The Roles Of Cofilin In The Regulation Of Apoptosis And Mitophagy In Cancer Cells

BackgroundCancer is a class of serious diseases which are harmful to human health. The incidence and death rate of cancer have increased year by year. Therefore, it is important and urgent to look for novel therapeutic targets for cancer treatment. In recent years, development of novel agents targeting cancers has attracted a great deal of attentions worldwide.Cofilin is a member of ADF(actin depolymerizing factor)/Cofilin family, which plays a key role in cytoskeletal dynamics, mitosis, cell adhesion and cell pseudopod formation. Cofilin also plays an important role in tumor cell invasion and metastasis, proliferation and apoptosis. The expression and activation status of cofilin in tumor tissues is associated with the tumor malignancy. Therefore, cofilin is expected to become a new target for prediction of tumor metastasis and prognosis. Development of novel chemotherapeutic agents targeting cofilin are expected to provide a new strategy for treatment of cancer.In recent years, great advances have been made in study on the structure and biological effects of cofilin. However, the molecular mechanisms of cofilin in the regulation of apoptosis in tumor cells remains unclear. Our recent studies indicate that the natural compounds allyl isothiocyanate(AITC) and 4-methylthiobutyl isothiocyanate(Erucin), the active components found in consumable cruciferous vegetables, induce mitochondrial damge and apoptosis in dose- and time-dependent manners in human leukemia and breast cancer cells respectively. These events were accompanied by translocation of cofilin from the cytosol to mitochondria. We found that erucin-induced apoptosis could be related to mitochondrial fission. It has been reported that mitochondrial fission is closely related to mitophagy. Mitochondrial fission appears to be required for mitophagy, and actin plays an important role in autophagy process. Cofilin is best known as a regulator of actin filament nonequilibrium assembly and disassembly. Because of the possible role of cofilin in the regulation of mitochondrial fission, cofilin may be involved in modulating mitophagy. In the present study, we investigated the molecular mechanisms by which cofilin modulates mitochondrial fission, mitophagy, as well as mitochondrial injury and apoptosis through its mitochondrial translocation by using a variety of agents which induce mitochondrial translocation of cofilin, and elucidated the molecular mechanisms of the anti-tumor activities mediated by these agents.Methods and Results1. Study on molecular mechanism by whcih cofilin mitochondrial translocation regulates mitochondrial injury and apoptosis1.1 AITC induces mitochondrial injury and apoptosis in human leukemia cells via dephosphorylation of cofilin in vitro and in vivo.Western blot and flow cytometry showed that AITC triggers loss of mitochondrial membrane potential and apoptosis in human transformed and primary leukemia cells in dose- and time-dependent manners. Exposure of leukemia cells to AITC resulted in cleavage/activation of caspase-9 and caspase-3, and degradation of PARP. These events were also accompanied by release of cytochrome c into the cytosolic fraction and dephosphorylation of cofilin. In vivo study also showed that AITC inhibits tumor growth of U937 xenograft mouse model through induction of apoptosis and cofilin dephosphorylation.1.2 AITC induces actin depolymerization and mitochondrial translocation of G-actin and cofilinExposure of cells to AITC resulted in decrease in levels of the filamentous actin(F-actin) and increase in levels of the globular actin(G-actin). Treating cells with AITC also resulted in translocation of G-actin and cofilin from the cytosol to the mitochondria. Confocal microscopy showed that only colocalization of cofilin with G-actin, but not with F-actin, was observed in the mitochondria in response to AITC treatment. These findings indicate that AITC induces translocation of cofilin and G-actin to mitochondria, leading to interaction of cofilin and G-actin in the mitochondria, culminating in mitochondrial injury and apoptosis.1.3 ROCK1/PTEN/PI3K/PPs signaling pathway is involved in cofilin-mediated mitochondrial injury and apoptosis.We used PP1/PP2 A inhibitor(calyculin), PI3 K inhibitor(LY294002), ROCK1 inhibitor(Y-27632) to evaluate the role of ROCK1/PTEN/PI3K/PPs signaling pathway in the regulation of cofilin dephosphorylation and mitochondrial translocation.2. Study on the role of cofilin in the regulation of mitochondrial fission2.1 Erucin induces mitochondrial injury and apoptosis in human breast cells through mitochondrial fissionExposure of human breast cells(MDA-MB-231, MCF-7) to erucin resulted in loss of mitochondrial membrane potential and apoptosis in dose- and time-dependent manners. These events were also accompanied by release of cytochrome c into the cytosolic fraction. Confocal and electron microscopy also revealed