RESUMO
The number of opioid-related overdose deaths and individuals that have suffered from opioid use disorders have significantly increased over the last 30 years. FDA approved maintenance therapies to treat opioid use disorder may successfully curb drug craving and prevent relapse but harbor adverse effects that reduce patient compliance. This has created a need for new chemical entities with improved patient experience. Previously our group reported a novel lead compound, NAT, a mu-opioid receptor antagonist that potently antagonized the antinociception of morphine and showed significant blood-brain barrier permeability. However, NAT belongs to thiophene containing compounds which are known structural alerts for potential oxidative metabolism. To overcome this, 15 NAT derivatives with various substituents at the 5'-position of the thiophene ring were designed and their structure-activity relationships were studied. These derivatives were characterized for their binding affinity, selectivity, and functional activity at the mu opioid receptor and assessed for their ability to antagonize the antinociceptive effects of morphine in vivo. Compound 12 showed retention of the basic pharmacological attributes of NAT while improving the withdrawal effects that were experienced in opioid-dependent mice. Further studies will be conducted to fully characterize compound 12 to examine whether it would serve as a new lead for opioid use disorder treatment and management.
Assuntos
Receptores Opioides mu , Animais , Relação Estrutura-Atividade , Camundongos , Receptores Opioides mu/metabolismo , Receptores Opioides mu/antagonistas & inibidores , Humanos , Estrutura Molecular , Tiofenos/química , Tiofenos/farmacologia , Tiofenos/síntese química , Tiofenos/uso terapêutico , Masculino , Relação Dose-Resposta a Droga , Analgésicos Opioides/farmacologia , Analgésicos Opioides/química , Antagonistas de Entorpecentes/farmacologia , Antagonistas de Entorpecentes/química , Morfina/farmacologiaRESUMO
In the present work, we reported the application of a nitrogen-walk approach on developing a series of novel opioid ligands containing an azaindole moiety at the C6-position of the epoxymorphinan skeleton. In vitro study results showed that introducing a nitrogen atom around the indole moiety not only retained excellent binding affinity, but also led to significant functional switch at the mu opioid receptor (MOR). Further computational investigations provided corroborative evidence and plausible explanations of the results of the in vitro studies. Overall, our current work implemented a series of novel MOR ligands with high binding affinity and considerably low efficacy, which may shed light on rational design of low efficacy MOR ligands for opioid use disorder therapeutics.
Assuntos
Naltrexona/análogos & derivados , Nitrogênio/química , Receptores Opioides mu/efeitos dos fármacos , Sítios de Ligação , Humanos , Ligantes , Modelos Moleculares , Simulação de Acoplamento Molecular , Naltrexona/síntese química , Naltrexona/farmacologia , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Conformação ProteicaRESUMO
In the present study, the role of 3-hydroxy group of a series of epoxymorphinan derivatives in their binding affinity and selectivity profiles toward the opioid receptors (ORs) has been investigated. It was found that the 3-hydroxy group was crucial for the binding affinity of these derivatives for all three ORs due to the fact that all the analogues 1a-e exhibited significantly higher binding affinities compared to their counterpart 3-dehydroxy ones 6a-e. Meanwhile most compounds carrying the 3-hydroxy group possessed similar selectivity profiles for the kappa opioid receptor over the mu opioid receptor as their corresponding 3-dehydroxy derivatives. [35S]-GTPγS functional assay results indicated that the 3-hydroxy group of these epoxymorphinan derivatives was important for maintaining their potency on the ORs with various effects. Further molecular modeling studies helped comprehend the remarkably different binding affinity and functional profiles between compound 1c (NCP) and its 3-dehydroxy analogue 6c.
