RESUMEN
First generation silver(I) phosphines have garnered much interest due to their vast structural diversity and promising anticancer activity. Increasing incidences of cancer, side-effects to chemotherapeutic agents and redevelopment of tumors due to resistance prompts the exploration of alternative compounds showing anticancer activity. This study revealed the effective induction of cell death by a silver(I) thiocyanate 4-methoxyphenyl phosphine complex in a malignant esophageal cell line. Apoptotic cell death was confirmed in treated cells. Moreover, mitochondrial targeting via the intrinsic cell death pathway was evident due to low levels of ATP, altered ROS activity, mitochondrial membrane depolarization, cytochrome c release and caspase-9 cleavage. The complex displayed low cytotoxicity towards two human non-malignant, skin and kidney, cell lines. The findings reported herein give further insight into the selective targeting of silver(I) phosphines and support our belief that this complex shows great promise as an effective chemotherapeutic drug.
Asunto(s)
Apoptosis/efectos de los fármacos , Complejos de Coordinación/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Caspasa 9/genética , Línea Celular Tumoral , Complejos de Coordinación/uso terapéutico , Citocromos c/genética , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Mitocondrias/metabolismo , Fosfinas/farmacología , Especies Reactivas de Oxígeno/metabolismo , Plata/química , Tiocianatos/farmacologíaRESUMEN
BACKGROUND: 2-Methoxyestradiol (2ME2) is an endogenous metabolite of 17-ß-estradiol with anti-proliferative and anti-angiogenic properties. Due to 2ME2's rapid metabolism and low oral bioavailability in in vivo settings, 2ME2 analogues have been designed to alleviate these issues. One of these compounds is 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (ESE-16). A previous work alluded to the ability of ESE-16 to induce autophagic cell death. Therefore, we investigated the mode of action of ESE-16 by studying its effects on autophagy, vesicle formation, and lysosomal organisation. SUMMARY: Vesicle formation and autophagy induction were analysed by transmission electron microscopy (TEM), monodansylcadaverine (MDC) staining and Lysotracker staining, while autophagosome turnover was analysed using microtubule-associated protein 1A/1B-light chain 3 (LC3 lipidation) analysis. MDC staining of acidic vesicles revealed an increase both in the number and size of vesicles after ESE-16 exposure. This was confirmed by TEM. Lysotracker staining indicated an increase in the size of lysosomes, as well as changes in their distribution within the cell. However, autophagy was not induced, since LC3 lipidation did not increase after exposure to ESE-16. Key -Messages: This study showed that ESE-16 exposure leads to the aggregation of acidic vesicles, identified as lysosomes, not accompanied by an induction of autophagy. Therefore, ESE-16 disrupts normal endocytic vesicle maturation likely through the inhibition of the microtubule function.
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Autofagia/efectos de los fármacos , Estrenos/farmacología , Lisosomas/efectos de los fármacos , Sulfonamidas/farmacología , Línea Celular Tumoral , Humanos , Microscopía Electrónica de Transmisión , Proteínas Asociadas a Microtúbulos/metabolismoRESUMEN
There is much interest currently in the design of metal compounds as drugs and various metal compounds are already in clinical use. These include gold(I) compounds such as auranofin and the anti-cancer platinum(II) complex, cisplatin. Bis-chelated gold(I) phosphine complexes have also shown great potential as anticancer agents, however, their efficacy has been limited by their high toxicity. In this study, silver(I) thiocyanate compounds linked to four specific ligands, were synthesized and characterized. These silver-phosphine adducts included [AgSCN{P(4-MeC6H4)3}2]2 (1); [AgSCN{P(4-ClC6H4)3}2]2 (2); [AgSCN{P(4-MeOC6H4)3}2]2 (3); [AgSCN(PPh3)2]2 (4). The compounds were found to be toxic to MCF-7 breast cancer cells while the ligands on their own were not toxic. Our findings further indicate that the silver(I) phosphine compounds induce apoptotic cell death in these breast cancer cells. In addition, the compounds were not toxic to nonmalignant fibroblast cells at the IC50 concentrations. This is an indication that the compounds show selectivity towards the cancer cells.
