Inhibition of USP10 induces myeloma cell apoptosis by promoting cyclin D3 degradation.

The cell cycle regulator cyclin D3 (CCND3) is highly expressed in multiple myeloma (MM) and it promotes MM cell proliferation. After a certain phase of cell cycle, CCND3 is rapidly degraded, which is essential for the strict control of MM cell cycle progress and proliferation. In the present study, we investigated the molecular mechanisms regulating CCND3 degradation in MM cells. By utilizing affinity purification-coupled tandem mass spectrometry, we identified the deubiquitinase USP10 interacting with CCND3 in human MM OPM2 and KMS11 cell lines. Furthermore, USP10 specifically prevented CCND3 from K48-linked polyubiquitination and proteasomal degradation, therefore enhancing its activity. We demonstrated that the N-terminal domain (aa. 1-205) of USP10 was dispensable for binding to and deubiquitinating CCND3. Although Thr283 was important for CCND3 activity, it was dispensable for CCND3 ubiquitination and stability modulated by USP10. By stabilizing CCND3, USP10 activated the CCND3/CDK4/6 signaling pathway, phosphorylated Rb, and upregulated CDK4, CDK6 and E2F-1 in OPM2 and KMS11 cells. Consistent with these findings, inhibition of USP10 by Spautin-1 resulted in accumulation of CCND3 with K48-linked polyubiquitination and degradation that synergized with Palbociclib, a CDK4/6 inhibitor, to induce MM cell apoptosis. In nude mice bearing myeloma xenografts with OPM2 and KMS11 cells, combined administration of Spautin-l and Palbociclib almost suppressed tumor growth within 30 days. This study thus identifies USP10 as the first deubiquitinase of CCND3 and also finds that targeting the USP10/CCND3/CDK4/6 axis may be a novel modality for the treatment of myeloma.

TRIM25 activates AKT/mTOR by inhibiting PTEN via K63-linked polyubiquitination in non-small cell lung cancer.

The PTEN/AKT/mTOR signaling pathway is frequently dysregulated in non-small cell lung cancer (NSCLC), but the mechanisms are not well-understood. The present study found that the ubiquitin ligase TRIM25 is highly expressed in NSCLC tissues and promotes NSCLC cell survival and tumor growth. Mechanistic studies revealed that TRIM25 binds to PTEN and mediates its K63-linked ubiquitination at K266. This modification prevents the plasma membrane translocation of PTEN and reduces its phosphatase activity therefore accumulating PI(3,4,5)P3. TRIM25 thus activates the AKT/mTOR signaling. Moreover, we found that the antibacterial nitroxoline can activate PTEN by reducing its K63-linked polyubiquitination and sensitizes NSCLC to cisplatin-induced apoptosis. This study thus identified a novel modulation on the PTEN signaling pathway by TRIM25 and provides a potential target for NSCLC treatment.

Suppression of the USP10/CCND1 axis induces glioblastoma cell apoptosis.

Recent studies show that the expression of CCND1, a key factor in cell cycle control, is increased following the progress and deteriotation of glioma and predicts poor outcomes. On the other hand, dysregulated deubiquitinase USP10 also predicts poor prognosis for patients with glioblastoma (GBM). In the present study, we investigated the interplay between CCND1 protein and USP10 in GBM cells. We showed that the expression of CCND1 was significantly higher in both GBM tissues and GBM-derived stem cells. USP10 interacted with CCND1 and prevented its K48- but not K63-linked polyubiquitination in GBM U251 and HS683 cells, which led to increased CCND1 stability. Consistent with the action of USP10 on CCND1, knockdown of USP10 by single-guided RNA downregulated CCND1 and caused GBM cell cycle arrest at the G1 phase and induced GBM cell apoptosis. To implement this finding in the treatment of GBMs, we screened a natural product library and found that acevaltrate (AVT), an active component derived from the herbal plant Valeriana jatamansi Jones was strikingly potent to induce GBM cell apoptosis, which was confirmed by the Annexin V staining and activation of the apoptotic signals. Furthermore, we revealed that AVT concentration-dependently suppressed USP10-mediated deubiquitination on CCND1 therefore inducing CCND1 protein degradation. Collectively, the present study demonstrates that the USP10/CCND1 axis could be a promising therapeutic target for patients with GBMs.

