Apoptotic and Anti-Proliferative Effects of Licorice Extract (Licochalcone A) and Paclitaxel Chemotherapy on Human Oral Squamous Cell Carcinoma Cell Line
Phase: PHASE1
Conditions: Squamous Cell Carcinoma
Sponsor: Cairo University

Apoptotic and Anti-Proliferative Effects of 
Licorice Extract (Licochalcone A) and Paclitaxel 
Chemotherapy on Human Oral Squamous Cell 
Carcinoma Cell Line 
(In Vitro Study) 
September, 22 2017  
 
Presented by 
Mohamed Osama Mostafa 
      
 
 
 
 
 
 
 
 
 
 
 
 

List of Contents: 
Contents 
Page 
Administrative information 
5 
Title 
5 
Protocol Version 
5 
Roles and Responsibilities  
5 
Steering Committees  
6 
Assessment and Auditing 
6 
Research Ethics Approval 
6 
Access to Data 
7 
Dissemination Policy 
7 
Funding 
7 
Background and Scientific Rationale 
8 
Objectives 
10 
PICO 
11 
Research question 
12 
Material and Methods 
12 
Intervention 
12 
Materials used 
12 
Methods 
13 
Collecting samples 
13 
Sample Preparation 
13 

Testing the Samples 
15 
Outcomes 
16 
Sample size 
16 
Randomization 
16 
Statistical Methodology 
16 
Protocol Amendments 
16 
References 
17 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Administrative Information: 
1- Title: 
Apoptotic and Anti-Proliferative Effects of Licorice 
Extract (Licochalcone A) and Paclitaxel Chemotherapy 
on Human Oral Squamous Cell Carcinoma Cell Line. 
 
2- Protocol Version: 
Version (1) 
 
3- Roles and Responsibilities: 
The Principle Supervisor: 
Prof.Dr. SawsanNaguib Abdel-Bari, Professor of Oral and 
Maxillofacial Pathology, Faculty of Dentistry, Cairo University 
The Assistant Supervisor: 
Ass. Prof. Dr. Nermine Raouf Amin, Ass.Professor of Oral and 
Maxillofacial Pathology, Faculty of Dentistry, Cairo University 
The Principle Investigator: 
Mohamed Osama Mostafa, Assistant Lecturer, Oral Pathology, 
Faculty of Dentistry, Beni-Suef University. 
Investigator of cell viability assay and fluorescence microscopy: 
Dr. L. Rashed, Professor of Medical Biochemistry, Faculty of 
Medicine, Cairo University. 
Investigator of cell culturing technique: 
Dr. E.Rashwan, Head of the Confirmatory Diagnostic Unit 
(VACSERA-EGYPT). 
 
 

Steering Committees: 
1 - Oral & Maxillofacial Pathology Department Board, Faculty of 
Dentistry, Cairo University. 
2 -
 Research Plan Committee, Faculty of Dentistry, Cairo 
University. 
3 -
 Evidence Based Committee, Faculty of Dentistry, Cairo 
University. 
4 - Ethics Committee, Faculty of Dentistry, Cairo University. 
5 - Higher Education and Research Committee. 
6- 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. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Background and Scientific Rationale: 
Oral squamous cell carcinoma (OSCC) is considered the most 
common human body malignant tumor. It has poor prognosis regarding its 
distant lymph node metastasis and local destruction which lead to low 
survival rate (Pereira et al., 2007). 
 Paclitaxel is a chemotherapeutic drug of the taxols group that is used 
in the treatment of lung, ovarian and prostate cancer. It is also used to treat 
breast, head and neck cancer as well as sarcoma and leukemia. Recent 
studies and researches explored that Paclitaxel has an important role in 
mitotic arrest activation and induction, which subsequently lead to cell 
death (Weaver, 2014). 
Regarding the effect of Paclitaxel on head and neck cancer cell lines, 
Maushagen et al. (2016) noticed a time-dependent decrease in the number 
of viable cancer cells (24-48 hours) upon a fixed dose of Paclitaxel (10µM). 
Inhibition of microtubule union and assembly by Paclitaxel induces 
apoptosis and cell cycle arrest in the G2/M-phase and this leads to 
inhibition of cell replication and cancer growth (Rowinsky and 
Donehower, 1995; Zhang et al., 2014). 
 
