Embed Size (px)
Citation preview
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
ONLINEFIRST
ThisisaprovisionalPDFonly.Copyeditedandfullyformattedversiónwillbemadeavailableatfinalpublication
Thisarticlehasbeenpeerreviewedandpublishedimmdediatelyuponacceptance.Articlesin“HistologyandHistopathology”arelistedinPubmed.
Pre-printauthor´sversion
ISSN:0213-3911e-ISSN:1699-5848
SubmityourarticletothisJournal(http://www.hh.um.es/Instructions.htm)
Stigmasterolinhibitstheprogressionoflungcancerbyregulatingretinoicacid-relatedorphanreceptorC
Authors: Yuan Dong, Congcong Chen, Chen Chen, Chunxia Zhang, Lei Zhang, YanZhang,YongliangLiandZhiqiangDongDOI:10.14670/HH-18-388Articletype:ORIGINALARTICLEAccepted:2021-10-29Epubaheadofprint:2021-10-29
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
1
Stigmasterol inhibits the progression of lung cancer by regulating retinoic acid-1
related orphan receptor C 2
3
Yuan Dong, Congcong Chen, Chen Chen, Chunxia Zhang, Lei Zhang, Yan Zhang, 4
Yongliang Li, Zhiqiang Dong * 5
6
Department of Pharmacy, The First Affiliated Hospital of Baotou Medical College of 7
Inner Mongolia Scientific and Technological University, Baotou city, Inner Mongolia 8
autonomous, China. 9
10
* Correspondence to: Dr Zhiqiang Dong, Department of Pharmacy, The First 11
Affiliated Hospital of Baotou Medical College of Inner Mongolia Scientific and 12
Technological University, No. 41, Linyin Road, Kundulun District, Baotou city, Inner 13
Mongolia autonomous, China. E-mail: [email protected]. 14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
2
Abstract 31
Objective: This study aims to investigate the role of stigmasterol in lung cancer. The 32
study aims to investigate the role of stigmasterol in lung cancer and further explore its 33
possible mechanisms. 34
35
Methods: Cell Counting Kit-8 assay, 5-ethynyl-2-deoxyuridine (EdU), TUNEL and 36
Flow cytometry were conducted to detect the proliferation and apoptosis of lung 37
cancer cell lines. qRT-PCR and western blot were conducted to detect mRNA and 38
protein levels of caspase-3 and caspase-9. In addition, Gene Ontology, STRING, 39
SWISSMODEL, cellular thermal shift assay (CETSA) and Swiss Target Prediction 40
were used to predict the targets of stigmasterol. 41
42
Results: Behavioral studies showed that stigmasterol inhibited the proliferation and 43
promoted the apoptosis of lung cancer cells. Further research revealed that retinoic 44
acid-related orphan receptor C (RORC) directly targeted stigmasterol in lung cancer. 45
Interestingly, rescue experiments indicated that RORC overexpression reversed the 46
inhibitory effect of stigmasterol on lung cancer. 47
48
Conclusion: In our study, we confirmed the functional role of the stigmasterol-RORC 49
axis in lung cancer progression, which provides a latent target for lung cancer 50
treatment. 51
52
Key Words: Lung cancer; Stigmasterol; Retinoic acid-related orphan receptor C; 53
Network pharmacology; Caspase; 54
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
3
Introduction 55
Lung cancer occurs in the epithelium of the bronchial mucosa, and the incidence of 56
lung cancer has increased significantly in the past 50 years (Mao et al., 2019). 57
According to the latest data released by the World Health Organization, the number of 58
new lung cancers worldwide accounts for approximately 13.0% of all tumor 59
incidence, and lung cancer deaths account for approximately 19.4% of all tumor 60
mortality (Cheng et al., 2016; Didkowska et al., 2016). In developed countries in 61
Europe and the United States and large cities in our country, the incidence of lung 62
cancer has ranked first among various tumors in men, and the incidence in women has 63
also increased rapidly, accounting for the second or third most common malignant 64
tumors in women. Thus, it has become a significant disease endangering life and 65
health (Hsu et al., 2011). The lack of appropriate and reliable biomarkers and 66
therapeutic targets, the late diagnosis time and the low drug efficiency have formed a 67
bottleneck in lung cancer treatment (Miller et al., 2016). In addition, the development 68
of chemotherapeutic resistance in tumor cells has made its treatment more difficult 69
(Jemal et al., 2011). 70
Phytosterols are plant lipids with chemical structures similar to cholesterol and have 71
been attributed various biological effects, such as anti-cancer, anti-pyretic, anti-72
inflammatory, and immune-modulating effects (Haque and Moon, 2018). 73
Stigmasterol, steroid alcohol, one of such phytosterols found in a number of medicinal 74
plants, vegetables and nuts, has been shown to have a variety of biological functions, 75
either alone or as a component of phytosterol mixtures (Chen et al., 2012; Antwi et 76
al., 2018). For example, Cabral et al. reported that regular consumption of 77
stigmasterol effectively reduces cholesterol density in the blood and reduces the 78
incidence of cardiovascular disease (Cabral and Klein, 2017). Moon et al. revealed 79
that stigmasterol up-regulates immediate early genes and promotes neuronal 80
cytoarchitecture in primary hippocampal neurons (Haque and Moon, 2018). Tantratian 81
et al. proved that stigmasterol provided significant protection to the yeast cell when 82
subjected to slow freezing. 83
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
4
Furthermore, it increased the survival rate of the culture subjected to subzero 84
temperature storage (Tantratian et al., 2019). Besides, the data also showed that 85
stigmasterol inhibited the occurrence and development of a variety of cancers. For 86
example, Kim et al. demonstrate that stigmasterol effectively induces apoptosis of 87
HepG2 cells, thereby inhibiting HCC progression (Kim et al., 2014). In addition, 88
Thaned et al. show that stigmasterol exerts anti-cancer effects by inhibiting tumor 89
angiogenesis in mice and inhibiting the growth of cholangiocarcinoma 90
(Kangsamaksin et al., 2017). However, the anti-cancer effect of stigmasterol on lung 91
cancer had not been studied, and the underlying mechanism of its anti-cancer activity 92
remained largely unknown. 93
The current study aimed to investigate the impacts of stigmasterol on lung cancer, 94
followed by an investigation of the underlying mechanism. As a result, we discovered 95
