Baicalein sensitizes triple negative breast cancer MDA-MB-231 cells to doxorubicin via autophagy-mediated down-regulation of CDK1.

Triple negative breast cancer (TNBC) is a kind of refractory cancer with poor response to conventional chemotherapy. Recently, the combination of baicalein and doxorubicin was reported to exert a synergistic antitumor effect on breast cancer. However, the underlying mechanism how baicalein sensitizes breast cancer cells to doxorubicin remains to be elucidated. Here, it was found that 20 µM baicalein increased the autophagy markers including the ratio of LC3B II/I, GFP-LC3 punctate aggregates and down-regulation of p62 expression, and up-regulated mitophagy marker PINK1 and Parkin in TNBC MDA-MB-231 cells as well. In contrast, doxorubicin decreased the levels of autophagy markers, and significantly up-regulated CDK1 in MDA-MB-231 cells. Pretreatment with baicalein markedly inhibited the doxorubicin-induced decrease in autophagy markers and up-regulation of CDK1, which was reversed by the autophagy inhibitor 3-Methyladenine. Moreover, baicalein alleviated the doxorubicin-induced expression and phosphorylation (at Ser616) of mitochondrial fission protein Drp1. Intriguingly, the autophagy inhibitor 3-Methyladenine also significantly weakened the effect of baicalein on doxorubicin-induced viability decrease and apoptosis in MDA-MB-231 cells. Taken together, our data indicate that baicalein improves the chemosensitivity of TNBC cells to doxorubicin through promoting the autophagy-mediated down-regulation of CDK1, also suggest a novel strategy for prevention of TNBC in the future.

Curcumin protects retinal neuronal cells against oxidative stress-induced damage by regulating mitochondrial dynamics.

Oxidative stress plays a crucial role in the damage of retinal neuronal cells. Curcumin, the phytocompound, has anti-inflammatory and antioxidative properties. It was shown that curcumin exerted a beneficial effect on retinal neuronal cell survival. However, the role of mitochondrial dynamics in curcumin-mediated protective effect on retinal neuronal cells remains to be elucidated. Here, H2O2 was used to mimic the oxidative stress in retinal neuronal R28 cells. Drp1 and Mfn2 are key regulators of mitochondrial fission and fusion. 100 µM of H2O2 significantly increased the cleavage of caspase-3 and Drp1 expression, but downregulated the expression of Mfn2. Pretreatment with 5 µM curcumin effectively alleviated H2O2-induced alterations in the expression of Drp1 and Mfn2 and mitochondrial fission in R28 cells. In addition, curcumin and Drp1 knockdown prevented H2O2-induced intracellular ROS increment and mitochondrial membrane potential disruption. On the contrary, knockdown of Mfn2 diminished curcumin-mediated protection against ROS increment and mitochondrial membrane potential disruption after H2O2. Moreover, curcumin protected R28 cells against H2O2-induced PINK1 expression, mitophagy, caspase-3 cleavage and apoptosis. Knockdown of Mfn2 significantly alleviated the protective effect of curcumin on R28 cells after H2O2. Taken together, our data indicate that curcumin protects against oxidative stress-induced injury in retinal neuronal cells by promoting mitochondrial fusion.

Characterization of Pantoea ananatis from rice planthoppers reveals a clade of rice-associated P. ananatis undergoing genome reduction.

Pantoea ananatis is a bacterium that is found in many agronomic crops and agricultural pests. Here, we isolated a P. ananatis strain (Lstr) from the rice planthopper Laodelphax striatellus, a notorious pest that feeds on rice plant sap and transmits rice viruses, in order to examine its genome and biology. P. ananatis Lstr is an insect symbiont that is pathogenic to the host insect and appears to mostly inhabit the gut. Its pathogenicity thus raises the possibility of using the Lstr strain as a biological agent. To this end, we analysed the genome of the Lstr strain and compared it with the genomes of other Pantoea species. Our analysis of these genomes shows that P. ananatis can be divided into two mono-phylogenetic clades (clades one and two). The Lstr strain belongs to clade two and is grouped with P. ananatis strains that were isolated from rice or rice-associated samples. A comparative genomic analysis shows that clade two differs from clade one in many genomic characteristics including genome structures, mobile elements, and categories of coding proteins. The genomes of clade two P. ananatis are significantly smaller, have much fewer coding sequences but more pseudogenes than those of clade one, suggesting that clade two species are at the early stage of genome reduction. On the other hand, P. ananatis has a type VI secretion system that is highly variable but cannot be separated by clades. These results clarify our understanding of P. ananatis' phylogenetic diversity and provide clues to the interactions between P. ananatis, host insect, and plant that may lead to advances in rice protection and pest control.

