RESUMO
More than 90% of ovarian cancer deaths are due to relapse following development of chemoresistance. Our main objective is to better understand the molecular mechanism underlying paclitaxel resistance (taxol resistance, Txr) in ovarian cancer. Here, we observed that the linker histone H1.0 is upregulated in paclitaxel-resistant ovarian cancer cells. Knockdown of H1.0 significantly downregulates the androgen receptor (AR) and sensitizes paclitaxel-resistant SKOV3/Txr and 2774/Txr cell lines to paclitaxel. Conversely, ectopic expression of H1.0 upregulates AR and increases Txr in parental SKOV3 and MDAH2774 cells. Notably, H1.0 upregulation is associated with disease recurrence and poor survival in a subset of ovarian cancer subjects. Inhibition of PI3K significantly reduces H1.0 mRNA and protein levels in paclitaxel-resistant cells, suggesting the involvement of the PI3K/AKT signaling pathway. Knockdown of H1.0 and AR also downregulates the Txr genes ABCB1 and ABCG2 in paclitaxel-resistant cells. Our data show that H1.0 induces GCN5 expression and histone acetylation, thereby enhancing Txr gene transactivation. These findings suggest that Txr in ovarian cancer involves the PI3K/AKT pathway and leads to upregulation of histone H1.0, recruitment of GCN5 and AR, followed by upregulation of a subgroup of Txr genes that include ABCB1 and ABCG2. This study is the first report describing the relationship between histone H1.0 and GCN5 that cooperate to induce AR-dependent Txr in ovarian cancer cells.
Assuntos
Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas , Paclitaxel , Receptores Androgênicos , Fatores de Transcrição de p300-CBP , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Recidiva Local de Neoplasia/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Paclitaxel/farmacologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/genética , Receptores Androgênicos/metabolismo , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
We report here that the androgen receptor (AR) and ABCB1 are upregulated in a model of acquired taxol resistance (txr) in ovarian carcinoma cells. AR silencing sensitizes txr cells to taxol threefold, whereas ectopic AR expression in AR-null HEK293 cells induces resistance to taxol by 1.7-fold. AR activation using the agonist dihydrotestosterone (DHT) or sublethal taxol treatment upregulates ABCB1 expression in both txr cells and AR-expressing HEK293 cells. In contrast, AR inactivation using the antagonist bicalutamide downregulates ABCB1 expression and enhances cytotoxicity to taxol. A functional ABCB1 promoter containing five predicted androgen-response elements (AREs) is cloned. Deletion assays reveal a taxol-responsive promoter segment which harbors ARE4. Notably, DHT- or taxol-activated AR potentiates binding of the AR to ARE4 as revealed by the chromatin immunoprecipitation. On the other hand, txr cells display an increase in chromatin remodeling. AR/H3K9ac and AR/H3K14ac complexes bind specifically to ARE4 in response to taxol. Furthermore, acetyltransferase protein levels (p300 and GCN5) are upregulated in txr cells. Silencing of p300 or GCN5 reduces chromatin modification and enhances cytotoxicity in both parental and txr SKOV3 cells. While the phosphatidylinositol 3-kinase (PI3K)/serine/threonine protein kinase (AKT) pathway is significantly activated by taxol, taxol-induced ABCB1 expression, histone posttranslational modifications, and p300 binding to ARE4 are suppressed following inhibition of the PI3K/AKT cellular pathway. These results demonstrate that the AKT/p300/AR axis can be activated to target ABCB1 gene expression in response to taxol, thus revealing a new treatment target to counter taxol resistance.
