RESUMO
Microtubules are a proven target for anticancer drug development because they are critical for mitotic spindle formation and the separation of chromosomes at mitosis. 2-(Naphthalene-1-yl)-6-pyrrolidinyl-4-quinazolinone (HL66) induced cell death with the large cells and multiple micronuclei in M21 skin cancer cells. We demonstrated that HL66-induced cell death is caspase-independent and accompanied by the failure of cell cycle progression. Therefore, HL66-induced cell death may be a mitotic catastrophe. HL66 inhibits the dephosphorylation on Thr14 or Tyr15 of cyclin-dependent kinase (Cdk) 1 and the formation of Cdk1/cyclin B1 complex, which might be associated with cell cycle arrest at the S and G(2)/M phases. HL66 is an antimicrotubule agent by molecular modeling on the basis of ligand binding to tubulin molecule. Furthermore, we also demonstrated that HL66, like vinblastine, is a tubulin-destabilizing agent via microtubule disruption in M21 cells. These results describe a novel pharmacological property of HL66 as a microtubule inhibitor, which may make it an attractive new agent for the treatment of skin cancer.
Assuntos
Antineoplásicos/farmacologia , Ciclo Celular/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Pirrolidinas/farmacologia , Quinazolinonas/farmacologia , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Anexina A5/metabolismo , Western Blotting , Proteína Quinase CDC2/metabolismo , Caspases/metabolismo , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Corantes , Ciclina B1/metabolismo , DNA de Neoplasias/biossíntese , DNA de Neoplasias/efeitos dos fármacos , Desenho de Fármacos , Fase G2/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Mitose/efeitos dos fármacos , Modelos Moleculares , Fosforilação , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Azul Tripano , Tubulina (Proteína) , Moduladores de Tubulina/farmacologiaRESUMO
Humans and higher primates are unique in that they lack uricase, the enzyme capable of oxidizing uric acid. As a consequence of this enzyme deficiency, humans have high serum uric acid levels. In some people, uric acid levels rise above the solubility limit resulting in crystallization in joints, acute inflammation in response to those crystals causes severe pain; a condition known as gout. Treatment for severe gout includes injection of non-human uricase to reduce serum uric acid levels. Krystexxa® is a hyper-PEGylated pig-baboon chimeric uricase indicated for chronic refractory gout that induces an immunogenic response in 91% of treated patients, including infusion reactions (26%) and anaphylaxis (6.5%). These properties limit its use and effectiveness. An innovative approach has been used to develop a therapeutic uricase with improved properties such as: soluble expression, neutral pH solubility, high E. coli expression level, thermal stability, and excellent activity. More than 200 diverse uricase sequences were aligned to guide protein engineering and reduce putative sequence liabilities. A single uricase lead candidate was identified, which showed low potential for immunogenicity in >200 human donor samples selected to represent diverse HLA haplotypes. Cysteines were engineered into the lead sequence for site specific PEGylation and studies demonstrated >95% PEGylation efficiency. PEGylated uricase retains enzymatic activity in vitro at neutral pH, in human serum and in vivo (rats and canines) and has an extended half-life. In canines, an 85% reduction in serum uric acid levels was observed with a single subcutaneous injection. This PEGylated, non-immunogenic uricase has the potential to provide meaningful benefits to patients with gout.