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1.
Cancers (Basel) ; 14(1)2022 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-35008385

RESUMO

Tumors deploy various immune-evasion mechanisms that create a suppressive environment and render effector T-cells exhausted and inactive. Therefore, a rational utilization of checkpoint inhibitors may alleviate exhaustion and may partially restore antitumor functions. However, in high-tumor-burden models, the checkpoint blockade fails to maintain optimal efficacy, and other interventions are necessary to overcome the inhibitory tumor stroma. One such strategy is the use of radiotherapy to reset the tumor microenvironment and maximize systemic antitumor outcomes. In this study, we propose the use of anti-PD1 and anti-TIGIT checkpoint inhibitors in conjunction with our novel RadScopal technique to battle highly metastatic lung adenocarcinoma tumors, bilaterally established in 129Sv/Ev mice, to mimic high-tumor-burden settings. The RadScopal approach is comprised of high-dose radiation directed at primary tumors with low-dose radiation delivered to secondary tumors to improve the outcomes of systemic immunotherapy. Indeed, the triple therapy with RadScopal + anti-TIGIT + anti-PD1 was able to prolong the survival of treated mice and halted the growth of both primary and secondary tumors. Lung metastasis counts were also significantly reduced. In addition, the low-dose radiation component reduced TIGIT receptor (PVR) expression by tumor-associated macrophages and dendritic cells in secondary tumors. Finally, low-dose radiation within triple therapy decreased the percentages of TIGIT+ exhausted T-cells and TIGIT+ regulatory T-cells. Together, our translational approach provides a new treatment alternative for cases refractory to other checkpoints and may bring immunotherapy into a new realm of systemic disease control.

2.
Cancer Res ; 82(5): 916-928, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-34965932

RESUMO

Squamous cell carcinoma driven by human papillomavirus (HPV) is more sensitive to DNA-damaging therapies than its HPV-negative counterpart. Here, we show that p16, the clinically used surrogate for HPV positivity, renders cells more sensitive to radiotherapy via a ubiquitin-dependent signaling pathway, linking high levels of this protein to increased activity of the transcription factor SP1, increased HUWE1 transcription, and degradation of ubiquitin-specific protease 7 (USP7) and TRIP12. Activation of this pathway in HPV-positive disease led to decreased homologous recombination and improved response to radiotherapy, a phenomenon that can be recapitulated in HPV-negative disease using USP7 inhibitors in clinical development. This p16-driven axis induced sensitivity to PARP inhibition and potentially leads to "BRCAness" in head and neck squamous cell carcinoma (HNSCC) cells. Thus, these findings support a functional role for p16 in HPV-positive tumors in driving response to DNA damage, which can be exploited to improve outcomes in both patients with HPV-positive and HPV-negative HNSCC. SIGNIFICANCE: In HPV-positive tumors, a previously undiscovered pathway directly links p16 to DNA damage repair and sensitivity to radiotherapy via a clinically relevant and pharmacologically targetable ubiquitin-mediated degradation pathway.


Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Infecções por Papillomavirus , Carcinoma de Células Escamosas/patologia , Proteínas de Transporte , Inibidor p16 de Quinase Dependente de Ciclina/genética , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Dano ao DNA , DNA Viral/genética , Neoplasias de Cabeça e Pescoço/genética , Humanos , Papillomaviridae/genética , Transdução de Sinais , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina , Ubiquitina-Proteína Ligases/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo
3.
J Nanobiotechnology ; 19(1): 416, 2021 Dec 11.
Artigo em Inglês | MEDLINE | ID: mdl-34895262