Drp1-dependent mitochondrial fission mediates corneal injury induced by alkali burn.

Corneal alkali burn, one of the most serious ophthalmic emergencies, is difficult to be cured by conservative treatments. It is well known that oxidative stress, inflammation and neovascularization are the main causes of corneal damage after alkali burn, but its underlying mechanism remains to be elucidated. Here, we reported that the expression and phosphorylation (Ser616) of mitochondrial fission protein Drp1 were up-regulated at day 3 after alkali burn, while mitochondrial fusion protein Mfn2 was down-regulated. The phosphorylation of ERK1/2 in corneas was increased at day 1, 3, 7 and peaked at day 3 after alkali burn. In human corneal epithelial cells (HCE-2), NaOH treatment induced mitochondrial fission, intracellular ROS production and mitochondrial membrane potential disruption, which was prevented by Drp1 inhibitor Mdivi-1. In corneas, Mdivi-1 or knockdown of Drp1 by Lenti-Drp1 shRNA attenuated alkali burn-induced ROS production and phosphorylation of IκBα and p65. In immunofluorescence staining, it was detected that Mdivi-1 also prevented NaOH-induced nuclear translocation of p65 in HCE-2 cells. Moreover, the expression of NADPH oxidase NOX2 and NOX4 in corneas peaked at day 7 after alkali burn. Mdivi-1, Lenti-Drp1 shRNA or the mitochondria-targeted antioxidant mito-TEMPO efficiently alleviated activation of NF-κB, expression of NOX2/4 and inflammatory cytokines including IL-6, IL-1ß and TNF-α in corneas after alkali burn. In pharmacological experiments, both Mdivi-1 and NADPH oxidases inhibitor Apocynin protected the corneas against alkali burn-induced neovascularization. Intriguingly, the combined administration of Mdivi-1 and Apocynin had a synergistic inhibitory effect on corneal neovascularization after alkali burn. Taken together, these results indicate that Drp1-dependent mitochondrial fission is involved in alkali burn-induced corneal injury through regulating oxidative stress, inflammatory responses and corneal neovascularization. This might provide a novel therapeutic target for corneal injury after alkali burn in the future.

Blockage of Drp1 phosphorylation at Ser579 protects neurons against Aß1­42­induced degeneration.

Alzheimer's disease (AD), one of the most common types of chronic neurodegenerative diseases, is pathologically characterized by the formation of amyloid ß (Aß) peptide­containing plaques and neurofibrillary tangles. Among Aß peptides, Aß1­42 induces neuronal toxicity and neurodegeneration. In our previous studies, Cdk5 was found to regulate Aß1­42­induced mitochondrial fission via the phosphorylation of dynamin­related protein 1 (Drp1) at Ser579. However, whether blockage of Drp1 phosphorylation at Ser579 protects neurons against Aß1­42­induced degeneration remains to be elucidated. Thus, the aim the present study was to examine the effect of mutant Drp1­S579A on neurodegeneration and its underlying mechanism. First, the phosphorylation­defect (phospho­defect) mutant, Lenti­Drp1­S579A was constructed. Phospho­defect Drp1­S579A expression was detected in primary cultures of mouse cortical neurons infected with Lenti­Drp1­S579A using western blotting and it was found to successfully attenuate the phosphorylation of endogenous Drp1 at Ser579. In primary neuronal cultures, the neuronal processes were evaluated under microscopy. Treatment with 10 µM Aß1­42 significantly decreased dendritic density and length, spine outgrowth and synapse number. As expected, infection of neurons with Lenti­Drp1­S579A efficiently alleviated the inhibitory effect of Aß1­42 on neurite outgrowth and synapse density. In addition, infection with Lenti­Drp1­S579A abolished the cleavage of caspase­3 and apoptosis in neurons exposed to Aß1­42. Thus, the current data demonstrated that blockage of Drp1 phosphorylation at Ser579 may be an effective strategy to protect neurons against Aß1­42­induced degeneration and apoptosis. These findings underline the therapeutic potential of targeting Drp1 in the treatment of AD.

Mitochondrial Dynamics Mediated by DRP1 and MFN2 Contributes to Cisplatin Chemoresistance in Human Ovarian Cancer SKOV3 cells.

