Background Anti-mitotic compounds (microtubule de-stabilizers) such as vincristine and vinblastine have been shown clinically successful in treating various cancers. assembly and dynamic instability in buy Salvianolic acid D KB-cells and this predicted model was supported by an increased microtubule assembly and reduced microtubule dynamic instability in KB-cells, as shown by Western blot analysis. Conclusions and Significance Our study exhibited that these novel mutations in exon four of the I-tubulin induced resistance to microtubule de-stabilizers and hyper-sensitivity to microtubule stabilizer through an alteration in the microtubule assembly and mechanics in cancer cells. Importantly, the current study reveals that cancer cells may acquire drug resistance ability to anti-mitotic compounds through multiple changes in the microtubule networks. This study further provided molecular information in drug selection for patients with specific tubulin mutations. Introduction Microtubules are protein filaments of the cytoskeleton composed of -tubulin and -tubulin molecules. In cells, microtubule filaments rapidly alternate between phases of growth and shrinkage (dynamic instability) during cell cycle [1]. Since microtubules play crucial functions in the rules of the mitotic apparatus, disruption of microtubules can induce cell cycle buy Salvianolic acid D arrest in M phase, the formation of abnormal mitotic spindles, and finally triggering of signals for apoptosis. The finding that the cytotoxic activity of various compounds is usually through the interference with the mitotic spindle apparatus has drawn much attention buy Salvianolic acid D within the past two decades, and microtubules have become an attractive pharmacological target for anticancer drug finding. Anti-mitotic compounds such as vincristine, vinblastine (microtubule-destabilizing alkaloid) and paclitaxel (microtubule-stabilizing taxane) have been developed to target cancers clinically [2], [3]. Although the taxanes and alkaloids are effective for the management of different malignancies, their potential is usually limited by the development of multidrug resistance (MDR) [4], [5]. MDR is usually multi-factorial, with buy Salvianolic acid D one pathway leading to resistance mediated by the over-expression of transmembrane efflux pumps, namely, the alkaloid [7]. On the other hand, cells made up of mutations such as P173A, Q292E and Y422C in the class I beta-tubulin have been found resistant to epothilone (microtubule stabilizing agent) [9]. Oddly enough, over-expression of III-tubulin has been shown in paclitaxel-resistant cells [10], [11], [12]. However, combined changes (alternations in in tubulin isotype manifestation and mutations in the -tubulin gene) in the microtubule networks are seldom exhibited in the anti-mitotic drug resistance malignancy cells. In this study, a microtubule de-stabilizer-resistant cancer cell line was used to investigate novel changes present in cells that were able to induce resistance to anti-mitotic compounds. KB-is a KB-derived BPR0L075 (microtubule de-stabilizer)-resistant cancer cell line. Our published study revealed that KB-cells over-expressed survivin, leading to the stabilization of microtubule networks and resulting in resistance to microtubule de-stabilizing compounds [13]. However, down-regulation of survivin only partially restored the drug-sensitivity to microtubule de-stabilizers KEL colchicine and BPR0L075, suggesting that additional drug-resistant mechanism is present in this cell line [13]. Here, we investigated additional mechanisms that may be responsible for drug-resistance to microtubule de-stabilizers in KB-cells. Results KB-cells show drug-resistance to microtubule de-stabilizers and hyper-sensitivity to microtubule stabilizer A KB-derived BPR0L075-resistant cancer cell line, KB-was a monoclonal cell line selected for resistance by continual exposure of the parental cancer cell line, KB, to increasing concentrations of the microtubule de-stabilizing compound, BPR0L075. KB-cells are cultured in the medium with 30 nM of BPR0L075 to maintain its drug resistant characteristic. This specific drug-resistant cancer cell line is 6-fold more resistant to BPR0L075 as compared to its parental cells (Table 1) (Figure 1A). In addition, KB-cells exhibit cross-resistance with another microtubule de-stabilizing agents, colchicine (7-fold resistant) and vincristine (7-fold resistant) (Table 1) (Figure 1B and C). Surprisingly, this drug-resistant cancer cell line is 5-fold more sensitive to the microtubule stabilizing agent paclitaxel as compare to KB cells (Table 1) (Figure 1D). Figure 1 KB-cells show resistant to microtubule de-stabilizers and hyper-sensitive to microtubule stabilizer. Table 1 Characterization of KB and KB-cells. Multiple-drug resistant protein (MDR) is not present in KB-cells Previous study demonstrated that the microtubule de-stabilizing compound BPR0L075 was equally effective in both MDR/MRP negative and positive cancer cells [14], indirectly suggesting that the BPR0L075-resistance KB-cells exhibit drug resistance properties through MDR/MRP-independent mechanism. buy Salvianolic acid D To further support the above findings, RT-PCR was performed to determine whether KB-cells over-expressed MDR-1. RT-PCR analysis revealed that neither KB nor KB-cells express MDR-1 (Figure 2A). In contrast, MDR-1 was expressed in the vincristine-resistant cancer cell line, KB-VIN10 (positive control) (Figure 2A). Furthermore, results from the quantitative real-time PCR revealed that another important drug efflux pump, MPR-1, was not over-expressed in KB-cells as compared to its parental KB cells (Figure 2B). In contrast, MRP-1 was over-expressed.