RESUMO
RNA therapeutics has advanced into the third milestone in pharmaceutical drug development, following chemical and protein therapeutics. RNA itself can serve as therapeutics, carriers, regulators, or substrates in drug development. Due to RNA's motile, dynamic, and deformable properties, RNA nanoparticles have demonstrated spontaneous targeting and accumulation in cancer vasculature and fast excretion through the kidney glomerulus to urine to prevent possible interactions with healthy organs. Furthermore, the negatively charged phosphate backbone of RNA results in general repulsion from negatively charged lipid cell membranes for further avoidance of vital organs. Thus, RNA nanoparticles can spontaneously enrich tumor vasculature and efficiently enter tumor cells via specific targeting, while those not entering the tumor tissue will clear from the body quickly. These favorable parameters have led to the expectation that RNA has low or little toxicity. RNA nanoparticles have been well characterized for their anticancer efficacy; however, little detail on RNA nanoparticle pathology and safety is known. Here, we report the in vitro and in vivo assessment of the pathology and safety aspects of different RNA nanoparticles including RNA three-way junction (3WJ) harboring 2'-F modified pyrimidine, folic acid, and Survivin siRNA, as well as the RNA four-way junction (4WJ) harboring 2'-F modified pyrimidine and 24 copies of SN38. Both animal models and patient serum were investigated. In vitro studies include hemolysis, platelet aggregation, complement activation, plasma coagulation, and interferon induction. In vivo studies include hematoxylin and eosin (H&E) staining, hematological and biochemical analysis as the serum profiling, and animal organ weight study. No significant toxicity, side effect, or immune responses were detected during the extensive safety evaluations of RNA nanoparticles. These results further complement previous cancer inhibition studies and demonstrate RNA nanoparticles as an effective and safe drug delivery vehicle for future clinical translations.
Assuntos
Nanopartículas , Neoplasias , Animais , Humanos , RNA Interferente Pequeno/genética , Sistemas de Liberação de Medicamentos , Neoplasias/metabolismo , Nanopartículas/química , PirimidinasRESUMO
The microenvironment for tumor growth and developing metastasis should be essential. This study demonstrated that the hyaluronic acid synthase 3 (HAS3) protein and its enzymatic product hyaluronic acid (HA) encompassed in the subcutaneous extracellular matrix can attenuate the invasion of human breast tumor cells. Decreased HA levels in subcutaneous Has3-KO mouse tissues promoted orthotopic breast cancer (E0771) cell-derived allograft tumor growth. MDA-MB-231 cells premixed with higher concentration HA attenuate tumor growth in xenografted nude mice. Human patient-derived xenotransplantation (PDX) experiments found that HA selected the highly migratory breast cancer cells with CD44 expression accumulated in the tumor/stroma junction. In conclusion, HAS3 and HA were detected in the stroma breast tissues at a high level attenuates effects for induced breast cancer cell death, and inhibit the cancer cells invasion at the initial stage. However, the highly migratory cancer cells were resistant to the HA-mediated effects with unknown mechanisms.
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Neoplasias da Mama/metabolismo , Hialuronan Sintases/metabolismo , Tecido Parenquimatoso/metabolismo , Animais , Neoplasias da Mama/patologia , Feminino , Humanos , Hialuronan Sintases/deficiência , Hialuronan Sintases/genética , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Tecido Parenquimatoso/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Apiole was isolated from the leaves of various plants and vegetables and has been demonstrated to inhibit human colon cancer cell (COLO 205 cells) growth through induction of G0/G1 cell cycle arrest and apoptotic cell death. This study further explored the antitumor effects of apiole derivatives AP-02, 04, and 05 in COLO 205 cancer cells. METHODS: Human breast (MDA-MB-231, ZR75), lung (A549, PE089), colon (COLO 205, HT 29), and hepatocellular (Hep G2, Hep 3B) cancer cells were treated with apiole and its derivatives in a dose-dependent manner. Flow cytometry analysis was subsequently performed to determine the mechanism of AP-02-induced G0/G1 cell cycle arrest. The in vivo antitumor effect of AP-02 (1 and 5 mg/kg, administered twice per week) was examined by treating athymic nude mice bearing COLO 205 tumor xenografts. The molecular mechanisms of AP-02-induced antitumor effects were determined using western blot analysis. RESULTS: AP-02 was the most effective compound, especially for inhibition of COLO 205 colon cancer cell growth. The cytotoxicity of AP-02 in normal colon epithelial (FHC) cells was significantly lower than that in other normal cells derived from the breast, lung or liver. Flow cytometry analysis indicated that AP-02-induced G0/G1 cell cycle arrest in COLO 205 cells but not in HT 29 cells (< 5 µM for 24 h, **p < 0.01). Tumor growth volume was also significantly inhibited in AP-02 (> 1 mg/kg)-treated athymic nude mice bearing COLO 205 tumor xenografts compared to control mice (*p < 0.05). Furthermore, G0/G1 phase regulatory proteins (p53 and p21/Cip1) and an invasion suppressor protein (E-cadherin) were significantly upregulated, while cyclin D1 was significantly downregulated, in AP-02-treated tumor tissues compared to the control group (> 1 mg/kg, *p < 0.05). CONCLUSIONS: Our results provide in vitro and in vivo molecular evidence of AP-02-induced anti-proliferative effects on colon cancer, indicating that this compound might have potential clinical applications.