RNF6 promotes myeloma cell proliferation and survival by inducing glucocorticoid receptor polyubiquitination.

RNF6, a RING-type ubiquitin ligase, has been identified as an oncogene in various cancers but its role in multiple myeloma (MM) remains elusive. In the present study we first showed that the expression levels of RNF6 in MM were significantly elevated compared with the bone marrow cells of healthy donors. Overexpression of RNF6 in LP1 and PRMI-8266 MM cell lines promoted cell proliferation, whereas knockdown of RNF6 led to apoptosis of MM cells. Furthermore, we revealed that RNF6, as a ubiquitin ligase, interacted with glucocorticoid receptor (GR) and induced its K63-linked polyubiquitination. Different from current knowledge, RNF6 increased GR stability at both endogenous and exogenous contexts. Such an action greatly promoted GR transcriptional activity, which was confirmed by luciferase assays and by the increased expression levels of prosurvival genes including Bcl-xL and Mcl-1, two typical downstream genes of the GR pathway. Consistent with these findings, ectopic expression of RNF6 in MM cells conferred resistance to dexamethasone, a typical anti-myeloma agent. In conclusion, we demonstrate that RNF6 promotes MM cell proliferation and survival by inducing atypical polyubiquitination to GR, and RNF6 could be a promising therapeutic target for the treatment of MM.

Mebendazole elicits potent antimyeloma activity by inhibiting the USP5/c-Maf axis.

c-Maf is a critical oncogenic transcription factor that contributes to myelomagenesis. Our previous studies demonstrated that the deubiquitinase USP5 stabilizes c-Maf and promotes myeloma cell proliferation and survival; therefore, the USP5/c-Maf axis could be a potential target for myeloma therapy. As a concept of principle, the present study established a USP5/c-Maf-based luciferase system that was used to screen an FDA-approved drug library. It was found that mebendazole, a typical anthelmintic drug, preferentially induced apoptosis in c-Maf-expressing myeloma cells. Moreover, oral administration of mebendazole delayed the growth of human myeloma xenografts in nude mice but did not show overt toxicity. Further studies showed that the selective antimyeloma activity of mebendazole was associated with the inhibition of the USP5/c-Maf axis. Mebendazole downregulated USP5 expression and disrupted the interaction between USP5 and c-Maf, thus leading to increased levels of c-Maf ubiquitination and subsequent c-Maf degradation. Mebendazole inhibited c-Maf transcriptional activity, as confirmed by both luciferase assays and expression measurements of c-Maf downstream genes. In summary, this study identified mebendazole as a USP5/c-Maf inhibitor that could be developed as a novel antimyeloma agent.

A novel STAT3 inhibitor negatively modulates platelet activation and aggregation.

The signal transducer and activator of transcription 3 (STAT3) plays a critical role in platelet functions. This study sought to understand the effects of the STAT3 inhibitor SC99 on platelet activation and aggregation. Immunoblotting assays were applied to measure the effects of SC99 on the STAT3 signaling pathway. A ChronoLog aggregometer was used to evaluate platelet aggregation. A flow cytometer was used to evaluate P-selectin expression in the presence of SC99. AlamarBlue and Annexin-V staining were used to evaluate platelet viability and apoptosis, respectively. A fluorescence microscope was applied to analyze platelet spreading. SC99 inhibited the phosphorylation of JAK2 and STAT3 in human platelets but had no effects on the phosphorylation of AKT, p65 or Src, all of which are involved in platelet activation. Further studies revealed that SC99 inhibited human platelet aggregation induced by collagen and thrombin in a dose-dependent manner. SC99 inhibited thrombin-induced P-selectin expression and fibrinogen binding to single platelets. Moreover, SC99 inhibited platelet spreading on fibrinogen and clot retraction mediated by outside-in signaling. SC99 inhibited platelet aggregation in mice but it did not significantly prolong the bleeding time. Taken together, the present study revealed that SC99 inhibited platelet activation and aggregation as a STAT3 inhibitor. This agent can be developed as a promising treatment for thrombotic disorders.

[Effects of endogenous dopamine on two-state voltage oscillations of striatum medium spiny neurons].