Asunto(s)
Neoplasias de la Mama/patología , Compuestos Organometálicos/farmacología , Plata/farmacología , Tiocianatos/farmacología , Ciclo Celular/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Células HEK293 , Humanos , Células MCF-7 , Compuestos Organometálicos/síntesis química , Compuestos Organometálicos/química , Fosfinas/química , Fosfinas/farmacología , Plata/química , Relación Estructura-Actividad , Tiocianatos/químicaRESUMEN
Esophageal cancer is one of the least studied cancers and is found to be prominent in black South African males. It is mainly diagnosed in the late stages, and patients tend to have a low 5-year survival rate of only 10%. Silver is generally used as an antimicrobial agent, with limited reports on anticancer studies. In this study, dimeric silver(I) thiocyanate complexes were used containing a variation of 4-substitued triphenylphosphines, including [AgSCN(PPh(3))(2)](2) (1), [AgSCN{P(4-MeC(6)H(4))(3)}(2)](2) (2), [AgSCN{P(4-FC(6)H(4))(3)}(2)](2) (3) and [AgSCN{P(4-ClC(6)H(4))(3)}(2)](2) (4). All four complexes, with their respective phosphine ligands, PPh(3) (L1), P(4-MeC(6)H(4))(3) (L2), P(4-FC(6)H(4))(3) (L3) and P(4-ClC(6)H(4))(3) (L4), were subjected to in vitro toxicity studies in SNO-esophageal cancer cells, using an alamarBlue(®) assay. Morphological changes, including blebbing and apoptotic body formation, were observed. Phosphatidylserine externalization, a marker of apoptosis, was quantified by flow cytometry. The phosphine ligands L1-L4, on their own, had minimal effect on the malignant while complexes 1-4 resulted in significant cell death. A 10x decreased concentration of these complexes had similar effects than cisplatin, used as the positive control. These complexes show promise as anticancer agents.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Fosfinas/química , Plata/química , Tiocianatos/química , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Citometría de Flujo , Humanos , Fosfatidilserinas/metabolismoRESUMEN
2-Methoxyestradiol, a natural metabolite of estradiol, exerts antiproliferative and antitumour properties in vitro and in vivo. Because of its low oral bioavailability, several promising analogues of 2-methoxyestradiol have been developed. In this study, the in vitro influence of the compound, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10)16-tetraene (C19), a non-commercially available 17-ß-estradiol analogue, was tested on the breast adenocarcinoma MCF-7 cell line. The in vitro influence of 24 h exposure to 0.18 µM of C19 on MCF-7 cells was evaluated on cell morphology, cell cycle progression and possible induction of apoptosis and autophagy. Polarization-optical transmitted light differential interference contrast and fluorescence microscopy revealed the presence of cells blocked in metaphase, occurrence of apoptotic bodies and compromised cell density in C19-treated cells. Hallmarks of autophagy, namely an increase in the number of acidic vacuoles and lysosomes, were also observed in C19-treated samples. An increase in the number of cells present in the sub-G1 fraction, as well as a reduction in mitochondrial membrane potential was observed. No significant alterations in caspase 8 activity were observed. A twofold increase in aggresome formation was observed in C19-treated cells. C19 induced both apoptosis and autophagy in MCF-7 cells.
