RESUMO
The commitment of mesenchymal stem cells (MSCs) to preadipocytes and the termination of differentiation to adipocytes are critical for maintaining systemic energy homeostasis. However, our knowledge of the molecular mechanisms governing the commitment of MSCs to preadipocytes and the subsequent termination of their differentiation into adipocytes remain limited. Additionally, the role of Sox6 sex-determining region Y (SRY)-box6 (Sox6), a transcription factor that regulates gene transcription, is reportedly involved in various cellular processes, including adipogenesis; however, its function in regulating preadipocyte development and the factors involved in the termination of adipogenic differentiation remain unexplored. Therefore, we investigated the role of Sox6 in regulating the differentiation of adipocytes by monitoring the effects of its overexpression in C3H10T1/2 cells (in vitro) and C57BL/6J mouse (in vivo) models of adipogenesis. We observed lower Sox6 expression in the adipose tissue of obese mice than that in control mice. Sox6 overexpression inhibited the differentiation of MSC by directly binding to the lysyl oxidase (Lox) and preadipocyte factor 1 (Pref1) promoters, which was potentiated by histone deacetylase-1(HDAC1). Our findings suggest that Sox6 is a key regulator of MSC commitment to adipocytes; therefore, targeting the Sox6-mediated regulation of this process could offer potential therapeutic avenues for addressing obesity and related metabolic disorders.
Assuntos
Adipogenia , Células-Tronco Mesenquimais , Animais , Camundongos , Adipogenia/genética , Diferenciação Celular/genética , Camundongos Endogâmicos C57BL , Proteína-Lisina 6-Oxidase/genética , Proteína-Lisina 6-Oxidase/metabolismo , Fatores de Transcrição SOXD/genética , Fatores de Transcrição SOXD/metabolismoRESUMO
Patients with ovarian cancer frequently develop acquired drug resistance after the long-term chemotherapy, leading to disease progression. Enhanced epithelial-mesenchymal transition (EMT) has been implicated in chemoresistance of ovarian cancer cells; however, the molecular mechanisms involved are largely undefined. Pyruvate dehydrogenase kinase 1 (PDK1), a key regulatory enzyme in glucose metabolism, has been recognized as a gatekeeper of the Warburg effect, a hallmark of cancer. In this study, the function of PDK1 in cisplatin resistance of ovarian cancer in terms of growth and EMT was investigated. PDK1 was upregulated in cisplatin-resistant ovarian cancer cells. PDK1 knockdown in resistant cells led to increased sensitivity to cisplatin-induced cell death and apoptosis. PDK1 downregulation also reversed the EMT and cell motility in cisplatin-resistant cells. In a mouse xenograft model, tumors derived from PDK1-silenced ovarian cancer cells exhibited decreased tumor growth and EMT compared with control after the cisplatin treatment. Mechanistically, PDK1 overexpression led to increased phosphorylation of EGFR, and blocking EGFR kinase activity by erlotinib reversed cisplatin resistance induced by PDK1 overexpression. Furthermore, in patients with ovarian cancer, higher PDK1 and p-EGFR levels were associated with chemoresistance. These results supported that PDK1 contributes to chemoresistance of ovarian cancer by activating EGFR. Therefore, PDK1 may serve as a promising target to combat chemoresistance of ovarian cancer.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Ovarianas/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino/farmacologia , Transição Epitelial-Mesenquimal/fisiologia , Receptores ErbB/metabolismo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias Ovarianas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Polycystic ovary syndrome is a common endocrine disorder and a major cause of anovulatory sterility in women at reproductive age. Most patients with polycystic ovary syndrome have hyperandrogenism, caused by excess androgen synthesis. Bone morphogenetic protein 4 (BMP4) is an essential regulator of embryonic development and organ formation, and recent studies have also shown that BMP4 may be involved in female steroidogenesis process. However, the effect of BMP4 on hyperandrogenism remains unknown. Here, using a female mouse model of hyperandrogenism, we found that ovarian BMP4 levels were significantly decreased in hyperandrogenism. Elevated androgens inhibited BMP4 expression via activation of androgen receptors. Moreover, BMP4 treatment suppressed androgen synthesis in theca cells and promoted estrogen production in granulosa cells by regulating the expression of steroidogenic enzymes, including CYP11A, HSD3B2, CYP17A1, and CYP19A1 Consistently, knockdown of BMP4 augmented androgen levels and inhibited estrogen levels. Mechanistically, Smad signaling rather than the p38 MAPK pathway regulated androgen and estrogen formation, thereby mediating the effect of BMP4. Of note, BMP4-transgenic mice were protected against hyperandrogenism. Our observations clarify a vital role of BMP4 in controlling sex hormone levels and offer new insights into intervention for managing hyperandrogenism by targeting the BMP4-Smad signaling pathway.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Modelos Animais de Doenças , Hiperandrogenismo/etiologia , Ovário/metabolismo , Síndrome do Ovário Policístico/fisiopatologia , Transdução de Sinais , Proteína Smad4/metabolismo , Androgênios/metabolismo , Androgênios/farmacologia , Animais , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Proteína Morfogenética Óssea 4/genética , Células Cultivadas , Desidroepiandrosterona , Regulação para Baixo/efeitos dos fármacos , Estrogênios/metabolismo , Feminino , Regulação Enzimológica da Expressão Gênica , Técnicas de Silenciamento de Genes , Células da Granulosa/efeitos dos fármacos , Células da Granulosa/metabolismo , Células da Granulosa/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovário/efeitos dos fármacos , Ovário/patologia , Síndrome do Ovário Policístico/metabolismo , Síndrome do Ovário Policístico/patologia , Interferência de RNA , Receptores Androgênicos/química , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad4/antagonistas & inibidores , Proteína Smad4/genética , Células Tecais/efeitos dos fármacos , Células Tecais/metabolismo , Células Tecais/patologiaRESUMO
