Development of new combination strategies for fist-line chemotherapy with selective cyclin-dependent kinases (CDK) 4/6 inhibitors in colorectal cancer and glioblastoma

Favorite this paper
How to cite this paper?
Details
  • Presentation type: DR - Doctoral Student
  • Track: Drugs and/or Natural Products Therapy
  • Keywords: Cyclin-Dependent Kinases (CDK) Inhibitors; Colorectal cancer; Glioblastoma; High Content Screening;
  • 1 Universidade Federal do Rio de Janeiro
  • 2 Programa de Imunologia e biologia tumoral, Instituto Nacional do Cancer (INCA), Rio de Janeiro

Please log in to watch the video

Log in
Abstract

INTRODUCTION AND OBJECTIVES: The retinoblastoma (RB) pathway is one of the major regulators of the cell cycle. The RB protein is phosphorylated by cyclin-dependent kinases 4/6 (CDK4/6), releasing the E2F transcription factor and promoting G1-to-S phase transition. RB1, the gene codifying pRB, is classically defined as a tumor suppressor gene. However, it has been shown that hyperphosphorylated pRB has an anti-apoptotic role in cell lines displaying aberrant RB pathway activity. Recently, new CDK4/6 inhibitors (CDK4/6i), Palbociclib and Abemaciclib, which inhibit phosphorylation of pRB and lead to cell cycle arrest at G1, have been approved for advanced breast cancer treatment. Colorectal cancer (CRC) is one of the most common types of cancer in Brazil. Glioblastoma (GBM) is the most common and aggressive primary tumor of the central nervous system. Both CRC and GBM have changes in the RB pathway (amplification of CCNE1, CDK4 and CDK6, such as loss of CDKN2A) that may provide sensitivity to CDK4/6i. Therefore, this work aims to evaluate the efficacy of new CDK4/6i agents as monotherapy or in combination with first-line chemotherapy in CRC and GBM. MATERIAL AND METHODS: CRC (SW480 and HCT116) and GBM (T98G) cell lines were cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum and incubated at 37ºC and 5% CO2. Dose-response curve analysis of commonly used chemotherapy drugs (Temozolomide – TMZ in GBM; SN-38, oxaliplatin and 5-fluorouracil – 5-FU in CRC) was performed to determine low doses (EC20) to be combined with effective CDK4/6i doses in the High-content screening platform using the Live/Dead kit. RESULTS AND CONCLUSION: Western Blotting analysis showed that both CDK4/6i efficiently decrease pRB levels in 24-hour treatment at nanomolar concentrations for CRC lines, but no further decrease was seen after 48 hours. In the SW480 line, 24-hour treatment reduced the percentage of live cells only with the highest doses of oxaliplatin, but no effect was observed with SN-38 and 5-FU. Duration of treatment was increased to 48 hours to allow for effective action under lower drug concentrations. After 24 hours, low doses reduced the percentage of living cells of the HCT116 line. Blotting analysis showed reductions in pRB levels in 24h and 72h for the T98G cell line and 72h treatment with CDK4/6i reduced cell viability compared to control, even at the lowest concentration in this cell line. Moreover, the combination of 500nM abemaciclib and 200µM temozolomide showed a significant decrease in viable cells compared to either drug alone. These data suggest the efficacy of new CDK4/6i agents in GBM cell lines as single therapy and indicate new combination strategies for GBM treatment that could decrease TMZ resistance. We now aim to test drug combinations in the CRC cell lines and validate these results in RB-silenced lineages to propose new combination strategies that might efficiently promote tumor cell death while reducing toxicity levels.

Questions (2 topics)

Share your ideas or questions with the authors!

Did you know that the greatest stimulus in scientific and cultural development is curiosity? Leave your questions or suggestions to the author!

Sign in to interact

Have a question or suggestion? Share your feedback with the authors!

Sorry, this topic is private. Only the topic creator and the authors can view comments and interact with the topic.
Author

Alana Souza

Olá Mariana! Não testamos. Mas essas drogas alvo apenas inibem a proliferação celular, assim, acredito que fariam o mesmo efeito em células saudáveis, porém esse efeito de inibição será muito maior em células que proliferam muito rápido, como as tumorais.

MARIANA STELLING

Obrigada pela resposta, Alana, deixo aqui uma sugestão de experimento em que você marque células sadias com um traçador e células tumorais com outro traçador (algo como live/dead que você possa analisar pelo Operetta, por exemplo), combine elas em cultura e aplique os fármacos em teste. A porcentagem de cada traçador pode te dar uma informação valiosa do quanto esses inibidores de CDK afetam as células tumorais e sadias em condições de co-cultura ;) 

MARIANA STELLING

Obrigada pela resposta, Alana, deixo aqui uma sugestão de experimento em que você marque células sadias com um traçador e células tumorais com outro traçador (algo como live/dead que você possa analisar pelo Operetta, por exemplo), combine elas em cultura e aplique os fármacos em teste. A porcentagem de cada traçador pode te dar uma informação valiosa do quanto esses inibidores de CDK afetam as células tumorais e sadias em condições de co-cultura ;) 

Author

Alana Souza

Obrigada pela sugestão Mariana. Certamente iremos fazer. Acho que é importante ter esse resultado.