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1.
Cancers (Basel) ; 16(5)2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38473298

RESUMO

The Epstein-Barr virus (EBV) is accepted as a primary risk factor for certain nasopharyngeal carcinoma (NPC) subtypes, where the virus persists in a latent stage which is thought to contribute to tumorigenesis. Current treatments are sub-optimal, and recurrence occurs in many cases. An alternative therapeutic concept is aimed at triggering the lytic cycle of EBV selectively in tumor cells as a means to add clinical benefit. While compounds able to stimulate the lytic cascade have been identified, their clinical application so far has been limited. We are developing a novel anticancer molecule, NEO212, that was generated by covalent conjugation of the alkylating agent temozolomide (TMZ) to the naturally occurring monoterpene perillyl alcohol (POH). In the current study, we investigated its potential to trigger the lytic cycle of EBV in NPC cells in vitro and in vivo. We used the established C666.1 cell line and primary patient cells derived from the brain metastasis of a patient with NPC, both of which harbored latent EBV. Upon treatment with NEO212, there was an increase in EBV proteins Zta and Ea-D, key markers of the lytic cycle, along with increased levels of CCAAT/enhancer-binding protein homologous protein (CHOP), a marker of endoplasmic reticulum (ER) stress, followed by the activation of caspases. These effects could also be confirmed in tumor tissue from mice implanted with C666.1 cells. Towards a mechanistic understanding of these events, we used siRNA-mediated knockdown of CHOP and inclusion of anti-oxidant compounds. Both approaches blocked lytic cycle induction by NEO212. Therefore, we established a sequence of events, where NEO212 caused reactive oxygen species (ROS) production, which triggered ER stress and elevated the levels of CHOP, which was required to stimulate the lytic cascade of EBV. Inclusion of the antiviral agent ganciclovir synergistically enhanced the cytotoxic impact of NEO212, pointing to a potential combination treatment for EBV-positive cancers which should be explored further. Overall, our study establishes NEO212 as a novel agent able to stimulate EBV's lytic cycle in NPC tumors, with implications for other virus-associated cancers.

2.
Autophagy ; 19(12): 3169-3188, 2023 12.
Artigo em Inglês | MEDLINE | ID: mdl-37545052

RESUMO

Glioblastoma (GBM) is the most aggressive primary brain tumor, exhibiting a high rate of recurrence and poor prognosis. Surgery and chemoradiation with temozolomide (TMZ) represent the standard of care, but, in most cases, the tumor develops resistance to further treatment and the patients succumb to disease. Therefore, there is a great need for the development of well-tolerated, effective drugs that specifically target chemoresistant gliomas. NEO214 was generated by covalently conjugating rolipram, a PDE4 (phosphodiesterase 4) inhibitor, to perillyl alcohol, a naturally occurring monoterpene related to limonene. Our previous studies in preclinical models showed that NEO214 harbors anticancer activity, is able to cross the blood-brain barrier (BBB), and is remarkably well tolerated. In the present study, we investigated its mechanism of action and discovered inhibition of macroautophagy/autophagy as a key component of its anticancer effect in glioblastoma cells. We show that NEO214 prevents autophagy-lysosome fusion, thereby blocking autophagic flux and triggering glioma cell death. This process involves activation of MTOR (mechanistic target of rapamycin kinase) activity, which leads to cytoplasmic accumulation of TFEB (transcription factor EB), a critical regulator of genes involved in the autophagy-lysosomal pathway, and consequently reduced expression of autophagy-lysosome genes. When combined with chloroquine and TMZ, the anticancer impact of NEO214 is further potentiated and unfolds against TMZ-resistant cells as well. Taken together, our findings characterize NEO214 as a novel autophagy inhibitor that could become useful for overcoming chemoresistance in glioblastoma.Abbreviations: ATG: autophagy related; BAFA1: bafilomycin A1; BBB: blood brain barrier; CQ: chloroquine; GBM: glioblastoma; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MGMT: O-6-methylguanine-DNA methyltransferase; MTOR: mechanistic target of rapamycin kinase; MTORC: MTOR complex; POH: perillyl alcohol; SQSTM1/p62: sequestosome 1; TFEB: transcription factor EB; TMZ: temozolomide.