In another study on gastric cancer, Paclitaxel induced a higher 
inhibitory effect on tumor cell proliferation than other types of 
chemotherapy (90.7% for Paclitaxel, 72.6%for Oxaliplatin and 67.7% for 
Epirubicin (Zhang et al., 2013). 
                  Regarding the apoptotic effect, a study of multiple 
concentrations of Paclitaxel on breast cancer cells has explored high levels 
of apoptotic cells (80 % – 87%) at doses of 500nM and 700nM, 
respectively for 48 hours (Armat et al., 2016). 
The conventional treatment strategies such as surgery, radiotherapy 
and chemotherapy come with low levels of expectations regarding 
prognosis and survival rate (Jemal et al., 2006). On the other hand, serious 
side effects of chemotherapy such as renal toxicity, gastrointestinal upset 
and bone marrow suppression remain hazards which cannot be avoided 
(Carr et al., 2008).Thus, there is a great need to discover new agents and 

strategies for treating OSCC or even enhancing the effect of chemotherapy 
in order to reduce the dose given. 
Complementary alternative medicine (CAM) is an additional 
modality for treating various types of cancers (Brauer et al., 2010; Lewith 
et al., 2010). Traditional medicinal plants and their derivatives have been 
widely used as effective therapeutic agents for OSCC. 
Licorice extract, Licochalcone A, is one of many natural extracts of 
Glycyrrhiza inflata plant. It has been used in treatment of inflammation 
(Shibata et al.,1991; Kwon et al.,2008), infections (Chen et al., 2001; 
Ziegler et al., 2004)and cancer (Rafi et al., 2002).  
Regarding its antitumor effect, Licochalcone A reveals interesting 
results in cell cycle arrest and apoptosis in cancer cells such as prostate (Yo 
et al., 2009), bladder (Yuan et al., 2013), colon (Lee et al., 2008) and 
gastric cancer (Xiao et al., 2011). 
 
 
 
 
 
 
 
 
 
 
 
 
 

Aim and objectives of the study 
1-  To study the effect of Licochalcone A on cell viability, cell 
proliferation and apoptosis compared with the effect of Paclitaxel 
chemotherapy in cultured OSCC cell line (SCC-15). 
 
2- To determine the role of Licochalcone A in enhancing the ant-
proliferative and apoptosis inducing effects of Paclitaxel in an 
attempt to reduce the chemotherapeutic dose in the clinical 
practice. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
PICO: 
Pobulation: Cultured human oral squamous cell carcinoma cell line 
(SCC-15). 
Intervention1: Application of Licochalcone A alone on the cultured 
(SCC-15) cell line. 
Intervention2: Application of Paclitaxel chemotherapy alone on the 
cultured (SCC-15) cell line. 
Intervention3: Application of Licochalcone A and Paclitaxel 
chemotherapy together on the cultured (SCC-15) cell line. 
Control: Cultured (SCC-15) cell line without treatment. 
Outcome: Assessment of cell viability, proliferation and apoptosis. 
 
Outcome 
Measuring 
Device/technique 
Measuring 
Unit 
 
Primary  
 
 
 
 
Cell viability 
 
 
 
 
MTT assay and Eliza reader 
  (Meerloo JV et al., 2011) 
 
Stained Viable 
Cell counts 
 
 
 
Secondary 
 
 
Proliferation 
IPO-38 
immunohistochemical 
proliferation marker via 
Image analyzer computer 
system. (Prasad K., 2011) 
Area % of  
immuno-stain. 
 
 
Tertiary 
 
 
 
 
Apoptosis  
 
 
 
Fluorescent microscopy for  
Annexin-V binding protein 
(Rieger AM et al., 2011) 
 
 
 
Stained 
apoptotic Cell 
counts 
 
 
 

Research Question: 
Does Licochalcone A enhance the anti-proliferative and apoptosis 
inducing effects of Paclitaxel chemotherapy on cultured oral squamous 
cell carcinoma cell line? 
 
Material and Methods 
A- Intervention: 
Squamous cell carcinoma cell line (SCC-15) cultured with 
Licorice extract (Licochalcone A) and chemotherapeutic drug 
(Paclitaxel). 
 