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Antineoplásicos/farmacología , Caspasa 7/metabolismo , Caspasa 8/metabolismo , Estradiol/análogos & derivados , Estradiol/farmacología , Estrenos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Sulfonamidas/farmacología , Adenocarcinoma , Apoptosis/efectos de los fármacos , Autofagia/efectos de los fármacos , Neoplasias de la Mama , Femenino , Humanos , Células MCF-7 , Pliegue de ProteínaRESUMEN
BACKGROUND: A broad range of metal-coordinated complexes have been studied for their anticancer activities. However, some of these complexes display high toxicity profiles to non-malignant cells, therefore limiting their use in cancer therapeutics. Aims/Method: Several silver(I) triphenylphosphine adducts were prepared as 1:1 to 1:4 ratios of silver nitrate to triphenylphosphine. They were further used to determine their anticancer activity in a malignant SNO esophageal cell line. The silver(I) phosphine adducts include: [Ag(PPh3)]NO3 (1); [Ag(PPh3)2]NO3 (2); [Ag(PPh3)3]NO3 (3) and [Ag(PPh3)4]NO3 (4). In addition, the activity of complexes 1-4 was compared to previously reported complexes [Ag(Ph2P(CH2)2PPh2)2]NO3 (5) and [Ag(Ph2P(CH2)3PPh2)2]NO3 (6). The cytotoxicity of complexes 1-6 was also evaluated in non-malignant human dermal fibroblast cells (HDF-a). RESULTS: The majority of the complexes (specifically those containing PPh3) were found to be highly toxic to the SNO cells and less toxic towards HDF-a cells, as determined by the alamarBlue® assay. Morphological studies and flow cytometry confirmed that the silver(I) complexes induced apoptosis in the malignant cells. CONCLUSION: These results may have an impact on research related to drug discovery and silver(I) phosphine complexes could be added to the arsenal of anticancer agents in addition to the silver-bis-diphenylphosphinoethane and silver-bis-diphenylphosphinopropane adducts.
Asunto(s)
Complejos de Coordinación/farmacología , Neoplasias Esofágicas/tratamiento farmacológico , Fosfinas/farmacología , Plata/farmacología , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Esofágicas/patología , Humanos , Estructura Molecular , Fosfinas/química , Plata/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Carcinogenesis occurs via mutation of critical genes conferring enhanced survival and protection to the ensuing tissue. Current therapies in use garner success due to their specificity for certain intracellular targets. This particularity, whist beneficial in identifying tumorigenic from normal tissue states, is limited by the variations in geno/phenotypic profiles displayed between tumor tissue types. As such, tissue-specific therapeutic combinations and adjuvants are often required for adequate effect, but present symptomatic complications and occasionally generate secondary carcinogenesis displaying multi-drug resistance (MDR). An accumulation of research over the recent years has suggested that photodynamic therapy (PDT) with macrocycle photosensitizers are a promising alternative. Its administration method and toxicity mechanism present attractive features for potentially overcoming MDR cancers of multiple tissue origins with limited symptomatic onsets. Herein, we highlight these potentials as referenced against existing therapeutics and consider the impact of macrocycle-PDT for broad spectrum application regardless of tumorigenic resistance profiles.
Asunto(s)
Neoplasias/clasificación , Neoplasias/tratamiento farmacológico , Fotoquimioterapia , Resistencia a Antineoplásicos , Genotipo , Homeostasis , Humanos , Neoplasias/genética , Fármacos Fotosensibilizantes/uso terapéuticoRESUMEN
A longstanding obstacle to cancer eradication centers on the heterogeneous nature of the tissue that manifests it. Variations between cancer cell resistance profiles often result in a survival percentage following classic therapeutics. As an alternative, photodynamic therapys' (PDT) unique non-specific cell damage mechanism and high degree of application control enables it to potentially deliver an efficient treatment regime to a broad range of heterogeneous tissue types thereby overcoming individual resistance profiles. This study follows on from previous design, characterization and solubility analyses of three novel carbohydrate-ligated zinc-porphyrazine (Zn(II)Pz) derivatives. Here we report on their PDT application potential in the treatment of five common cancer tissue types in vitro. Following analyses of metabolic homeostasis, toxicity and cell death induction, overall Zn(II)Pz-PDT proved comparably efficient between all cancer tissue populations. Differential localization patterns of Zn(II)Pz derivatives between cell types did not appear to influence the overall PDT effect. All cell types exhibited significant disruptions to mitochondrial activity and associated ATP production levels. Toxicity and chromatin structure profiles revealed indiscernible patterns of damage between Zn(II)Pz derivatives and cell type. The subtle differences observed between individual Zn(II)Pz derivatives is most likely due to a combination of carbohydrate moiety characteristics on energy transfer processes and associated dosage optimization requirements per tissue type. Collectively, this indicates that resistance profiles are negated to a significant extent by Zn(II)Pz-PDT making these derivatives attractive candidates for PDT applications across multiple tissue types and subtypes.