BACKGROUND: Cervical lesions caused by integrated human papillomavirus (HPV) infection are highly dangerous because they can quickly develop into invasive cancers. However, clinicians are currently hampered by the lack of a quick, convenient and precise technique to detect integrated/mixed infections of various genotypes of HPVs in the cervix. This study aimed to develop a practical tool to determine the physical status of different HPVs and evaluate its clinical significance. METHODS: The target population comprised 1162 women with an HPV infection history of > six months and an abnormal cervical cytological finding. The multiple E1-L1/E6E7 ratio analysis, a novel technique, was developed based on determining the ratios of E1/E6E7, E2/E6E7, E4E5/E6E7, L2/E6E7 and L1/E6E7 within the viral genome. Any imbalanced ratios indicate integration. Its diagnostic and predictive performances were compared with those of E2/E6E7 ratio analysis. The detection accuracy of both techniques was evaluated using the gold-standard technique "detection of integrated papillomavirus sequences" (DIPS). To realize a multigenotypic detection goal, a primer and probe library was established. RESULTS: The integration rate of a particular genotype of HPV was correlated with its tumorigenic potential and women with higher lesion grades often carried lower viral loads. The E1-L1/E6E7 ratio analysis achieved 92.7% sensitivity and 99.0% specificity in detecting HPV integration, while the E2/E6E7 ratio analysis showed a much lower sensitivity (75.6%) and a similar specificity (99.3%). Interference due to episomal copies was observed in both techniques, leading to false-negative results. However, some positive results of E1-L1/E6E7 ratio analysis were missed by DIPS due to its stochastic detection nature. The E1-L1/E6E7 ratio analysis is more efficient than E2/E6E7 ratio analysis and DIPS in predicting precancerous/cancerous lesions, in which both positive predictive values (36.7%-82.3%) and negative predictive values (75.9%-100%) were highest (based on the results of three rounds of biopsies). CONCLUSIONS: The multiple E1-L1/E6E7 ratio analysis is more sensitive and predictive than E2/E6E7 ratio analysis as a triage test for detecting HPV integration. It can effectively narrow the range of candidates for colposcopic examination and cervical biopsy, thereby lowering the expense of cervical cancer prevention.
Assuntos
Técnicas de Genotipagem/métodos , Proteínas Oncogênicas Virais/genética , Papillomaviridae/genética , Neoplasias do Colo do Útero/virologia , Adulto , Fatores Etários , Sequência de Bases , Feminino , Dosagem de Genes , Genótipo , Humanos , Reprodutibilidade dos Testes , Neoplasias do Colo do Útero/patologia , Integração ViralRESUMO
Polycystic ovary syndrome (PCOS) is the leading cause of anovulatory infertility. Inadequate understanding of the ovulation drivers hinders PCOS intervention. Herein, we report that follicle stimulating hormone (FSH) controls follicular fluid (FF) glutamine levels to determine ovulation. Murine ovulation starts from FF-exposing granulosa cell (GC) apoptosis. FF glutamine, which decreases in pre-ovulation porcine FF, elevates in PCOS patients FF. High-glutamine chow to elevate FF glutamine inhibits mouse GC apoptosis and induces hormonal, metabolic, and morphologic PCOS traits. Mechanistically, follicle-development-driving FSH promotes GC glutamine synthesis to elevate FF glutamine, which maintain follicle wall integrity by inhibiting GC apoptosis through inactivating ASK1-JNK apoptotic pathway. FSH and glutamine inhibit the rapture of cultured murine follicles. Glutamine removal or ASK1-JNK pathway activation with metformin or AT-101 reversed PCOS traits in PCOS models that are induced with either glutamine or EsR1-KO. These suggest that glutamine, FSH, and ASK1-JNK pathway are targetable to alleviate PCOS.
Assuntos
Hormônio Foliculoestimulante , Glutamina , Células da Granulosa , Ovulação , Síndrome do Ovário Policístico , Animais , Feminino , Células da Granulosa/metabolismo , Células da Granulosa/efeitos dos fármacos , Glutamina/metabolismo , Camundongos , Hormônio Foliculoestimulante/metabolismo , Síndrome do Ovário Policístico/metabolismo , Síndrome do Ovário Policístico/patologia , Humanos , Apoptose/efeitos dos fármacos , MAP Quinase Quinase Quinase 5/metabolismo , MAP Quinase Quinase Quinase 5/genética , Suínos , Camundongos Endogâmicos C57BLRESUMO
Polycystic ovary syndrome (PCOS), a prevalent reproductive disorder in women of reproductive age, features androgen excess, ovulatory dysfunction, and polycystic ovaries. Despite its high prevalence, specific pharmacologic intervention for PCOS is challenging. In this study, we identified artemisinins as anti-PCOS agents. Our finding demonstrated the efficacy of artemisinin derivatives in alleviating PCOS symptoms in both rodent models and human patients, curbing hyperandrogenemia through suppression of ovarian androgen synthesis. Artemisinins promoted cytochrome P450 family 11 subfamily A member 1 (CYP11A1) protein degradation to block androgen overproduction. Mechanistically, artemisinins directly targeted lon peptidase 1 (LONP1), enhanced LONP1-CYP11A1 interaction, and facilitated LONP1-catalyzed CYP11A1 degradation. Overexpression of LONP1 replicated the androgen-lowering effect of artemisinins. Our data suggest that artemisinin application is a promising approach for treating PCOS and highlight the crucial role of the LONP1-CYP11A1 interaction in controlling hyperandrogenism and PCOS occurrence.
