human thyroid cancer cell lines bcpap  (Procell Inc)

Journal: International Journal of Molecular Sciences

Article Title: Curcumin-Mediated Apoptotic Cell Death in Papillary Thyroid Cancer and Cancer Stem-Like Cells through Targeting of the JAK/STAT3 Signaling Pathway

doi: 10.3390/ijms21020438

Figure Lengend Snippet: Curcumin-mediated inhibition of cell proliferation and apoptosis in PTC cells. ( A , B ) Curcumin inhibited the cell viability of PTC cell lines BCPAP and TPC-1. PTC cells were treated with 0 µM, 5 µM, 10 µM, 20 µM, 40 µM, and 80 µM of curcumin for 24 h. A cell proliferation assay was performed using a Cell Counting Kit-8 (CCK-8), as described in “Materials and Methods”. Values are expressed as the mean +/− the SD (standard deviation) of at least six replicates, with a p -value of *** p <0.001. ( C , D ) Real-time cell proliferation (cell index) analysis of BCPAP and TPC-1 cells, respectively, treated with different doses of curcumin. Cells were cultured on electrodes and treated with curcumin followed by the monitoring of real-time cell growth, as mentioned in the material and methods section. ( E , F ) Curcumin mediated the inhibition of BCPAP and TPC-1 cell growth and proliferation through apoptosis. PTC cell lines were treated with different doses of curcumin for 24 h, followed by cell lysis and immunoblotting. Equal amount of proteins were separated using SDS-PAGE, transferred to a Polyvinylidene fluoride or polyvinylidene difluoride (PVDF) membrane, and immunoblotted with antibodies against caspase-3, cleaved caspase-3, cleaved caspase-8, caspase-9, cleaved caspase-9, poly (ADP-ribose) polymerase (PARP), phosphor-histone family member X (p-H2AX), heat shock protein 60 (Hsp60), and Gapdh.

Article Snippet: The human papillary thyroid carcinoma cell (PTC) cancer cell lines, BCPAP and TPC-I, were obtained from DSMZ (Braunschh, Germany) and EMD Millipore, MA, USA, respectively, and were cultured using RPMI 1640 medium supplemented with 10% ( v / v ) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

Techniques: Inhibition, Proliferation Assay, Cell Counting, CCK-8 Assay, Standard Deviation, Cell Culture, Lysis, Western Blot, SDS Page, Membrane

Journal: International Journal of Molecular Sciences

Article Title: Curcumin-Mediated Apoptotic Cell Death in Papillary Thyroid Cancer and Cancer Stem-Like Cells through Targeting of the JAK/STAT3 Signaling Pathway

doi: 10.3390/ijms21020438

Figure Lengend Snippet: Curcumin suppressed the constitutive JAK/STAT3 signaling pathway and upregulated reactive oxygen species (ROS) content to induce apoptosis. ( A ) BCPAP and ( B ) TPC-1 cells were treated with 10 µM, 20 µM, and 40 µM of curcumin for 24 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies against p-STAT3, STAT3, p-JAK2, JAK2, Cyclin D, pCXCR4, Bcl2, Bclxl, and Hsp60. ( C ) Curcumin induced ROS generation and oxidative stress, which are involved in cell cycle arrest and apoptotic cell death in PTC cells. N -acetyl cysteine (NAC) pretreatment with abrogated curcumin induced an increase in the SubG0 fraction in PTC cells. BCPAP cells were pretreated with 10 mM NAC followed by 40 µM curcumin for 24 h, and the cell cycle fraction was measured by flow cytometry. ( D ) BCPAP cells were pretreated with 10 mM NAC followed by 40 µM curcumin for 24 h, and apoptosis was measured by staining with fluorescein-conjugated Annexin V and propidium iodide (PI) and analyzed by flow cytometry. Data are expressed as the mean ± the SD. ( E ) NAC pretreatment in PTC cells reversed curcumin-mediated apoptosis. BCPAP cells were pretreated with 10 mM NAC and then subsequently treated with 40 µM curcumin. Cells were lysed and separated using SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies such as caspase-3, cleaved caspase-3, Bcl2, Bax, and Hsp60. Curcumin, CUR; cisplatin, CIS.