Asunto(s)
Carbohidratos/química , Proliferación Celular/efectos de los fármacos , Metaloporfirinas/química , Fármacos Fotosensibilizantes/toxicidad , Células A549 , Adenosina Trifosfato/metabolismo , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Línea Celular Tumoral , Proliferación Celular/efectos de la radiación , Cromatina/química , Cromatina/efectos de los fármacos , Humanos , Luz , Células MCF-7 , Metaloporfirinas/uso terapéutico , Metaloporfirinas/toxicidad , Microscopía Fluorescente , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neoplasias/tratamiento farmacológico , Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/uso terapéutico , Zinc/químicaRESUMEN
Research within the field of photodynamic therapy has escalated over the past 20 years. The required conjunctional use of photosensitizers, particularly of the macrocycle structure, has lead to a vast repertoire of derivatives that branch classes and subclasses thereof. Each exhibits a differential range of physiochemical properties that influence their potential applications within the larger phototherapy field for use in either diagnostics, photodynamic therapy, both or none. Herein, we provide an overview of these properties as they relate to photodynamic therapy and to a lesser extent diagnostics. By summarizing the mechanistics of photodynamic therapy coupled to the photo-energetics displayed by macrocycle photosensitizers, we aimed to highlight the critical aspects any researcher should be aware of and consider when selecting and performing research for therapeutic application purposes. These include photosensitizer, photophysical and structural properties, synthesis design and subsequent attributes, main applications within research, common shortcomings exhibited and the current methods practiced to overcome them.
Asunto(s)
Compuestos Macrocíclicos/química , Compuestos Macrocíclicos/farmacología , Fotoquimioterapia/métodos , Fármacos Fotosensibilizantes/química , Fármacos Fotosensibilizantes/farmacología , Colorantes/química , Humanos , Luz , Metales/química , Estructura Molecular , Procesos FotoquímicosRESUMEN
Metal-based drugs have shown early promise as anticancer agents suggesting the potential application of silver(I) complexes as apoptosis-inducing agents. The ability of a silver(I) cyanide containing phosphine complex to induce cell death was evaluated in both a malignant (SNO esophageal cancer) and non-malignant (HDF-a skin and HEK293 kidney) cell lines. A dose-dependent decrease in cell viability was observed in the SNO cells. Light microscopy revealed morphological features indicative of apoptotic cell death. The mode of cell death was confirmed as apoptosis by phosphatidylserine externalization, DNA fragmentation and nuclear condensation. Furthermore, both the non-malignant cell lines showed morphological features indicative of apoptosis when exposed to complex 1. We propose the use of this silver(I) cyanide phosphine complex as an highly effective positive apoptosis control for use in anticancer studies of phosphine complexes.
RESUMEN
Sutherlandia frutescens is a medicinal plant indigenous to Southern Africa and is commonly known as the "cancer bush." This plant has traditionally been used for the treatment of various ailments, although it is best known for its claims of activity against "internal" cancers. Here we report on its effect on melanoma cells. The aim of this study was to investigate whether an extract of S. frutescens could induce apoptosis in the A375 melanoma cell line and to outline the basic mechanism of action. S. frutescens extract induced apoptosis in A375 cells as evidenced by morphological features of apoptosis, phosphatidylserine exposure, nuclear condensation, caspase activation, and the release of cytochrome c from the mitochondria. Studies in the presence of a pan-caspase inhibitor allude to caspase-independent cell death, which appeared to be mediated by the apoptosis inducing factor. Taken together, the results of this study show that S. frutescens extract is effective in inducing apoptosis in malignant melanoma cells and indicates that further in vivo mechanistic studies may be warranted.
