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DOI: 10.1039/C6RA08370F (Communication) RSC Adv., 2016, 6, 84810-84814

Photoluminescence distinction of lung adenocarcinoma cells A549 and squamous cells H520 using metallothionein expression in response to Cd-doped Mn3[Co(CN)6]2 nanocubes†

Jian Chenab, Mengni Hea, Pengping Xua, Huihui Wuc, Zhen Guo*c, Bingyang Shi*b and Qianwang Chen*a
aHefei National Laboratory for Physical Sciences at Microscale, Department of Materials Science & Engineering, CAS High Magnetic Field Laboratory, University of Science and Technology of China, Hefei, 230026, China. E-mail: cqw@ustc.edu.cn; Fax: +86-551-63603005
bSchool of Life Sciences, Henan University, Kaifeng, 475004, China
cAnhui Key Laboratory for Cellular Dynamics and Chemical Biology, School of Life Sciences, University of Science and Technology of China, Hefei, 230027, China

Received 1st April 2016 , Accepted 23rd August 2016

First published on 31st August 2016

Abstract

Luminescent Mn3[Co(CN)6]2 nanocubes doped with Cd2+ can induce human lung adenocarcinoma cells A549 generating more metallothioneins (MTs) than squamous cells H520. The nanocubes can be bound to MTs and be excluded from cells for detoxification. Then A549 cells have a lower fluorescence imaging intensity than H520 cells, which can distinguish the two kinds of cells.

Lung carcinomas are the most frequent malignant tumors and the predominant cause of cancer deaths worldwide.1 According to histological types of the tumors, lung carcinomas can be classified as non-small cell lung carcinomas (NSCLC) and small cell lung carcinomas (SCLC). These two types of cancers can be distinguished decisively based on clinicopathological features. The NSCLC can be further subcategorized as large-cell carcinomas, squamous cell carcinomas and adenocarcinomas.2 Accurately identifying the types of the NSCLC is very important for the prediction of patient outcome, development of rational clinical therapy plan, and selection of proper molecular targets for chemotherapy.3 However, the histopathological subclassification of NSCLC is challenging, especially for the small samples obtained through puncture. And the identification of the types of NSCLC is currently depended on the experience of the doctors. Then, the diagnosis accuracy is unreliable and the patients with NSCLC have low survival rate (8–10%).4 Nowadays, immunohistochemistry has been employed to distinguish lung adenocarcinoma and squamous cell carcinoma.5,6 However, as single immunohistochemical marker doesn't have the absolute specificity and sensitivity, immunoglobulin combination is always needed for the clinical application of immunohistochemistry. Then, applying immunohistochemistry for differentiation of lung adenocarcinoma and squamous is still inaccurate, and the test cost is very high. Thus, development of methods which can aid to distinguish the kinds of NSCLC accurately and easily is very significant.

Metallothioneins (MTs) are cysteine-rich proteins with low molecular weight. Due to the thiol(–SH) groups from cysteine, MTs have specific ability to bind metal ions of group II1,7 (Fig. S1†). Such capacity can protect cells from the toxicity of heavy metals, including Cd2+, Hg2+, Cu2+ and Zn2+.8 The prevailing mechanism for heavy metal resistance is that the metal ions can chelate MTs and the formed metal complexes can be pumped out of the cells.9 MTs expression level has a relationship with the types of cancer cells,7 and it has been reported that MTs expression level in breast adenocarcinomas is statistically significantly high.10

It is shown that distinct categories of cancer cells expressed different level of MTs,7 and Cd2+ can induce the synthesis of MTs in cancer cells,8 we suggest that the phenomenon of different MTs expression level in response to the stimulation of Cd2+ can be employed to distinguish the lung adenocarcinoma cells and squamous cells. However, as the direct detection of MTs is vexing task, methods which are effective to reflect the different expression level of MTs in cancer cells, even though indirect, are worth to be taken into consideration. Fluorescence imaging is a characterization method commonly used in the biomedical field. Thus, cell probes which can integrate the tasks of providing Cd2+ to induce the generation of MTs and displaying fluorescence effect to reflect the different expression level of MTs, are ideal candidates for distinguishing the types of NSCLC.