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Antineoplásicos/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Dioxóis/administração & dosagem , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Petroselinum/química , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Neoplasias do Colo/fisiopatologia , Ciclina D1/genética , Ciclina D1/metabolismo , Dioxóis/efeitos adversos , Dioxóis/química , Feminino , Humanos , Camundongos , Camundongos Nus , Fase de Repouso do Ciclo Celular/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Breast cancer (BC) is the most common cancer affecting women worldwide and has been associated with active tobacco smoking. Low levels of nicotine (Nic) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have been detected in cases of second-hand smoke (SHS). However, the correlation between SHS and BC risk remains controversial. In this study, we investigated whether the physiological SHS achievable dose of Nic and tobacco specific nitrosamine, NNK act together to induce breast carcinogenesis using an in vitro breast cell carcinogenesis model. Immortalized non-tumorigenic breast epithelial cell line, HBL-100 used for a time-course assay, was exposed to very low levels of either Nic or NNK, or both. The time-course assay consisted of 23 cycles of nitrosamines treatment. In each cycle, HBL-100 cells were exposed to 1pM of Nic and/or 100 femtM of NNK for 48 hours. Cells were passaged every 3 days and harvested after 10, 15, and 23 cycles. Our results demonstrated that the tumorigenicity of HBL-100, defined by soft agar colony forming, proliferation, migration and invasion abilities, was enhanced by co-exposure to physiologically SHS achievable doses of Nic and NNK. In addition, α9-nAChR signaling activation, which plays an important role in cellular proliferation and cell survival, was also observed. Importantly, an increase in stemness properties including the prevalence of CD44+/CD24- cells, increase Nanog expression and mammosphere-forming ability were also observed. Our results indicate that chronic and long term exposure to environmental tobacco smoke, may induce breast cell carcinogenesis even at extremely low doses.
Assuntos
Neoplasias da Mama/induzido quimicamente , Transformação Celular Neoplásica/efeitos dos fármacos , Glândulas Mamárias Humanas/efeitos dos fármacos , Nicotina/toxicidade , Nitrosaminas/toxicidade , Acetilcolina/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinógenos/toxicidade , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/fisiologia , Feminino , Humanos , Glândulas Mamárias Humanas/patologia , Glândulas Mamárias Humanas/fisiologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Fatores de Tempo , Testes de Toxicidade CrônicaRESUMO
PURPOSE: Glutathione S-transferase mu 3 (GSTM3) is an enzyme involving in the detoxification of electrophilic compounds by conjugation with glutathione. Higher GSTM3 mRNA levels were reported in patients with ERα-positive breast cancer who received only tamoxifen therapy after surgery. Thus, this study aimed to clarify the oncogenic characteristics of GSTM3 in breast cancer and the mechanism of tamoxifen resistance. METHODS: GSTM3 expression in human breast tumour tissues (n = 227) was analysed by RT-PCR and quantitative PCR. Western blot, promoter activity assays, and chromatin immunoprecipitation (ChIP) assays were used to investigate the mechanism of GSTM3 gene regulation. Hydrogen peroxide (H2O2)-induced cytotoxicity in breast cancer cells was detected by MTT assays and flow cytometry. The oncogenic characteristics of GSTM3 in MCF-7 cells were examined by siRNA knockdown in soft agar assays and a xenograft animal model. RESULTS: GSTM3 mRNA was highly expressed in ER- and HER2-positive breast cancers. Moreover, patients who received adjuvant Herceptin had increased GSTM3 mRNA levels in tumour tissue. Oestrogen-activated GSTM3 gene expression through ERα-mediated recruitment of SP1, EP300, and AP-1 complexes. GSTM3-silenced MCF-7 cells were more sensitive to H2O2, with significantly inhibited proliferation and colony formation abilities. Tamoxifen-resistant (Tam-R) cells lacking GSTM3 showed enhanced sensitivity to H2O2, but this result was contrary to that obtained after short-term tamoxifen exposure. The animal model suggested that GSTM3 silencing might suppress the tumourigenic ability of MCF-7 cells and increase tumour cell apoptosis. CONCLUSIONS: ROS production is one mechanism by which cancer drugs kill tumour cells, and according to our evidence, GSTM3 may play an important role in preventing breast cancer treatment-induced cellular cytotoxicity.