human nsclc cell lines calu- 6  (Procell Inc)

Journal: Cancer Cell International

Article Title: CircRNA CDR1as/miR-641/HOXA9 pathway regulated stemness contributes to cisplatin resistance in non-small cell lung cancer (NSCLC)

doi: 10.1186/s12935-020-01390-w

Figure Lengend Snippet: The expression patterns of circRNA CDR1as, miR-641 and HOXA9 in human NSCLC cell lines (A549, H1299 and Calu6) and their paired descendent cisplatin-resistant sub-lines (A549/DDP, H1299/DDP and Calu6/DDP). The above cells were cultured in standard conditions and subsequently stimulated with high-dose cisplatin for 48 h, cell proliferation was measured by a CCK-8 assay and b trypan blue staining method was employed to detect cell viability. The results indicated that A549/DDP, H1299/DDP and Calu6/DDP were more resistant to high-dose cisplatin treatment compared to the parental DDP-sensitive NSCLC cells. The expression levels of c circRNA CDR1as and e HOXA9 mRNA were upregulated, while d miR-641 was downregulated in DDP-resistant NSCLC cells compared to DDP-sensitive cells, determined by using Real-Time qPCR. f , g Western Blot results showed that HOXA9 protein levels were increased in DDP-resistant NSCLC cells. Each experiment repeated at least 3 times. ** P < 0.01

Article Snippet: Human NSCLC cell lines (A549, H1299 and Calu6) and HEK-293T cells were purchased from American Type Culture Collections (ATCC, USA), and cisplatin resistant sub-line A549/DDP were obtained from the Resistant Cancer Cell Line (RCCL) collection ( http://www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html ).

Techniques: Expressing, Cell Culture, CCK-8 Assay, Staining, Western Blot

Journal: Cancer Cell International

Article Title: CircRNA CDR1as/miR-641/HOXA9 pathway regulated stemness contributes to cisplatin resistance in non-small cell lung cancer (NSCLC)

doi: 10.1186/s12935-020-01390-w

Figure Lengend Snippet: Overexpression of circRNA CDR1as increased DDP chemoresistance in DDP-sensitive NSCLC cells. The a circRNA CDR1as overexpression vectors, b miR-641 mimic and c vectors for HOXA9 ablation were pre-transfected into NSCLC cells, examined by Real-Time qPCR. Subsequently, the NSCLC cells (A549, H1299 and Calu6) were subjected to high-dose DDP for 48 h. d CCK-8 assay and e trypan blue assay results indicated that upregulated circRNA CDR1as increased DDP chemoresistance in DDP-sensitive NSCLC cells by regulating miR-641/HOXA9 axis. f , g Colony formation assay results showed that circRNA CDR1as rescued colony formation abilities in DDP-treated NSCLC cells by regulating miR-641/HOXA9 axis. (Note: “D” represented “DDP”, “C” and “OE-C” represented “circRNA CDR1as overexpression”, “Mic” represented “miR-641 mimic” and “KD-H” meant “Knock-down of HOXA9″). * P < 0.05, ** P < 0.01. “NS” represented no statistical significance

Article Snippet: Human NSCLC cell lines (A549, H1299 and Calu6) and HEK-293T cells were purchased from American Type Culture Collections (ATCC, USA), and cisplatin resistant sub-line A549/DDP were obtained from the Resistant Cancer Cell Line (RCCL) collection ( http://www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html ).

Techniques: Over Expression, Transfection, CCK-8 Assay, Colony Assay, Knockdown

Journal: Cancer Cell International

Article Title: CircRNA CDR1as/miR-641/HOXA9 pathway regulated stemness contributes to cisplatin resistance in non-small cell lung cancer (NSCLC)

doi: 10.1186/s12935-020-01390-w

Figure Lengend Snippet: Knock-down of circRNA CDR1as sensitized DDP-resistant NSCLC cells to by regulating miR-641/HOXA9 axis. The a circRNA CDR1as downregulation vectors, b miR-641 inhibitor and c HOXA9 overexpression vectors were successfully delivered into DDP-resistant NSCLC cells, examined by Real-Time qPCR. d CCK-8 assay and e trypan blue assay results indicated that circRNA CDR1as deficiency aggravated the inhibiting effects of DDP on DDP-resistant NSCLC cell proliferation and viability by downregulating HOXA9 through upregulating miR-641. f Annexin V-FITC/PI double stain method combined with flow cytometer (FCM) revealed that knock-down of circRNA CDR1as increased apoptosis ratio in DDP treated A549/DDP, H1299/DDP and Calu6/DDP cells by regulating miR-641/HOXA9 axis. g The xenograft tumor bearing mice models were established to evaluate the tumorigenicity of NSCLC cells in vivo. (Note: “D” represented “DDP”, “KD-C” represented “circRNA CDR1as silence”, “KD-miR” represented “miR-641 ablation” and “OE-H” meant “overexpression of HOXA9”). Each experiment repeated at least 3 times. * P < 0.05, ** P < 0.01. “NS” represented no statistical significance

