Title

 

Authors

P. Mosillo¹, P. Spinsanti¹, F. Serone¹ , F. Azzollini¹, L. Cinque¹, B. Turriziani¹, I. Cappuccio², G. Battaglia ³, L. Ricci-Vitiani⁴, F. Nicoletti^1,3, D. Melchiorri^1,5
1Dept. Physiology & Pharmacology, The University of Rome, “Sapienza”,²I.R.C.C.S. G. Mondino, Pavia,  ³I.R.C.C.S. Neuromed, Pozzilli, ⁴I.S.S. Rome, Italy,  ⁵I.R.C.C.S. San Raffaele Pisana, Rome.

 

Abstract

Therapy of colorectal cancer (CC) is complicated because of its tendency to be chemotherapy resistant. The isolation of CC cells from human bioptical specimens has led to the hypothesis that CC is generated and propagated by a population of tumorigenic stem cells responsible of both tumor spreading and chemoresistance. CC stem cells (CSCs) CD133⁺, were cultured in vitro under non differentiating conditions and exposed to either 5-fluorouracil (5-FU), the mainstay treatment for CC, or irinotecan (IR), a drug with significant clinical activity in advanced CC patients. In addition, cisplatin (CS), a chemotherapic drug with reduced activity towards CC. Exposure of CSCs to different concentrations of  5-FU (0.05, 0.5 and 5 µg/ml)  for 48 h or IR (0.5 or 5 µg/ml) for 72 h induced a dose-related cell death as assessed by MTT assay. No cell death was detected following CS (0.5 or 5 µg/ml) treatment. In the attempt to increase chemosensitivity of CSCs, we focused on metabotropic glutamate receptors (mGluRs), which were recently shown to be expressed by a number of tumors, and to regulate cell proliferation and survival. WB and RT-PCR analysis showed that mGluR2/3 were the only mGluR subtype consistently expressed by cultured CSCs- CD133⁺. We exposed CSCs to a combination of the selective mGluR2/3 agonist, LY379268 (1 µM) the selective mGluR2/3 antagonist, LY341495 (1 µM), and the three cytotoxic drugs. LY341495, which was ineffective by itself, significantly increased cell death when added in combination with either 5-FU and IR, but did not induce cell death when combined with CS. No amplification of cell death was detected when LY379268 was combined with any of the three chemotherapic drugs. However, administration of LY379268 to CSCs treated with LY341495 and 5-FU or IR, reverted LY341495-induced amplification of cell death, indicating the involvement of mGluR2/3 in the increased chemosensitivity elicited by LY341495. Type 2/3-mGluRs are coupled to activation of the phosphatidylinositide-3 kinase (PI-3K) pathway. Co-administration of the selective inhibitor of PI-3K activity, LY290042 (100 nM), with 5-FU lead to an increase in cell death similar to what observed following the co-administration of LY341495 and 5-FU. No effect on cell death was observed when LY290042 was administered by itself. To confirm the involvement of PI-3K pathway in cell death induced by association of 5-FU and LY341495 cells were transfected with either a plasmid encoding wild type AKT or a plasmid encoding a costitutively active form of AKT, and exposed to drug treatment. In cells overexpressing AKT or expressing a constitutively active form of AKT, 5-FU combined with LY341495 failed to reduce cell viability. These results suggest that mGlu2/3 receptor blockade enhances the toxic action of 5-FU trough the inhibition of the PI-3K pathway.Increased expression of dihydrouracil dehydrogenase(DPD) is known to contribute to 5-FU resistance.Exposure of cells to LY341495 + 5-FU greatly reduced DPD expression as compared to treatment with 5-FU, suggesting that amplification of 5-FU-induced cell death by LY341495 is due, at least in part, to a decreased metabolism of 5-FU. To explore the molecular mechanisms underlying the reduced expression of DPD in cells treated with 5-FU and LY341495, cultured CSCs were exposed to LY294002 in the presence or absence of 5-FU for 48 h. LY294002, when combined with 5-FU decresead DPD levels similarly to what observed following coadministration of 5-FU and LY341495, suggesting that the decrease of DPD expression induced by the combination of 5-FU and LY341495 is mediated by the inhibition of the PI-3K pathway. These results were confirmed in cells transfected with AKT. These data show that mGlu2/3 receptors are potential for innovative antineoplastic therapies aiming at  overcoming resistance to chemotoxic drugs.