the idea that stigmasterol can be deemed as a potential agent for the management of 96
lung cancer via modulating retinoic acid-related orphan receptor C (RORC). 97
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
5
Methods 98
99
Identification of active ingredients 100
Swiss Target Prediction (http://www.swisstargetprediction.ch/) was used to predict 101
and screen the targets of stigmasterol for lung cancer. 102
103
Chemical structure analysis 104
The chemistry of stigmasterol, including molecular formula, 2D and 3D structural 105
information, were searched, collected and confirmed with PubChem (https:// 106
pubchem.ncbi.nlm.nih.gov/). 107
108
Chemicals 109
Stigmasterol (95% purity) was procured from Sigma-Aldrich Ltd (S2424, St. Louis, 110
Missouri, USA). In analyzing the cytotoxic effect of stigmasterol, we treated cells 111
with three different concentrations of stigmasterol (2.5 µ mol / L, 5 µ mol / L, 10 µ 112
mol / L, 20 µ mol / L, and 40 µ mol / L). In experiments investigating the effect of 113
stigmasterol on lung cancer progression, we treated lung cancer cells with three 114
different concentrations of stigmasterol (5 µg/mL, 10 µg/mL and 20 µg/mL). In the 115
experiment to clarify the regulatory mechanism of stigmasterol in lung cancer, the 116
dose of stigmasterol we used was 20 µg/mL. 117
118
Cell culture 119
Lung cancer cells (PLA-801D, A-549, H661 and SK-SEM-1) and normal lung 120
bronchial epithelial cell line (BEAS-2B) were obtained from American Type Culture 121
Collection (ATCC, Manassas, VA, USA). After all the cells were resuscitated, they 122
were cultured in RPMI-1640 medium containing 10% FBS in a humidified 123
environment, and the medium was changed every other day. When cells reached 70% 124
to 80% confluence, transfection experiments were conducted. 125
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
6
126
Cell Counting Kit-8 assay 127
In short, the cells adjusted to the appropriate concentration (5×103cells/well) were 128
inoculated on 96-well plates and treated accordingly (5µg/mL, 10µg/mL and 129
20µg/mL stigmasterol and 40 µM cisplatin). Then, each well was added with CCK-8 130
solution and incubated for 2 h in the dark. Finally, the optical density at 450 nm was 131
measured. 132
133
5-ethynyl-2-deoxyuridine (EdU) assay 134
Briefly, PLA-801D and A-549 cells were inoculated in 96-well plates for 48 h. Then, 135
washed with PBS (Beyotime, Beijing, China), they were incubated with 10 µM EdU 136
(Beyotime, Beijing, China) for 2 h at 37�. EdU-positive cells were detected by 137
Apollo staining and DAPI staining, and the percentage of positive cells was defined as 138
proliferation rate. 139
140
TUNEL assay 141
TUNEL detection kit (Beyotime, Beijing, China) was used to detect the apoptosis of 142
PLA-801D and A-549 cells. First, lung cancer cells were fixed with 4% formaldehyde 143
and then washed in PBS containing proteinase K (20 µg/mL) at 37°C. Afterward, 144
PLA-801D and A-549 cells were incubated overnight with the two (1 and 2, 1:10) 145
TUNEL reagents. Finally, TUNEL staining was observed under an optical microscope 146
(Olympus, Tokyo, Japan). 147
148
Flow cytometry 149
PLA-801D and A-549 cells were harvested and washed with ice-cold PBS. After re-150
suspension, Annexin V (1 µg/ml; Invitrogen) was added to cells and incubated in the 151
dark for 20 min. Following, propidium iodide (PI; 1 µg/ml) was then added. The 152
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
7
apoptosis rate of PLA-801D and A-549 cells was determined by the FACScan flow 153
cytometer (BD Biosciences, CA, USA). All experiments in the present study were 154
performed in triplicate. 155
156
Cell transfection 157
pc-DNA-NC and pc-DNA-RORC were constructed by Ribobio corporation 158
(Guangzhou, China) and the plasmid usage per transfection was 100 ng. After 6 h of 159
incubation, a fresh culture medium was added to the PLA-801D and A-549 cells with 160
a confluence rate of 70%. After 48 h, the transfected cells were collected for further 161
experiments. 162
163
Quantitative real-time PCR 164
TRIpure reagent (Invitrogen, USA) was used to extract the total RNA from PLA-165
801D, and A-549 cells and PrimeScript RT kit (TaKaRa, Otsu, Japan) was used for 166
reverse transcription. After the sample was prepared, the expression level was 167
detected with SYBR green, and β-actin was controlled as an internal parameter. The 168
primer sequences for this experiment are shown below: caspase-3 sense, 169
TTAATAAAGGTATCCATGGAGAACACT and antisense, 170
TTAATAAAGGTATCCATGGAGAACACT; caspase-9 sense, 171
GCTCTTCCTTTGTTCATCTCC and antisense CATCGGCTCGGGGTTACTGC; β-172
actin sense, TCACCAACTGGGACGACATG and antisense, 173
GTCACCGGAGTCCATCACGAT. 174
175
Western blot assay 176
According to the manufacture’s instructions, the proteins were extracted, and their 177
concentration was measured. Subsequently, the prepared protein was separated by 178
polyacrylamide-SDS gels and then transferred onto PVDF membranes (Roche, 179
Switzerland). Blocked with 5% skimmed milk for 2.5 h, the PVDF membrane was 180
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
8
subjected to incubation with primary antibodies against caspase-3, caspase-9 RORC 181
and β-actin (1:1000) Proteintech Group Inc., Wuhan, China) at 4°C overnight. On the 182
following day, protein samples were incubated with the secondary antibody at 37°C 183
for 45 min, and the intensity of protein expression was detected by ECL 184
chemiluminescence. The levels of proteins were quantified using Quantity One 185
software (Bio-Rad, Hercules, CA, USA). 186
187
Prediction of putative targets of stigmasterol 188
To identify the potential targets of stigmasterol, the functional annotation including 189
Gene Ontology (GO) enrichment analysis for biological process (BP) and molecular 190
function (MF), and STRING database (https://string-db.org/cgi/input.pl) containing 191
Protein-protein interactions (PPI) data were used. 192
193
Three-dimensional modeling of RORC 194
First, the amino acid sequence of RORC was obtained with the NCBI BLAST search 195
program. Then, the amino acid sequence was input into SWISSMODEL to construct 196
the 3D models of RORC by the SWISSMODEL Protein Modelling Server. In 197
addition, the sequence was also input into Protein Data Bank (PDB) database to query 198