Assuntos
Cromatina/metabolismo , Genes MDR/genética , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Receptores Androgênicos/metabolismo , Transcrição Gênica/efeitos dos fármacos , Subfamília B de Transportador de Cassetes de Ligação de ATP , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cromatina/genética , Resistencia a Medicamentos Antineoplásicos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores Androgênicos/genéticaRESUMO
We report here that toll-like receptor 4 (TLR4) and ABCB1 are upregulated in SKOV3 ovarian carcinoma cells that acquired resistance to the anticancer drug taxol. Silencing of TLR4 using short-hairpin RNA sensitized taxol-resistant SKOV3 cells to taxol (4.6 fold), whereas ectopic expression of TLR4 in parental, taxol-sensitive SKOV3 cells or TLR4-null HEK293 cells induced taxol resistance (â¼2 fold). A sub-lethal dose of taxol induced ABCB1 protein expression in taxol-resistant SKOV3 cells. Inactivation of TLR4 using chemical inhibitors (CLI-095 and AO-I) downregulated ABCB1 protein expression and enhanced the cytotoxic activity of taxol in taxol-resistant SKOV3 cells. While the sensitization effect of TLR4 inactivation was also detected in TOV21G ovarian cancer cells, which express moderate level of TLR4, ectopic expression of ABCB1 prevented the sensitization effect in these cells. Notably, the NFκB pathway was significantly activated by taxol, and inhibition of this pathway suppressed TLR4-regulated ABCB1 expression. Furthermore, taxol-induced NFκB signaling was reduced following TLR4 silencing in taxol-resistant SKOV3 cells. Consistent with these results, ectopic expression of TLR4 in taxol-sensitive SKOV3 cells enhanced ABCB1 expression and conferred resistance to taxol. The protective effect of exogenous TLR4 expression against taxol was reduced by treatment with NFκB inhibitor in these cells. These results demonstrate that taxol activates the TLR4-NFκB pathway which in turn induces ABCB1 gene expression. This cellular pathway thus represents a novel target to limit resistance to taxol in ovarian cancer cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Carcinoma/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos , NF-kappa B/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Paclitaxel/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Sítios de Ligação , Carcinoma/genética , Carcinoma/metabolismo , Carcinoma/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Concentração Inibidora 50 , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Regiões Promotoras Genéticas , Interferência de RNA , Sulfonamidas/farmacologia , Receptor 4 Toll-Like/antagonistas & inibidores , Receptor 4 Toll-Like/genética , Transfecção , Regulação para CimaRESUMO
Endometrial carcinoma (EC) is one of the main gynecologic malignancies affecting women, but effective treatments are currently lacking. In the present study, we investigated the effect of sorafenib, a general kinase inhibitor, on several EC cell lines (HEC1A, HEC1B, and RL95-2). Sorafenib induced cell death in EC cells with the following order of sensitivity: HEC1A > HEC1B > RL95-2. Sorafenib suppressed several anti-apoptotic proteins in HEC1A cells, including myeloid cell leukemia 1 (Mcl-1). Ectopic overexpression of Mcl-1 prevented the cell killing effect of sorafenib. Sorafenib suppressed Mcl-1 at the gene transactivation level by inactivating the ERK/Elk-1 pathway. Accordingly, the inhibitory effect of sorafenib on Mcl-1 expression decreased following knockdown of Elk-1 using short-hairpin RNA (shRNA). Elk-1 overexpression rescued both the inhibitory effect of sorafenib on Mcl-1 expression and the cell killing effect of sorafenib. Furthermore, sorafenib reduced the stability of the Mcl-1 protein by enhancing its ubiquitination and degradation by the proteasome via the AKT/GSK3ß and the ERK pathways. Similar results were detected in other EC cell lines. These results indicate that sorafenib induces apoptosis in EC cells by down-regulating the anti-apoptotic protein Mcl-1 via transcriptional inhibition and protein degradation. Our results thus support the notion that sorafenib may be used in endometrial cancer therapy.
Assuntos
Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias do Endométrio/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Niacinamida/análogos & derivados , Compostos de Fenilureia/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Transcrição Gênica/efeitos dos fármacos , Proteínas Elk-1 do Domínio ets/metabolismo , Apoptose/genética , Linhagem Celular Tumoral , Neoplasias do Endométrio/tratamento farmacológico , Neoplasias do Endométrio/genética , Neoplasias do Endométrio/patologia , Feminino , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides , Niacinamida/farmacologia , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Sorafenibe , Transcrição Gênica/genética , Proteínas Elk-1 do Domínio ets/genéticaRESUMO
In the present study, we observed that the Golgi-SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) GS28 forms a complex with p53 in HEK (human embryonic kidney)-293 cells. Given that p53 represents a tumour suppressor that affects the sensitivity of cancer cells to various chemotherapeutic drugs, we examined whether GS28 may influence the level of sensitivity to the DNA-damaging drug cisplatin. Indeed, knockdown of GS28 using short-hairpin RNA (shGS28) induced resistance to cisplatin in HEK-293 cells. On the other hand, overexpression of GS28 sensitized HEK-293 cells to cisplatin, whereas no sensitization effect was noted for the mitotic spindle-damaging drugs vincristine and taxol. Accordingly, we observed that knockdown of GS28 reduced the accumulation of p53 and its pro-apoptotic target Bax. Conversely, GS28 overexpression induced the accumulation of p53 and Bax as well as the pro-apoptotic phosphorylation of p53 on Ser(46). Further experiments showed that these cellular responses could be abrogated by the p53 inhibitor PFT-α (pifithrin-α), indicating that GS28 may affect the stability and activity of p53. The modulatory effects of GS28 on cisplatin sensitivity and p53 stability were absent in lung cancer H1299 cells which are p53-null. As expected, ectopic expression of p53 in H1299 cells restored the modulatory effects of GS28 on sensitivity to cisplatin. In addition, GS28 was found to form a complex with the p53 E3 ligase MDM2 (murine double minute 2) in H1299 cells. Furthermore, the ubiquitination of p53 was reduced by overexpression of GS28 in cells, confirming that GS28 enhances the stability of the p53 protein. Taken together, these results suggest that GS28 may potentiate cells to DNA-damage-induced apoptosis by inhibiting the ubiquitination and degradation of p53.