Article Snippet: Human NSCLC cell lines (A549, H1299 and Calu6) and HEK-293T cells were purchased from American Type Culture Collections (ATCC, USA), and cisplatin resistant sub-line A549/DDP were obtained from the Resistant Cancer Cell Line (RCCL) collection ( http://www.kent.ac.uk/stms/cmp/RCCL/RCCLabout.html ).

Techniques: Knockdown, Over Expression, CCK-8 Assay, Staining, Flow Cytometry, In Vivo

Journal: Neoplasia (New York, N.Y.)

Article Title: Induction of Activating Transcription Factor 3 Is Associated with Cisplatin Responsiveness in Non–Small Cell Lung Carcinoma Cells 1 2

doi: 10.1016/j.neo.2016.07.004

Figure Lengend Snippet: (A) MTT analysis of Calu6, Calu6cisR1-3, H23, and H23cisR1-3 following 48-hour treatments with cisplatin, carboplatin, and doxorubicin. Cisplatin and carboplatin treatments showed reduced cytotoxicity in the Calu6cisR1-3 and H23cisR1 compared to their parental cell lines. With the exception of Calu6cisR3, response to doxorubicin treatment was similar in the parental and cisR1 sublines. *Significant difference in the viability curves of the cisR sublines compared to their parental counterparts ( P < .05). (B) No significant morphological differences were observed between the parental and resistant sublines (phase microscopy); however, proliferation rates as determined by cell doubling times were reduced in the Calu6cisR sublines, and this was most pronounced in the Calu6cisR3 cell line.

Article Snippet: The human NSCLC tumor–derived cell lines Calu6 and NCI-H23 (H23), the breast cancer cell lines MCF7 and T47D, and the prostate cancer cell lines LNCAP and PC3 were obtained from the ATCC (Rockville, MD, USA).

Techniques: Microscopy

Journal: Neoplasia (New York, N.Y.)

Article Title: Induction of Activating Transcription Factor 3 Is Associated with Cisplatin Responsiveness in Non–Small Cell Lung Carcinoma Cells 1 2

doi: 10.1016/j.neo.2016.07.004

Figure Lengend Snippet: (A) RNA-seq analysis of Calu6 and H23 parental versus their respective cisR1 resistant clones identified 42 commonly differentially expressed genes in both cell lines following 2-μg/ml cisplatin treatment for 24 hours. (B) The cellular stress response gene ATF3 was differentially induced in cisplatin-treated sensitive parental lines but not their cisR1 sublines as indicated by the RNA-seq reads determined in this analysis (2-μg/ml cisplatin treatments for 24 hours). (C) Q-RT-PCR confirmed and expanded upon the differential expression of ATF3 in cisplatin-treated Calu6 cells compared to their Calu6cisR1-3 cisplatin-resistant sublines following the cytotoxic 4-μg/ml treatments of cisplatin for up to 24 hours. (D) Western blot analysis of ATF3 expression in cisplatin-treated Calu6 and H23 parental cell lines compared to their Calu6cisR1-3 and H23cisR1-3 cisplatin-resistant sublines following the cytotoxic 4-μg/ml treatments of cisplatin for 24 hours.

Article Snippet: The human NSCLC tumor–derived cell lines Calu6 and NCI-H23 (H23), the breast cancer cell lines MCF7 and T47D, and the prostate cancer cell lines LNCAP and PC3 were obtained from the ATCC (Rockville, MD, USA).

Techniques: RNA Sequencing, Clone Assay, Reverse Transcription Polymerase Chain Reaction, Quantitative Proteomics, Western Blot, Expressing

Journal: Neoplasia (New York, N.Y.)

Article Title: Induction of Activating Transcription Factor 3 Is Associated with Cisplatin Responsiveness in Non–Small Cell Lung Carcinoma Cells 1 2

doi: 10.1016/j.neo.2016.07.004

Figure Lengend Snippet: (A) Following 4-μg/ml cisplatin treatments for up to 24 hours, Western blot analysis showed differential induction of JNK MAPKinase activation (through phosphorylated c-jun) but not ERK (phospho-ERK) or p38 (through phosphorylated HSP27) in both Calu6 and H23 cells. This induction was abrogated in their respective cisR1 sublines. (B) MTT analysis of Calu6 cells following 48-hour treatments of (0, 1, and 4 μg/ml) cisplatin with or without 10-μM treatment of the JNK inhibitor SP600125. *Significant differences between columns highlighted ( P < .05). (C) Exogenous expression of a JNK-activated construct enhanced cisplatin cytotoxicity in the Calu6cisR1 subline as assessed by MTT cell viability assay following 48-hour cisplatin treatments. * P < .05 between columns highlighted. (D) Exogenous expression of activated JNK in Calu6cisR1 also rescued cisplatin-inducible ATF3 expression in these cells assessed by Western blot analysis (24-hour treatments).