RESUMO

BACKGROUND: Combining radiotherapy with PD1 blockade has had impressive antitumor effects in preclinical models of metastatic lung cancer, although anti-PD1 resistance remains problematic. Here, we report results from a triple-combination therapy in which NBTXR3, a clinically approved nanoparticle radioenhancer, is combined with high-dose radiation (HDXRT) to a primary tumor plus low-dose radiation (LDXRT) to a secondary tumor along with checkpoint blockade in a mouse model of anti-PD1-resistant metastatic lung cancer. METHODS: Mice were inoculated with 344SQR cells in the right legs on day 0 (primary tumor) and the left legs on day 3 (secondary tumor). Immune checkpoint inhibitors (ICIs), including anti-PD1 (200 µg) and anti-CTLA4 (100 µg) were given intraperitoneally. Primary tumors were injected with NBTXR3 on day 6 and irradiated with 12-Gy (HDXRT) on days 7, 8, and 9; secondary tumors were irradiated with 1-Gy (LDXRT) on days 12 and 13. The survivor mice at day 178 were rechallenged with 344SQR cells and tumor growth monitored thereafter. RESULTS: NBTXR3 + HDXRT + LDXRT + ICIs had significant antitumor effects against both primary and secondary tumors, improving the survival rate from 0 to 50%. Immune profiling of the secondary tumors revealed that NBTXR3 + HDXRT + LDXRT increased CD8 T-cell infiltration and decreased the number of regulatory T (Treg) cells. Finally, none of the re-challenged mice developed tumors, and they had higher percentages of CD4 memory T cells and CD4 and CD8 T cells in both blood and spleen relative to untreated mice. CONCLUSIONS: NBTXR3 nanoparticle in combination with radioimmunotherapy significantly improves anti-PD1 resistant lung tumor control via promoting antitumor immune response.


Assuntos
Inibidores de Checkpoint Imunológico/farmacologia , Memória Imunológica/efeitos dos fármacos , Neoplasias Pulmonares , Nanopartículas/química , Radiossensibilizantes , Animais , Resistencia a Medicamentos Antineoplásicos , Feminino , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Camundongos , Neoplasias Experimentais , Radiossensibilizantes/química , Radiossensibilizantes/farmacologia , Radioimunoterapia
4.
Nat Commun ; 12(1): 6340, 2021 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-34732714

RESUMO

Despite radiation forming the curative backbone of over 50% of malignancies, there are no genomically-driven radiosensitizers for clinical use. Herein we perform in vivo shRNA screening to identify targets generally associated with radiation response as well as those exhibiting a genomic dependency. This identifies the histone acetyltransferases CREBBP/EP300 as a target for radiosensitization in combination with radiation in cognate mutant tumors. Further in vitro and in vivo studies confirm this phenomenon to be due to repression of homologous recombination following DNA damage and reproducible using chemical inhibition of histone acetyltransferase (HAT), but not bromodomain function. Selected mutations in CREBBP lead to a hyperacetylated state that increases CBP and BRCA1 acetylation, representing a gain of function targeted by HAT inhibition. Additionally, mutations in CREBBP/EP300 are associated with recurrence following radiation in squamous cell carcinoma cohorts. These findings provide both a mechanism of resistance and the potential for genomically-driven treatment.


Assuntos
Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Mutação com Ganho de Função , Histona Acetiltransferases/metabolismo , Recombinação Homóloga , Acetilação , Animais , Apoptose , Proteína BRCA1/metabolismo , Biomarcadores Tumorais , Linhagem Celular Tumoral , Histona Acetiltransferases/química , Histona Acetiltransferases/genética , Humanos , Masculino , Camundongos Nus , Mutação , Neoplasias/genética , Neoplasias/terapia , Domínios Proteicos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
5.
Int J Radiat Oncol Biol Phys ; 111(3): 647-657, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34242713

RESUMO

PURPOSE: Radiation combined with PD1 blockade offers significant treatment benefits in several tumor types; however, anti-PD1 resistance precludes such benefits in many cases. Here we attempted to overcome anti-PD1 resistance by combining localized radiation with a radioenhancing nanoparticle (NBTXR3) and systemic anti-PD1 treatment to achieve abscopal effects in an anti-PD1-resistant mouse model of lung cancer. METHODS AND MATERIALS: Female 129Sv/Ev mice were inoculated with 344SQ anti-PD1-resistant (344SQR) or anti-PD1-sensitive (344SQP) metastatic lung cancer cells in the right leg on day 0 ("primary" tumor) and the left leg on day 4 ("secondary" tumor). Primary tumors were injected intratumorally with NBTXR3 on day 7 and were irradiated with 12 Gy on days 8, 9, and 10. Mice were given 6 intraperitoneal injections of anti-PD1. T cell receptor repertoire was analyzed in tumor samples with RNA sequencing, infiltration of CD8 T cells with immunohistochemical staining, and activities of various immune pathways with NanoString analysis. RESULTS: The triple combination of NBTXR3 with localized radiation and systemic anti-PD1 significantly delayed the growth of both irradiated and unirradiated tumors in both 344SQP and 344SQR tumor models. NBTXR3 remodeled the immune microenvironment of unirradiated tumors by triggering the activation of various immune pathways, increasing the number of CD8+ T cells, and modifying the T cell receptor repertoire in the 344SQR tumor model. CONCLUSIONS: The ability of NBTXR3 to evoke significant abscopal effects in both anti-PD1-sensitive and anti-PD1-resistant lung cancers could open the possibility of its use for treating patients with metastatic lung cancer regardless of sensitivity (or resistance) to immunotherapies.


