Tashinone IIA Can Trigger the Apoptosis of a Megakaryocytic Cell Line CHRF- 288 through the TNFRSF9 Mediated Caspase-3 Activation Pathway

Tanshinone IIA (TIIA), a purified compound and an active component of Salvia miltiorrhiza (DanShen) has previously been shown to induce apoptosis on several human cancer cell lines. In the current study, we studied the cytotoxic effect of TIIA on a megakaryocytic leukemic cell line CHRF-288. First, we tested the cytotoxic effect of TIIA on CHRF-288 cells by Trypan blue assay and the cell viability was reduced to 80.8% after 72hrs of TIIA treatment (30ug/ml). Using Annexin V/PI staining and flow cytometry, we confirmed that TIIA induced apoptosis on CHRF-288 cells in a dose dependent manner. At the concentrations of 1, 3, 10 and 30ug/ml, the early apoptosis cells proportion showed a stepwise increment of 11.8±1.2%, 13.2±2.8%, 16.3±0.8% and 22.4±1.5% at 72 hrs respectively. This was verified by Caspase 3 assay which showed the activation of Caspase 3 increased from 5.1% to 16.2% after 10ug/ml of TIIA treatment. Activation of Caspase 3 implied the involvement of common apoptotic pathway. Then, we studied the mechanisms of apoptosis. Using JC-1 assay, we found that the depolarized cells increased from 9.06% to 16.6% after 10ug/ml TIIA treatment suggestive the involvement of intrinsic apoptotic pathway. To further determine the molecular mechanisms involved in the pro-apoptosis effect, Microarray studies using Affymetrix 133 plus genechips were conducted to identify the genes that were differentially expressed after TIIA treatment. Several groups of genes involved in apoptosis, calcium regulation and cell cycle checkpoints were found to be differentially expressed after the treatment. The differential expressions of these genes were validated using quantitative PCR. The most significantly upregulated genes (5.9±0.333 folds) was TNF Receptor Super-Family 9 (TNFRSF9). In addition, Receptor Interacting Protein Kinase (RIPK), a protein that likely interacts with TNFRSF9, was upregulated to 1.7±0.167 folds. And lastly, Jun, a transcriptional factor of TNFRSF9 was up-regulated to 1.7±0.03 folds. Since TNF and RIPK1 both involve in caspase-8 activation, this was suggestive of the activation of external apoptotic pathway. Our findings suggested that TIIA could induce significant apoptosis on mekakaryoctic leukemic cells via both intrinsic and extrinsic apoptotic pathways. Its role in treating this poor prognostic leukemia deserves further exploration.