OBJECTIVE: To explore the effects of endogenous dopamine (DA) on striatum medium spiny neurons (MSNs) in acute brain slices by the means of electrophysiology and elucidate the impact of nigrostriatal circuit loop on the functional status of MSNs. METHODS: Various structural combinations of striatum and cortex or/and substantia nigra were used to explore the conditions with appearance of spontaneous two-state voltage oscillations. And the blockage of dopaminergic or glutamic acid receptor was employed to examine how amino glutaminic acid from cortex and dopamine from substantia nigra influenced two-state voltage oscillations. RESULTS: It was found that 65.2% (30/46) MSNs recorded in corticostriatonigral slices displayed spontaneous and two-state voltage oscillations.92.3% (24/26) of MSNs in dorsal striatum close to cortex. And the percentage was only 30% (6/20) in ventral striatum. The amplitude of depolarized plateau potential (up state) decreased through a blockage of dopamine receptor (P < 0.05). The potential level of up state decreased through a blockage of D1 receptor (P < 0.05) and action potentials were stopped. The results of blocking D2 receptors were the increased potentials level of two states (P < 0.05). The membrane potential of MSNs showed a stable resting level in corticostriatonigral (-67 ± 3 mV, n = 10), corticostriatal (-70 ± 3 mV, n = 10), striatal (-73 ± 3 mV, n = 10) and nigrostriatal (-71 ± 3 mV, n = 10) slices. There was no significant difference (P > 0.05) . CONCLUSION: Endogenous dopamine from substantia nigra may regulate the instantaneous integration and coding of information from cortex by tonic effect on short-term plasticity of paired and short pulses in corticostriatal synapses.

[Electrophysiological characteristics of medium spiny neurons in neocortex-striatum-substantia nigra brain slices of rats].

OBJECTIVE: To establish the neocortex-striatum-substantia nigra brain slices of rats and observe the medium spiny neurons of striatum under a visible condition so as to explore their electrophysiological characteristics. METHODS: The brain slices containing the neocortex-striatum-substantia nigra were prepared from SD rats of postnatal 7 - 10 days. With infrared differential interference contrast (IR-DIC) microscope and patch clamp amplifier whole-cell recording technique, the medium spiny neurons were located in striatum and their spontaneous electrical activity was recorded in the current clamp mode. By infusing the step current, we observed the variation of membrane potentials. RESULTS: There were three types of conditions in the 92 medium spiny neurons successfully recorded. Among them, 14 were in persistent down state without action potential firing; 61 displayed persistent down state accompanied with interval temporal depolarizing to the threshold potential with action potential firing; and the remaining 17 showed persistent down state with the interval emergent up state. There was no statistical significance in the difference of mean resting and threshold potentials in three neuronal types (P > 0.05). When the neurons received an infused current, their membrane potentials displayed some delays. The variance of electric potentials showed a tendency of reduction along with the reinforcement of infusing current. CONCLUSION: The medium spiny neurons of striatum in parasagittal section brain slices reserve their in vivo electrophysiological characteristics. It establishes rationales for an in-depth study of the role of electrical generation and transmission of nigrostriatal access in the pathogenesis of Parkinson's disease.

[Electrophysiological characteristics of neurons in rat substantia nigra by visual patch clamp technique].

OBJECTIVE: To establish the brain slices of substantia nigra and striatum in rats and study the properties and functional significance of substantia nigra neurons. METHODS: The technique of infrared differential interference contrast (IR-DIC) and CCD-Camera system with visual patch clamp whole-cell recording were combined. One hundred healthy substantia nigra neurons were located and the measurements of active potential and Ih current recorded and analyzed. RESULTS: Visual patch clamp technique could be used to make a direct localization of substantia nigra and to identify the active neurons. Spontaneous active potential was shown in 94/100 neurons. Among them, 85 (Group A) had a frequency less than 10 Hz and 9 more than 10Hz (Group B). The means were (5.0 +/- 2.7) Hz and (14.0 +/- 2.6) Hz. respectively. As compared with the former, the latter had no obvious hyperpolarization during active potential period. Apparent hyperpolarizing current could be induced in 92.9% (79/85) of Group A neurons (-120 mV, 47.5 pA +/- 5.8 pA). They were classified as dopaminergic neurons. However, the Group B neurons had no or little Ih current and were classified as GABA neurons (P < 0.001). CONCLUSION: The types of nigra neurons can be distinguished by observing the fundamental electrical characteristic of dopaminergic neurons in nigrostriatal brain slices under visual inspections. And it may provide rationales for an in-depth knowledge of the occurrence and transmission of signal pathways between substantia nigra and

[The effect of four kinds of traditional Chinese herbal compound on the experimental acute stress behaviors and the hypothalamus-pituitary-adrenal gland axis].