Assuntos
Antineoplásicos , Neoplasias Pulmonares , Nanopartículas , Animais , Linfócitos T CD8-Positivos , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Pulmonares/radioterapia , Camundongos , Receptor de Morte Celular Programada 1 , Receptores de Antígenos de Linfócitos T , Microambiente Tumoral
6.
Int J Radiat Biol ; 97(8): 1121-1128, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32073931

RESUMO

PURPOSE: Head and neck cancers (HNSCC) are routinely treated with radiotherapy; however, normal tissue toxicity remains a concern. Therefore, it is important to validate treatment modalities combining molecularly targeted agents with radiotherapy to improve the therapeutic ratio. The aim of this study was to assess the ability of the PARP inhibitor niraparib (MK-4827) alone, or in combination with cell cycle checkpoint abrogating drugs targeting Chk1 (MK-8776) or Wee1 (MK-1775), to radiosensitize HNSCCs in the context of HPV status. MATERIALS AND METHODS: PARP1, PARP2, Chk1 or Wee1 shRNA constructs were analyzed from an in vivo shRNA screen of HNSCC xenografts comparing radiosensitization differences between HPV(+) and HPV(-) tumors. Radiosensitization by niraparib alone or in combination with MK-8776 or MK-1775 was assessed by clonogenic survival in HPV(-) and HPV(+) cells; and the role of p16 in determining response was explored. Relative expressions of DNA repair genes were compared by PCR array in HPV(+) and HPV(-) cells, and following siRNA-mediated knockdown of TRIP12 in HPV(-) cells. RESULTS: In vivo shRNA screening showed a modest preferential radiosensitization by Wee1 and PARP2 in HPV(-) and Chk1 in HPV(+) tumor models. Niraparib alone enhanced the radiosensitivity of all HNSCC cell lines tested. However, HPV(-) cells were sensitized to a greater degree, as suggested by the shRNA screen. When combined with MK-8776 or MK-1775, radiosensitization was further enhanced in an HPV dependent manner with HPV(+) cells enhanced by MK-8776 and HPV(-) cells enhanced by MK-1775. A PCR array for DNA repair genes showed PARP and HR proteins BRCA1 and RAD51 were much lower in HPV(+) cells than in HPV(-). Similarly, directly knocking down p16-dependent TRIP12 decreased expression of these same genes. Overexpressing p16 decreased TRIP12 expression and increased radiosensitivity in HPV(-) HN5. However, while PARP inhibition led to significant radiosensitization in the control, it led to no further significant radiosensitization in p16 overexpressing cells. Forced p16 expression in HPV(-) HN5 increased accumulation in G1 and subG1 and limited progression to S phase, thus reducing effectiveness of PARP inhibition. CONCLUSIONS: Niraparib effectively radiosensitizes HNSCCs with a greater benefit seen in HPV(-). HPV status also plays a role in response to MK-8776 or MK-1775 when combined with niraparib due to differences in DNA repair mechanisms. This study suggests that using cell cycle abrogators in combination with PARP inhibitors may be a beneficial treatment option in HNSCC, but also emphasizes the importance of HPV status when considering effective treatment strategies.


Assuntos
Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Dano ao DNA , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/patologia , Pontos de Checagem do Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Humanos , Indazóis/farmacologia , Piperidinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Pirazóis/farmacologia , Pirimidinonas/farmacologia , Tolerância a Radiação/efeitos dos fármacos
7.
Transl Oncol ; 14(2): 100983, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33340886