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/genética , Receptor alfa de Estrogênio/genética , Glutationa Transferase/genética , Animais , Antineoplásicos Hormonais/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Proliferação de Células/efeitos dos fármacos , Estrogênios/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Peróxido de Hidrogênio/toxicidade , Células MCF-7 , Camundongos , Espécies Reativas de Oxigênio/metabolismo , Receptor ErbB-2/genética , Transdução de Sinais/efeitos dos fármacos , Tamoxifeno/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The field of RNA therapeutics has been emerging as the third milestone in pharmaceutical drug development. RNA nanoparticles have displayed motile and deformable properties to allow for high tumor accumulation with undetectable healthy organ accumulation. Therefore, RNA nanoparticles have the potential to serve as potent drug delivery vehicles with strong anti-cancer responses. Herein, we report the physicochemical basis for the rational design of a branched RNA four-way junction (4WJ) nanoparticle that results in advantageous high-thermostability and -drug payload for cancer therapy, including metastatic tumors in the lung. The 4WJ nanostructure displayed versatility through functionalization with an anti-cancer chemical drug, SN38, for the treatment of two different cancer models including colorectal cancer xenograft and orthotopic lung metastases of colon cancer. The resulting 4WJ RNA drug complex spontaneously targeted cancers effectively for cancer inhibition with and without ligands. The 4WJ displayed fast renal excretion, rapid body clearance, and little organ accumulation with undetectable toxicity and immunogenicity. The safety parameters were documented by organ histology, blood biochemistry, and pathological analysis. The highly efficient cancer inhibition, undetectable drug toxicity, and favorable Chemical, Manufacturing, and Control (CMC) production of RNA nanoparticles document a candidate with high potential for translation in cancer therapy.
Assuntos
Antineoplásicos , Neoplasias Pulmonares , Nanopartículas , Humanos , RNA , Eliminação Renal , Sistemas de Liberação de Medicamentos/métodos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Nanopartículas/química , Linhagem Celular TumoralRESUMO
Triple-negative breast cancer (TNBC) is highly aggressive with a poor prognosis because of a lack of cell markers as drug targets. α9-Nicotinic acetylcholine receptor (nAChR) is expressed abundantly in TNBC; thus, it is a valuable biomarker for TNBC detection and treatment. In this study, we utilized thermodynamically stable three-way junction (3WJ) packaging RNA (pRNA) as the core to construct RNA nanoparticles with an α9-nAChR RNA aptamer as a targeting ligand and an anti-microRNA-21 (miR-21) as a therapeutic module. We compared the configuration of the two RNA nanoparticles and found that 3WJ-B-α9-nAChR-aptamer fluorescent RNA nanoparticles (3WJ-B-α9-apt-Alexa) exhibited better specificity for α9-nAChR in TNBC cells compared with 3WJ-C-α9-nAChR. Furthermore, 3WJ-B-α9-apt-Alexa bound more efficiently to TNBC patient-derived xenograft (PDX) tumors than 3WJ fluorescent RNA nanoparticles (3WJ-Alexa) with little or no accumulation in healthy organs after systemic injection in mice. Moreover, 3WJ-B-α9-nAChR-aptamer RNA nanoparticles carrying anti-miR-21 (3WJ-B-α9-apt-anti-miR-21) significantly suppressed TNBC-PDX tumor growth and induced cell apoptosis because of reduced miR-21 gene expression and upregulated the phosphatase and tensin homolog (PTEN) and programmed cell death 4 (PDCD4) proteins. In addition, no pathological changes were detected upon toxicity examination of treated mice. In conclusion, the 3WJ-B-α9-nAChR-aptamer RNA nanoparticles established in this study efficiently deliver therapeutic anti-miR-21, indicating their potential as a novel TNBC therapy.
