Abstract
Purpose: To evaluate the effect of bryostatin I on proliferation of pancreatic cancer cells as well as tumor growth in mice tumor xenograft model. Methods: Activation of NF-κB was evaluated by preparing nuclear material extract using nuclear extract kit (Carlsbad, CA, USA) followed by enzyme-linked immunosorbent assay (ELISA). Mice were injected with 3 x 10 5 MIApaCa 2 cells in 100 μL volume of PBS. The animals in the treatment group were injected with 50 μg/kg of bryostatin 1 daily for 1 month in the morning whereas those in the untreated group received an equal volume of normal saline. Results: Treatment of the MIApaCa 2 cells with bryostatin I caused a significant reduction in the activity of NF-κB in nucleoplasm (p = 0.0002). The increase in the concentration of bryostatin I from 10 to 50 μM reduced MIApaCa 2 cell proliferation from 87 to 26 %. Bryostatin I treatment also led to increase in the proportion of cells in M1 phase with subsequent reduction in sub-G1 phase of cell cycle. Examination of the cell lysates revealed a higher expression level of cleaved caspase-8 in bryostatin Itreated MIApaCa 2 cells. Mean tumor diameter in the treatment and untreated groups was 5.34 ± 2.16 and 19.45 ± 5.71 mm, respectively, after 2 months of treatment (p < 0.0002). The mean weight of the tumors in the treatment and untreated groups was 123.67 ± 22.56 and 939.14 ± 213.51 mg, respectively, after 2 months of treatment. Conclusion: Bryostatin I inhibits growth and proliferation of pancreatic cancer through inhibition of NF- κB expression, and therefore, needs to be further investigated for therapeutic application in pancreatic cancer. Keywords: Bryostatin I, NF-κB expression, Proliferation, Apoptosis, Pancreatic cancer, Tumor volume
Highlights
Pancreatic cancer is one of the most aggressive and common causes of deaths caused by cancer with a five year survival rate of less than 4 % [1,2]
Treatment of the MIApaCa 2 cells with bryostatin I caused a significant reduction in the activity of NF-κB in nucleoplasm compared to untreated cells (p = 0.0002, Fig. 1A)
The results from MTT assay revealed that bryostatin I exhibited concentration and time dependent inhibitory effects on the proliferation of MIApaCa-2 cells
Summary
Pancreatic cancer is one of the most aggressive and common causes of deaths caused by cancer with a five year survival rate of less than 4 % [1,2]. Activation of the factors involved in apoptosis has a great impact on the inhibition of the cancer cell proliferation and in turn carcinoma treatment [11]. The present study was aimed to investigate the effect of bryostatin I on pancreatic cancer cell proliferation and tumor xenograft mice model. The nuclear extract of the cells was analyzed by enzyme-linked immunosorbent assay (ELISA) kit (TransAMTM NF-κB; Active Motif) For this purpose, 5 μg samples of the nuclear material were incubated with oligonucleotide containing NF-κB p65-binding sequence for a period of 45 min at 30 oC in a microwell. MIApaCa 2 cells after incubation with bryostatin 1 were washed with PBS and treated with lysis buffer (Beyotime). The membranes were washed three times with TBS-T and incubated with a secondary antibody (Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Differences were considered statistically significant at p < 0.05
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