Assuntos
Proteases Dependentes de ATP , Artemisininas , Enzima de Clivagem da Cadeia Lateral do Colesterol , Proteínas Mitocondriais , Síndrome do Ovário Policístico , Animais , Feminino , Humanos , Camundongos , Ratos , Androgênios/metabolismo , Artemisininas/uso terapêutico , Artemisininas/farmacologia , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Modelos Animais de Doenças , Hiperandrogenismo/tratamento farmacológico , Hiperandrogenismo/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Ovário/efeitos dos fármacos , Ovário/metabolismo , Síndrome do Ovário Policístico/tratamento farmacológico , Proteólise , Camundongos Endogâmicos C57BL , Adulto Jovem , Adulto , Ratos Sprague-Dawley , Proteases Dependentes de ATP/genética , Proteases Dependentes de ATP/metabolismoRESUMO
OBJECTIVE: To observe the gene expression of herpes simplex virus type 1 thymidine kinase (HSVl-tk) in rat malignant ovarian tumor tissues and the therapeutic effect of ganciclovior (GCV) after intra-arterial infusion of HSVl-tk gene therapy mediated by GE7-delivery system. METHODS: A GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 4-element complex was constructed. Eighteen rats with DMBA-induced ovarian tumor were divided into 3 groups as Atk, ANS and Vtk groups. The 4-element complex GE7-polylysine/pCMV-HSV1-tk/polylysine-HA20 was injected via the ovarian artery into the rats of Atk group, saline buffer was injected in the ANS groups, and the 4-element complex was injected via the tail vein into the rats of Vtk group. All rats received intraperitoneal injection of GCV in a dose of 50 mg/kg daily for 10 days. The rats were sacrificed 3 days after the final dose of GCV, and the tumor weight was measured and tumor growth inhibition rate was calculated. Flow cytometry was used to assess the cell cycle and apoptosis. RESULTS: The tumor weight in the rats of Atk group was (4.77 ± 2.31) g, significantly lower than that of ANS group [(14.66 ± 6.26) g, P < 0.01] and Vtk group [(17.53 ± 7.19) g, P < 0.01]. The tumor growth inhibition rate of the Atk group was 67.5%, while that of Vtk group was -19.6%. The flow cytometry showed that S-phase tumor cells in the Atk group were (54.32 ± 9.65)%, significantly higher than that in the ANS (27.43 ± 9.22)% and (30.16 ± 11.57)% in the Vtk group (both P < 0.01). The tumor cell apoptosis rate in the Atk group was (39.15 ± 12.16)%, significantly higher than that in the ANS group [(11.86 ± 5.28)%, P < 0.01] and Vtk group [(14.32 ± 6.43)%, P < 0.01]. CONCLUSION: HSV1-tk/GCV gene therapy system mediated by GE7 non-viral delivery system via ovarian arterial infusion effectively causes cell cycle arrest at S phase and enhances cell apoptosis, therefore, exerts an inhibitory effect on tumor growth.
Assuntos
Ganciclovir/farmacologia , Técnicas de Transferência de Genes , Terapia Genética , Herpesvirus Humano 1/genética , Neoplasias Ovarianas/terapia , Timidina Quinase/metabolismo , 9,10-Dimetil-1,2-benzantraceno , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Animais , Antivirais/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Feminino , Herpesvirus Humano 1/metabolismo , Infusões Intra-Arteriais , Neoplasias Ovarianas/induzido quimicamente , Neoplasias Ovarianas/patologia , Distribuição Aleatória , Ratos , Ratos Wistar , Timidina Quinase/genéticaRESUMO
Background: Circulating tumor cells (CTCs) have considered to be promising liquid biopsy in cancer due to the intact information of whole cells and the potential to reflect micrometastasis. However, CTCs research are extremely limited in ovarian cancer, probably due to their rarity. The predictive value of CTCs and circulating tumor microemboli (CTM) in metastasis remains to be elucidated in ovarian cancer. This study tried to identify CTCs/CTM in ovarian cancer with considerably positive rate. To preliminarily identify the invasive capacity of CTCs/CTM, the epithelial-mesenchymal transition (EMT) patterns of CTCs/CTM was evaluated. Moreover, for comprehensive understanding of invasiveness of disseminated cells in ovarian cancer, EMT pattern of exfoliated tumor cells in ascites were also confirmed in this study. Methods: Peripheral blood samples and ascites samples were collected from 22 ovarian cancer patients. The Microfiltration combined with morphological analysis was used to detect CTC single cells or cell clusters. Microfiltration combined with morphological analysis was applied in the CTC isolation and identification. EMT was evaluated by immunofluorescence via markers including vimentin and cytokeratin. Results: Microfiltration combined with morphological analysis was introduced to detect CTCs/CTM with a positivity rate of 40.9% in ovarian cancer patients. The number of CTC varied from 1 to 8, with CTM number from 4 to 30. CTCs/CTM of all samples have experienced EMT process. Vimentin was expressed in all CTC samples and all tumor cells in ascites, while cytokeratin was expressed in 44.4% (4/9) of CTC samples. There were no significant differences of the clinical parameters between the CTC-positive and CTC-negative patients. Conclusions: This study showed that both CTCs/CTM and detached tumor cells in ascites might have undergone complete or partial EMT in ovarian cancer. Moreover, microfiltration combined with cytomorphological analysis showed a considerable CTC detection rate.
