectedly followed by a small increase of VEGFR2 mRNA without altering VEGFR2 proteinexpression and phosphorylation.Clinical studies have shown that anti-angiogenic therapies have an ephemeral effect since tumorsbecome eventually refractory to therapy. Treatment-induced hypoxia emerges as a major mechanismof resistance. We investigated whether severe hypoxia mediates resistance to metronomic vinorelbinetreatment. We showed that severe hypoxia did not alter the ability of metronomic vinorelbine to inhibitendothelial cell migration, tube formation and sprouting. However severe hypoxia mediated resistanceto its anti-proliferative action.To investigate this hypoxic resistance we examined cell cycle and apoptosis. Severe hypoxiaattenuated the ability of metronomic vinorelbine to induce G2/M arrest as it shifted the cell population tothe G1 phase and decreased the fraction of the cells in the DNA synthesis S phase throughupregulation of the cyclin-dependent kinase (cdk) inhibitor p27kip. Furthermore severe hypoxialessened the pro-apoptotic action of metronomic vinorelbine. We examined the intrinsic mitochondrialapoptotic pathway as it is implicated in the cell death caused by vinca alkaloids. We found thatmetronomic vinorelbine decreased the Bcl-2/Bax ratio in normoxia. Surprisingly the Bcl-2/Bax ratio wasalso reduced in severe hypoxia without simultaneous induction of the cell death and metronomicvinorelbine did not further reduce the ratio. Metronomic vinorelbine failed to regulate the Bcl-2/Bax ratioin severe hypoxia and this may account for its decreased pro-apoptotic activity. We also questioned themechanism of survival of endothelial cells in severe hypoxia despite the low Bcl-2/Bax ratio.Mitochondrial reactive oxygen species are required for cardiolipin oxidation which results in a looseattachment of cytochrome C. The latter renders cytochrome C able to pass through bax pore. Weshowed that severe hypoxia decreased mitochondrial superoxide production. Low superoxide mayexplain decreased cytochrome C release even with low Bcl-2/Bax ratio.Finally we sought to find ways to overcome this hypoxic resistance. At first we examined the impact oftwo major pathways of sprouting angiogenesis. We investigated the combination of metronomicvinorelbine with sunitinib which is a VEGFR2 inhibitor and DBZ which is γ-secretase inhibitor thatsuppresses Notch signaling. Sunitinib inhibited proliferation to the same extent in normoxia and severehypoxia and to the same extent as metronomic vinorelbine in normoxia. However the combination hadno additive effect. DBZ did not alter the sensitivity of endothelial cells to the anti-proliferative action ofmetronomic vinorelbine. We next investigated the role of HIF-1α the master regulator of hypoxia.Suppression of HIF-1α protein levels by RNA interference did not alter the sensitivity of HUVECs to theanti-proliferative action of metronomic vinorelbine. Finally, we inhibited Akt and ERK which play criticalrole in cell survival, apoptosis and angiogenesis. The MEK inhibitor U0126 and the Akt inhibitor Vincreased the anti-proliferative effect of metronomic vinorelbine in normoxia and severe hypoxia andreversed the hypoxic resistance.In conclusion, we provide evidence for the anti-angiogenic basis of metronomic vinorelbine and itsmechanism of action. We show that severe hypoxia confers resistance to its anti-proliferative activity___________________________________________________