2.1. Characterisation of GO
2.1.1. Characterisation of GO flakes
GO, observed with TEM, created single-layered, slightly wrinkled flakes that ranged from 100 nm to 2.9 µm in size (Fig. 1A). GO suspended in ultra-pure water at a concentration of 50 mg/L showed high stability and no tendency for agglomeration. The zeta potential of the GO was -27.5 mV.
Chemical groups present on GO were identified by the Fourier transform infrared (FTIR) method (Fig. 1B). The most characteristic features of the nanomaterial spectra were the intense, broad bands at 3457 cm–1, which correspond to O–H stretching rotations. The O–H stretch together with the shoulder bands located at 1728 cm–1 confirm the presence of at least some oxygen attached to carbon. We observed four characteristic bands generated by C–H stretching at approximately 2959–2642 cm–1. Bands generated by aromatic ring carbon–carbon double bonds were present at 1621 cm–1 in the GO spectra (Thygesen et al. 2003). This location corresponds to the sp2 character of GO (Kurantowicz et al. 2017). The presence of only seven bands in the spectrum of the GO material confirms its simple composition of only three atoms (carbon, oxygen, and hydrogen).
2.1.2. Characterisation of GO nanofilm
GO nanofilm was formed by applying a GO colloid to the bottom of a plastic cell culture plate and drying it. The topography of the surface of the plastic culture plate vs the nanofilm GO coated plate was determined by atomic force microscopy (AFM) (Fig. 2 A,B). It can be seen that the morphology differed significantly between plastic and plastic coated with GO surfaces. The average roughness of the uncoated surface was Ra = 1.5 nm (Fig. 2A) and the roughness of the coated surface was Ra = 9.8 nm (Fig. 2B). Moreover, the maximum height of the roughness of the nfGO coated plate was 67.7 nm. Some dense, transverse inequalities of GO were seen, corresponding to flakes arrangement. This structure was created through self-organisation and generated a structurally varied surface, formed by GO flakes. The average roughness of the surface increased about six times and its greatest height was less than 100 nm, which entitles it to be called a nanofilm.
The hydrophilicity of the surface covered with nanofilm GO vs the polystyrene surface was measured. Generally, the contact surface of a water droplet on the nfGO was higher than on the uncoated plate (Fig. 2C). As shown in Fig. 2D, the area of the water droplet was increased about two times on the modified surface compared to the control; thus, the hydrophilicity of the surface increased after coating with GO.
2.2. Protein composition of the liver extract
1735 proteins were identified in the CELE by mass spectrometry analysis. The exact formulation of these proteins has been registered in PRoteomics IDentifications Database. The functions and scope of activity of the identified proteins were determined on the basis of the UniProt database, and on this basis 57 key proteins were selected that could be involved in adhesion, ECM organisation, migration, epithelial-mesenchymal transition (EMT), proliferation, cell cycle, and apoptosis of cancer cells of the liver (Table 1).
Table 1.
The top 57 proteins from the chicken liver extract divided into functional activities according to UniProt database, including the name of the gene and the molecular mass of the protein (kDa)
No.