Assuntos
Apoptose/fisiologia , Cisplatino/farmacologia , Complexos Multiproteicos/metabolismo , Proteólise , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteínas Qb-SNARE/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Ubiquitinação/fisiologia , Sequência de Aminoácidos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA/genética , Sinergismo Farmacológico , Células HEK293 , Humanos , Dados de Sequência Molecular , Proteólise/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Qb-SNARE/genética , Ubiquitinação/efeitos dos fármacosRESUMO
The therapeutic activity of paclitaxel against ovarian carcinoma is relatively low due to the frequent occurrence of chemoresistance and disease recurrence. We found earlier that a combination of curcumin and paclitaxel reduces cell viability and promotes apoptosis in paclitaxel-resistant (i.e., taxol-resistant, Txr) ovarian cancer cells. In the present study, we first used RNA sequencing (RNAseq) analysis to identify genes that are upregulated in Txr cell lines but downregulated by curcumin in ovarian cancer cells. The nuclear factor kappa B (NFκB) signaling pathway was shown to be upregulated in Txr cells. Furthermore, based on the protein interaction database BioGRID, we found that Smad nuclear interacting protein 1 (SNIP1) may be involved in regulating the activity of NFκB in Txr cells. Accordingly, curcumin upregulated SNIP1 expression, which in turn downregulated the pro-survival genes Bcl-2 and Mcl-1. Using shRNA-guided gene silencing, we found that SNIP1 depletion reversed the inhibitory effect of curcumin on NFκB activity. Moreover, we identified that SNIP1 enhanced NFκB protein degradation, thereby suppressing NFκB/p65 acetylation, which is involved in the inhibitory effect of curcumin on NFκB signaling. The transcription factor early growth response protein 1 (EGR1) was shown to represent an upstream transactivator of SNIP1. Consequently, we show that curcumin inhibits NFκB activity by modulating the EGR1/SNIP1 axis to attenuate p65 acetylation and protein stability in Txr cells. These findings provide a new mechanism to account for the effects of curcumin in inducing apoptosis and reducing paclitaxel resistance in ovarian cancer cells.
Assuntos
Carcinoma , Curcumina , Neoplasias Ovarianas , Feminino , Humanos , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , NF-kappa B/metabolismo , Curcumina/farmacologia , Curcumina/uso terapêutico , Linhagem Celular Tumoral , Recidiva Local de Neoplasia , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Resistencia a Medicamentos Antineoplásicos , Proteínas de Ligação a RNARESUMO
Ovarian cancer is poorly treatable due, at least in part, to induced drug resistance to taxol- and cisplatin-based chemotherapy. Recent studies showed that ectopic overexpression of toll-like receptor 4 (TLR4) in ovarian cancer cells leads to upregulation of the androgen receptor (AR) and transactivation of taxol resistance genes, thereby causing chemoresistance. In the present study, we examined the signaling pathways involving TLR4 and interleukin 6 (IL-6) that enhance AR expression. Based on transcriptomic analysis, we show that IL-6 functions as a hub gene among the upregulated genes in taxol-treated TLR4-overexpressing ovarian cancer cells. Both the TLR4 activator taxol and IL-6 can induce AKT phosphorylation, whereas TLR4 knockdown or inhibition of the IL-6 signal transducer GP130 abrogates AKT activation. Furthermore, expression of AR and IL-6 is downregulated in TLR4-knockdown, taxol-resistant cells. In addition, TLR4 knockdown inhibits GP130 and IL-6 receptor alpha (IL6Rα) activities, indicating that TLR4 plays a critical role in IL-6 signaling. On the other hand, nuclear translocation of AR is induced by IL-6 treatment, whereas knockdown of endogenous IL-6 reduces AR and TLR4 expression in taxol-resistant ovarian cancer cells. These results indicate that TLR4 and IL-6 play a crucial role in AR gene regulation and function. We also identify interferon regulatory factor 1 (IRF1) as a downstream target of IL-6 signaling and as a regulator of AR expression. Moreover, analysis of clinical samples indicates that high IL-6 expression correlates with poor progression-free survival in ovarian cancer patients treated with taxol. Overall, our findings indicate that the TLR4/IL-6/IRF1 signaling axis represents a potential therapeutic target to overcome AR-based taxol resistance in ovarian cancer.