the tertiary structure of RORC protein. The final RORC model was built by 199
combining PDB and SWISSMODEL through the homology modeling method and the 200
primary structure of amino acids. Finally, the best model was selected according to 201
the score. 202
203
Docking studies 204
Docking studies were performed by MGL tools (version 1.5.6) and Autodock 4.2. 205
First, MGL tools were used to convert the file type, and Pymol was used to preprocess 206
the protein model file. Then Autodock was conducted to calculate the possibility of 207
binding between stigmasterol molecules and RORC protein molecules and concluded 208
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
9
that there were 10 possible binding modes. Finally, all 10 configurations of the 209
receptor and ligand complexes were analyzed by Software company Accelrys, with 210
each color bar representing a binding pattern of the chemical molecules. 211
212
Cellular thermal shift assay (CETSA) 213
Cells treated with stigmasterol or DMSO at 37°C for 24 h were collected, and the cell 214
suspension was distributed into 0.2 ml PCR tubes, with 200 µl cell suspension in each 215
tube. The PCR tubes were heated at the designated temperature (40, 60, 64, 67, 70, 72 216
and 75°C) for 3 min. They were then removed and incubated at 4°C immediately after 217
the heating. Cells were then lysed using cell lysis buffer for western (Beyotime 218
Institute of Biotechnology) and analyzed by western blotting as described in the 219
western blotting methods above. 220
221
Prediction of RORC expression in lung cancer 222
We searched the RNA sequencing data of the tumor genome Altas (TCGA) project 223
and used the RORC transcript per million (TPM) value as the gene expression level. 224
Also, the expression levels of the RORC gene at different stages of lung cancer were 225
analyzed using TCGA data. Online KM plotter software (http://kmplot.com/analysis/) 226
was used to generate the Kaplan-Meier Plot to study the potential effect of RORC on 227
the overall survival (OS). 228
229
Statistical analysis 230
All the data were analyzed by Statistical Package for Social Sciences19.0 (SPSS, 231
Chicago, IL, USA) and presented as mean ± standard deviation (SD). The comparison 232
of proliferation, apoptosis and gene differential expression changes among the groups 233
was analyzed by one One-way ANOVA followed by Dunnett’s multiple comparisons. 234
In addition, the overall survival under different RORC expressions was evaluated by 235
the Kaplan-Meier method. P<0.05 was considered a significant difference between 236
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
10
groups. 237
238
Results 239
240
Target and chemical structure of stigmasterol on lung cancer 241
To investigate whether stigmasterol acts on lung cancer, we conducted a Swiss Target 242
Prediction analysis. In detail, Swiss Target Prediction predicted 19 targets related to 243
lung cancer, speculating that stigmasterol might act on lung cancer (Fig. 1A). The 244
molecular structure of stigmasterol is shown in Fig. 1B. 245
246
Stigmasterol inhibits proliferation and promotes apoptosis of lung cancer cells 247
To further study the role of stigmasterol on lung cancer progression, we examined the 248
effects of stigmasterol on the proliferative activity and apoptotic ability of PLA-801D 249
and A-549 cells. As shown by the results of CCK-8, stigmasterol significantly 250
inhibited the activity of lung cancer cells, like the traditional anti-cancer drug 251
cisplatin, and the cytotoxicity of stigmasterol presents in a dose-dependent manner 252
(Fig. 2A, all P˂0.05). Of note, stigmasterol exerted less toxicity on the SK-SEM-1 253
cell and normal BEAS-2B cell (Fig. 3). Consistently, EdU analysis also showed that 254
stigmasterol inhibited lung cancer progression (Fig. 2B, all P˂0.05). Furthermore, 255
TUNEL assay and flow cytometry results illustrated that compared with the control 256
group, both stigmasterol (5 µg/mL, 10 µg/mL and 20 µg/mL) and cisplatin induced 257
apoptosis significantly, and their apoptosis rates were 15%, 20%, 25% and 30%, 258
respectively (Fig. 4,B, P˂0.01). 259
As is known, caspase-3 and caspase-9 are both apoptosis-related genes. Therefore, to 260
test the biological effects of the addition of stigmasterol on these factors, qRT-PCR 261
and western bolt were performed. As shown in Fig. 4C, the transcriptional levels of 262
caspase-3 and caspase-9 increased remarkably after the treatment with stigmasterol (5 263
µg/mL, 10 µg/mL and 20 µg/mL) compared to the control group. Interestingly, at the 264
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
11
protein level, we observed the same expression pattern (Fig. 4D). 265
266
RORC is the target of stigmasterol on lung cancer 267
To clarify the regulatory mechanism of stigmasterol in lung cancer, we conducted a 268
GO and STRING bioinformatics analysis. BP and MF analyses revealed that RORC 269
was primarily associated with lipid binding, oxysterol binding, steroid binding and 270
responded to oxygen-containing compounds (Fig. 5A). Fig. 5B is the PPI network 271
constructed by STRING. Each solid circle represents a target protein, and the center 272
of the point represents the protein structure. In the PPI network, the link of each node 273
represents the homology of proteins, the co-expression of genes and the coevolution 274
of genes. Based on the degree value of algorithm STRING, we deduced that RORC 275
might be the critical anti-cancer target of stigmasterol, and its 3D structure is 276
presented in Fig. 5C. In addition, we used Autodock to calculate the possibility of 277
binding between stigmasterol molecules and RORC protein molecules, and the results 278
showed that there might be 10 binding modes for the two. Fig. 5D shows partial 279
molecular binding patterns, with each color bar representing a binding pattern of 280
chemical molecules. 281
Further, CETSA results confirmed that stigmasterol can bind to RORC and promote 282
the degradation of RORC protein. As shown in Fig. 6, under the same temperature 283
conditions, the stigmasterol group was more likely to promote RORC degradation 284
than the control group. In addition, under the same temperature conditions, RORC 285
degradation was accelerated with increased stigmasterol concentration. 286
