Multifunctional selenium nanoparticles with Galangin-induced HepG2 cell apoptosis through p38 and AKT signalling pathway

The morbidity and mortality of hepatocellular carcinoma, the most common cancer, are increasing continuously worldwide. Galangin (Ga) has been demonstrated to possess anti-cancer effect, but the efficacy of Ga was limited by its low permeability and poor solubility. To develop aqueous formulation and improve the anti-cancer activity of Ga, surface decoration of functionalized selenium nanoparticles with Ga (Se@Ga) was synthesized in the present study. The aim of this study was to evaluate the anti-cancer effect of Se@Ga and the mechanism on HepG2 cells. Se@Ga-induced HepG2 cell apoptosis was confirmed by depletion of mitochondrial membrane potential, translocation of phosphatidylserine and caspase-3 activation. Furthermore, Se@Ga enhanced the anti-cancer activity of HepG2 cells through ROS-mediated AKT and p38 signalling pathways. In summary, these results suggest that Se@Ga might be potential candidate chemotherapy for cancer.

YL, 0000-0002-6271-8283; BZ, 0000-0001-7695-6082 The morbidity and mortality of hepatocellular carcinoma, the most common cancer, are increasing continuously worldwide. Galangin (Ga) has been demonstrated to possess anti-cancer effect, but the efficacy of Ga was limited by its low permeability and poor solubility. To develop aqueous formulation and improve the anti-cancer activity of Ga, surface decoration of functionalized selenium nanoparticles with Ga (Se@Ga) was synthesized in the present study. The aim of this study was to evaluate the anti-cancer effect of Se@Ga and the mechanism on HepG2 cells. Se@Ga-induced HepG2 cell apoptosis was confirmed by depletion of mitochondrial membrane potential, translocation of phosphatidylserine and caspase-3 activation. Furthermore, Se@Ga enhanced the anti-cancer activity of HepG2 cells through ROS-mediated AKT and p38 signalling pathways. In summary, these results suggest that Se@Ga might be potential candidate chemotherapy for cancer.

Background
Hepatocellular carcinoma (HCC) is considered as one of the most common malignancies and ranks third in cancer-associated deaths around the world. [1,2]. However, the early stage diagnosis of HCC is difficult and the prognosis is less satisfactory [3,4]. Moreover, HCC is metastatic or advanced at the time of diagnosis, and the local therapies are unsuitable [5,6]. Furthermore, due to the insensitivity of HCC to chemotherapy and high metastatic potential, the survival of patients with HCC is very difficult [7].

Preparation and characterization of Se@Ga
Se@Ga nanoparticles were synthesized as follows: briefly, 0.25 ml of stock solution (0.1 M) of Na 2 SeO 3 was gradually added into 2 ml stock solution (50 mM) of Vitamin C. Then, 2 ml of Ga solution (44 mM) was added into the SeNP solution. The Se@Ga complex was purified overnight by dialysis. Se@Ga nanoparticles were sonicated and then filtered through 0.2 mm pore size. They were characterized by various methods. The concentration of SeNPs was measured by ICP-AES. Se@Ga nanoparticle samples were prepared by dispersing the particles onto a holey carbon film on copper grids. The micrographs were obtained for TEM operated at an accelerating voltage of 80 kV. EDX analysis was carried out on an EX-250 system to examine the elemental composition of Se@Ga. FT-IR samples were recorded on an Equinox 55 IR spectrometer (in the range of 4000-500 cm 21 ) using the KBr-disc method. The particle size distribution and zeta potential were determined by Zetasizer Nano ZS particle analyser.

Cell culture and viability assay
The cell proliferative inhibition by Se@Ga nanoparticles was measured, as previously described [28].

Scratch assay
The anti-cell migration effect of Se@Ga on HepG2 cells was detected by a scratch assay [30]. In brief, after the cell confluence reached 80%, cell monolayers were wounded with a sterile microtip and washed with PBS to discard detached cells. Then, the cells were treated with Se, Ga, Se@Ga and incubated for 24 h. After that, the wound closure was observed and photographed by an Olympus microscope at 0 and 24 h.

Mitochondrial membrane potential measurement (DC m )
JC-1 was used to detect the mitochondrial membrane potential by Se@Ga in HepG2 cells, as previously reported [31]. The cells cultured in six-well plates were released by trypsinization, resuspended in PBS buffer with 10 mg ml 21 JC-1 and then incubated at 378C for 30 min. The cells were then harvested by centrifugation, resuspended in PBS and analysed by flow cytometry. JC-1 fluorescence was measured with excitation (485 nm) and dual emission (shift from green at 530 nm to red at 590 nm).