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Although various HER2-targeted therapies have been approved clinically, drug resistance remains a considerable challenge. Studies have found that the cause of drug resistance is related to the expression of genes co-amplified with HER2 in breast cancer cells. Our study found that STARD3 was highly expressed in tumor tissues (n = 130, P < 0.001), especially in the HER2+ subtype (n = 35, P < 0.05), and correlated with poorer overall survival (HR = 1.47, P < 0.001). We discovered the interaction mechanism between STARD3 and HER2 proteins. We found that STARD3 overexpression increases HER2 levels by directly interacting with the HSP90 protein and inducing phosphorylated SRC, which may protect HER2 from degradation. Conversely, loss of STARD3 attenuates HER2 expression through lysosomal degradation. In addition, STARD3 overexpression induced cell cycle progression by inducing cyclin D1 and reducing p27. Therefore, the development of STARD3-specific targeted anti-cancer drugs would be helpful in the treatment of HER2+ patients. We further found that curcumin (15 µM) is a potent STARD3 inhibitor. STARD3-knockdown cells treated with curcumin (5 µM) showed a significant synergistic effect in inhibiting cancer cell growth and migration. The results suggest that targeting STARD3 would aid in treating HER2-positive breast cancer patients. This article uses curcumin as an example to prove that the targeted inhibition of STARD3 expression can be an option for the clinical treatment of HER2+ breast cancer patients.
RESUMO
This study demonstrated for the first time that curcumin effectively inhibits the growth of triple-negative breast cancer (TNBC) tumors by inhibiting the expression of salt-induced kinase-3 (SIK3) protein in patient-derived xenografted tumor mice (TNBC-PDX). For TNBC patients, chemotherapy is the only option for postoperative adjuvant treatment. In this study, we detected the SIK3 mRNA expression in paired-breast cancer tissues by qPCR analysis. The results revealed that SIK3 mRNA expression was significantly higher in tumor tissues when compared to the normal adjacent tissues (73.25 times, n = 183). Thus, it is proposed for the first time that the antitumor effect induced by curcumin by targeting SIK3 can be used as a novel strategy for the therapy of TNBC tumors. In vitro mechanism studies have shown that curcumin (>25 µM) inhibits the SIK3-mediated cyclin D upregulation, thereby inhibiting the G1/S cell cycle and arresting TNBC (MDA-MB-231) cancer cell growth. The SIK3 overexpression was associated with increased mesenchymal markers (i.e., Vimentin, α-SMA, MMP3, and Twist) during epithelial-mesenchymal transition (EMT). Our results demonstrated that curcumin inhibits the SIK3-mediated EMT, effectively attenuating the tumor migration. For clinical indications, dietary nutrients (such as curcumin) as an adjuvant to chemotherapy should be helpful to TNBC patients because the current trend is to shrink the tumor with preoperative chemotherapy and then perform surgery. In addition, from the perspective of chemoprevention, curcumin has excellent clinical application value.