Assuntos
Glioblastoma , Glioma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Autofagia/genética , Rolipram/metabolismo , Rolipram/farmacologia , Rolipram/uso terapêutico , Morte Celular , Monoterpenos/farmacologia , Monoterpenos/metabolismo , Monoterpenos/uso terapêutico , Glioma/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Sirolimo/farmacologia , Cloroquina/farmacologia , Cloroquina/uso terapêutico , Lisossomos/metabolismo
3.
J Neurosurg ; 139(3): 822-830, 2023 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36738464

RESUMO

OBJECTIVE: Immune checkpoint-inhibitory therapeutic antibodies have shown striking activity against several types of cancers but are less effective against brain-localized malignancies, in part due to the protective effect of the blood-brain barrier (BBB). The authors hypothesized that intraarterial (IA) delivery of a novel compound, NEO100, has the potential to safely and reversibly open the BBB to enable brain-targeted therapeutic activity of checkpoint-inhibitory antibodies. METHODS: Immunocompetent mice with syngeneic glioblastoma or melanoma cells implanted into their brains were subjected to a single IA injection of NEO100 to open their BBB. One dose of murine anti-PD-1/PD-L1 antibody was either coinjected with NEO100 or separately injected intravenously. Brain penetration of these antibodies and levels of CD8+ T cell infiltrate into the tumor microenvironment were quantitated and animal survival was monitored. RESULTS: IA NEO100 enabled the increased accumulation of checkpoint-inhibitory antibodies in the brain, along with greater numbers of T cells. In both malignancy models, a single intervention of IA NEO100 combined with antibody resulted in the long-term survival of animals. Antibody treatment in the absence of NEO100 was far less effective. CONCLUSIONS: BBB opening by IA NEO100 facilitates brain tumor access by checkpoint-inhibitory antibodies and enables their therapeutic activity, along with increased levels of T-cell recruitment.


Assuntos
Neoplasias Encefálicas , Carcinoma , Animais , Camundongos , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais/farmacologia , Neoplasias Encefálicas/tratamento farmacológico , Imunoterapia/métodos , Encéfalo , Microambiente Tumoral
4.
Cancers (Basel) ; 14(24)2022 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-36551551

RESUMO

Many patients with acute myeloid leukemia (AML) are still dying from this disease. In the past, the alkylating agent temozolomide (TMZ) has been investigated for AML and found to be partially effective; however, the presence of O6-methylguanine DNA methyltransferase (MGMT; a DNA repair enzyme) in tumor cells confers profound treatment resistance against TMZ. We are developing a novel anticancer compound, called NEO212, where TMZ was covalently conjugated to perillyl alcohol (a naturally occurring monoterpene). NEO212 has revealed robust therapeutic activity in a variety of preclinical cancer models, including AML. In the current study, we investigated its impact on a panel of human AML cell lines and found that it exerted cytotoxic potency even against MGMT-positive cells that were highly resistant to TMZ. Furthermore, NEO212 strongly stimulated the expression of a large number of macrophage-associated marker genes, including CD11b/ITGAM. This latter effect could not be mimicked when cells were treated with TMZ or an equimolar mix of individual agents, TMZ plus perillyl alcohol. The superior cytotoxic impact of NEO212 appeared to involve down-regulation of MGMT protein levels. In a mouse model implanted with TMZ-resistant, MGMT-positive AML cells, two 5-day cycles of 25 mg/kg NEO212 achieved an apparent cure, as mice survived >300 days without any signs of disease. In parallel toxicity studies with rats, a 5-day cycle of 200 mg/kg NEO212 was well tolerated by these animals, whereas animals that were given 200 mg/kg TMZ all died due to severe leukopenia. Together, our results show that NEO212 exerts pleiotropic effects on AML cells that include differentiation, proliferation arrest, and eventual cell death. In vivo, NEO212 was well tolerated even at dosages that far exceed the therapeutic need, indicating a large therapeutic window. These results present NEO212 as an agent that should be considered for development as a therapeutic agent for AML.

5.
Cancers (Basel) ; 13(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298603

RESUMO

Despite progress in the treatment of acute myeloid leukemia (AML), the clinical outcome remains suboptimal and many patients are still dying from this disease. First-line treatment consists of chemotherapy, which typically includes cytarabine (AraC), either alone or in combination with anthracyclines, but drug resistance can develop and significantly worsen prognosis. Better treatments are needed. We are developing a novel anticancer compound, NEO212, that was created by covalent conjugation of two different molecules with already established anticancer activity, the alkylating agent temozolomide (TMZ) and the natural monoterpene perillyl alcohol (POH). We investigated the anticancer activity of NEO212 in several in vitro and in vivo models of AML. Human HL60 and U937 AML cell lines, as well as different AraC-resistant AML cell lines, were treated with NEO212 and effects on cell proliferation, cell cycle, and cell death were investigated. Mice with implanted AraC-sensitive or AraC-resistant AML cells were dosed with oral NEO212, and animal survival was monitored. Our in vitro experiments show that treatment of cells with NEO212 results in growth inhibition via potent G2 arrest, which is followed by apoptotic cell death. Intriguingly, NEO212 was equally potent in highly AraC-resistant cells. In vivo, NEO212 treatment strikingly extended survival of AML mice and the majority of treated mice continued to thrive and survive without any signs of illness. At the same time, we were unable to detect toxic side effects of NEO212 treatment. All in all, the absence of side effects, combined with striking therapeutic activity even in an AraC-resistant context, suggests that NEO212 should be developed further toward clinical testing.

