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Log inINTRODUCTION 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.
Leandro Miranda Alves
Vocês vão testar em algum modelo animal de cancer colon e de glioblastoma?
MARIANA STELLING
Olá Alana, tudo bem? Gostaria de saber se você ou alguém no mesmo projeto já testou o efeito dos inibidores de CDK4/6 sobre células sadias. Caso já tenha testado, qual foi o resultado? Caso não tenha testado, qual seriam os efeitos esperados?
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 ;)
Alana Souza
Obrigada pela sugestão Mariana. Certamente iremos fazer. Acho que é importante ter esse resultado.
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