B- Materials: 
1. Squamous cell carcinoma cell line (SCC-15) from 
Veterinary Serum & Vaccine Research Institute 
(VACSERA) (51 Wezaret EL Zeraa Street, Agouza, Giza, 
Egypt). 
2. Licorice extract ( Licochalcone A) obtained from Sigma-
Aldrich company ( Eschenstrasse 5,82024, Taufkirchen, 
Germany). 
3. Paclitaxel chemotherapeutic reagent from The Egyptian 
Pharmaceutical Trading Company (1353 Nile Kornish, 
Shobra, Cairo, Egypt). 
4. Annexin V-FITC Apoptosis Staining / Detection Kit from 
abcam company, KEMET Medical (14a El Emam Aly St Off 
Orouba St - Ismailia Square, Heliopolis, 11341). 
5. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium 
bromide (MTT) solution from Sigma-Aldrich company  
(Eschenstrasse 5,82024, Taufkirchen, Germany). 
6. IPO-38 proliferation marker from US Biological life science 
company (4 Technology Way Salem, MA 01970, Boston, 
United States). 
 
 
 
 
 

C- Methods: 
Squamous cell carcinoma cell line will be cultured alone, with 
Licochalcone A,with Paclitaxel chemotherapeutic agent and 
with Licochalcone A and Paclitaxel together. 
 
D- Collecting samples: 
Squamous cell carcinoma cell lines will be purchased from 
Veterinary Serum & Vaccine Research Institute(VACSERA). 
 
• Inclusion criteria: 
Human squamous cell carcinoma cell line 
 
• Exclusion criteria: 
1.Animal cell lines  
2. Cell lines other than squamous cell carcinoma cell lines. 
 
E- Cell culture (Sample) Preparation: 
• The stored cell line will be removed from the liquid nitrogen 
container and thawed in a water bath at 37ºC. 
• Then cells will be cultured in modified Eagle’s 
medium(MEM) supplemented with heat inactivated fetal 
bovine serum (FBS), sodium bicarbonate and 2% 
streptomycin penicillin in flasks at 37ºC in a 5% CO2 
incubator. 
• The culture flasks were examined under the inverted phase 
contrast microscope to assure viability and sterility. 
Sub-culturing: 
• Trypsin will be added to the flask and turned over for 15 
seconds- 2 minutes. 
• (MEM) medium then will be added to neutralize the action 
of trypsin. 
• Cultured cells will be re-suspended in newly fresh medium 
and centrifuged at 2000 rpm for 10 minutes to obtain a cell 
pellet. 
• Cell pellet will then be dispersed and re-suspended in fresh 
complete culture media and diluted to the appropriate 
seeding concentration in the culture flask. 
 

• Cells will be sub-cultured in a 96-well plate and incubated at 
37ºC in the 5% CO2 incubator. 
• Then well – plates will be treated with two doses of 
Licohalcone A (3mg, 6mg) and Paclitaxel chemotherapy 
(8mg, 16mg) for 24, 48 and 72 hours.  
• The control group will be left without treatment. 
• Study Design Table: 
 
Intervention 
 
 
Groups 
 
Dose (mg) 
 
Duration of Treatment (hours) 
 
 
 
 
Control 
group 
 
 
    Untreated 
 
 
                  No duration 
 
 
 
 
LicochalconeA 
group 
 
 
 
 
 
3 mg 
 
 
 
6 mg 
 
 
 
24 
 
 
 
48 
 
 
 
72 
 
 
 
Paclitaxel 
group 
 
 
8 mg 
 
 
16 mg 
 
 
24 
 
 
48 
 
 
72 
 
Licochalcone A 
and Paclitaxel 
group 
 
3 mg +    
8 mg 
 
 
6 mg +    
16 mg 
 
 
 
24 
 
48 
 
72 
 
 
 
 
 
 

F- Paraffin embedded cell line pellet preparation: 
• Cell suspension will be spanned at 1800 rpm for 10 minutes 
at room temperature. 
• Supernatant decanted 
•  5 ml of 70% ethanol is added for 30 minutes and vortexed 
mildly. 
• Suspension is spanned at 1800 rpm for 10 minutes at room 
temperature. 
• Supernatant decanted 
• 5 ml of 100% ethanol will be added for 30 minutes and 
vortexed mildly. Optional: Overnight incubation in 100% 
ethanol at 4°C makes a very solid pellet 
• Suspension will be spanned 1800 rpm for 10 minutes at 
room temperature. 
• Supernatant decanted. 
• Using a clean wooden applicator stick, cell pellet will be 
pulled out carefully of the tube and into a cassette lined with 
black biopsy filter paper. 
• The cassette will be placed in a tissue processor. 
• The pellet sample is processed and embedded in paraffin 
within 24 hours of preparation. 
 
