Potent Antitumor Activity Of Nampt Inhibitor FK866 On Astrocytoma

Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme to convert nicotinamide to nicotinamide mononucleotide (NMN), which can be further converted to nicotinamide adenine dinucleotide (NAD) in the salvage pathway of NAD+ biosynthesis. By synthesizing NAD, Nampt participates in various important physiological and pathophysiological processes. Recently, Nampt has been found to be functionally important in cancers and is becoming a novel therapeutic target for some cancers. FK866 (APO866) is a specific Nampt inhibitor that was previously shown a novel potent anticancer agent. A phase II clinical trial of FK866 in patients with advanced solid tumors was started. Astrocytomas are the most common and lethal intracranial tumors. These cancers exhibit a relentless malignant progression and resistance to traditional chemotherapeutic approaches. It was reported that Nampt is not expressed in normal astrocyte, but it was induced in astrocytomas cells. Meanwhile, the expression level of Nampt is positively correlated with the malignance of astrocytomas. Thus, our current study was set to study whether Nampt is a novel therapeutic target for astrocytomas and to see whether FK866 is a novel anti-astrocytomas agent. We used rat glioma C6 cells and observed the effects of FK866 on C6 cell viability, apoptosis, autophagy, cell cycle and proliferation. We also investigated the mechanisms under the effects of FK866. The results include:(1) By using MTT assay, we found FK866 time-and concentration-dependently inhibited C6 cell viability. Such inhibition was reversed by 0.01-1 mM NMN. It took FK866 48-72 h to inhibit C6 cell viability and the cell viability at 72 h after FK866 adiministration was 86.3%. The IC50 of FK866 was around 10 nM (-8.07 logM), which is much lower than the IC50 of CDDP (-5.83 logM) and TMZ (-2.82 logM), two clinically using chemotherapeutic agent on astrocytomas. FK866 also dose-dependently inhibited the viability of SHG44, U251 and BT325, three human astrocytomas cell lines. The inhibitory efficiency on these cell lines was similar as that on C6 cell. By using Western blot, we found that all these four cell line expresses Nampt. In addition, FK866 decreased the cell viability of primary cultured rat astrocyte, which also expresses Nampt. However, by using immunostaining, we found there was no Nampt expression in astrocyte in mouse brain.(2) By using flow cytometry, we found that FK866 at 100 nM induced a few apoptosis, while FK866 at 10 and 100 nM induced LC3 accumulation representing autophagy. However, autophagy inhibitors, LY294002 and 3-methyladenine showed no effect on FK866-induced cell viability decline. These implied that FK866-induced autophagy may not be a factor responsible for the FK866-induced strong reduction of cell viability.(3) By using MTT assay and cellular counting, FK866 for 48 h dose-dependently inhibited C6 cell proliferation. The IC50 was around 40 nM. This inhibition was also reversed by 1 mM NMN. Moreover, by using flowcytometry, we found that FK866 40 nM for 48 h increased the amount of the cells in G2 phase from 8.78%to 21.25% (P<0.001), while decreased the amount of the cells in G1 phase from 61.93%to 48.78% (P<0.001) and did not affect that in S phase. These imply that FK866 induced G2/M arrest on C6 cell. FK866-induced G2/M arrest was also reversed by 1 mM NMN.(4) By using Western blot, we found that ERK1/2 was activated during growth and was inhibited by FK866 40 nM for 48 h. FK866 showed no effect on p38 and JNK. ERK1/2 inhibitor, U0126, dose-dependently inhibited the cell viability of C6. The combination of FK866 and U0126 did not show further inhibition, which suggested that FK866 and U0126 may share the same inhibitory pathway on cell viability. In addition, U0126 at 20μM did not affect the cell cycle, while U0126 at 40μM increased the amount of the cells in G1 phase and decreased the cells in S phase. PFT, a p53 inhibitor, also did not affect FK866-induced G2/M arrest.In conclusion, our results showed that FK866 inhibits the enzamatic role of Nampt →decreases NAD synthesis→inhibits ERK activation→inhibits cell proliferation→inhibits cell viability. Meanwhile, FK866 can induce C6 cell autophagy at relative low and high concentration and induce apoptosis at high concentration. FK866 also induces p53-and ERK-independent G2/M arrest. It is the first time that we show the regulation of FK866 on cell proliferation and cell cycle. These results imply that Nampt may be a novel therapeutic target for astrocytomas and FK866 may be a novel anti-astrocytomas agent. However, the effects and the action mechanisms of FK866 on astrocytomas might be different than on other tumors.