RESUMEN
Complexing of ligands to photosensitizers (Ps) has gained popularity by enhancing solubility, cell-surface recognition and tissue specificity for applications in Photodynamic Therapy (PDT) and fluorescence-based diagnostics. Here we report on nine carbohydrate-functionalized porphyrazine (Pz-galactopyranose/methyl-ribose) derivatives bearing either H2 , Zn(II) or Ni(II) cores for potential use in PDT. Derivatives proved soluble only in organic solvents; dichloromethane (DCM) and tetrahydrofuran (THF). Derivatives were subsequently solubilized using DCM-based PEG-DSPE5000 -PBS encapsulation for biological studies due to THF cytotoxicity. Absorption spectra analyses viewed no correlation between core ion, carbohydrate type and peripheral position though encapsulation efficiency (%EE) followed a general order of Zn(II) (60-92%) > H2 (5-34%) > Ni(II) (4-21%). As such, phototoxicity of Zn(II)Pz derivatives were far superior to H2 Pz and Ni(II)Pz counterparts following 631.4 nm excitation of MCF-7 breast cancer cells. Variation was attributed to persistent aggregation and low %EE when regarding the absorption properties recorded. It is therefore believed that revision of the encapsulation method for H2 Pz and Ni(II)Pz derivatives would render improved phototoxicity. Zn(II)Pz derivatives show promise as agents for PDT of cancer.
Asunto(s)
Fotoquimioterapia , Fármacos Fotosensibilizantes/química , Interacciones Hidrofóbicas e Hidrofílicas , SolubilidadRESUMEN
Apoptosis is induced in MCF-7 breast cancer cells following treatment with salicylic acid (20 mM), either in the presence or absence of a heat shock (42°C for 30 min). In order to study the alterations of apoptotic genes with quantitative real-time PCR (qPCR), suitable genes with unchanged expression following the treatments is required for normalizing the gene expression levels. In this study, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ß-actin (ACTB), Histone H2A (HIST), constitutively expressed heat shock protein 70 (HSC70) and tyrosine 3-monooxygenase/trytophan 5 monooxygenase activation protein, 14-3-3 (YWHAZ) were evaluated as appropriate reference genes. Analysis of gene expression data with one-way ANOVA, geNorm and NormFinder identified HIST and YWHAZ as the least affected during the induction of apoptosis by the different treatments, and is the most suitable gene-pair for normalization during qPCR analysis in MCF-7 breast cancer cells undergoing apoptosis following treatment with SA and/or HS.
Asunto(s)
Apoptosis/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Proteínas 14-3-3/genética , Actinas/genética , Análisis de Varianza , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Femenino , Marcadores Genéticos , Gliceraldehído-3-Fosfato Deshidrogenasas/genética , Proteínas del Choque Térmico HSC70/genética , Respuesta al Choque Térmico/genética , Histonas/genética , Humanos , Ácido Salicílico/farmacología , Tirosina 3-Monooxigenasa/genéticaRESUMEN
BACKGROUND: Cancer cells possess an innate resistance to inducers of the death program. Novel phthalocyanines with improved physiochemical properties harbor the potential for use in photodynamic therapy (PDT); a rising treatment alternative preferred for its mostly asymptomatic application and unique mechanism of action. METHODS: This study aimed to determine whether in vitro PDT with two new metallo-phthalocyanines (metallo-Pcs), AlPcSmix and GePcSmix, are similarly effective in overcoming resistance to cell death in MCF-7 cells with a brief comparison to an established chemotherapeutic agent, etoposide. Optimum induction of cell death in these cancer cells was initially determined via measurement of cellular respiration and energy production levels. Indications of cytotoxicity and cell stress were evaluated and resultant levels compared to those in cells treated with etoposide. RESULTS: Initial findings report AlPcSmix is predominantly more detrimental to cellular function and homeostasis when excited via red light irradiation of 15 J/cm(2). It appears GePcSmix requires higher dosage administrations to achieve similar responses within identical populations. However, due to the mechanism of PDT application, our findings indicate a greater toxic effect with both phthalocyanines when compared to etoposide of higher dosage within MCF-7 cells. CONCLUSION: Both phthalocyanines, despite similarity in structure, indicate induction of different cell death response pathways based on their toxicity potential. These therefore show great promise as potential PDT agents for the treatment of cancer.
