To investigate the effects of cocaine and alcohol on tubulin dynamics, we performed a MT-based assay (Cytoskeleton, Inc., Denver, CO, USA)[25-27] in SH-SY5Y neuroblastoma cells treated with 100 mM EtOH[10] and 1 mM cocaine[8] (n=6/group) for 3 days. This commercially available kit separates large complexes of polymerized MTs attached to nuclei and Golgi bodies into bound and non-polymerized free tubulins. After differential centrifugation, the high-speed pellet supernatant and low-speed pellet samples were isolated and used for western blot analysis. An enhanced Chemiluminescence kit was used to visualize the tubulin bands,[12,11] (Fig. 1a and 2a). Bound/stabilized MT content was significantly higher and unbound/free α/β MTs lower in EtOH-treated cells than control cells treated with PBS. Cells treated with cocaine showed no significant difference in bound MTs and slightly fewer free α/β MTs than untreated cells possibly due to the accrued rate of MT polymerization with substances. Therefore, both EtOH and cocaine compromise MT integrity i.e., increase in bound and decrease in free tubulin units. Having demonstrated these drug-induced changes in MT integrity in SH-SY5Y cells, we next aimed to determine whether [11C]MPC-6827 could also detect similar MT alterations using the same cells.
We performed cell binding assays in vitro in SH-SY5Y cells with [11C]MPC-6827 following our previously published protocols.[28-30] The cells were treated with 100 mM EtOH or 1 mM cocaine[8] (n = 6/group) for 3 days. We then measured radiotracer binding by adding [11C]MPC-6827 (1-2 µCi/well) and incubating the cells for 5, 30, 60, and 90 min at room temperature (n = 6/time point). To demonstrate tracer specificity, a subgroup of cells (n = 3) was pre-treated with non-radioactive MPC-6827 (1.0 µM), adding radiotracer 60 min later and incubating for 30 min. To demonstrate tracer sensitivity to length of drug exposure, cells were treated with 100 mM EtOH or 1 mM cocaine for 1 h, 1 day, or 3 days and incubated with [11C]MPC-6827 for 30 min at room temperature. All the cells were then washed with PBS and lysed with 1N NaOH. Finally, the lysate from each well was g-counted (PerkinElmer, Waltham, MA, USA) and counts-per-minute (cpm) values were normalized to the amount of radioactivity added to each well. Cpm values were then matched with the protein concentration per well, and the data expressed as %ID/mg of protein present in each well.
EtOH- (Fig 1b) and cocaine-treated (Fig 2b) cells demonstrated an ~30(±2) and ~24(±6) percent decrease respectively in radioactive uptake versus non-treated controls over the 30-90 min incubation times. Additionally, uptake in EtOH-treated and cocaine-treated cells increased ~13(±3) and ~12(±2) percent from 5 to 30 min of incubation times respectively and decreased ~53(±2) and ~19(±3) percent by 90 min in EtOH- and cocaine-treated cells; thus demonstrating favorable pharmacokinetics. For the self-blocking assays (Fig 1b), uptake was ~78(±1) percent lower after addition of nonradioactive MPC-6827, demonstrating high specificity. Radioactive uptake was decreased ~21(±1) and 28(±1) percent from 1 h to 3 days EtOH and cocaine exposures (Fig 3) respectively. Therefore, [11C]MPC-6827 uptake decreased selectively with increased exposure to EtOH or cocaine. Moreover, since no significant decrease in radioactivity was observed after 3 days of drug exposure we used the same 3 days exposure in all our assays. MPC-6827 primarily targets the β tubulin site at pharmacological doses.[31-33] The lowered radioactive uptake in EtOH- and cocaine-treated SH-SY5Y cells indicates that [11C]MPC-6827 uptake correlate well with observed bound/free tubulin changes and may be tracking free β tubulin units, as both substance treatments decreased free tubulin content in the same cells.
MTAs are categorized as either stabilizing agents (paclitaxel, laulimalide, and EpoD),[34-36] which favor polymerization of tubulin units and inhibit cell proliferation, or destabilizing agents (vinblastine and mertasine),[37-40] which increase free/unbound tubulins and promote apoptotic cell death. To distinguish their effect on MT integrity in SH-SY5Y cells, we performed the same tubulin-based western blot assays on paclitaxel- and vinblastine-treated cells.[41] The paclitaxel-treated cells had more bound/stabilized MTs, and vinblastine-treated cells had more unbound/free MTs than the untreated cells (Fig 4a). To confirm the free tubulin-based binding mechanism of [11C]MPC-6827, SH-SY5Y cells were pretreated with different MTAs at 1.0 µM concentration, 3.0 h prior to addiction of [11C]MPC-6827. Paclitaxel, laulimalide and EpoD decreased radioactive uptake by ~58(±3), ~40(±4), and ~66(±7) percent respectively, while vinblastine, and mertasine increased it by ~77(±6), and 64(±5) percent respectively (Fig 4b), confirming that [11C]MPC-6827 primarily targets primarily free tubulin units.