human non small cell lung cancer nsclc cell a549  (ATCC)

Journal: Drug Design, Development and Therapy

Article Title: Rhein Alleviates Cisplatin-Induced Acute Kidney Injury via Downregulation of NOX4-COX2/PGFS Signaling Pathway

doi: 10.2147/DDDT.S515409

Figure Lengend Snippet: Rhein reduces CDDP-induced nephrotoxicity without affecting the antitumor efficacy. CDDP (10μg/mL)-treated human non-small-cell lung cancer (NSCLC) A549 cells were incubated with 40 μM Rhein for 24 h. ( A ) The cell viability was determined by the CCK8 analysis (n=6). ( B ) The CDDP-sensitive Lewis lung carcinoma (LLC) cells (2×10 5 ) were inoculated subcutaneously into C57BL/6 mice for 14 days, and the mice were treated with CDDP alone (4 mg/kg, the drugs were administered every other day for 10 days, resulting in an accumulative dose of 20 mg/kg) or combination with Rhein (80mg/kg, the drugs were administered 1 hour after CDDP injection). ( C and D ) The xenograft tumor weights and tumor volume of C57BL/6 mice injected subcutaneously with LLC cells with the treatment of CDDP, Rhein alone or in combination with Rhein (n=5). Data are shown as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001: CDDP vs Control. ## P < 0.01 CDDP+Rhein vs CDDP.

Article Snippet: To determine whether Rhein might affect the anti-tumor activity of CDDP in vitro, CDDP-sensitive human non-small-cell lung cancer (NSCLC) cell line A549 (catalog no. CCL-185, RRID: CVCL_0023; ATCC, Manassas, VA, USA) was exposed to CDDP (10 μg/mL) alone or in combination with Rhein for 24h, cell viability was then measured.

Techniques: Incubation, Injection, Control

Journal: Cancers

Article Title: Augmented Concentration of Isopentyl-Deoxynyboquinone in Tumors Selectively Kills NAD(P)H Quinone Oxidoreductase 1-Positive Cancer Cells through Programmed Necrotic and Apoptotic Mechanisms

doi: 10.3390/cancers15245844

Figure Lengend Snippet: IP-DNQ induces potent cytotoxicity in various NQO1-overexpressing cancer cells. ( A – D ) Breast cancer cell line MCF-7 ( A ), NSCLC cell line A549 ( B ), breast cancer cell line MDA-MB-231 WT and NQO1-overexpressing MDA-MB-231 NQO1 + cells ( C ) , and A549- NQO1 -KO cells ( D ) were treated with various doses of β-lap (0–20 µM), DNQ (0–1.0 µM), or IP-DNQ (0–1.0 µM) ± DIC (dicoumarol, 50 µM, an NQO1 inhibitor) for 2 h. After this, drugs were removed, and cell viability was determined with a relative survival assay (DNA assay) 7 days later. ( E – H ) All of the aforementioned cell lines were treated with IP-DNQ (0.2 µM) at the indicated time points, and long-term relative survival assays were performed. Each group has six replicates for every biological repeat. Results (mean ± SD) in ( A – H ) represent experiments performed at least three times. *** p < 0.001 ( t tests).

Article Snippet: Endogenous NQO1-overexpressing human A549 non-small-cell lung cancer (NSCLC) cells, and MCF-7 and MDA-MB-231 breast cancer cells were obtained from the American Tissue Culture Collection (ATCC).

Techniques: Clonogenic Cell Survival Assay

Journal: Cancers

Article Title: Augmented Concentration of Isopentyl-Deoxynyboquinone in Tumors Selectively Kills NAD(P)H Quinone Oxidoreductase 1-Positive Cancer Cells through Programmed Necrotic and Apoptotic Mechanisms