Assuntos
Fator Regulador 1 de Interferon/biossíntese , Interleucina-6/biossíntese , Neoplasias Ovarianas/metabolismo , Paclitaxel/administração & dosagem , Receptores Androgênicos/biossíntese , Receptor 4 Toll-Like/biossíntese , Antineoplásicos Fitogênicos/administração & dosagem , Biomarcadores Tumorais/biossíntese , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Fator Regulador 1 de Interferon/genética , Interleucina-6/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/genética , Receptores Androgênicos/genética , Receptor 4 Toll-Like/genéticaRESUMO
Toll-like receptor 4 (TLR4) is often overexpressed in taxol-resistant cancer cells. Here we used whole-genome transcriptomic analysis to identify 787 upregulated genes in SKOV3 ovarian carcinoma cells that ectopically express TLR4. Using chromatin immunoprecipitation enrichment analysis, we observed that 27.8% of the TLR4-upregulated genes identified were androgen receptor (AR)-regulated genes. Accordingly, AR expression was induced in taxol-resistant SKOV3 cells overexpressing TLR4, whereas depletion of TLR4 by shRNA repressed AR expression. Activation of AR by androgens or silencing of AR using shRNA also regulated expression of AR-related genes. We found that expression of DCDC2, ANKRD18B, ALDH1A1, c14orf105, ITGBL1 and NEB was overexpressed in taxol-resistant cells, suggesting the involvement of these AR-related genes in taxol resistance. Pathway enrichment analysis confirmed that the expression of several upregulated genes enriched in steroid biosynthesis pathways was inducible by androgens, supporting the results of previous studies. We also observed that genistein inhibits AR activation, leading to suppression of AR-driven genes and reduced taxol resistance in ovarian cancer cells. Overall, we identified six TLR4- and AR-regulated genes involved in taxol resistance. Our results reveal that the TLR4/AR axis plays a critical role in taxol resistance and that genistein is a candidate compound to limit chemoresistance and improve cancer treatment in ovarian cancer.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Genisteína/farmacologia , Neoplasias Ovarianas/genética , Paclitaxel/farmacologia , Receptores Androgênicos/genética , Transdução de Sinais/genética , Receptor 4 Toll-Like/genética , Anticarcinógenos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Interferência de RNA , Receptores Androgênicos/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
The role of Epstein-Barr viruses (EBVs) in lymphoepithelioma-like carcinoma (LELC) of the uterine cervix is controversial. We aimed to investigate the existence of EBV and human papillomavirus (HPV) in LELC of the cervix. Nine patients of LELC of the cervix, treated at Chang Gung Memorial Hospital between 1996 and 2000, with complete clinicopathologic findings and follow-up data were studied. Twenty-five patients with squamous cell carcinoma were recruited as controls. The EBV genome was measured by real-time quantitative polymerase chain reaction (PCR) and EBV-encoded RNA in situ hybridization from formalin-fixed, paraffin-embedded tissues. HPV genotyping was carried out by SPF1/GP6+ PCR and hybridization with a GeneChip. Type-specific E6 PCR of the 18 most commonly found HPV genotypes in Taiwan was also performed. HPV-16 was found in 3 cases, HPV-18, HPV-31, and HPV-35, and HPV-58 in 1 case each. One case showed positive for both HPV-16 and HPV-58. Low copy number of EBV DNA was found in 9 cases of LELC (1-14.7 copies/microg) and 7 cases of squamous cell carcinoma (3.8-1586 copies/microg) using real-time quantitative PCR Bam H1 W fragment probe, but EBV-encoded RNA-in situ hybridization was negative in tumor cells. Therefore, positive rates for EBV and HPV were 0% and 88.9% (8/9) in LELC of the cervix, respectively. All patients with LELC of the cervix had no evidence of disease for more than 5 years from diagnoses. Our results suggest that EBV is not involved in the carcinogenesis of so-called LELC of the cervix but the EBV sequences might exist in a florid inflammatory stromal component.