Next, we analyzed the relationship between RORC and lung cancer. The Cancer 287
Genome Altas showed that in lung cancer RORC was highly-expressed compared 288
with normal tissues (P˂0.05). Survival analysis showed that lung cancer samples with 289
high RORC expression had poor overall survival. In addition, RORC expression 290
levels were detected at different tumor stages, and no significant statistical differences 291
were found at each stage (Fig. 5E). 292
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
12
293
RORC overexpression reverses the inhibitory effect of stigmasterol on lung cancer 294
To further explore the relationship between stigmasterol and RORC in lung cancer 295
progression, we first constructed the RORC overexpression vector. Next, PLA-801D 296
and A-549 cells were co-transfected with pc-DNA-NC or pc-DNA-RORC to detect 297
the effects on proliferation and apoptosis. As exhibited in Fig. 7A, compared with 298
transfection pc-DNA-NC, transfection pc-DNA-RORC partially offset the toxic effect 299
of stigmasterol on PLA-801D and A-549 cells (P˂0.01). Consistently, EdU results 300
showed that the number of cell clones in group stigmasterol + pc-DNA-RORC also 301
increased relatively (Fig. 7B, P˂0.05). In the verification of apoptosis experiments, 302
the flow cytometry results showed that the Annexin V positive cells of group 303
stigmasterol + pc-DNA-RORC decreased, and the results of TUNEL results showed 304
that the fluorescence intensity of group stigmasterol + pc-DNA-RORC was weaker 305
than that of group stigmasterol + pc-DNA-NC (Fig. 8A,B, P˂0.05). Furthermore, the 306
same expression pattern was observed at both caspase-3 and caspase-9 transcriptional 307
and protein levels (Fig. 8A,B, P˂0.05). 308
309
Discussion 310
Stigmasterol, a kind of phytosterol, has been reported to have many physiological 311
functions (Newill et al., 2007; Miras-Moreno et al., 2016). Recently, multitudes of 312
data have shown that stigmasterol has a practical anti-cancer effect on a variety of 313
cancers. For example, stigmasterol inhibits skin cancer by increasing lipid peroxide 314
levels and causing DNA damage (Ali et al., 2015) and inhibits the occurrence of 315
cholangiocarcinoma by down-regulating TNF-A and VEGF2 (Kangsamaksin, 2017). 316
Additionally, stigmasterol inhibits prostate cancer by inducing the expression of p53 317
protein and simultaneously inhibiting the expression of p21 and P27 proteins 318
(Scholtysek et al., 2009). In gastric cancer, stigmasterol has been shown to have a 319
substantial anti-tumor effect by inhibiting cell migration, cell cycle arrest, 320
mitochondria-mediated apoptosis, and inhibiting the JAK/STAT signaling pathway 321
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
13
(Li et al., 2018). In this study, the anti-lung cancer effects of stigmasterol were 322
determined by CCK-8, EdU, flow cytometry, and TUNEL. It was confirmed that 323
stigmasterol inhibited the proliferation activity and promoted the apoptosis of lung 324
cancer cell lines, which was consistent with the previous results. Caspases are 325
activated in the process of apoptosis in many cells and play a vital role in the initiation 326
and execution of apoptosis. Among them, caspase-3 and -9 are two essential proteins 327
involved in apoptosis (Liu et al., 2009; Hsu, 2011). A previous study has shown that 328
stigmasterol promotes the mitochondrial apoptotic signaling pathways via up-329
regulating caspase-9 and-3 (Kim, 2014). Similarly, in the presented study, we 330
revealed that stigmasterol elevated the activity of apoptotic proteins, including 331
cleaved caspase-3 and cleaved caspase-9. 332
Go can define and describe the function of genes and proteins. It involves three kinds 333
of functional information: gene biological process, cell composition and molecular 334
function. The functional concepts of different concepts are organized into the 335
structure of DAG (Wang et al., 2020). STRING is an online database for searching 336
known protein interactions. It can be used to filter and evaluate functional genomics 337
data and provide a relatively intuitive platform for annotating the structure, function, 338
and evolution of proteins (Chandran and Patwardhan, 2017). To understand the anti-339
cancer effects of stigmasterol, we used the above two databases to construct a 340
compound-target network related to stigmasterol’s anti-cancer effects. GO enrichment 341
analysis and STRING analysis showed that RORC might be the direct target of 342
stigmasterol influencing lung cancer progression. CETSA is one of the drug target 343
screening methods. It directly monitors the interaction between the target protein and 344
the drug in the cell. The change in the protein’s thermal stability caused by the drug-345
binding protein is used to study the drug’s mechanism of action. Ligand-induced 346
stability can be assessed by the remaining level of soluble protein (Zhang et al., 2020). 347
In the current study, CETSA results indicated that stigmasterol was able to bind to 348
RORC and promote the degradation of RORC protein. To further validate this 349
prediction, we conducted RORC expression detection and survival analysis in lung 350
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
14
cancer patients. As expected, RORC was highly expressed in lung cancer patients, 351
and survival rates were relatively lower compared with patients with low-expression 352
of RORC. In addition, RORC expression varied in different lung cancer stages. Taken 353
together, all the above data indicated that RORC was the downstream target of 354
stigmasterol involvement in lung cancer. 355
Retinoic acid-related orphan receptor C (RORC), a transcription factor that binds to 356
DNA, is a family of receptors for nuclear orphans(Alhassan Mohammed et al., 2018). 357
It has attracted much attention because it plays a critical regulatory role in the cell 358
proliferation, metastasis and chemoresistance of various types of malignant tumors. 359
Muscat et al. (Oh et al., 2014) demonstrated that the expression of RORC is 360
significantly reduced in invasive basal-like breast cancer, and there is a negative 361
correlation with the histological grade of the human breast cancer cohorts. Brozyna et 362
