Fanconi anemia (FA) is a fatal heterogeneous autosomal recessive disorder, characterized by progressive bone marrow failure, congenital defect and cancer predisposition. Cell culture from FA fibroblast (FAF) displays ...Fanconi anemia (FA) is a fatal heterogeneous autosomal recessive disorder, characterized by progressive bone marrow failure, congenital defect and cancer predisposition. Cell culture from FA fibroblast (FAF) displays certain abnormalities as compared to normal human dermal fibroblast (NHDF). This prompted us to investigate the effect of a specific nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract, which has demonstrated a broad spectrum of pharmacological activities, on FAF compared to NHDF. We investigated the in vitro effect of NM on FAF and NHDF cell proliferation by MTT assay, MMPs secretion by zymography, morphology by H&E staining and apoptosis by green caspase assay. FAF (FA-A: PD20, FA-A: PD220) and NHDF were cultured in modified Dulbecco Eagle media. At near confluence, the cells were treated with different concentrations of NM (0, 50, 100, 250, 500 and 1000 μg/ml) in triplicate. The cells were also treated with PMA to induce MMP-9 activity. NM had no effect on FAF cell viability in both cell lines compared to control. In contrast NM exhibited 20% at 50 and 100, 50% at 250, 60% at 500 and 70% toxicity at 1000 μg/ml on NHDF cells. Zymography demonstrated MMP-2 and MMP-9 on PMA stimulation in FAF and NM inhibited the activity of both MMP-2 and MMP-9 in a dose response fashion with total block at 500 μg/ml. In contrast, NHDF exhibited only MMP-2, both active and inactive forms, and NM inhibited their activities in a dose-dependent manner with total block at 1000 μg/ml. H&E staining did not indicate any morphological changes in FAF nor induced apoptosis at higher concentrations, as seen by caspases assay. However, although no morphological changes in NHDF were noted up to NM 100 μg/ml, progressive changes in cell shrinkage, rounding and nuclear condensation, pertaining to apoptosis, were observed at higher concentrations. These changes were consistent with the results from the green caspases apoptosis assay. Our data demonstrate that NM exhibited different responses toward FAF and NHDF. This may in part be due to elevated chromosomal break, deletion and hypersensitivity to cross linking agents, a DNA repair disorder in FAF that is lacking in NHDF.展开更多
Objective:To estimate electroporation(EP) influence on malignant and normal cells.Methods: Two cell lines including human malignant melanoma(Me-43) and normal human gingival fibroblast(HCFs) were used.EP parameters we...Objective:To estimate electroporation(EP) influence on malignant and normal cells.Methods: Two cell lines including human malignant melanoma(Me-43) and normal human gingival fibroblast(HCFs) were used.EP parameters were the following:230,1000,1 730,2 300 V/cm;30 μ s by 3 impulses for every case.The viability of cells after EP was estimated by MTT assay. The ullrastructural analysis was observed by transmission electron microscope(Zeiss EM 900). Results:In the current study we observed the intracellular effect following EP on Me-43 and HGF cells.At the conditions applied,we did not observe any significant damage of mitochondrial activity in both cell lines treated by EP.Conversely,we showed that EP in some conditions can stimulate cells to proliferation.Some changes induced by EP were only visible in electron microscopy.In fibroblast cells we observed significant changes in lower parameters of EP(230 and 1 000 V/cm).After applying higher electric field intensities(2 300 V/cm) we detected many vacuoles,myelin-like bodies and swallowed endoplasmic reticulum.In melanoma cells such strong pathological modifications after EP were not observed,in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters of EP.Conclusions:We can claim that EP conditions are cell line dependent.In terms of the intracellular morphology,human fibroblasts are more sensitive to electric field as compared with melanoma cells.Optimal conditions should be determined for each cell line.Summarizing our study,we can conclude that EP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.展开更多