RESUMO

Radiotherapy (RT) has been used to control tumors by physically damaging DNA and inducing apoptosis; it also promotes antitumor immune responses via neoantigens release and augmenting immune-oncology agents to elicit systemic response. Tumor regression after RT can recruit inflammatory cells, such as tumor-associated macrophages and CD11b+ myeloid cell populations, a major subset of which may actually be immunosuppressive. However, these inflammatory cells also express Toll-like receptors (TLRs) that can be stimulated to reverse suppressive characteristics and promote systemic antitumor outcomes. Here, we investigated the effects of adding CMP-001, a CpG-A oligodeoxynucleotide TLR9 agonist delivered in a virus-like particle (VLP), to RT in two murine models (344SQ metastatic lung adenocarcinoma and CT26 colon carcinoma). High-dose RT (12Gy x 3 fractions) significantly increased the percentages of plasmacytoid dendritic cells within the tumor islets 3- and 5-days post-RT; adding CMP-001 after RT also enhanced adaptive immunity by increasing the proportion of CD4+ and CD8+ T cells. RT plus CMP-001-mediated activation of the immune system led to significant inhibition of tumor growth at both primary and abscopal tumor sites, thereby suggesting a new combinatorial treatment strategy for systemic disease.

8.
J Immunother Cancer ; 8(1)2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32581056

RESUMO

BACKGROUND: Despite outstanding responses to anti-PD-1 agents in a subset of non-small cell lung cancer (NSCLC) patients, approximately 80% of patients fail to have prolonged favorable response. Recent studies show that tumor cell oxidative metabolism is a barrier to PD-1 immunotherapy and radiotherapy could overcome PD-1 resistance, so it is urgent to determine if combination treatment with radiotherapy and a novel oxidative phosphorylation (OXPHOS) inhibitor (IACS-010759) is an effective strategy against PD-1 resistance in NSCLC. METHODS: The antitumor effect of this combinational treatment was evaluated in vitro and in vivo. For in vivo experiments, we treated 129Sv/Ev mice with anti-PD1-sensitive and anti-PD1-resistant 344SQ NSCLC adenocarcinoma xenografts with oral IACS-010759 combined with radiotherapy (XRT). In vitro experiments included PCR, seahorse bioenergetic profiling, flow cytometry phenotyping, and clonogenic survival assay. RESULTS: In the current study, we found that our PD-1-resistant model utilized OXPHOS to a significantly greater extent than the PD-1-sensitive model and XRT increased OXPHOS in vitro and in vivo. Thus, we explored the effect of the novel OXPHOS inhibitor IACS-010759 on PD-1-resistant NSCLC in an effort to overcome XRT-induced immunosuppression and maximize response to PD-1. Additionally, combined XRT and IACS-010759 promoted antitumor effects in the PD-1-resistant model, but not in the sensitive model. After elucidation of the most optimal dose/fractionation scheme of XRT with IACS-010759, the combinatorial therapy with this regimen did not increase the abscopal antitumor effect, although IACS-010549 did not decrease CD45+, CD4+, and CD8+ immune cells. Finally, triple therapy with IACS-010759, XRT, and anti-PD-1 promoted abscopal responses and prolonged survival time. CONCLUSION: OXPHOS inhibition as part of a combinatorial regimen with XRT is a promising strategy to address PD-1-resistant NSCLC, and this combination is being tested clinically.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Quimiorradioterapia/métodos , Neoplasias Pulmonares/terapia , Oxidiazóis/farmacologia , Piperidinas/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral/transplante , Modelos Animais de Doenças , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos da radiação , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Oxidiazóis/uso terapêutico , Fosforilação Oxidativa/efeitos dos fármacos , Piperidinas/uso terapêutico , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Receptor de Morte Celular Programada 1/imunologia
9.
Cancer Immunol Res ; 8(7): 883-894, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32299915

RESUMO

Immune checkpoint inhibitors, such as anti-PD-1/PD-L1, have emerged as promising therapies for advanced non-small cell lung cancer (NSCLC). However, approximately 80% of patients do not respond to immunotherapy given alone because of intrinsic or acquired resistance. Radiotherapy (XRT) can overcome PD-1 resistance and improve treatment outcomes, but its efficacy remains suboptimal. The tyrosine phosphatase SHP-2, expressed in some cancers and in immune cells, has been shown to negatively affect antitumor immunity. Our hypothesis was that SHP-2 inhibition in combination with anti-PD-L1 would enhance immune-mediated responses to XRT and synergistically boost antitumor effects in an anti-PD-1-resistant mouse model. We treated 129Sv/Ev mice with anti-PD-1-resistant 344SQ NSCLC adenocarcinoma with oral SHP099 (a SHP-2 inhibitor) combined with XRT and intraperitoneal anti-PD-L1. Primary tumors were treated with XRT (three fractions of 12 Gy each), whereas abscopal (out-of-field) tumors were observed but not treated. XRT in combination with SHP099 and anti-PD-L1 promoted local and abscopal responses, reduced lung metastases, and improved mouse survival. XRT also increased SHP-2+ M1 tumor-associated macrophages in abscopal tumors (P = 0.019). The addition of SHP099 also associated with a higher M1/M2 ratio, greater numbers of CD8+ T cells, and fewer regulatory T cells. This triple-combination therapy had strong antitumor effects in a mouse model of anti-PD-1-resistant NSCLC and may be a novel therapeutic approach for anti-PD-1-resistant NSCLC in patients.


