Therapeutic Effect Of Luteolin Natural Extract Versus Its Nanoparticles On Tongue Squamous Cell Carcinoma Cell Line: In Vitro Study
Phase: EARLY_PHASE1
Conditions: Tongue Neoplasms, Carcinoma
Sponsor: Cairo University

 
1  
 
 
 
 
Therapeutic Effect Of Luteolin Natural Extract Versus 
Its Nanoparticles On Tongue Squamous Cell 
Carcinoma Cell Line: In Vitro Study 
 
September, 18 2017 
 
Presented by: 
Safaa Mohamed Mohamed Ibrahim Baz 
 
 
 
 
 
 
 

 
2  
 
List of Contents: 
Administrative Information……………………………………………… 
4
 
 
 
1. Title………………………………………………………............ 
4
 
 
 
2. Protocol Version………………………………………………… 
4
 
 
 
3. Roles and Responsibilities………………………………………. 
4
 
 
 
Steering Committees……………………………………………………... 
4
 
 
 
Assessment and Auditing.………………………………………………... 
5
 
 
 
Research Ethics Approval ……………………………………….............. 
5
 
 
 
Access to Data .……………………………………………………........... 
5
 
 
 
Dissemination Policy .…………………………………………………… 
5
 
 
 
Funding.………………………………………………………………….. 
5
 
 
 
1. Introduction.…………………………………………………………… 
6
 
 
 
1. 1. Background and Scientific Rationale………………………… 
6
 
 
 
1. 2. Objectives…………………………………………………….. 
8
 
 
 
2. PICO…………………………………………………………………… 
9
 
 
 
3. Research Question.……………………………………………............. 
9
 
 
 
4. Material and Methods…………………………………………………. 
9
 
 
 
4. 1. Collecting samples……………………………………………. 
9
 
 
 
4. 2. Materials …………………..………………………………….. 10 
 
 
4. 3. Methods.……………………………………………………… 
10
 
 
4. 4. Sample size.……………………………………………........... 13 
 
 
4. 5. Randomization……………...…..…………………………….. 
14
 
 
4. 6. Blinding and assessment………..…………………………….. 14
 
 
4. 7. Statistical methodology……………………………………….. 14
 
 

 
3  
 
5. Results.………………………………………………………………… 14 
 
 
5. 1. Outcome(s) (O)..………………………………………............ 
14
 
 
5. 2. Testing the samples.…………………………………………... 14
 
 
6. Protocol amendments.…………………………………………………. 15
 
 
7. Search strategy………………………………………………………… 15
 
 
7. 1. The PRISMA Statement………………………………………. 15
 
 
7. 2. Summary of finding tables (SOFT)…………………………… 24
 
 
8. References.…………………………………………………………….. 29
 
 
 
 
 
 
 
 
 
 
 
 
 

 
4  
 
Administrative Information: 
 Title: 
Therapeutic Effect of Luteolin Natural Extract Versus Its Nanoparticles on Tongue 
Squamous Cell Carcinoma Cell Line: In Vitro Study 
 Protocol Version:  
   Version (1) 
 Roles and Responsibilities:  
    1-The Principle Supervisor:  
 Prof. Dr. Heba Ahmad Farag (H.F.), Professor of Oral & Maxillofacial 
Pathology, Faculty of Oral & Dental Medicine, Cairo University. 
    2-The Assistant Supervisor: 
 Dr. Safa Fathy Abd-Elghany (S.F.), Assistant Professor of Oral & Maxillofacial 
Pathology, Faculty of Oral & Dental Medicine, Cairo University.  
    3-The Investigators: 
 Dr. Esam Rashwan (E.R.), Assistant Professor and Head of Confirmatory 
Diagnostic Unit, VACSERA-Egypt 
 Safaa Mohamed Mohamed Ibrahim Baz (S.B.), (The Principle Investigator) 
Assistant Lecturer of Oral & Maxillofacial Pathology, Faculty of Oral & Dental 
Medicine, BUE. 
Steering Committees:  
1. Oral & Maxillofacial Pathology Department Board, Faculty of Dentistry, Cairo 
University.  
2. Evidence Based Committee, Faculty of Dentistry, Cairo University. 

 
5  
 
3. Ethics Committee, Faculty of Dentistry, Cairo University. 
4. Higher Education and Research Committee.  
5. Faculty Board. 
Assessment and Auditing:  
The assessment and auditing of the study design was done by: 
1.  Oral & Maxillofacial Pathology Department Board, Faculty of Dentistry, Cairo 
University. 
2. Evidence Based Committee, Faculty of Dentistry, Cairo University.  
Research Ethics Approval:  
This protocol will be reviewed by:  
Ethical Committee, Faculty of Dentistry, Cairo University. 
Access to Data:  
All Investigators of the study will be given access to the data.  
Dissemination Policy:  
Study results will be published as a partial fulfillment of the requirements for the PhD 
degree in Oral & Maxillofacial Pathology. 
Funding: 
Self-funding. 
 
 

 
6  
 
1. Introduction: 
1. 1. Background and Scientific Rationale: 
Cancer still one of the most lethal diseases causing an expansive number of deaths 
globally. In 2015, the WHO stated that the number of individuals died from cancer all over 
the world was about 8.8 million and indicated that by 2030 this number will show a marked 
elevation exceeding 13 million 1. Among malignancies, oral cancer is the sixth most 
common neoplasm around the world. Oral squamous cell carcinoma (OSCC) is the most 
widely recognized oral malignancy representing almost 90% of the oral cancers worldwide 
2, with a variable global incidence which differs in each area according to the exposure to 
specific risk factors 3. 
For treatment of OSCC, the primary therapeutic modalities are surgical intervention, 
radiotherapy, and chemotherapy 4,5. Chemotherapeutic agents play a vital role in the 
treatment of malignancy by killing cancerous cells. Nevertheless, they cause cytotoxic 
impacts to the adjacent normal cells and leave behind tissue resistance which is considered 
to be serious obstructions encountered the chemotherapy treatment. Therefore, the scientific 
community has the responsibility to discover an alternative naturally occurring drug with 
minimal side effects and maximal efficacy in cancer therapy 6. 
Among 
plant-derived 
compounds, 
flavonoids 
are 
accepted 
as 
natural 
chemotherapeutic agents 7 with many points of interest over the traditional chemotherapy, 
(e.g. better bioavailability, lower toxicological profile, as well as affordability) 8. Luteolin, 
3',4',5,7-tetrahydroxyflavone, is a naturally occurring flavonoid isolated from a wide variety 
of medicinal herbs, fruits, and vegetables (e.g. parsley, artichoke, celery, onion leaves, 
broccoli, carrots, green and sweet bell pepper, perilla leaves and chrysanthemum flowers) 9–
12. Luteolin has been appeared to display several significant biological properties, ranging 
from 
antioxidant 
and 
anti‑inflammatory 
effects 
to 
cancer 
chemo-
therapeutic/chemoprevention activities 13. 

