




































CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

44  

  

 
 

Abstract: Our goal is to learn how Qingjie Fuzheng Granule (QFG) and 5-fluorouracil (5-FU) work together to treat colorectal 

cancer. Research Tools and Procedures: Human colorectal cells and xenograft mice were utilized to study the synergistic anti-

cancer capacity of QFG and 5-FU, which targets the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) signaling 

pathway. Chou-Talalay was employed to generate this index. Findings: The growth of HCT-8 cells was suppressed by a 

synergistic effect of QFG and 5-FU. The combination of QFG with 5-FU in HCT-8 cells and mice resulted in apoptosis induced 

by regulation of associated proteins, inhibition of proliferation, migration, and invasion. In addition, enzymes involved in 5-FU 

metabolism, including thymidylate synthase and dihydropyrimidine dehydrogenase, were down-regulated by QFG and 5-FU. 

Furthermore, by inhibiting the PI3K/AKT signaling pathway, it eased cancer-related behaviors such as migration, invasion, and 

proliferation. Findings: By inhibiting the PI3K/AKT signaling pathway, the anti-cancer actions of QFG and 5-FU are enhanced.  

 

Keywords: Phosphatidylinositol 3-kinase/protein kinase B signaling pathway, Qingjie Fuzheng granule, 5-fluorouracil, 

colorectal cancer, synergistic impact 

 

 

Chinese Traditional Medical Journal 

 

Qingjie Fuzheng Granule and 5-fluorouracil Inhibit Colorectal Cancer Growth in a 

Synergistic Manner via the Phosphatidylinositol 3-Kinase/Protein Kinase B Pathway 

Rajesh Kumar and Ravi Prasad 
1,2 Academy of Acupuncture and Moxibustion, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian 

350122, China 

Received on:  21 Oct 2024   Revised on: 20 Nov 2024    Accepted Date: 25 Dec 2024  
Published on: 18 Jan 2025 

 

 

 

INTRODUCTION  

A malignant tumor of the gastrointestinal tract, colorectal 

cancer (CRC) has a high incidence rate and ranks second or 

third in terms of fatality. [1] Although surgery is now the 

gold standard for treating CRC, individuals who have 

postoperative recurrence, missed surgical opportunities, or 

metastatic disease still primarily get chemotherapy. (2, 3). 

One of the most frequent chemotherapy medications used to 

treat colorectal cancer is 5-fluorouracil (5-FU). As a 

chemotherapeutic treatment that depends on the cell cycle, 

5-FU prevents tumor cell growth by interfering with DNA 

synthesis, which in turn stops cell cycles. [5] Although 5-

FU is very effective against cancer, it has been used with 

caution due to its adverse effects, which may harm healthy 

organisms and lower patients' quality of life when taken in 

large quantities over an extended length of time. In addition, 

therapeutic efficacy is further diminished as a result of 

primary and secondary chemotherapy resistance. [6] 

Therefore, there is an immediate need to develop better 

therapies to increase the effectiveness of chemotherapy and 

enhance the quality of life for patients with colorectal 

cancer.  

 

The growth and chemoresistance of colorectal cancer cells 

are regulated by the phosphatidylinositol-3-kinase/protein 

kinase B (PI3K/AKT) signaling system. [7] In a typical 

scenario, extracellular signals trigger PI3K activity, which 

in turn activates AKT. Finally, CRC cell survival is 

enhanced by blocking apoptosis via the negative regulation 

of B-cell lymphoma-2-associated X protein (Bax) by 

activated AKT. [8] At the same time, AKT activation 

triggers EMT, which improves CRC cell migration and 

invasion and makes tumor spread easier. [9] Therefore, 

reducing PI3K/AKT activity is helpful for many reasons, 

including making CRC cells more sensitive to 

chemotherapy, reducing their ability to proliferate and 

metastasize, and triggering cell death. [10] 

 
 

 

 

 



45 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

45  

  

  

Utilizing two or more medications in conjunction with one 

another or in a certain order to accomplish therapeutic 

objectives is known as combination therapy, and it is a 

commonly used method in the treatment of many disorders. 

the eleventh This approach not only improves results by 

making the treatments more effective, but it also lessens their 

side effects and slows the development of drug resistance. A 

synergistic effect occurs when the effects of several 

medications work together. One way to classify a synergistic 

impact is by its strength; there are two main types: additive 

and reinforcing. The combined impact of the two 

medications is what is meant by an additive effect. To have 

an augmentation effect, all of the parts working together have 

a stronger impact than each one working alone. Drug 

combinations may be quantitatively defined by Chou-

Talalay's combination index (CI), which measures the 

additive (CI = 1), synergistic (CI < 1), and antagonistic (CI > 

1) effects. [12] Finding effective chemoprotectors and 

potentiators, decreasing chemo toxicities, and increasing 

chemotherapy sensitivity are all critical issues that must be 

addressed immediately.  

