HSPA8-mediated stability of the CLPP protein regulates mitochondrial autophagy in cisplatin-resistant ovarian cancer cells.

Currently, platinum agents remain the mainstay of chemotherapy for ovarian cancer (OC). However, cisplatin (DDP) resistance is a major reason for chemotherapy failure. Thus, it is extremely important to elucidate the mechanism of resistance to DDP. Here, we establish two DDP-resistant ovarian cancer cell lines and find that caseinolytic protease P (CLPP) level is significantly downregulated in DDP-resistant cell lines compared to wild-type ovarian cancer cell lines (SK-OV-3 and OVcar3). Next, we investigate the functions of CLPP in DDP-resistant and wild-type ovarian cancer cells using various assays, including cell counting kit-8 assay, western blot analysis, immunofluorescence staining, and detection of reactive oxygen species (ROS) and apoptosis. Our results show that CLPP knockdown significantly increases the half maximal inhibitory concentration (IC 50) and mitophagy of wild-type SK-OV-3 and OVcar3 cells, while CLPP overexpression reduces the IC 50 values and mitophagy of DDP-resistant SK-OV-3 and OVcar3 cells. Next, we perform database predictions and confirmation experiments, which show that heat shock protein family A member 8 (HSPA8) regulates CLPP protein stability. The dynamic effects of the HSPA8/CLPP axis in ovarian cancer cells are also examined. HSPA8 increases mitophagy and the IC 50 values of SK-OV-3 and OVcar3 cells but inhibits their ROS production and apoptosis. In addition, CLPP partly reverses the effects induced by HSPA8 in SK-OV-3 and OVcar3 cells. In conclusion, CLPP increases DDP resistance in ovarian cancer by inhibiting mitophagy and promoting cellular stress. Meanwhile, HSPA8 promotes the degradation of CLPP protein by regulating its stability.

Oxidative Radical NHC Catalysis: Divergent Difunctionalization of Olefins through Intermolecular Hydrogen Atom Transfer.

Oxidative N-heterocyclic carbene (NHC) organocatalysis, typically leading to the formation of acyl azolium reactive intermediates, constitutes one of the most important activation strategies for the NHC-catalyzed chemical transformations. Here, we report an unprecedented oxidative radical NHC catalysis by using peroxyester as the external single-electron oxidant to realize divergent difunctionalization of olefins. The key to success of this chemistry is the catalytic generation of oxygen radicals that could trigger an intermolecular hydrogen atom transfer to activate the inert C-H bonds, thereby enabling the productive radical relay process. With this protocol, commonly used general chemicals could serve as radical precursors to allow efficient synthesis of value-added products in a straightforward and cost-effective manner. Preliminary mechanistic investigations, including control experiments and DFT calculations, shed light on the NHC organocatalytic radical reaction mechanism.

Radical Acylalkylation of 1,3-Enynes To Access Allenic Ketones via N-Heterocyclic Carbene Organocatalysis.

An N-heterocyclic carbene organocatalytic 1,4-difunctionalization of 1,3-enynes was developed. This organocatalytic strategy was suitable for a broad spectrum of substrates to efficiently synthesize allenic ketones bearing diverse substituents. Preliminary mechanistic studies suggest a radical reaction pathway for this organocatalytic acylalkylation process.

Caseinolytic protease P (CLPP) activated by ONC201 inhibits proliferation and promotes apoptosis in human epithelial ovarian cancer cells by inducing mitochondrial dysfunction.

BACKGROUND: Caseinolytic protease P (CLPP) is a mitochondrial specific protein which has been reported to be related to tumor cell apoptosis. This study aims to explore the roles of CLPP in human epithelial ovarian cancer (EOC). METHODS: We determined CLPP expression in paracancerous tissues and cancer tissues obtained from 20 EOC patients, and also in 4 EOC cell lines, and one normal ovarian cell line (IOSE-80). We knocked CLPP expression down in SK-OV-3 and A2780 cells and overexpressed it in SW626 and OVcar3 cells. The effect of CLPP expression on cell proliferation, mitochondrial membrane potential, and apoptosis was then assessed by flow cytometry assay. Furthermore, the effect of ONC201 (agonist of CLPP) on the EOC cell lines was also investigated. RESULTS: The CLPP expression was markedly down-regulated in EOC cancer tissues, and the Kaplan-Meier Plotter database revealed its low expression was linked to poor prognosis in EOC patients. Low expression of CLPP up-regulated the expression of NADH: ubiquinone oxidoreductase subunit A12 (NDUFA12), succinate dehydrogenase complex flavoprotein subunit A (SDHA), and succinate dehydrogenase complex iron sulfur subunit B (SDHB), which are key members of the mitochondrial respiratory chain, and these up-regulated proteins further led to the increase of mitochondrial membrane potential, cell proliferation promotion and neoplasm metastasis. Conversely, while overexpression of CLPP led to the opposite results, including inducing the decrease of mitochondrial membrane potential and apoptosis. In addition, stimulation with ONC201 enhanced the function of CLPP in SW626 and OVcar3 cells, and silencing of CLPP could neutralize the effect of ONC201. CONCLUSIONS: Our findings suggest that CLPP mediated mitochondrial dysfunction inhibits the proliferation and migration of EOC cells.

