Fig 1.
Effect of fisetin, acacetin and doxorubicin on cell growth of NSCLC cells.
Total live cell number of A549 and H1299 cells treated with different concentrations of Fs (A, B), Ac (C, D) and Dox (E, F) were counted at 24, 48 and 72 h using hemocytometer by trypan blue assay. The data are presented as means of triplicate samples for each treatment. Fs, fisetin; Ac, acacetin; Dox, doxorubicin. Bars, SE; P<0.0001 (#), P<0.001 (***), P<0.01 (**), P< 0.05 (*).
Table 1.
Effects of fisetin, acacetin and doxorubicin on cytotoxicity IC50 values of A549 and H1299 cells.
Fig 2.
Combinatorial effects of fisetin or acacetin with doxorubicin on cell viability in NSCLC cells.
A549 (A, B) and H1299 cells (C, D) were treated with 10 and 25 μM of Fs and Ac in combination with Dox (10 nM for A549 and 25 nM for H1299 cells) for 48 and 72 h. At the end of treatments, cells were processed for trypan blue assay. The data are presented as means of triplicate samples for each treatment. Fs, fisetin; Ac, acacetin; Dox, doxorubicin. Bars, SE; P<0.0001 (#), P<0.001 (***), P<0.01 (**), P< 0.05 (*).
Table 2.
Combination index (CI) of the combination treatment of fisetin-doxorubicin and acacetin-doxorubicin in NSCLC cells.
Fig 3.
Effect of fisetin and acacetin in combination with doxorubicin on A549 and H1299 cell cycle progression.
A549 (A, B) and H1299 (A, C) cells were treated for 72 h as indicated in the figure and analyzed by flow cytometry. Immunoblot analysis of cell cycle regulatory molecules involved in G2/M phase were done after 72 h of treatments in A549 (D) and H1299 cells (E). Tubulin and β-actin were used as loading control. The data is presented as means of triplicate samples for each treatment. Fs, fisetin; Ac, acacetin; Dox, doxorubicin. P<0.001 (***), P<0.01 (**), P< 0.05 (*).
Fig 4.
Effect of combination of fisetin and acacetin with doxorubicin on the clonogenic potential of A549 and H1299 cells.
A549 (A, B) and H1299 cells (C, D) were treated with indicated concentrations of Fs, Ac and Dox in regular growth medium for 8 days. After that cells were processed/stained with crystal violet dye. Colonies size between 50–200 cells were counted and plotted (C, D). The data is presented as means of triplicate samples for each treatment. Fs, fisetin; Ac, acacetin; Dox, doxorubicin. P<0.0001 (#), P<0.01 (**), P< 0.05 (*).
Fig 5.
Doxorubicin efflux measurement in the cytoplasm of A549 and H1299 cells.
(A) Standard curve of Dox was prepared in PBS to measure its fluorescence by spectrofluorophotometer. (B) A549 and (C) H1299 cells were pre-treated with 10 μM Dox for 2 h and washed with PBS to remove extracellular Dox. Thereafter, cells were incubated with 25 μM Ac and 25 μM SB at 37°C and 50 rpm. Cell supernatant was processed to measure effluxed Dox at different time points. Dox ex = 488 nm and em = 597 nm. Ac, acacetin; SB, silibinin; Dox, doxorubicin. The data shown are means of three independent plates. P<0.0001 (#), P<0.001 (***), P<0.01 (**), P< 0.05 (*).
Fig 6.
Flow cytometry analysis of doxorubicin uptake in A549 and H1299 cells.
(A) A549 and (B) H1299 cells were pre-treated with 25 μM Ac and 25 μM SB for 6 h and post-incubated with 10 μM Dox. The cells were then collected at different time points and proceed for FACS analysis. (C) Representative figure of flow cytometry histogram of fluorescence intensity after respective treatments. The data is presented as means of triplicate samples for each treatment. Groups at different time points have been compared with respective Dox alone treatment. Ac, acacetin; SB, silibinin; Dox, doxorubicin. P<0.001 (***), P<0.01 (**), P< 0.05 (*).
Fig 7.
Doxorubicin uptake in A549 and H1299 cells by confocal microscopy.
A549 and H1299 cells (A) cells were treated with 25 μM Ac and 25 μM SB for 6 h with 2 h posttreatment of 10 μM Dox. Afterward, cells were stained with DAPI followed by confocal imaging. Quantitative analysis of Dox fluorescence intensity in A549 (B) and H1299 (C). Treatment groups have been compared with Dox alone exposure. Ac, acacetin; SB, silibinin; Dox, doxorubicin. The quantitative data shown are means of two samples for each treatment. P<0.001(***), P<0.01(**).
Fig 8.
Expression of MDR1 gene by RT-PCR.
A549 and H1299 cells were treated with respective treatments for 72 h and processed by RNA isolation. Semi quantitative PCR reaction of amplified MDR1 product were analysed by 1% agarose gel electrophoresis. (A) and (B) are representing the RNA expression of MDR1 gene in A549 and H1299 cells, respectively. (C) and (D) are bar diagram of densitometric value of bands representing the relative fold change in RNA expression. GAPDH was used as loading control.