Assuntos
Antineoplásicos Imunológicos/farmacologia , Antígeno B7-H1/antagonistas & inibidores , Carcinoma Pulmonar de Células não Pequenas/terapia , Neoplasias Pulmonares/terapia , Piperidinas/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Pirimidinas/farmacologia , Animais , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Quimiorradioterapia , Resistencia a Medicamentos Antineoplásicos , Feminino , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL
10.
Clin Cancer Res ; 24(3): 600-607, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29113987

RESUMO

Purpose: This study examined the potential role of the nuclear deubiquitinating enzyme BRCA1-associated protein-1 (BAP1) in radioresistance in head and neck squamous cell cancer (HNSCC).Experimental Design: We overexpressed, knocked down, and rescued BAP1 expression in six HNSCC cell lines, three human papillomavirus (HPV)-negative and three HPV-positive, and examined the effects on radiosensitivity in vitro and in an HNSCC mouse xenograft model. Radiosensitivity was assessed by clonogenic cell survival and tumor growth delay assays; changes in protein expression were analyzed by immunofluorescence staining and Western blotting. We also analyzed The Cancer Genome Atlas HNSCC database to test for associations between BAP1 expression and outcome in patients.Results: Overexpression of BAP1 induced radioresistance in both cell lines and xenograft models; conversely, BAP1 knockdown led to increased ubiquitination of histone H2A, which has been implicated in DNA repair. We further found that BAP1 depletion suppressed the assembly of constitutive BRCA1 foci, which are associated with homologous recombination (HR), but had minimal effect on γ-H2AX foci and did not affect proteins associated with nonhomologous end joining, suggesting that BAP1 affects radiosensitivity in HNSCC by modifying HR. Finally, in patients with HNSCC, overexpression of BAP1 was associated with higher failure rates after radiotherapy.Conclusions: BAP1 can induce radioresistance in HNSCC cells, possibly via deubiquitination of H2Aub and modulation of HR, and was associated with poor outcomes in patients with HNSCC. BAP1 may be a potential therapeutic target in HNSCC. Clin Cancer Res; 24(3); 600-7. ©2017 AACR.


Assuntos
Biomarcadores Tumorais , Neoplasias de Cabeça e Pescoço/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Animais , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Neoplasias de Cabeça e Pescoço/etiologia , Neoplasias de Cabeça e Pescoço/patologia , Neoplasias de Cabeça e Pescoço/terapia , Histonas/metabolismo , Recombinação Homóloga , Humanos , Camundongos , Infecções por Papillomavirus/complicações , Infecções por Papillomavirus/virologia , Prognóstico , Tolerância a Radiação/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Ubiquitinação , Ensaios Antitumorais Modelo de Xenoenxerto
11.
Methods Mol Biol ; 1165: 3-9, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24839014

RESUMO

Metformin is a commonly utilized antidiabetic agent, which has been associated with improved clinical outcomes in cancer patients. The precise mechanism of action remains unclear, but preclinical evidence suggests that metformin can sensitize tumor cells to the effects to conventional chemotherapeutic agents and ionizing radiation (IR). In this chapter we discuss the general background of an approach to evaluate the effects of metformin on conventional chemotherapeutic agent toxicity in a preclinical model.


Assuntos
Antineoplásicos/farmacologia , Metformina/farmacologia , Neoplasias/tratamento farmacológico , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica , Morte Celular/efeitos dos fármacos , Humanos , Metformina/administração & dosagem , Neoplasias/metabolismo , Neoplasias/patologia , Resultado do Tratamento
12.
Methods Mol Biol ; 1165: 11-8, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24839015

RESUMO

Metformin is a commonly utilized antidiabetic agent, which has been associated with improved clinical outcomes in cancer patients. The precise mechanism of action remains unclear, but preclinical evidence suggests that metformin can sensitize tumor cells to the effects to conventional chemotherapeutic agents and ionizing radiation (IR). In this chapter we describe two assays to investigate the effects of combination of metformin and a chemotherapeutic agent (in this case cisplatin) in head and neck cancer squamous cell carcinoma (HNSCC) cell lines.


Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Escamosas/patologia , Cisplatino/farmacologia , Neoplasias de Cabeça e Pescoço/patologia , Metformina/farmacologia , Antineoplásicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Glicólise/efeitos dos fármacos , Humanos , Metformina/administração & dosagem , Mitocôndrias/enzimologia , Carcinoma de Células Escamosas de Cabeça e Pescoço
13.
PLoS Pathog ; 7(4): e1002025, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21552325

RESUMO

Activation of I-κB kinases (IKKs) and NF-κB by the human T lymphotropic virus type 1 (HTLV-1) trans-activator/oncoprotein, Tax, is thought to promote cell proliferation and transformation. Paradoxically, expression of Tax in most cells leads to drastic up-regulation of cyclin-dependent kinase inhibitors, p21(CIP1/WAF1) and p27(KIP1), which cause p53-/pRb-independent cellular senescence. Here we demonstrate that p21(CIP1/WAF1)-/p27(KIP1)-mediated senescence constitutes a checkpoint against IKK/NF-κB hyper-activation. Senescence induced by Tax in HeLa cells is attenuated by mutations in Tax that reduce IKK/NF-κB activation and prevented by blocking NF-κB using a degradation-resistant mutant of I-κBα despite constitutive IKK activation. Small hairpin RNA-mediated knockdown indicates that RelA induces this senescence program by acting upstream of the anaphase promoting complex and RelB to stabilize p27(KIP1) protein and p21(CIP1/WAF1) mRNA respectively. Finally, we show that down-regulation of NF-κB by the HTLV-1 anti-sense protein, HBZ, delay or prevent the onset of Tax-induced senescence. We propose that the balance between Tax and HBZ expression determines the outcome of HTLV-1 infection. Robust HTLV-1 replication and elevated Tax expression drive IKK/NF-κB hyper-activation and trigger senescence. HBZ, however, modulates Tax-mediated viral replication and NF-κB activation, thus allowing HTLV-1-infected cells to proliferate, persist, and evolve. Finally, inactivation of the senescence checkpoint can facilitate persistent NF-κB activation and leukemogenesis.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/fisiologia , Senescência Celular/genética , Produtos do Gene tax/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , NF-kappa B/metabolismo , Proteínas Virais/fisiologia , Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Inibidor de Quinase Dependente de Ciclina p27/fisiologia , Regulação para Baixo , Produtos do Gene tax/genética , Células HeLa , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Dados de Sequência Molecular , Inibidor de NF-kappaB alfa , RNA Mensageiro/metabolismo , Proteínas dos Retroviridae , Replicação Viral/efeitos dos fármacos
14.
J Virol ; 85(6): 3001-9, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21209109

RESUMO

Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia/lymphoma (ATL), a malignancy of CD4(+) T cells whose etiology is thought to be associated with the viral trans-activator Tax. We have shown recently that Tax can drastically upregulate the expression of p27(Kip1) and p21(CIP1/WAF1) through protein stabilization and mRNA trans-activation and stabilization, respectively. The Tax-induced surge in p21(CIP1/WAF1) and p27(Kip1) begins in S phase and results in cellular senescence. Importantly, HeLa and SupT1 T cells infected by HTLV-1 also arrest in senescence, thus challenging the notion that HTLV-1 infection causes cell proliferation. Here we use time-lapse microscopy to investigate the effect of Tax on cell cycle progression in two reporter cell lines, HeLa/18x21-EGFP and HeLa-FUCCI, that express enhanced green fluorescent protein (EGFP) under the control of 18 copies of the Tax-responsive 21-bp repeat element and fluorescent ubiquitin cell cycle indicators, respectively. Tax-expressing HeLa cells exhibit elongated or stalled cell cycle phases. Many of them bypass mitosis and become single senescent cells as evidenced by the expression of senescence-associated ß-galactosidase. Such cells have twice the normal equivalent of cellular contents and hence are enlarged, with exaggerated nuclei. Interestingly, nocodazole treatment revealed a small variant population of HeLa/18x21-EGFP cells that could progress into mitosis normally with high levels of Tax expression, suggesting that genetic or epigenetic changes that prevent Tax-induced senescence can occur spontaneously at a detectable frequency.