The Chinese herbal remedy Qingjie Fuzheng Granules 

(QFG) contains roasted malt, Astragalus membranaceus, 

Hedyotis diffusa, and Scutellaria barbata. Some of the 

qualities possessed by these herbs include the ability to 

remove heat, eliminate toxins, boost vitality, and eliminate 

food. Inducing apoptosis and reducing proliferation via the 

PI3K/AKT signaling pathway, prior research has shown that 

QFG has an anti-cancer effect on CRC cells. In the treatment 

of colorectal cancer, clinical findings have shown that the 

chemotherapeutic effectiveness is enhanced when 

5-FU-based chemotherapy regimens are coupled with QFG. 

References [13,14]: Nevertheless, the mechanism of 5-FU 

coupled with QFG in CRC is not yet the subject of any 

investigation. This study's overarching goal is to learn more 

about the possible mechanism of action of 5-FU with QFG in 

CRC and their synergistic anti-tumor impact. 

 

MATERIALS AND METHODS 
Roswell Park Memorial Institute (RPMI) 1640 medium 

(c11875500bt), fetal bovine serum (FBS) (10099141), 

penicillin and streptomycin (15140122) were obtained from 

Life Technologies; crystalline violet (g1062, Solarbio); Cell 

Counting Kit 8 (CCK8) kit (KGA3111018), Annexin‑V 

FITC/PI kit (KGA108) and terminal deoxynucleotidyl 

transferase‑mediated dUTP‑biotin nick‑end labeling 

(TUNEL) kit (KGA704) were required from KeyGene 

Biotech Co., Ltd. (Nanjing, China); Transwell chamber 

(3422, Corning Inc.); Antibodies to Bax (50599-2), 

Bcl‑2 (12789‑1), CDK4 (11026‑1‑AP), cyclin D1 (60186), 

p21 (10355‑1‑AP), Vimentin (10366‑1‑AP),  PI3K 

(60225‑1), and GAPDH (60004) were purchased from 

Proteintech (Rosemount, IL, USA). N‑cadherin (14215s), 

E‑cadherin (14472s), cleaved caspase‑3 (9664), AKT (4685S), 

p‑AKT (4060S) were purchased from Cell Signaling 

Technology, Inc., (Danvers, MA, USA). The enhanced 

chemiluminescent substrate for horseradish peroxidase (HRP) 

activity was acquired from US Everbright (S6009S, U.S. 

Everbright, China). Other reagents, except for explicitly 

mentioned, were obtained from Nest (Wuxi, China). 

Cell culture 
HCT‑8 and HCT‑116 cells were cultured in RPMI 1640 

complete medium which contained 10% FBS, 100 U/mL 

penicillin and 100 g/mL streptomycin. When the cell 

confluence reached 80%~90% and the cells were subcultured. 

Animals 
A total of 40 SPF‑grade male BALB/c nude mice, acquired 

from Slike Co. Ltd., (Shanghai, China), weighting 18–20 g, 

were kept independently ventilated animal cage. The mice 

were allowed to eat and drink ad libitum. All experimental 

procedures were performed in accordance with the Guidelines 

for Animal Experimentation of Fujian University of 

Traditional Chinese Medicine and were approved by the 

Fujian Institute of Traditional Chinese Medicine Animal Ethics 

Committee (FJTCM IACUC 2021018). 

Cell viability assay 
HCT-8 and HCT-116 cells were inoculated in 96-well 

plates (100 L/well) at a density of 1.0 × 105 cells/mL, and 

there are 6 replicates in each group. The cells were cultured 

overnight at 37°C, 5% CO2, and saturated humidity. Afterward, 

treatment with different drugs for 24 h or 48 h, respectively. 