The application of extracellular vesicles,particularly exosomes(EXs),is rapidly expanding in the field of medicine,owing to their remarkable properties as natural carriers of biological cargo.This study investigates u...The application of extracellular vesicles,particularly exosomes(EXs),is rapidly expanding in the field of medicine,owing to their remarkable properties as natural carriers of biological cargo.This study investigates utilization of exosomes derived from stromal cells of tumor adjacent normal tissues(NAF-EXs)for personalized medicine,which can be derived at the time of diagnosis by endoscopic ultrasound.Herein,we show that exosomes(EXs)derived from NAFs demonstrate differential bio-physical characteristics,efficient cellular internalization,drug loading efficiency,pancreatic tumor targeting and delivery of payloads.NAF-derived EXs(NAF-EXs)were used for loading ormeloxifene(ORM),a potent anti-cancer and desmoplasia inhibitor as a model drug.We found that ORM maintains normal fibroblast cell phenotype and renders them incompatible to be triggered for a CAF-like phenotype,which may be due to regulation of Ca^(2+) influx in fibroblast cells.NAF-EXs-ORM effectively blocked oncogenic signaling pathways involved in desmoplasia and epithelial mesenchymal transition(EMT)and repressed tumor growth in xenograft mouse model.In conclusion,our data suggests preferential tropism of NAF-EXs for PDAC tumors,thus imply feasibility of developing a novel personalized medicine for PDAC patients using autologous NAF-EXs for improved therapeutic outcome of anti-cancer drugs.Additionally,it provides the opportunity of utilizing this biological scaffold for effective therapeutics in combination with standard therapeutic regimen.展开更多
目的探讨趋化因子受体(chemokine receptors,CRs)、基质金属蛋白酶(matrix metalloproteinases,MMPs)、血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达与胰腺癌肿瘤相关成纤维细胞(cancer associated fibroblasts,CA...目的探讨趋化因子受体(chemokine receptors,CRs)、基质金属蛋白酶(matrix metalloproteinases,MMPs)、血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达与胰腺癌肿瘤相关成纤维细胞(cancer associated fibroblasts,CAFs)生物学行为的关系。方法用原代培养的方法建立胰腺癌CAFs和正常成纤维细胞(normal fibroblasts,NFs)。用Real-time PCR方法检测CRs(CXCR1、CXCR2、CXCR4、CXCR7)、MMPs(MMP1、MMP2、MMP7、MMP9)和VEGF m RNA的表达。结果成功建立胰腺癌CAFs和正常胰腺NFs。在CAFs和NFs中,趋化因子受体CXCR1、CXCR2、CXCR4、CXCR7 m RNA的表达均存在差异,CAFs表达量高于NFs。其中CXCR2、CXCR4 m RNA在两株细胞中的表达差异具有统计学意义(P=0.010,P=0.049)。MMPs和VEGF m RNA的表达同样存在差异,CAFs中MMP7和VEGF m RNA表达明显高于NFs,差异具有统计学意义(P=0.017,P=0.049);CAFs中MMP9 m RNA的表达明显低于NFs,差异具有统计学意义(P=0.012)。结论在胰腺癌CAFs中CRs、MMPs和VEGF m RNA的表达有差异,可能与其促侵袭转移能力存在一定的相关性。展开更多
AIM: To investigate telomerase activity and human telomerase reverse transcriptase (hTERT) expression in normal human gastric mucosal epithelial cells (nhGMECs) and fibroblasts (nhGMFs).
文摘Fanconi anemia (FA) is a fatal heterogeneous autosomal recessive disorder, characterized by progressive bone marrow failure, congenital defect and cancer predisposition. Cell culture from FA fibroblast (FAF) displays certain abnormalities as compared to normal human dermal fibroblast (NHDF). This prompted us to investigate the effect of a specific nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract, which has demonstrated a broad spectrum of pharmacological activities, on FAF compared to NHDF. We investigated the in vitro effect of NM on FAF and NHDF cell proliferation by MTT assay, MMPs secretion by zymography, morphology by H&E staining and apoptosis by green caspase assay. FAF (FA-A: PD20, FA-A: PD220) and NHDF were cultured in modified Dulbecco Eagle media. At near confluence, the cells were treated with different concentrations of NM (0, 50, 100, 250, 500 and 1000 μg/ml) in triplicate. The cells were also treated with PMA to induce MMP-9 activity. NM had no effect on FAF cell viability in both cell lines compared to control. In contrast NM exhibited 20% at 50 and 100, 50% at 250, 60% at 500 and 70% toxicity at 1000 μg/ml on NHDF cells. Zymography demonstrated MMP-2 and MMP-9 on PMA stimulation in FAF and NM inhibited the activity of both MMP-2 and MMP-9 in a dose response fashion with total block at 500 μg/ml. In contrast, NHDF exhibited only MMP-2, both active and inactive forms, and NM inhibited their activities in a dose-dependent manner with total block at 1000 μg/ml. H&E staining did not indicate any morphological changes in FAF nor induced apoptosis at higher concentrations, as seen by caspases assay. However, although no morphological changes in NHDF were noted up to NM 100 μg/ml, progressive changes in cell shrinkage, rounding and nuclear condensation, pertaining to apoptosis, were observed at higher concentrations. These changes were consistent with the results from the green caspases apoptosis assay. Our data demonstrate that NM exhibited different responses toward FAF and NHDF. This may in part be due to elevated chromosomal break, deletion and hypersensitivity to cross linking agents, a DNA repair disorder in FAF that is lacking in NHDF.