Hsa-miR-105-1 Regulates Cisplatin-Resistance in Ovarian Carcinoma Cells by Targeting ANXA9.

PURPOSE: Cisplatin is one of the most effective drugs for treating ovarian carcinoma (OC), which is among the most lethal types of carcinoma. However, the chemoresistance to cisplatin that develops over time leads to a poor clinical outcome for many OC patients. Therefore, it is necessary to clearly understand the molecular mechanisms of chemoresistance. In this study, we examined how Hsa-miR-105-1 functions in cisplatin-resistant OC cells. METHODS: The levels of Hsa-miR-105-1 expression in cisplatin-sensitive and resistant OC cell lines were detected by qRT-PCR. The target gene of Hsa-miR-105-1 was predicted by using the TargetScan and Starbase databases and verified by the double luciferase reporter gene assay. The target gene of Hsa-miR-105-1 was identified as ANXA9, and ANXA9 expression was evaluated by qRT-PCR, western blotting, and immunofluorescence. To validate the function of Hsa-miR-105-1 in OC cells, we silenced or overexpressed Hsa-miR-105-1 in cisplatin-sensitive or resistant OC cell lines, respectively. Furthermore, the expression levels of several apoptosis-related proteins, including P53, P21, E2F1, Bcl-2, Bax, and caspase-3, were examined by western blot analysis. RESULTS: The levels of Hsa-miR-105-1 expression were abnormally downregulated in cisplatin-resistant OC cells, while ANXA9 expression was significantly upregulated in those cells. Treatment with an Hsa-miR-105-1 inhibitor promoted the expression of ANXA9 mRNA and protein, enhanced the resistance to cisplatin, and attenuated the cell apoptosis induced by cisplatin in cisplatin-sensitive OC cells. Moreover, treatment with Hsa-miR-105-1 mimics inhibited ANXA9 expression, which further increased the levels of P53, P21, and Bax expression and decreased the levels of E2F1 and Bcl-2 expression, finally resulting in an increased sensitivity to cisplatin in cisplatin-resistant OC cells. CONCLUSION: We found that a downregulation of Hsa-miR-105-1 expression enhanced cisplatin-resistance, while an upregulation of Hsa-miR-105-1 restored the sensitivity of OC cells to cisplatin. The Hsa-miR-105-1/ANXA9 axis plays an important role in the cisplatin-resistance of OC cells.

Leptin siRNA promotes ovarian granulosa cell apoptosis and affects steroidogenesis by increasing NPY2 receptor expression.

Leptin has been found to be involved in the ovarian granulosa cell apoptosis and steroidogenesis. Loss of neuropeptide Y (NPY) can correct the obesity syndrome of mutant mice lacking of leptin (ob/ob). However, the association of NPY and leptin in ovarian granulosa cells and ovarian steroidogenesis has not been investigated. Here, C57BL/6J ob/ob mice and C57BL/6J (control) mice were intraperitoneally injected with PBS, leptin (0.4µg/g bodyweight) or BIIE0246 (NPY2 receptor [NPY2R] antagonist, 30µg/kg bodyweight) every day for 15days. We found that NPY2R mRNA expression in mouse ovary was suppressed by leptin treatment, but increased by leptin deficiency. Leptin or BIIE0246 treatment significantly increased E2, but notably decreased progesterone in both mice. A lower level of E2 and a higher level of progesterone was observed in ob/ob mice than in control mice. Further, we then knocked down leptin expression in human ovarian granulosa cells by siRNA transfection and treated the cells with DMSO or BIIE0246. In vitro experiments confirmed the findings in mice. siLeptin treatment decreased the secretion of E2, anti-Mullerian hormone (AMH), insulin-like growth factor (IGF)-1 and transforming growth factor (TGF)-ß, and the cell proliferation, but increased the secretion of progesterone and cell apoptosis. Western blotting analysis of PCNA, Bcl-2 and Bax confirmed the results of cell proliferation and apoptosis. Activation of JAK2 and STAT3 was also suppressed by knocking down leptin. All the effects of siLeptin on ovarian granulosa cells were partially reversed by BIIE0246. In conclusion, knockdown of leptin significantly affected ovarian steroidogenesis and ovarian function through NPY. siLeptin transfection impaired the activation of JAK2/STAT3 and contributed to ovarian granulosa cell apoptosis partially through up-regulating NPY2R expression.

