Autophagy stimulates apoptosis in HER2-overexpressing breast cancers treated by lapatinib.

HER2-overexpressing breast cancers often show hyperactivation of the HER2/AKT/mTOR signaling pathway. Lapatinib is an oral dual tyrosine kinase inhibitor (TKI) that targets both EGFR and HER2 to inhibit the proliferation of breast cancer cells. However, it is obscure whether and how lapatinib could induce autophagy in breast cancer cells, an important cell response with drug treatment. In this study, we investigated the apoptosis and the autophagy in the HER2-overexpressing breast cancer cells BT474 and AU565 treated with lapatinib, and further examined their relationship. Lapatinib inhibited the proliferation and the rate of DNA synthesis in HER2-positive cells, as observed by MTT, colony formation and EDU assays. Lapatinib not only induced apoptosis accompanied by an increased expression of cleaved Caspase-3 and cleaved PARP, but it also induced autophagy in vitro, as confirmed by electron microscopy (EM), acridine orange (AO) staining and LC3-II expression. Meanwhile, lapatinib inhibited the phosphorylation of HER2, AKT, mTOR, and p70S6K, whereas that of AMPK was activated. When the cells were pre-incubated with 3-Methyladenine (3-MA), the specific autophagy inhibitor, the growth inhibitory ratio and apoptosis rate were frustrated, whereas colony formation and DNA synthesis ability were encouraged. In addition, 3-MA application could up-regulate Caspase-3 and PARP expression, compared with the treatment with lapatinib alone. The addition of 3-MA could attenuate the inhibitory role on HER2/AKT/mTOR pathway and the active role on AMPK that was raised by lapatinib. Therefore, lapatinib simultaneously induced both apoptosis and autophagy in the BT474 and AU565 cells, and in these settings, autophagy facilitates apoptosis.

Ras homolog gene family, member A promotes p53 degradation and vascular endothelial growth factor-dependent angiogenesis through an interaction with murine double minute 2 under hypoxic conditions.

BACKGROUND: Tumor neovascularization (TNV) is a common pathologic basis for malignant growth and metastasis. However, the mechanism of TNV pathogenesis is not fully understood. Ras homolog gene family, member A (RhoA), a Rho guanosine triphosphatase (GTPase) family member, may be involved in a hypoxia-induced vascular endothelial growth factor (VEGF) pathway that regulates TNV angiogenesis through an unclear mechanism. METHODS: The regulation of RhoA on p53, the p53 binding protein homolog murine double minute 2 (MDM2), and VEGF was analyzed in hypoxic MCF-7 cells using Western blot analysis, real-time polymerase chain reaction (PCR) analysis, coimmunoprecipitation, and immunofluorescence staining assays. Changes in proliferation, invasion, migration, stress fiber formation, and tube formation were detected in an MCF-7 human umbilical vein endothelial cell (HUVEC) coculture system. Correlations of RhoA expression with MDM2, wild-type p53 (wt-p53), and VEGF expression in breast cancer tissues and relations between RhoA and breast cancer clinical features were analyzed by immunohistochemistry. RESULTS: Activated RhoA down-regulated p53 protein, which increased VEGF expression in hypoxic MCF-7 cells; whereas p53 messenger RNA levels were not altered. In addition, the ubiquitin-mediated degradation of p53 was enhanced by active RhoA. RhoA and MDM2 colocalized in the cytoplasm of hypoxic MCF-7 cells and interacted with each other physically. Furthermore, nutlin-3, a specific MDM2 inhibitor, was capable of reducing activated RhoA-induced p53 protein stability and attenuating VEGF accumulation. In an MCF-7-HUVEC coculture system, nutlin-3 effectively inhibited HUVEC proliferation, invasion, migration, stress fiber formation, and tube formation mediated by activated RhoA under hypoxic conditions. Data from 129 clinical breast cancer specimens with wt-p53 revealed that high RhoA expression was correlated with high MDM2 expression, low wt-p53 expression, and high VEGF expression. CONCLUSIONS: The current data suggested that activated RhoA promotes VEGF expression and hypoxia-induced angiogenesis through the up-regulation of MDM2 to decrease p53 stability.