Assuntos
Morfinanos/química , Morfinanos/farmacologia , Receptores Opioides/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Estrutura Molecular , Ligação Proteica , Receptores Opioides/químicaRESUMO
While there are approved therapeutics to treat opioid overdoses, the need for treatments to reverse overdoses due to ultrapotent fentanyls remains unmet. This may be due in part to an adrenergic mechanism of fentanyls in addition to their stereotypical mu-opioid receptor (MOR) effects. Herein, we report our efforts to further understanding of the functions these distinct mechanisms impart. Employing the known MOR neutral antagonist phenylfentanil as a lead, 17 analogues were designed based on the concept of isosteric replacement. To probe mechanisms of action, these analogues were pharmacologically evaluated in vitro and in vivo, while in silico modeling studies were also conducted on phenylfentanil. While it did not indicate MOR involvement in vivo, phenylfentanil yielded respiratory minute volumes similar to those caused by fentanyl. Taken together with molecular modeling studies, these results indicated that respiratory effects of fentanyls may also correlate to inhibition of both α1A- and α1B-adrenergic receptors.
Assuntos
Adrenérgicos , Fentanila , Fentanila/farmacologia , Receptores Opioides mu , Antagonistas de Entorpecentes , Analgésicos Opioides/farmacologiaRESUMO
Despite the availability of numerous pain medications, the current array of Food and Drug Administration-approved options falls short in adequately addressing pain states for numerous patients and consequently worsens the opioid crisis. Thus, it is imperative for basic research to develop novel and nonaddictive pain medications. Toward addressing this clinical goal, nalfurafine (NLF) was chosen as a lead and its structure-activity relationship (SAR) systematically studied through design, syntheses, and in vivo characterization of 24 analogues. Two analogues, 21 and 23, showed longer durations of action than NLF in a warm-water tail immersion assay, produced in vivo effects primarily mediated by KOR and DOR, penetrated the blood-brain barrier, and did not function as reinforcers. Additionally, 21 produced fewer sedative effects than NLF. Taken together, these results aid the understanding of NLF SAR and provide insights for future endeavors in developing novel nonaddictive therapeutics to treat pain.
Assuntos
Morfinanos , Compostos de Espiro , Relação Estrutura-Atividade , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Compostos de Espiro/síntese química , Animais , Morfinanos/farmacologia , Morfinanos/química , Morfinanos/síntese química , Morfinanos/uso terapêutico , Camundongos , Masculino , Humanos , Receptores Opioides kappa/agonistas , Receptores Opioides kappa/metabolismo , Manejo da Dor/métodos , Dor/tratamento farmacológico , Analgésicos/farmacologia , Analgésicos/química , Analgésicos/síntese química , Analgésicos/uso terapêuticoRESUMO
BACKGROUND: Although oral squamous cell carcinomas (OSCCs) commonly overexpress the epidermal growth factor receptor (EGFR), EGFR tyrosine kinase inhibitors (TKIs) exhibit poor efficacy clinically. Activation of the insulin-like growth factor-1 receptor (IGF1R) induces resistance of OSCC cells to EGFR-TKIs in vitro. This study seeks to evaluate the changes in cell cycle status in OSCC cells in response to gefitinib and IGF1R activation. METHODS: SCC-25 OSCC cells were used for in vitro analyses. RESULTS: Gefitinib caused a 50% reduction in S-phase population, and IGF1R activation caused a 2.8-fold increase; combined treatment yielded a baseline S-phase population. Gefitinib treatment increased the cyclin-dependent kinase inhibitor p27, and this was not abrogated by IGF1R activation. pT157-p27 was noted by immunoblot to be decreased on gefitinib treatment, but this was reversed with IGF1R activation. T157 phosphorylation contributes to cytoplasmic localization of p27 where it can promote cell proliferation and cell motility. Using both subcellular fractionation and immunofluorescence microscopy techniques, IGF1R stimulation was noted to increase the relative cytoplasmic localization of p27; this persisted when combined with gefitinib. CONCLUSIONS: IGF1R activation partially reverses the cell cycle arrest caused by gefitinib in OSCC cells. While IGF1R stimulation does not eliminate the gefitinib-induced increase in total p27, its phosphorylation state and subcellular localization are altered. This may contribute to the ability of the IGF1R to rescue OSCC cells from EGFR-TKI treatment and may have important implications for the use of p27 as a biomarker of cell cycle arrest and response to therapy.