Assuntos
Ciclo Celular/fisiologia , Produtos do Gene tax/metabolismo , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Fusão Gênica Artificial , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Microscopia de Vídeo
15.
J Antimicrob Chemother ; 65(5): 931-8, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20167587

RESUMO

OBJECTIVES: The objective of this study was to identify existing clinical compounds that either possess a fungicidal activity alone or can act synergistically with fungistatic antifungals. METHODS: We screened a clinical compound library for drugs that exhibited anti-Aspergillus activity. Among selected compounds, the cationic peptide antibiotic polymyxin B was chosen for further characterization because it can be used parenterally and topically. The fungicidal effect of polymyxin B and its synergistic interactions with azole antifungals were tested against a variety of fungal species. The toxicity of the drug combination of polymyxin B and fluconazole was compared with that of each drug alone in mammalian cell cultures. RESULTS: We found that polymyxin B possesses a broad-spectrum antifungal activity at relatively high concentrations. However, because of its synergistic interactions with azole antifungals, polymyxin B at much lower concentrations exerts a potent fungicidal effect against Cryptococcus neoformans, Candida albicans and non-albicans Candida species and moulds when combined with azoles. The combination of polymyxin B and fluconazole at concentrations within susceptible breakpoints is particularly potent against C. neoformans isolates, including fluconazole-resistant strains. The drug combination displayed no additional toxicity compared with polymyxin B alone when tested in cell culture. CONCLUSIONS: The combination of polymyxin B and fluconazole has the potential to be used in the clinic to treat systemic cryptococcosis. Our findings suggest that combining cationic peptide antibiotics with azole antifungals could provide a new direction for developing novel antifungal therapies.


Assuntos
Antifúngicos/farmacologia , Fluconazol/farmacologia , Fungos/efeitos dos fármacos , Viabilidade Microbiana/efeitos dos fármacos , Polimixina B/farmacologia , Sinergismo Farmacológico
16.
J Virol ; 82(17): 8442-55, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18596104

RESUMO

Infection by the human T-cell leukemia virus type 1 (HTLV-1) is thought to cause dysregulated T-cell proliferation, which in turn leads to adult T-cell leukemia/lymphoma. Early cellular changes after HTLV-1 infection have been difficult to study due to the poorly infectious nature of HTLV-1 and the need for cell-to-cell contact for HTLV-1 transmission. Using a series of reporter systems, we show that HeLa cells cease proliferation within one or two division cycles after infection by HTLV-1 or transduction of the HTLV-1 tax gene. HTLV-1-infected HeLa cells, like their tax-transduced counterparts, expressed high levels of p21(CIP1/WAF1) and p27(KIP1), developed mitotic abnormalities, and became arrested in G(1) in senescence. In contrast, cells of a human osteosarcoma lineage (HOS) continued to divide after HTLV-1 infection or Tax expression, albeit at a reduced growth rate and with mitotic aberrations. Unique to HOS cells is the dramatic reduction of p21(CIP1/WAF1) and p27(KIP1) expression, which is in part associated with the constitutive activation of the phosphatidylinositol-3-kinase (PI3K)-protein kinase B (Akt) pathway. The loss of p21(CIP1/WAF1) and p27(KIP1) in HOS cells apparently allows HTLV-1- and Tax-induced G(1) arrest to be bypassed. Finally, HTLV-1 infection and Tax expression also cause human SupT1 T cells to arrest in the G(1) phase of the cell cycle. These results suggest that productive HTLV-1 infection ordinarily leads to Tax-mediated G(1) arrest. However, T cells containing somatic mutations that inactivate p21(CIP1/WAF1) and p27(KIP1) may continue to proliferate after HTLV-1 infection and Tax expression. These infected cells can expand clonally, accumulate additional chromosomal abnormalities, and progress to cancer.


Assuntos
Ciclo Celular/fisiologia , Fase G1/fisiologia , Vírus Linfotrópico T Tipo 1 Humano/fisiologia , Linhagem Celular , Técnicas de Cocultura , Infecções por HTLV-I/virologia , Células HeLa , Humanos , Rim/citologia
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