It is reported that Prussian blue analogue can be doped with positive-valent metal elements into their frameworks,11 and have a good fluorescence performance.12 Herein, Cd-doped Mn3[Co(CN)6]2 nanocubes (Fig. 1) were prepared as probes for distinguishing the type of NSCLC. Fluorescence imaging of human lung adenocarcinoma cells A549 and squamous cells H520 cultured with the nanoprobes was performed to identify the two types of cancers through indirectly reflecting the MTs expression level.


image file: c6ra08370f-f1.tif
Fig. 1 Schematic illustration of the method for distinguishing NSCLC based on Cd-doped Mn3[Co(CN)6]2 nanocubes. When Cd-doped Mn3[Co(CN)6]2 nanocubes were endocytosed by lung adenocarcinoma cells (A549) and squamous cells (H520), the two types of cancer cells will have different MTs expression level in response to the stimulation of Cd2+. The two types of cancer cells will show different luminescence intensity because Cd-doped Mn3[Co(CN)6]2 nanocubes can chelate MTs and the formed adducts can be pumped out of the cells, resulting in different concentrations of Cd-doped Mn3[Co(CN)6]2 nanocubes in the two types of cells.

The incorporation of Cd2+ ions into Mn3[Co(CN)6]2 nanocubes was confirmed by XPS (Fig. 2), in which Cd 3d3/2 peaks at 412.1 eV and Cd 3d5/2 peaks at 405.3 eV were clearly observed. This result indicated that Cd2+ ions were doped in the crystal lattice of Mn3[Co(CN)6]2, which is important for the nanocubes to interact with MTs. In addition, the other characteristic peaks of C 1s, O 1s, Mn 2p, and Co 2p had no obvious change compared with the undoped samples. Given that Cd2+ ions can also be connected with cyanide linkers, forming nanoporous Prussian blue analogues Cd3[Co(CN)6]2,13 we suggested that the doped Cd2+ ions partially replaced the Mn2+ ions in the crystal lattice. This conclusion can be further confirmed by the result of inductively coupled plasma mass spectrometry, which demonstrated that the molar ratio of Mn, Co, and Cd in the Cd-doped Mn3[Co(CN)6]2 nanocubes was about 10.57[thin space (1/6-em)]:[thin space (1/6-em)]7.96[thin space (1/6-em)]:[thin space (1/6-em)]1. XRD patterns (Fig. S2†) and SEM images (Fig. S3†) demonstrated that the doped Cd2+ had no apparent effects on the crystal structure and morphology of Mn3[Co(CN)6]2 nanocubes. In addition, elemental mapping image analysis (Fig. S4†) revealed that the doped Cd2+ has a homogenous distribution. The photoluminescence properties of Cd-doped Mn3[Co(CN)6]2 nanocubes were investigated with the excitation wavelength of 514 nm. Fig. S5† shows a blue emission peak centered at 477 nm. However, pure Cd3[Co(CN)6]2 nanocubes showed no obvious emission peaks under the same test conditions. Thus, the synthesis of Cd-doped Mn3[Co(CN)6]2 nanocubes was badly needed for finding the distinction of lung adenocarcinoma cells and squamous cells.


image file: c6ra08370f-f2.tif
Fig. 2 XPS spectra of Mn3[Co(CN)6]2 and Cd-doped Mn3[Co(CN)6]2 nanocubes.

Given that Cd2+ can induce cancer cells to synthesize MTs,8 the amount of metallothionein in the supernatant of A549 cells and H520 cells (from American Type Culture Collection) cultured with or without Cd-doped Mn3[Co(CN)6]2 nanocubes was analyzed by the Human MT1 (Metallothionein 1) ELISA Kit. As shown in Fig. 3, the amount of MTs in the culture supernatant of H520 cells is higher than that in the culture supernatant of A549 cells. However, with the stimulation of Cd-doped Mn3[Co(CN)6]2 nanocubes, the supernatant of A549 cells showed a significant increment of MTs, the rate of increase can reach 83.70%. On the contrary, the amount of MTs in the supernatant of H520 cells has no obvious change. The rate of increase is only 9.56% under the same culture condition. This result indicated that the basal level of MTs expression in A549 cells was lower than that in H520 cells. But A549 cells were more sensitive to Cd2+ and could generate more MTs than H520 cells in response to the stimulation of Cd-doped Mn3[Co(CN)6]2 nanocubes.


image file: c6ra08370f-f3.tif
Fig. 3 Increment of metallothionein from culture supernatant of A549 cells or H520 cells under the stimulation of Cd-doped Mn3[Co(CN)6]2 nanocubes.

