Apoptosis and necrosis induced by novel realgar quantum dots in human endometrial cancer cells via endoplasmic reticulum stress signaling pathway

Abstract

Realgar (AS4S4) has been used in traditional medicines for malignancy, but the poor water solubility is still a major hindrance to its clinical use. Realgar quantum dots (RQDs) were therefore synthesized with improved water solubility and bioavailability. Human endometrial cancer JEC cells were exposed to various concentrations of RQDs to evaluate their anticancer effects and to explore mechanisms by the MTT assay, transmission electron microscopy (TEM), flow cytometry, real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. Results revealed that the highest photoluminescence quantum yield of the prepared RQDs was up to approximately 70%, with the average size of 5.48 nm. RQDs induced antipro-liferative activity against JEC cells in a concentration-dependent manner. In light microscopy and TEM examinations, RQDs induced vacuolization and endoplasmic reticulum (ER) dilation in JEC cells in a concentration-dependent manner. ER stress by RQDs were further confirmed by increased expression of GADD153 and GRP78 at both mRNA and protein levels. ER stress further led to JEC cell apoptosis and necrosis, as evidenced by flow cytometry and mitochondrial membrane potential detection. Our findings demonstrated that the newly synthesized RQDs were effective against human endometrial cancer cells. The underlying mechanism appears to be, at least partly, due to ER stress leading to apoptotic cell death and necrosis.

Keywords: apoptosis; endometrial cancer cells; endoplasmic reticulum stress; necrosis; quantum dots; realgar.

Figure 1

The expression of ER stress marker in JEC cells treated with different concentrations of RQDs. Notes: (A) Primer sequences for real-time PCR. (B) The expression of GRP78 and GADD153 mRNA. (C) The expression of GRP78 and GADD153 protein. Abbreviations: RQDs, realgar quantum dots; ER, endoplasmic reticulum; PCR, polymerase chain reaction.

Figure 2

The characters of RQDs. Notes: (A) UV-vis absorption and photoluminescence emission spectra of the as-prepared RQDs using ethanolamine and citric acid. Inset shows fluorescence photo under 365 nm UV light. (B) TEM and HRTEM images (inset) of the above RQDs, with the arrows emphasizing the 0.31 nm distance of parallel between them. (C) Gives the corresponding size histogram and curve. Abbreviations: UV-vis, ultravoilet-visible emission spectra; PL, photoluminescence emission spectra; RQDs, realgar quantum dots; TEM, transmission electron microscopy; HRTEM, high-resolution TEM.

Figure 3

Concentration-dependent effects of RQDs on the cell viability of human endometrial cancer cells JEC. Notes: After treatment of RQDs (10, 20, 40, 80, and 160 µg/mL) for 48 hours, the cell viability was analyzed using the MTT assay. The data are expressed as the mean ± SD of three experiments. *P<0.05 indicates statistically significant differences from the control group. Abbreviations: RQDs, realgar quantum dots; SD, standard deviation.

Figure 4

Morphological change of JEC cells treated with RQDs. Notes: (A) Vacuolization induced by RQDs, with the arrows indicating the vacuolization and ER dilation. (B) Ultrastructural analysis of JEC cells by ultrathin section transmission electron microscopy. Abbreviations: Ctrl, control; RQDs, realgar quantum dots.

Figure 5

Induction of apoptosis in JEC cells following the RQDs treatment. Notes: (A) Cells were treated with different concentrations of RQDs for 24 hours, and stained with Annexin V-FITC and PI, and then apoptotic cells were quantified by flow cytometry. In order to quantify the apoptotic rate, different subpopulations were distinguishable: D1, Annexin V-negative but PI-positive, ie, necrotic cells; D2, Annexin V/PI-double positive, ie, late apoptotic cells; D3, Annexin V/PI-double negative, ie, live cells; D4, Annexin V-positive but PI-negative, ie, early apoptotic cells. (B) The apoptotic rate was determined as the percentage of D2 + D4. (C) Mitochondrial membrane potential (ΔΨm) was analyzed using JC-1 mitochondrial membrane dye. JEC cells were treated with DMSO (control) or RQDs (15 µg/mL) for 24 hour. The drug caused an increase in the green (JC-1 monomers) and decrease in the red fluorescence (JC-1 aggregates), indicative of loss of ΔΨm. *P<0.05, **P<0.01. Abbreviations: RQDs, realgar quantum dots; PI, propidium iodide; DMSO, dimethyl sulfoxide; FITC, fluorescein isothiocyanate; ctrl, control; AV, annexin V.