Proliferation, apoptosis and senescence differential modulation by combining the retinoid ATRA and Protein Kinase C pharmacological inhibitors, in a bicellular murine mammary tumor cell line

DAMIAN E. BERARDI; MARÍA INÉS DÍAZ BESSONE; CAROLINA FLUMIAN; STÉFANO M. CIRIGLIANO; ELISA D. BAL DE KIER JOFFÉ; ALEJANDRO J. URTREGER; LAURA B. TODARO

Washington

Congreso; 104th Annual Meeting of the American Associaton for Cancer Research; 2013

American Associaton for Cancer Research (AACR)

Retinoids exert some of their effects on cell differentiation and malignant phenotype reversion through interactions with specific Protein Kinase C (PKC) isoforms. In this work we studied: A)- the modulation of PKCa and PKCd expression when cells were treated with ATRA (1 uM), B)- The expression profile of Retinoic Acid Receptors (RAR) when cells are treated with ATRA and/or PKCa / PKCd inhibitors (Gö6976 2.5 uM / Rottlerin 1 uM) and C)- the effect of combining ATRA and PKC inhibitors on in vitro cell growth, cell cycle distribution and senescence. We used as model the murine mammary tumor-derived cell line LM38-LP, composed by luminal (LEP) and myoepithelial (MEP) cells. By RT-PCR we could determine that 48 h ATRA treatment increased PKCd only in the LM38-LP LEP population and decreased PKCa expression levels in both cellular components. Regarding RARs profile, the treatment with PKCa inhibitor Gö6976 reduced RARg2 levels, while its combination with ATRA also increased RARb2. Furthermore, 96 h treatment with ATRA, Gö6976, or Rottlerin or their combinations induced cell growth inhibition (cell number x105: control 22±1.1; ATRA 11±0.1; Gö6976 3.6±0.6; Rottlerin 9±0.1; ATRA/Gö6976 2.3±0.03; ATRA/Rottlerin 6.7±1.3, significant vs control in all treatments). By flow cytometry we could determine that both ATRA and Rottlerin treatments induce LM38-LP cell cycle arrest in G1 phase, while Gö6976 increased the number of cells in the subG1 phase, correlating with increased apoptosis and confirmed by Annexin V-FITC staining. Employing b-galactosidase activity assay and bright field microscopy, we determined that 96 h ATRA treatment induced senescence mainly in the MEP component while Gö6976 does it in the LEP population. Interestingly, Rottlerin treated cells showed morphological alterations compatible with autophagy. We conclude that cell growth inhibition exerted by retinoids may be enhanced by the reduction in PKCa expression and / or activity. In addition the combination of ATRA plus Gö6976, induced differential responses on the different LM38-LP cell components, showing senescence in MEP cells, together with cell cycle arrest and apoptosis mainly in the LEP compartment. Consistently, all these biological effects correlated with an increase in RARb2 (known to induce cell differentiation) and a decrease in RARg2 (implicated in proliferation) mRNAs level.