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POF (premature ovarian failure) is a distressing condition that is a common cause of infertility. No effective treatment is available to overcome the loss of fertility. A method to derive oestrogen from miPSCs (mouse-induced pluripotent stem cells) was explored as a potential treatment for POF. In this study, C57BL/6 female mice were injected with PMSG (pregnant mare's serum gonadotropin) to obtain ovarian GCs (granulosa cells) and then co-cultured with miPSCs. The morphological changes in the miPSCs co-cultured with GCs were observed by light microscopy. The expression of FSHR (follicle-stimulating hormone receptor) was detected by immunocytochemistry and flow cytometry. Radioimmunoassay was used to analyse the level of E2 (oestradiol) in culture supernatants. The results showed that the proportion of GCs expressing FSHR in GCs was over 90%. The E2 concentration of the culture supernatant of the GC group was 62.4 pg/ml on day 1 and decreased in a time-dependent manner. The opposite situation was observed in the miPSCs-GC co-cultured group with an E2 concentration of 87.9 pg/ml on day 1 that increased in a time-dependent manner to reach a concentration of 328.4 pg/ml on day 7. The data indicate that GC-like cells were effectively induced from miPSCs through indirect cell-to-cell contact. Our method provides a novel in vitro system to study miPSC differentiation, particularly the interactions between miPSCs and GCs. The ultimate goal of this approach would be to provide a treatment for POF in the future.
Assuntos
Estradiol/metabolismo , Estrogênios/metabolismo , Células da Granulosa/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Técnicas de Cocultura , Feminino , Gonadotropinas Equinas/farmacologia , Células da Granulosa/citologia , Imuno-Histoquímica , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores do FSH/metabolismoRESUMO
OBJECTIVE: To observe the gene and protein expression of herpes simplex virus type I-thymidine kinase (HSV(1)-tk) in the ovarian tumor tissues and other organs after arterial infusion of HSV(1)-tk gene mediated by GE7 delivery system. METHODS: GE7-polylysine/pCMV-HSV(1)-tk/polylysine-HA20 complexes were constructed. Nine rats with induced ovarian tumor were divided into 3 groups, injecting the 4-element complexes or saline buffer through the ovarian artery and complexes through the tail vein, respectively. The ovarian tumors, hearts, livers, spleens, lungs and kidneys were obtained at 72 hours after injection. RT-PCR and Western Blot were preceeded to determine the expression of HSV(1)-tk gene and protein in the tumor tissues and other organs. RESULTS: In the group of arterial injection with 4-element complexes, the HSV(1)-tk gene and protein were expressed strongly in the tumor tissues, while little or none was detected in other organs. In the group of arterial injection with saline buffer, no HSV(1)-tk gene and protein was detected in both tumor tissues and other organs. In the group of tail vein injection, none was detected in tumor tissues and only little was found in the livers, spleens, lungs and kidneys. CONCLUSION: High target and gene transfer rates can be obtained when HSV(1)-tk gene is transferred via the artery route mediated by GE7 delivery system. HSV(1)-tk protein can be expressed after the gene transfer. The results may provide a new strategy for target killing of HSV(1)-tk/GCV system in ovarian tumors.
Assuntos
Adenocarcinoma/metabolismo , Técnicas de Transferência de Genes , Herpesvirus Humano 1/genética , Neoplasias Ovarianas/metabolismo , Timidina Quinase/biossíntese , 9,10-Dimetil-1,2-benzantraceno , Adenocarcinoma/induzido quimicamente , Adenocarcinoma/genética , Animais , Feminino , Infusões Intra-Arteriais , Neoplasias Ovarianas/induzido quimicamente , Neoplasias Ovarianas/genética , RNA Mensageiro/metabolismo , Distribuição Aleatória , Ratos , Ratos Wistar , Timidina Quinase/genéticaRESUMO
OBJECTIVE: To investigate the effects and possible mechanisms of platelet-activating factor (PAF) on the invasion of ovarian cancer cells and to provide a potential target for ovarian cancer therapy. METHODS: (1)Serous type ovarian cancer cell line OVCA429 with platelet-activating factor receptor (PAFR) positive and mucinous type cell line RMUG-L (PAFR negative) were treated with 100 nmol/L of the PAF, cell invasion ability was determined by transwell cell migration assay. (2) For determination of the optimal PAF concentration, ovarian cancer cell OVCA429 was treated by 0, 0.1, 1, 10, 100, and 1000 nmol/L of PAF for 10 minutes or 24 hour, respectively. To observe the different time point of protein changes, OVCA429 were treated by 100 nmol/L of PAF for 0, 5 minutes, 10 minutes, 30 minutes, 1 hour or 12 hours, respectively. The total proteins of treated cells were extracted according to standard protocol. The expression of p38 mitogen-activated protein kinase (p38 MAPK), phosphorylated p38 MAPK (p-p38 MAPK), transcription factor response element-binding protein (CREB), phosphorylated CREB (p-CREB) and matrix metalloproteinase-2 (MMP-2) were detected by western blot. (3) To verify the pathway involved in the PAF induction of the cancer cell invasion, we repeated the experiments by adding the inhibitors when treating cells with PAF. The inhibitors used were as follows, PAFR inhibitor-WEB2076 (50 µmol/L), p-p38 MAPK inhibitor-SB203580 (10 µmol/L), CREB binding protein (CBP)-CREB interaction inhibitor-217505(25 µmol/L). All treated cells were divided into 6 groups: control group, PAF group, PAF + WEB2076 group, PAF + SB203580 group, PAF + 217505 group and PAF + SB203580 + 217505 group. RESULTS: (1) By transwell assay, 100 nmol/L of PAF could improve the invasion ability of OVCA429 cell significantly [PAF: (118 ± 23) cells vs. control: (36 ± 8) cells, P < 0.01], while the same treatment had no effect on RMUG-L cells [PAF: (45 ± 13) cells vs. control: (53 ± 9) cells, P > 0.05]. (2) Even a very low concentration of PAF (0.1 