基金Suppoted by statutory funds of Medical Wroelaw University andresearch Tellowship within"Development Program of Wroclaw Medical University"funded from European Social Fund.Human CapitalNational Cohesion Strategy(Contract No.UDA-POKL.04.01.01-00-010/08-00)
文摘Objective:To estimate electroporation(EP) influence on malignant and normal cells.Methods: Two cell lines including human malignant melanoma(Me-43) and normal human gingival fibroblast(HCFs) were used.EP parameters were the following:230,1000,1 730,2 300 V/cm;30 μ s by 3 impulses for every case.The viability of cells after EP was estimated by MTT assay. The ullrastructural analysis was observed by transmission electron microscope(Zeiss EM 900). Results:In the current study we observed the intracellular effect following EP on Me-43 and HGF cells.At the conditions applied,we did not observe any significant damage of mitochondrial activity in both cell lines treated by EP.Conversely,we showed that EP in some conditions can stimulate cells to proliferation.Some changes induced by EP were only visible in electron microscopy.In fibroblast cells we observed significant changes in lower parameters of EP(230 and 1 000 V/cm).After applying higher electric field intensities(2 300 V/cm) we detected many vacuoles,myelin-like bodies and swallowed endoplasmic reticulum.In melanoma cells such strong pathological modifications after EP were not observed,in comparison with control cells. The ultrastructure of both treated cell lines was changed according to the applied parameters of EP.Conclusions:We can claim that EP conditions are cell line dependent.In terms of the intracellular morphology,human fibroblasts are more sensitive to electric field as compared with melanoma cells.Optimal conditions should be determined for each cell line.Summarizing our study,we can conclude that EP is not an invasive method for human normal and malignant cells. This technique can be safely applied in chemotherapy for delivering drugs into tumor cells.
基金UTRGV School of medicine start up,CPRIT TREC Award RP230419 and Integrated Cancer Research Core(ICRC)-RP210180Herb Kosten foundation for pancreatic cancer researchNational Institutes of Health grants R01CA206069,SC1GM139727,SC2GM139715,R01CA210192 and R01CA204552.
文摘The application of extracellular vesicles,particularly exosomes(EXs),is rapidly expanding in the field of medicine,owing to their remarkable properties as natural carriers of biological cargo.This study investigates utilization of exosomes derived from stromal cells of tumor adjacent normal tissues(NAF-EXs)for personalized medicine,which can be derived at the time of diagnosis by endoscopic ultrasound.Herein,we show that exosomes(EXs)derived from NAFs demonstrate differential bio-physical characteristics,efficient cellular internalization,drug loading efficiency,pancreatic tumor targeting and delivery of payloads.NAF-derived EXs(NAF-EXs)were used for loading ormeloxifene(ORM),a potent anti-cancer and desmoplasia inhibitor as a model drug.We found that ORM maintains normal fibroblast cell phenotype and renders them incompatible to be triggered for a CAF-like phenotype,which may be due to regulation of Ca^(2+) influx in fibroblast cells.NAF-EXs-ORM effectively blocked oncogenic signaling pathways involved in desmoplasia and epithelial mesenchymal transition(EMT)and repressed tumor growth in xenograft mouse model.In conclusion,our data suggests preferential tropism of NAF-EXs for PDAC tumors,thus imply feasibility of developing a novel personalized medicine for PDAC patients using autologous NAF-EXs for improved therapeutic outcome of anti-cancer drugs.Additionally,it provides the opportunity of utilizing this biological scaffold for effective therapeutics in combination with standard therapeutic regimen.
文摘目的探讨趋化因子受体(chemokine receptors,CRs)、基质金属蛋白酶(matrix metalloproteinases,MMPs)、血管内皮生长因子(vascular endothelial growth factor,VEGF)的表达与胰腺癌肿瘤相关成纤维细胞(cancer associated fibroblasts,CAFs)生物学行为的关系。方法用原代培养的方法建立胰腺癌CAFs和正常成纤维细胞(normal fibroblasts,NFs)。用Real-time PCR方法检测CRs(CXCR1、CXCR2、CXCR4、CXCR7)、MMPs(MMP1、MMP2、MMP7、MMP9)和VEGF m RNA的表达。结果成功建立胰腺癌CAFs和正常胰腺NFs。在CAFs和NFs中,趋化因子受体CXCR1、CXCR2、CXCR4、CXCR7 m RNA的表达均存在差异,CAFs表达量高于NFs。其中CXCR2、CXCR4 m RNA在两株细胞中的表达差异具有统计学意义(P=0.010,P=0.049)。MMPs和VEGF m RNA的表达同样存在差异,CAFs中MMP7和VEGF m RNA表达明显高于NFs,差异具有统计学意义(P=0.017,P=0.049);CAFs中MMP9 m RNA的表达明显低于NFs,差异具有统计学意义(P=0.012)。结论在胰腺癌CAFs中CRs、MMPs和VEGF m RNA的表达有差异,可能与其促侵袭转移能力存在一定的相关性。
基金Supported by National Natural Science Foundation of China,No.30270609
文摘AIM: To investigate telomerase activity and human telomerase reverse transcriptase (hTERT) expression in normal human gastric mucosal epithelial cells (nhGMECs) and fibroblasts (nhGMFs).
