K. Majumder1, B. Giri1, N. Arora1, S. Modi1, V. Dudeja1, A. Saluja1, S. Banerjee1 1University Of Minnesota,Surgery,Minneapolis, MN, USA
Introduction: Gallbladder cancer (GBC) is the 5th most common gastrointestinal cancer in the US. Despite a dismal 5 year survival of advanced GBC (Stage III= 8%), therapeutic regimes for the same are ill-defined. Current treatment strategies for unresectable disease offer a survival benefit of less than a year. Triptolide, a derivative of a Chinese herb and its water soluble pro-drug Minnelide, which was developed by our laboratory, has been previously shown to be effective against various solid organ cancers. We have previously show that one of the mechanisms of triptolide/Minnelide’s anti-cancer action is SP1 (Specificity protein 1, a transcriptional factor) inhibition. Minnelide is currently in Phase I clinical trial against advanced gastrointestinal malignancies.
Methods: The effect of triptolide on cell viability of three patient derived GBC cell lines SNU 308, NOZ, and GBD1 was assessed. Cells were treated with triptolide (0-200nM) and protein and mRNA were extracted and cell cycle analysis was performed using flow cytometry. Xenograft mouse models using GBC cell lines (GbD1and NOZ) in athymic nude mice were used to analyze the efficacy of Minnelide against GBC.
Results: In vitro, triptolide treatment (0-200 nM) resulted in a significant dose and time dependent decrease in cell viability in all three cell lines (p<0.05). As opposed to our previous studies in pancreatic cancer cells in which triptolide induced apoptotic cell death, western blotting did not demonstrate apoptosis. Flow cytometry analysis revealed a G1/S phase arrest following treatment with triptolide as compared to control treated cells in all three cell lines.
Triptolide treatment results in a significant dose and time dependent decrease in the protein and mRNA levels of transcription factor SP1 in all three cell lines. As a proof of principle, GBC cell lines were treated with SP1 inhibitor mithramycin (0-200nM) and treatment resulted in a significant dose and time dependent decrease in viability of all three cell lines. Moreover, on flow cytometry analysis, mithramycin treatment led to a G1/S phase arrest.
In vivo, tumor weights ( GbD1: 1.5± 0.3 vs 0.7 ± 0.1 vs 0.6 ± 0.1 p< 0.05) as well as tumor volumes ( GbD1: 1515 ± 329 vs 510 ± 133 vs 419 ± 49, p < 0.05) were significantly smaller with Minnelide treatment as compared to saline treatment. [Saline vs Minnelide 0.21 mg/kg/day vs Minnelide 0.42 mg/kg/day]
Conclusion: Minnelide markedly reduces tumor growth in animal model of gallbladder cancer. Our mechanistic studies demonstrate that triptolide/ Minnelide induces cell cycle arrest via SP1 inhibition. Given that Minnelide is already in Phase I clinical trials against advanced GI cancers, it can emerge as a potential therapeutic option for advanced gallbladder cancer.