|
Gene name
|
Protein name
|
Molecular mass [kDa]
|
ADHESION
|
1
|
TJP2
|
Tight junction protein ZO-2
|
130.7
|
2
|
LARP1
|
La-related protein 1
|
118.6
|
3
|
CDH1
|
Cadherin-1
|
97.8
|
4
|
CD2AP
|
CD2-associated protein
|
71.2
|
5
|
tr|A0A1D5PRE3|A0A1D5PRE3_CHICK
|
N-myc downstream regulated
|
39.0
|
6
|
EPCAM
|
Epithelial cell adhesion molecule
|
34.4
|
7
|
MTDH
|
Metadherin
|
14.2
|
8
|
BSG
|
Basigin
|
Few isoforms
|
ECM ORGANISATION
|
9
|
LAMA1
|
Laminin subunit alpha-1
|
339.1
|
10
|
COL12A1
|
Collagen alpha-1(XII) chain
|
333.5
|
11
|
FN1
|
Fibronectin
|
273.2
|
12
|
COL5A1
|
Collagen alpha-1(V) chain
|
184.2
|
13
|
LAMC1
|
Laminin subunit gamma-1
|
176.5
|
14
|
PEPD
|
Xaa-Pro dipeptidase
|
55.1
|
15
|
VTN
|
Vitronectin
|
51.7
|
16
|
PRDX4
|
Peroxiredoxin-4
|
29.6
|
MIGRATION AND EMT
|
17
|
PTPN23
|
Tyrosine-protein phosphatase non-receptor type 23
|
179.0
|
18
|
FAM98A
|
Protein FAM98A
|
58.6
|
19
|
TWF1
|
Twinfilin-1
|
55.2
|
20
|
MGLL
|
Monoglyceride lipase
|
33.3
|
21
|
TPM1
|
Tropomyosin alpha-1 chain
|
32.8
|
22
|
PBLD
|
Phenazine biosynthesis-like domain-containing protein
|
32.2
|
23
|
ADI1
|
1,2-dihydroxy-3-keto-5-methylthiopentene dioxygenase
|
21.7
|
PROLIFERATION AND CELL CYCLE
|
24
|
GOLGA2
|
Golgin subfamily A member 2
|
140.1
|
25
|
MOV10
|
Putative helicase MOV-10
|
109.1
|
26
|
ASMTL
|
Probable bifunctional dTTP/UTP pyrophosphatase/methyltransferase protein
|
70.2
|
27
|
RPA1
|
Replication protein A 70 kDa DNA-binding subunit
|
68.0
|
28
|
ARID3A
|
AT-rich interactive domain-containing protein 3A
|
67.4
|
29
|
RIC8B
|
Guanine Nucleotide Exchange Factor B
|
60.5
|
30
|
CERS2
|
Ceramide synthase 2
|
55.1
|
31
|
KRT18
|
Keratin, type I cytoskeletal 18
|
46.9
|
32
|
MRPS27
|
28S ribosomal protein S27, mitochondrial
|
46.7
|
33
|
TARDBP
|
DNA-binding protein 43
|
44.6
|
34
|
CA9
|
Carbonic anhydrase 9
|
43.8
|
35
|
DRG1
|
Developmentally regulated GTP-binding protein 1
|
40.5
|
36
|
SLC35F6
|
Solute carrier family 35 member F6
|
40.2
|
37
|
AIMP2
|
Aminoacyl tRNA synthase complex-interacting multifunctional protein 2
|
34.5
|
38
|
FBXO6
|
F-box only protein 6
|
30.7
|
39
|
HPGD
|
15-hydroxyprostaglandin dehydrogenase [NAD(+)]
|
29.0
|
40
|
PSMD9
|
26S proteasome non-ATPase regulatory subunit 9
|
22.7
|
41
|
NIT2
|
Nitrilase NIT2
|
22.1
|
42
|
DYNLL1
|
Dynein light chain
|
10.3
|
APOPTOSIS
|
43
|
HTT
|
Huntingtin
|
344.0
|
44
|
PARP1
|
Poly [ADP-ribose] polymerase
|
113.1
|
45
|
DNMT3A
|
DNA (cytosine-5)-methyltransferase 3A
|
99.0
|
46
|
ATAD3A
|
ATPase family AAA domain-containing protein 3A
|
67.2
|
47
|
SGPL1
|
Sphingosine-1-phosphate lyase 1
|
61.1
|
48
|
CTSC
|
Dipeptidyl peptidase 1
|
59.4
|
49
|
FKBP8
|
Peptidylprolyl isomerase
|
44.3
|
50
|
AIFM2
|
Apoptosis-inducing factor 2
|
40.6
|
51
|
APIP
|
Methylthioribulose-1-phosphate dehydratase
|
26.8
|
52
|
PDCD10
|
Programmed cell death protein 10
|
24.7
|
53
|
ENDOG
|
Endonuclease G, mitochondrial
|
22.2
|
54
|
FAM162A
|
Protein FAM162A
|
17.1
|
55
|
HIGD1A
|
HIG1 domain family member 1A, mitochondrial
|
10.7
|
56
|
DYNLL1
|
Dynein light chain
|
10.3
|
57
|
NQO1
|
NAD(P)H dehydrogenase [quinone] 1
|
Few isoforms
|
2.3. Influence of nfGO and CELE on cell morphology
The morphological picture of non-tumour HS-5 cells after 48h of incubation on GO nanofilm, with the addition of CELE and using both factors, did not indicate pathological changes, however, some differences could be found (Fig. 3). Cells growing on the nfGO were extended and possessed large cell bodies, long protrusions, and distinct lamellipodia. Cell clusters and high cell-cell adhesion were observed on the nfGO (Fig. 3B). The CELE induced severe morphological changes in HS-5 cells, such as elongation of filopodia, reduction of cell bodies (shrunken forms), and formation of small clusters (pieces of extract) (Fig. 3C). In the nfGO + CELE group, the HS-5 cell size was reduced and no tendency of cells to form clusters was observed (Fig. 3D).