Postoperative external beam irradiation with and without brachytherapy in pelvic node-positive IB1-IIA2 cervical cancer patients: a retrospective clinical study.

BACKGROUND: This study assessed the survival outcomes and recurrent patterns in pelvic node-positive IB1-IIA2 cervical cancer patients treated with postoperative external beam irradiation with or without vaginal brachytherapy. METHODS: The records of 1149 cervical cancer patients received radical surgery between February 2008 and March 2010 were retrospectively reviewed. 126 stages IB1-IIA2 patients with positive pelvic lymph node (LN) were included and a total of 113 patients who received different postoperative radiation therapy were identified and analyzed. Of the enrolled patients, 55 patients received pelvic external beam radiotherapy (EBRT) without vaginal brachytherapy and 58 patients received pelvic EBRT with vaginal brachytherapy. Treatment-related toxicities were evaluated. Progression-free survival (PFS) and overall survival (OS) were analyzed using Kaplan-Meier estimates and statistical significance was determined using the log-rank test. RESULTS: With a median follow-up of 47 months (range: 10-61 months), the group which had pelvic EBRT with brachytherapy had a significantly improved 5-year PFS rate (P = 0.044), but no significant difference in 5-year overall survival was found between the two groups (P = 0.437). In patients treated without brachytherapy, the most common site of relapse was the pelvis. No significant differences were found regards to acute and chronic radiation toxicities, including myelosuppression, dermatitis, enterocolitis, proctitis and cystitis (P = 0.485, 0.875, 0.671, 0.459 and 0.969 respectively) between the groups of pelvic EBRT with and without vaginal brachytherapy. CONCLUSIONS: Treated with pelvic EBRT in combination with vaginal brachytherapy, cervical cancer patients with positive pelvic lymph node had a reduced risk of locoregional recurrence without increased side effects compared with patients treated with pelvic EBRT without vaginal brachytherapy.

Clinical application of sartorius tendon transposition during radical vulvectomy: a case control study of 58 cases at a single institution.

OBJECTIVE: The aim of this study was to investigate the clinical effects of sartorius tendon transposition versus sartorius transposition during bilateral inguinal lymphadenectomy of radical vulvectomy. METHODS: A total of 58 vulvar cancer patients who had surgery from May 2007 to October 2013, in which 30 patients received sartorius transposition and 28 patients received sartorius tendon transposition. All patients were matched by age, body mass index, stage, histology, and grade. Intraoperative variables and postoperative complications, recurrence, progression-free survival (PFS), and overall survival (OS) and postoperative life quality were compared and analyzed. RESULTS: No significant differences were found at median surgical times and amounts of bleeding (p=0.316 and p=0.249, respectively), neither at the incidences of groin cellulitis and lymphocele (p=0.673 and p=0.473, respectively), but the recovery times of the inguinal wounds were shorter (p=0.026) and the incidences of wound break and chronic lymphedema were significantly decreased in the tendon transposition group (p=0.012 and p=0.022, respectively). Postoperative quality of life in tendon transposition group was significantly improved as indicated by the EORTC QLQ-C30 questionnaire. Recurrences were similar (p=0.346) and no significant differences were found at PFS and OS (p=0.990 and p=0.683, respectively). CONCLUSION: Compared to sartorius transposition, sartorius tendon transposition during inguinal lymphadenectomy led to improved patient recovery, reduced postoperative complications, and improved life quality without compromising the outcomes.

Altering intercomponent interactions in a photochromic multi-state [2]rotaxane.

Rotaxanes have attracted much attention because of their challenging constructions and potential applications. In this paper, a multi-state [2]rotaxane, in which a dithienylethene-functionalized dibenzo-24-crown-8 macrocycle was interlocked onto a thread component bearing a 4-morpholin-naphthalimide fluorescent stopper and two distinct recognition sites, namely, dibenzylammonium and N-methyltriazolium recognition sites, was prepared and studied. By introducing a dithienylethene photochrome into the macrocycle component, multi-mode alteration of the intercomponent interactions, such as energy transfer, electron transfer, and charge transfer interaction between the photochrome and the fluorescent naphthalimide stopper could be altered in this multi-state rotaxane system in response to the combination of chemical and photochemical stimuli.