Assuntos
Curcumina , Proteínas Serina-Treonina Quinases , Neoplasias de Mama Triplo Negativas , Animais , Linhagem Celular Tumoral , Curcumina/farmacologia , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , RNA Mensageiro/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Triple-negative breast cancers (TNBCs) lack specific targeted therapy options and have evolved into highly chemo-resistant tumors that metastasize to multiple organs. The present study demonstrated that the proline dehydrogenase (PRODH) mRNA level in paired (tumor vs. normal) human breast tissue samples (n=234) was 6.6-fold greater than normal cells (*p=0.021). We established stable PRODH-overexpressing TNBC (HS578T) cells, and the malignant phenotypes were evaluated using soft agar colony formation and Transwell migration assays. The results demonstrated that PRODH induced epithelial-mesenchymal transition in cancer cells and increased cell proliferation. The present study found that the tea polyphenol epigallocatechin-3-gallate (EGCG) significantly inhibited PRODH and its regulated proteins, such as alpha-smooth muscle actin (alpha-SMA) expression in TNBC cells. These findings support the targeting of the PRODH signaling pathway as a potential therapeutic strategy in preventing cancer cell metastasis. The patient-derived xenograft (PDX) mouse model is highly relevant to real human tumor growth. We established a TNBC-PDX (F4, n=4 in each group)mouse model. The PDX mice were treated with EGCG (50 mg/kg), and the results indicated that EGCG significantly inhibited PDX tumor growth (*p = 0.013). These experiments provide additional evidence to evaluate the antitumor effects of EGCG-induced PRODH inhibition for clinical therapeutic application, especially in TNBC patients.
Assuntos
Polifenóis , Neoplasias de Mama Triplo Negativas , Animais , Catequina/análogos & derivados , Linhagem Celular Tumoral , Proliferação de Células , Modelos Animais de Doenças , Xenoenxertos , Humanos , Camundongos , Polifenóis/farmacologia , Prolina/farmacologia , Prolina Oxidase , Chá , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismoRESUMO
Enolase-alpha (ENO-1) is a key glycolytic enzyme that has been used as a diagnostic marker to identify human lung cancers. To investigate the role of ENO-1 in breast cancer diagnosis and therapy, the mRNA levels of ENO-1 in 244 tumor and normal paired tissue samples and 20 laser capture-microdissected cell clusters were examined by quantitative real-time PCR analysis. Increased ENO-1 mRNA expression was preferentially detected in estrogen receptor-positive (ER+) tumors (tumor/normal ratio >90-fold) when compared to ER-negative (tumor/normal ratio >20-fold) tumor tissues. The data presented here demonstrate that those patients whose tumors highly expressed ENO-1 had a poor prognosis with greater tumor size (>2 cm, *P = .017), poor nodal status (N > 3, *P = .018), and a shorter disease-free interval (<==1 year, *P < .009). We also found that higher-expressing ENO-1 tumors confer longer distance relapse (tumor/normal ratio = 82.8-92.4-fold) when compared to locoregional relapse (tumor/normal ratio = 43.4-fold) in postsurgical 4-hydroxy-tamoxifen (4-OHT)-treated ER+ patients (*P = .014). These data imply that changes in tumor ENO-1 levels are related to clinical 4-OHT therapeutic outcome. In vitro studies demonstrated that decreasing ENO-1 expression using small interfering RNA (siRNA) significantly augmented 4-OHT (100 nM)-induced cytotoxicity in tamoxifen-resistant (Tam-R) breast cancer cells. These results suggest that downregulation of ENO-1 could be utilized as a novel pharmacological approach for overcoming 4-OHT resistance in breast cancer therapy.
Assuntos
Antineoplásicos Hormonais/farmacologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/tratamento farmacológico , Proteínas de Ligação a DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Fosfopiruvato Hidratase/metabolismo , Tamoxifeno/farmacologia , Proteínas Supressoras de Tumor/metabolismo , Adulto , Idoso , Biomarcadores Tumorais/antagonistas & inibidores , Neoplasias da Mama/patologia , Estudos de Casos e Controles , Células Cultivadas , Proteínas de Ligação a DNA/antagonistas & inibidores , Ensaios de Seleção de Medicamentos Antitumorais , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Fosfopiruvato Hidratase/antagonistas & inibidores , Prognóstico , RNA Mensageiro/análise , RNA Interferente Pequeno , Análise de Sobrevida , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
It is well-known that human epidermal growth factor receptor 2 (HER2) is critical for breast cancer (BC) development and progression. Several studies have revealed the role of the ubiquitin/proteasome system (UPS) in cancer. In this study, we investigated the expression level of Proteasome 26S subunit, non-ATPase 3 (PSMD3) in BC using BC cell lines, human BC tissue samples, Oncomine, and TCGA databases and studied the PSMD3-HER2 protein interaction. PSMD3 was upregulated in BC, particularly in the HER2+ subtype. PSMD3 immunostaining was detected in the cytoplasm and nucleus of BC tumor tissues. Strong interaction between PSMD3 and HER2 at the protein level was observed. Knockdown of PSMD3 significantly impaired the stability of HER2, inhibited BC cell proliferation and colony formation, and induced cell apoptosis. Ubiquitination process was strongly enhanced after knockdown of PSMD3 in association with decreased HER2 level. Accumulation and Localization of LAMP-1 in the cell membrane with decreased HER2 immunostaining was observed after knockdown of PSMD3. High expression level of PSMD3 was associated with HER2 expression (p < 0.001), tumor size (p < 0.001), and clinical stage (p = 0.036). High expression level of PSMD3 predicted a short overall survival (OS), particularly for HER2+. Overall, we provide a novel function for PSMD3 in stabilizing HER2 from degradation in HER2+ BC, which suggests that PSMD3 is a novel target for HER2+ BC.