that increased mitochondrial fission was observed in cells treated with erucin.2.2 Mitochondrial translocation of cofilin and Drp1 is required for erucin-mediated mitochondrial fission and apoptosisTreatment of MDA-MB-231 cells with erucin significantly increased mitochondrial translocation of cofilin and Drp1. By using proteinase K, which digest the outer mitochondrial membrane, we found that both cofilin and Drp1 are localized at the outer mitochondrial membrane after their translocate from the cytosol to mitochondria. Immunoprecipitation and immunofluorescence studies revealed the interaction and colocalization of cofilin and Drp1 at the outer mitochondrial membrane. Knockdown of cofilin or Drp1 markedly reduced the interaction between cofilin and Drp1 and attenuated the mitochondrial fission and apoptosis induced by erucin.2.3 ROCK1 is involved in erucin-induced dephosphorylation and mitochondrial translocation of cofilin and Drp1By generating cofilin and Drp1 mutants plasmid to mimick either the dephosphorylated or phosphorylated forms, we found that only dephosphorylation of cofilin(Ser 3) and Drp1(Ser 637) translocate from the cytosol to the mitochondria. Both ROCK1 inhibitor Y-27632 and knockdown of ROCK1 with si RNA blocked erucin-mediated dephosphorylation and mitochondrial translocation of cofilin and Drp1, mitochondrial fission, and apoptosis.2.4 Erucin inhibits tumor growth of MDA-MB-231 xenograft mouse model through mitochondrial translocation of cofilin and Drp1, mitochondrial fission, and apoptosis.3. Study on the role of cofilin in the regulation of mitophagy3.1 Mitochondrial translocation of cofilin and mitochondrial fission are required for PINK1/Park2-mediated mitophagy.MDA-MB-231 cells were treated with a variety of positive agents which induce mitochondrial fission, mitochondrial translocation of cofilin, mitochondrial fission and mitophagy were observed by using confocal and electron microscopy. Western blot also showed that mitochondrial translocation of cofilin and activation of PINK1/Park2 signaling pathway were observed in cells treated with mitochondrial fission inducers.3.2 Mitochondrial translocation of cofilin is involved in the regulation of mitophagy.Knockdown of cofilin significantly blocked mitochondrial fission, activation of PINK1/Park2 signaling pathway, and mitophagy mediated by mitochondrial fission inducers. Overexpression of cofilin significantly enhanced these agents-mediated mitochondrial fission and mitophagy.3.3 Mitophagy mediated by cofilin is dependent on mitochondrial membrane potentialKnockdown of cofilin significantly blocked the loss of mitochondrial membrane potential and the downregulation of PINK1 cleaving proteases(MPPβ/PARL/AFG3L2) induced by positive mitochondrial fission inducers(STS、ETO、CCCP), resulting in the degradation of PINK1. In contrast, overexpression of cofilin decreased mitochondrial membrane potential and the expression of MPPβ/PARL/AFG3L2, leading to blockage of PINK1 degradation. These findings indicate that cofilin mediated-PINK1 activation and mitophagy mainly depend on its regulation of mitochondrial membrane potential activity.3.4 Cofilin play an essential role in mitochondrial fission and mitophagy through regulation of the actin dynamics at the mitochondrial fission site.A variety of positive mitochondrial fission inducers can induce mitochondrial translocation of cofilin and actin, leading to interact between cofilin and actin at the outer mitochondrial membrane. Both the actin polymerization inhibitor(Latrunculin B) and the depolymerization inhibitor(Jasplakinolide) blocked these agents-induced mitochondrial fission. Confocal microscopy showed that cofilin mediated-F-actin/G-actin conversion at the mitochondrial fission site plays an important role in the regulation of mitochondrial fission and mitophagy.ConlusionsIn the present study, we evaluated the role of cofilin in the regulation of mitophagy and apoptosis. The main results are as follows:1. Mitochondrial translocation of cofilin and G-actin is a early step which is involved in AITC-induced mitochondrial injury, cytochrome c release, and apoptosis in leukemia cells. ROCK1/PTEN/PI3K/PPs signaling pathway plays a key role in this process.2. Erucin induces mitochondrial translocation of cofilin and Drp1 in breast cancer cells. Drp1 is best known as a regulator of mitochondrial fission. Cofilin-mediated mitochondrial injury induced by erucin mainly dependent on the interaction of cofilin and Drp1 at the mitochondrial fission site, which result in mitochondrial fission, cytochrome c release, and apoptosis.3. We investigated the role of cofilin in the regulation of mitophagy by using a variety of positive mitochondrial fission inducers(STS、ETO、CCCP). Our results revealed that cofilin-mediated mitochondrial fission through the regulation of actin dynamics at the mitochondrial fission site, thereby affecting the mitochondrial membrane potential and PINK1/Park2 dependent mitophagy.