Article Snippet: The human papillary thyroid carcinoma cell (PTC) cancer cell lines, BCPAP and TPC-I, were obtained from DSMZ (Braunschh, Germany) and EMD Millipore, MA, USA, respectively, and were cultured using RPMI 1640 medium supplemented with 10% ( v / v ) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

Techniques: Lysis, SDS Page, Membrane, Flow Cytometry, Staining

Journal: International Journal of Molecular Sciences

Article Title: Curcumin-Mediated Apoptotic Cell Death in Papillary Thyroid Cancer and Cancer Stem-Like Cells through Targeting of the JAK/STAT3 Signaling Pathway

doi: 10.3390/ijms21020438

Figure Lengend Snippet: Curcumin synergistically enhanced the antiproliferative response of cisplatin in PTC cells. BCPAP cells were treated with various combinations of curcumin and cisplatin for 24 h, and graphs were generated using Calcusyn software, as described by Chou and Talalay. Briefly, curcumin and cisplatin were combined, and at least four different combinations were used to treat PTC cells for 24 h. A cell proliferation assay was performed using a CCK8 kit, as described in the materials and methods section, and data are expressed as the mean +/− the SD of at least six replicates. Different types of graphs, using Calcusyn software (as described by Chou and Talalay), including ( A ) a median dose effect graph, where the y axis represents the scale of fa (the fraction affected by the dose) and fu (the fraction unaffected by dose); ( B ) a graph representing the dose effect curve of curcumin and cisplatin alone and in combination; ( C , D ) graphs representing the combination index (CI) value of the curcumin and cisplatin combination; and ( E ) a graph representing an isobologram of the curcumin and cisplatin interaction, were generated. CUR: curcumin; CIS: cisplatin.

Article Snippet: The human papillary thyroid carcinoma cell (PTC) cancer cell lines, BCPAP and TPC-I, were obtained from DSMZ (Braunschh, Germany) and EMD Millipore, MA, USA, respectively, and were cultured using RPMI 1640 medium supplemented with 10% ( v / v ) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

Techniques: Generated, Software, Proliferation Assay

Journal: International Journal of Molecular Sciences

Article Title: Curcumin-Mediated Apoptotic Cell Death in Papillary Thyroid Cancer and Cancer Stem-Like Cells through Targeting of the JAK/STAT3 Signaling Pathway

doi: 10.3390/ijms21020438

Figure Lengend Snippet: Cotreatment of curcumin with cisplatin augmented the inhibition of cell growth and proliferation and suppressed the motility of PTC cells. Curcumin synergistically enhanced the growth inhibitory potential of cisplatin in BCPAP ( A ) and TPC-1 ( B ) cells. BCPAP and TPC-1 cells were treated with 0 µM or 20 µM of curcumin and 10 µM cisplatin alone and in combination for 24 h. A cell proliferation assay was performed using a CCK8 kit, as described in the materials and methods section. Values were expressed as the mean +/− the SD of at least six replicates, with a p -value od *** p < 0.001. ( C , D ) Real-time cell proliferation (cell index) analysis of BCPAP and TPC-1 cells treated with curcumin and cisplatin, respectively. BCPAP and TPC-1 cells were cultured on electrodes and treated with 20 µM curcumin and 10 µM cisplatin alone and in combination followed by the monitoring of real-time cell growth, as mentioned in the material and methods section. ( E – G ) Curcumin synergistically inhibited the colony formation potential of cisplatin. BCPAP cells were cultured in a six-well plate with and without agar and treated with 0 µM or 20 µM curcumin and 10 µM of cisplatin alone and in combination for one to two weeks. Colonies were stained with crystal violet, and fluorescence intensity using CyQuant dye was measured at 485/530 nm. Data are presented as the mean +/− the SD, *** p < 0.001. Curcumin (CUR), cisplatin (CIS).