nmol/L) could increase the expression of p-CREB and MMP-2, while the most effective concentration of PAF was 100 nmol/L. The highest p-CREB protein expression was detected at 10 minutes after administration of 100 nmol/L PAF, as well as the expression of p-p38 MAPK protein. Even 12 hours after treatment the p-p38 MAPK protein could be detected, while there was no significant difference in the expression of CREB (P > 0.05). (3) As compared with PAF group, both in PAF + WEB2076 group and PAF + SB203580 group, the expressions of p-p38 MAPK, p-CREB and MMP-2 protein were decreased significantly; in PAF + 217505 group, although the expression of p-p38 MAPK and p-CREB protein was significantly higher than the control group, the expression of MMP-2 protein was significantly lower; in PAF + SB203580 + 217505 group, the expression of these three proteins were also significantly lower, but there was no significant difference as compared with that in the PAF + WEB2076 or PAF + SB203580 group. CONCLUSION: PAF could induce MMP-2 expression and contributed to PAFR positive ovarian cancer cell invasion via activation of CREB by phosphorylating of p38 MAPK.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Fator de Ativação de Plaquetas/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Plaquetas , Western Blotting , Carcinoma Epitelial do Ovário , Ensaios de Migração Celular , Feminino , Humanos , Imidazóis , Técnicas In Vitro , Neoplasias Epiteliais e Glandulares , Neoplasias Ovarianas , Fosforilação/efeitos dos fármacos , Glicoproteínas da Membrana de Plaquetas , Piridinas , Receptores Acoplados a Proteínas G , Fatores de TempoRESUMO
BACKGROUND: CA125 is the most widely used serum marker for preoperative diagnosis of ovarian cancer. However, CA125 elevation is not specific to ovarian cancer. More than 60% of patients who have elevated CA125 levels do not have ovarian cancer. To overcome the low specificity of CA125, we identified a CA125 glycoform that was specifically elevated in ovarian cancer and that may help in the further triage of patients with elevated serum CA125 levels. METHODS: We used antibody-lectin ELISA to detect various CA125 glycoforms. Among 21 lectins tested, VVA, a plant lectin that preferentially binds Tn antigen, showed significantly stronger binding with ovarian cancer-derived CA125 than benign condition-derived CA125. CA125-Tn levels were tested among patients with elevated CA125 levels (n=328, including 68 ovarian cancer, 15 ovarian borderline tumors, and 245 benign conditions). The efficacy of CA125-Tn in diagnosing ovarian cancer was evaluated using ROC analysis. RESULTS: Medians and 25th to 75th quartiles of CA125-Tn levels were 0.31 (0.18-0.65) in ovarian cancer, 0.07 (0.02-0.12) in ovarian borderline tumor, and 0.07 (0.01-0.12) in benign conditions. AUC of the ROC curve was 0.890 (95% CI: 0.845, 0.935) for CA125-Tn to discriminate ovarian cancer cases from nonmalignant cases (borderline tumors and benign conditions). Its performance was even better among patients older than 45 y (AUC: 0.905, 95% CI: 0.841, 0.969). Specificity was improved from 35.1% for CA125 to 75.7% for CA125-Tn among patients older than 45 y when sensitivity was fixed at 90%. CONCLUSIONS: CA125-Tn ELISA assay can improve specificity of the preoperative diagnosis of ovarian cancer and serve as a further triage strategy for patients with elevated CA125 levels.
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OBJECTIVE: To establish a satisfactory lung metastasis model of human choriocarcinoma using severe combined immunodeficient (SCID) mice and explore the appropriate cell concentration for the model. METHODS: Forty SCID mice aged between 5 - 6 weeks were randomly divided into four groups. 1 × 10(7) cells/ml × 0.1 ml, 5 × 10(6) cells/ml × 0.2 ml and 1 × 10(6) cells/ml × 0.1 ml of human choriocarcinoma cells JEG-3 were respectively injected in SCID mice of experimental groups by lateral tail vein, the remain group was assigned to the control group. The status and weight of mice were observed every three days. When these mice were being dying, the size and the number of the lesions of lung metastasis in every mouse were inspected with Micro CT. After Micro CT inspection, the SCID mice were executed dissected to note whether there were tumors on all organ surfaces with naked eyes, then made pathological sections from the metastatic foci of fresh lung tissues, and cultured primarily cells and purified cells and passaged cells isolated from the same metastastic foci. The pathological sections were observed under the microscope. The special antigen human chorionic gonadotropin-beta subunit (ß-hCG) of the choriocarcinoma cells was immunohistochemically detected in the pathological sections and the cells out of cultured primarily cells. The chromosomes of the cells out of cultured primarily cells were analysed. RESULTS: Of the group inoculated 1 × 10(7) cells/ml × 0.1 ml, all mice died when inoculating. In the group of 5 × 10(6) cells/ml × 0.2 ml, when inoculating, 3 mice died; the remain 7 mice were being dying on (18.0 ± 2.0) days after injection. 5 of them, there were 1 - 3 lesions of lung metastasis after Micro CT inspection in each mice, and the diameter of the tumors lesions reached 1.5 - 3.5 mm, which was choriocarcinoma confirmed by pathological sections. The special antigen ß-hCG was detected by immunohistochemical method in the pathological sections of pulmonary tissue with tumor and in the cells, which were purified and passaged from being cultured primarily cells isolated from metastastic foci of fresh lung tissues from the SCID mice. The chromosome numbers of these cells out of cultured primarily cells were variety from 19 to 128, and modal numbers were variety from 70 to 79. CONCLUSIONS: We successfully established the lung metastatic model of human choriocarcinoma in SCID mice by injecting JEG-3 cells into lateral tail vein, of which 5 × 10(6) cells/ml × 0.2 ml is the suitable concentration and volume for the model.