Assuntos
Carcinoma/virologia , Infecções por Vírus Epstein-Barr/epidemiologia , Herpesvirus Humano 4/isolamento & purificação , Neoplasias do Colo do Útero/virologia , Adulto , Idoso , DNA Viral/isolamento & purificação , Infecções por Vírus Epstein-Barr/complicações , Feminino , Humanos , Hibridização In Situ , Pessoa de Meia-Idade , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/epidemiologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
We conducted a population-based cohort study to evaluate the complementary value of HPV testing to Papanicolaou (Pap) smear and the prevalence and genotype distribution of HPV in Taiwan. In this report, we described the design of the whole study and analyzed the cross-sectional results. Female residents (age >or= 30 years) of Taoyuan, Taiwan were invited. After signing informed consent, every participant had a Pap smear and a HPV testing. Patients with Pap >or= atypical squamous cell of undetermined significance (Group I) or those with HPV-positive but normal cytology (Group II) were referred for a colposcopic examination. A total of 10,014 women were eligible. The overall HPV prevalence was 10.8% (95% confidence interval 10.5%-11.4%) in the study population. A total of 37 types of HPV were identified and the leading three were HPV-52, -18 and -58. There was a significant positive correlation of HPV prevalence with older age, postmenopausal status, current-user of oral contraceptives and never-user of hormone replacement therapy. Past users of oral contraceptives and never users of Pap were associated with higher risk of abnormal Pap, while age 40-49 strata had lower risk. Fifty-nine cases of cervical intraepithelial neoplasia (CIN) 2 from Group I and additional 11 from Group II were identified. The improvement of sensitivity with additional HPV testing was 15.3%. Besides, no specific subgroup was found to most benefit from the combined strategy. The value of adding HPV test to conventional Pap smear has to be evaluated after longer-term follow-up of this population-based cohort.
Assuntos
DNA Viral/análise , Teste de Papanicolaou , Papillomaviridae/isolamento & purificação , Neoplasias do Colo do Útero/diagnóstico , Esfregaço Vaginal , Adulto , Estudos de Coortes , Feminino , Humanos , Pessoa de Meia-Idade , Papillomaviridae/genética , Reação em Cadeia da Polimerase , Neoplasias do Colo do Útero/virologiaRESUMO
Squamous cell carcinoma (SCC), the most common primary malignant tumor of the conjunctiva, has a variable clinical presentation and immunohistochemical profile. Abundant cell cycles exist, including MIB-1 (Ki67 antigen), p16, p53, and p63, within the conjunctiva SCC. This investigation first reports the expressions of cell cycle markers in SCC. A retrospective study was conducted between December 1976 and June 2004, comprising 13 consecutive patients with conjunctiva SCC who were treated with surgical excision. Detailed clinical parameters were also reviewed. Overexpression of MIB-1, p16, p53, and p63 genes were studied by immunohistochemistry. Genechip containing 39 subtypes was used to elucidate human papillomavirus (HPV). The study group contained 13 (100%) men, with a mean age of 68+/-18 years and follow-up period of 20+/-17 months. The sample included four (33%) SCC located in the left eye and two (17%) recurrent SCC. Overexpression of the p53 and p63 was considerably higher than that of the p16 (P<0.01). HPV DNA was not detected in any of the 13 cases. This work first examined the immunohistochemical overexpression of cell cycle (MIB-1, p16, p53, and p63) in SCC. This investigation then showed that the expression of cell cycles in SCC was associated with key tumor clinicopathological features. This approach can help distinguish the potential roles of cell cycle in the development of SCC.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Neoplasias da Túnica Conjuntiva/metabolismo , Papillomaviridae/isolamento & purificação , Infecções por Papillomavirus/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/virologia , Contagem de Células , Proteínas de Ciclo Celular/genética , Neoplasias da Túnica Conjuntiva/genética , Neoplasias da Túnica Conjuntiva/virologia , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , DNA Viral/análise , Feminino , Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos , Papillomaviridae/genética , Infecções por Papillomavirus/patologia , Estudos Retrospectivos , Transativadores/metabolismo , Fatores de Transcrição , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismoRESUMO
A previous genome-wide screening analysis identified a panel of genes that sensitize the human non-small-cell lung carcinoma cell line NCI-H1155 to taxol. However, whether the identified genes sensitize other cancer cells to taxol has not been examined. Here, we silenced the taxol-sensitizer genes identified (acrbp, atp6v0d2, fgd4, hs6st2, psma6, and tubgcp2) in nine other cancer cell types (including lung, cervical, ovarian, and hepatocellular carcinoma cell lines) that showed reduced cell viability in the presence of a sub-lethal concentration of taxol. Surprisingly, none of the genes studied increased sensitivity to taxol in the tested panel of cell lines. As observed in H1155 cells, SKOV3 cells displayed induction of five of the six genes studied in response to a cell killing dose of taxol. The other cell types were much less responsive to taxol. Notably, four of the five inducible taxol-sensitizer genes tested (acrbp, atp6v0d2, psma6, and tubgcp2) were upregulated in a taxol-resistant ovarian cancer cell line. These results indicate that the previously identified taxol-sensitizer loci are not conserved genetic targets involved in inhibiting cell proliferation in response to taxol. Our findings also suggest that regulation of taxol-sensitizer genes by taxol may be critical for acquired cell resistance to the drug.
RESUMO
A systematic analysis of the genes involved in taxol resistance (txr) has never been performed. In the present study, we created txr ovarian carcinoma cell lines to identify the genes involved in chemoresistance. Transcriptome analysis revealed 1,194 overexpressed genes in txr cells. Among the upregulated genes, more than 12 cryptic transcription factors were identified using MetaCore analysis (including AR, C/EBPß, ERα, HNF4α, c-Jun/AP-1, c-Myc, and SP-1). Notably, individual silencing of these transcription factors (except HNF4`)sensitized txr cells to taxol. The androgen receptor (AR) and its target genes were selected for further analysis. Silencing AR using RNA interference produced a 3-fold sensitization to taxol in txr cells, a response similar to that produced by silencing abcb1. AR silencing also downregulated the expression of prominent txr gene candidates (including abcb1, abcb6, abcg2, bmp5, fat3, fgfr2, h1f0, srcrb4d, and tmprss15). In contrast, AR activation using the agonist DHT upregulated expression of the target genes. Individually silencing seven out of nine (78%) AR-regulated txr genes sensitized txr cells to taxol. Inhibition of AKT and JNK cellular kinases using chemical inhibitors caused a dramatic suppression of AR expression. These results indicate that the AR represents a critical driver of gene expression involved in txr.
Assuntos
Carcinoma/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Ovarianas/metabolismo , Paclitaxel/química , Receptores Androgênicos/metabolismo , Antineoplásicos Fitogênicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Concentração Inibidora 50 , Masculino , Análise de Sequência com Séries de Oligonucleotídeos , Oligonucleotídeos/genética , Fenótipo , Reação em Cadeia da Polimerase , Neoplasias da Próstata/metabolismo , Interferência de RNA , Fatores de Transcrição/metabolismo , Transcriptoma , Regulação para CimaRESUMO
Taxol is a mitotoxin widely used to treat human cancers, including of the breast and ovary. However, taxol resistance (txr) limits treatment efficacy in human patients. To study chemoresistance in ovarian cancer, we established txr ovarian carcinoma cells derived from the SKOV3 cell lineage. The cells obtained were cross-resistant to other mitotoxins such as vincristine while they showed no resistance to the genotoxin cisplatin. Transcriptomic analysis identified 112 highly up-regulated genes in txr cells. Surprisingly, FK506-binding protein 5 (FKBP5) was transiently up-regulated 100-fold in txr cells but showed decreased expression in prolonged culture. Silencing of FKBP5 sensitized txr cells to taxol, whereas ectopic expression of FKBP5 increased resistance to the drug. Modulation of FKBP5 expression produced similar effects in response to vincristine but not to cisplatin. We observed that a panel of newly identified txr genes was trancriptionally regulated by FKBP5 and silencing of these genes sensitized cells to taxol. Notably, immunoprecipitation experiments revealed that FKBP5 forms a protein complex with the androgen receptor (AR), and this complex regulates the transcriptional activity of both proteins. Furthermore, we found that the Akt kinase pathway is regulated by FKBP5. These results indicate that the FKBP5/AR complex may affect cancer cell sensitivity to taxol by regulating expression of txr genes. Our findings suggest that mitotoxin-based treatment against ovarian cancer should be avoided when the Akt/FKBP5/AR axis is activated.
