SRC-3 Ubiquination, Mediated By Cullin3, Potentiates Gambogic Acid-Induced Apoptosis in Non-Hodgkin's Lymphoma

Steroid receptor coactivator-3 (SRC-3) mediates the transcriptional functions of nuclear receptors and other transcription factors, regulating the cell proliferation, survival and growth. SRC-3 amplification or overexpression, detected in many hormone-dependent or -independent tumors, has been demonstrated its important role in promoting carcinogenesis. It was suggested that development of an effective SRC-3 inhibitor could offer a promising therapeutic strategy.

Gambogic acid (GA), the major active ingredient in gamboges, is secreted from Garcinia hanburyi. In recent decades, accumulating evidence has been discovered to elucidate its potent anti-tumor efficacies. In our study, we investigated the effects and molecular mechanisms of GA on non-Hodgkin's lymphoma (NHL) cell lines.

GA was demonstrated to induce cell growth inhibition, G1 phase cell cycle arrest and apoptosis in NHL. The IC₅₀ for Daudi or Raji at 24 h was 0.15±0.01 μΜ or 0.79±0.23 μΜ, respectively. In control, the IC₅₀ for Jurkat or normal human lymphocytes was 1.324±0.02 μΜ or 2.54±0.21 μΜ, respectively. After incubation with 0.2 μΜ GA for 12 h, the rate of Daudi cells in G1 phase increased from 33.8±1.83% to 64.4±4.4%, with rate of S phase from 53.0±0.14% to 17.99±3.87%. For Raji cells (0.6 μΜ for 12 h), the rate of cells in G1 phase increased from 44.34±1.55% to 57.07±0.5%, with rate of S phase from 44.75±2.2% to 38.16±0.87%. The G1 phase cell cycle arrest was accompanied by the down-regulation of cyclin D3 and p-Rb and up-regulation of P21 and P27. GA (0.2 μΜ for 24 h) greatly induced apoptosis in Daudi cells (from 6.2±2.8% to 46.8±3.2%), but almost none in Raji cells (from 9.5±0.5% to 9±0.72%). When treated with 0.4 μΜ or 0.6 μΜ GA, the rate of apoptosis in Raji cells increased to 40.5±5.5% or 76.5±4.5%. The apoptosis was further confirmed by evaluating the activation of caspase3 with western blot assay. After GA treatment, cyto C and Apaf-1 increased, and Bcl-2 decreased, all indicating GA induced apoptosis in mitochondrial pathway.

The effects of GA on the regulations of five oncoproteins (Bcl-6, c-Myc, NF-κB, SRC-3 and Bcl-2) were measured by western blot analysis. These oncoproteins have been considered to be strongly implicated in B-cell lymphomagenesis. SRC-3, Bcl-2 and c-Myc were down-regulated. NF-κB was down-regulated directly or inhibited via up-regulation of IκB-α. The effects of GA on Bcl-6 were contradictory in Daudi and Raji.

Because of the pivotal role of SRC-3 in promoting carcinogenesis, we explored whether the above effects of GA were mainly mediated by the regulation of SRC-3. Western blot analysis showed an obvious down-regulation of SRC-3 while the mRNA levels measured by RT-PCR analysis had no marked decline. SRC-3 protein stability examined by cycloheximide assay showed an accelerated degradation of SRC-3. Since SRC-3 has been reported to be degraded in proteasome pathway, we doubted whether SRC-3 was degraded similarly in response to GA. Cullin3, the component of the ubiquitin E3 ligase for SRC-3, was demonstrated to be up-regulated. The proteasome inhibitor MG132 could weaken GA-induced SRC-3 degradation. SRC-3 was shown to be ubiquitinated with immunoprecipitation method, and the ubiquitinated SRC-3 accumulated in the presence of MG132. All the data suggested that GA induced down-regulation of SRC-3 mainly through ubiquitin-dependent proteasome degradation pathway mediated by cullin3.

According to GA-induced apoptosis in five cell lines (Daudi, Raji, Pfeiffer, SU-DHL6 and Jurkat), cells with higher SRC-3 level were observed the higher sensitivity. To figure out the relationship of SRC-3 level and cell sensitivity, stable cell clones expressing lower SRC-3 by lentiviral shRNA transfection was established. We compared the antitumor efficacies of GA in SRC-3 siRNA clones with control clones. After treated with 0.6 μΜ GA for 24 h, the rate of apoptosis in SRC-3 siRNA clones or control clones increased from 14.6±3.3% to 44.3±3.7% or from 16.6±2.24% to 63.6±1.99%, respectively (44.3±3.7% vs. 63.6±1.99%, P<0.05). The result showed SRC-3 knockdown was accompanied with reduction of sensitivity, further confirming SRC-3 as a critical target of GA.

Taken together, our results demonstrated the potent antitumor efficacies of GA on NHL and revealed that SRC-3 ubiquitination, mediated by Cullin3, played a critical role in this process, providing the molecular basis for the clinical trial in lymphoma.