HepG2 and C3A liver tumour cells form natural clusters as observed in the experiment with the control groups (Fig 4A, Fig 5A). After 48h of culture, HepG2 cancer cells were also not pathologically altered due to surface modification with GO nanofilm and CELE addition. Moreover, none of the experimental factors significantly changed their morphology (Fig. 4). Although it was observed that the cells were willingly located on the GO coated surface, they appeared to be more shrunken. The CELE additive to the cells caused some loosening of cell clusters. In the nfGO + CELE group there were more cells not associated in clusters but migrating outside the clusters.
C3A cells also did not undergo pathological changes under the influence of experimental factors (Fig. 5). C3A cells were visible on the GO surface as well as outside it. Under the influence of CELE addition, a number of cells could be seen separated from clusters. In the nfGO + CELE group, a greater number of individual cells that migrated outside of the clusters was observed.
The morphology of the cells was also observed using scanning electron microscopy (SEM). Cells were grown on plates, which were only partially covered with GO nanofilm. This enabled cells to be visualised on the GO surface and, above all, on the border of the GO surface and the surface of the plastic dish. The image of the cells after 7 days of culture allowed an assessment of their preferences for their placement on nfGO or outside of nfGO.
HS-5 cells were evenly located both on nfGO and outside nfGO as well as on the plastic/nfGO border. Cell settlement topography did not reflect the plastic/nfGO border line, although cells on nfGO had slightly longer protrusions and were more elongated (Fig. 6).
Liver cancer cells exhibit different culture behaviour, forming characteristic clusters that have been observed with HepG2. Cluster formation was observed both on the plastic surface of the culture vessel as well as on nfGO and at the plastic/nfGO border. Observation of the image of cells colonising on nfGO showed that the cells formed looser clusters with single cells more visible. The tendencies to create groups (clusters) were clearly smaller, and the adherence of individual cells to the GO substrate seemed larger (Fig. 7).
C3A cells, like HepG2, formed clusters, colonising both the GO surface and the plastic/nfGO border. In the nfGO group, slightly more single cells not bound into clusters were also observed (Fig. 8).
2.4. Influence of nfGO and CELE on cell proliferation
Cell proliferation was measured by using the test based on the measurement of BrdU incorporation during DNA synthesis (Fig. 9). The experimental factors did not affect the proliferation of non-tumour HS-5 cells. In contrast with HS-5 cells, nfGO, CELE, and to the greatest extent the combined factors (nfGO + CELE) reduced the proliferation of HepG2 cells. However, none of the experimental factors significantly influenced the proliferation of C3A cells.
2.4.1. Influence of nfGO and CELE on proliferation-associated gene expression at the mRNA level
In order to clarify the molecular basis of proliferation under the influence of the signal induced by the GO nanofilm and the addition of CELE, the mRNA expression of the pcna, ki67, and mcm2 genes was studied (Fig. 10).
The cultivation of HS-5 cells on nfGO as well as on nfGO with CELE addition resulted in a decrease in pcna expression, an increase in ki67 expression, and a tendency towards an increase in mcm2 expression. However, the addition of CELE to the medium did not change the expression of the mitotic index markers.
HepG2 tumour cells showed a slightly different response to the factors tested. Surface modification by GO nanofilm was the reason for the increased expression of ki67 and mcm2. This was in contrast to the CELE, which reduced the expression of all genes (pcna, ki67 and mcm2). The effect of the GO surface was to reduce the CELE interaction on pcna expression. Moreover, the GO nanofilm eliminated the effects of CELE on ki67, and the combined use of these factors was the cause of overexpression of the ki67 gene. A similar picture was observed in the case of mcm2, where the CELE effect was levelled by the surface treatment.