doi: 10.3390/cancers15245844

Figure Lengend Snippet: IP-DNQ leads to ROS formation, PARP1 hyperactivation, and NAD + /ATP depletion. ( A , B ) A549 cells were treated with DMSO or IP-DNQ (0.1 or 0.25 µM) ± DIC (50 µM) for 2 h. Real-time oxygen consumption rates (OCRs) and extracellular acidification rates (ECARs) after various drug treatments (added at t = 20 min, solid arrow) were monitored at 9 min intervals for 200 min. Dashed arrow indicates the addition of 2-DG at 80 min. ( C ) A549 and A549- NQO1 -KO cells were treated with DMSO or IP-DNQ (0.1 or 0.25 µM) ± DIC (50 µM) for 2 h. ROS formation was assessed using H 2 O 2 level kit. ( D ) Same cells as C were treated with DMSO or IP-DNQ (0.25 µM), and cell extracts were prepared at the indicated time points. Cells were also exposed to H 2 O 2 (500 µM, 15 min in PBS) as a positive control. Samples were assessed for PAR formation, γ-H2AX, total H2AX, NQO1, and β-actin (loading control). ( E ) A549 cells were exposed to DMSO and various dosages of IP-DNQ (0.02–0.1 µM) for 5 min. Samples were assessed for PAR formation and β-actin (loading control). ( F ) MCF-7 cells were treated with DMSO or IP-DNQ (0.2 µM), and cell extracts were prepared at the indicated time points. Cells were also exposed to H 2 O 2 (500 µM, 15 min in PBS) as a positive control. Samples were assessed for PAR formation, γ-H2AX, total H2AX, NQO1, and β-actin (loading control). ( G , H ) The same cells as those in C were treated with DMSO or IP-DNQ (0.1 or 0.25 µM) ± DIC (50 µM) for 2 h. Then, the relative NAD + and ATP levels were monitored. Results were separately repeated at least three times in triplicate. ( I , J ) Same cells as those in C were treated with DMSO or IP-DNQ (0.05, 0.1, 0.15, 0.2, or 0.25 µM) for 2 h. Then, the relative NAD + and ATP levels were monitored. Results were separately repeated at least three times in triplicate. ( K , L ) Same cells as those in C were treated with DMSO or IP-DNQ (0.25 µM) for different time points (0 -120 min). Cells were collected and assessed for the relative NAD + and ATP levels. For panels ( A – C ) and ( I – L ), each group had three replicates for every biological repetition. All error bars (mean ± SD) are derived from three independent experiments. *** p < 0.001, * p < 0.05 ( t tests). The uncropped blots are shown in .

Article Snippet: Endogenous NQO1-overexpressing human A549 non-small-cell lung cancer (NSCLC) cells, and MCF-7 and MDA-MB-231 breast cancer cells were obtained from the American Tissue Culture Collection (ATCC).

Techniques: Positive Control, Control, Derivative Assay

Journal: Cancers

Article Title: Augmented Concentration of Isopentyl-Deoxynyboquinone in Tumors Selectively Kills NAD(P)H Quinone Oxidoreductase 1-Positive Cancer Cells through Programmed Necrotic and Apoptotic Mechanisms

doi: 10.3390/cancers15245844

Figure Lengend Snippet: NQO1 is required for IP-DNQ-mediated DNA damage in A549 cells. ( A ) Representative images of A549 cells exposed to DMSO, IP-DNQ (0.25 µM), or IP-DNQ + DIC (50 µM); cells were assessed for DSB breaks over time (30, 60, and 120 min) using γ-H2AX as the surrogate marker (in green). Cells were also stained for nuclear DNA using DAPI (in blue). Scale bar, 10 μm. ( B ) Graphical representation of data presented in A. ( C ) Total DNA damage by alkaline comet assay in A549 cells after IP-DNQ (0.1 or 0.25 µM) ± DIC (50 µM) for 2 h. ( D ) Comet tail lengths (arbitrary units) were assessed using NIH Image J software. Points or means were counted from 100 cells per treatment group. Each group had three replicates for every biological repetition. Results (mean ± SD) were derived from three independent experiments. *** p < 0.001 ( t tests).

Article Snippet: Endogenous NQO1-overexpressing human A549 non-small-cell lung cancer (NSCLC) cells, and MCF-7 and MDA-MB-231 breast cancer cells were obtained from the American Tissue Culture Collection (ATCC).

Techniques: Marker, Staining, Alkaline Single Cell Gel Electrophoresis, Software, Derivative Assay

Journal: Cancers

Article Title: Augmented Concentration of Isopentyl-Deoxynyboquinone in Tumors Selectively Kills NAD(P)H Quinone Oxidoreductase 1-Positive Cancer Cells through Programmed Necrotic and Apoptotic Mechanisms

doi: 10.3390/cancers15245844

Figure Lengend Snippet: Ca 2+ plays a pivotal role in IP-DNQ-induced cytotoxicity. ( A ) Long-term survival of A549 cells pretreated with or without BAPTA-AM (5 µM, 60 min), then cotreated with IP-DNQ (0–1 µM) for 2 h. ( B ) Western blot assays confirmed PAR formation in A549 cells pretreated with or without BAPTA-AM (5 µM, 60 min) ± IP-DNQ (0.25 µM) at indicated times (0–120 min). Cells were treated with H 2 O 2 (500 µM, 15 min in PBS) as a positive control. Loading was controlled by β-actin levels. ( C , D ) A549 cells pretreated with or without BAPTA-AM (5 µM, 60 min) for 1 h, then cotreated with IP-DNQ (0–1 µM) at the indicated time points; cells were assessed for NAD + ( C ) and ATP ( D ) levels. For panel A, each group had six replicates for every biological repetition; for panels C-D, each group had three replicates for every biological repetition. Results (mean ± SD) were derived from three independent experiments. *** p < 0.001 ( t tests). The uncropped blots are shown in .

Article Snippet: Endogenous NQO1-overexpressing human A549 non-small-cell lung cancer (NSCLC) cells, and MCF-7 and MDA-MB-231 breast cancer cells were obtained from the American Tissue Culture Collection (ATCC).