Annexin-V/PI double-staining assay
Translocation of phosphatidylserine in HepG2 cells treated with Se@Ga was detected, as previously described [32]. In brief, the cells were seeded into six-well plates till 70% confluence and then incubated with SeNPs, Ga and Se@Ga for 24 h. The cells were then washed three times by PBS and stained with Annexin-V/PI for 30 and subjected to flow cytometric analysis.

Caspase-3 activity
Caspase-3 activity was determined by a fluorometric method, as described in our previous paper [33].
Harvested cell pellets were suspended in cell lysis buffer and incubated on ice for 1 h. After centrifugation at 11 000 Â g for 30 min, supernatants were collected and immediately measured for protein concentration and caspase activity. For determination of caspase activity, cell lysates were added in 96-well plates and then incubated with specific caspase-3 substrates for 1 h at 378C. Caspase-3 activity was determined by fluorescence intensity with an excitation of 380 nm and an emission of 460 nm.

Transmission electron microscopic analysis Se@Ga-treated HepG2
Se@Ga-treated HepG2 was negatively stained and morphologically detected by TEM, as previously described [34]. The HepG2 cell was treated with Se@Ga at various time points and then was attached to the carbon-coated collodion grid for 10 min. The grids were stained with 2% phosphotungstic acid in Sorensen phosphate buffer for 2 min. The grids were examined by TEM after rinsing and air-drying the slides. 2.9. Determination of ROS generation ROS accumulation induced by Se@Ga was estimated, as previously described [35]. In brief, HepG2 cells were harvested and suspended in PBS containing 10 mM of DCFH-DA for 30 min. ROS lever was determined by measuring the fluorescence intensity using a microplate reader. ROS generation was indicated by green florescence which was measured with an excitation of 488 nm and an emission of 525 nm. Experiments were performed in triplicate.

Western blotting analysis
Western blotting was performed, as previously reported [36]. Briefly, total intracellular proteins in HepG2 cells treated with Se@Ga were extracted. The protein concentration was examined by the  bicinchoninic acid assay. An equal amount of protein was electrophoresed in 12% tricine gels and blocked with 5% non-fat milk in Tris-buffered saline Tween-20 buffer for 1 h. The membranes were incubated with primary antibodies at 1 : 1000 dilutions overnight at 48C with continuous agitation. Then, the membranes were incubated with secondary antibody conjugates with horseradish peroxides at 1 : 1000 dilutions for 2 h at room temperature, followed by washing three times with Tris-buffered saline Tween-20 buffer. The proteins were visualized on the X-ray film. The densitometry analysis of band intensity was detected by ImageJ.

Statistical analysis
All data were processed using the SPSS 19.0 software. The difference between three and more groups was analysed by one-way ANOVA multiple comparisons. Differences between two groups were evaluated by two-tailed Student's t-test. A probability of p , 0.05 (*) or p , 0.01 (**) indicates statistically significant values.

Preparation and characterization of Se@Ga
Galangin-modified SeNPs (Se@Ga) enhanced anti-cancer effect (scheme 1). Light image of Ga, SeNPs and Se@Ga is shown in figure 1a. Owing to the modified of SeNPs, the colour of SeNPs was deeper than that of Se@Ga. As shown in figure 1b, the Tyndall effect of Se@Ga indicated that Se@Ga nanoparticles were synthesized. TEM images showed that Se@Ga presented spherical and monodisperse particle (figure 2a). As shown in figure 2b, EDX indicated the signal of C (15%), O (2%) than from Ga, Se atoms were 32% and Cu (51%) comes from copper grids. Compared with SeNPs (164 nm), Se@Ga presented high uniformity with a minimum diameter of 71 nm (figure 2c,d). Ga may reduce the surface-free energy, and the size was lower than that of SeNP. The zeta potential of SeNPs was 224.8 mV and decreased to 236 mV after capping with Galangin (figure 2e), which indicated that Se@Ga with positive charge was easier to cross into the cell membrane. Furthermore, size distribution of Se@Ga revealed that the decorated SeNPs were stable at least for 30 days (figure 2f ),