The culture supernatant was then removed, and a 10% CCK8 

solution was prepared by mixing 1 mL CCK8 stock solution 

with 9 mL phosphate‑buffered saline (PBS). Then 100 L of 

the CCK8 solution was added to each well and incubated for 

2 h at 37°C. Finally, the absorbance at 450 nm was measured 

using an enzyme marker. Cell viability (%) was calculated as 

follows: Cell viability (%) = A value of experimental group/A 

value of control group × 100% 

Drug interaction analysis 
According to the unified theory of Chou-Talalay, the drug 

interaction between QFG and 5‑FU was calculated with 

CompuSyn software (ComboSyn, Inc., New York, NY, 

USA).[15] Different ratio of QFG and 5‑FU [Table 1] were used to 

quantify the CI, which provides a quantitative representation of 

pharmacological interactions to assess drug synergy combination 

treatments. CI <1 indicates the synergistic effect of QFG and 5‑FU. 

In vivo nude mouse transplantation tumor study 
A total of 4 × 106 HCT-8 cells, mixed 1:1 (V:V) with 

Matrigel™, were subcutaneously injected in the right flank of 

the mice to initiate tumor growth. When the tumors reached 

3 mm in diameter, the mice were randomly divided into four 

groups (n = 10) according to the size of the tumors: Control 

group, QFG group (1 g/kg), 5‑FU group (25 mg/kg), and 

QFG + 5‑FU group (1 g/kg + 25 mg/mL). The drugs were 

administered immediately according to the group assignment. 

The QFG group was given QFG by gavage at a dose of 



46 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

46  

  

  

 
160:1 0.125:7.81×10−4 

0.25:1.56×10−3 

80:1 0.125:1.56×10−3 

0.25:3.13×10−3 

40:1 0.125:3.13×10−3 

0.25:6.25×10−3 

20:1 0.125:6.25×10−3 

0.25:1.25×10−2 
 

QFG: Qingjie Fuzheng Granule, FU: Fluorouracil 

 

1 g/kg. The 5‑FU group was given 5‑FU intraperitoneally 

at 25 mg/kg. QFG + 5‑FU group was given QFG at 1 g/kg 

orally, meanwhile, given 5‑FU intraperitoneally at 25 mg/ 

kg. The control group was given the same volume of saline. 

QFG was administered once a day for 6 consecutive days per 

week; 5‑FU was injected once every other day. The length (L) 

and width (W) of the tumors were recorded every other day, 

and calculated the tumor volume as the following formula: 

Tumor volume = /6 × L × W2. During the experiment, the 

weight of each nude mouse was recorded. At the end of the 

experiment, the animals were anesthetized, the tumors were 

excised and weighed. 

Colony formation 
Treatment with QFG (0.25 mg/mL), 5‑FU (3.13 g/mL) and 

the combination for 24 h. Control cells (untreated) were also 

processed in parallel. Then 500 cells/well were collected and 

seeded into 12‑well plates and incubated for 8 days, fixed with 

4% paraformaldehyde (1 mL/well) for 20 min and stained with 

0.1% crystal violet for another 20 min. Finally, the number of 

clones was counted. 

Flow cytometry 
Treatment with different drugs for 24 h, 1 × 106 cells were 

collected, and re-suspended in 500 L of 1× binding buffer 

followed by washed twice with cold PBS. Annexin V‑FITC 

and PI (both 5 L) were added and incubated for an additional 

15 min in the dark. For each sample, 10,000 events were 

collected and analyzed. 

4’,6‑Diamidino‑2‑phenylindole staining assay 
Twenty-four hours after treated with different drugs, 

4’,6‑Diamidino‑2‑phenylindole (DAPI) staining was 

performed. In brief, the cells were fixed with 4% 

paraformaldehyde for 15 min, followed by three washes 

with PBS. Next, 10% DAPI staining solution was added 

and incuated for 15 min in the dark, then imaged using a 

fluorescence microscope (×200). 

Migration and invasion assay 
Twenty‑four hours after treated with different drugs, 50,000 

HCT-8 cells were seeded in the upper chamber of transwell 

plates. Matrigel coating was applied for the invasion assay, 

while no Matrigel was used for the migration assay. The upper 

chamber contained RPMI‑1640 basic midium, and the lower 

chamber contained 700 L of RPMI‑1640 with FBS. The plates 

were incubated at 37°C for 14 h. Then, the both the upper and 

lower chambers were fixed with 4% paraformaldehyde for 

20 min, and stained with crystal violet for 15 min. Finally, 

cells in the lower layer of chamber was photographed using 

an inverted microscope (×200). 

Western blot 
The proteins were extracted using radioimmunoassay buffer 

containing inhibitor cocktail (Thermo Fisher Scientific, USA). 

The samples were then centrifuged and obtain final supernatants. 

The concentration of total protein was detected with BCA (bovine 

serum albumin) protein assay kit (Thermo Fisher Scientific, USA). 