Article Snippet: The human papillary thyroid carcinoma cell (PTC) cancer cell lines, BCPAP and TPC-I, were obtained from DSMZ (Braunschh, Germany) and EMD Millipore, MA, USA, respectively, and were cultured using RPMI 1640 medium supplemented with 10% ( v / v ) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

Techniques: Inhibition, Proliferation Assay, Cell Culture, Staining, Fluorescence, CyQUANT Assay

Journal: International Journal of Molecular Sciences

Article Title: Curcumin-Mediated Apoptotic Cell Death in Papillary Thyroid Cancer and Cancer Stem-Like Cells through Targeting of the JAK/STAT3 Signaling Pathway

doi: 10.3390/ijms21020438

Figure Lengend Snippet: Curcumin and cisplatin synergistically enhanced the inhibition of JAK/STAT3 and Inbibitor of apoptosis proteins (IAPs) and also modulated apoptotic regulators. ( A , B ) The cotreatment of curcumin and cisplatin enhanced the downregulation of JAK/STAT3 in BCPAP and TPC-1 cells. PTC cell lines were treated with 0 µM or 20 µM of curcumin and 10 µM of cisplatin alone and in combination, and after cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies of p-STAT3, STAT3, p-JAK2, JAK2, and Gapdh ( C ) Curcumin and cisplatin modulated the expression of antiapoptotic and proapoptotic proteins in PTC cells. BCPAP cells were treated with 0 µM and 20 µM of curcumin and 10 µM of cisplatin alone and in combination, and after cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies of Bcl2, Bclxl, Bax, and Gapdh. ( D ) Curcumin and cisplatin synergistically downregulated IAP expression in PTC cells. BCPAP cells were treated with 0 µM or 20 µM of curcumin and 10 µM of cisplatin alone and in combination, and after cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies of XIAP, CIAP1, CIAP2, Survivin, and Gapdh.

Article Snippet: The human papillary thyroid carcinoma cell (PTC) cancer cell lines, BCPAP and TPC-I, were obtained from DSMZ (Braunschh, Germany) and EMD Millipore, MA, USA, respectively, and were cultured using RPMI 1640 medium supplemented with 10% ( v / v ) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

Techniques: Inhibition, Lysis, SDS Page, Membrane, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Curcumin-Mediated Apoptotic Cell Death in Papillary Thyroid Cancer and Cancer Stem-Like Cells through Targeting of the JAK/STAT3 Signaling Pathway

doi: 10.3390/ijms21020438

Figure Lengend Snippet: Cotreatment of curcumin with cisplatin-enhanced apoptosis of PTC cells via STAT3 downregulation. Curcumin potentiated cisplatin-induced growth arrest and inhibited the proliferation of BCPAP ( A ) and TPC-1 ( B ) cells through apoptosis. PTC cells were cultured and treated with 0 µM or 20 µM of curcumin and 10 µM of cisplatin alone and in combination for 24 h. Cells were lysed and separated using SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies such as caspase-9, cleaved caspase-9, cleaved caspase-8, caspase-3, cleaved caspase-3, PARP, p-H2AX, and Gapdh. ( C ) Curcumin synergistically potentiated cisplatin-induced growth inhibition by stimulating the expression of cyclin-dependent kinase inhibitor proteins (CDKIs) in PTC cells. BCPAP cells were treated with 0 µM and 20 µM of curcumin and 10 µM of cisplatin alone and in combination for 24 h, followed by cell lysis and immunoblotting. Equal amounts of proteins were separated using SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies against p27, p21, and Gapdh. ( D ) The effect of siRNA-mediated knockdown of STAT3 on p-STAT3, CDKIs, and caspases. BCPAP cells were transfected with either control (100 pmol) or STAT3-specific siRNA (50 or 100 pmol). Cell extracts were separated using SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies against p-STAT3, caspase-3, cleaved caspase-3, Bax, p21, and Gapdh.