al. expound that in melanoma, RORC expression levels are relatively lower than in 363
normal tissue, and there is a downward trend during tumor progression (Brozyna et 364
al., 2016). Additionally, RORC significantly reduces the glucose metabolism of 365
bladder cancer and inhibits the proliferation of cancer cells, and its low expression is 366
related to the improvement of survival results (Cao et al., 2019). In our study, 367
bioinformatics data showed that RORC expression was relatively high in lung cancer 368
cells, and its high expression led to lower survival rates in lung cancer patients. 369
Moreover, the rescue experiment results showed that RORC and stigmasterol were 370
negatively regulated, and its overexpression reversed the inhibitory effect of 371
stigmasterol on lung cancer cells to a certain extent. Notably, this finding was 372
different from previous results. Thus, we speculated that the reason for this 373
occurrence was that in different cancer progressions, the physiological functions of 374
RORC were different. 375
376
377
378
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
15
Conclusion 379
In this study, we demonstrated the anti-lung cancer effect of stigmasterol, which was 380
able to effectively inhibit the proliferation activity of lung cancer cells and promote 381
their apoptosis. Furthermore, we proved that RORC was the target of stigmasterol and 382
overexpression of RORC reversed the inhibitory effect of stigmasterol on lung cancer. 383
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
16
References 384
Alhassan Mohammed H., Saboor-Yaraghi A.A., Vahedi H., Panahi G., Hemmasi 385
G., Yekaninejad M.S. and Mirshafiey A. (2018). Immunotherapeutic Effects of beta-D 386
Mannuronic Acid on IL-4, GATA3, IL-17 and RORC Gene Expression in the PBMC 387
of Patients with Inflammatory Bowel Diseases. Iran J. Allergy Asthma Immunol. 17, 388
308-317. 389
Ali H., Dixit S., Ali D., Alqahtani S.M., Alkahtani S. and Alarifi S. (2015). Isolation 390
and evaluation of anticancer efficacy of stigmasterol in a mouse model of DMBA-391
induced skin carcinoma. Drug Des. Devel. Ther. 9, 2793-2800. 392
Antwi A.O., Obiri D.D., Osafo N., Essel L.B., Forkuo A.D. and Atobiga C. (2018). 393
Stigmasterol Alleviates Cutaneous Allergic Responses in Rodents. Biomed Res Int. 394
3984068. 395
Brozyna A.A., Jozwicki W., Skobowiat C., Skobowiat C., Jetten A. and Slominski 396
A.T. (2016). RORalpha and RORgamma expression inversely correlates with human 397
melanoma progression. Oncotarget. 7, 63261-63282. 398
Cabral C.E. and Klein M. (2017). Phytosterols in the Treatment of 399
Hypercholesterolemia and Prevention of Cardiovascular Diseases. Arq. Bras. Cardiol. 400
109, 475-482. 401
Cao D., Qi Z., Pang Y., Li H., Xie H., Wu J., Huang Y., Zhu Y., Shen Y., Zhu Y., Dai 402
B., Hu X., Ye D. and Wang Z. (2019). Retinoic Acid-Related Orphan Receptor C 403
Regulates Proliferation, Glycolysis, and Chemoresistance via the PD-404
L1/ITGB6/STAT3 Signaling Axis in Bladder Cancer. Cancer Res. 79, 2604-2618. 405
Chandran U. and Patwardhan B. (2017). Network ethnopharmacological evaluation of 406
the immunomodulatory activity of Withania somnifera. J. Ethnopharmacol. 197, 250-407
256. 408
Chen W.P., Yu C., Hu P.F., Bao J.-P., Tang J.-L. and Wu L.-D. (2012). Stigmasterol 409
blocks cartilage degradation in rabbit model of osteoarthritis. Acta Biochim. Pol. 59, 410
537-541. 411
Cheng T.Y., Cramb S.M., Baade P.D., Youlden D.R., Nwogu C. and Reid M.E. 412
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
17
(2016). The International Epidemiology of Lung Cancer: Latest Trends, Disparities, 413
and Tumor Characteristics. J. Thorac. Oncol. 11, 1653-1671. 414
Didkowska J., Wojciechowska U., Manczuk M. and Łobaszewski J. (2016). Lung 415
cancer epidemiology: contemporary and future challenges worldwide. Ann. Transl. 416
Med. 4, 150. 417
Haque M.N. and Moon I.S. (2018). Stigmasterol upregulates immediate early genes 418
and promotes neuronal cytoarchitecture in primary hippocampal neurons as revealed 419
by transcriptome analysis. Phytomedicine. 46, 164-175. 420
Hsu H.F., Huang K.H., Lu K.J., Chiou S.-J., Yen J.-H., Chang C.-C. and Houng J.-Y. 421
(2011). Typhonium blumei extract inhibits proliferation of human lung 422
adenocarcinoma A549 cells via induction of cell cycle arrest and apoptosis. J. 423
Ethnopharmacol. 135, 492-500. 424
Jemal A., Bray F., Center M.M., Ferlay J., Ward E. and Forman D. (2011). Global 425
cancer statistics. CA Cancer J. Clin. 61, 69-90. 426
Kangsamaksin T., Chaithongyot S., Wootthichairangsan C., Hanchaina R., 427
Tangshewinsirikul C. and Svasti J. (2017). Lupeol and stigmasterol suppress tumor 428
angiogenesis and inhibit cholangiocarcinoma growth in mice via downregulation of 429
tumor necrosis factor-alpha. PLoS One. 12, e0189628. 430
Kim Y.S., Li X.F., Kang K.H., Ryu B. and Kim S.K. (2014). Stigmasterol isolated 431
from marine microalgae Navicula incerta induces apoptosis in human hepatoma 432
HepG2 cells. BMB Rep. 47, 433-438. 433
Li K., Yuan D., Yan R., Meng L., Zhang Y. and Zhu K. (2018). Stigmasterol exhibits 434
potent antitumor effects in human gastric cancer cells mediated via inhibition of cell 435
migration, cell cycle arrest, mitochondrial mediated apoptosis and inhibition of 436
JAK/STAT signalling pathway. J. BUON. 23, 1420-1425. 437
Liu Z.B., Hou Y.F., Min D., Di G.-H., Wu J., Shen Z.-Z. and Shao Z.-M. (2009). PA-438
MSHA inhibits proliferation and induces apoptosis through the up-regulation and 439
activation of caspases in the human breast cancer cell lines. J. Cell Biochem. 108, 440
195-206. 441
Mao Z., Shen X., Dong P., Liu G., Pan S., Sun X., Hu H., Pan L. and Huang J. (2019). 442
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
18
Fucosterol exerts antiproliferative effects on human lung cancer cells by inducing 443
apoptosis, cell cycle arrest and targeting of Raf/MEK/ERK signalling pathway. 444
Phytomedicine. 61, 152809. 445
Miller K.D., Siegel R.L., Lin C.C., Mariotto A.B., Kramer J.L., Rowland J.H., Stein 446
K.D., Alteri R. and Jemal A. (2016). Cancer treatment and survivorship statistics, 447
2016. CA Cancer J. Clin. 66, 271-289. 448
Miras-Moreno B., Sabater-Jara A.B., Pedreno M.A. and Almagro L. (2016). 449
Bioactivity of phytosterols and their production in plant in Vitro cultures. J. Agric 450