In vitro cytoxicity of Se@Ga
The inhibition of HepG2 cell proliferation by SeNPs, Galangin and Se@Ga was measured through the MTT assay. As shown in figure 4a, the cell viability of HepG2 was dramatic lower than LO2 cells. The cell viability of HepG2 by SeNPs and Ga was 87% and 67%, respectively, but when treated with Se@Ga, the cell viability decreased to 52%. Compared with SeNPs and Galangin, Se@Ga significantly inhibited the growth of HepG2 cells. As shown in figure    The caspase family of aspartate-specific cysteine proteases plays important roles in the initiation and execution of apoptosis. As shown in figure 6a, compared with the control group (100%), SeNPs (642%) and Galangin (913%), treatments of HepG2 cells with Se@Ga (1000%) significantly increased the activity of caspase-3. Meanwhile, to determine whether caspase family was activated in HepG2 cells exposed to Se@Ga, the activities of caspase 3 were measured by Western blotting, as shown in figure 6b,c. The protein expression level of caspase-3 (control 100%, SeNPs 96%, Ga 83% and Se@Ga 31%) was downregulated with different treatments. The results show that Se@Ga significantly strengthened the activation of caspase-3 and induced the HepG2 cell apoptosis.

Se@Ga inhibited the migration of HepG2 cells and TEM image of thin sections
As presented in figure 7a, the HepG2 cells were exposed to SeNPs, Ga and Se@Ga, and the cellular migration was analysed by a scratch assay. In the control group, the wounded gap was almost completely occupied by the migrating cells after 24 h, while in the Se@Ga-treated group, this gap was not occupied by migrating cells. This result suggested that Se@Ga significantly inhibited the migration ability of HepG2 cells. The morphology change of HepG2 cells treated with Se@Ga was observed by TEM. As shown in figure 7b, when incubated with Se@Ga, TEM image indicates few cells with the disappearance of microvilli, a shrinking cytoplasm, distorted organelles and condensed chromatin.
The percentage of cells that lost adhesion and shrunk was decreased after treatment with Se@Ga.

Induction of ROS generation by Se@Ga
ROS generation was determined by the DCF fluorescence assay to reveal its role in the action mechanisms of Se@Ga. As shown in figure 8a (control 100%, SeNPs 150%, Ga 200% and Se@Ga 260%), the ROS generation of HepG2 cells increased significantly after treatment with Se@Ga. As shown in figure 8b, the fluorescent intensity of DCF in HepG2 cell exposure to Se@Ga was the most strongest in treatment groups. The results indicate the involvement of ROS in the anti-cancer action of Se@Ga.

Activation of ROS-mediated signalling pathways by Se@Ga
Intracellular ROS overproduction could trigger DNA damage and cause a series of different signalling pathways, such as AKT and MAPK signalling pathways. Western blot analysis was used to examine the effects of Se@Ga on the expression of AKT and p38. As shown in figure 9a,c, the expression of total AKT was downregulated after treatment with Se@Ga (control 100%, SeNPs 61%, Ga 70% and Se@Ga 22%). Meanwhile, as shown in figure 9b,d, HepG2 cells treated with Se@Ga effectively increased the expression of total p38 in HepG2 cells (control 100%, SeNPs 746%, Ga 995% and Se@Ga 1382%). The dates suggest that MAPK pathways were involved in cancer cell apoptosis induced by Se@Ga. The results reveal that nanosystem induces HepG2 cells apoptosis through regulation of ROSmediated AKT and p38 signalling pathways.

Conclusion
Galangin-modified SeNPs were successfully fabricated in the study. Se@Ga enhanced the drug sensitivity and induced apoptosis of cancer cells rather than normal cells. The underlying molecular mechanisms indicated that Se@Ga activated caspase-3-mediated HepG2 cell apoptosis via ROS generation. Furthermore, our results indicated the apoptotic signalling pathway through ROSmediated triggered by the Se@Ga in HepG2 cells, including AKT and p38 signalling pathways. Taken together, for achieving anti-cancer activity, the strategy to use SeNPs as a carrier of Ga could be a highly efficient way. Se@Ga may be a candidate for the next assessment as chemotherapeutics of cancers, especially HCC.
Ethics. This study was conducted in accordance with the Declaration of Helsinki and with approval from the Ethics Committee of Guangzhou Women and Children's Medical Center.
Data accessibility. We have conducted our experiments systematically and reported their experimental procedure clearly in the experimental section and provided all necessary data in the results and discussion section in the main manuscript.