 
7  
 
A recent in vitro and in vivo study has demonstrated that luteolin and its nanoparticles 
have anticancer activity against cells of lung cancer and squamous cell carcinoma of head 
and neck 14. 
Regarding the therapeutic effect of luteolin, a study on hepatic carcinoma cells has 
revealed a high level of apoptotic cells (reached to 58.18±2.11%) after 50 μM luteolin 
treatment for 24 hours 15. 
To provide a magnificent enhancement in the treatment effectiveness and overcome 
the undesired current cancer therapy toxicity, the researchers have potentially turned to 
nanotechnology-based regimens incorporation with several promising qualities over the 
conventional chemotherapy 16. Furthermore, the anticancer activity of nano-luteolin 
(nanoparticle delivery of luteolin) has suggested many advantages superior to luteolin and 
may have potential application in clinical settings 17. 
 
 
 
 
 
 
 
 
 

 
8  
 
1. 2. Objectives: 
In vitro study to examine whether luteolin and nano-luteolin exert an inhibitory effect on 
tongue squamous cell carcinoma cell line by inducing apoptosis, and to assess if nano-luteolin 
has more efficient apoptotic activity than luteolin on tongue squamous cell carcinoma cell 
line. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
9  
 
2. PICO: 
Population (P): Cultured tongue squamous cell carcinoma cell line. 
Intervention (I): 
 I 1: Application of  luteolin 
 I 2: Application of nano-luteolin 
Control (C): No treatment. 
Outcome(s) (O):   
 Primary O: Apoptosis (Gene expression of Caspase 3). 
 Secondary O: Cytotoxicity/Cell viability. 
Measuring Unit 
Measuring Device 
Outcome name 
Outcome 
fold-changes 
RT-qPCR system 18. 
Apoptosis  
(Gene expression of Caspase 3) 
Primary 
Micromolar (μM) 
MTT assay using an ELISA 
Reader 19. 
Cytotoxicity/Cell viability 
Secondary 
Study design:  
In vitro study 
3. Research Question: 
Do luteolin and nano-luteolin natural extracts exert an inhibitory effect on tongue squamous 
cell carcinoma cell lines by inducing apoptosis? Is nano-luteolin more efficient than luteolin 
in inducing apoptosis on tongue squamous cell carcinoma cell line?    
4. Material and Methods: 
4. 1. Collecting samples: 
Tongue squamous cell carcinoma cell line will be purchased from veterinary serum & 
vaccine research institute (VACSERA), supplied with compatible nutrient media. 

 
10  
 
4. 2. Materials: 
4. 2. 1. Intervention (I): 
1. Luteolin (Sigma-Aldrich) 
2. Nano-luteolin (Luteolin nanoparticles prepared using ultrasonication    
nanotechnology) 
4. 2. 2. Check list for the used drug: 
Item
Description
Name, common names and family 
name
Luteolin, 3′,4′,5,7-Tetrahydroxyflavone; Flavones, 
Flavonoids
Used product
Luteolin (Sigma-Aldrich)
Form
Powder
Color
Yellow
The type and concentration of 
solvent used
Ethanol (4.90 - 5.10 mg/ml)
Nano-luteolin
Luteolin nanoparticles prepared using ultrasonication 
nanotechnology
4. 3. Methods: 
All the steps of the current experiment will take place in Confirmatory Diagnostic Unit, R 
and D sector, VACSERA-Egypt, by E.R. and S.B. 
4. 3. 1. Sample preparation: (According to ATCC® protocol) 
[Continuous Cell Line Cell Culture Protocol]  
1. Examination: 
E.R. and S.B. will examine the culture prior to subcultivation using an inverted phase 
contrast microscope, and will check the general appearance of the culture.  

 
11  
 
2. Cell harvesting: 
When using 0.1% Trypsin solution for enzymatic dissociation: 
a) E.R. and S.B. will remove the old medium and discard it. 
b) We will rinse the cell monolayer gently. 
c) Then we will add the enzyme solution and incubate the culture until the cells are 
freleased. 
3. Cell counting: 
To determine growth rates or set up cultures at known concentrations it is necessary to 
count the cell suspension. 
a) E.R. and S.B. will remove 0.5mL sample and place it in a tube for counting, with 
adding the vital stain Trypan blue. We will withdraw 20μL of the sample with a 
wide tip pipettor and carefully load a clean hemacytometer. 
b) We will count a viable cell count. We will calculate the number of viable cells/mL 
and the total cell number. 
4. Plating: 
After making the appropriate dilutions, E.R. and S.B. will add the correct amount of cells 
to each culture vessel. Then we will add fresh medium to bring the cell culture vessel to 
its recommended working volume. 
5. Incubation:  
a) E.R. and S.B. will leave caps on flasks slightly loosened and place them on a shelf 
in a 37○C, humidified CO2 incubator*. 
b) We will examine culture daily and will change medium as needed. 
 
 
 
* Thermo Scientific Heraeus, UK. 