Article Snippet: The human NSCLC tumor–derived cell lines Calu6 and NCI-H23 (H23), the breast cancer cell lines MCF7 and T47D, and the prostate cancer cell lines LNCAP and PC3 were obtained from the ATCC (Rockville, MD, USA).

Techniques: Western Blot, Activation Assay, Expressing, Construct, Viability Assay

Journal: Neoplasia (New York, N.Y.)

Article Title: Induction of Activating Transcription Factor 3 Is Associated with Cisplatin Responsiveness in Non–Small Cell Lung Carcinoma Cells 1 2

doi: 10.1016/j.neo.2016.07.004

Figure Lengend Snippet: (A) MTT analysis of wild-type and ATF3−/− MEFs following 48-hour treatments of cisplatin, carboplatin, doxorubicin, and docetaxel. Loss of ATF3 inhibits cisplatin-, carboplatin-, and doxorubicin-induced cytotoxicity but had no effect on docetaxel cytotoxicity. *Significant difference in the viability curves comparing the wild-type and the ATF3−/− MEFs responses ( P < .05). (B) MTT analysis of Calu6, Calu6cisR1, H23, and H23cisR1 following 48-hour treatments with docetaxel showing no significant differences in sensitivity (note: cisplatin, carboplatin, and doxorubicin treatments were presented in A ). (C) Western blot analysis shows significant ATF3 induction restricted to the parental cell lines with cisplatin and carboplatin treatments. Doxorubicin treatments showed similar ATF3 induction in both the parental and their cisR1 sublines. No significant ATF3 induction was observed with the docetaxel treatments in any of the lines tested, with a weak induction in the H23 series (24-hour treatments, IC50 dose).

Article Snippet: The human NSCLC tumor–derived cell lines Calu6 and NCI-H23 (H23), the breast cancer cell lines MCF7 and T47D, and the prostate cancer cell lines LNCAP and PC3 were obtained from the ATCC (Rockville, MD, USA).

Techniques: Western Blot

Journal: Neoplasia (New York, N.Y.)

Article Title: Induction of Activating Transcription Factor 3 Is Associated with Cisplatin Responsiveness in Non–Small Cell Lung Carcinoma Cells 1 2

doi: 10.1016/j.neo.2016.07.004

Figure Lengend Snippet: (A) MTT analysis of wild-type and ATF3−/− MEFs following 48-hour treatments with vorinostat showing that loss of ATF3 inhibits vorinostat-induced cytotoxicity in MEFs. (B), MTT analysis of wild-type (wt) and ATF3−/− deficient MEFs following 24-hour pretreatments of 1 and 2 μM vorinostat followed by cisplatin treatment in combination for a further 48 hours. Enhanced cytotoxicity with the combination was observed only in the wild-type MEFs. (C) MTT analysis comparing response of Calu6 to Calu6cisR1 and H23 to H23cisR1 following 0-, 1-, 2-, and 5-μM vorinostat treatment for 72 hours. The H23cisR1 showed significant resistance to vorinostat. *Significant differences between columns highlighted ( P < .05). (D) Western blot analysis shows ATF3 induced by vorinostat treatments (0-10 μM, 48 hours) in the Calu6 and the H23 parental lines that was evident but attenuated in their cisR1 sublines. (E) MTT analysis of Calu6 and Calu6cisR1 and (F) H23 and H23cisR1 following 24-hour pretreatments of 0, 1, 5, and 5 μM vorinostat followed by cisplatin treatment in combination for a further 48 hours. Enhanced cytotoxicity with the combination was observed in all four lines compared to either agent alone. (G) Western blot analysis shows ATF3 induced by 2- and 4-mg/ml cisplatin treatment (24 hours) was enhanced by vorinostat treatment (5 μM, 24-hour pretreatment) in the Calu6cisR1 and the H23cisR1 resistant sublines.

Article Snippet: The human NSCLC tumor–derived cell lines Calu6 and NCI-H23 (H23), the breast cancer cell lines MCF7 and T47D, and the prostate cancer cell lines LNCAP and PC3 were obtained from the ATCC (Rockville, MD, USA).

Techniques: Western Blot