IntroductionHeat shock proteins (HSPs) are normally induced under environmental stress to serve as chaperones for maintenance of correct protein folding but they are often overexpressed in many cancers, including breast cancer. The expression of Hsp27, an ATP-independent small HSP, is associated with cell migration and drug resistance of breast cancer cells. Breast cancer stem cells (BCSCs) have been identified as a subpopulation of breast cancer cells with markers of CD24-CD44+ or high intracellular aldehyde dehydrogenase activity (ALDH+) and proved to be associated with radiation resistance and metastasis. However, the involvement of Hsp27 in the maintenance of BCSC is largely unknown.MethodsMitogen-activated protein kinase antibody array and Western blot were used to discover the expression of Hsp27 and its phosphorylation in ALDH + BCSCs. To study the involvement of Hsp27 in BCSC biology, siRNA mediated gene silencing and quercetin treatment were used to inhibit Hsp27 expression and the characters of BCSCs, which include ALDH+ population, mammosphere formation and cell migration, were analyzed simultaneously. The tumorigenicity of breast cancer cells after knockdown of Hsp27 was analyzed by xenograftment assay in NOD/SCID mice. The epithelial-mesenchymal transition (EMT) of breast cancer cells was analyzed by wound-healing assay and Western blot of snail, vimentin and E-cadherin expression. The activation of nuclear factor kappa B (NF-κB) was analyzed by luciferase-based reporter assay and nuclear translocation.ResultsHsp27 and its phosphorylation were increased in ALDH+ BCSCs in comparison with ALDH- non-BCSCs. Knockdown of Hsp27 in breast cancer cells decreased characters of BCSCs, such as ALDH+ population, mammosphere formation and cell migration. In addition, the in vivo CSC frequency could be diminished in Hsp27 knockdown breast cancer cells. The inhibitory effects could also be observed in cells treated with quercetin, a plant flavonoid inhibitor of Hsp27, and it could be reversed by overexpression of Hsp27. Knockdown of Hsp27 also suppressed EMT signatures, such as decreasing the expression of snail and vimentin and increasing the expression of E-cadherin. Furthermore, knockdown of Hsp27 decreased the nuclear translocation as well as the activity of NF-κB in ALDH + BCSCs, which resulted from increasing expression of IκBα. Restored activation of NF-κB by knockdown of IκBα could reverse the inhibitory effect of Hsp27 siRNA in suppression of ALDH+ cells.ConclusionsOur data suggest that Hsp27 regulates the EMT process and NF-κB activity to contribute the maintenance of BCSCs. Targeting Hsp27 may be considered as a novel strategy in breast cancer therapy.

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We previously reported that heat stroke induces autophagy as a protection mechanism against neurodegeneration in the brain. Heme oxygenase (HO)-1 is a stress protein and can be induced by heat stress (HS). Cerebellar Purkinje cells are selectively vulnerable to heat-induced injury. In this study, we first validated an animal model of HS (38°C for 4 h) in which sustained increase of Purkinje cell injury, HO-1 expression up to 24 h post HS (HS₂₄), and hyperthermia reaching a rectal temperature 41.52 ± 0.32 were observed. In subsequent experiments, we investigated the effects of HO-1 on HS-induced Purkinje cell injury. Rats were divided into four groups: one normothermic control group receiving saline vehicle (1 mL/kg, intraperitoneal [i.p.]) and exposed to 25 for 4 h; and three HS groups receiving saline, or HO-1 inducer haemin (30 mg/kg, i.p.) or HO-1 inhibitor tin protoporphyrin (SnPP, 30 mg/kg, i.p.), respectively, at 12 h prior to HS. HS-induced Purkinje cell injury was further enhanced by HO-1 inducer but attenuated by HO-1 inhibitor as evaluated by immunoreactivity of apoptosis marker (active caspase-3) as well as Fluoro-Jade B histochemistry (staining for degenerating neurons), suggesting a detrimental role of HO-1. Interestingly, the protective autophagy was reduced by HO-1 inducer but enhanced by HO-1 inhibitor as demonstrated by autophagy markers including Beclin-1 and microtubule-associated protein light chain 3 in Purkinje cells. Double immunofluorescent labelling of Beclin-1 or 8-hydroxydeoxyguanosine (an oxidative DNA damage marker) with HO-1 immunoreactivity not only demonstrated their co-localization, but also confirmed that HO-1 negatively regulated Beclin-1 but increased oxidative stress in the same Purkinje cell. Taken together, our results indicate that HO-1 aggravates HS injury in cerebellar Purkinje cells. Our findings shed new light on cell damage mechanisms by HS in central nervous system and may help to provide potential therapeutic foci.

Heat stroke (HS) is defined clinically as a condition when core body temperature rises above 40°C and is accompanied by central nervous system abnormalities. In this study, we established a rat model of HS by exposing anesthetized rats to elevated ambient temperature (40°C) until core temperature reaching 40.5°C (HS onset). The rat was immediately removed from heating chamber, allowed recovery for various time periods, and killed for histological and biochemical studies. Our results indicated neuronal shrinkage and pyknosis of the nucleus and sustained up to 12 h recovery time in cerebral cortex. Elevated expression of autophagy-related proteins, including microtubule associated protein light chain 3 and beclin 1 in cortical tissue at various times (3, 6, 12 h) of recovery was observed. In addition, the number of autophagosomes stained by monodansylcadaverine, a specific autophagosome marker, increased after heat exposure but was reduced by pretreatment with 3-methyladenine, an autophagy inhibitor. Furthermore, heat exposure increased neuronal degeneration in cortical tissue, as evidenced by staining with the fluorescent dye Fluoro-Jade B for degenerating neuron. Pretreatment with 3-methyladenine in HS rats aggravated neurodegeneration. Taken together, these results suggest that HS induces autophagy as a protection mechanism against neurodegeneration. Modulation of autophagy may provide a potential therapeutic approach for HS and await further research.

3,4-Methylenedioxymethamphetamine (MDMA), a common recreational drug, is known to cause serotonergic neurotoxicity in the brain. Dextromethorphan (DM) is a widely used antitussive reported to exert anti-inflammatory effect in vivo. In this study, we examined the long-term effect of MDMA on the primate serotonergic system and the protective property of DM against MDMA-induced serotonergic abnormality using single photon emission computed tomography (SPECT). Nine monkeys (Macaca cyclopis) were divided into three groups, namely control, MDMA and co-treatment (MDMA/DM). [123I]-ADAM was used as the radioligand for serotonin transporters (SERT) in SPECT scans. SERT levels of the brain were evaluated and presented as the uptake ratios (URs) of [123I]-ADAM in several regions of interest of the brain including midbrain, thalamus and striatum. We found that the URs of [123I]-ADAM were significantly lower in the brains of MDMA than control group, indicating lower brain SERT levels in the MDMA-treated monkeys. This MDMA-induced decrease in brain SERT levels could persist for over four years. However, the loss of brain SERT levels was not observed in co-treatment group. These results suggest that DM may exert a protective effect against MDMA-induced serotonergic toxicity in the brains of the non-human primate.