The proteins (30 g) that were denatured at 100°C in a metal 

bath were resolved by sodium dodecyl sulfate-polyacrylamide 

gel electrophoresis and electro-transferred to polyvinylidene 

difluoride membranes, sealed with 5% milk and incubated 

with primary antibodies (1:1000) at 4°C overnight. After the 

membranes were incubated with the corresponding secondary 

antibodies (HRP‑conjugated 1:5000) for 1 h at 25°C, the Image 

Lab (Bio‑Rad Laboratories, Inc., Berkley, California, USA) was 

used to detect proteins. 

Immunohistochemistry 
After inactivation of endogenous peroxidase with 

3% hydrogen peroxide and blocked with goat serum 

(G9023, Sigma), the slides were incubated with primary 

antibodies against Ki‑67 (1:200) at 4°C overnight. On the 

following day, the slides were incubated with biotinylated 

secondary antibodies (KIT‑9710, MXB Biotechnologies, 

China) and streptavidin peroxidase (KIT‑9710, MXB 

Biotechnologies China) at 37°C, respectively. After for a 

30 min incubation, the slides were washed 3 times with 

PBS before each reagent change. Finally, the sections were 

stained using a DAB kit (DAB-0031, Solarbio, China) and 

hematoxylin was used for restaining for 1 min (G1140, 

Solarbio, China). Five random fields from each slice were 

imaged at ×400 magnification. The positive expression 

in tumor tissues was quantified using a true-color 

multifunctional cellular image analysis system (Image‑Pro 

Plus, Media Cybernetics). 

TUNEL staining 
The tumor tissues, fixed with 4% paraformaldehyde, were treated 

with Triton X-100, and then sealed with goat serum. They were 

subsequently incubated with TUNEL immunofluorescence reagent. 

After stained with DAPI, the tissues were photografted using a 

confocal laser scanning microscope (ZEISS, Germany). 

Optimal time for phosphatidylinositol 3‑kinase/protein kinase 

B pathway activation 
After 10 h’ culture, HCT‑8 cells were changed into RPMI 1640 blank 

medium for 24 h. Subsequently, 10% FBS was added at 0, 5, 15, 

30, 60 and 120 min, respectively, then p‑AKT was analyzed by 

Western blot. 

 

 

 

    

      

 



47 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

47  

  

  

 

The combined effect of Qingjie Fuzheng Granule and 
5‑fluorouracil on survival, apoptosis, migration, and 
invasion of HCT‑8 cells after phosphatidylinositol 
3‑kinase/protein kinase B pathway activation 
HCT-8 cells were pretreated for 24 h, followed by a 30-min 

culture to activate PI3K/AKT pathway. Subsequently, 

morphological observation, colony formation, DAPI staining, 

migration, and invasion assays were performed. 

Statistical analysis 
The data are presented as mean ± standard deviation, and 

statistical analyses were performed using Graphpad Prism 

(version 8.2.1 Windows version, GraphPad Software, San 

Diego). The unpaired Student’s t-test was applied to compare 

two groups, and one-way analysis of variance, followed 

by post hoc Fisher’s least significant difference testing was 

applied to compare multiple groups. P < 0.05 was considered 

statistically significant. 

 

RESULTS 
Synergistic effects of Qingjie Fuzheng Granule and 
5‑fluorouracil in colorectal cancer cells 
Treatment with QFG and 5‑FU for 24 h or 48 h, Chou‑Talalay 

was used to calculate the CI. As shown in Figure 1, the CI 

values were all <1, that indicated a synergistic anti-cancer 

effect of QFG and 5‑FU. The synergistic effect was more 

significant in HCT‑8 cells. The synergistic effect of QFG/5‑FU 

at a ratio of 80:1 was stronger in CRC HCT-8 cells. Therefore, 

the best concentrations of QFG and 5‑FU were 0.25 mg/mL 

and 3.13 × 10−3 mg/mL respectively, in HCT-8 cells. 

Qingjie Fuzheng Granule combined with 5‑fluorouracil 

synergistically inhibited intestinal cancer growth 
The synergistic activity of QFG combined with 5‑FU on 

HCT-8 cells was assesses in vitro and in vivo. As shown 

in Figure 2a, treatment with QFG and 5‑FU significantly 

reduced the viability of HCT-8 cells in a time-dependent 

manner (P < 0.05). Furthermore, the combination of QFG 

and 5‑FU induced to a decrease in the number of attached 

and detached HCT-8 cells after 24 h [Figure 2b]. Moreover, 

we explored the synergistic effect of QFG and 5‑FU using 

xenograft mice, as shown in Figure 2c and d, compared 

with treatment with QFG or 5‑FU alone, the combination 

significantly inhibited the tumor volume (P < 0.05). In addition, 

the combination of QFG and 5‑FU resulted in a reduction in 

tumor weight as shown in Figure 2e (P < 0.05). Taken together, 

these data suggest that QFG combined with 5‑FU effectively 

synergizes to inhibit the growth of intestinal cancer both 

in vitro and in vivo. 

