Prohibitin silencing reverses stabilization of mitochondrial integrity and chemoresistance in ovarian cancer cells by increasing their sensitivity to apoptosis.

Current approaches to the treatment of ovarian cancer are limited because of the development of resistance to chemotherapy. Prohibitin (Phb1) is a possible candidate protein that contributes to development of drug resistance, which could be targeted in neoplastic cells. Phb1 is a highly conserved protein that is associated with a block in the G0/G1 phase of the cell cycle and also with cell survival. Our study was designed to determine the role of Phb1 in regulating cellular growth and apoptosis in ovarian cancer cells. Our results showed that Phb1 content is differentially overexpressed in papillary serous ovarian carcinoma and endometrioid ovarian adenocarcinoma when compared to normal ovarian epithelium and was inversely related to Ki67 expression. Immunofluorescence microscopy and Western analyses revealed that Phb1 is primarily associated with the mitochondria in ovarian cancer cells. Over-expression of Phb1 by adenoviral Phb1 infection resulted in an increase in the percentage of ovarian cancer cells accumulating at G0/G1 phase of the cell cycle. Treatment of ovarian cancer cells with staurosporine (STS) induced apoptosis in a time-dependent manner. Phb1 over-expression induced cellular resistance to STS via the intrinsic apoptotic pathway. In contrast, silencing of Phb1 expression by adenoviral small interfering RNA (siRNA) sensitized ovarian cancer cells to STS-induce apoptosis. Taken together, these results suggest that Phb1 induces block at G0/G1 phase of the cell cycle and promotes survival of cancer cells. Furthermore, silencing of the Phb1 gene expression may prove to be a valuable therapeutic approach for chemoresistant ovarian cancer by increasing sensitivity of cancer cells to apoptosis.

Mitochondrial DNA sequence variants in epithelial ovarian tumor subtypes and stages.

BACKGROUND: A majority of primary ovarian neoplasms arise from cell surface epithelium of the ovaries. Although old age and a positive family history are associated risk factors, the etiology of the epithelial ovarian tumors is not completely understood. Additionally, knowledge of factors involved in the histogenesis of the various subtypes of this tumor as well as those factors that promote progression to advanced stages of ovarian malignancy are largely unknown. Current evidence suggests that mitochondrial alterations involved in cellular signaling pathways may be associated with tumorigenesis. METHODS: In this study, we determined the presence of polymorphisms and other sequence variants of mitochondrial DNA (mtDNA) in 102 epithelial ovarian tumors including 10 matched normal tissues that paired with some of the tumors. High-resolution restriction endonucleases and PCR-based sequencing were used to assess the mtDNA variants spanning 3.3 kb fragment that comprised the D-Loop and 12S rRNA-tRNAphe, tRNAval, tRNAser, tRNAasp, tRNAlys, ATPase 6, ATPase 8, cytochrome oxidase I and II genes. RESULTS: Three hundred and fifty-two (352) mtDNA sequence variants were identified, of which 238 of 352 (68%) have not been previously reported. There were relatively high frequencies of three mutations in the 12S rRNA gene at np 772, 773, and 780 in stage IIIC endometrioid tumors, two of which are novel (773delT and 780delC), and occurred with a frequency of 100% (7/7). Furthermore, two mutations were observed in serous tumors only at np 1657 in stage IV (10/10), and at np 8221delA in benign cystadenomas (3/3) and borderline tumors (4/4). A high frequency, 81% (13/16) of TC insertion at np 310 was found only in early stages of serous subtype (benign cystadenomas, 3/3; borderline tumors, 4/4; stage I tumors, 2/5 and matched normal tissues 4/4). CONCLUSION: Our findings indicate that certain mtDNA mutations can reliably distinguish the different histologic subtypes of epithelial ovarian tumors. In addition, these data raise the possibility that certain mtDNA mutations may be useful biomarkers for predicting tumor aggressiveness and may play a potential role in tumorigenesis.

Influence of a leptin deficiency on testicular morphology, germ cell apoptosis, and expression levels of apoptosis-related genes in the mouse.

Leptin-deficient (ob/ob) male mice are morbidly obese and exhibit impaired reproductive function. The objective of this study was to assess the effect of a leptin deficiency on testicular morphology, germ cell development, apoptotic activity within germ cells, and expression levels of apoptosis-related genes in the testis. Sixteen week-old ob/ob male mice (n = 8) and controls (n = 8) were killed, and their reproductive organs were weighed. Testes were processed for either histomorphological analysis (hematoxylin and eosin [H&E] staining), germ cell apoptosis assessment (deoxy-UTP-digoxigenin nick end labeling [TUNEL] method), or apoptosis-related gene expression analysis (microarray). Cross sections of the testes of leptin-deficient animals showed reduced seminiferous tubule area, fewer pachytene spermatocytes, and fewer tubules exhibiting elongated spermatids/mature spermatozoa. Condensation of germ cell nuclei and Sertoli cell vacuolization were evident in the testes of some ob/ob animals. Overall there was an elevation of apoptotic activity in the germ cells of ob/ob mice, particularly within the pachytene spermatocytes. With microarray technology, we identified 9 proapoptosis-related genes that were expressed at a significantly higher level in the testes of ob/ob mice than in the testes of the controls. Among these were members of the tumor necrosis factor receptor super family 1A and 5 (TNFR1 and 5) and peptidoglycan recognition proteins (associated with the extrinsic apoptotic pathway), and granzymes A and B, growth arrest and DNA damage inducible 45 gamma, sphingosine phosphate lyase 1, and caspase 9 (associated with the intrinsic apoptotic pathway). The results of the current study show that a leptin deficiency in mice is associated with impaired spermatogenesis, increased germ cell apoptosis, and up-regulated expression of proapoptotic genes within the testes.