 
12  
 
4. 3. 2. MTT assay: (According to Sigma-Aldrich protocol) 
[IC50 Calculation and MTT Assay with ELISA reader* Protocol]  
 IC50 calculation:  
 After plating and 24 hours incubation of the cell culture, E.R. and S.B. will remove the 
cell culture from incubator into laminar flow hood, and then apply to a 96-well plate (about 
25,000-30,000 cells per well). 
 E.R. and S.B. will add the drug with five serial concentrations (100, 10, 1, 0.1, and 
0.01µg/mL) to calculate the IC50% concentration in Micromolar (μM). 
 MTT Assay should include: 
 Blank wells containing medium only. 
 Untreated control cells. 
 Test cells treated with either luteolin or nano-luteolin with IC50% concentration. 
1) After plating and 24 hours incubation of the cell culture, E.R. and S.B. will remove the 
cell culture from incubator into laminar flow hood, and then apply to a 96-well plate 
(about 25,000-30,000 cells per well). 
2) E.R. and S.B. will reconstitute each vial of MTT to be used with 3 ml of medium or 
balanced salt solution without phenol red and serum. We will add the reconstituted 
MTT in an amount equal to 10% of the culture medium volume.  
3) We will return cultures to incubator for 2-4 hours until purple precipitate is visible. 
4) At the end of the incubation period, we will remove the cultures from incubator and 
dissolve the resulting purple formazan crystals by adding an amount of MTT 
Solubilization solution equal to the original culture medium volume. 
5) Well mixing will improve dissolution. 
6) We will measure absorbance spectrophotometrically at a wavelength of 450nm. 
 
* Bioline Technologies, India. 

 
13  
 
4. 3. 3. RT-PCR: (According to Bio-Rad Laboratories protocol) 
[One-step Rotor-Gene Q Real-Time PCR System* Protocol] 
1. Treatment of cell culture:  
After plating and 24 hours incubation of the cell culture, E.R. and S.B. will treat the flask 
with the IC50% concentration of the drug for the treatment incubation time. 
2. We will harvest cells, then lysis cells. 
3. E.R. and S.B. will prepare all the required components (including SYBR® Green one-
step kit, SYBR® Green Supermix, sequence-specific primers for Caspase, necessary 
enzymes and a buffer solution) except RNA on ice reaction set up. 
4. We will mix the reaction set up thoroughly then dispense into the PCR tube.  
5. Then we will add the template RNA to the PCR tube, seal and shake the tube. 
6. The thermal cycling protocol will be programmed according to the steps which are 
summarized in the following table: 
The first cycle
 
The second cycle
 
The next 35-40 cycles
 
Reverse 
Transcription 
Reaction
 
Polymerase Activation and 
 
cDNA Denaturation
 
Amplification
 
Denaturation
 
Annealing/Extension
10 min. at 50ºC
 
1 min. at 95ºC
 
15 sec. at 95ºC
 
30 sec. at 60ºC
 
7. We will load the PCR tube onto RT-PCR instrument and allow the RT-qPCR to run. 
8. E.R. and S.B. will then analyze the RT-PCR products by Melt-Curve Analysis using 
instrument default setting. 
4. 4. Sample size: 
No sample size could be applied on cell line experiments as we generate millions of naturally 
randomized cells. 
 
*Qiagen Rotor-Gene®, USA. 

 
14  
 
4. 5. Randomization: 
Randomization (with sequence generation and allocation concealment mechanism) is not 
applied on cell line experiments, as we generate millions of naturally randomized cells, where 
the amount of cells in each plate will be standardized and each of the 96-well plates will 
contain the same number of cells (about 25,000-30,000 cells/well).  
4. 6. Blinding and assessment: 
Blinding is not applied on cell line experiments. 
4. 7. Statistical methodology: 
Data will be analyzed using IBM SPSS advanced statistics (Statistical Package for Social 
Sciences), version 21 (SPSS Inc., Chicago, IL). Numerical data will be described as mean 
and standard deviation or median and range. Categorical data will be described as numbers 
and percentages. 
5. Results: 
5. 1. Outcome(s) (O): 
 Primary O: Apoptosis (Gene expression of Caspase 3). 
 Secondary O: Cytotoxicity/Cell viability. 
5. 2. Testing the samples: 
1) After treatment incubation period, E.R. and S.B. will measure the color intensity to 
estimate cell viability using an ELISA Reader. 
2) After treatment incubation period, E.R. and S.B. will detect the fold-changes of 
Caspase 3 gene expression to estimate apoptosis using Rotor-Gene RT-PCR System. 
 

 
15  
 
6. Protocol amendments: 
Any adjustments to the protocol which might affect the conduct of the study, patient’s 
potential benefit or might impact patient safety, including any change in the design of the 
study, study objectives, methodology, sample sizes, or significant modification in the 
administrative aspects will require amendment to the protocol formally. Such amendments 
should be agreed upon by the Council of Oral & Maxillofacial Pathology department. 
7. Search strategy: 
7. 1. The PRISMA Statement 
7. 1. 1. Structured summary: 
Objective: To estimate the inhibitory effect of luteolin and nano-luteolin as a promoter of 
cancer cell death in tongue squamous cell carcinoma. 
Data Sources: In vitro and in vivo articles using PubMed and Cochrane databases. An 
additional study was identified by contacting the Egyptian Universities Libraries Consortium 
website (www.eulc.edu.eg). Search terms included OSCC, TSCC, HNSCC, luteolin and 
nano-luteolin. 
Study Selection: Only in vivo and in vitro studies of luteolin (luteolin 5,7,3',4'-tetrahydroxy-
flavone) activity regarding apoptosis, cytotoxicity/cell viability on cell lines of oral squamous 
cell carcinoma (OSCC), tongue squamous cell carcinoma (TSCC) or head and neck squamous 
cell carcinoma (HNSCC) were included. 
Data Extraction: Extraction of articles by the author using predefined data fields. 
Data Synthesis: Out of all pooled analyses three articles met the inclusion criteria. All trials 
used luteolin. Apoptosis, cytotoxicity/cell viability were observed to be increased with 
different doses of luteolin (10 ϻM for 12 hours) (20-100 ϻM for 24, 48, 72 hours) (50 ϻM 
for 12, 24 hours), separately. Luteolin of 10 ϻM selectively decreased cell viability and 