Assuntos
Coriocarcinoma/patologia , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Modelos Animais de Doenças , Neoplasias Pulmonares/secundário , Animais , Índice de Massa Corporal , Linhagem Celular Tumoral , Aberrações Cromossômicas , Feminino , Humanos , Imuno-Histoquímica , Cariotipagem , Pulmão/patologia , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Camundongos , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias , Coloração e RotulagemRESUMO
OBJECTIVE: To investigate the expression of exogenous gene transferred by piggyBac (PB) transposon in various gynecological malignant cell lines and reveal its potential application of gene therapy in gynecological cancer. METHODS: Amplified herpes simplex virus thymidine kinase (HSV-tk) gene coding region by PCR and integrated it into PB expression vector, PB[Act-RFP]DS, for reconstructing PB[Act-RFP, HSV-tk]DS (pPB/TK). By using different transfection reagents: FuGENE HD, jetPEI, lipofectamine 2000, pPB/TK together with helper plasmid Act-PBase were cotransfected into four mostly common gynecological malignant tumor cell lines HeLa, JEG-3, SKOV3 and HEC-1B. The mRFP1 report gene expressions was observed and detected by fluorescence microscope and flow cytometry to analyze transfection efficiency. The expressions of HSV-tk and mRFP1 gene were detected by reverse transcription PCR (RT-PCR). The cytotoxic effect of various concentration of pro-drug ganciclovir (GCV) for transfected cells was detected by methyl thiazole tetrazolium assay. The transfected cells were positive sorted by flow cytometry and limiting diluted to obtain the stable transfected cell line. The insertion sites of foreign gene transferred by PB transposon in genome were analyzed by inverse PCR. RESULTS: (1) Double digests analysis and sequences test demonstrated that pPB/TK vector was reconstructed successfully. (2) Using three different transfective reagents, PB transposon transferred HSV-tk gene and mRFP1 gene into HeLa, HEC-1B, SKOV3 and JEG-3 cell efficiently, and the transfection efficiency of pPB/TK for the same cell was different by using different transfective reagents; in Hela cell, the transfection efficiency of FuGENE HD [(78.7+/-9.2)%] was higher than that of lipofectamine 2000 [(54.1+/-11.4)%] and jetPEI [(46.5+/-7.4)%, all P<0.05]; using the same transfective reagent, the transfection efficiency of pPB/TK was also different on various cell lines, using FuGENE HD, the transfection efficiency of pPB/TK on HeLa, JEG-3 and SKOV3 cell was (78.7+/-9.2)%, (74.4+/-8.9)% and (83.2+/-9.7)% respectively, which all were higher than that on HEC-1B [(39.5+/-8.7)%, P<0.05]. (3) RT-PCR showed that there were the mRNA expression of HSV-tk and mRFP1 in all cell lines. (4) 50% inhibitory concentration of GCV for transfected cells, HeLa, JEG-3, SKOV3 and HEC-1B, was 1.29, 3.35, 0.09 and 13.28 microg/ml respectively. Inhibitory effect of GCV (10 microg/ml) on SKOV3 transfected with pPB/TK was (86+/-9)%, which was superior to that transfected with pORF-HSVtk alone [(52+/-12)%, P<0.05]. (5) The insertion sites of PB transposon in the target cells genome were located at TTAA sites. mRFP1 expression still could be detected in three months after transfected. CONCLUSIONS: PB transposon could transfer exogenous gene into various gynecological malignant cells, which could integrated into genome and obtain a long-term and stable expression. It is expected that PB transposon may supply a more efficient and safer transgene technology platform for gene therapy in gynecological cancer.
Assuntos
Elementos de DNA Transponíveis/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Proteínas Luminescentes/genética , Timidina Quinase/genética , Sobrevivência Celular , Feminino , Citometria de Fluxo , Ganciclovir/administração & dosagem , Ganciclovir/farmacologia , Expressão Gênica , Terapia Genética , Neoplasias dos Genitais Femininos/genética , Neoplasias dos Genitais Femininos/metabolismo , Neoplasias dos Genitais Femininos/terapia , Células HeLa , Humanos , Proteínas Luminescentes/metabolismo , Plasmídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simplexvirus/genética , Simplexvirus/metabolismo , Timidina Quinase/metabolismo , Transfecção , Transgenes , Proteína Vermelha FluorescenteRESUMO
PURPOSE: While numerous epidemiological studies have indicated that omega-3 polyunsaturated fatty acids have anticancer properties in various cancers, the effects and mechanisms of eicosapentaenoic acid (EPA) in ovarian cancer cell growth are poorly understood. MATERIALS AND METHODS: ES2 ovarian clear cell carcinoma cells and SKOV3 adenocarcinoma cells were treated with palmitic acid or EPA, followed by flow cytometry and cell counting to measure apoptosis and proliferation, respectively. A modified protein lipid overlay assay was used to further verify whether EPA was a ligand of G protein-coupled receptor 30 (GPR30) in ES2 cells. The levels of apoptosis-related genes, phosphorylated AKT, and phosphorylated ERK1/2 were detected to explore the underlying mechanism. Finally, inhibitory effect of EPA on tumor growth via GPR30 was determined in vitro and in vivo. RESULTS: EPA suppressed ES2 ovarian clear cell carcinoma cells growth via GPR30, a novel EPA receptor, by inducing apoptosis. As a ligand of GPR30, EPA activated the GPR30-cAMP- protein kinase A signaling pathway. When GPR30 was suppressed by siRNA or its inhibitor G15, the antiproliferative action of EPA was impaired. Furthermore, EPA inhibited tumor growth by blocking the activation of AKT and ERK. In the mouse xenograft model, EPA decreased tumor volume and weight through GPR30 by blocking tumor cell proliferation. CONCLUSION: These results confirm that EPA is a tumor suppressor in human ovarian clear cell carcinoma cells and functions through a novel fatty acid receptor, GPR30, indicating a mechanistic linkage between omega-3 fatty acids and cancers.