Food Chem. 64, 7049-7058. 451
Newill H., Loske R., Wagner J., Johannes C., Lorenz R.L. and Lehmann L. (2007). 452
Oxidation products of stigmasterol interfere with the action of the female sex 453
hormone 17beta-estradiol in cultured human breast and endometrium cell lines. Mol. 454
Nutr. Food Res. 51, 888-898. 455
Oh T.G., Bailey P., Dray E., Smith A.G., Goode J., Eriksson N., Funder J.W., Fuller 456
P.J., Simpson E.R., Tilley W.D., Leedman P.J., Clarke C.L., Grimmond S., Dowhan 457
D.H. and Muscat G.E.O. (2014). PRMT2 and RORgamma expression are associated 458
with breast cancer survival outcomes. Mol. Endocrinol. 28, 1166-1185. 459
Scholtysek C., Krukiewicz A.A., Alonso J.L., Sharma K.P., Sharma P.C. and 460
Goldmann W.H. (2009). Characterizing components of the Saw Palmetto Berry 461
Extract (SPBE) on prostate cancer cell growth and traction. Biochem. Biophys. Res. 462
Commun. 379, 795-798. 463
Tantratian S., Sae-Ngow A., Pradistsuwan C., Prakitchaiwattana C. and Pukahuta C. 464
(2019). Survival of Kluyveromyces marxianus with stigmasterol as subjected to 465
freezing stress. J. Biosci. Bioeng. 128, 39-43. 466
Wang F., Chen S., Ren L., Wang Y., Li Z., Song T., Zhang H. and Yang Q. (2020). The 467
effect of silibinin on protein expression profile in white adipose tissue of obese mice. 468
Front Pharmacol. 11, 55. 469
Zhang J., Zhou Y., Li N., Liu W.-T., Liang J.-Z., Sun Y., Zhang W.-X., Fang R.-D., 470
Huang S.-L., Sun Z.-H., Wang Y., and He Q.-Y. (2020). Curcumol Overcomes TRAIL 471
Resistance of Non-Small Cell Lung Cancer by Targeting NRH:Quinone 472
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
19
Oxidoreductase 2 (NQO2). Adv. Sci. (Weinh). 7, 2002306. 473
474
Figure Legends 475
476
Fig. 1. Target and chemical structure of stigmasterol on lung cancer. A) Target 477
prediction of Chaihu and stigmasterol on lung cancer B) Chemical structure of 478
stigmasterol. 479
480
Fig. 2. Stigmasterol inhibits the proliferation of lung cancer cells. The 481
proliferation ability of PLA-801D and A-549 after treatment with stigmasterol (5 482
µg/mL, 10 µg/mL and 20 µg/mL) was detected by A) CCK-8 assay and B) EdU assay. 483
*indicated compared with the control group, P˂0.05; **indicated compared with the 484
control group, P˂0.01; ***indicated compared with the control group, P˂0.001. CCK-485
8, Cell Counting Kit-8 assay; EdU, 5-ethynyl-2-deoxyuridine. 486
487
Fig 3. Effect of stigmasterol on BEAS-2B, PLA-801D, A-549, H661 and SK-SEM-488
1 cell viability. The Figure shows that stigmasterol significantly inhibited the viability 489
of PLA-801D, A-549, SK-SEM-1 and H661 cells while it exerted less toxicity on the 490
normal BEAS-2B cells. 491
492
Fig. 4. Stigmasterol promotes apoptosis of lung cancer cells. The apoptosis of 493
PLA-801D and A-549 cells after treatment with stigmasterol (5 µg/mL, 10 µg/mL and 494
20 µg/mL) was determined by A) TUNEL assay B) flow cytometry. In addition, the 495
mRNA level and protein level of apoptosis-related proteins (caspase-3 and caspase-9) 496
after treatment with stigmasterol (5 µg/mL, 10 µg/mL and 20 µg/mL) were detected 497
by C) qRT-PCR and D) western blot, respectively. *indicated compared with the 498
control group, P˂0.05; **indicated compared with the control group, P˂0.01. 499
500
Fig. 5. RORC is the target of stigmasterol on lung cancer. A) GO functional 501
enrichment analysis for BP and MF, and B) STRING database were used to predict 502
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
20
the target genes of stigmasterol; C) The 3D structure model of RORC constructed by 503
the SWISSMODEL Protein Modelling Server; D) Autodock was used to calculate the 504
possibility of binding between stigmasterol molecules and RORC protein molecules. 505
E) TCGA data showed the expression changes of RORC and the Kaplan Meier 506
survival curve. *indicated compared with normal tissues, P˂0.05. GO, gene ontology; 507
BP, biological process; MF, molecular function. 508
509
Fig. 6. RORC is identified as a potential target of stigmasterol on lung cancer. 510
CETSA curves comparing RORC thermal stability change between stigmasterol 511
group and control group. 512
513
Fig. 7. RORC overexpression reverses the inhibitory effect of stigmasterol on 514
lung cancer. After co-transfection with stigmasterol (20 µg/mL) and pc-DNA-RORC, 515
the proliferation ability of PLA-801D and A-549 was detected by A) CCK-8 assay 516
and B) and EdU assay. #indicated compared with pcDNA-NC, P˂0.05; **indicated 517
compared with stigmasterol + pcDNA-RORC, P˂0.01; **indicated compared with 518
pcDNA-NC, P˂0.01; #indicated compared with stigmasterol + pcDNA-RORC, 519
P˂0.05. 520
521
Fig. 8. RORC overexpression reverses the inhibitory effect of stigmasterol on 522
lung cancer. The apoptosis of PLA-801D and A-549 cells was determined by A) 523
TUNEL assay and B) Flow cytometry after co-treated with stigmasterol (20 µg/mL) 524
pc-DNA-RORC. **indicated compared with pcDNA-NC, P˂0.01; #indicated 525
compared with stigmasterol + pcDNA-RORC, P˂0.05. The mRNA level and protein 526
level of apoptosis-related proteins (caspase-3 and caspase-9) were detected by C) 527
qRT-PCR and D) western blot, respectively. **indicated compared with pcDNA-NC, 528
P˂0.01; #indicated compared with stigmasterol + pcDNA-RORC, P˂0.05; ##indicated 529
compared with stigmasterol + pcDNA-RORC, P˂0.01. 530
531
532
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
A Chaihu
B
Lung Cancer
4 145 (90. 6\)
Stigmasterol Lung Cancer
4226 (97. 7\)
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
A
0.8 Q) ::J i6 > 0.6 e o ->- 0.4 := :e ra ·:;; 0.2 Qj
-+- Control
..... Stigmasterol (5 µg/ml)
""'*"" Stigmasterol ( 1 O µg/ml)
....... Stigmasterol (20 µg/ml) Cisplatin
PLA-801 D *
** **
***
u ...... 0.0--..-------.---"""'T""---..---
B
-~ o
~ Qj CJ Q)
> :¡::::; ·¡¡; o Q.
1
:::::> "'C UJ
e
e e: o
ü
:::¡-* E Cll Oi E ::i .Ql l!) U5 ~ o~ ..... _J
2 E Cf) -Cll Ol E ::i Ol o (¡) ~ o ~ ..... _J
2 E Cf) -Cll Ol E ::i Ol o
:;::; N (/) ~
e ~ D.. Cf)
ü
100
80
60
40
20
o
Oh 24h 48h 72h
EdU DAPI Merge
PLA-8010
CJ Control
CJ Stigmasterol C5µg/ml )
CJ Stigmasterol (10µg/ml )
CJ Stigmasterol C20µg/ml ) * Cisplatin
** **
**
PLA-8010
0.8 Q) ::J i6
0.6 > e o -~ 0.4 :e ra ·:;; 0.2 Qj u
o.o
100 -~ o 80 ~ Qj CJ 60 Q)
> E 1/) 40 o Q.