6.
PLoS One ; 15(9): e0238238, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32881880

RESUMO

The prognosis for patients with glioblastoma (GB) remains grim. Concurrent temozolomide (TMZ) radiation-the cornerstone of glioma control-extends the overall median survival of GB patients by only a few months over radiotherapy alone. While these survival gains could be partly attributed to radiosensitization, this benefit is greatly minimized in tumors expressing O6-methylguanine DNA methyltransferase (MGMT), which specifically reverses O6-methylguanine lesions. Theoretically, non-O6-methylguanine lesions (i.e., the N-methylpurine adducts), which represent up to 90% of TMZ-generated DNA adducts, could also contribute to radiosensitization. Unfortunately, at concentrations attainable in clinical practice, the alkylation capacity of TMZ cannot overwhelm the repair of N-methylpurine adducts to efficiently exploit these lesions. The current therapeutic application of TMZ therefore faces two main obstacles: (i) the stochastic presence of MGMT and (ii) a blunted radiosensitization potential at physiologic concentrations. To circumvent these limitations, we are developing a novel molecule called NEO212-a derivatization of TMZ generated by coupling TMZ to perillyl alcohol. Based on gas chromatography/mass spectrometry and high-performance liquid chromatography analyses, we determined that NEO212 had greater tumor cell uptake than TMZ. In mouse models, NEO212 was more efficient than TMZ at crossing the blood-brain barrier, preferentially accumulating in tumoral over normal brain tissue. Moreover, in vitro analyses with GB cell lines, including TMZ-resistant isogenic variants, revealed more potent cytotoxic and radiosensitizing activities for NEO212 at physiologic concentrations. Mechanistically, these advantages of NEO212 over TMZ could be attributed to its enhanced tumor uptake presumably leading to more extensive DNA alkylation at equivalent dosages which, ultimately, allows for N-methylpurine lesions to be better exploited for radiosensitization. This effect cannot be achieved with TMZ at clinically relevant concentrations and is independent of MGMT. Our findings establish NEO212 as a superior radiosensitizer and a potentially better alternative to TMZ for newly diagnosed GB patients, irrespective of their MGMT status.


Assuntos
Dacarbazina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos , Glioma/tratamento farmacológico , Radiossensibilizantes/uso terapêutico , Temozolomida/uso terapêutico , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Dacarbazina/análise , Dacarbazina/metabolismo , Dacarbazina/farmacologia , Dacarbazina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/genética , Cromatografia Gasosa-Espectrometria de Massas , Glioma/patologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , O(6)-Metilguanina-DNA Metiltransferase/metabolismo , Radiossensibilizantes/análise , Radiossensibilizantes/metabolismo , Radiossensibilizantes/farmacologia , Temozolomida/análise , Temozolomida/metabolismo , Temozolomida/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Ther Adv Med Oncol ; 11: 1758835919891567, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31839810

RESUMO

BACKGROUND: Mycosis fungoides (MF) and Sézary syndrome (SS) are subtypes of primary cutaneous lymphomas and represent complex diseases regarding their physiopathology and management. Depending on the stage of the disease, different treatment regimens are applied, but there is no consensus on an optimal approach. Prognosis for patients with early stage MF is favorable, but significantly worsens in advanced disease and in SS, where patients frequently relapse and require multiple therapies. METHODS: We investigated the potential anticancer effects of NEO212, a novel compound generated by covalently conjugating perillyl alcohol (a natural monoterpene) to temozolomide (an alkylating agent), on MF and SS cell lines in vitro. HUT-78, HUT-102, and MyLa cells were treated with NEO212 under different conditions, and drug effects on proliferation, viability, and apoptosis were characterized. RESULTS: NEO212 inhibited proliferation, diminished viability, and stimulated apoptosis in all cell lines, although with varying degrees of potency in the different cell lines. It down-regulated c-myc and cyclin D1 proteins, which are required for cell proliferation, but triggered endoplasmic reticulum stress and activation of caspases. Pretreatment of cells with antioxidants ascorbic acid and beta-mercaptoethanol prevented these NEO212-induced effects. CONCLUSIONS: NEO212 exerted promising anticancer effects on SS and MF cell lines. The generation of reactive oxygen species (ROS) appears to play a key role in the NEO212-induced cell death process, because the blockage of ROS with antioxidants prevented caspase activation. We propose that NEO212 should be investigated further toward clinical testing in these tumor types.