OBJECTIVE: To study the effects of four kinds of traditional Chinese herbal compound, Sini powder, Banxiahoupu soup, Ganmaidazao soup and Guizhigancaolonggumuli soup, on the experimental acute stress behaviors and the hypothalamus-pituitary-adrenal gland axis. METHOD: Rats were divide into 7 groups: normal group, model 1 and 2 groups, Sini powder group, Banxiahoupu soup group, Ganmaidazao soup group, and Guizhigancaolonggumuli soup group. The behaviors of the rats and their CRH of the hypothalamus, ACTH of plasma and CORT of serum (by the means of radio-immunity) were detected. RESULT: Sini powder could reduce the crossing times of open-field test( P < 0.01) and the contents of CRH and CORT (P < 0.01, P < 0.05); Banxiahoupu soup could extent the still time of tail-hanging test ( P < 0.05) and the content of CRH (P < 0.05); Ganmaidazao soup could prolong the still time (P < 0.05) and reduce the struggle times (P < 0.01) of tail-hanging test and reduce the contents of CRH, ACTH and CORT (P < 0.01, P < 0.05); Guizhigancaolonggumuli soup could reduce the crossing times of open-field test (P < 0.01) , extent the still time (P < 0.05) and reduce the struggle times ( P < 0.01) of tail-hanging test of tail-hanging test, also reduce the contents of CRH, ACTH and CORT (P < 0.01, P < 0.05). CONCLUSION: The four kinds of traditional Chinese herbal compound can increase the ability of anti stress by affect the different taches of the hypothalamus-pituitary-adrenal gland axle and change the stress behaviors, which are based on their respective functions of regulating Qi, dispersing phlegm, tonifying Qi and warming Yang.

Effects of Erlong Zuoci pill and its disassembled prescriptions on gentamicin-induced ototoxicity model in vitro.

OBJECTIVE: To study the effects of Erlong Zuoci Pill (, ELZCP) and its disassembled: prescriptions on gentamicin (GM)-induced ototoxicity model in vitro. METHODS: After the spiral organ of cochleae: of newborn mice (postnatal days: 2-3) cultured for 24 h, GM alone or combined with water extracting-alcohol precipitating solution of ELZCP or with its disassembled prescriptions was added. Hair cells were observed under a fluorescence microscope after TRITC-phalloidin staining, and the cochlear hair cell loss rate was calculated by counting the whole cochlear hair cells and analyzed by whole cochlear hair cells analyzing software. RESULTS: GM induced cochlear outer hair cells (OHCs) and inner hair cells (IHCs) injuries in a dose-dependent manner, and they were significantly different as compared with those in the normal control group (P<0.05, P<0.01). ELZCP at the concentration of 0.003-3 mg/mL could decrease the hair cells loss induced by the 0.3 mmol/L GM (P<0.05, P<0.01), the effects was in a dose-dependent manner, and the concentration of 0.3 mg/mL showed the optimal protective effect. For the ELZCP disassembled prescriptions, Liuwei-Dihuang could decrease OHC loss rate than that in the 0.3 mmol/L GM model group (P<0.05), but the OHC loss rate was still higher than that in the ELZCP group (P<0.01), which indicated that the protective effect of hair cells by Liuwei-Dihuang was not better than that of ELZCP. Poria decreased OHC loss rate from 72.1 % +/-3.7 % to 58.8 %+/- 8.2 % (P<0.05). CONCLUSIONS: ELZCP could play a role in antagonizing the injury of cochlear hair cells induced by GM ototoxicity,: and its disassembled prescriptions, Liuwei-Dihuang was the main component to protect the cochlear hair cells from GM-induced ototoxicity, and Magnetitum combined with Radix Bupleurui could strengthen the action of the whole prescription; Poria could reduce GM-induced OHC loss.