 
16  
 
increased cytotoxicity and apoptosis on tongue squamous cell carcinoma cell line (SCC-25) 
while increased cell viability on human keratinocyte-derived cell line (HaCaT) versus 
standard-of-care. When luteolin was used in a dose-dependent and time-dependent pattern the 
apoptotic cells increased (71%) meanwhile the cell viability decreased (29%) and induced 
apoptosis selectively on tongue squamous cell carcinoma cell line (SCC-4) and oral cancer 
cell line (OC-2) versus control. Application of 50 ϻM of luteolin increased apoptosis of 
laryngeal HNSCC cell line (Hep-2 cells) versus control. 
Limitations: Data of results of the included studies is insufficient. 
Conclusions: Luteolin between 10 to 100 ϻM appears to decrease viability and increase 
cytotoxicity and apoptosis selectively to OSCC, TSCC and HNSCC cell lines. 
7. 1. 2. Eligibility criteria: 
7. 1. 2. 1. Inclusion criteria: 
 Squamous cell carcinoma cell lines (OSCC, TSCC and HNSCC). 
 Luteolin 5,7,3',4'-tetrahydroxy-flavone (MeSH term). 
 Application of luteolin as a chemotherapeutic drug. 
 Detection of luteolin activity in apoptosis or cytotoxicity/cell viability. 
7. 1. 2. 2. Exclusion criteria: 
 Any cancer cell line other than OSCC, TSCC and HNSCC cell lines. 
 Luteolin glycosides. 
 Any use of luteolin other than chemotherapy. 
 Detection of luteolin activities other than apoptosis or cytotoxicity/cell viability. 
7. 1. 2. 3. Report characteristics:  
 No year limitation. 
 No language limitation. 

 
17  
 
7. 1. 3. Information sources: 
Studies were identified by searching electronic databases and scanning reference lists of 
articles. No limits were applied for language or date of search. This search was first started 
on 6 May 2017 applied to PubMed and Cochrane electronic databases. The last search was 
run on 15 June 2017. In addition, the hand search was done through: searching in the 
department of oral and maxillofacial pathology of Cairo University, checking reference lists 
of the included articles, searching in the Egyptian Universities Libraries Consortium website 
www.eulc.edu.eg as well as contacting the authors of the included articles to acquire missing 
results and there is no reply till now. A limited update literature search was performed from 
the date of inception till now. 
7. 1. 4. Synonyms of PICO items: 
The following search terms and synonyms were used to search all databases: 
Index Terms 
PICO items 
Synonyms 
Tongue squamous cell carcinoma cell line 
Tongue squamous cell carcinom* 
Tongue squamous cell tumour* 
Tongue squamous cell tumor* 
Tongue squamous cell neoplasm* 
Tongue squamous cell malignan* 
Tongue squamous cell cancer* 
TSCC 
Tongue SCC 
Oral tongue squamous cell carcinom* 
Oral squamous cell carcinom* 
Oral squamous cell tumour* 
Oral squamous cell tumor* 
Oral squamous cell neoplasm* 
Oral squamous cell malignan* 
Oral squamous cell cancer* 

 
18  
 
Oral cavity squamous cell carcinom* 
OSCC 
OSCC cell 
OSCC cell line 
OSCC cancer 
Head and neck squamous cell carcinom* 
Head and neck squamous cell tumour* 
Head and neck squamous cell tumor* 
Head and neck squamous cell neoplasm* 
Head and neck squamous cell malignan* 
Head and neck squamous cell cancer* 
HNSCC 
SCCHN 
Luteolin 
Luteolin 
Nano-luteolin 
Nano-luteolin 
Nano luteolin 
Nanoluteolin 
Luteolin nanoparticles 
Luteolin nanotechnology 
7. 1. 5. Databases: 
Search Strategy in PubMed and Cochrane, was run on 6 May 2017 till now: 
 
Index Term 
PubMed Cochrane 
#1 
“Tongue Neoplasms” [MeSH] 
9190 
56 
#2 
tongue squamous cell carcinom* 
781 
95 
#3 
tongue squamous cell tumour* 
1 
9 
#4 
tongue squamous cell tumor* 
7 
53 
#5 
tongue squamous cell neoplasm* 
36 
54 
#6 
tongue squamous cell malignan* 
3 
5 
#7 
tongue squamous cell cancer* 
16 
70 
#8 
tscc 
289 
6 
#9 
tongue scc 
827 
11 
#10 oral tongue squamous cell carcinom* 
202 
54 
#11 oral squamous cell carcinom* 
7720 
816 
#12 oral squamous cell tumour* 
8 
98 
#13 oral squamous cell tumor* 
42 
324 
#14 oral squamous cell neoplasm* 
228 
504 
#15 oral squamous cell malignan* 
10 
75 

 
19  
 
#16 oral squamous cell cancer* 
112 
615 
#17 oral cavity squamous cell carcinom* 
420 
278 
#18 oscc 
4492 
51 
#19 oscc cell 
4448 
51 
#20 oscc cell line 
1066 
4 
#21 oscc cancer 
4171 
32 
#22 head and neck squamous cell carcinom* 
7849 
1923 
#23 head and neck squamous cell tumour* 
9 
166 
#24 head and neck squamous cell tumor* 
6 
616 
#25 head and neck squamous cell neoplasm* 
377 
1029 
#26 head and neck squamous cell malignan* 
3 
84 
#27 head and neck squamous cell cancer* 
497 
1178 
#28 hnscc 
5763 
303 
#29 scchn 
1355 
277 
#30 #1 or #2 or #3 or #4 or #5 or #6 or #7 or #8 or #9 or #10 or #11 or #12 or #13 
or #14 or #15 or #16 or #17 or #18 or #19 or #20 or #21 or #22 or #23 or #24 
or #25 or #26 or #27 or #28 or #29 
 
(tongue squamous cell carcinom*) OR (tongue squamous cell tumour*) OR 
(tongue squamous cell tumor*) OR (tongue squamous cell neoplasm*) OR 
(tongue squamous cell malignan*) OR (tongue squamous cell cancer*) OR 
(tscc) OR (tongue scc) OR (oral tongue squamous cell carcinom*) OR (oral 
squamous cell carcinom*) OR (oral squamous cell tumour*) OR (oral 
squamous cell tumor*) OR (oral squamous cell neoplasm*) OR (oral 
squamous cell malignan*) OR (oral squamous cell cancer*) OR (oral cavity 
squamous cell carcinom*) OR (oscc) OR (oscc cell) OR (oscc cell line) OR 
(oscc cancer) OR (head and neck squamous cell carcinom*) OR (head and 
neck squamous cell tumour*) OR (head and neck squamous cell tumor*) OR 
(head and neck squamous cell neoplasm*) OR (head and neck squamous cell 
malignan*) OR (head and neck squamous cell cancer*) OR (hnscc) OR 
(scchn) 
 
