Purpose: Small-cell lung cancer (SCLC) is a rapidly-progressive and highly fatal disease, and new treatments are needed. Increased expression of ß-III tubulin (TUBB3) correlates with decreased response to paclitaxel in multiple cancers. We have discovered that TUBB3 is highly expressed in SCLC pathology samples. CK2 is a serine/threonine kinase with over 300 substrates and is overexpressed in many cancers. CK2 interacts with the microtubule apparatus and may be related to TUBB3-mediated drug resistance in cancer cells. Our hypothesis is that simultaneous targeting of microtubules and CK2 will be an effective strategy in decreasing SCLC proliferation.
Methods: Cell proliferation experiments were conducted as follows. SCLC cell lines H69 and H209 were maintained in appropriate media at 5% CO2 and 37°C. A 96-well microtiter plate was seeded with 100 μL of cell suspension at 1 x 104 cells/well. After 20 hours or 68 hours of incubation with Ixabepilone (LC Laboratories), 10 μL of Cell Counting Kit-8 (Dojindo) reagent was added to each well, followed by incubation for 4 hours for total incubations of 24 hours or 72 hours. Absorbance was read at 450 nm. All experiments were performed in triplicate. Cells were lysed in lysis buffer and cleared by centrifugation at 4°C. Proteins were resolved on 10% or 12% SDS PAGE gels, transferred onto PVDF membranes, and blocked with 5% nonfat dry milk in TBS-T followed by incubation with primary antibody diluted in TBS-T (mouse TUBB3 [MMS-435P, Covance] at a dilution of 1:2,000 or rabbit CK2α [A300-197A, Bethyl Laboratories]) plus Anti-CK2α’ (A300-199A, Bethyl Laboratories) at a dilution of 1:3,000. Blots were washed and incubated with horseradish peroxidase-conjugated secondary antibody diluted in TBS-T (Anti-mouse [Santa Cruz Biotech] at 1:25,000 and Anti-rabbit at 1:1,000). Antibody complexes were visualized, using an enhanced chemiluminescent Western blot detection system (Thermo Fisher Scientific).
Results: Previously, we demonstrated that TUBB3 is highly expressed in about 85% of SCLC cases. In the current study, multiple SCLC cell lines were evaluated for TUBB3 and CK2 expression via immunoblotting. In all cell lines, bands corresponding to the molecular weights of TUBB3 and CK2 were observed at multiple protein lysate concentrations. Ixabepilone is a microtubule-stabilizing analogue of epothilone B that is thought to preferentially bind the TUBB3 isotype. Incubation of ixabepilone with H69 and H209 SCLC cell lines at 25 mM resulted in 8.6% and 5.3% inhibition, respectively, after 24 hours and 14.0% and 20.6% inhibition, respectively, after 72 hours. Incubation of ixabepilone with H69 and H209 SCLC cell lines at 100 mM resulted in 29.2% and 7.0% inhibition, respectively, after 24 hours and 47.5% and 32.2% inhibition, respectively, after 72 hours.
Conclusions: TUBB3 and CK2 were expressed in SCLC cell lines. Ixabepilone has modest activity against SCLC cell lines and will be further evaluated to obtain IC50 values. We will further evaluate the effect of CK2 inhibition in the presence and absence of ixabepilone and paclitaxel. The work described here may contribute to new therapeutic strategies for SCLC.