The cytotoxicity of Cd-doped Mn3[Co(CN)6]2 nanocubes against A549 cells or H520 cells was shown in Fig. 4. It can be seen that the cell viability of A549 cells cultured with Cd-doped Mn3[Co(CN)6]2 nanocubes was high. However, H520 cells cultured with the nanocubes displayed low cell viability, which was 20–25% less than A549 cells cultured with the nanocubes at the same concentration. In addition, the cytotoxicity of Mn3[Co(CN)6]2 nanocubes against A549 cells or H520 cells was also tested as a control. The result showed that the two types of cells have similar cell viability. And the cytotoxicity of Mn3[Co(CN)6]2 was low even the concentration used here is 100 μg mL−1. These results indicated that Cd2+ can cause different cell viability between A549 cells and H520 cells due to the different expression of MTs under the stimulation Cd2+. Because Cd2+ can induce A549 cells generating more MTs than H520 cells, and MTs can bind Cd2+ and form complexes for the detoxification of Cd2+,9 A549 cells have a stronger ability to resist the toxicity of Cd2+. Furthermore, Cd-doped Mn3[Co(CN)6]2 nanocubes had lower cytotoxicity against A549 cells than that of Mn3[Co(CN)6]2 nanocubes at the same concentration, which further confirmed that the cell viability has a relationship with the expression of MTs under the stimulation Cd-doped Mn3[Co(CN)6]2 nanocubes.


image file: c6ra08370f-f4.tif
Fig. 4 Cytotoxicity of Mn3[Co(CN)6]2 nanocubes and Cd-doped Mn3[Co(CN)6]2 nanocubes (25, 50, 75, 100 μg mL−1) against A549 cells or H520 cells.

The fluorescence imaging of A549 cells or H520 cells cultured with Cd-doped Mn3[Co(CN)6]2 nanocubes for 24 h was performed and the results were shown in Fig. 5. It can be seen that fluorescence from H520 cells is brighter than that from A549 cells no matter which excitation wavelength was adopted. Table 1 lists the mean luminescence intensity corresponding to the above images to quantitatively determine the imaging differences between A549 cells and H520 cells. The data further showed that H520 cells had stronger luminescence intensity than A549 cells with the same excitation wavelength. These results indicated that the lung adenocarcinoma cells A549 and squamous cells H520 could be distinguished through fluorescence imaging accurately.


image file: c6ra08370f-f5.tif
Fig. 5 Confocal microscopy of bright-field, fluorescence excited with 488 nm and 403 nm laser beams, and two-photon fluorescence excited with 730 nm femtosecond laser pulses images for A549 cells or H520 cells after incubation with 50 μg mL−1 Cd-doped Mn3[Co(CN)6]2 nanocubes for 24 h.
Table 1 Mean luminescence intensity corresponding to the excitation wavelength of 488 nm and 403 nm laser beams, and two-photon fluorescence excited with 730 nm femtosecond laser pulses for A549 cells or H520 cells after incubation with 50 μg mL−1 Cd-doped Mn3[Co(CN)6]2 nanocubes for 24 h
  Mean intensity
Ch1-T1 403 nm Ch2-T1 488 nm Ch1-T3 730 nm
A549 cell 38.40098 31.44917 47.59175
H520 cell 61.09226 34.24072 73.35901

Because the fluorescence is originated from Cd-doped Mn3[Co(CN)6]2 nanocubes,12 the luminescence intensity has a relationship with the concentration of the nanocubes. The stronger luminescent intensity means more nanocubes remain in cells. As Cd2+ has serious cytotoxicity,14 more Cd-doped Mn3[Co(CN)6]2 nanocubes in cells will lead to lower cell viability. On the other hand, Cd-doped Mn3[Co(CN)6]2 nanocubes can chelate MTs and be pumped out of the cells for detoxication. Thus, more MTs generated in cells will reduce the cytotoxicity of Cd-doped Mn3[Co(CN)6]2 nanocubes more greatly. Integrated the results of fluorescence imaging, MTs analysis and cytotoxicity mentioned above, we suggested that Cd2+ could affect the expression level of MTs and cause different amounts of Cd-doped Mn3[Co(CN)6]2 nanocubes to remain in A549 and H520 cells. In addition, we also performed fluorescence imaging for A549 and H520 cells cultured with Cd-doped Mn3[Co(CN)6]2 nanocubes for 6 h and 48 h, respectively. Fig. S6† revealed that after culturing for 6 h, A549 cells displayed higher fluorescence intensity than H520 cells. This result indicated that A549 cells have a higher efficiency of taking up Cd-doped Mn3[Co(CN)6]2 nanocubes than H520 cells. However, with the extension of incubation time, fluorescence intensity from H520 cells became higher than that from A549 cells. And compared with the results of culturing for 24 h, fluorescence intensities from both A549 cells and H520 cells have no significant change after cultured for 48 h. Thus, we suggested that more nanocubes could be excluded from A549 cells than from H520 cells. All of the results confirmed that the different fluorescence intensity between the two types of cancer cells was caused by the different exclusion efficacy of nanocubes from the cancer cells rather than the different rate for the nanocubes entering into the cancer cells.