Qingjie Fuzheng Granule and 5‑fluorouracil synergisticly 

inhibited the proliferation of HCT‑8 cells 
To investigate the effects of QFG combined with 5‑FU on 

the proliferation of HCT-8 cells in vitro and in vivo, colony 

formation assays and immunohistochemical staining were 

performed. As shown in [Figures 3a and b], QFG combined 

with 5 FU inhibited the ability of clonal goblet formation 

compared with single drug intervention (P < 0.05). Similarly, 

QFG combined with 5‑FU inhibited Ki‑67 expression in tumor 

tissues [Figure 3e and f]. Moreover, as shown in Figure 3c, d, g 

and h, QFG combined with 5‑FU decreased CDK4 and Cyclin 

 

 
Figure 1: Synergistic effects of Qingjie Fuzheng Granule (QFG) and 5‑fluorouracil (5‑FU) on colorectal cancer. (a) 24‑h combination index (CI) values 
of different ratios of QFG combined with 5‑FU intervention in HCT‑8 cells. (b) 48‑h CI values of different ratios of QFG combined with 5‑FU intervention 
in HCT‑8 cells. (c) 24‑h CI values of different ratios of QFG and 5‑FU intervention in HCT‑116 cells. (d) 48‑h CI values of different ratios of QFG 
combined with 5‑FU intervention in HCT‑116 cells 

 a   b  

 c   d  



48 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

48  

  

  

 

 
Figure 2: Synergistic inhibition of intestinal cancer growth by QingjieFuzheng Granule (QFG) and 5‑fluorouracil (5‑FU) in vitro and in vivo. (a) HCT‑8 cells 
viability. (b) Morphology of HCT‑8 cells (×200, scale bar = 100 m). (c) Tumor volume in xenograft mice. (d) Representative photographs of tumors 
in xenograft mice. (e) Tumor weight. *P < 0.05 compared with the control group; ^ P < 0.05 compared with the QFG group; #P < 0.05 compared 
with the 5‑FU group. QFG: QingjieFuzheng Granule, 5‑FU: 5‑Fluorouracil 

 

D1 protein expression while increased p53 and p21 levels. 

Taken together, these results suggest that QFG combined with 

5‑FU synergistically inhibits the proliferation of HCT‑8 cells 

both in vitro and in vivo. 

Qingjie Fuzheng Granule and 5‑fluorouracil synergisticly 

promoted apoptosis of HCT‑8 cells 
As shown in Figure 4a‑d, QFG combined with 5‑FU promoted 

apoptosis in HCT-8 cells compared to the single drug groups, 

as demonstrated by Annexin V/PI staining (P < 0.05) and 

DAPI staining assay (P < 0.05). Furthermore, in xenograft 

mice, treatment with QFG and 5‑FU increased the 

expression of TUNEL positive cells (P < 0.05) as shown by 

immunofluorescence staining [Figure 5a and b]. In addition, 

apoptosis-related proteins expression was determined. As 

shown in Figures 4e and f and 5c and d, QFG combined 

with 5‑FU increased Bax and cleaved‑caspase‑3 protein 

expression while decreased of Bcl-2 protein expression, both 

in vitro and in vivo (P < 0.05). These results suggest that 

QFG and 5‑FU synergistically promotes apoptosis in vitro 

and in vivo. 

Qingjie Fuzheng Granule combined with and 5‑fluorouracil 

synergistically inhibited migration and invasion of HCT‑8 cells 
As shown in Figure 6a-d, compared to the individual 

treatment, QFG and 5‑FU inhibited migration and invasion 

of HCT-8 cells (both P < 0.05). Meanwhile, we detected 

EMT-related proteins expression. As shown in Figure 6e-h, 

QFG combined with 5‑FU increased E‑cadherin protein 

expression and decreased N‑cadherin and Vimentin protein 

expression in both HCT-8 cells and tumor tissues. These results 

further support the synergistic effect of QFG and 5‑FU on the 

metastasis of intestinal cancer cells. 