The influence of experimental factors on C3A cells was smaller. Some overexpression of the ki67 and mcm2 genes was observed under the influence of the GO nanofilm as well as the GO nanofilm together with the CELE (Table S3).
2.5. The cell-ECM and cell-cell connections
2.5.1. Integrin expression profile
The modification of the culture plate surface by using GO nanofilm increased the expression of α1, α2, α3, α5, α6, and β4 and decreased the expression of β1 integrins in non-cancer cells HS-5 (Fig. 11, Table S1). In turn, the addition of CELE elevated the expression of integrin αV and β1 and reduced the α2, α3, and β4 integrin mRNA level. The use of both factors (nfGO + CELE) caused overexpression of α1, α3, α5, and especially α4, αV, and β1 in HS-5 cells.
Integrin expression in HepG2 cancer cells was completely different. A reduction in the α3 and α5 mRNA level was found, while an increase in α1, α6, αV, and β4 was influenced by the use of GO surfaces. The opposite effect was observed under the influence of CELE for integrins α1, α5, α6, and αV. The use of both factors was clearly the cause of integrin overexpression α2, α5, αV, and β4 in HepG2 cells (Table S2).
A different effect was observed in C3A tumour cells than in HepG2. First of all, no factor influenced downregulation of integrin expression. The surface of the nanofilm induced the integrin expression α1, α2, α3, α6, αV, β1, and β4, while CELE induced an increase in α1, α2, α3, α6, αV, and β1 integrins (Table S3). Interestingly, the combined use of both factors (nfGO + CELE) resulted in a decrease in the expression of most integrins compared to their use separately.
Thus, mutual enhancement of the surface effect and addition of CELE to the culture medium was observed in non-tumour cells for α4 and αV integrins and above all in HepG2 tumour cells for α2, α5, αV, and β4 integrins.
2.5.2. Focal adhesion kinase, cadherins and β-catenin
Gene expression for key proteins involved in the integration of mechano- and chemo-signalling between ECM-cell and cell-cell, as fak, e-cadherin, n-cadherin, and β-catenin are presented in Fig. 12. By analysing gene expression in HS-5 cells, it was found that the surface modification by GO only significantly increased the expression of n-cadherin, while the CELE did not affect the results. The combined use of nfGO + CELE resulted in significant overexpression of all adhesion markers (Table S1).
In the HepG2 liver cancer line, the use of GO nanofilm resulted in a significant increase in the expression of genes responsible for cell-ECM adhesion (fak) as well as cell-cell adhesion (cadherins). Moreover, nfGO decreased the expression of β-catenin. Unlike other genes, the expression of β-catenin was also downregulated by CELE. The use of both factors (nfGO + CELE) clearly increased the expression of fak, e-cadherin, and n-cadherin and reduced the expression of β-catenin (Table S2).
C3A cells cultured on nfGO showed higher expression of fak compared to the control group. Furthermore, we found that the CELE supplementation increased n-cadherin expression. Nanofilm GO with additive CELE increased n-cadherin and β-catenin expression of C3A cells (Table S3).
2.6. Effect on cell cycle
To investigate whether the mechanical signal from nfGO and a molecular signal from a CELE influence cell cycle, flow cytometric analysis was performed (Fig. 13). HS-5 cells cultured on GO nanofilm as well as cultured with additives of CELE but also treated nfGO + CELE for 7 days showed a decrease in the population of cells in S phase with a concomitant slight increase in the G0/G1 phase. Simultaneously, a reduction in the cell population in the G2/M phase was observed under the influence of CELE and nfGO + CELE.
In HepG2 cells, in contrast to non-tumour cells, a decrease in the G0/G1 phase population, slight increase the S population, and an increase in the G2/M phase population under the influence of nfGO + CELE were observed.
Observation of C3A cells showed no effect of the GO surface on the cell cycle, although the CELE and nfGO + CELE slightly increased the population of cells in the S phase and decreased the population of cells in the G2/M phase.