Assuntos
Biomarcadores Tumorais/análise , Resistencia a Medicamentos Antineoplásicos/fisiologia , Neoplasias Ovarianas/metabolismo , Receptores Androgênicos/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Linhagem Celular Tumoral , Feminino , Humanos , Immunoblotting , Camundongos , Camundongos SCID , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Ovarianas/genética , Paclitaxel/uso terapêutico , Reação em Cadeia da Polimerase em Tempo Real , Transcriptoma , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Our aim was to evaluate the prognostic significance of human papillomavirus (HPV) genotype in early-stage cervical carcinoma primarily treated with surgery in a large tertiary referral medical center. PATIENTS AND METHODS: Consecutive patients who underwent primary surgery for invasive cervical carcinoma of International Federation of Gynecology and Obstetrics (FIGO) stage I to IIA between 1993 and 2000 were retrospectively reviewed. Polymerase chain reaction (PCR) using a general primer set followed by reverse-blot detection of 38 types of HPV DNA in a single reaction was performed for genotyping. E6 type-specific PCR was performed to validate multiple types. RESULTS: A total of 1,067 eligible patients were analyzed. HPV DNA sequences were detected in 95.1% of the specimens, among which 9.6% contained multiple types. HPV 16 was detected in 63.8% of the samples, and HPV 18 was detected in 16.5% of the samples. The median follow-up time of surviving patients was 77 months. By multivariate analysis, FIGO stage, lymph node metastasis, depth of cervical stromal invasion, grade of differentiation, and HPV 18 positivity were significantly related to cancer relapse. FIGO stage II, deep stromal invasion, parametrial extension, HPV 18 positivity, and age older than 45 years were significant predictors for death. Using the seven selected variables from either recurrence-free or overall survival analysis, death-predicting (P < .0001) and relapse-predicting (P < .0001) models classifying three risk groups (low, intermediate, and high risk) were constructed and endorsed by internal validation. CONCLUSION: The independent prognostic value of HPV genotype is confirmed in this study. The prognostic models could be useful in counseling patients and stratifying patients in future clinical trials.
Assuntos
Carcinoma/virologia , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Infecções por Papillomavirus/complicações , Neoplasias do Colo do Útero/virologia , Adulto , Carcinoma/cirurgia , Terapia Combinada , Intervalo Livre de Doença , Feminino , Genótipo , Humanos , Histerectomia , Pessoa de Meia-Idade , Infecções por Papillomavirus/virologia , Prognóstico , Taxa de Sobrevida , Neoplasias do Colo do Útero/mortalidade , Neoplasias do Colo do Útero/cirurgiaRESUMO
Our aim was to investigate the human papillomavirus (HPV) genotype distribution and correlation between HPV parameters and clinicopathological variables in cervical carcinoma treated in a large tertiary referral medical center in Taiwan. Consecutive patients treated for cervical carcinoma (Stages I-IV according to the International Federation of Gynecology and Obstetrics) between 1993 and 2000 were included. HPV genotyping using SPF1/GP6+ PCR was performed, followed by hybridization with a genechip (Easychip HPV Blot, King Car, Taiwan). E6 type-specific PCR was performed to validate multiple-type. HPV-negative samples were further verified by type-specific PCR and a repeat HPV Blot. A total of 2,118 patients were eligible for analysis. HPV DNA sequences were detected in 96.6% (95% CI, 95.8-97.4%) of the specimens, among which 82% harbored single-type and 18% contained multiple-type HPV sequences. Thirty-five types of HPV were identified and the leading 8 were HPV16 (50.0%), HPV18 (17.8%), HPV58 (16.3%), HPV33 (8.7%), HPV52 (6.8%), HPV39 (3.0%), HPV45 (2.5%) and HPV31 (2.3%). HPV58 or 33 or 52 was detected in 30.3% (641/2,118). By multivariate analysis, HPV58- or 33- or 52-infection was significantly associated with older age (p < 0.001) and primary radiotherapy or concurrent chemoradiation (RT/CCRT) (p < 0.001). Among HPV-positive cases, multiple-type was more frequently seen in those receiving primary RT/CCRT (p < 0.001). The knowledge of HPV genotype distribution will form a basis for guidelines in HPV-based cervical cancer screening and cost-effective multivalent HPV vaccine policy in Taiwan and in the world. The association between HPV parameters and clinicopathological variables warrants further investigations.