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Neuroblastoma is the second most common pediatric malignancy and has a high rate of spontaneous remission. Uncovering the mechanisms underlying neuroblastoma cell differentiation is critical for therapeutic purposes. A neuroblastoma cell line (N2a) treated with either serum withdrawal (<2.5%) or melatonin (>0.1 nmol/L) for 24 hours was used as a cell differentiation research model. Interestingly, the hyaluronan synthase 3 (HAS3) protein was induced in differentiated N2a cells. N2a-allografted nude mice received an intraperitoneal injection of melatonin (40 or 80 mg/kg/day for 3 weeks). The mean tumor volume in mice treated with 80 mg/kg melatonin was smaller than that in PBS-treated mice (1416.3 and 3041.3 mm3 , respectively, difference = 1625 mm3 , *P = 0.0003, n = 7 per group). Compared with the vector control group, N2a cells with forced HAS3 overexpression showed significantly increased neuron length (*P = 0.00082) and neurite outgrowth (*P = 0.00059). Intracellular changes in autophagy, including distorted mitochondria with abnormal circular inner membranes, were detected by transmission electron microscopy (TEM). Our study demonstrated that HAS3-mediated signaling activated by physiological concentrations of melatonin (>0.1 nmol/L) triggered significant N2a cell differentiation. These results provide molecular data with potential clinical relevance for therapeutic drug development.
Assuntos
Hialuronan Sintases/metabolismo , Melatonina/administração & dosagem , Neuroblastoma/tratamento farmacológico , Animais , Autofagia , Diferenciação Celular , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Melatonina/farmacologia , Camundongos , Camundongos Nus , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The function of small G protein signalling modulators (SGSM1/2/3) in cancer remains unknown. Our findings demonstrated that SGSM2 is a plasma membrane protein that strongly interacted with E-cadherin/ß-catenin. SGSM2 downregulation enhanced the phosphorylation of focal adhesion kinase (FAK; Y576/577), decreased the expression of epithelial markers such as E-cadherin, ß-catenin, and Paxillin, and increased the expression of Snail and Twist-1, which reduced cell adhesion and promoted cancer cell migration. Oestrogen and fibronectin treatment was found to promote the colocalization of SGSM2 at the leading edge with phospho-FAK (Y397). The BioGRID database showed that SGSM2 potentially interacts with cytoskeleton remodelling and cell-cell junction proteins. These evidences suggest that SGSM2 plays a role in modulating cell adhesion and cytoskeleton dynamics during cancer migration.
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Antígenos CD/metabolismo , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/patologia , Caderinas/metabolismo , Adesão Celular , Movimento Celular , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Antígenos CD/genética , Apoptose , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Caderinas/genética , Proliferação de Células , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Metástase Neoplásica , Fosforilação , Receptor ErbB-2/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Transdução de Sinais , Células Tumorais Cultivadas , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Cigarette smoking is associated with an increased risk of melanoma metastasis. Smokers show higher PD-L1 expression and better responses to PD-1/PD-L1 inhibitors than nonsmokers. Here, we investigate whether nicotine, a primary constituent of tobacco, induces PD-L1 expression and promotes melanoma cell proliferation and migration, which is mediated by the α9 nicotinic acetylcholine receptor (α9-nAChR). α9-nAChR overexpression in melanoma using melanoma cell lines, human melanoma tissues, and assessment of publicly available databases. α9-nAChR expression was significantly correlated with PD-L1 expression, clinical stage, lymph node status, and overall survival (OS). Overexpressing or knocking down α9-nAChR in melanoma cells up- or downregulated PD-L1 expression, respectively, and affected melanoma cell proliferation and migration. Nicotine-induced α9-nAChR activity promoted melanoma cell proliferation through stimulation of the α9-nAChR-mediated AKT and ERK signaling pathways. In addition, nicotine-induced α9-nAchR activity promoted melanoma cell migration via activation of epithelial-mesenchymal transition (EMT). Moreover, PD-L1 expression was upregulated in melanoma cells after nicotine treatment via the transcription factor STAT3 binding to the PD-L1 promoter. These results highlight that nicotine-induced α9-nAChR activity promotes melanoma cell proliferation, migration, and PD-L1 upregulation. This study may reveal important insights into the mechanisms underlying nicotine-induced melanoma growth and metastasis through α9-nAChR-mediated carcinogenic signals and PD-L1 expression.