8.
Molecules ; 23(11)2018 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-30413113

RESUMO

We developed a bacterial expression system to produce a recombinant disintegrin, vicrostatin (VCN), whose structure is based on a natural disintegrin isolated from southern copperhead snake venom. Our goal is to develop VCN for potential clinical translation as an anti-cancer agent. VCN is a peptide of 69 amino acids with a single tyrosine residue. We have employed VCN as integrin-targeted radionuclide therapy (brachytherapy) for treatment of glioblastoma (GBM, glioma). GBM is a deadly brain cancer that doesn't discriminate between sexes and knows no age limit. We established that the tyrosine residue in VCN can be radioiodinated with full retention of bioactivity. 131I-VCN was utilized for integrin-targeted radionuclide therapy using mouse models of glioma. The combination of radioiodinated VCN plus temozolomide (a DNA alkylating agent) significantly prolonged survival of glioma-bearing mice. We also obtained similar results using an immunocompetent mouse model and a murine glioma cell line. In summary, as demonstrated in studies reported here we have shown that VCN as targeted radionuclide therapy for GBM has significant translational potential for therapy of this deadly disease.


Assuntos
Neoplasias Encefálicas/radioterapia , Glioblastoma/radioterapia , Radioisótopos do Iodo/química , Proteínas Recombinantes/química , Venenos de Serpentes/genética , Animais , Braquiterapia , Neoplasias Encefálicas/tratamento farmacológico , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Desintegrinas/química , Desintegrinas/genética , Desintegrinas/metabolismo , Sinergismo Farmacológico , Glioblastoma/tratamento farmacológico , Humanos , Camundongos , Venenos de Serpentes/metabolismo , Temozolomida/administração & dosagem , Temozolomida/farmacologia , Tirosina/química , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Cancers (Basel) ; 10(10)2018 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-30274152

RESUMO

The chemotherapeutic agent temozolomide (TMZ) kills tumor cells preferentially via alkylation of the O6-position of guanine. However, cells that express the DNA repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), or harbor deficient DNA mismatch repair (MMR) function, are profoundly resistant to this drug. TMZ is in clinical use for melanoma, but objective response rates are low, even when TMZ is combined with O6-benzylguanine (O6BG), a potent MGMT inhibitor. We used in vitro and in vivo models of melanoma to characterize the early events leading to cellular TMZ resistance. Melanoma cell lines were exposed to a single treatment with TMZ, at physiologically relevant concentrations, in the absence or presence of O6BG. Surviving clones and mass cultures were analyzed by Western blot, colony formation assays, and DNA methylation studies. Mice with melanoma xenografts received TMZ treatment, and tumor tissue was analyzed by immunohistochemistry. We found that MGMT-negative melanoma cell cultures, before any drug treatment, already harbored a small fraction of MGMT-positive cells, which survived TMZ treatment and promptly became the dominant cell type within the surviving population. The MGMT-negative status in individual cells was not stable, as clonal selection of MGMT-negative cells again resulted in a mixed population harboring MGMT-positive, TMZ-resistant cells. Blocking the survival advantage of MGMT via the addition of O6BG still resulted in surviving clones, although at much lower frequency and independent of MGMT, and the resistance mechanism of these clones was based on a common lack of expression of MSH6, a key MMR enzyme. TMZ treatment of mice implanted with MGMT-negative melanoma cells resulted in effective tumor growth delay, but eventually tumor growth resumed, with tumor tissue having become MGMT positive. Altogether, these data reveal stochastic expression of MGMT as a pre-existing, key determinant of TMZ resistance in melanoma cell lines. Although MGMT activity can effectively be eliminated by pharmacologic intervention with O6BG, additional layers of TMZ resistance, although considerably rarer, are present as well and minimize the cytotoxic impact of TMZ/O6BG combination treatment. Our results provide rational explanations regarding clinical observations, where the TMZ/O6BG regimen has yielded mostly disappointing outcomes in melanoma patients.