Variation of NDRG2 and c-Myc expression in rat heart during the acute stage of ischemia/reperfusion injury.

N-Myc downstream regulated gene 2 (NDRG2), a Myc-repressed gene, is highly expressed in heart tissue. NDRG2 increases in response to hypoxia-induced stress and is involved in hypoxia-induced radioresistance. However, little is known about the expression changes and possible roles of NDRG2 in the heart under hypoxia condition. Here, the authors show that NDRG2, mainly localized in cardiomyocyte cytoplasm, was significantly reduced in myocardial tissue after acute ischemia/reperfusion (I/R) injury. Meanwhile, c-Myc was up-regulated following acute I/R injury, and the expression of c-Myc was significantly inversely correlated with that of NDRG2. In addition, overexpression of c-Myc in primary cultured cardiomyocyte repressed NDRG2 expression. Furthermore, the increase of cardiomyocyte apoptosis was correlated with the decrease of NDRG2 protein during the acute phase of reperfusion. These data suggested for the first time that I/R injury-induced up-regulation of pro-apoptotic c-Myc expression may contribute to the down-regulation of anti-apoptotic NDRG2. This stress response might be involved in the novel mechanism of myocardial apoptosis induced by I/R injury in rat.

Depletion of Survivin suppresses docetaxel-induced apoptosis in HeLa cells by facilitating mitotic slippage.

The anticancer effects of taxanes are attributed to the induction of mitotic arrest through activation of the spindle assembly checkpoint. Cell death following extended mitotic arrest is mediated by the intrinsic apoptosis pathway. Accordingly, factors that influence the robustness of mitotic arrest or disrupt the apoptotic machinery confer drug resistance. Survivin is an inhibitor of apoptosis protein. Its overexpression is associated with chemoresistance, and its targeting leads to drug sensitization. However, Survivin also acts specifically in the spindle assembly checkpoint response to taxanes. Hence, the failure of Survivin-depleted cells to arrest in mitosis may lead to taxane resistance. Here we show that Survivin depletion protects HeLa cells against docetaxel-induced apoptosis by facilitating mitotic slippage. However, Survivin depletion does not promote clonogenic survival of tumor cells but increases the level of cellular senescence induced by docetaxel. Moreover, lentiviral overexpression of Survivin does not provide protection against docetaxel or cisplatin treatment, in contrast to the anti-apoptotic Bcl-xL or Bcl-2. Our findings suggest that targeting Survivin may influence the cell response to docetaxel by driving the cells through aberrant mitotic progression, rather than directly sensitizing cells to apoptosis.

NDRG2 in rat liver regeneration: role in proliferation and apoptosis.

Liver regeneration is a complex process that is orchestrated by the precise interplay of cell proliferation, differentiation control, and molecular pathways, but this complicated molecular signaling network is not fully understood. In this study, we showed that N-Myc downstream-regulated gene 2 (NDRG2) is involved in this process. The mRNA and protein levels of NDRG2 were strongly reduced when liver regeneration reached a peak of activity. In addition, we found that rat NDRG2 expression and C-Myc expression were inversely correlated during this process. A low level of NDRG2 was observed as the C-Myc expression increased during regeneration. Moreover, a dramatic cell cycle arrest was found in normal rat liver-derived BRL cells 48 hours after being infected by adenoviral vectors expressing rat NDRG2. Meanwhile, the apoptotic rates were increased from 9.4% in control group to 64.7% in adenoviral vectors expressing rat NDRG2 group. These phenomena could also be observed in BRL 3A and L-02 cells. Further analysis revealed that NDRG2 overexpression may mediate the antiproliferative effect by inducing p53 and p21 regulated Bax/Bcl-2 increase and cyclin E-Cdk2 inhibition. In conclusion, our findings point to physiological roles for NDRG2 in liver regeneration.