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α-L-fucosidase 1 (FUCA1) is a lysosomal enzyme that catalyzes the hydrolytic cleavage of the terminal fucose residue in breast cancer cells. FUCA1 mRNA levels were detected by real-time PCR, and there was a greater than 139-fold increase in FUCA1 mRNA expression in breast tumor samples compared with normal breast tissue samples (*P = 0.005, n = 236). Higher FUCA1 mRNA expression was preferentially detected in early-stage tumors (stage 0 to 2) compared with advanced-stage tumors (stage 3 to 4) (stage 0-1 versus stage 3, *P = 0.015; stage 0-1 versus stage 4, *P = 0.024). FUCA1 protein levels were higher in advanced-stage tumors concomitant with decreased fucosylated Lewis-x antigen expression, as evidenced using the immunohistochemical staining H-score method (*P < 0.001). Statistical analysis revealed that lower FUCA1 levels significantly predicted an inferior overall survival rate among triple-negative breast cancer (TNBC) patients compared with non-TNBC patients (*P = 0.009). Two stable FUCA1 siRNA knock-down MDA-MB-231 cell lines were established, and the results suggest that transient FUCA inhibition creates a selective pressure that triggers the metastasis of primary tumor cells, as detected by wound healing and invasion assays (*P < 0.01). The results suggest that FUCA1 may be a potential prognostic molecular target for clinical use, especially in TNBC patients.
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Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , alfa-L-Fucosidase/metabolismo , Idoso , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular , Fragmentação do DNA , Feminino , Fucose/química , Perfilação da Expressão Gênica , Glicosilação , Humanos , Hidrólise , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Células MCF-7 , Pessoa de Meia-Idade , Metástase Neoplásica , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: To quantify the mitochondrial DNA contents in human embryos with good or poor quality at the 8-cell stage. DESIGN: Prospective study. SETTING: Private infertility clinic. PATIENT(S): Five women aged 24 to 34 years. INTERVENTION(S): Embryos obtained in standard superovulation and embryo culture procedures. MAIN OUTCOME MEASURE(S): Mitochondrial DNA (mtDNA) copy numbers in human embryos at cleavage stage were quantified by real-time polymerase chain reaction, in an effort to correlate with morphology. RESULT(S): The grade 8A embryos contained a mean mtDNA copy number at 1163937 (n = 8, from three patients); grade 8B embryos, at 939345 (n = 5, from two patients); grade 8C(+) embryos, at 637872 (n = 12, from 5 patients); and grade 8C(+) embryos derived from 3PN zygotes, at 300429 (n = 3, from a single patient). CONCLUSION(S): Great variations were found among blastomeres from a single embryo and among embryos from a single patient. The native variations of mtDNA copy number may affect developmental ability irrespective of morphology.