20055 
2633 
#31 #1 or #30 
 
("Tongue Neoplasms" [Mesh]) OR (tongue squamous cell carcinom*) OR 
(tongue squamous cell tumour*) OR (tongue squamous cell tumor*) OR 
(tongue squamous cell neoplasm*) OR (tongue squamous cell malignan*) OR 
(tongue squamous cell cancer*) OR (tscc) OR (tongue scc) OR (oral tongue 
squamous cell carcinom*) OR (oral squamous cell carcinom*) OR (oral 
squamous cell tumour*) OR (oral squamous cell tumor*) OR (oral squamous 
cell neoplasm*) OR (oral squamous cell malignan*) OR (oral squamous cell 
cancer*) OR (oral cavity squamous cell carcinom*) OR (oscc) OR (oscc cell) 
OR (oscc cell line) OR (oscc cancer) OR (head and neck squamous cell 
carcinom*) OR (head and neck squamous cell tumour*) OR (head and neck 
squamous cell tumor*) OR (head and neck squamous cell neoplasm*) OR 
(head and neck squamous cell malignan*) OR (head and neck squamous cell 
cancer*) OR (hnscc) OR (scchn) 
27867 
2660 
#32 "Luteolin"[Mesh] 
1263 
2 

 
20  
 
#33 Luteolin 
3224 
18 
#34 
or #33
#32 
 
("Luteolin"[Mesh]) or (Luteolin) 
3224 
18 
#35 nano-luteolin 
1 
0 
#36 nano luteolin 
11 
0 
#37 Nanoluteolin 
0 
0 
#38 luteolin nanoparticles 
19 
0 
#39 luteolin nanotechnology 
8 
0 
#40 
#39
 
or
 
#38
 
or
 
#37
 
or
 
#36
 
or
 
#35
 
(nano-luteolin) OR (nano luteolin) OR (nanoluteolin) OR (luteolin 
nanoparticles) OR (luteolin nanotechnology) 
31 
0 
#41 #34 and #40
 
("Luteolin"[Mesh]) OR (luteolin) AND (nano-luteolin) OR (nano luteolin) 
OR (nanoluteolin) OR (luteolin nanoparticles) OR (luteolin nanotechnology) 
31 
0 
#42 #31 and #34
 
("Tongue Neoplasms" [Mesh]) OR (tongue squamous cell carcinom*) OR 
(tongue squamous cell tumour*) OR (tongue squamous cell tumor*) OR 
(tongue squamous cell neoplasm*) OR (tongue squamous cell malignan*) OR 
(tongue squamous cell cancer*) OR (tscc) OR (tongue scc) OR (oral tongue 
squamous cell carcinom*) OR (oral squamous cell carcinom*) OR (oral 
squamous cell tumour*) OR (oral squamous cell tumor*) OR (oral squamous 
cell neoplasm*) OR (oral squamous cell malignan*) OR (oral squamous cell 
cancer*) OR (oral cavity squamous cell carcinom*) OR (oscc) OR (oscc cell) 
OR (oscc cell line) OR (oscc cancer) OR (head and neck squamous cell 
carcinom*) OR (head and neck squamous cell tumour*) OR (head and neck 
squamous cell tumor*) OR (head and neck squamous cell neoplasm*) OR 
(head and neck squamous cell malignan*) OR (head and neck squamous cell 
cancer*) OR (hnscc) OR (scchn) AND ("Luteolin"[Mesh]) or (Luteolin) 
6 
0 
#43 #31 and #40
 
("Tongue Neoplasms" [Mesh]) OR (tongue squamous cell carcinom*) OR 
(tongue squamous cell tumour*) OR (tongue squamous cell tumor*) OR 
(tongue squamous cell neoplasm*) OR (tongue squamous cell malignan*) OR 
(tongue squamous cell cancer*) OR (tscc) OR (tongue scc) OR (oral tongue 
squamous cell carcinom*) OR (oral squamous cell carcinom*) OR (oral 
squamous cell tumour*) OR (oral squamous cell tumor*) OR (oral squamous 
cell neoplasm*) OR (oral squamous cell malignan*) OR (oral squamous cell 
cancer*) OR (oral cavity squamous cell carcinom*) OR (oscc) OR (oscc cell) 
OR (oscc cell line) OR (oscc cancer) OR (head and neck squamous cell 
carcinom*) OR (head and neck squamous cell tumour*) OR (head and neck 
squamous cell tumor*) OR (head and neck squamous cell neoplasm*) OR 
(head and neck squamous cell malignan*) OR (head and neck squamous cell 
cancer*) OR (hnscc) OR (scchn) AND (nano-luteolin) OR (nano luteolin) OR 
(nanoluteolin) OR (luteolin nanoparticles) OR (luteolin nanotechnology) 
1 
0 

 
21  
 
#44 #31 and #34 and #40
("Tongue Neoplasms" [Mesh]) OR (tongue squamous cell carcinom*) OR 
(tongue squamous cell tumour*) OR (tongue squamous cell tumor*) OR 
(tongue squamous cell neoplasm*) OR (tongue squamous cell malignan*) OR 
(tongue squamous cell cancer*) OR (tscc) OR (tongue scc) OR (oral tongue 
squamous cell carcinom*) OR (oral squamous cell carcinom*) OR (oral 
squamous cell tumour*) OR (oral squamous cell tumor*) OR (oral squamous 
cell neoplasm*) OR (oral squamous cell malignan*) OR (oral squamous cell 
cancer*) OR (oral cavity squamous cell carcinom*) OR (oscc) OR (oscc cell) 
OR (oscc cell line) OR (oscc cancer) OR (head and neck squamous cell 
carcinom*) OR (head and neck squamous cell tumour*) OR (head and neck 
squamous cell tumor*) OR (head and neck squamous cell neoplasm*) OR 
(head and neck squamous cell malignan*) OR (head and neck squamous cell 
cancer*) OR (hnscc) OR (scchn) AND ("Luteolin"[Mesh]) or (Luteolin) AND 
(nano-luteolin) OR (nano luteolin) OR (nanoluteolin) OR (luteolin 
nanoparticles) OR (luteolin nanotechnology) 
1 
0 
 
7. 1. 6. Study selection: 
The search started with 7 articles. The retrieved records were screened on the basis of title 
and abstract and sequentially two out of them were excluded according to the exclusion 
criteria. (The used cell lines were human breast cancer adenocarcinoma, human colon 
colorectal carcinoma and human hepatic carcinoma cell lines, not OSCC, TSCC or HNSCC 
cell lines (n=1), and luteolin glycosides were used with different molecular formula than 
luteolin 5,7,3',4'-tetrahydroxy-flavone (n=1)). Manuscript review and application of inclusion 
criteria of the remaining five articles with full text publication were done. This resulted in 
Reasonable exclusion of two additional articles. (Luteolin was used in chemoprevention not 
as a chemotherapeutic drug (n=1), and luteolin was used to detect different outcomes other 
than apoptosis or cytotoxicity/cell viability (n=1)). The three remaining full-text articles met 
the eligibility criteria and included to extract data out of them. For note, each stage of these 
steps was carried out only by the author. 
 