Article Snippet: The human papillary thyroid carcinoma cell (PTC) cancer cell lines, BCPAP and TPC-I, were obtained from DSMZ (Braunschh, Germany) and EMD Millipore, MA, USA, respectively, and were cultured using RPMI 1640 medium supplemented with 10% ( v / v ) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

Techniques: Cell Culture, SDS Page, Membrane, Inhibition, Expressing, Lysis, Western Blot, Knockdown, Transfection, Control

Journal: International Journal of Molecular Sciences

Article Title: Curcumin-Mediated Apoptotic Cell Death in Papillary Thyroid Cancer and Cancer Stem-Like Cells through Targeting of the JAK/STAT3 Signaling Pathway

doi: 10.3390/ijms21020438

Figure Lengend Snippet: Curcumin potentiated the cisplatin-mediated apoptosis of PTC cells via enhanced oxidative stress. ( A ) NAC pretreatment prevented the curcumin- and cisplatin-mediated apoptosis of PTC cells. BCPAP cells were treated with 0 µM and 20 µM of curcumin and 10 µM cisplatin, 20 µM curcumin plus 10 µM cisplatin, NAC (10 mM) and NAC plus 20 µM curcumin and 10 µM cisplatin for 24 h; cells were subsequently stained with fluorescein-conjugated Annexin-V and propidium iodide (PI) and analyzed by flow cytometry; apoptosis was measured; and the percentage of apoptosis relative to untreated cells was calculated. ( B ) NAC pretreatment prevented curcumin- and cisplatin-induced increased ROS generation in PTC cells. BCPAP cells were treated with 0 µM or 20 µM of curcumin and 10 µM of cisplatin alone and in combination or were treated with NAC (10 mM) and NAC + curcumin and cisplatin for 24 h; cells were fixed and subsequently stained with 2′,7′ –dichlorofluorescin diacetate (DCFDA); and images were taken through confocal microscopy at 60X magnification (scale bar: 25 µm). DCFDA is well known for measuring the various ROS species: once it diffuses inside the cells, it gets deacetylated by cellular esterases, leading to the formation of a non-fluorescent compound. Next, cellular ROS oxidized DCFDA into a highly fluorescent compound known as dichlorofluorescein (DCF), which can be detected by fluorescence spectroscopy. ( C ) NAC pretreatment attenuated curcumin- and cisplatin-induced damage in PTC cells. We observed increased nuclear condensation, distorted membrane integrity, moderate chromatin condensation, and nuclear fragmentation in the cells challenged with both cisplatin and curcumin, while the control and NAC-treated cells depicted diffuse nuclear staining with intact membrane integrity. BCPAP cells were treated with 0 µM or 20 µM of curcumin and 10 µM of cisplatin alone and in combination or NAC (10 mM) and NAC + curcumin and cisplatin for 24 h, and cells were fixed and subsequently stained with 4′,6-diamidino-2-phenylindole (DAPI). Images were taken through an EVOS FLc Cell Imaging System from Invitrogen (Thermo Fisher Scientific) at 40× magnification (scale bar: 100 µm). ( D ) NAC prevented curcumin- and cisplatin-induced modulation in the expression of antiapoptotic and proapoptotic proteins in PTC cells. BCPAP cells were pretreated with NAC (10 mM), followed by a combination treatment of 20 µM curcumin and 10 µM cisplatin, and after cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies of Bcl2, Bcl-xL, Bax, and Hsp60. ( E ) NAC prevented curcumin- and cisplatin-induced activation of caspase-mediated apoptosis in PTC cells. BCPAP cells were pretreated with NAC (10 mM), followed by a combination treatment of 20 µM curcumin and 10 µM cisplatin, and after cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies of caspase-9, cleaved caspase-8, cleaved caspase-3, PARP, and Hsp60. CUR: curcumin; CIS: cisplatin.