Assuntos
Adenocarcinoma de Células Claras/tratamento farmacológico , Biomarcadores Tumorais/metabolismo , Ácido Eicosapentaenoico/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adenocarcinoma de Células Claras/metabolismo , Adenocarcinoma de Células Claras/patologia , Animais , Apoptose , Biomarcadores Tumorais/genética , Ciclo Celular , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Prognóstico , Receptores de Estrogênio/genética , Receptores Acoplados a Proteínas G/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Gene-directed enzyme prodrug therapy (GDEPT) is a strategy developed to selectively target cancer cells. However, the clinical benefit is limited due to its poor gene transfer efficiency. To overcome this obstacle, we took advantage of piggyBac (PB) transposon, a natural non-viral gene vector that can induce stable chromosomal integration and persistent gene expression in vertebrate cells, including human cells. To determine whether the vector can also mediate stable gene expression in ovarian cancer cells, we constructed a PB transposon system that simultaneously expresses the Herpes simplex virus thymidine kinase (HSV-tk) gene and the monomeric red fluorescent protein (mRFP1) reporter gene. The recombinant plasmid, pPB/TK, was transfected into ovarian adenocarcinoma cells SKOV3 with FuGENE HD reagent, and the efficiency was given by the percentage of mRFP1-positive cells detected by flow cytometry and confocal microscopy. The specific expression of HSV-tk in transfected cells was confirmed by RT-PCR and western blotting. The sensitivity of transfected cells to pro-drug ganciclovir (GCV) was determined by methylthiazoletetrazolium (MTT) assay. A total of 56.4 +/- 8.4% cells transfected with pPB/TK were mRFP1 positive, compared to no measurable mRFP1 expression in pORF-HSVtk-transfected cells. The expression level of HSV-tk in pPB/TK-transfected cells was 10 times higher than in pORF-HSVtk-transfected cells. The results show that pPB/TK transfection increases the sensitivity of cells to GCV in a dose-dependent manner. Our data indicate that the PB transposon system could enhance the anti-tumor efficiency of GDEPT in ovarian cancer.
Assuntos
Adenocarcinoma/terapia , Elementos de DNA Transponíveis/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Neoplasias Ovarianas/terapia , Pró-Fármacos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Feminino , Ganciclovir/farmacologia , Humanos , Proteínas Luminescentes/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Timidina Quinase/genética , Transfecção , Proteína Vermelha FluorescenteRESUMO
BACKGROUND: Decidual γδ T cells are known to regulate the function of trophoblasts at the maternal-fetal interface; however, little is known about the molecular mechanisms of cross talk between trophoblast cells and decidual γδ T cells. METHODS: Expression of chemokine C-X-C motif ligand 6 (CXCL16) and its receptor CXCR6 was evaluated in first-trimester human villus and decidual tissues by immunohistochemistry. γδ T cells were isolated from first-trimester human deciduae and cocultured with JEG3 trophoblast cells. Cell proliferation and apoptosis-related molecules, together with cytotoxicity factor and cytokine production, were measured by flow cytometry analysis. RESULTS: Expression of CXCL16 and CXCR6 was reduced at the maternal-fetal interface in patients who experienced unexplained recurrent spontaneous abortion as compared to healthy pregnancy women. With the administration of pregnancy-related hormones or coculture with JEG3 cells, CXCR6 expression was upregulated on decidual γδ T cells. CXCL16 derived from JEG3 cells caused a decrease in granzyme B production of decidual γδ T cells. In addition, decidual γδ T cells educated by JEG3-derived CXCL16 upregulated the expression of Bcl-xL in JEG3 cells. CONCLUSION: This study suggested that the CXCL16/CXCR6 axis may contribute to maintaining normal pregnancy by reducing the secretion of cytotoxic factor granzyme B of decidual γδ T cells and promoting the expression of antiapoptotic marker Bcl-xL of trophoblasts.
