Investigation of autophagy mechanism in serum-starved BT-474 breast cancer cells


Cılızlar M., Turk M., Tatlı Ö. , Dinler Doğanay G.

7th International Congress of the Molecular Biology Association of Turkey, İstanbul, Türkiye, 27 - 29 Eylül 2019, cilt.43, no.5, ss.37

  • Cilt numarası: 43
  • Basıldığı Şehir: İstanbul
  • Basıldığı Ülke: Türkiye
  • Sayfa Sayısı: ss.37

Özet

Background/aim: Autophagy is an intracellular self-degradation mechanism responsible for maintaining cellular homeostasis. Beclin 1 and Bcl-2 are the main players in the regulation of autophagy/apoptosis decision. Bcl-2 associated athanogene 1 (BAG-1), a well-known binding partner of Bcl-2, is a pro-survival molecule involved in chaperone-assisted proteasomal degradation to tune proteostasis. Hence, BAG-1 might have a role in the autophagy-apoptosis toggle switch, in concert with Beclin 1 and Bcl-2. Here, we studied the starvation-induced autophagic response in BT-474 human breast ductal carcinoma cells to understand how BAG-1 acts in autophagy/apoptosis decision.

Materials and methods: BT-474 cells were cultured under serum starvation (0.1% instead of 10%) for 3 to 24 hours to induce autophagy. Total protein was extracted from serum-starved cells. The levels of autophagy marker proteins including LC3, ATG5-12 complex, Beclin 1, p-Beclin 1 (T119), Bcl-2, p-Bcl-2(S70) were analyzed by western blotting to monitor autophagic flux within the given time period. Further, BAG-1 levels were also assessed under serum starvation-induced autophagy. Statistical analyses were performed by GraphPad Prism version 8.0.1.

Results: Autophagy markers were significantly upregulated in 3 to 6 hours of starvation in BT-474 cells. Serum starvation of the cells resulted in increased levels of p-Beclin 1 (T119), and conversely, decreased levels of p-Bcl-2 (S70) within this time period. BAG-1 levels, especially BAG-1S isoform, were started to increase after 6 hours of treatment. 

Conclusion: We conclude that autophagic response is induced between 3 to 5 hours of serum starvation in BT-474 cells. Disruption of Bcl-2/Beclin 1 interaction likely occurs through phosphorylation of Beclin 1. The increase in BAG-1 levels at the end of autophagic flux suggested that BAG-1, in particular BAG-1S isoform, may have a role in tipping the balance toward proteasomal activation.