 
 

 
22  
 
PRISMA flow diagram of study selection: 
Identification 
 
Screening 
Eligibility 
Included 
 
Cochrane search 
(N=0) 
PubMed search 
(N=6) 
# of records screened on basis of 
title and abstract 
# of records after duplicates removed 
(N=7) 
# of records excluded  
(N=2) 
Any cancer cell line other than OSCC, TSCC or 
HNSCC cell lines: 1 
Luteolin glycosides: 1 
# of full-text articles assessed for 
eligibility 
(N=3)
# of full-text articles excluded with 
reasons (N=2) 
Any use of luteolin other than chemotherapy: 1 
Detection of different luteolin activities: 1 
Studies included 
Cytotoxicity/Cell viability and 
Apoptosis  
(N=3) 
# of records identified through 
database searching 
(N=6) 
# of additional records identified 
through other sources 
(N=1) 
Manuscript review and 
application of inclusion criteria 
# of records included 
(N=5) 

 
23  
 
7. 1. 7. Data collection process:  
Information was extracted from each included study and refined accordingly. The authors of 
the included studies were contacted for information that is unclearly reported. However, there 
is no answer till now. 
7. 1. 8. Data items: 
Information were extracted from each included study on: (1) characteristics of trial population 
(including number of groups), and the trial’s inclusion and exclusion criteria; (2) type of 
intervention (including type, dose, duration versus control, standard-of-care or versus no 
treatment), (3) type of outcome measure (including apoptosis and cytotoxicity/cell viability). 
 
 
 
 
 
 
 
 
 
 
 
 

 
24  
 
7. 2. Summary of finding tables (SOFT): 
7. 2. 1. Demographics of included studies: 
No. 
of 
study 
Study 1st 
Author 
(Year/ 
country) 
Name of journal 
Title of the 
study 
Type of 
samples 
Control 
Type of 
study 
Source 
1 
K.C. 
Tjioe, 
France 
2016 
Nutrition And 
Cancer 
Luteolin 
Impacts on the 
DNA Damage 
Pathway in 
Oral 
Squamous 
Cell 
Carcinoma 
1- Tongue 
squamous cell 
carcinoma cell 
line (SCC-25 
cells)  
2- Human 
keratinocyte-
derived cell 
line (HaCaT) 
DMSO 
In vitro  
PubMed 
2 
S. F. 
Yang, 
Taiwan 
2008 
J Dent Res 
Luteolin 
Induces 
Apoptosis in 
Oral 
Squamous 
Cancer Cells 
1- Tongue 
squamous cell 
carcinoma cell 
line (SCC-4 
cells)  
2-Oral cancer 
cell line (OC-
2 cells) 
DMSO 
In vitro 
and In 
vivo 
(Animal) 
PubMed 
3 
H. 
Zhang, 
China 
2014 
Eur Arch 
Otorhinolaryngol 
Luteolin 
induces 
apoptosis by 
activating Fas 
signaling 
pathway at the 
receptor level 
in laryngeal 
squamous cell 
line Hep.2 
cells 
- Laryngeal 
HNSCC cell 
line (Hep-2 
cells) 
DMSO 
In vitro  
PubMed 

 
 
25  
 
7. 2. 2. Material and Methods: 
 
No. of study 
Cell line types 
Culture media 
Drug 
MTT assay 
Crystal violet staining 
Western blot 
 
DAPI staining 
Flowcytometric analysis 
RT-PCR 
Groups 
Control 
Standard-of-
Dose 
Duration 
Material 
Groups 
Control 
Dose 
Duration 
Material 
Control 
Dose 
Duration 
Antibodies 
Material 
Control 
Dose 
Duration 
Material 
Dose 
Duration 
Protein 
expression
Material 
Housekeeping 
Primer 
Antibodies 
Material 
1 
1-SCC-25  2-HaCaT 
DMEM/F12 
10% FBS 
1% penicillin 
and 
streptomycin  
400ng/ml of 
hydro-
cortisone  
15mM N-2 
HEPES 
Luteolin 
NA 
DMSO 
 
5, 10,20  ϻM 
72, 24, 48, 72 h 
Colorimetric 
assay 1-(4,5-
dimethyl-
thiazol-2-yl)-
3 
5-diphenyl 
formazan 
Thiazolyl 
blue 
formazan 
 25ml of 
MTT reagent 
6-well plate (25,000 cells/well) 
DMSO 
10 ϻM 
72 h 
Glutaral-
dehyde 
2%, 
PBS, 
Crystal 
violet 
0.05%, 
Acetic 
acid  1% 
 
Nil 
10 ϻM 
30 min 
p-JNK p-EGFR p-AKT p- p38 p-ERK1/2 
p-ATM p-ATR p- H2AX p-CHK2 p-
BRCA1 ERK1/2 JNK1/2 ERK2 CHK2 
TNT buffer, 
MicroBCA kit 
(30 ϻg/well) 
NuPAGE 
electrophoresis 
Nitrocellulose 
membranes 
Not used 
Not used 
Not used 
CP 
5, 10 ϻm 
AG1478 
10 ϻM 
2 
1-SCC-4  2-OC-2 
DMEM/F12 
10% FBS 
1% penicillin 
and 
streptomycin  
400ng/ml of 
hydro-
cortisone  
2 mM 
glutamine 
Luteolin 
24-well plate 
DMSO 
 
5, 10 ϻM 
24, 48, 72 h 
(5 7 d)
0.5 mg/mL 
MTT 
reagent, Blue 
formazan 
crystals 
 
