PG-Priming Enhances Doxorubicin Influx to Trigger Necrotic and Autophagic Cell Death in Oral Squamous Cell Carcinoma

Abstract

Synergistic effects between natural compounds and chemotherapy drugs are believed to have fewer side effects with equivalent efficacy. However, the synergistic potential of prodigiosin (PG) with doxorubicin (Dox) chemotherapy is still unknown. This study explores the synergistic mechanism of PG and Dox against oral squamous cell carcinoma (OSCC) cells. Three OSCC cell lines were treated with different PG/Dox combinatory schemes for cytotoxicity tests and were further investigated for cell death characteristics by cell cycle flow cytometry and autophagy/apoptosis marker labelling. When OSCC cells were pretreated with PG, the cytotoxicity of the subsequent Dox-treatment was 30% higher than Dox alone. The cytotoxic efficacy of PG-pretreated was found better than those of PG plus Dox co-treatment and Dox-pretreatment. Increase of Sub-G1 phase and caspase-3/LC-3 levels without poly (ADP-ribose) polymeras (PARP) elevation indicated both autophagy and necrosis occurred in OSCC cells. Dox flux after PG-priming was further evaluated by rhodamine-123 accumulation and Dox transporters analysis to elucidate the PG-priming effect. PG-priming autophagy enhanced Dox accumulation according to the increase of rhodamine-123 accumulation without the alterations of Dox transporters. Additionally, the cause of PG-triggered autophagy was determined by co-treatment with endoplasmic reticulum (ER) stress or AMP-activated protein kinase (AMPK) inhibitor. PG-induced autophagy was not related to nutrient deprivation and ER stress was proved by co-treatment with specific inhibitor. Taken together, PG-priming autophagy could sensitize OSCC cells by promoting Dox influx without regulation of Dox transporter. The PG-priming might be a promising adjuvant approach for the chemotherapy of OSCC.

Keywords: autophagy; doxorubicin; influx; priming; prodigiosin.

Figure 1

Alteration of cytotoxicity in sequential PG (prodigiosin)/Dox (doxorubicin) and PG/cisplatin combination in oral squamous cell carcinoma (OSCC) and BEAS-2b cells. (A) OSCC and (B) normal bronchus cells-BEAS-2b were treated with various schemes of PG and Dox, and (C) Cisplatin substituted Dox for 12 h and analyzed cell viability by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. The results were represented as mean ± SD from three individual experiments. * p < 0.05 as compared with Dox alone.

Figure 2

Alteration of cell cycle in (A) OECM1, (B) SAS, and (C) FaDu cells. OSCC cells were treated with PG/Dox for 12/12 h prior to staining with propidium iodide (PI) and fluorescent intensity was analyzed by flow cytometry. The results were represented as mean ± SD from three individual experiments. * p < 0.05 as compared with DOX alone.

Figure 3

Expression of (A) LC3, (B) PARP1, and (C) Caspase3 in OSCCs after PG-priming. OSCC cells were treated with PG/Dox for 12/12 h and then desired protein levels were analyzed by Western blotting. The results were normalized with GAPDH and represented as mean ± SD from three individual experiments. Molecular weight: PARP1, 116 KDa; GAPDH, 37 KDa; Caspase3, 34 KDa; LC3, 18 KDa. # p < 0.05 compared with untreated control; * p < 0.05 as compared with Dox alone.

Figure 4

Alteration of cell viability combined with autophagy inhibitor. OSCC cells were treated with PG + inhibitor/DOX or PG/Dox + inhibitor and cell viability was analyzed. Table under figure was the scheme of treatment. “X” meant incubated with complete medium without PG or Dox. The results were represented as mean ± SD from three individual experiments. * p < 0.05 as compared with PG/Dox.

Figure 5

Rhodamine 123 accumulation after PG pretreatment. OSCC cells were treated with PG/R123 for (A) short term (1/1 h) and (B) long term (12/1 h) followed by analyzed fluorescent intensity within cells. The results were represented as mean ± SD from three individual experiments. # p < 0.05 as compared with R123 alone; * p < 0.05 compared with PG/R123 combination.

Figure 6

Protein levels of Doxorubicin-related importer and exporter after PG-priming. OSCC cells were treated with PG/Dox for 12/12 h and then were analyzed for Importer OCT-6 (A) and exporter MDR-1 (B) and ABCG2 (C) protein levels by Western blotting. The results were normalized with GAPDH and represented as mean ± SD from three individual experiments. Molecular weight: MDR-1, 170 KDa; ABCG2, 72 KDa; OCT-6, 58 KDa; GAPDH, 37 KDa. * p < 0.05 compared with Dox alone.

Figure 7

Cell viability change when PG/Dox combined with ER stress and energy deprivation inhibitors. OSCC cells were treated with PG + inhibitor/Dox or PG/Dox + inhibitor and were analyzed for cell viability. The inhibitors contained tauroursodeoxycholic acid (TUDC) and dorsomorphin (compound C, CC). The results were represented as mean ± SD from three individual experiments. * p < 0.05 compared with PG/Dox.

Figure 8

Potential mechanism of PG-priming doxorubicin cytotoxicity enhancement. “X” indicates not according to this mechanism. “?” illustrates still unknown. ER, endoplasmic reticulum; ROS, reactive oxygen species.