G- Testing the samples: 
 
1- Cell viability will be measured by the MTT assay by adding 
MTT solution to every treated well plate then incubated with 
5 % CO2 at 37 °C. Records of viable cells will be obtained 
by the ELISA reader. 
 
2- Area percent of proliferation marker IPO-38 
immunohistochemical staining will be measured by image 
analyzer computer system. IPO-38 will be applied on slides 
sectioned from paraffin embedded cell line pellet 
 
3- Apoptosis will be measured by Annexin-V binding protein 
to apoptotic cells in a multi well fluorescence plate reader. 
Fluorescence of each well will be recorded and images will 
be taken under the fluorescent microscope. 
 

H- Outcome: 
• Primary: Cell viability by MTT assay measured by the 
ELIZA reader 
• Secondary: Cell proliferation by measuring the area percent 
of immunohistochemical staining of proliferation marker 
IPO-38. 
• Tertiary:  percentage of apoptotic cells to the total count of 
all viable cells via Annexin V binding protein measured by 
fluorescence microscopy. 
 
I- Sample size: 
             Based on (Cumming et al., 2007) and (Lazic SE., 2010), when 
cell lines are used, there isn't any biological replication, only technical 
replication, and it is important to have this replication at the right level in 
order to have valid inferences. For every single cell line intervention, 6 
times applications of drug dose in 6 well plates will be sufficient for every 
outcome. 
J- Randomization: 
Randomization of the cell line will not be done as the count of 
cells in each well plate is standardized and contains the same 
number of cells (Amiralaet al., 2013). 
 
 
K- 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. 
 
 
L- Protocol amendments: 
Any modifications to the protocol which may affect the 
progress of the study, including changes of study objectives, 
study design, sample sizes or study procedures will require a 
formal amendment to the protocol. Such amendment will be 
agreed upon by the Council of Oral pathology department. 
 

Search strategy: 
1.Title: 
Apoptotic and Anti Proliferative Effects of Licorice Extract 
(Licochalcone A) and Paclitaxel Chemotherapy on Human Oral 
Squamous Cell Carcinoma Cell Line. 
2. Structured Summary: 
Objective: To study the effects of Licochalcone A on cell 
proliferation and apoptosis in cultured OSCC cell lines. 
Data Sources:   In-vitro study articles in PubMed and Cochran 
data bases. In addition, using of Egyptian universities libraries web site: 
www.eulc.edu.eg. Several search items or terms were used as: 
Licochalcone , licorice extract , OSCC and Paclitaxel.  
Study Selection: the selected articles include In-vitro studies only 
that discuss the uses and activities of licorice extract (Licochalcone) in 
treating any type of head and neck squamous cell carcinoma cell lines by 
apoptotic induction or cytotoxic effect of cancer cells and to compare that 
with similar actions of traditional chemotherapeutics.  
 Data Extraction: achieved according to the study selection criteria 
from predefines data bases. 
Data Synthesis: after searching and date analysis, 4 articles were 
obtained and they met the selection criteria of data search. 3 studies of 
them reveal that licorice extract (Licochalcone) has an apoptotic effect on 
different types of OSCC cell lines by inducing cytotoxicity to the cancer 
cells and subsequent reduction in its viability. By using different 
successive doses of Liochalcone A, (25, 50 ,100 ,125 ,200 µg/mL or µM) 
, the apoptotic effect increase on time dependent manner ( 12, 24 ,48 

hours), with clear decrease in viable cancer cells (78%, 80%, 48% 
and19%) respectively . the last fourth article explain the apoptotic effect 
of paclitaxel chemotherapy, and it will be used as a reference in our 
comparison between Licochalcone A extract and conventional 
chemotherapy. 
Limitations: limited included studies with insufficient data. 
Conclusions: on higher concentrations, Licochalcone A appear to 
be effective apoptotic and cytotoxic natural extract for OSCC cell lines. 
Funding: self – funded. 
3: Rationale: 
Oral squamous cell carcinoma (OSCC) are considered as a one of 
the most malignant cancers. It has prognosis due to its distant metastasis 
and obvious destruction which lead to low survival rate.  
The conventional treatment modalities such as surgery, radiotherapy 
and chemotherapy are only the suitable strategies until now. We cannot 
ignore the serious side effects of chemotherapy such as gastrointestinal 
upset, bone marrow suppression which cannot be overcome. For all these 
complications, there is a great need and demand to discover a new agents 
and strategies for treating OSCC. 
Licorice extract, especially Licochalcone A, is one of many natural 
extracts that have used as a traditional in treatment of inflammation, 
microbial infections. Regarding antitumor effect, Licochalcone A reveals 
interesting results in programmed cell death and apoptosis in cancer cells 
such as prostate, bladder, colon and gastric cancer. 
 