Not used 
β-actin 
0-100 ϻM 
NA 
Bcl-2 Bax Caspase9 Caspase 3 PARP 
NA 
Nil 
0-100 ϻM 
48 h 
4’,6-
Diamidino 
-2-
phenylindol
e (DAPI) 
(1 ϻg/ml ) 
0-100 ϻM 
24 h 
NA 
NA 
Not used 
0-100 ϻM 
24 h 
Paclita
0.3 
24, 48, 
72 h
3 
Hep-2 cells 
DMEM/F12 
penicillin 
and 
streptomycin  
 
Luteolin 
NA 
DMSO 
No 
20-100 ϻM 
NA 
3-(4,5-
dimethyl-
thiazol- 
2-yl)-2,5-
diphenyl 
tetrazolium 
bromide 
(MTT) 
Not used 
β-actin 
0, 30, 50 ϻM 
6, 12, 24 h 
anti-c-FLIPS/L anti-Fas  
per-oxidase-conjugated goat 
anti-rabbit 2ry antibody 
SDS/PAGE
 
 (12 % w/v)
 
Nitrocellulose 
membranes
 
non-fat milk
 
 (5 %)
 
TBST
 
DMSO 
0, 30, 50 ϻM 
24 h 
4’,6-
Diamidino-
2-
phenylindol
e(DAPI)
 
50 μM
6, 12, 24 h 
Caspase3, Caspase 8, Caspase 9 
Annexin V-
FITC 
Apoptosis 
Detection 
Kit
 
Becton–
Dickinson 
CELL Quest 
software 
 
β-actin 
8-12 
c-FLIP  Fas
 
IgG peroxidase-conjugated 2ry 
antibody
 
Aliquots 
of PCR 
products 
agarose 
gel (1.5 
%)
 
electropho
resis
 
ethidium 
bromide
 
 

 
 
26  
 
7. 2. 3. Results:
 
 
 
No. of the study
Drug
 
Apoptosis 
Antibodies 
Duration 
Dose 
Effect 
p-JNK 
Caspase-3 
Caspase-9 
cFLIPL 
6 h 
12 h 
24 h 
48 h 
72 h 
1 
Luteolin 
p-JNK 
_
 
_
 
_
 
_ 
_ 
_ 
_ 
72 h 
10  μM 
0.34 fold 
2 
Luteolin 
_
 
Caspase-3 
_
 
_
 
_ 
_ 
_ 
48 h 
_ 
60-100  μM 
32 kDa 
Luteolin 
_
 
_
 
Caspase-9 
_
 
_
 
_
 
_
 
48 h 
_
 
60-100  μM 
47 kDa 
3 
Luteolin 
_
 
_
 
_
 
cFLIPL 
6 h 
_ 
_ 
_ 
_ 
30 μM 
0.65 fold 
Luteolin 
_
 
_
 
_
 
cFLIPL 
6 h 
_
 
_
 
_
 
_
 
50 μM 
0.40 fold 
Luteolin 
_
 
_
 
_
 
cFLIPL 
_ 
12 h 
_ 
_ 
_ 
30 μM 
0.38 fold 
Luteolin 
_
 
_
 
_
 
cFLIPL 
_ 
12 h 
_ 
_ 
_ 
50 μM 
0.19 fold 
Luteolin 
_
 
_
 
_
 
cFLIPL 
_ 
_ 
24 h 
_ 
_ 
30 μM 
0.15 fold 
Luteolin 
_
 
_
 
_
 
cFLIPL 
_ 
_ 
24 h 
_ 
_ 
50 μM 
0.13 fold 

 
 
27  
 
 
No. of the study
 
Drug
 
Cytotoxicity/Cell viability (%)
 
Duration 
Dose 
Effect 
6 h 
12 h 
24 h 
48 h 
72 h 
1 
Luteolin 
_ 
_ 
_ 
_ 
72 h 
20  μM 
83.33% 
2 
Luteolin 
_ 
_ 
24 h 
_ 
_ 
60  μM 
0.12 *104 % 
Luteolin
 
_
 
_ 
24 h 
_ 
_ 
80  μM 
0.15*104 % 
Luteolin
 
_
 
_ 
24 h 
_ 
_ 
100  μM 
0.17*104 % 
Luteolin
 
_
 
_ 
_ 
48 h 
_
 
60  μM 
0.63*104 % 
Luteolin
 
_
 
_ 
_ 
48 h 
_ 
80  μM 
1.08*104 % 
Luteolin
 
_
 
_ 
_ 
48 h 
 
100  μM 
1.60*104 % 
Luteolin
 
_
 
_ 
_ 
_ 
72 h 
60  μM 
1.17*104 % 
Luteolin
 
_
 
_ 
_ 
_ 
72 h 
80  μM 
1.75*104 % 
Luteolin
 
_
 
_ 
_ 
_ 
72 h 
100  μM 
2.35*104 % 
3 
Luteolin 
6 h 
_ 
_ 
_ 
_ 
50 μM 
4.33 % 
Luteolin
 
_
 
12 h 
_ 
_ 
_ 
50 μM 
4.66 % 
Luteolin
 
_
 
_ 
24 h 
_ 
_ 
50 μM 
16.00 % 
 

 
 
28  
 
7. 2. 4. Table of excluded studies: 
No. of 
study
 
Study 1st 
Author 
(Country, 
Year)
 
Journal
 
Title of study
 
Reason of exclusions
 
Source
 
1
 
L. Gohar, 
Egypt 2016
 
International 
Journal of 
Pharmacognosy 
and 
Phytochemistry
 
Phytochemical and 
Biological Studies of 
Certain Plants 
Possessing Cytotoxic 
Activity
 
Human breast cancer 
adenocarcinoma, Human 
colon colorectal carcinoma 
and Human hepatic 
carcinoma cell lines. NOT 
OSCC, TSCC or HNSCC 
cell lines
 
www.eulc.edu.eg
 
2
 
T. Matsuta, 
Japan 2011
 
In Vivo
 
Biological Activity of 
Luteolin Glycosides 
and Tricin from Sasa 
senanensis Rehder
 
Application of Luteolin 
glycosides. NOT the 
molecular formula Luteolin 
5,7,3',4'-tetrahydroxy-
flavone
 
PubMed
 
3
 
D. Majumdar, 
USA 2014
 
Cancer Prev 
Res
 
Luteolin nanoparticle 
in chemoprevention – 
in vitro and in vivo 
anticancer activity
 
Application of luteolin in 
chemopevention. NOT as a 
chemotherapeutic drug
 
PubMed
 
4
 
R. B. Selvi, 
India 2015
 
Oncotarget
 
Inhibition of p300 
lysine 
acetyltransferase 
activity by luteolin 
reduces tumor growth 
in head and neck 
squamous cell 
carcinoma (HNSCC) 
xenograft mouse 
model
 
Detection of p300 
KAT/p300 acetyl-
transferase and cancer 
progression activity. NOT 
detecting apoptosis or 
cytotoxicity/cell viability.
 