10.
Biomacromolecules ; 15(7): 2347-58, 2014 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-24871936

RESUMO

Recombinant protein therapeutics have increased in number and frequency since the introduction of human insulin, 25 years ago. Presently, proteins and peptides are commonly used in the clinic. However, the incorporation of peptides into clinically approved nanomedicines has been limited. Reasons for this include the challenges of decorating pharmaceutical-grade nanoparticles with proteins by a process that is robust, scalable, and cost-effective. As an alternative to covalent bioconjugation between a protein and nanoparticle, we report that biologically active proteins may themselves mediate the formation of small multimers through steric stabilization by large protein polymers. Unlike multistep purification and bioconjugation, this approach is completed during biosynthesis. As proof-of-principle, the disintegrin protein called vicrostatin (VCN) was fused to an elastin-like polypeptide (A192). A significant fraction of fusion proteins self-assembled into multimers with a hydrodynamic radius of 15.9 nm. The A192-VCN fusion proteins compete specifically for cell-surface integrins on human umbilical vein endothelial cells (HUVECs) and two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Confocal microscopy revealed that, unlike linear RGD-containing protein polymers, the disintegrin fusion protein undergoes rapid cellular internalization. To explore their potential clinical applications, fusion proteins were characterized using small animal positron emission tomography (microPET). Passive tumor accumulation was observed for control protein polymers; however, the tumor accumulation of A192-VCN was saturable, which is consistent with integrin-mediated binding. The fusion of a protein polymer and disintegrin results in a higher intratumoral contrast compared to free VCN or A192 alone. Given the diversity of disintegrin proteins with specificity for various cell-surface integrins, disintegrin fusions are a new source of biomaterials with potential diagnostic and therapeutic applications.


Assuntos
Neoplasias da Mama/tratamento farmacológico , Polímeros/química , Polímeros/farmacologia , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/farmacologia , Animais , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Desintegrinas/química , Desintegrinas/farmacologia , Elastina/química , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Integrinas/metabolismo , Camundongos Nus , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Peptídeos/química , Ensaios Antitumorais Modelo de Xenoenxerto
11.
PLoS One ; 5(6): e10929, 2010 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-20532165

RESUMO

Similar to other integrin-targeting strategies, disintegrins have previously shown good efficacy in animal cancer models with favorable pharmacological attributes and translational potential. Nonetheless, these polypeptides are notoriously difficult to produce recombinantly due to their particular structure requiring the correct pairing of multiple disulfide bonds for biological activity. Here, we show that a sequence-engineered disintegrin (called vicrostatin or VCN) can be reliably produced in large scale amounts directly in the oxidative cytoplasm of Origami B E. coli. Through multiple integrin ligation (i.e., alphavbeta3, alphavbeta5, and alpha5beta1), VCN targets both endothelial and cancer cells significantly inhibiting their motility through a reconstituted basement membrane. Interestingly, in a manner distinct from other integrin ligands but reminiscent of some ECM-derived endogenous anti-angiogenic fragments previously described in the literature, VCN profoundly disrupts the actin cytoskeleton of endothelial cells (EC) inducing a rapid disassembly of stress fibers and actin reorganization, ultimately interfering with EC's ability to invade and form tubes (tubulogenesis). Moreover, here we show for the first time that the addition of a disintegrin to tubulogenic EC sandwiched in vitro between two Matrigel layers negatively impacts their survival despite the presence of abundant haptotactic cues. A liposomal formulation of VCN (LVCN) was further evaluated in vivo in two animal cancer models with different growth characteristics. Our data demonstrate that LVCN is well tolerated while exerting a significant delay in tumor growth and an increase in the survival of treated animals. These results can be partially explained by potent tumor anti-angiogenic and pro-apoptotic effects induced by LVCN.


Assuntos
Inibidores da Angiogênese/farmacologia , Apoptose/efeitos dos fármacos , Desintegrinas/antagonistas & inibidores , Integrinas/antagonistas & inibidores , Neovascularização Patológica/prevenção & controle , Linhagem Celular Tumoral , Desintegrinas/farmacologia , Humanos , Fosforilação
12.
Biochem Biophys Res Commun ; 388(2): 240-6, 2009 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-19665997

RESUMO

For internalization experiments that use fluorescent antibody (Ab) staining to distinguish between inside versus outside cellular localization of various receptor targeting ligands, it is critical that there be efficient removal of all residual surface-bound fluorescent Ab. To achieve this, a fluorescent Ab removal technique is commonly employed in receptor internalization assays that utilizes low pH glycine-based buffers to wash off the residual non-internalized fluorescent Ab retained on cell surfaces. In this study, we highlight the shortcomings of this technique and propose an alternative in situ proteolytic approach that we found to be non-deleterious to the cells and significantly more effective in removing the residual fluorescence resulting from non-internalized surface-bound Ab.


Assuntos
Anticorpos/química , Endocitose , Imunofluorescência , Pepsina A/química , Linhagem Celular Tumoral , Humanos , Concentração de Íons de Hidrogênio , Ligantes
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