 
 

4.Objectives: 
1- To study the effects of Licochalcone A on cell proliferation and 
apoptosis in cultured OSCC cell lines trying to support the basis 
for possible potential application in the clinical practice of OSCC 
treatment. 
2- To determine and distinguish between different effects of natural 
extract (Licochalcone A) and conventional chemotherapy 
(taxols). 
 
5: Protocol and Registration: 
 
6: Eligibility criteria: 
 
Type of the study : In-vitro study of the therapeutic effect of 
natural extract (Licochalcone A) on the oral squamous cell 
carcinoma cell lines and comparing it with the conventional 
paclitaxel chemotherapy. 
 
Type of participants: cultured oral squamous cell carcinoma cell 
line. Any type other than head and neck squamous cell carcinoma 
and oral squamous cell carcinoma will be excluded. 
 
Type of intervention 1: Application of Licochalcone A. 
Type of intervention 2: Application of Paclitaxel chemotherapy. 
Type of intervention 3: Application of Licochalcone A with 
Paclitaxel chemotherapy 
Type of control:   Cultured cells without intervention. 
Type of outcome measures: 
• Apoptosis: gene expression of annexin -V 
• Proliferation: by cell viability and immuonexpression of  
IPO38 marker. 
 
 
 

Inclusion criteria: 
1- All head and neck squamous cell carcinoma cell lines  
2- Licochalcone extracts  
3- Taxols group of chemotherapy  
4- English language  
 
Exclusion criteria: 
1- Animal experimental studies  
2- Any cancer cell line other than squamous cell carcinoma cell 
lines. 
3- Any chemotherapeutics other than Taxols group. 
4- Non-English studies. 
5- Systematic review 
 
7: Information sources: 
 
The search done through electronic data bases: PubMed and 
Cochrane data bases according to the inclusion and exclusion 
criteria, the date of search was 10/1/2017. In addition to that, 
hand search was done by searching in the library of the faculty 
of oral and dental medicine, Cairo university. Also, searching 
using the website of the Egyptian Universities Libraries 
Consortium www.eulc.edu.eg to figure out any related articles 
to this study. 
 
 
 
 
 
 
 
 
 
 
 

 
8: Search: 
 
Index Term  (synonyms) 
PubMed Cochrane 
#1 
Oral squamous cell carcinoma  
1624 
5 
#2 
Head and neck squamous cell carcinoma 
2139 
4 
#3 
Oral cancer 
6002 
36 
#4 
HNSCC 
654 
63 
#5 
Licochalcone A 
17 
3 
#6 
licorice 
106 
13 
#7 
paclitaxel 
236 
4 
#8 
Taxol 
239 
2 
#9 
(oral squamous cell carcinoma) or (head and neck 
squamous cell carcinoma) or (oral cancer) or 
(HNSCC) 
7445 
33 
#10 
(Licochalcone A) or (licorice) 
110 
15 
#11 
(Paclitaxel) or (taxol) 
240 
4 
#12 
(oral squamous cell carcinoma) or (head and neck 
squamous cell carcinoma) or (oral cancer) or 
(HNSCC) AND (Licochalcone A) or (licorice) 
91 
39 
#13 
(oral squamous cell carcinoma) or (head and neck 
squamous cell carcinoma) or (oral cancer) or 
(HNSCC) AND (paclitaxel) or (taxol) 
 
150 
39 
#14 
(oral squamous cell carcinoma) or (head and neck 
squamous cell carcinoma) or (oral cancer) or 
(HNSCC) AND (Licochalcone A) or (licorice) OR 
(oral squamous cell carcinoma) or (head and neck 
squamous cell carcinoma) or (oral cancer) or 
(HNSCC) AND (paclitaxel) or (taxol) 
 
241 
39 
 
 
 
 
 
 

 
 
 
 
 
 
 
 
 
 
 

 
         PRISMA flow diagram of study selection: 
 
 
 