Furthermore, it can be found that the distinction of blue-fluorescence intensity between A549 cells and H520 cells is more obvious than the corresponding distinction of green-fluorescence intensity (Table 1). Such difference may be caused by the different luminescence mechanism of the nanocubes with different excitation wavelength. It has been reported that there exists carbon cluster in Mn3[Co(CN)6]2 nanocubes. And the form of cluster was described as cobalt atom together with six hexa-coordinated –C[double bond, length as m-dash]N– skeletons (Fig. S7†).15 The emission of blue fluorescence was ascribed to the energy level transition in carbon cluster under the short wavelength excitation, which is similar to the luminescent mechanism of carbon dots. While the emission of green fluorescence was ascribed to the energy level transition from carbon clusters to Mn2+ with a long excitation wavelength.15 Since the MT molecule contained a large number of cysteines, and sulfhydryl in the cysteines is an electrondrawing group, fluorochrome connected with MTs may have extensive fluorescence quenching.16 And because both electron acceptor and electron donor can induce carbon dots fluorescence quenching,17 blue fluorescence from Cd-doped Mn3[Co(CN)6]2 nanocubes will be weakened due to the connection of MTs. In view of this, Cd-doped Mn3[Co(CN)6]2 nanocubes incubated with A549 cells will have a greater degree of blue fluorescence quenching than the nanocubes incubated with H520 cells due to the different amount of MTs generated in the two types of cells under the stimulation of Cd2+. On the other hand, green fluorescence from the nanocubes was not obviously affected by the MTs. Thus, the significant distinction of blue-fluorescence intensity between A549 and H520 cells could be ascribed to two factors: (1) More MTs can be generated in the cytoplasm of A549 cells than in the cytoplasm of H520 cells with the stimulation of Cd-doped Mn3[Co(CN)6]2 nanocubes. The formed MTs can bind with the nanocubes and remove the nanocubes from cells. As a result, the amount of Cd-doped Mn3[Co(CN)6]2 nanocubes remained in A549 cells is lower than that in H520 cells. (2) The amount of generated MTs in A549 cells is higher than that in H520 cells. Then, blue fluorescence from the residual nanocubes in A549 cells will be quenched more significantly than that in H520 cells. Such two factors worked together to weaken the blue fluorescence intensity from A549 cells.

Bio-TEM imaging for ultrathin sections of A549 cells or H520 cells cultured with Cd-doped Mn3[Co(CN)6]2 nanocubes was performed to further identify the amount of nanocubes remained in the cells. As shown in Fig. 6, the quantity of Cd-doped Mn3[Co(CN)6]2 nanocubes in A549 cells was fewer than that in H520 cells. Based on a statistical evaluation of at least 8 cells (Fig. S8 and Table S1†), the average quantity of Cd-doped Mn3[Co(CN)6]2 nanocubes in A549 cells and H520 cells was 6.5 and 10.25 per cell, separately. Interestingly, it can be also found that some nanocubes were located in the frontier of A549 cells and even in the intercellular space (circled with green solid line in Fig. 6a), while such phenomenon didn't appear in H520 cells. These results further confirmed that more Cd-doped Mn3[Co(CN)6]2 nanocubes could be excluded from A549 cells than from H520 cells.


image file: c6ra08370f-f6.tif
Fig. 6 Bio-TEM images of A549 cells (a) and H520 cells (b) uptake Cd-doped Mn3[Co(CN)6]2 nanocubes. The black dots circled with red dashed line indicate the existence of Cd-doped Mn3[Co(CN)6]2 nanocubes in different cancer cells. The black dots circled with green solid line indicate some Cd-doped Mn3[Co(CN)6]2 nanocubes are located in the frontier of A549 cells and even in the intercellular space.