Asunto(s)
Apoptosis/efectos de los fármacos , Indoles/administración & dosificación , Metales/administración & dosificación , Fotoquimioterapia/métodos , Humanos , Isoindoles , Células MCF-7 , Fármacos Fotosensibilizantes/administración & dosificaciónRESUMEN
AIM OF STUDY: Oesophageal cancer is the ninth most common cancer in the world and the second most common cancer among South African men. It also has one of the lowest possibilities of cure, with the 5-year survival rate estimated to be only 10% overall. Sutherlandia frutescens, or the "cancer bush", is a medicinal plant indigenous to southern Africa that is believed to have anti-cancer and anti-proliferative properties. The aim of this study was to investigate the potential apoptosis-inducing effects of two S. frutescens extracts and one Sutherlandia tomentosa extract on the SNO oesophageal cancer cell line. MATERIALS AND METHODS: Cell viability and morphology of SNO cells were evaluated following exposure to the extracts. Apoptotic markers including cytochrome c translocation and phosphatidylserine externalisation were quantified by flow cytometry. The activity of caspases 3 and 7 was evaluated with spectrofluorometry. Apoptosis was evaluated in the presence of the pan-caspase inhibitor, Z-VAD-fmk. The effect of the extracts was compared to non-cancerous peripheral blood mononuclear cells (PBMCs). RESULTS: Time- and dose-response studies were conducted to establish treatment conditions of 2.5 and 5mg/ml of crude plant extracts. Microscopy studies revealed that S. frutescens- and S. tomentosa-treated SNO cells had morphological features characteristic of apoptosis. Annexin V/propidium iodide flow cytometry confirmed that the extracts do, in fact, induce apoptosis in the SNO cells. Caspase inhibition studies seem to indicate that extracts A (S. frutescens (L.) R. Br. subsp. microphylla from Colesberg), B (S. frutescens (L.) R. Br. subsp. microphylla from Platvlei) and C (S. tomentosa Eckl. & Zeyh from Stil Bay) are able to induce caspase-dependent as well as -independent cell death. The S. frutescens and S. tomentosa extracts were found to be more cytotoxic to cancerous SNO cells when compared to the PBMCs. CONCLUSIONS: S. frutescens and S. tomentosa extracts show promise as apoptosis-inducing anti-cancer agents.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias Esofágicas/patología , Fabaceae , Extractos Vegetales/farmacología , Adenosina Trifosfato/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Antineoplásicos Fitogénicos/aislamiento & purificación , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Inhibidores de Caspasas , Línea Celular Tumoral , Forma de la Célula/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Neoplasias Esofágicas/metabolismo , Fabaceae/química , Citometría de Flujo , Humanos , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/patología , Fosfatidilserinas/metabolismo , Extractos Vegetales/aislamiento & purificación , Plantas Medicinales , Transporte de Proteínas , Espectrometría de Fluorescencia , Factores de TiempoRESUMEN
OBJECTIVE: The objective of this study was to establish the influence of two metallophthalocyanine photosensitizers, in their inactive and activated forms, on the cellular reactions of esophageal cancer cells. BACKGROUND DATA: Photodynamic therapy (PDT) is an alternative used in the treatment of cancer. During PDT, the activated compound produces cytotoxic singlet molecular oxygen ((1)O(2)), which ultimately leads to cell death. Esophageal cancer has become one of the most common cancers to occur in the world, and the incidence in South Africa is high, especially within the black male population. METHODS: Optimal photosensitizer concentration was determined by following the viability of esophageal cancer (SNO) cells treated with a range of concentrations of two metallophthalocyanine photosensitizers, GePcSmix and AlPcSmix, activated by irradiation at a fluence of 20 J/cm(2). Changes in cell morphology were observed after treatment with optimal photosensitizer concentrations, and the effect of the treatment on cell proliferation and cytotoxicity were studied. RESULTS: Cell viability decreased in a dose-dependent manner after PDT, while the photosensitizers in their inactive forms did not have an effect on the cells. The altered morphology of cells after PDT was indicative of a necrotic mode of cell death. The optimal photosensitizer concentrations reduced cell proliferation by more than 50% and a significant reduction in cytotoxicity, as detected by lactate dehydrogenase release, was observed following PDT. CONCLUSION: Under the studied parameters PDT using GePcSmix and AlPcSmix in vitro could be a useful therapy for esophageal cancer since the photosensitizers alone caused no damage, but cell death is imminent post-PDT.