Assuntos
Carcinoma de Células Escamosas/patologia , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Receptores ErbB/fisiologia , Neoplasias Bucais/patologia , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor IGF Tipo 1/fisiologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Núcleo Celular/ultraestrutura , Ciclina D/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/efeitos dos fármacos , Citoplasma/ultraestrutura , Relação Dose-Resposta a Droga , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Gefitinibe , Humanos , Fator de Crescimento Insulin-Like I/farmacologia , Proteína Oncogênica v-akt/fisiologia , Fragmentos de Peptídeos/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Pirimidinas/farmacologia , Quinazolinas/administração & dosagem , Receptor IGF Tipo 1/efeitos dos fármacos , Fase S/efeitos dos fármacos , Frações Subcelulares/ultraestruturaRESUMO
The search for selective opioid ligands with desired pharmacological potency and improved safety profile has always been an area of interest. Our previous effort yielded a potent opioid modulator, NAN, a 6α-N-7'-indolyl-substituted naltrexamine derivative, which exhibited promising pharmacological activities both in vitro and in vivo. However, significant human ether-a-go-go-related gene (hERG) liability limited its further development. Therefore, a systematic structural modification on NAN was conducted in order to alleviate hERG toxicity while preserving pharmacological properties, which led to the discovery of 2'-methylindolyl derivative compound 21. Compared to NAN, compound 21 manifested overall improved pharmacological profiles. Follow-up hERG channel inhibition evaluation revealed a seven-fold decreased potency of compound 21 compared to NAN. Furthermore, several fundamental drug-like property evaluations suggested a reasonable ADME profile of 21. Collectively, compound 21 appeared to be a promising opioid modulator for further development as a novel therapeutic agent toward opioid use disorder treatments.
Assuntos
Analgésicos Opioides , Receptores Opioides , Humanos , Analgésicos Opioides/farmacologia , Canais de Potássio Éter-A-Go-Go , LigantesRESUMO
Opioid-induced constipation (OIC) is a common adverse effect of opioid analgesics. Peripherally acting µ opioid receptor antagonists (PAMORAs) can be applied in the treatment of OIC without compromising the analgesic effects. NAP, a 6ß-N-4-pyridyl-substituted naltrexamine derivative, was previously identified as a potent and selective MOR antagonist mainly acting peripherally but with some CNS effects. Herein, we introduced a highly polar aromatic moiety, for example, a pyrazolyl or imidazolyl ring to decrease CNS MPO scores in order to reduce passive BBB permeability. Four compounds 2, 5, 17, and 19, when administered orally, were able to increase intestinal motility during morphine-induced constipation in the carmine red dye assays. Among them, compound 19 (p.o.) improved GI tract motility by 75% while orally administered NAP and methylnaltrexone showed no significant effects at the same dose. Thus, this compound seemed a promising agent to be further developed as an oral treatment for OIC.
Assuntos
Constipação Induzida por Opioides , Analgésicos Opioides/efeitos adversos , Constipação Intestinal/induzido quimicamente , Constipação Intestinal/tratamento farmacológico , Humanos , Ligantes , Naltrexona/farmacologia , Naltrexona/uso terapêutico , Antagonistas de Entorpecentes/farmacologia , Antagonistas de Entorpecentes/uso terapêutico , Receptores Opioides muRESUMO
Discovery of analgesics void of abuse liability is critical to battle the opioid crisis in the United States. Among many strategies to achieve this goal, targeting more than one opioid receptor seems promising to minimize this unwanted side effect while achieving a reasonable therapeutic profile. In the process of understanding the structure-activity relationship of nalfurafine, we identified a potential analgesic agent, NMF, as a dual kappa opioid receptor/delta opioid receptor agonist with minimum abuse liability. Further characterizations, including primary in vitro ADMET studies (hERG toxicity, plasma protein binding, permeability, and hepatic metabolism), and in vivo pharmacodynamic and toxicity profiling (time course, abuse liability, tolerance, withdrawal, respiratory depression, body weight, and locomotor activity) further confirmed NMF as a promising drug candidate for future development.