Cisplatin (DDP) is the first-line chemotherapeutic agent for ovarian cancer. However, the development of DDP resistance seriously influences the chemotherapeutic effect and prognosis of ovarian cancer. It was reported that DDP can directly impinge on the mitochondria and activate the intrinsic apoptotic pathway. Herein, the role of mitochondrial dynamics in DDP chemoresistance in human ovarian cancer SKOV3 cells was investigated. In DDP-resistant SKOV3/DDP cells, mitochondrial fission protein DRP1 was down-regulated, while mitochondrial fusion protein MFN2 was up-regulated. In accordance with the expression of DRP1 and MFN2, the average mitochondrial length was significantly increased in SKOV3/DDP cells. In DDP-sensitive parental SKOV3 cells, downregulation of DRP1 and upregulation of mitochondrial fusion proteins including MFN1,2 and OPA1 occurred at day 2~6 under cisplatin stress. Knockdown of DRP1 or overexpression of MFN2 promoted the resistance of SKOV3 cells to cisplatin. Intriguingly, weaker migration capability and lower ATP level were detected in SKOV3/DDP cells. Respective knockdown of DRP1 in parental SKOV3 cells or MFN2 in SKOV3/DDP cells using siRNA efficiently reversed mitochondrial dynamics, migration capability and ATP level. Moreover, MFN2 siRNA significantly aggravated the DDP-induced ROS production, mitochondrial membrane potential disruption, expression of pro-apoptotic protein BAX and Cleaved Caspase-3/9 in SKOV3/DDP cells. In contrast, DRP1 siRNA alleviated DDP-induced ROS production, mitochondrial membrane potential disruption, expression of pro-apoptotic protein BAX and Cleaved Caspase-3/9 in SKOV3 cells. Thus, these results indicate that mitochondrial dynamics mediated by DRP1 and MFN2 contributes to the development of DDP resistance in ovarian cancer cells, and will also provide a new strategy to prevent chemoresistance in ovarian cancer by targeting mitochondrial dynamics.

Mitochondrial Fission Is Required for Blue Light-Induced Apoptosis and Mitophagy in Retinal Neuronal R28 Cells.

Light emitting diodes (LEDs) are widely used to provide illumination due to their low energy requirements and high brightness. However, the LED spectrum contains an intense blue light component which is phototoxic to the retina. Recently, it has been reported that blue light may directly impinge on mitochondrial function in retinal ganglion cells (RGCs). Mitochondria are high dynamic organelles that undergo frequent fission and fusion events. The aim of our study was to elucidate the role of mitochondrial dynamics in blue light-induced damage in retinal neuronal R28 cells. We found that exposure to blue light (450 nm, 1000 lx) for up to 12 h significantly up-regulated the expression of mitochondrial fission protein Drp1, while down-regulating the expression of mitochondrial fusion protein Mfn2 in cells. Mitochondrial fission was simultaneously stimulated by blue light irradiation. In addition, exposure to blue light increased the production of reactive oxygen species (ROS), disrupted mitochondrial membrane potential (MMP), and induced apoptosis in R28 cells. Notably, Drp1 inhibitor Mdivi-1 and Drp1 RNAi not only attenuated blue light-induced mitochondrial fission, but also alleviated blue light-induced ROS production, MMP disruption and apoptosis in cells. Compared with Mdivi-1 and Drp1 RNAi, the antioxidant N-acetyl-L-cysteine (NAC) only slightly inhibited mitochondrial fission, while significantly alleviating apoptosis after blue light exposure. Moreover, we examined markers for mitophagy, which is responsible for the clearance of dysfunctional mitochondria. It was found that blue light stimulated the conversion of LC3B-I to LC3B-II as well as the expression of PINK1 in R28 cells. Mdivi-1 or Drp1 RNAi efficiently inhibited the blue light-induced expression of PINK1 and co-localization of LC3 with mitochondria. Thus, our data suggest that mitochondrial fission is required for blue light-induced mitochondrial dysfunction and apoptosis in RGCs.

The role of Cdk5-mediated Drp1 phosphorylation in Aß1-42 induced mitochondrial fission and neuronal apoptosis.

