PMC

A schematic model for the molecular mechanism of ALT-induced anti-cancer activity in A549 lung adenocarcinoma cells.

ALT induces ER stress and mitochondrial dysfunction through oxidative stress. (A) A549 cells were treated with indicated concentrations of ALT for 12 h. Total cell lysates were extracted and subjected to Western blots for the expression of TrxR1, p-eIF2α, eIF2α, ATF4 and CHOP. Columns not sharing same superscript letters within the group differ significantly (P < 0.05). (B) A549 cells were treated with ALT as indicated for 12 h. Total cell lysates were extracted and subjected to Western blots for the expression of Bcl-2, Bax, and Bad. Columns not sharing same superscript letters within the group differ significantly (P < 0.05). (C) A549 cells were treated with 45 μM ALT in the presence or absence of 3 mM NAC for 12 h. Total cell lysates were then subjected to immunoblotting for the expression of Bcl-2, Bax, ATF4 and CHOP. Columns not sharing same superscript letters within the group differ significantly (P < 0.05).

Effect of ALT on morphology and viability of cells. (A) Chemical structure of ALT. (B) A549 cells were treated with indicated concentrations of ALT for 12 h and cells morphological changes were observed under phase-contrast microscope. Scale bar = 100 μm. Pretreatment of cells with 3 mM NAC and/or 3 mM TCEP completely suppressed the toxic effect of ALT. (C) Cells were treated with 10–100 μM ALT for 12 h and cell viability was measured by MTT assay. Data are expressed as Mean ± SEM (n = 3). Columns not sharing same superscript letters differ significantly (P < 0.05). (D) A549 cells were treated with indicated concentrations of ALT for 12 h and live and dead cells were quantified using Live/dead assay. Pretreatment of cells with NAC and/or TCEP reversed ALT-induced death in A549 cells. Data are expressed as Mean ± SEM (n = 3). Columns not sharing same superscript letters differ significantly (P < 0.05).

ALT augments doxorubicin toxicity in doxorubicin sensitive (A549) and doxorubicin resistant (A549/DR) lung cancer cells. (A) A549/DR cells were treated with doxorubicin or a combination of doxorubicin and ALT for 12 h. The expression of p-STAT3, STAT3, Cox-2, MMP-9 and p-gp was determined by Western blot. Columns not sharing same superscript letters within the group differ significantly (P < 0.05). (B) A549 cells were treated with doxorubicin or a combination of doxorubicin and ALT for 12 h. The expression of Bcl-2, Bax, Survivin, Xiap, cleaved-caspase-3 and cleaved-PARP was determined by Western blot. Columns not sharing same superscript letters within the group differ significantly (P < 0.05). (C) A549/DR cells were treated with doxorubicin, ALT and S31-201 either as single agent or in combination for 12 h and cell viability was determined by MTT assay. (D) A549/DR cells were incubated with or without 30 μM ALT for 12 h. The cells were then incubated with 2.5 μM doxorubicin for 2 h at 37 °C. After washing with PBS, the fluorescence of doxorubicin was measured at 488 nm by spectrophotometer. Columns not sharing same superscript letters differ significantly (P < 0.05).

Effect of ALT on apoptosis, ROS generation, GSH/GSSG ratio and MMP dissipation. (A) A549 cells were treated with indicated concentration of ALT in the presence or absence of 3 mM NAC and 1 mM diamide for 12 h. Cells were stained with Annexin V/PI and apoptosis was determined by flow cytometery. (B) Statistical analysis of data from A. Columns not sharing same superscript letters within the same group differ significantly (P < 0.05). (C) A549 cells were incubated with ALT in a dose- and time-dependent manner as indicated and ROS generation was determined by staining the cells with DCFH-DA. (D) A549 cells were treated with ALT for 12 h and intracellular GSH/GSSG ratio was measured according to kit instructions. (E) A549 cells were treated with ALT for 12 h and MMP was measured using JC-1 kit according to manufacturer instructions. Columns not sharing same superscript letters (Fig. C,D, and E) differ significantly (P < 0.05). (F) A549 cells were treated with indicated concentrations of ALT for 12 h and expressions of xiap, survivin, cleaved caspases-9, cleaved caspases-3 and cleaved PARP were measured by Western blot analysis.

