Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 10 (8), 467-477

Piperlongumine Induces Reactive Oxygen Species (ROS)-Dependent Downregulation of Specificity Protein Transcription Factors

Affiliations

Piperlongumine Induces Reactive Oxygen Species (ROS)-Dependent Downregulation of Specificity Protein Transcription Factors

Keshav Karki et al. Cancer Prev Res (Phila).

Abstract

Piperlongumine is a natural product found in the plant species Piper longum, and this compound exhibits potent anticancer activity in multiple tumor types and has been characterized as an inducer of reactive oxygen species (ROS). Treatment of Panc1 and L3.6pL pancreatic, A549 lung, 786-O kidney, and SKBR3 breast cancer cell lines with 5 to 15 μmol/L piperlongumine inhibited cell proliferation and induced apoptosis and ROS, and these responses were attenuated after cotreatment with the antioxidant glutathione. Piperlongumine also downregulated expression of Sp1, Sp3, Sp4, and several pro-oncogenic Sp-regulated genes, including cyclin D1, survivin, cMyc, EGFR and hepatocyte growth factor receptor (cMet), and these responses were also attenuated after cotreatment with glutathione. Mechanistic studies in Panc1 cells showed that piperlongumine-induced ROS decreased expression of cMyc via an epigenetic pathway, and this resulted in downregulation of cMyc-regulated miRNAs miR-27a, miR-20a, and miR-17 and induction of the transcriptional repressors ZBTB10 and ZBTB4. These repressors target GC-rich Sp-binding sites to decrease transactivation. This pathway observed for piperlongumine in Panc1 cells has previously been reported for other ROS-inducing anticancer agents and shows that an important underlying mechanism of action of piperlongumine is due to downregulation of Sp1, Sp3, Sp4, and pro-oncogenic Sp-regulated genes. Cancer Prev Res; 10(8); 467-77. ©2017 AACR.

Figures

Figure 1
Figure 1
Piperlongumine inhibits cancer cell proliferation. 786-O (A), SKBR3 (B), Panc1 (C), A549 (D) and L3.6pL (E) cancer cell lines were treated with different concentrations of piperlongumine or 5 mM GSH alone or in combination for 24 and 48 hr and cell numbers were determined as outlined in the Materials and Methods. Results are means ± SEM for at least 3 replicate determinations and significant (p<0.05) growth inhibition by piperlongumine (*), growth induction or reversal of piperlongumine-dependent growth inhibition by GSH (**) are indicated.
Figure 2
Figure 2
Piperlongumine induces ROS in cancer cell lines. 786-O (A), SKBR3 (B), Panc1 (C), A549 (D) and L3.6pL (E) cells were treated with piperlongumine or 5 mM GSH alone or in combination and ROS was determined by FACS analysis of the cell permeant dye. CM-H2DFCDA as outlined in the Materials and Methods. Results are expressed as means ± SEM for at least 3 replicate determinations and significant (p<0.05) induction of ROS by piperlongumine (*) and inhibition by GSH (**) are indicated.
Figure 3
Figure 3
Piperlongumine induces apoptosis in cancer cells. 786-O (A), SKBR3 (B), Panc1 (C), A549 (D) and L3.6pL (E) cells were treated with 5 mM GSH and different concentration of piperlongumine alone and in combination and after 24 hr, effects on Annexin V staining and PARP cleavage were determined fluorimetrically or by western blot analyses of whole cell lysates, respectively, as outlined in the Materials and Methods. Results of Annexin V staining are expressed as means ± SEM of at least 3 replicate determinations and significant (p<0.05) induction of Annexin V (*) and inhibition by GSH (**) are indicated.
Figure 4
Figure 4
Piperlongumine downregulates Sp1, Sp3, Sp4 and Sp-regulated genes: effects of GSH. 786-O (A), SKBR3 (B), Panc1 (C), A549 (D), and L3.6pL (E) cells were treated with 5 mM GSH or different concentrations of piperlongumine alone and in combination for 24 hr, and whole cell lysates were analyzed by western blots. Effects on Sp proteins and Sp-regulated gene expression and PARP cleavage (Fig. 3) were all obtained in the same experiment and have the same GAPDH loading control. Similar results were observed in duplicate experiments.
Figure 5
Figure 5
Mechanism of piperlongumine induced Sp downregulation. (A) Proposed mechanism of piperlongumine-induced Sp downregulation by initial induction of ROS. (B) Panc1 cells were treated with 5 μM piperlongumine for up to 24 hr, and whole cell lysates were analyzed by western blots. (C) Panc1 cells were treated with 5 μM piperlongumine or 5 mM GSH alone for 4 hr, and whole cell lysates were analyzed by western blots. Panc1 cells with 5 μM piperlongumine or 5 mM GSH alone and in combination for up to 24 hr, and the extracted RNA (D) or protein (E) was analyzed by real time PCR or western blots, respectively. Results in (D) are means ± SEM for at least 3 replicates and significant (p<0.05) miR downregulation by piperlongumine (*) and reversal by cotreatment with GSH (**) are indicated.
Figure 6
Figure 6
Piperlongumine-dependent Sp downregulation is cMyc-dependent and in vivo studies. Panc1 cells were treated with 5 μM piperlongumine or 5 mM GSH alone or in combination for 3 hr, and interactions with the cMyc (A) and Sp1 (B) promoters were determined in ChIP assays. Quantitation of the bands was carried out by quantitative PCR and results are illustrated in Supplemental Figure S3. (C) Panc1 cells were treated with DMSO or 5 μM piperlongumine alone or after transfection with a cMyc expression plasmid and after 3 hr, whole cell lysates were analyzed by western blots. Athymic nude mice bearing L3.6pL cells as xenografts were treated with piperlongumine (30 mg/kg/d), and effects on tumor weights and body weights (D) and expression of various gene products (E) in tumors from control (corn oil) and piperlongumine-treated mice were determined by western blot analysis of tumor lysates. Expression levels of various proteins in control vs. piperlongumine-treated mice were determined (normalized to GAPDH). Significant (p<0.05) changes in protein levels in tumors from piperlongumine-treated mice compared to controls (*) are indicated.

Similar articles

See all similar articles

Cited by 12 articles

See all "Cited by" articles
Feedback