Alzheimer's disease, one of the most common neurodegenerative diseases, is pathologically characterized by Amyloid beta containing plaques and neurofibrillary tangles. Amyloid beta (Aß) induces neuronal apoptosis through the intracellular Ca2+ increase, subsequent hyperactivation of cyclin-dependent kinase 5 (Cdk5) and mitochondrial abnormality. Recently, Cdk5 was identified as an upstream regulator of mitochondrial fission during neuronal apoptosis, but the underlying mechanism remains unclear. Here, in vitro phosphorylation assays showed that Cdk5 could phosphorylate the recombinant Drp1 at Serine 579. Aß1-42 stimulation increased the phosphorylation level of Drp1 at Serine 579 in mouse cortical neurons. Cdk5 inhibitor roscovitine and knockdown of Cdk5 by a lentiviral vector expressing shRNA targeting Cdk5 (Lenti-Cdk5-shRNA) efficiently prevented Aß1-42 induced Drp1 phosphorylation in neurons. In addition, Aß1-42 stimulation induced markedly mitochondrial fission in neurons. Roscovitine, Lenti-Cdk5-shRNA and expression of phospho-defect mutatant GFP-Drp1-S579A in neurons attenuated Aß1-42 induced mitochondrial fission, whereas expression of phospho-mimetic mutant GFP-Drp1-S579D alone resulted in mitochondiral fission similar to Aß1-42 stimulation. Moreover, Roscovitine and Lenti-Cdk5-shRNA suppressed the cleavage of caspase-3 and protected neurons against Aß1-42 induced neuronal apoptosis.Thus, our data indicate that Drp1 is a direct target of Cdk5, and Cdk5-mediated phosphorylation of Drp1 at Serine 579 regulates Aß1-42 induced mitochondrial fission and neuronal toxicity.

Amyloid ß-42 induces neuronal apoptosis by targeting mitochondria.

Alzheimer's disease (AD), with a typical pathological hallmark of amyloid­beta (Aß)­containing plaques and neurofibrillary tangles, is one of the most common types of chronic neurodegenerative diseases. Aß oligomers serve a crucial role in the pathogenesis of AD, and lead to neuronal loss. However, the precise mechanism of Aß oligomers in AD remains to be elucidated. The present study demonstrated that 10 µM Aß­42 activated the caspase signaling pathway, and induced significant apoptosis in primary cultured mouse cerebral cortical neurons. The results of reverse transcription­quantitative polymerase chain reaction and western blotting demonstrated that Aß­42 (10 µM) also significantly upregulated the transcription and expression of the mitochondrial fission protein dynamin­related protein 1 (Drp1), and downregulated the transcription and expression of mitochondrial fusion proteins, including mitofusin 1/2 (Mfn1/2) and mitochondrial dynamin like GTPase (OPA­1). Neurons were transfected with pDsRed2­Mito for mitochondrial imaging, which revealed that 10 µM Aß­42 induced mitochondrial fission in cortical neurons. In addition, 2',7'­dichlorodihydrofluorescein diacetate and tetramethylrhodamine ethyl ester staining indicated that Aß­42 increased the reactive oxygen species (ROS) level and reduced mitochondrial membrane potential in neurons. Inhibition of Drp1 activity by Mdivi­1 efficiently prevented Aß­42­induced ROS production and disruption of mitochondrial membrane potential. Loss of mitochondrial membrane potential may activate PTEN­induced putative kinase 1 (Pink1), the prominent sensor for mitochondrial damage, and trigger the process of mitophagy to remove the damaged mitochondria. In the present study, western blotting revealed that the levels of autophagy marker microtubule­associated proteins 1A/1B light chain 3B (LC3B) and Pink1 were upregulated after Aß­42 stimulation. In conclusion, these data indicated that Aß­42 induces neuronal apoptosis by targeting mitochondria, including promotion of mitochondrial fission, disruption of mitochondrial membrane potential, increasing intracellular ROS level and activation of the process of mitophagy. Therefore, mitochondria may represent a potential therapeutic target for AD in the future.

Involvement of mitochondrial dynamics in the antineoplastic activity of cisplatin in murine leukemia L1210 cells.

Leukemia is a type of hematopoietic stem cell malignant cloned disease with high mortality. Cisplatin-based chemotherapy is one of the most common treatments for leukemia. Similar to other chemotherapeutic agents, cisplatin resistance has become a serious issue in cancer therapy. In the present study, we investigated the role of mitochondrial dynamics in the antineoplastic activity of cisplatin in murine leukemia L1210 cells. Firstly, the L1210 cell line resistant to cisplatin (L1210/DDP) was established. Compared to its parental cell line, the IC50 value of cisplatin in the L1210/DDP cells was increased 10-fold. Mitofusins (Mfn1 and Mfn2), mitochondrial outer membrane fusion proteins, were markedly upregulated in the L1210/DDP cells, whereas the expression of fission protein Drp1 and inner membrane fusion protein OPA1 were not significantly altered. In addition, mitofusins were also upregulated in the parental L1210 cells subjected to cisplatin stress. To investigate the role of mitochondrial dynamics in the antineoplastic activity of cisplatin, the effect of mitochondrial division inhibitor (Mdivi)-1 on cisplatin­induced cell death, caspase-3 cleavage and ROS production was examined in L1210 cells. We found that 5 µM of Mdivi-1 efficiently attenuated cisplatin-induced cell death, caspase activation and intracellular ROS increase in L1210 cells. Our data indicated that mitochondrial dynamics play an important role in the antineoplastic activity of cisplatin, and mitofusin-mediated mitochondrial fusion may be involved in the process of cisplatin resistance in leukemia cells. Therefore, the present study revealed that mitochondrial dynamics may be a potential target used to improve the antineoplastic activity of cisplatin in leukemia in the future.

