Abstract 982: Silibinin reverses drug resistance in human small-call lung carcinoma cells.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Small-cell lung carcinoma (SCLC) has a dismal prognosis in part because of multidrug resistance (MDR). Silibinin is a flavonolignan extracted from milk thistle (Silybum marianum), extracts of which are used in traditional medicine. Recently, silibinin has shown anti-cancer activity in cell and animal models, in particular prostate cancer where a clinical trial is underway. Here, we report the effects of silibinin on SCLC, particularly cells showing MDR. SCLC cells studied were NCI-H69 and a derived cell line VPA17 showing resistance to etoposide (9-fold), doxorubicin (15-fold) and vincristine (10-fold). VPA17 cells did not show resistance to silibinin (IC50 = 60μM for H69 and VPA17). Flow cytometry analysis after incubation in 30 μM silibinin showed no changes in cell cycle phases in VPA17 or H69 cells (G1 = 49%, S = 29%, G2/M = 18%) compared with untreated cells (p>0.05). Silibinin (30 μM) incubation was pro-apoptotic in VPA17 cells after >3 days, as measured by ELISA of BUdR labeled DNA fragments. Apoptosis was also indicated by an increase in caspase-3 specific activity (to 16 enzyme units/104 cells vs. 5 units/104 cells for untreated controls (p < 0.05). The anti-apoptotic protein survivin was elevated as measured by ELISA in VPA17 compared to H69 cells (11.8 vs.1.8 pg/μg protein, p < 0.01). Incubation for 96 h in 30μM silibinin significantly reduced survivin in VPA17 cells (3.4 pg/μg protein, p < 0.01 compared with untreated cells) but not in H69 cells. VPA17 cells incubated in 30 μM silibinin for 5 days showed an inhibition of pgp-mediated efflux of calcein acetoxymethyl ester (calcein AM): Compared with drug-sensitive H69 cells, VPA17 drug-resistant cells had significantly reduced calcein AM retention (21 vs. 59 fluorescence units, p<0.01). However, silibinin incubation reversed this drug efflux, with increased retention of calcein AM only in VPA17 cells (88 fluorescence units, p<0.001 compared with untreated cells). Silibinin did not inhibit pgp-mediated efflux directly, since there was no effect on calcein AM efflux after 45 min. incubation of untreated VPA17 cells. Pre-incubation of VPA17 cells in 30 μM silibinin for 5 days reduced the IC50 for etoposide (5.50 uM to 0.65 μM, p < 0.05) and doxorubicin (0.620 μM to 0.035 μM, p < 0.05). Synergism between silibinin and chemotherapy drugs was determined by exposure of VPA17 cells to 1:1 ratios at the appropriate IC50 values of silibinin: etoposide or silibinin: doxorubicin at 0.25 x IC50, 0.5 x IC50, 2 x IC50 and 4 x IC50. After 4 days, the combination index (CI) was determined using CalcuSyn software. Silibinin and etoposide showed synergism (CI = 0.46 at ED50 and CI = 0.95 at ED95); silibinin and doxorubicin also showed synergism (CI = 0.24 at ED50 and CI = 0.96 at ED95). These data indicate that in SCLC, silibinin is pro-apoptotic, reverses MDR and acts synergistically with chemotherapy drugs. Silibinin, a non-toxic natural product may be useful in treatment of drug-resistant SCLC. Citation Format: David Sadava, Susan Kane. Silibinin reverses drug resistance in human small-call lung carcinoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 982. doi:10.1158/1538-7445.AM2013-982