Garcinol Sensitizes NSCLC Cells to Standard Therapies by Regulating EMT-Modulating miRNAs

Abstract

Garcinol, a dietary factor obtained from Garcinia indica, modulates several key cellular signaling pathways as well as the expression of miRNAs. Acquired resistance to standard therapies, such as erlotinib and cisplatin, is a hallmark of non-small cell lung cancer (NSCLC) cells that often involves miRNA-regulated epithelial-to-mesenchymal transition (EMT). We used A549 cells that were exposed to transforming growth factor beta 1 (TGF-β1), resulting in A549M cells with mesenchymal and drug resistant phenotype, and report that garcinol sensitized resistant cells with mesenchymal phenotype to erlotinib as well as cisplatin with significant decrease in their IC₅₀ values. It also potentiated the apoptosis-inducing activity of erlotinib in A549M and the endogenously mesenchymal H1299 NSCLC cells. Further, garcinol significantly upregulated several key EMT-regulating miRNAs, such as miR-200b, miR-205, miR-218, and let-7c. Antagonizing miRNAs, through anti-miRNA transfections, attenuated the EMT-modulating activity of garcinol, as determined by mRNA expression of EMT markers, E-cadherin, vimentin, and Zinc Finger E-Box Binding Homeobox 1 (ZEB1). This further led to repression of erlotinib as well as cisplatin sensitization, thus establishing the mechanistic role of miRNAs, particularly miR-200c and let-7c, in garcinol-mediated reversal of EMT and the resulting sensitization of NSCLC cells to standard therapies.

Keywords: EMT; NSCLC; cisplatin; erlotinib; garcinol; miRNAs.

Figure 1

Structure of garcinol.

Figure 2

Garcinol sensitizes transforming growth factor beta 1 (TGF-β1)-induced epithelial-to-mesenchymal transition (EMT) cells, A549M to therapy. A549M cells were treated with increasing doses of erlotinib (A) and cisplatin (B) in the absence (garcinol 0 μM) as well as presence of increasing doses of garcinol (5 and 20 μM) for 72 h and then subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (C,D) The effect of such treatment on anchorage-independent colony formation was also observed. The number of colonies are represented as %, relative to the control conditions with no erlotinib/cisplatin or garcinol. * p < 0.05 and ** p < 0.01, compared to erlotinib alone.

Figure 3

Garcinol potentiates apoptosis induction. A549M and H1299 cells were treated with increasing doses of garcinol for 24 h/72 h, and the induction of apoptosis was measured by Histone/DNA enzyme-linked immunosorbent assay (ELISA) (A) and homogeneous caspase-3/7 assay (B). * p < 0.05 and ** p < 0.01, compared to 0 μM garcinol. A549M (C,D) and H1299 (E,F) cells were treated with 25 and 50 μM erlotinib for 24 h in the absence (erlotinib alone) or presence of 5 and 20 μM garcinol, and the apoptosis was detected by Histone/DNA ELISA (C,E) and homogenous caspase-3/7 assay (D,F). * p < 0.05 and ** p < 0.01, compared to erlotinib alone.

Figure 4

Garcinol upregulates EMT-regulating miRNAs. A549M cells were treated with 20 μM garcinol for 72 h and the expression of select miRNAs was evaluated relative to control cells (garcinol 0 μM). * p < 0.05, ** p < 0.01.

Figure 5

Expression of EMT markers. Expression of epithelial marker E-cadherin (A) and mesenchymal markers vimentin (B) and ZEB1 (Zinc Finger E-Box Binding Homeobox 1) (C) was measured in A549M cells, relative to parental A549 cells. G0: control untreated cells, G20: cells treated with 20 μM garcinol for 72 h, G20+miR-200b: cells treated with 20 μM garcinol for 72 h after transfections with anti-miR-200b, G20+miR-205: cells treated with 20 μM garcinol for 72 h after transfections with anti-miR-205, G20+miR-218: cells treated with 20 μM garcinol for 72 h after transfections with anti-miR-218, G20+let7c: cells treated with 20 μM garcinol for 72 h after transfections with anti-Let-7c. # p < 0.01, compared to G0, * p < 0.05, relative to G20, ** p < 0.01, relative to G20.

Figure 6

Mechanistic role of miRNAs in drug sensitization. Effect of anti-miRNAs was evaluated on garcinol-induced sensitization of A549M cells to erlotinib (A) and cisplatin (B), through MTT assay. Treatment with drugs in the absence (garcinol 0 μM) or presence of 20 μM garcinol was for 72 h, after transfections with non-specific or specific anti-miRNAs. Effect of anti-miRNAs was also evaluated on potentiation of apoptosis induction as assayed by Histone/DNA ELISA (C) and homogeneous caspase-3/7 assay (D). * p < 0.05 and ** p < 0.01, compared to garcinol 20 μM.