Article Snippet: The human papillary thyroid carcinoma cell (PTC) cancer cell lines, BCPAP and TPC-I, were obtained from DSMZ (Braunschh, Germany) and EMD Millipore, MA, USA, respectively, and were cultured using RPMI 1640 medium supplemented with 10% ( v / v ) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

Techniques: Staining, Flow Cytometry, Confocal Microscopy, Fluorescence, Spectroscopy, Membrane, Control, Imaging, Expressing, Lysis, SDS Page, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: Curcumin-Mediated Apoptotic Cell Death in Papillary Thyroid Cancer and Cancer Stem-Like Cells through Targeting of the JAK/STAT3 Signaling Pathway

doi: 10.3390/ijms21020438

Figure Lengend Snippet: IL6 attenuated the curcumin- and cisplatin-induced inhibition of STAT3 and enhanced the proliferation of PTC cells. ( A ) BCPAP cells were treated with 0 µM or 20 µM of curcumin and 10 µM of cisplatin alone and in combination in reduced serum-containing media, followed by IL6 (100 ng/mL) treatment. Cells were lysed and separated using SDS-PAGE, transferred to a PVDF membrane, and immunoblotted with antibodies such as p-STAT3, STAT3, cleaved caspase-8, and Gapdh. ( B ) IL6 stimulated the cell viability of BCPAP cells and protected cells from curcumin- and cisplatin-mediated inhibition of cell proliferation. BCPAP cells were stimulated with IL6 (100 ng/mL) and subsequently treated with curcumin and cisplatin, as indicated in B, for 24 h. A cell proliferation assay was performed using a CCK8 kit, as described in the materials and methods section. Values are expressed as the mean +/− the SD of at least six replicates, with a p -value of * p < 0.05, *** p < 0.001.

Article Snippet: The human papillary thyroid carcinoma cell (PTC) cancer cell lines, BCPAP and TPC-I, were obtained from DSMZ (Braunschh, Germany) and EMD Millipore, MA, USA, respectively, and were cultured using RPMI 1640 medium supplemented with 10% ( v / v ) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

Techniques: Inhibition, SDS Page, Membrane, Proliferation Assay

Journal: International Journal of Molecular Sciences

Article Title: Curcumin-Mediated Apoptotic Cell Death in Papillary Thyroid Cancer and Cancer Stem-Like Cells through Targeting of the JAK/STAT3 Signaling Pathway

doi: 10.3390/ijms21020438

Figure Lengend Snippet: Cotreatment of curcumin and cisplatin potentiated the inhibition of cancer stem cell (CSC) growth and stemness features. ( A ) Treatment with curcumin and cisplatin inhibited thyrosphere formation. BCPAP cells were grown and treated in ultralow attachment plates with 0 µM or 20 µM of curcumin and 10 µM of cisplatin alone and in combination for 7 days, and images of thyrospheres were taken on day 3 and day 7 using an EVOS FLc Cell Imaging System from Invitrogen (Thermo Fisher Scientific) at a magnification of 4×(scale bar 1000 µm). ( B ) BCPAP cells were grown and treated in ultralow attachment plates with 0 µM and 20 µM of curcumin and 10 µM of cisplatin alone and in combination for 7 days, and images of thyrospheres were taken on day 7 through a confocal microscope at a magnification of 20×(scale bar 100 µm). ( C ) Cotreatment of curcumin and cisplatin potentiated the inhibition of stemness features in PTC cells. Thyrospheres were grown and treated in ultralow attachment plates with 0 µM or 20 µM of curcumin and 10 µM of cisplatin alone and in combination, followed by cell lysis and western blotting against ALDH2, CD44, Nanog, c-Myc, SOX-2, and Gapdh. ( D ) Cotreatment with curcumin and cisplatin potentiated the apoptosis of CSCs. Thyrospheres were grown and treated in ultralow attachment plates with 0 µM and 20 µM of curcumin and 10 µM of cisplatin alone and in combination for 7 days, followed by cell lysis and western blotting against p-STAT3, cleaved caspase-3, p-H2AX, and Hsp60. CUR: curcumin; CIS: cisplatin.

Article Snippet: The human papillary thyroid carcinoma cell (PTC) cancer cell lines, BCPAP and TPC-I, were obtained from DSMZ (Braunschh, Germany) and EMD Millipore, MA, USA, respectively, and were cultured using RPMI 1640 medium supplemented with 10% ( v / v ) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 U/mL streptomycin at 37 °C in a humidified atmosphere containing 5% CO2.

Techniques: Inhibition, Imaging, Microscopy, Lysis, Western Blot