Assuntos
Papillomaviridae/classificação , Neoplasias do Colo do Útero/virologia , Adulto , Idoso , Feminino , Genótipo , Humanos , Modelos Logísticos , Pessoa de Meia-Idade , Análise Multivariada , Papillomaviridae/genética , Reação em Cadeia da PolimeraseRESUMO
In an attempt to understand the molecular mechanisms for the different clinical features between adenocarcinoma/adenosquamous carcinoma (AC/ASC) and squamous carcinoma (SC) of the uterine cervix, we analyzed gene expression profiles of different histological subtypes of cervical cancer. Cancer specimens and the surrounding normal tissue counterparts were separately collected from cervical cancer patients undergoing type III radical hysterectomy. Paired total RNA (cancer and normal tissues) was isolated and analyzed with cDNA microarrays containing duplicate spots of 7 334 sequence-verified human cDNA clones. Selected differentially expressed genes specific for AC or SC were further verified using real-time quantitative polymerase chain reaction (RTQ-PCR) and immunohistochemistry. Genes, including CEACAM5, TACSTD1, S100P and MSLN were upregulated in AC. Contrarily, genes involved in epidermal differentiation complex such as S100A9 and ANXA8 were upregulated in SC. Cross-validation of the results using an independent but comparable group of patients with known long-term outcomes (n = 63, median follow-up 70.3 months; range, 4-208 months) showed that the correlation between the selected 6 differentially expressed genes and histology was highly significant. CEACAM5 (p < 0.0001) and TACSTD1 (p = 0.009) were significant prognostic factors by multivariate Cox proportional hazards regression analysis. The combination of cDNA microarray, RTQ-PCR and immunohistochemical results of this study showed that it is possible to define different gene profiles for AC and SC. Moreover, TACSTD1 expression may be a novel poor prognostic factor.
Assuntos
Adenocarcinoma/genética , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Análise em Microsséries , Neoplasias do Colo do Útero/genética , Adenocarcinoma/patologia , Adulto , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Imuno-Histoquímica , Metástase Linfática , Mesotelina , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Reprodutibilidade dos Testes , Neoplasias do Colo do Útero/patologiaRESUMO
We compared the efficacy of human papillomavirus (HPV) DNA detection between a PCR-based genechip (Easychip HPV Blot [hereafter referred to as HPV Blot]; King Car, Taiwan) method and Hybrid Capture II (HCII; Digene, Gaithersburg, MD) in women with previous normal (n = 146) or abnormal (> or =atypical squamous cells of undetermined significance [ASCUS] [n = 208]) cytology. A total of 354 cervical swab samples were collected for HPV DNA assay by both HCII and SPF1/GP6+ PCR followed by HPV Blot tests. Colposcopy-directed biopsy was performed if clinically indicated. Of the 354 samples, HPV-positive rates by these two methods (HCII and HPV Blot) were 12.6% and 18.2% in 143 normal samples, 36.2% and 45.7% in 105 ASCUS samples, 57.4% and 57.4% in 94 low-grade squamous intraepithelial lesion samples, and 83.3% and 75.0% in 12 high-grade squamous intraepithelial lesion samples, respectively. The concordance of HPV Blot and HCII was 80.8% (286/354), and the agreement between the methods (kappa value, 0.68) was substantial. Discrepancies were further investigated by at least one of the following three methods: direct sequencing, type-specific PCR, and HPV Blot genotyping of cervical biopsy tissue. In the 15 HCII-positive samples, HPV Blot detected only non-HCII HPV genotypes; results of further verification methods were consistent with the latter test in the 15 samples. Of the 20 samples with HCII-negative and HPV Blot-positive results, 18 were found to contain the 13 HCII high-risk genotypes by verification methods. In only 16.7% (3/18) of the HCII-positive but HPV Blot-negative samples, further studies detected the 13 HCII genotypes. We conclude that HPV Blot seemed comparable to HCII for detection of HPV DNA in cervical swab samples.