RESUMEN

Here, we present a methodology based on multiplexed fluorescence screening of two- or three-dimensional cell cultures in a newly designed multichambered microwell chip, allowing direct assessment of drug or immune cell cytotoxic efficacy. We establish a framework for cell culture, formation of tumor spheroids, fluorescence labeling, and imaging of fixed or live cells at various magnifications directly in the chip together with data analysis and interpretation. The methodology is demonstrated by drug cytotoxicity screening using ovarian and non-small cell lung cancer cells and by cellular cytotoxicity screening targeting tumor spheroids of renal carcinoma and ovarian carcinoma with natural killer cells from healthy donors. The miniaturized format allowing long-term cell culture, efficient screening, and high-quality imaging of small sample volumes makes this methodology promising for individualized cytotoxicity tests for precision medicine.

Asunto(s)

Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Humanos , Técnicas de Cultivo de Célula , Esferoides Celulares

RESUMEN

Chemotherapy has been the predominant treatment modality for cancer patients, but its overall performance is still modest. Difficulty in penetration of tumor tissues, a toxic profile in high doses, multidrug resistance in an array of tumor types, and the differential architecture of tumor cells as they grow are some of the bottlenecks associated with the clinical usage of chemotherapeutics. Recent advances in tumor biology understanding and the emergence of novel targeted drug delivery tools leveraging various nanosystems offer hope for developing effective cancer treatments. Topotecan is a topoisomerase I inhibitor that stabilizes the transient TOPO I-DNA cleavable complex, leading to single-stranded breaks in DNA. Due to its novel mechanism of action, TOPO is reported to be active against various carcinomas, namely small cell lung cancer, cervical cancer, breast cancer, and ovarian cancer. Issues of cross-resistance with numerous drugs, rapid conversion to its inactive form in biological systems, appended adverse effects, and higher water solubility limit its therapeutic efficacy in clinical settings. Topotecan nanoformulations offer several benefits for enhancing the therapeutic action of this significant class of chemotherapeutics. The likelihood that the target cancer cells will be exposed to the chemotherapeutic drug while in the drug-sensitive s-phase is increased due to the slow and sustained release of the chemotherapeutic, which could provide for a sustained duration of exposure of the target cancer cells to the bioavailable drug and result in the desired therapeutic outcome. This article explores nanoenabled active and passive targeting strategies and combinatorial therapy employing topotecan to ameliorate various cancers, along with a glimpse of the clinical studies utilizing the said molecule.

RESUMEN

FMS-like tyrosine kinase 3 (FLT3) is the most frequently-mutated gene in acute myeloid leukemia and a target for tyrosine kinase inhibitors (TKI). FLT3 TKI have yielded limited improvements to clinical outcomes. One reason for this is TKI inhibition by endogenous factors. We characterized plasma protein binding of FLT3 TKI, specifically staurosporine-derivatives (STS-TKI) by alpha-1-acid glycoprotein (AGP); simulating its effects upon drug efficacy. Human AGP inhibits the anti-proliferative activity of STS-TKI in FLT3-ITD-dependent cells, with IC50 shifts higher than clinically achievable. This is not seen with non-human plasma. Mifepristone co-treatment, with its higher AGP affinity, improves TKI activity despite AGP, yielding IC50s predicted to be clinically effective. In a mouse model of AGP drug inhibition, mifepristone restores midostaurin activity. This suggests combinatorial methods for overcoming plasma protein inhibition of existing TKIs for leukemia as well as providing a platform for investigating the drug-protein interaction space for developing more potent small-molecule agents.

Asunto(s)

Leucemia Mieloide Aguda , Inhibidores de Proteínas Quinasas , Animales , Proteínas Sanguíneas/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Inhibidores de Proteínas Quinasas/farmacología , Tirosina Quinasa 3 Similar a fms/genética

RESUMEN

Designing optimal combinatorial drug therapies is challenging, because the drug interactions depend not only on the drugs involved, but also on their doses. With recent advances, combinatorial drug therapy is closer than ever to clinical application. Herein, we summarize approaches and advances over the past decade for identifying and optimizing drug combination therapies, with innovations across research fields, covering physical laboratory platforms for combination screening to computational models and algorithms designed for synergism prediction and optimization. By comparing different types of approach, we detail a three-step workflow that could maximize the overall optimization efficiency, thus enabling the application of personalized optimization of combinatorial drug therapy.

Asunto(s)

Simulación por Computador , Interacciones Farmacológicas , Quimioterapia Combinada , Medicina de Precisión , Desarrollo de Medicamentos , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Humanos , Flujo de Trabajo

RESUMEN

Therapeutics targeting the phosphatidylinositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway initially produce potent antitumor effects, but resistance frequently occurs. Using a phosphoproteome analysis, we found that colorectal cancer (CRC) cells exhibit resistance against PI3K/mTOR inhibition through feedback activation of multiple receptor tyrosine kinases, and their downstream focal adhesion kinase, Src and extracellular signal-regulated kinases signaling. Unexpectedly, PI3K/mTOR blockade causes senescence, mediated by the activation of the stress kinase p38. The senescent cancer cells induce the secretion of various cytokines and this senescence-associated secretome increases migration and invasion capabilities of CRC cells. We found that cotargeting PI3K/mTOR and bromodomain and extra-terminal domain can suppress activation of many oncogenic kinases involved in resistance to the PI3K/mTOR inhibition, induce cell death in vitro and tumor regression in vivo, and further prolong the survival of xenograft models. Our findings provide a rationale for a novel therapeutic strategy to overcome resistance to the PI3K/mTOR inhibitors in CRC.