Assuntos
Analgésicos Opioides , Morfinanos , Humanos , Analgésicos Opioides/química , Receptores Opioides kappa/agonistas , Morfinanos/farmacologia , Analgésicos/farmacologia , Relação Estrutura-Atividade , Receptores Opioides mu/agonistasRESUMO
The µ opioid receptor (MOR) has been an intrinsic target to develop treatment of opioid use disorders (OUD). Herein, we report our efforts on developing centrally acting MOR antagonists by structural modifications of 17-cyclopropylmethyl-3,14-dihydroxy-4,5α-epoxy-6ß-[(4'-pyridyl) carboxamido] morphinan (NAP), a peripherally acting MOR-selective antagonist. An isosteric replacement concept was applied and incorporated with physiochemical property predictions in the molecular design. Three analogs, namely, 25, 26, and 31, were identified as potent MOR antagonists in vivo with significantly fewer withdrawal symptoms than naloxone observed at similar doses. Furthermore, brain and plasma drug distribution studies supported the outcomes of our design strategy on these compounds. Taken together, our isosteric replacement of pyridine with pyrrole, furan, and thiophene provided insights into the structure-activity relationships of NAP and aided the understanding of physicochemical requirements of potential CNS acting opioids. These efforts resulted in potent, centrally efficacious MOR antagonists that may be pursued as leads to treat OUD.
Assuntos
Morfinanos , Transtornos Relacionados ao Uso de Opioides , Analgésicos Opioides/química , Sistema Nervoso Central , Humanos , Morfinanos/química , Naloxona , Antagonistas de Entorpecentes/farmacologia , Antagonistas de Entorpecentes/uso terapêutico , Transtornos Relacionados ao Uso de Opioides/tratamento farmacológico , Receptores Opioides muRESUMO
The modulation and selectivity mechanisms of seven mixed-action kappa opioid receptor (KOR)/mu opioid receptor (MOR) bitopic modulators were explored. Molecular modeling results indicated that the 'message' moiety of seven bitopic modulators shared the same binding mode with the orthosteric site of the KOR and MOR, whereas the 'address' moiety bound with different subdomains of the allosteric site of the KOR and MOR. The 'address' moiety of seven bitopic modulators bound to different subdomains of the allosteric site of the KOR and MOR may exhibit distinguishable allosteric modulations to the binding affinity and/or efficacy of the 'message' moiety. Moreover, the 3-hydroxy group on the phenolic moiety of the seven bitopic modulators induced selectivity to the KOR over the MOR.
Assuntos
Receptores Opioides kappa/metabolismo , Receptores Opioides mu/metabolismo , Regulação Alostérica , Sítio Alostérico , Sítios de Ligação , Humanos , Ligantes , Simulação de Acoplamento Molecular , Morfinanos/química , Morfinanos/metabolismo , Naltrexona/análogos & derivados , Naltrexona/química , Naltrexona/metabolismo , Ligação Proteica , Receptores Opioides kappa/química , Receptores Opioides mu/química , Compostos de Espiro/química , Compostos de Espiro/metabolismo , TermodinâmicaRESUMO
EGFR inhibitors have shown poor efficacy in head and neck squamous cell carcinoma (HNSCC) with demonstrated involvement of the insulin-like growth factor-1 receptor (IGF1R) in resistance to EGFR inhibition. IGF1R activates the PI3K-Akt pathway, which phosphorylates proline-rich Akt substrate of 40 kDa (PRAS40) to cease mTOR inhibition resulting in increased mTOR signaling. Proliferation assays separated six HNSCC cell lines into two groups: sensitive to EGFR inhibition or resistant; all sensitive cell lines demonstrated reduced sensitivity to EGFR inhibition upon IGF1R activation. Reverse phase protein