Mitochondrial dynamics regulates hypoxia-induced migration and antineoplastic activity of cisplatin in breast cancer cells.

Mitochondria are high dynamic organelles with frequent fission and fusion. Here, we found hypoxia stimulated Drp1 expression, mitochondrial fission and migration in metastatic MDA-MB­231 cells, but not in non-metastatic MCF-7 cells. Inhibition of Drp1-dependent mitochondrial fission by Mdivi-1 or silencing Drp1 attenuated hypoxia-induced mitochondrial fission and migration in MDA-MB­231 cells. On the other hand, cisplatin induced significant apoptosis and mitochondrial fission in MDA-MB­231 cells, but not in MCF-7 cells. Mdivi-1 and silencing Drp1 also efficiently prevented cisplatin-induced MMP decrease, ROS production and apoptosis in MDA-MB­231 cells. Our data suggest that Drp1-dependent mitochondrial fission not only regulates hypoxia-induced migration of breast cancer cells, but also facilitates its sensitivity to chemotherapeutic agents. Thus, targeting Drp1-dependent mitochondrial dynamics may provide a novel strategy to suppress breast cancer metastasis and improve the chemotherapeutic effect in the future.

Development of a novel liposomal nanodelivery system for bioluminescence imaging and targeted drug delivery in ErbB2-overexpressing metastatic ovarian carcinoma.

Liposomes as targeted drug delivery systems are an emerging strategy in the treatment of cancer to selectively target tumors or genes. In this study, we generated the recombinant protein, EC1-GLuc, by fusing the EC1 peptide, an artificial ligand of ErbB2, with Gaussia luciferase (GLuc). The purified EC1-GLuc was conjugated with a nickel-chelating liposome to construct the EC1-GLuc-liposome. In vitro experiments revealed that the EC1-GLuc-liposome selectively targeted and internalized into ErbB2-overexpressing SKOv3 cells for bioluminescence imaging. A cell-impermeable fluorescence dye (HPTS) encapsulated in the EC-GLuc-liposome was efficiently delivered into the SKOv3 cells. In addition, the EC1-GLuc-liposome also targeted metastatic SKOv3 tumors for bioluminescence imaging and effectively delivered HPTS into metastatic tumors in vivo. Therefore, the present study demonstrates the novel EC1-GLuc-liposome to be an effective theranostic system for monitoring and treating ErbB2-overexpressing metastatic ovarian carcinoma through a combination of targeted molecular imaging and DDS.

Involvement of Drp1 in hypoxia-induced migration of human glioblastoma U251 cells.

Glioblastoma is one of the most aggressive brain tumors with high morbidity and mortality. Hypoxia is often the common characteristic of tumor microenvironment, and hypoxia-inducible factor-1α (HIF-1α) is an essential factor regulating the migratory activity of cancer cells including glioblastoma. Recently, mitochondrial dynamics was found to be involved in the aggression of cancer cells. However, whether dynamin-related protein 1 (Drp1) contributes to the migration of human glioblastoma cells under hypoxia remains unknown. In the present study, hypoxia was found to upregulate the transcription and expression of Drp1, and stimulated mitochondrial fission in glioblastoma U251 cells. Inhibition of HIF-1α with echinomycin blocked hypoxia­induced expression of Drp1. Notably, Drp1 inhibitor Mdivi-1 efficiently attenuated hypoxia-induced mitochondrial fission and migration of U251 cells. In addition, three U251 stable cell lines expressing GFP, GFP-Drp1 and dominant negative GFP-Drp1­K38A were established to examine the direct role of Drp1 in hypoxia-induced migration. MTT assay showed that there was no significant difference in proliferation of three cell lines. Compared with the GFP cell line, exogenously expressed GFP-Drp1-K38A inhibited hypoxia-induced migration of U251 cells, while stable expression of GFP-Drp1 enhanced the migration of U251 cells under hypoxia. Therefore, this study indicates the involvement of Drp1 in hypoxia-induced migration of human glioblastoma U251 cells, and suggests Drp1 to be a potential therapeutic target to suppress the aggression of glioblastoma in the future.