According to our experimental results and the reports about the relationship between MTs and Cd2+,8,9,18 a mechanism of A549 cells and H520 cells in response to Cd-doped Mn3[Co(CN)6]2 nanocubes was schematically illustrated in Fig. 7. When Cd-doped Mn3[Co(CN)6]2 nanocubes enter into cells, MTs in the cytoplasm will have a significant increase due to the stimulation of Cd2+.8 Because MTs expression level among various types of cancer cells is different,7 and the increment of MTs in A549 cells is higher than that in H520 cells, more Cd-doped Mn3[Co(CN)6]2 nanocubes in A549 cells can be bound by MTs and the combined MTs-nanocubes can be excluded from cells for the detoxification. Then the residual amount of Cd-doped Mn3[Co(CN)6]2 nanocubes in A549 cells is less than that of H520 cells. And the amount of MTs remained in A549 cells is higher than in H520 cells, which can induce fluorescence quenching. As a result, the H520 cells cultured with Cd-doped Mn3[Co(CN)6]2 nanocubes have a lower cell viability and brighter fluorescent intensity than the corresponding A549 cells.


image file: c6ra08370f-f7.tif
Fig. 7 Schematic illustration of a mechanism for A549 cells and H520 cells respond differently to Cd-doped Mn3[Co(CN)6]2 nanocubes. When Cd-doped Mn3[Co(CN)6]2 nanocubes were endocytosed by cells, the increment of MTs in A549 cells is higher than that in H520 cells due to the stimulation of Cd2+. Then more Cd-doped Mn3[Co(CN)6]2 nanocubes in A549 cells can be bound by MTs and the combined MTs-nanocubes can be excluded from cells for the detoxification. The residual amount of Cd-doped Mn3[Co(CN)6]2 nanocubes in A549 cells is less than H520 cells. And the amount of MTs remained in A549 cells are higher than that of H520 cells, which can induce fluorescence quenching. Thus, the H520 cells cultured with Cd-doped Mn3[Co(CN)6]2 nanocubes have a lower cell viability and brighter fluorescent intensity than the corresponding A549 cells.

Based on the above conclusion, Cd-doped Mn3[Co(CN)6]2 nanocubes can be used to distinguish lung adenocarcinoma cells A549 and squamous cells H520. To be specific, the two types of cells should firstly be incubated with Cd-doped Mn3[Co(CN)6]2 nanocubes for a period of time. Then, different amount of MTs will be generated in A549 cells and H520 cells under the stimulation of Cd2+. Finally, such difference can be observed through fluorescence imaging indirectly, especially with the short wavelength excitation. And the difference can be further reflected by the quantitative analysis of the fluorescence intensity. Cells with high blue fluorescence intensity can be regarded as A549 cells, and cells with low blue fluorescence intensity can be regarded as H520 cells. As the identification for lung adenocarcinoma cells and squamous cells in clinical is usually achieved through distinguishing the cell morphology, it is over-reliance on individual experience and may result in misdiagnosis. On the contrary, the identification method reported in this paper is simple and more accurate because the different fluorescence intensity can be analyzed quantitatively. However, because lung adenocarcinoma cells and squamous cells have many kinds of cell lines, more study should be performed to explore the response of other NSCLC cell lines caused by Cd2+ and identify whether the method mentioned by us has a generality among all NSCLC cell lines. In addition, an accuracy rule for the distinction of lung adenocarcinoma cells and squamous cells should be concluded. Even so, we have provided interesting glimmers for distinguishing lung adenocarcinoma cells and squamous cells based on nanotechnology, which is meaningful for the clinical treatment of lung cancers.

Acknowledgements

This work was supported by the National Natural Science Foundation of China, 21271163, U1232211 (QW. Chen), 31471268 (Z. Guo), Program for Changjiang Scholars and MOE Innovative Research Team IRT13038 (Z. Guo), Ph.D. Program Foundation of Ministry of Education of China 20113402120041 (Z. Guo).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra08370f
This journal is © The Royal Society of Chemistry 2016
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