PURPOSE: Pyrrolotetrazinones are a new class of azolotetrazinones endowed with a high, remarkable antiproliferative activity in human tumor cultured cells. They hold the deaza skeleton of the antitumor drug temozolomide, although preliminary investigations indicated a different mechanism of action. To understand their mechanism(s) of action along with their target at molecular level, four derivatives were selected on the basis of their activity on a panel of human tumor cell lines and they were investigated in depth in a T leukemia cell line (Jurkat). METHODS AND RESULTS: Flow cytometric analysis of cell cycle after treatment with pyrrolotetrazinones has demonstrated that they were able to induce an arrest of the cell cycle in G2/M phase. This effect was accompanied by apoptosis of the treated cells which is further characterized by exposure of phosphatidylserine on the external surface of the cell membranes. Mitochondria were strongly involved in the apoptotic pathway as demonstrated by the induced mitochondrial depolarization, generation of reactive oxygen species, and activation of caspase-3. Western blot analysis showed that Bcl-2 expression was down regulated whereas the proapototic protein Bax was upregulated in a time dependent manner. Moreover, these compounds induced a clear increase in the mitotic index, and inhibited microtubule assembly in vitro indicating that pyrrolotetrazinones, at variance with temozolomide, involved an efficacious inhibition of tubulin polymerization in their mechanism of action. Interestingly compound 3 at the concentration of 50 mg/kg body weight significantly inhibited in vivo the growth of a syngeneic hepatocellular carcinoma in Balb/c mice. CONCLUSION: These results suggest that pyrrolotetrazinones inhibit microtubule polymerization, induce G2/M arrest of cell cycle and cause apoptosis through the mitochondrial pathway identifying them as novel effective antimitotic agents with potential for clinical development.
Pyrrolotetrazinones deazaanalogues of temozolomide induce apoptosis in Jurkat cell line: involvement of tubulin polymerization inhibition
VIOLA, GIAMPIETRO;BASSO, GIUSEPPE;BRUN, PAOLA;SALVADOR, ALESSIA;DALL'ACQUA, FRANCESCO;
2009
Abstract
PURPOSE: Pyrrolotetrazinones are a new class of azolotetrazinones endowed with a high, remarkable antiproliferative activity in human tumor cultured cells. They hold the deaza skeleton of the antitumor drug temozolomide, although preliminary investigations indicated a different mechanism of action. To understand their mechanism(s) of action along with their target at molecular level, four derivatives were selected on the basis of their activity on a panel of human tumor cell lines and they were investigated in depth in a T leukemia cell line (Jurkat). METHODS AND RESULTS: Flow cytometric analysis of cell cycle after treatment with pyrrolotetrazinones has demonstrated that they were able to induce an arrest of the cell cycle in G2/M phase. This effect was accompanied by apoptosis of the treated cells which is further characterized by exposure of phosphatidylserine on the external surface of the cell membranes. Mitochondria were strongly involved in the apoptotic pathway as demonstrated by the induced mitochondrial depolarization, generation of reactive oxygen species, and activation of caspase-3. Western blot analysis showed that Bcl-2 expression was down regulated whereas the proapototic protein Bax was upregulated in a time dependent manner. Moreover, these compounds induced a clear increase in the mitotic index, and inhibited microtubule assembly in vitro indicating that pyrrolotetrazinones, at variance with temozolomide, involved an efficacious inhibition of tubulin polymerization in their mechanism of action. Interestingly compound 3 at the concentration of 50 mg/kg body weight significantly inhibited in vivo the growth of a syngeneic hepatocellular carcinoma in Balb/c mice. CONCLUSION: These results suggest that pyrrolotetrazinones inhibit microtubule polymerization, induce G2/M arrest of cell cycle and cause apoptosis through the mitochondrial pathway identifying them as novel effective antimitotic agents with potential for clinical development.
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