Techniques: Western Blot, Positive Control, Derivative Assay

Journal: Cancers

Article Title: Augmented Concentration of Isopentyl-Deoxynyboquinone in Tumors Selectively Kills NAD(P)H Quinone Oxidoreductase 1-Positive Cancer Cells through Programmed Necrotic and Apoptotic Mechanisms

doi: 10.3390/cancers15245844

Figure Lengend Snippet: IP-DNQ causes NQO1-dependent apoptosis and necrosis. ( A ) A549 cells were treated with DMSO or IP-DNQ (0.1 or 0.25 µM) ± DIC (50 µM) for 2 h. Cells were also treated with 1 µM staurosporine (STS) for 18 h as an apoptotic positive control. Cells were collected after 48 h and assessed by flow cytometry. ( B ) MCF-7 cells were treated with IP-DNQ (0.25 μM) ± ZVAD-fmk (pan-caspase inhibitor, 75 μM) ± DIC (50 μM) for 2 h. Cells were also treated with 1 μM STS for 18 h or STS (1 μM, 18 h) ± ZVAD-fmk (75 μM, 2 h) to detect cell death pathways. The cells were collected after 24 h, and Western blot analysis of PARP1, p53, or α-tubulin for loading control was conducted. ( C ) A549 or MDA-MB-231 NQO1 + cells were treated as described in ( B ), and cells were monitored for caspase-3/7 activation after 48 h. For panels A and C, each group had three replicates for every biological repetition. Results (mean ± SD) were derived from three independent experiments. **** p < 0.0001, *** p < 0.001, ** p < 0.01, and ns, not significant ( t tests). The uncropped blots are shown in .

Article Snippet: Endogenous NQO1-overexpressing human A549 non-small-cell lung cancer (NSCLC) cells, and MCF-7 and MDA-MB-231 breast cancer cells were obtained from the American Tissue Culture Collection (ATCC).

Techniques: Positive Control, Flow Cytometry, Western Blot, Control, Activation Assay, Derivative Assay

Journal: Cancers

Article Title: Augmented Concentration of Isopentyl-Deoxynyboquinone in Tumors Selectively Kills NAD(P)H Quinone Oxidoreductase 1-Positive Cancer Cells through Programmed Necrotic and Apoptotic Mechanisms

doi: 10.3390/cancers15245844

Figure Lengend Snippet: IP-DNQ elicits antitumor efficacy against A549 NSCLC orthotopic Xenografts. ( A – D ) A549 and NQO1 knockout (A549- NQO1 -KO) orthotopic lung tumors were established in 18–20 g female NSG mice (n = 5/group) by injecting ~1 × 10 6 cells (tail vein, iv). After seven days, mice were treated with vehicle (HPβCD, iv), or HPβCD-IP-DNQ (8 or 12 mg/kg, iv) every other day for a total of five treatments. Bioluminescence imaging (BLI) monitored relative tumor volumes. ( A ) Representative BLI images from days 0, 18, 26, 46 67, and 90 are displayed. ( B ) Average tumor volumes were quantified on the days indicated in A. ( C ) Kaplan–Meier survival curves for mice with A549 orthotopic tumors. ( D ) Kaplan–Meier survival curves for mice with A549 NQO1 -KO orthotopic tumors. ( E – G ) Orthotopic A549 tumor-bearing female NSG mice (n = 3/group) were treated as described in ( A – D ), but with only one dosing, and they were sacrificed at the given time points. ( E ) Two hours post-drug treatment, tumor tissues were harvested and analyzed for relative NAD + and ATP levels. ( F , G ) Pharmacokinetics (PK) of IP-DNQ/IB-DNQ were evaluated. ( I – K ) Orthotopic A549 tumor-bearing female NSG mice (n = 8/group) were treated as outlined in A-D, using the MTD doses of HPβCD-IP-DNQ (15 mg/kg), HPβCD-IB-DNQ (15 mg/kg), HPβCD-DNQ (5 mg/kg), or HPβCD-β-lap (25 mg/kg). ( I ) Mice body weights were tracked for a total of 30 days post tumor cell inoculation. ( J ) Kaplan–Meier survival curves for mice with A549 orthotopic tumors. ( K ) On day 18, three mice from each group were sacrificed for HE staining of the liver, scale bar = 10 μm. Results (mean ± SD) were derived from three independent experiments. For panels ( B , E – H ), *** p < 0.001, ** p < 0.01, and * p < 0.05 ( t tests); for panels ( C , D , J ), *** p < 0.001, ** p < 0.01, * p < 0.05, and ns, not significant (log-rank test).

Article Snippet: Endogenous NQO1-overexpressing human A549 non-small-cell lung cancer (NSCLC) cells, and MCF-7 and MDA-MB-231 breast cancer cells were obtained from the American Tissue Culture Collection (ATCC).

Techniques: Knock-Out, Imaging, Drug discovery, Staining, Derivative Assay