microarray analysis and immunoblot identified a correlation between increased PRAS40 phosphorylation and IGFR-mediated resistance to EGFR inhibition. In sensitive cell lines, PRAS40 phosphorylation decreased 44%-80% with EGFR inhibition and was restored to 98%-196% of control by IGF1R activation, while phosphorylation was unaffected in resistant cell lines. Possible involvement of mTOR in this resistance mechanism was demonstrated through a similar pattern of p70S6K phosphorylation. However, addition of temsirolimus, an mTORC1 inhibitor, was insufficient to overcome IGF1R-mediated resistance and suggested an alternative mechanism. Forkhead box O3a (FOXO3a), which has been reported to complex with PRAS40 in the cytoplasm, demonstrated a 6-fold increase in nuclear to cytoplasmic ratio upon EGFR inhibition that was eliminated with concurrent IGF1R activation. Transcription of FOXO3a-regulated TRAIL and PTEN-induced putative kinase-1 (PINK1) was increased with EGFR inhibition in sensitive cell lines; this effect was diminished with IGF1R stimulation. IMPLICATIONS: These data suggest PRAS40 may play an important role in IGF1R-based therapeutic resistance to EGFR inhibition, and this likely occurs via inhibition of FOXO3a-mediated proapoptotic gene transcription.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptor IGF Tipo 1/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Receptores ErbB/antagonistas & inibidores , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/antagonistas & inibidores , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Fosforilação , Transdução de Sinais , Sirolimo/análogos & derivados , Sirolimo/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Serina-Treonina Quinases TOR/metabolismoRESUMO
OBJECTIVES: The insulin-like growth factor-1 receptor (IGF1R) has been implicated in therapeutic resistance in head and neck squamous cell carcinoma (HNSCC), and small molecule tyrosine kinase inhibitors (TKIs) of IGF1R activity may have anticancer activity. Therefore, the relationship between survival and IGF1R expression was assessed for oral cavity (OC) cancer, and the antitumor effects of two IGF1R-TKIs, OSI-906 and BMS-754807, were evaluated in HNSCC cell lines in vitro. METHODS: Clinical outcome data and tissue microarray immunohistochemistry were used to generate IGF1R expression-specific survival curves. Immunoblot, alamarBlue proliferation assay, trypan blue exclusion viability test, clonogenic assay, flow cytometry, and reverse phase protein array (RPPA) were used to evaluate in vitro responses to IGF1R-TKIs. RESULTS: For patients with stage III/IV OCSCC, higher IGF1R expression was associated with poorer overall 5-year survival (P = 0.029). Both BMS-754807 and OSI-906 caused dose-dependent inhibition of IGF1R and Akt phosphorylation and inhibited proliferation; BMS-754807 was more potent than OSI-906. Both drugs reduced HNSCC cell viability; only OSI-906 was able to eliminate all viable cells at 10 µM. The two drugs similarly inhibited clonogenic cell survival. At 1 µM, only BMS-754807 caused a fourfold increase in the basal apoptotic rate. RPPA demonstrated broad effects of both drugs on canonical IGF1R signaling pathways and also inhibition of human epidermal growth factor receptor-3 (HER3), Src, paxillin, and ezrin phosphorylation. CONCLUSION: OSI-906 and BMS-754807 inhibit IGF1R activity in HNSCC cell lines with reduction in prosurvival and proliferative signaling and with concomitant antiproliferative and proapoptotic effects. Such antagonists may have utility as adjuvants to existing therapies for HNSCC. LEVEL OF EVIDENCE: NA Laryngoscope, 130:1470-1478, 2020.