Assuntos
Blastocisto/fisiologia , DNA Mitocondrial/análise , Diagnóstico Pré-Implantação/métodos , Adulto , Blastocisto/citologia , Blastocisto/patologia , Blastômeros/citologia , Blastômeros/fisiologia , Feminino , Humanos , Reação em Cadeia da Polimerase/métodos , Estudos Prospectivos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
OBJECTIVE: Assisted hatching may enhance embryo implantation. This study was conducted to examine the efficacy of the laser- and chemical-assisted hatching for promotion of implantation (IR), pregnancy (PR), and delivery rate (DR) in older women undergoing IVF cycles. DESIGN: Prospective study. SETTING: An IVF unit of a medical center. PATIENT(S): A total of 601 embryos from 141 women aged > or =38 years underwent controlled ovarian hyperstimulation (COH) and assisted hatching. INTERVENTION(S): The study population was divided into two groups: group 1 had laser-assisted hatching (n = 85) and group 2 had chemical-assisted hatching (n = 56). Before the transfer, the day 3 embryos were hatched by using a 1.48-microm noncontact diode laser or acid Tyrode's solution. MAIN OUTCOME MEASUREMENT(S): The IR, PR, and DR between the groups were compared. RESULT(S): There were no statistical differences between groups in age, E2 concentrations during hCG administration, gonadotrophin dosage, embryo grade, the numbers of oocytes retrieved, oocytes fertilized, and embryos transferred. Higher IR, PR, and DR were noted in the laser-assisted hatching group. The IR, PR, and DR were: group 1, 8.2%/31.8%/24.7% and group 2, 3.8%/16.1%/10.7%, respectively. CONCLUSION(S): Laser-assisted hatching of embryos is more effective than the chemical method in enhancing the IR and PR of women with advanced age. The laser system allows an easier, faster, and safer micromanipulation of the zona pellucida, which provided a better method in zona drilling.
Assuntos
Envelhecimento/fisiologia , Embrião de Mamíferos/efeitos da radiação , Fertilização in vitro , Terapia a Laser , Taxa de Gravidez , Punções/métodos , Adulto , Parto Obstétrico/estatística & dados numéricos , Implantação do Embrião , Embrião de Mamíferos/efeitos dos fármacos , Feminino , Humanos , Soluções Isotônicas/uso terapêutico , Gravidez , Estudos ProspectivosRESUMO
The aim of our study is to observe the relationship between the blastocyst morphology and the implantation rate for mice. Mouse embryos obtained from the superovulated-ICR mice were cultured in vitro from 1-cell zygotes to blastocysts. Mouse blastocysts were then classified into 3 grades: grade I, small blastocysts; grade II, large blastocysts; grade III, hatching blastocysts. They were independently transferred into the uterus of recipient females mated with vasectomized male mice on 96 hours after the zygotes were cultured in vitro. The successful implantation was checked by injection of Chicago Sky Blue 6B on the second day after embryo transfer. Although there was no significant difference in the implantation rates between the grade III and grade II, grade I was significantly decreased, as compared with grade III. Grade I and grade II was also significantly decreased in both the diameter of blastocysts and cell number of inner cell mass (ICM) and trophectoderm (TE), as compared with grade III. These findings indicate that the expanded and hatching blastocyst selections for embryo transfer in in vitro fertilization were evaluated with the high implantation rate.
Assuntos
Blastocisto/citologia , Blastocisto/fisiologia , Implantação do Embrião , Animais , Compostos Azo , Contagem de Células , Corantes , Técnicas de Cultura , Transferência Embrionária , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Coloração e Rotulagem , Fatores de Tempo , Azul Tripano , VasectomiaRESUMO
Here we report that 3'-hydroxypterostilbene (HPSB), a natural pterostilbene analogue, was more potent than pterostilbene against the growth of human cancer cells (COLO 205, HCT-116, and HT-29) with measured IC50 values of 9.0, 40.2, and 70.9 µM, respectively. We found that HPSB effectively inhibited the growth of human colon cancer cells by inducing apoptosis and autophagy. Autophagy occurred at an early stage and was observed through the formation of acidic vesicular organelles and microtubule-associated protein 1 light chain 3-II production. At the molecular levels, the results from western blot analysis showed that HPSB significantly down-regulated phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) signalings including decreased the phosphorylation of mammalian target of rapamycin (mTOR). Significant therapeutic effects were demonstrated in vivo by treating nude mice bearing COLO 205 tumor xenografts with HPSB (10 mg/kg i.p.). These inhibitory effects were accompanied by mechanistic down-regulation of the protein levels of cyclooxygenase-2 (COX-2), matrix metallopeptidase-9 (MMP-9), vascular endothelial growth factor (VEGF), and cyclin D1, as well as by the induction of apoptosis in colon tumors. Our findings suggest that HPSB could serve as a novel promising agent for colon cancer treatment.