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Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fármacos Fotosensibilizantes/farmacología , Línea Celular Tumoral , Neoplasias Esofágicas , Humanos , Indoles/farmacología , Isoindoles , FotoquimioterapiaRESUMEN
BACKGROUND: Elevated temperatures jeopardize plant disease resistance, as mediated by salicylic acid (SA). SA potentiates heat-induced expression of the 70-kDa heat shock protein (Hsp70) in tomato cells. In mammalian cells, Hsp70 suppresses apoptosis. We hypothesized that potentiation of heat-induced Hsp70 by SA contributes to a reduction in apoptosis in tobacco protoplasts. METHODS: Tobacco protoplasts (Nicotiana tabacum) were exposed to SA (70 microM) at normal temperatures or in combination with heat shock. Hsp70/Hsc70 accumulation and phosphatidylserine (PS) exposure, DNA fragmentation, as well as loss of mitochondrial membrane potential were quantified by flow cytometry. RESULTS AND CONCLUSIONS: SA at normal temperatures did not influence Hsp70/Hsc70 accumulation, but were found to induce apoptosis. In contrast, SA in combination with HS potentiated heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts that correlated negatively with apoptosis, illustrated by decreased PS exposure and DNA fragmentation and enhanced mitochondrial membrane potential. We propose that this correlation supports a possible role for apoptosis suppression by Hsp70 under elevated temperatures during pathogen infection.
Asunto(s)
Apoptosis/fisiología , Proteínas HSP70 de Choque Térmico/metabolismo , Nicotiana/fisiología , Protoplastos/efectos de los fármacos , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacología , Animales , Fragmentación del ADN , Proteínas HSP70 de Choque Térmico/genética , Calor , Potenciales de la Membrana/fisiología , Mitocondrias/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Nicotiana/citologíaRESUMEN
Current methods to determine heat shock protein (Hsp) synthesis or accumulation in plant cells, such as Western blotting and biometabolic labelling are either indirectly quantitative, labour-intensive or biohazardous. An optimal flow cytometric protocol was developed to measure the intracellular Hsp70/Hsc70 levels in tobacco protoplasts. After heat treatments, protoplasts were fixed in 2% paraformaldehyde-phosphate-buffered saline and dehydrated overnight in methyl cellusolve, followed by permeabilization with Triton X-100 (0.1% in Protoplast Wash Fluid). Immunolabelling of Hsp70/Hsc70 was done for 1 hour with a mouse monoclonal antibody and detected by R-Phycoerythrin-conjugated goat anti-mouse IgG using flow cytometry. Flow cytometry detected a significant 1.2-fold increase in Hsp70/Hsc70 accumulation (P < 0.001) in protoplasts, while Western blotting, quantified by image analysis, showed induction under similar conditions but at lower significance (P < 0.05). The coefficients of variance for flow cytometry and Western blotting were 30.7 and 49.8 respectively. Optimum temperature of heat-induced Hsp70/Hsc70 accumulation in tobacco protoplasts occurred at 40 degrees C. Flow cytometry is proposed as a quantitative, more reproducible and rapid alternative to Western blotting for the detection of Hsp70 accumulation in plant cells.