Assuntos
Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Imidazóis/uso terapêutico , Fator de Crescimento Insulin-Like I/antagonistas & inibidores , Pirazinas/uso terapêutico , Pirazóis/uso terapêutico , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Triazinas/uso terapêutico , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Imidazóis/farmacologia , Fator de Crescimento Insulin-Like I/biossíntese , Neoplasias Bucais/tratamento farmacológico , Estadiamento de Neoplasias , Pirazinas/farmacologia , Pirazóis/farmacologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Resultado do Tratamento , Triazinas/farmacologia , Células Tumorais CultivadasRESUMO
Epidermal growth factor receptor (EGFR) inhibitors have limited efficacy in head and neck squamous cell carcinoma (HNSCC) due to various resistance mechanisms, such as activation of the insulin-like growth factor-1 receptor (IGF1R), which initiates pro-survival signaling. Survivin, a member of the inhibitor of apoptosis proteins family, is expressed at relatively high levels in malignant tissues and plays a role in cell division. Expression of survivin in tumors has been shown to correlate with poor prognosis due to chemotherapy resistance and anti-apoptotic behavior. We previously demonstrated that activation of the IGF1R reduces sensitivity to EGFR-tyrosine kinase inhibitors (TKIs) via reduced apoptosis suggesting a role of survivin in this process. This study evaluates the role of survivin in IGF1R-mediated lapatinib resistance. Using HNSCC cell lines FaDu and SCC25, survivin expression increased and lapatinib sensitivity decreased with IGF1R activation. Further, these effects were reversed by the survivin inhibitor YM-155. Conversely, survivin expression and lapatinib sensitivity were unchanged with IGF1R activation in UNC10 cells. YM-155 enhanced the inhibitory effect of lapatinib on UNC10 cells, regardless of activation of the IGF1R. These results demonstrate that enhanced survivin expression correlates with IGF1R-mediated lapatinib resistance in HNSCC cells and suggest that regulation of survivin expression may be a key mechanistic element in IGF1R-based therapeutic resistance. Combinatorial treatment with survivin antagonists and EGFR-TKIs warrants further investigation.
RESUMO
Objective Patients with head and neck squamous cell carcinoma (HNSCC) have significant wound-healing difficulties. While adipose-derived stem cells (ASCs) facilitate wound healing, ASCs may accelerate recurrence when applied to a cancer field. This study evaluates the impact of ASCs on HNSCC cell lines in vitro and in vivo. Study Design In vitro experiments using HNSCC cell lines and in vivo mouse experiments. Setting Basic science laboratory. Subjects and Methods Impact of ASCs on in vitro proliferation, survival, and migration was assessed using 8 HNSCC cell lines. One cell line was used in a mouse orthotopic xenograft model to evaluate in vivo tumor growth in the presence and absence of ASCs. Results Addition of ASCs did not increase the number of HNSCC cells. In clonogenic assays to assess cell survival, addition of ASCs increased colony formation only in SCC9 cells (maximal effect 2.3-fold, P < .02) but not in other HNSCC cell lines. In scratch assays to assess migration, fluorescently tagged ASCs did not migrate appreciably and did not increase the rate of wound closure in HNSCC cell lines. Addition of ASCs to HNSCC xenografts did not increase tumor growth. Conclusion Using multiple in vitro and in vivo approaches, ASCs did not significantly stimulate HNSCC cell proliferation or migration and increased survival in only a single cell line. These findings preliminarily suggest that the use of ASCs may be safe in the setting of HNSCC but that further investigation on the therapeutic use of ASCs in the setting of HNSCC is needed.