Asunto(s)

Azepinas/administración & dosificación , Neoplasias Colorrectales/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Imidazoles/administración & dosificación , Proteómica/métodos , Quinolinas/administración & dosificación , Triazoles/administración & dosificación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Azepinas/farmacología , Células CACO-2 , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Neoplasias Colorrectales/metabolismo , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HCT116 , Humanos , Imidazoles/farmacología , Ratones , Fosfatidilinositol 3-Quinasa/metabolismo , Fosforilación/efectos de los fármacos , Quinolinas/farmacología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , Triazoles/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto

RESUMEN

With the advent of high-throughput technologies leading to big data generation, increasing number of gene signatures are being published to predict various features of diseases such as prognosis and patient survival. However, to use these signatures for identifying therapeutic targets, use of additional bioinformatic tools is indispensible part of research. Here, we have generated a pipeline comprised of nearly 15 bioinformatic tools and enrichment statistical methods to propose and validate a drug combination strategy from already approved drugs and present our approach using published pan-cancer epithelial-mesenchymal transition (EMT) signatures as a case study. We observed that histone deacetylases were critical targets to tune expression of multiple epithelial versus mesenchymal genes. Moreover, SRC and IKBK were the principal intracellular kinases regulating multiple signaling pathways. To confirm the anti-EMT efficacy of the proposed target combination in silico, we validated expression of targets in mesenchymal versus epithelial subtypes of ovarian cancer. Additionally, we inhibited the pinpointed proteins in vitro using an invasive lung cancer cell line. We found that whereas low-dose mono-therapy failed to limit cell dispersion from collagen spheroids in a microfluidic device as a metric of EMT, the combination fully inhibited dissociation and invasion of cancer cells toward cocultured endothelial cells. Given the approval status and safety profiles of the suggested drugs, the proposed combination set can be considered in clinical trials.

Asunto(s)

Biología Computacional , Histona Desacetilasas/metabolismo , Quinasa I-kappa B/metabolismo , Neoplasias/patología , Familia-src Quinasas/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/genética , Línea Celular Tumoral , Transición Epitelial-Mesenquimal , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/metabolismo

RESUMEN

Nanoparticulate systems have been receiving a significant attention especially for the treatment of cancer but one of the main hurdles is to produce these developed and high-tech nanosystems in large quantities. Anticancer drug formulations are generally designed for parenteral administrations but oral administration is still the most convenient route. In this study, orally applicable nano-sized chitosan nanoparticles (NPs) were successfully prepared using Nano Spray Dryer. It is possible to produce these NPs in large quantities by simply increasing the processing time using the machine without changing any parameter. A chemotherapeutic agent (imatinib mesylate; IMA) and nonsteroidal anti-inflammatory drug (dexketoprofen trometamol) were loaded together in these NPs. NPs were also functionalized with polyethylene glycol and folic acid to obtain long circulating NPs and tumor targeting. The antitumoral activities of formulations showed that these developed NPs can enhance the effectiveness. Animal experiments were performed on fibrosarcoma-bearing mice model, and the treatment with 0.8 mg/µL/kg IMA-loaded chitosan NPs was found to be successful to slow down the growth of tumors. The tumor tissues were removed from the animals and enzymatic activities were evaluated. The inhibitory effect of tyrosine kinase was found to be enhanced from 36.4% to 68.4% when IMA was used in combination with dexketoprofen trometamol. Furthermore, all dried NPs were found to be stable for more than a year at 25°C. Presented results show that these developed combinatorial drug-loaded NPs can be used for the treatment of fibrosarcoma, and these data can provide an insight, new strategies for productions or alternatives in cancer treatment.

Asunto(s)

Antiinflamatorios no Esteroideos/administración & dosificación , Antineoplásicos/administración & dosificación , Quitosano/química , Fibrosarcoma/tratamiento farmacológico , Mesilato de Imatinib/administración & dosificación , Cetoprofeno/análogos & derivados , Nanocápsulas/química , Trometamina/administración & dosificación , Administración Oral , Animales , Antiinflamatorios no Esteroideos/uso terapéutico , Antineoplásicos/uso terapéutico , Células CACO-2 , Composición de Medicamentos , Sistemas de Liberación de Medicamentos , Fibrosarcoma/patología , Humanos , Mesilato de Imatinib/uso terapéutico , Cetoprofeno/administración & dosificación , Cetoprofeno/uso terapéutico , Masculino , Ratones Endogámicos BALB C , Nanocápsulas/ultraestructura , Trometamina/uso terapéutico

RESUMEN

As novel heat shock protein 70 (HSP70) inhibitors, N, N'-disubstituted thiourea derivatives were designed and synthesized based on the X-ray structure of the ATPase domain (nucleotide binding domain, NBD). An ATPase activity inhibition assay revealed that these compounds effectively inhibited HSP70 ATPase activity. The results revealed that the compounds 370/371/374/379/380//392/394/397/404/405 and 407 can inhibit the HSP70 ATPase turnover with high percentages of inhibition: 50.42, 38.46, 50.45, 44.12, 47.13, 50.50, 40.95, 65.36, 46.23, 35.78, and 58.37 in 200 µM, respectively. Significant synergies with lapatinib were observed for compound 379 and compound 405 in the BT474 breast cancer cell line. A structure-function analysis revealed that most of the thiourea derivatives exhibited cooperative action with lapatinib in the BT474 cancer cell line and the BT/Lap(R)1.0 lapatinib-resistant cell line. HSP70 inhibitors may be developed as synergetic drugs in drug-resistant cancer therapy.

Asunto(s)

Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Diseño de Fármacos , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Tiourea/síntesis química , Tiourea/farmacología , Adenosina Trifosfatasas/metabolismo , Antineoplásicos/química , Línea Celular Tumoral , Técnicas de Química Sintética , Resistencia a Medicamentos , Sinergismo Farmacológico , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Modelos Moleculares , Dominios Proteicos , Tiourea/química