PubMed
 

 
  
  
8. References: 
1.  
WHO | Cancer. WHO [Internet]. 2017 [cited 2017 Apr 13]; Available from: 
http://www.who.int/mediacentre/factsheets/fs297/en/#.WO8mcUySM2c.men
deley&title=Cancer&description=Cancer is a generic term for a large group of 
diseases that can affect any part of the body. 
2.  
Warnakulasuriya S. Global epidemiology of oral and oropharyngeal cancer. 
Oral 
Oncol 
[Internet]. 
2009;45(4–5):309–16. 
Available 
from: 
http://dx.doi.org/10.1016/j.oraloncology.2008.06.002 
3.  
Aruna DS, Prasad KV V, Shavi GR, Ariga J, Rajesh G, Krishna M. 
Retrospective study on risk habits among oral cancer patients in karnataka 
cancer therapy and research institute, hubli, India. Asian Pacific J Cancer Prev. 
2011;12(6):1561–6.  
4.  
Huang S-H, O´sullivan B. Oral cancer: Current role of radiotherapy and 
chemotherapy. Med Oral Patol Oral Cir Bucal Mar Mar Med Oral S L CIF B 
Med Oral Patol Oral Cir Bucal Mar [Internet]. 2013;1181818(12):233–40. 
Available 
from: 
http://www.medicinaoral.com/medoralfree01/v18i2/medoralv18i2p233.pdf%
5Cnhttp://www.medicinaoral.com/:10.4317/medoral.18772%5Cnhttp://dx.do
i.org/doi:10.4317/medoral.18772 
5.  
Neville BW. Oral and maxillofacial pathology [Internet]. 3rd ed. Elsevier; 
2009. 
984 
p. 
Available 
from: 
https://evolve.elsevier.com/cs/product/9781416034353? 
6.  
Liu H, Dong Y, Gao Y, Du Z, Wang Y, Cheng P, et al. The fascinating effects 
of baicalein on cancer: A review. Int J Mol Sci. 2016;17(10).  
7.  
Romano B, Pagano E, Montanaro V, Fortunato AL, Milic N, Borrelli F. Novel 
insights into the pharmacology of flavonoids. Phyther Res. 2013;27(11):1588–
96.  
8.  
Russo M, Spagnuolo C, Tedesco I, Russo GL. Phytochemicals in cancer 
prevention and therapy: Truth or dare? Toxins (Basel). 2010;2(4):517–51.  

 
  
  
9.  
Chen Z, Kong S, Song F, Li L, Jiang H. Pharmacokinetic study of luteolin, 
apigenin, chrysoeriol and diosmetin after oral administration of Flos 
Chrysanthemi extract in rats. Fitoterapia [Internet]. 2012 [cited 2017 Apr 
1];83(8):1616–22. 
Available 
from: 
http://www.sciencedirect.com/science/article/pii/S0367326X12002584 
10.  Kim YA, Yu JG, Lee KW, Lee HJ. Luteolin suppresses UVB-induced 
photoageing by targeting. 2013;17(5):672–80.  
11.  Miean KH, Mohamed S. Flavonoid (Myricetin, Quercetin, Kaempferol, 
Luteolin, and Apigenin) Content of Edible Tropical Plants. J Agric Food Chem 
[Internet]. 
2001 
Jun 
1;49(6):3106–12. 
Available 
from: 
http://dx.doi.org/10.1021/jf000892m 
12.  Sun T, Xu Z, Wu CT, Janes M, Prinyawiwatkul W, No HK. Antioxidant 
activities of different colored sweet bell peppers (Capsicum annuum L.). J 
Food Sci. 2007;72(2):98–102.  
13.  Seelinger G, Merfort I, Wölfle U, Schempp CM. Anti-carcinogenic Effects of 
the Flavonoid Luteolin. 2008;2628–51.  
14.  Majumdar D, Jung KH, Zhang H, Nannapaneni S, Wang X, Ruhul Amin 
ARM, et al. Luteolin nanoparticle in chemoprevention: In vitro and in vivo 
anticancer activity. Cancer Prev Res. 2014;7(1):65–73.  
15.  Ding S, Hu A, Hu Y, Ma J, Weng P, Dai J. Anti-hepatoma cells function of 
luteolin through inducing apoptosis and cell cycle arrest. Tumour Biol J Int 
Soc Oncodevelopmental Biol Med [Internet]. 2014;35(4):3053–60. Available 
from: 
http://search.ebscohost.com/login.aspx?direct=true&db=cmedm&AN=24287
949&site=ehost-live 
16.  Calixto G, Fonseca-Santos B, Chorilli M, Bernegossi J. Nanotechnology-
based drug delivery systems for treatment of oral cancer: a review. Int J 
Nanomedicine 
[Internet]. 
2014;9(1):3719. 
Available 
from: 
http://www.dovepress.com/nanotechnology-based-drug-delivery-systems-
for-treatment-of-oral-cance-peer-reviewed-article-IJN 

 
  
  
17.  Chalovich JM, Eisenberg E. NIH Public Access. Biophys Chem. 
2005;257(5):2432–7.  
18.  Takahashi Y, Hamada J, Murakawa K, Takada M, Tada M, Nogami I, et al. 
Expression profiles of 39 HOX genes in normal human adult organs and 
anaplastic thyroid cancer cell lines by quantitative real-time RT-PCR system. 
2004;293:144–53.  
19.  Journal E. Comparison Of The Sulforhodamine-B Protein And Tetrazolium ( 
MTT ) Assays For Invitro Chemosensitivity Testing. 1991;5379(February).