Assuntos
Tecido Adiposo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Células-Tronco , Animais , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos NusRESUMO
BACKGROUND: In head and neck squamous cell carcinoma (HNSCC), resistance to single-agent targeted therapy may be overcome by co-targeting of compensatory signaling pathways. METHODS: A targeted drug screen with 120 combinations was used on 9 HNSCC cell lines. RESULTS: Multiple novel drug combinations demonstrated synergistic growth inhibition. Combining the insulin-like growth factor-1 receptor (IGF-1R) inhibitor, BMS754807, with either the human epidermal growth factor receptor (HER)-family inhibitor, BMS599626, or the Src-family kinase inhibitor, dasatinib, resulted in substantial synergy and growth inhibition. Depending on the cell line, these combinations induced synergistic or additive apoptosis; when synergistic apoptosis was observed, AKT phosphorylation was inhibited to a greater extent than either drug alone. Conversely, when additive apoptosis occurred, AKT phosphorylation was not reduced by the drug combination. CONCLUSION: Combined IGF-1R/HER family and IGF-1R/Src family inhibition may have therapeutic potential in HNSCC. AKT may be a node of convergence between IGF-1R signaling and pathways that compensate for IGF-1R inhibition.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carbamatos/administração & dosagem , Carcinoma de Células Escamosas/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Dasatinibe/administração & dosagem , Resistencia a Medicamentos Antineoplásicos , Sinergismo Farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Pirazóis/administração & dosagem , Triazinas/administração & dosagemRESUMO
OBJECTIVE: To demonstrate the feasibility of detecting and quantifying extracellular signal-related kinase (ERK) phosphorylation status using nanoimmunoassay (NIA). STUDY DESIGN: Analyses using Cal27, SCC25, and OSC19 head and neck squamous carcinoma cell lines in vitro and in a murine xenograft model. SUBJECTS AND METHODS: NIA and immunoblot were performed on whole-cell lysates, tumor lysates, and fine-needle aspirate biopsies to detect ERK phosphorylation states. RESULTS: Using NIA, all 6 isoforms of ERK1/2, including nonphosphorylated, monophosphorylated, and diphosphorylated species, could be reliably detected, distinguished, and quantified in a single assay using a single antibody. In vitro treatment of Cal27 cells with the epidermal growth factor receptor inhibitor gefitinib abolished phospho-ERK detection by immunoblot but resulted in residual detectable species by NIA. Residual phospho-ERK in gefitinib-treated cells could be further reduced by the addition of the insulin-like growth factor 1 receptor inhibitor OSI-906; this correlated with an additional decrease in proliferation over gefitinib alone. In a pilot study of 4 murine xenograft tumors, NIA performed on tumor lysates and fine-needle aspirate biopsies demonstrated altered ERK profiles after 2 days of gefitinib treatment compared with untreated mice. CONCLUSION: NIA offers a novel approach to quantitating the activation state of signaling molecules such as ERK in nanoscale in vitro and in vivo samples across a wide dynamic range. As such, it has potential to provide molecular diagnostic information before, during, and after treatment using a minimally invasive technique. Further study is warranted to determine its utility in assessing signaling proteins as biomolecular outcome predictors in clinical trials.
Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Células Escamosas/enzimologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Neoplasias de Cabeça e Pescoço/enzimologia , Imunoensaio , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Estudos de Viabilidade , Gefitinibe , Xenoenxertos , Imunoensaio/métodos , Técnicas In Vitro , Camundongos , Nanotecnologia , Quinazolinas/farmacologiaRESUMO
Therapies targeting oncogenic drivers rapidly induce compensatory adaptive responses that blunt drug effectiveness, contributing to therapeutic resistance. Adaptive responses are characteristic of robust cell signaling networks, and thus there is increasing interest in drug combinations that co-target the driver and the adaptive response. An alternative approach to co-inhibiting oncogenic and adaptive targets is to identify a critical node where the activities of these targets converge. Nodes of convergence between signaling modules represent potential therapeutic vulnerabilities because their inhibition could result in the collapse of the network, leading to enhanced cytotoxicity. In this report we demonstrate that p70S6 kinase (p70S6K) can function as a critical node linking HER-family and phosphoinositide-3-kinase (PI3K) pathway signaling. We used high-throughput combinatorial drug screening to identify adaptive survival responses to targeted therapies, and found that HER-family and PI3K represented compensatory signaling pathways. Co-targeting these pathways with drug combinations caused synergistic cytotoxicity in cases where inhibition of neither target was effective as a monotherapy. We utilized Reverse Phase Protein Arrays and determined that phosphorylation of ribosomal protein S6 was synergistically down-regulated upon HER-family and PI3K/mammalian target of rapamycin (mTOR) co-inhibition. Expression of constitutively active p70S6K protected against apoptosis induced by combined HER-family and PI3K/mTOR inhibition. Direct inhibition of p70S6K with small molecule inhibitors phenocopied HER-family and PI3K/mTOR co-inhibition. These data implicate p70S6K as a critical node in the HER-family/PI3K signaling network. The ability of direct inhibitors of p70S6K to phenocopy co-inhibition of two upstream signaling targets indicates that identification and targeting of critical nodes can overcome adaptive resistance to targeted therapies.