1
:::::> 20 "'C UJ
o
-+- Control
..... Stigmasterol (5 µg/ml)
""'*"" Stigmasterol ( 1 O ~tg/ml)
....... Stigmasterol (20 ~tg/ml)
Cisplatin
A549
Oh 24h 48h
* **
**
***
72h
EdU DAPI Merge
*
A549
**
A549
CJ Control
CJ Stigmasterol (5µg/ml)
CJ Stigmasterol (10µg/ml)
CJ Stigmasterol (20µg/ml)
Cisplatin
** **
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
...... BEAS-28
....... PLA-8010 100 ...... A549 -~ o .........
>-~ :e ca ·:;: 50 a; u
***
o o 2.5 5 10 20 40
Concentration (¡.mol/L)
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
A e +"" e o ü
º!..... _J
2 E (/)ro O"> E ::i. .Q>~ Ci5 o!.... _J
2 E (/)ro O"> E ::i.
.Q>~ Ci5 -o-!.... _J
2 E (/)-ro O"> E ::i.
.Q>~ Ci5 -
80 ~ o
~ 60 Q) (J
Q)
~ 40 ·¡¡; o c. ~ 20 e: ::::J 1-
o
. . ' . . . : ..
PLA-801 D
PLA-8010
. . . . . : ...
A549 100
~ 'if 80 Q; (J
Q) 60 ~ ·¡¡;
40 o 9-Q;
20 e: ::::J 1-
o
. . .
" .. " ..
' .
A549
'! . •
CJ Control
CJ Stigmasterol (5µg/ml)
CJ Stigmasterol (10µg/ml)
CJ Stigmasterol (20µg/ml)
Cisplatin
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
B Control
FL4-A :: APC-A
,,• FL4-A :: APC-A
20
-~ o 15 -!!J.
Q) (J
.~ 10 -o -c. o 5 c.
<C
o
**
Stigmasterol (Sµg/ml)
' .--------.----.., 10 01
106 0 .71
,,• 1031 " 1 ,1 " ] ,,' ]°' 03 o 92.7 3.65 10 ........ .,.-...... -y- .... ,,..,-,-... .,- ... . . ., ..... ,,.,-.-....
10° 102 104 106
101 04
10º 93.7
**
FL4-A :: APC-A
,,• FL4-A :: APC-A
**
**
0 3 3.17
,,•
PLA-801 D
' 10 01
106 0.79
Stigmasterol (1 Oµg/ml)
101 04 03 o 88.6 2.93
10 -····•"t_,., ... ~ ... ~ ....... -.... , .. ... ~-····1·., o 2 ' 8 10 10 10 10
FL4·A::Af>C.A
' 10 01
10ª 2.76
101 04 03 o 88.4 1.68
10 . ..... ,~~~~-......,-.-..,,
io0 102 10' 106
Fl4-A :: APC-A
**
**
**
A549
Stigmasterol (20µg/ml)
'j 10 01
6 1.76
" ,,•
10 1 04
o 82.2 10 • ., ........... ., ....... , 1""1 ............ -. ... ,.,
10° 102 io4
107 101
6 12.99
" 1
FL4-A :: APC-A
,,. ,_1 __ ~~
03 5.32
,,•
::1 10 1 -,04 03
10° ~:~.,-. .. ..,...-.-.-...,-.-.....,-,.,,,.,......- .,5~ 10° 102 104 106
Fl4·A :: APCA
**
Cisplatin , 01 la1
s 14.79
".1 " 1 ,,· ~\------,;,,
10 3 ~ 102 ~ 101 J 04 00
10° ~~~-n·.,.,~.· .. -rn·• ~.., ..::~ 10° 102 10' 106
:::l ,,' 101 04
10º ~:-~ ,,•
FL4-A :: APC-A
5.55
·~·-. ...,.-.-. .,... .... ,..... .. ~ .., ... ., ' . ,,• 10 10
FL4-A :: APC-A
CJ Control
CJ Stigmasterol (5µg/ml)
CJ Stigmasterol (10µg/ml)
CJ Stigmasterol (20µg/ml)
Cisplatin
....... o co <( _J
o...
Q)
""'" LO <(
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
e 6
PLA-8010
e: o ·¡¡;
~ 4 c.. >< Q)
Q)
~ 2 (ti
Q) e::::
Caspase 3
D
o 1--e o ü
Cleaved-j caspase3
Cleaved-1 caspase9
~-actin
Cleaved-caspase3
Cleaved-1 caspase9
j3-actin
Caspase 9
o o 1-
1- --a> -- 2 _J en _J CJ) E co E co -E- E °' O) O) O) :l.. ·-:l.. ·-o ........ l{) U5 :=, (f) ---
D Control
D Stigmasterol (5µg/ml)
D Stigmasterol (10µg/ml)
D Stigmasterol (20µg/ml)
Cisplatin
o !- --2 _J CJ) E co -E °' O) :l.. ·-o .......,N (f) ---
PLA-801 D
A549
5
e: .Q 4 1/)
A549
**
** D Control 1/) Q)
a. 3 >< Q)
g! 2 :; -¡¡; e::::
Caspase 3
PLA-8010
Caspase 9
** **
Cleaved-caspase-3 Cleaved-caspase-9
A549
Cleaved-caspase-3 Cleaved-caspase-9
D Stigmasterol (5µg/ml)
D Stigmasterol (10µg/ml)
D Stigmasterol (20µg/ml)
Cisplatin
D Control
CJ Stigmasterol (5µg/ml)
CJ Stigmasterol (10µg/ml)
CJ Stigmasterol (20µg/ml)
Cisplatin
CJ Control
CJ Stigmasterol (5µg/ml)
CJ Stigmasterol (10µg/ml)
CJ Stigmasterol (20µg/ml)
Cisplatin
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
A GO:MF
O Term name
O lipid bind inq
O oxysterol bindinq
O prostaqlandin receptor activity
O prostanoid receptor activity
O monocarboxylic acid bindinq
O icosanoid receptor activity
O steroid bindinq
GO:BP
O Terrn name
O response to stress
O response to externa! stimulus
O chemical homeostasis
O posit ive requlation of cytosolic calcium ion concentrati ...
O icosanoid biosynthetic process
O response to oxyqen-containinq compound
•• }}
;o o tD
n ;o J:
<1 n m
....
~ "C
d s: "C "C -4 "C '::'.1 -4 n -4 C\ -4 o "C "' C\ m ~ "C "C s: ::!l ~
)> m ;o ~
N .... VI .... ;o )> .... o
o
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
E
N
o
00 o
-e Q) '<!"
~ o Q)
11..
N o
o o
o
Stage 1
Overall Survival
50
Stage 11
- Low RORC TPM - High RORC TPM
Logrank p=0.6 HR(high)=0.92
p(HR)=0.6 n(high)=239 n(low)=239
·. <·. ·. ·. ·. ·. ·. ·. ·. ·. ·. ·. ·. ·. ·. ·. ~
100 150 200
Months
Stage 111
F value = 0.645 Pr(>F) = 0.586
Stage IV
250
N
o
¡----*
1 •
... • :J ..