Impact of gonadotropin administration on folliculogenesis in prepubertal ob/ob mice.

Female leptin deficient (ob/ob) mice exhibit abnormal ovarian folliculogenesis resulting in an impaired ability to reproduce. This effect may be related to the hypogonadotropic state of these animals, or leptin may directly modulate ovarian follicle development. In the present study we assessed whether exogenous gonadotropin administration would normalize folliculogenesis and induce ovulation in immature ob/ob animals. Eight 26-day-old ob/ob and eight control mice were injected sc with pregnant mare serum gonadotropin followed 48 h later with a sc injection of human chorionic gonadotropin. Animals were killed 24 h later. Gonadotropin (GTH) administration increased both ovarian and uterine weights in control mice, but this effect was attenuated in leptin deficient animals. The number of preantral follicles was greater in ob/ob mice than controls, but in GTH-treated animals the number of antral follicles was subnormal in the ovaries of leptin deficient animals. Ob/ob animals also failed to ovulate in response to GTH, and the protective actions of GTH against granulosa cell apoptosis and follicular atresia were attenuated in these animals. Interestingly, however, serum levels of estradiol and progesterone were higher in ob/ob mice than controls, regardless of whether or not the animals received GTH treatment. We conclude that the ovarian responsiveness to GTH is subnormal in leptin deficient animals suggesting that leptin may modulate the process of folliculogenesis by directly altering the sensitivity of the ovary to GTH.

Mitochondrial dysfunction and adipogenic reduction by prohibitin silencing in 3T3-L1 cells.

Increase in mitochondrial biogenesis has been shown to accompany brown and white adipose cell differentiation. Prohibitins (PHBs), comprised of two evolutionarily conserved proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are present in a high molecular-weight complex in the inner membrane of mitochondria. However, little is known about the effect of mitochondrial PHBs in adipogenesis. In the present study, we demonstrate that the levels of both PHB1 and PHB2 are significantly increased during adipogenesis of 3T3-L1 preadipocytes, especially in mitochondria. Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases. In addition, fragmentation of mitochondrial reticulum, loss of mitochondrial cristae, reduction of mitochondrial content, impairment of mitochondrial complex I activity and excessive production of ROS were observed upon PHB-silencing in 3T3-L1 cells. Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies.

Prohibitin (PHB) acts as a potent survival factor against ceramide induced apoptosis in rat granulosa cells.

AIM: Ceramide is a key factor in inducing germ cell apoptosis by translocating from cumulus cells into the adjacent oocyte and lipid rafts through gap junctions. Therefore studies designed to elucidate the mechanistic pathways in ceramide induced granulosa cell (GC) apoptosis and follicular atresia may potentially lead to the development of novel lipid-based therapeutic strategies that will prevent infertility and premature menopause associated with chemo and/or radiation therapy in female cancer patients. Our previous studies have shown that Prohibitin (PHB) is intimately involved in GCs differentiation, atresia, and luteolysis. MAIN METHODS: In the present study, we have examined the functional effects of loss-/gain-of-function of PHB using adenoviral technology in delaying apoptosis induced by the physiological ligand ceramide in rat GCs. KEY FINDINGS: Under these experimental conditions, exogenous ceramide C-8 (50 µM) augmented the expression of mitochondrial PHB and subsequently cause the physical destruction of GC by the release of mitochondrial cytochrome c and activation of caspase-3. In further studies, silencing of PHB expression by adenoviral small interfering RNA (shRNA) sensitized GCs to ceramide C8-induce apoptosis. In contrast, adenovirus (Ad) directed overexpression of PHB in GCs resulted in increased PHB content in mitochondria and delayed the onset of ceramide induced apoptosis in the infected GCs. SIGNIFICANCE: Taken together, these results provide novel evidences that a critical level of PHB expression within the mitochondria plays a key intra-molecular role in GC fate by mediating the inhibition of apoptosis and may therefore, contribute significantly to ceramide induced follicular atresia.

Proceedings from the conference on Reproductive Problems in Women of Color.

The purpose of the National Institutes of Health conference on Reproductive Problems in Women of Color that convened on July 25, 2009 was to bring investigators together to examine data related to reproductive health care access and ethnic disparities in reproductive problems, fertility treatments, and pregnancy outcomes. One of the goals discussed at this conference was to initiate a research network of investigators interested in studying these problems through the development of an American Society of Reproductive Medicine special interest group and Society of Assisted Reproductive Technology writing groups.

Primary primitive neuroectodermal tumor of the uterus: a report of two cases and review of the literature.

BACKGROUND: Primary primitive neuroectodermal (pPNET) tumors rarely occur in adults, and they very rarely present as primary tumors of the uterus. Only 12 reported cases of pPNET of the uterus have been published in the English literature. We report two additional cases treated at the Roswell Park Cancer Institute, Buffalo, NY, between 1999 and 2002. CASES: Two postmenopausal patients presenting with abnormal uterine bleeding underwent endometrial biopsy, and subsequently staging laparotomy. The diagnosis of pPNET in both cases was confirmed only by extensive immunohistochemical analysis of the tumors. One patient with disease confined to an endometrial polyp received no adjuvant therapy, while the second patient with extrauterine disease received adjuvant pelvic radiation followed by chemotherapy. CONCLUSIONS: The diagnosis of pPNET of the uterus may be a challenge. Features of diagnostic significance include positive staining with neuron-specific enolase, presence of neurosecretory granules, and positive staining with the MIC-2 gene. Currently, there is no uniformity in the treatment of these cases since the majority of the patients reported to date have had surgery, chemotherapy, and/or radiation therapy.