Assuntos
Quimiocina CXCL16/metabolismo , Decídua/metabolismo , Granzimas/metabolismo , Trofoblastos/metabolismo , Proteína bcl-X/metabolismo , Sobrevivência Celular , Células Cultivadas , Vilosidades Coriônicas/metabolismo , Feminino , Humanos , Gravidez , Primeiro Trimestre da Gravidez , Receptores CXCR6/metabolismoRESUMO
OBJECTIVE: To establish the high-performance liquid chromatography (HPLC) method for measuring concentrations of methotrexate (MTX) in rat lung and some other tissues through internal iliac artery infusion. METHODS: Fifty female Sprague-Dawley rats were included in this study. The rats were randomly assigned to two groups. Methotrexate was injected to group one through internal iliac artery, and was injected to group two through femoral vein. Blood and tissues were collected in each group at 15, 30, 60, 90 and 120 minutes for detection of the drug concentrations with HPLC. RESULTS: The area under the concentration time curve (AUC) in rat lung, ovary and uterus in the artery group were separately (3.77 +/- 0.28), (4.40 +/- 0.40), (9.97 +/- 0.89) microgxh(-1)xg(-1), which were significantly different from those of the vein group [(2.31 +/- 0.25), (3.91 +/- 0.19), (7.65 +/- 1.54) microgxh(-1)xg(-1); P < 0.05]. The AUC in the rat plasma, heart, kidney, liver and spleen in the artery group were separately (6.13 +/- 0.53), (1.90 +/- 0.11), (5.32 +/- 0.89), (14.16 +/- 1.96), (0.76 +/- 0.20) microgxh(-1)xg(-1). There were no significant differences from the vein group [(5.79 +/- 0.71), (1.64 +/- 0.29), (5.15 +/- 1.69), (14.29 +/- 3.47), (0.76 +/- 0.13) microgxh(-1)xg(-1); P > 0.05]. CONCLUSIONS: Through internal iliac artery infusion, there are higher drug concentrations in lung, uterus and ovarian compared to venous injection. The internal-arterial chemotherapy may be used to treat pulmonary metastasis of gynecological tumor.
Assuntos
Neoplasias dos Genitais Femininos/tratamento farmacológico , Infusões Intra-Arteriais , Pulmão/metabolismo , Metotrexato/farmacocinética , Ovário/metabolismo , Animais , Área Sob a Curva , Cromatografia Líquida de Alta Pressão , Feminino , Neoplasias dos Genitais Femininos/patologia , Artéria Ilíaca , Infusões Intravenosas , Rim/metabolismo , Fígado/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/secundário , Metotrexato/administração & dosagem , Metotrexato/sangue , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual , Útero/metabolismoRESUMO
OBJECTIVE: To prepare follicle stimulating hormone (FSH) polypeptide modified nanoparticles (NP) in order to achieve specific ovarian tumor targeting. METHODS: Expression of FSH receptor protein in human liver cancer and ovarian cancer cell lines BEL-7402, SKOV-3 and Caov-3 was detected by immunocytochemistry. The polypeptide fragment of FSH beta 81-95 amino acids (FSHL81-95) was synthesized and covalently coupled to NP. The specific binding of FSHL81-95 and FSHL81-95-NP was examined by fluorescence microscopy and flow cytometry. RESULTS: BEL-7402 and SKOV-3 cells were negative for FSH receptor staining, while Caov-3 cells were positive. The diameters of NP were about 100 nm, with a Zeta potential of -25 mV or so. Caov-3 cells showed a more specific interaction with FSHL81-95-NP than SKOV-3 cells (4.17 +/- 0.86 and 2.30 +/- 0.21; P < 0.05). The uptake of FSHL81-95-NP was more than NP in Caov-3 cells (4.17 +/- 0.86 and 0.41 +/- 0.32; P < 0.05). FSHL81-95-NP showed a selective targeting at Caov-3 cells compared with control NP. CONCLUSION: FSH polypeptide modified NP could selectively target ovarian cancer cells expressing FSH receptor, which might contribute to specific endocytosis mediated by FSH receptor.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Subunidade beta do Hormônio Folículoestimulante/farmacologia , Nanopartículas , Neoplasias Ovarianas/patologia , Peptídeos/farmacologia , Receptores do FSH/metabolismo , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Hormônio Foliculoestimulante Humano/administração & dosagem , Hormônio Foliculoestimulante Humano/farmacologia , Subunidade beta do Hormônio Folículoestimulante/administração & dosagem , Humanos , Neoplasias Hepáticas/patologia , Tamanho da Partícula , Peptídeos/administração & dosagemRESUMO
The high mortality rate of ovarian cancer is connected with the development of acquired resistance to multiple cancer drugs, especially cisplatin. Activation of cytoprotective autophagy has been implicated as a contributing mechanism for acquired cisplatin resistance in ovarian cancer cells. Hexokinase 2 (HK2) phosphorylates glucose to generate glucose-6-phosphate, the rate-limiting step in glycolysis. Higher HK2 expression has been associated with chemoresistance in ovarian cancer. However, whether HK2 functionally contributes to cisplatin resistance in ovarian cancer is unclear. In this study, we investigated the role of HK2 in regulating ovarian cancer cisplatin resistance. Increased HK2 levels were detected in drug-resistant human ovarian cancer cells and tissues. Cisplatin downregulated HK2 in cisplatin-sensitive but not in resistant ovarian cancer cells. HK2 knockdown sensitized resistant ovarian cancer cells to cisplatin-induced cell death and apoptosis. Conversely, HK2 overexpression in cisplatin-sensitive cells induced cisplatin resistance. Mechanistically, cisplatin increased ERK1/2 phosphorylation as well as autophagic activity. Blocking autophagy with the autophagy inhibitor 3-MA sensitized resistant ovarian cancer cells to cisplatin. HK2 overexpression enhanced cisplatin-induced ERK1/2 phosphorylation and autophagy while HK2 knockdown showed the opposite effects. Blocking the MEK/ERK pathway using the MEK inhibitor U0126 prevented cisplatin-induced autophagy enhanced by HK2 overexpression. Furthermore, HK2 knockdown sensitized resistance ovarian tumor xenografts to cisplatin in vivo. In conclusion, our data supported that HK2 promotes cisplatin resistance in ovarian cancer by enhancing drug-induced, ERK-mediated autophagy. Therefore, targeting HK2 may be a new therapeutic strategy to combat chemoresistance in ovarian cancer.