1 •
: 1 •
1.
:-'\ . ~ • ---1--
.·
,
LUAD (num(T)=483; num(N)=347)
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
40 60 64 67 70 72 75
Control
Stigmasterol
>--1.5 ·¡¡; ..... Control e: (IJ ....... Stigmasterol -.5
"'C 1.0 e:
ra .e < (J) 1-w 0.5 u (IJ
> ~ Qj o.o o:: 40 60 64 67 70 72 75
Temperature (°C)
o 2 10 50 250 1250
PLA-801 D
A549
>--·¡¡; 1.5 ~ 1.5 e: (IJ 1/)
:5 e: (IJ -"'C .5 e:
"'C 1.0 ra 1.0 .e e: ra o .e ..... en o co ...,.
<l: 0.5 ~ 0.5 ...J (IJ a.. .2: (IJ "l6 > ~ o.o Qj
o:: o.o (IJ o 2 10 50 250 1250 o 2 10 50 250 1250 o::
Concentration ( ¡J\11) Concentration ( ¡J\11)
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
A 0.8
Ci)' ::::s
~ 0.6 e o ~ 0.4 :e
C'tS ·s: 0.2 Q) (..)
-e- pcDNA-NC
..... Stigmasterol+pcDNA-NC
.......,. Stigmasterol+pcDNA-RORC
PLA-8010 #
**
0.0-------------.....-------.---Oh 24h 48h 72h
B pcDNA-NC
Stigmasterol +pcDNA-NC
Stigmasterol +pcDNA-RORC
80 -'?ft. (¡) 60 Q) (.)
(1)
-~ 40 ..... ·e;; o a.
1 20 ::::> "O w
PLA-801 D
Cl pcDNA-NC
Cl Stigmasterol+pcDNA-NC
CJ Stigmasterol+pcDNA-RORC
PLA-8010
#
**
o-------------------------
0.8 -(1)
::::s
~ 0.6 e o ~ 0.4
:e C'tS ·s: 0.2 Q) (..)
-e- pcDNA-NC
Stigmasterol+pcDNA-NC .......,. Stigmasterol+pcDNA-RORC
A549 #
**
0.0-------------.....-------.---Oh 24h 48h 72h
pcDNA-NC Stigmasterol +pcDNA-NC
Stigmasterol +pcDNA-RORC
80 -'?ft. (¡) 60 Q) (.)
(1)
-~ 40 ..... ·e;; o a.
1 20 ::::> "O w
A-549
CJ pcDNA-NC
Cl Stigmasterol+pcDNA-NC
CJ Stigmasterol+pcDNA-RORC
A549
#
**
o------------------------
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
A ü z <( z o (.)
+ o. oü Q)Z (¡) <(
EB O> (.)
:.¡::::; o. (/) + ü o a:: 2~ (/) 1
ro <( Ez ·º'º U5 g_
B 7
10 Q1
6 0.035 10
5 10
4 10
3 10
2 10
10 1 Q4
o 95.2 10
o 10
7 10 Q1
6 0.064 10
5 10
4 10
3 10
2 10
1 10
o 10
o 10
DAPI
pcDNA-NC
2 4 10 10
FL4-A : APC-A
2 4 10 10
FL4-A : APC-A
Tunel
PLA-8010
7 Q2 10 Q1
1.05 6 0.092 10
5 10
4 10
3 10
2 10
Q3 10 1 Q4
3.74 o 87.0 10
6 10 o
10
7 Q2 10 Q1
0.45
5 10
4 10
3 10
2 10
10 1 Q4
Q3
3.77 o 87.4 10
o 6 10
10
Merge
Stigmasterol+ pcDNA-NC
Q2
0.68
... ..
"·'":~i: . . .
Q3
12.2
2 4 6 10 10 10
FL4-A :APC-A
PLA-801 D
Q2
0.85
Q3
11 .7
2 4 6 10 10 10
FL4-A : APC-A
A549
7 10 Q1
106 0.038
5 10
4 10
3 10
2 10
3 10
2 10
o 10
101 Q4
o 91 .7
DAPI Tunel Merge
Stigmasterol+ pcDNA-RORC
2 10
4 10
FL4-A : APC-A
Q2
0.75
6 10
Q2
1.64
Q3
6.61
A549
Cl pcDNA-NC Cl Stigmasterol+pcDNA-NC
15 Cl Stigmasterol+pcDNA-Rq~c
!!l. 10 Qi ()
()
:g c.. o ~
e e: ·º 4 rn rn Q)
5. 3 >< Q)
~ 2 ~ ~ 1
#
PLA-8010
PLA-8010
Cl pcDNA-NC
A549
Cl Stigmasterol+pcDNA-NC Cl Stigmasterol+pcDNA-RORC
#
Caspase 3 Caspase 9
A549 Cl pcDNA-NC
5 Cl Stigmasterol+pcDNA-NC
e: Cl Sti~~asterol+pcDNA-RORC ·º 4 rn rn
Q)
5. 3 >< Q)
10 ""'r-rTTTm'l~.,.,.,-r-rmTI,,--r">Trmr-,.-rrlmr-r'""""...-nnnr-' ~ 2 ~ o
10 2 4
10 10
FL4-A : APC-A ~ 1
Caspase 3 Caspase 9
HISTOLO
GY AND H
ISTOPATHOLO
GY
(non-e
dited
man
uscri
pt)
D
ü z
1
<( z o ü a.
+ eo 2z en • ro <( EZ e>O
:¡::::; ü Cf) a.
+ ü o~ !..... o 2~ en • ro <( EZ e>O
:¡::::; ü Cf) a.
Cleaved- 1 caspase3~. _________ _ Cleaved- i~---------caspase9 ~. _________ _
13-actin I~---------PLA-8010
Cleavedcaspase3 ~----------
Cleaved- 1
caspase9 ::=· ===================== í3-actin
A549
CJ pcDNA-NC
CJ Stigmasterol+pcDNA-NC
e: 5 CJ Stigmasterol+pcDNA-RORC o ** ·~ PLA-8010 ~ 4 ** a. >< Cl) 3 ·~ # ... e 2 a. Cl)
.:::: 1 ... C'CS a; ~ o------------------------------
Cleaved-caspase-3 Cleaved-caspase-9
CJ pcDNA-NC
CJ Stigmasterol+pcDNA-NC
CJ Stigmasterol+pcDNA-RORC ** A549 **
##
##
Cleaved-caspase-3 Cleaved-caspase-9