Paeoniflorin, a potent natural compound, protects PC12 cells from MPP+ and acidic damage via autophagic pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeoniflorin (PF) is the principal bioactive component of Radix Paeoniae alba, which is widely used in Traditional Chinese Medicine for the treatment of neurodegenerative disorders such as Parkinson's disease (PD). AIM OF THE STUDY: To evaluate the neuroprotective effects of PF on MPP(+)- or acid- (pH 5.0) induced injury in cultured PC12 cells and to investigate the activity of autophagy-lysosome pathway (ALP). Amiloride (Ami), a non-selective blocker of acid-sensing ion channels (ASICs), as a positive control drug, since it is neuroprotective in rodent models of PD. MATERIALS AND METHODS: The cell viability was analyzed with MTT assay. The cell injury was assessed by lactate dehydrogenase (LDH) assay. Flow cytometry and Western blot analysis were used to study the apoptotic, calcium influx and autophagic mechanisms. RESULTS: Ami (100 microM) and PF (50 microM) both protected PC12 cells against MPP(+)- or acid-induced injury as assessed by MTT assay, lactate dehydrogenase release, and apoptosis rate. The concentrations of cytosolic free Ca(2+) were raised after exposure to MPP(+) or acidosis, while Ami and PF both reduced the influx of Ca(2+). More importantly, we found that the mechanisms of neuroprotective effects of Ami and PF were closely associated with the upregulation of LC3-II protein, which is specifically associated with autophagic vacuole membranes. Furthermore, application of MPP(+) or acid induced the overexpression of LAMP2a, which is directly correlated with the activity of the chaperone-mediated autophagy pathway. However, Ami and PF inhibited the overexpression of LAMP2a. CONCLUSIONS: Our data provide the first experimental evidence that PF modulates autophagy in models of neuron injury, as well as providing the first indication of a relationship between ASICs and ALP.

MPP+ impairs autophagic clearance of alpha-synuclein by impairing the activity of dynein.

Increasing evidence suggests that dynein has an important role in the clearance of misfolded proteins by autophagy. Here we show that treatment of cells with 1-methyl-4-phenylpyridinium (MPP) cause alpha-synuclein overexpression and aggregation, leading to the accumulation of autophagic vacuoles and the recruitment of LC3-II to these vacuoles in the cytoplasm. After MPP treatment, dynein expression decreased and was mainly aggregated at the periphery of cytoplasm and lost its colocalization with alpha-synuclein and lamp1, indicating that dynein lost its function in the aggresome formation and failed to return autophagosome and lysosomes to the center of the cell for degradation. We consider that dynein plays an important role in the autophagic clearance of aggregate-prone proteins.

Role of autophagy and proteasome degradation pathways in apoptosis of PC12 cells overexpressing human alpha-synuclein.

Parkinson's disease is a common neurodegenerative disease in the elderly. Its causes and mechanisms are not clearly understood. To explore the specific role of autophagy and the ubiquitin-proteasome pathway in apoptosis, a specific proteasome inhibitor and macroautophagy inhibitor and stimulator were selected to investigate pheochromocytoma (PC12) cell lines transfected with human mutant (A30P) and wild-type (WT) alpha-synuclein. The apoptosis ratio was assessed by flow cytometry. LC3, heat shock protein 70 (hsp70) and caspase-3 expression in cell culture were determined by Western blot. The hallmarks of apoptosis and autophagy were assessed with transmission electron microscopy. Compared to the control group or the rapamycin (autophagy stimulator) group, the apoptosis ratio in A30P and WT cells was significantly higher after treatment with inhibitors of the proteasome and macroautophagy. The results of Western blots for caspase-3 expression were similar to those of flow cytometry; hsp70 protein was significantly higher in the proteasome inhibitor group than in control, but in the autophagy inhibitor and stimulator groups, hsp70 was similar to control. These findings show that inhibition of the proteasome and autophagy promotes apoptosis, and the macroautophagy stimulator rapamycin reduces the apoptosis ratio. And inhibiting or stimulating autophagy has less impact on hsp70 than the proteasome pathway.