Records identified through 
PubMed searching  
(n = 241) 
        Screening 
           Included 
            Eligibility 
   Identification 
Records identified through 
Cochrane searching  
(n =39) 
Records after duplicates removed  
(n =251) 
Records screened 
according to the title and 
abstract 
(n =251) 
Records excluded  
(n = 246) 
Full-text articles assessed 
for eligibility  
(n = 5) 
Full-text articles excluded, 
with reasons  
(n = 1) 
Studies included in 
quantitative synthesis 
(meta-analysis) 
(n = 4) 
additional records 
identified through other 
sources 
(n =0) 

9: Study Selection:  
 
Data screening was started with 251 articles on the basics of title and 
abstract and 246 articles were excluded according to the exclusion 
criteria that restricted the choice only to the head and neck squamous 
cell carcinoma cell lines and exclude any other cell lines. The 
remaining five articles were screened by full text and only one article 
(systematic review) was excludes because it discusses other types of 
licorice extract and not Licochalcone extract groups. 
 
10: Data collection process: 
All data from the selected articles are refined and extracted into data 
search tables.  
11: Data Items: 
The extracted data include demographic data including article’s title, 
author name, journal and the year of publication. Methodology and 
intervention also extruded including the dose, material and duration. 
In addition, outcome measures are also assessed regarding Apoptosis, 
cytotoxicity and viability. 
 
 
 
 
 
 
 
 
 
 

Articles data:
 
Study
name
  
Author
 
Type of 
the study
Year
Journal
 
(1)
A polysaccharide from Glycyrrhiza inflata Licorice 
inhibits proliferation of human oral cancer cells by 
inducing apoptosis via 
mitochondrial pathway
Huan Shen. et al 
In vitro 
2015
International Society of 
Oncology and BioMarkers
(2)
Licochalcone-A Induces Intrinsic and Extrinsic 
Apoptosis via ERK1/2 and p38 Phosphorylation-
mediated TRAIL Expression in Head and Neck 
Squamous Carcinoma FaDu Cells
 
Mi-Ra Park. et al
 
In vitro
 
 
2015
 
Food Chem Toxicol
(3)
Licochalcone B induces apoptosis of human oral 
squamous cell carcinoma through the extrinsic- 
and intrinsic-signaling pathways
Hana Oh. et al
In vitro
2015
INTERNATIONAL 
JOURNAL OF 
ONCOLOGY
(4)
Effects of paclitaxel on permanent head and neck 
squamous cell carcinoma cell lines and 
identification of anti-apoptotic caspase 9b
Maushagen R. et al
In vitro
2016
Journal of Cancer 
Research and Clinical 
Oncology
 
 
 

• Material and methods: 
No. of 
the 
study
 
Type of samples    
(cell line)
 
control
 
Natural 
extract
 
Chemot
herapy
 
Western blot analysis
 
Immunohistochemistry 
 
Doses  µM 
 
Duration
 
groups
 
antibodies
 
Dose 
 
Mg /kg
 
Duration
 
Groups
 
Antibody
 
 
1 
 SCC-25
 
1
 
 
Glycyrrhiza 
inflata
 
nil
 
147
 
295
 
591
 
48 h
 
nil
 
Bax
 
Bcl 2
 
PARP
 
     
 
Not used
 
 
2 
FaDu -SCC cell 
 
1
 
Licochalcon
e- A
 
nil
 
 
50
 
100
 
24 h
 
nil
 
TRAIL
 
PARP
 
CASPASE-
3
 
10
 
3 times per 
week for 8 
weeks
 
5 µm thick 
sections
 
Caspase-3
 
 
3 
HN22 
HSC4 
 
2
 
Licochalcon
e-B
 
nil
 
10
 
20
 
30
 
NA
 
nil
 
Cyclin D
 
P21
 
P27
 
Bax
 
Bcl2
 
PARP
 
    
 
 
 
 
Not used
 
 
 
4 
UT-SCC:24-A,24-
B,60-A,60-B
 
 
 
4 
nil
 
paclitax
el
 
 
 
 
10 µM
 
 
NA
 
 
 
 
4
 
 
 
 
Caspase 9
 
Caspace 9b
 
 
 
 
 
 
Not used
 
 
 
 

No. of 
the 
study
 
Type of 
samples    
(cell 
line)
 
cont
rol
 
Natur
al 
extrac
t
 
Chem
othera
py
 
Cell viability assay
 
 
Cell apoptosis assay  
 
(Flow cytometric analysis)
 
 
(MTT assay)         
 
 
 
 
(Trypan blue assay) 
 