ALT abrogates STAT3 activation through oxidative stress. (A) A549 cells were incubated with ALT for 12 h. Cell lysates were collected and subjected to Western blot for the expression of p-JAK2, JAK2, SHP-2, p-SRC and SRC. (B) Protein expression was normalized to GAPDH. Each bar in graph represents Mean ± SEM of three repeated experiments. Columns not sharing same superscript letters within the group differ significantly (P < 0.05). (C) A549 cells were incubated with or without BaP (2 μM) and Doxorubicin (2 μM) for 7 days. The cells were then treated with ALT in the presence or absence of NAC for another 12 h. Cell extracts were collected and expression of p-STAT3/STAT3 was evaluated by Western blot. (D) Each bar in graph represents Mean ± SEM of three repeated experiments. Columns not sharing same superscript letters within the group differ significantly (P < 0.05). (E) A549 cells were treated with diamide and ALT in the presence or absence of NAC for 4 h. The total cell lysates were collected and subjected to immunoprecipitation (IP) for STAT3 and immunoblotting (IB) for protein linked GSH (PSSG) and STAT3. ALT increased the level of glutathionylated STAT3 as diamide while NAC inhibited ALT-induced STAT3 glutathionylation.

Effect of ALT on cell proliferation. (A) A549 cells were seeded in 96 well plates and incubated at 37 °C overnight. The cells were treated with indicated concentration of ALT for 12 h. Following treatment, drug containing medium was replaced with fresh medium and cells were allowed to grow in drug free medium for 24 h. The cell proliferation was measured by MTT assay. (B) A549 cells were treated with ALT for 12 h and expression of Ki-67, myc, cyclin D1 was measured by Western blot. (C) Statistical analysis of data from B. Columns not sharing same superscript letters within the same group differ significantly (P < 0.05). (D) Cells treated with indicated concentration of ALT for 12 h were seeded into 6 well plates and allowed to grow into colonies for 7 days. The colonies were fixed, stained with crystal violet, washed and photographed. (E) To quantify proliferation rate, methanol was added to each well to dissolve crystal violet stain and absorbance was measured at 595 nm. Columns not sharing same superscript letters within the group differ significantly (P < 0.05).

ALT inhibits STAT3 activation, translocation into nucleus and DNA binding activity in A549 cells. (A) ALT inhibits constitutive and inducible STAT3 activation (A1) A549 cells were treated with 45 and 60 μM ALT for 12 h, (A2 & A3) A549 cells were pretreated with 45 μM ALT for 4 h and then stimulated with 100 nM TPA and 10 ng/mL IL-6 for 1 h respectively, (A4) A549 cells were treated with 2 μM BaP for 7 days and then treated with 45 μM ALT for 12 h. Total cell lysates were extracted and subjected to Western blot for the expression of p-STAT3 and STAT3. (B) Statistical analysis of p-STAT3/STAT3 expression. Columns not sharing same superscript letters within the group differ significantly (P < 0.05). (C) A549 cells were treated with 45 and 60 μM ALT for 4 h. Nuclear proteins were isolated and subjected to Western blot for expression of p-STAT3 and STAT3. The expression of p-STAT3 and STAT3 was normalized with lamin B1. (D) Each bar in graph represents Mean ± SEM of three repeated experiments. Columns not sharing same superscript letters within the group differ significantly (P < 0.05). (E) A549 cells were incubated with or without ALT for 4 h and then further incubated with IL-6 for 1 h. The nuclear extracts were then collected and assayed for STAT3 DNA binding activity according to the instructions of kit. Columns not sharing same superscript letters within the group differ significantly (P < 0.05).

Effect of ALT on A549 cell migration. (A) A549 cells were cultured in 12 well plates. A wound was created in the middle using sterile micropipette tip. The cells were washed with PBS, exposed to ALT for 12 h. The drug containing medium was then replaced with fresh medium and allowed to grow at 37 °C for 24 h. The cells were photographed at 0 and 24 h under an inverted microscope. Scale bar = 100 μm (B). A549 cells were treated with ALT for 12 h. Drug containing medium was removed and cells were washed with PBS to remove floating cells. The adherent cells were harvested, counted and 2 × 10⁴ cells were seeded onto transwell chamber and incubated for 24 h as described in materials and methods. The migrated cells were fixed, stained with crystal violet and photographed. Scale bar = 100 μm (C) A549 cells were treated with ALT for 12 h and expression of iNOS, Cox-2, MMP-9 was measured by Western blot. (D) Statistical analysis of data from C. Columns not sharing same superscript letters differ significantly (P < 0.05). (E) A549 cells were treated with ALT in the presence or absence of TPA for 12 h. The culture medium was collected and the level of MMP-9 in culture medium was measured according to kit instructions.