Doses µM 
 
duration 
 
groups 
 
material 
 
Doses µM 
 
duration 
 
groups 
 
material 
 
Doses µM
 
Duration
 
groups
 
material
 
 
 
 
 
 
 
 
 
 
1 
 SCC-25
 
    1
 
 
Glycyr
rhiza 
inflata
 
nil
 
147
 
 
 
295 
 
 
591
 
 
48 h
 
96-
well 
plates
 
20 
µg/mL 
of MTT 
soloutio
n
 
 
 
 
 
 
Not used 
147
 
 
 
295 
 
 
591
 
 
48 h
 
nil
 
AnnexinV 
FITC/PI
 
 
2 
FaDu -
SCC cell 
 
1
 
Licoch
alcone
- A
 
nil
 
 
25
 
50
 
100
 
125
 
24 h
 
96-
well 
plates
 
20 
µg/mL 
of MTT 
soloutio
n
 
100 µM
 
12 h 
 
24 h 
6- well 
plates
 
AnnexinV 
FITC/PI
 
 
3 
HN22 
HSC4 
 
2
 
Licoch
alcone
-B
 
nil
 
 
10
 
20
 
30
 
 
 
 
 
10
 
20
 
30
 
48 h
 
12-
well 
plates
 
AnnexinV 
and dead 
cell kit
 
Not used  
 
 
24
h 
48
h 
12-
well 
plates 
Trypan 
blue 
solution 
 
 
4 
UT-
SCC:24-
A,24-
B,60-
A,60-B
 
 
 
4 
nil
 
paclitaxel
 
 
 
Not used
 
 
          10 µM
 
24 h
 
6 well 
plates
 
Annexin 
V/PI
 
• Results: 

 
No. 
of 
study 
 
Cytotoxicity /Viability 
Drug 
Duration 
(hours) 
Dose 
µM 
Effect 
(cells) 
Duration 
(hours)   
Dose 
µM 
Effect 
(cells) 
Duration 
(hours) 
Dose 
µM 
 
Effect 
(cells) 
Duration 
(hours) 
Dose 
µM 
Effect 
(cells) 
Control 
(cells) 
 
1 
Glycyrrhiza 
inflata 
 
48 
 
591 
 
4900 
 
48 
 
 
295  
 
 
6000 
 
48 
 
 
147  
 
7000 
 
- 
 
- 
 
- 
 
10000 
 
 
2 
 
 
Licochalcone- 
A 
24 
25 
78000 
24 
50  
80000 
24 
100  
48000 
24 
125  
19000 
1x105 
 
3 
Licochalcone-
B 
24 
10 
42000 
24 
20  
45000 
48 
 
20  
34800 
48 
 
30  
40800 
6x104 
 
4 
paclitaxel 
24 
10 
175000 
24 
10  
225000 
48 
 
10  
157500 
48 
 
10  
205000 
250000 
 

 
No. 
of 
study 
 
Apoptosis 
Drug 
Duration 
(hours) 
Dose 
µM 
Effect 
(cells) 
Duration 
(hours) 
Dose 
µM 
Effect 
(cells) 
Duration 
(hours) 
Dose 
µM 
Effect 
(cells) 
Duration 
(hours) 
Dose 
µM 
Effect 
(cells) 
Control 
(cells) 
 
1 
Glycyrrhiza 
inflata 
 
48 
147  
 
3270 
48 
295 
4140 
48 
591 
5200 
- 
- 
- 
10000 
 
 
2 
 
 
Licochalcone- 
A 
12 
100 
32120 
24 
100 
35650 
- 
- 
- 
- 
- 
- 
1x105 
 
3 
Licochalcone-
B 
48 
10 
7572 
48 
20 
28368 
48 
30 
50016 
- 
- 
- 
6x104 
 
4 
paclitaxel 
- 
10 
75000 
- 
10 
117500 
- 
- 
- 
- 
- 
- 
250000 
 

List of included studies: 
 
Author,date 
Database 
Park MR et al (2015) 
PubMed 
Huan Shen et al (2015) 
PubMed 
Oh H et al (2016) 
PubMed 
Maushagen R (2016) 
PubMed 
 
 Excluded studies: 
Title 
Author 
Reason for exclusion 
A Systematic Review of 
the Anticancer Properties 
of Compounds Isolated 
from 
Licorice (Gancao) 
Tang ZH et al (2015) 
Systematic review 
 
 
 
 
 
 
 
 
 
 
 
 

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