Qingjie Fuzheng Granule combined with 5‑fluorouracil 

inhibited of dihydropyrimidine dehydrogenase and 

thymidylate synthase expression of HCT‑8 cells 
To investigate the effect of 5‑FU in CRC cells, we examined 

the expression of 5‑FU metabolism‑related proteins. As 

shown in Figure 7, we found that QFG and 5‑FU inhibited 

dihydropyrimidine dehydrogenase (DPD) and thymidylate 

synthase (TS) protein expression of HCT-8 in vitro and 

in vivo (both P < 0.05) [Figure 7]. 

Qingjie Fuzheng Granule combined with 5‑fluorouracil 

synergistically inhibited phosphatidylinositol 3‑kinase/ 

protein kinase B signaling pathway of HCT‑8 cells 
To explore the effect of QFG combined with 5‑FU on 

the PI3K/AKT pathway, we examined it by western blot 

analysis and found that QFG and 5‑FU decreased p‑PI3K 

and p‑AKT protein expression both in HCT‑8 cells and 

tumor tissues [Figure 8]. These results suggest that QFG 

combined with 5‑FU synergistically inhibited intestinal 

cancer growth by suppressing the PI3K/AKT signaling 

pathway. 

 a   c  

 d  

 b   e  



49 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

49  

  

  

 

 
Figure 3: Qingjie Fuzheng Granule (QFG) and 5‑fluorouracil (5‑FU) synergistically inhibits the proliferation of HCT‑8 cells in vitro and in vivo. (a and b) 
Colony formation assay. (c and d) Proliferation‑related proteins expression in HCT‑8 cells. (e and f) Ki67 expression in xenograft mice (×400, scale 
bar = 50 m). (g and h) Expression of proliferation‑related proteins in xenograft mice. The arrow refers to Ki‑67 positive cell. *P < 0.05 compared 
with the control group; ^ P < 0.05 compared with the QFG group; #P < 0.05 compared with the 5‑FU group. QFG: Qingjie Fuzheng Granule, 5‑FU: 
5‑Fluorouracil 

 

Qingjie Fuzheng Granule combined with 5‑fluorouracil 

regulated proliferation, apoptosis, migration and invasion 

of HCT‑8 cells through blocking phosphatidylinositol 

3‑kinase/protein kinase B pathway 
FBS, as an important exogenous environmental factor 

containing lots of growth factors, plays an important role 

in cell signaling by binding to growth factor receptors on 

the cell membrane and inducing changes in the signaling 

pathway‑related proteins. To explore the effect of FBS on the 

PI3K/AKT pathway, we subjected HCT‑8 cells to 10% FBS 

intervention under starvation condition for different durations. 

The results are shown in Figure 9a and b demonstrate that 10% 

FBS intervention for 30 min significantly upregulated p‑AKT 

protein expression (P < 0.05), indicating the activation of 

the PI3K/AKT signaling pathway in HCT‑8 cells by 10% 

FBS intervention. 

 a   b  

 c   d  

 e   f  

 g   h  



50 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

50  

  

  

 

 
Figure 4: Qingjie Fuzheng Granule (QFG) combined with 5‑fluorouracil (5‑FU) synergisticly promoted apoptosis in HCT‑8 cells. (a and b) Annexin V/PI 
staining analyzed by flow cytometry. (c and d) DAPI staining (×200, bar = 100 m). (e and f) Expression of apoptosis‑related protein in HCT‑8 cells. 
*P < 0.05 compared to the control group; ^ P < 0.05 compared to the QFG group; #P < 0.05 compared to the 5‑FU group. QFG: Qingjie Fuzheng 
Granule, 5‑FU: 5‑Fluorouracil 

 

Next, we investigated whether QFG combined with 5‑FU 

synergistically regulated the proliferation, apoptosis, 

migration, and invasion of HCT‑8 cells via PI3K/AKT 

pathway. Moreover, QFG combined with 5‑FU blocked 

the activstion of AKT induced by FBS [Figure 9c and d]. 

As shown in Figure 10a, QFG and 5‑FU significantly 

inhibited cell growth in HCT-8 cells. Similarly, the results 

in Figure 10b and c demonstrated that QFG and 5‑FU 

reduced the number of colonies which was induced by 

PI3K/AKT pathway activation (P < 0.05). Furthermore, 

QFG and 5‑FU promoted apoptosis significantly (P < 0.05) 

as shown in Figure 10d and e. Moreover, QFG and 5 FU 

suppressed migration and invasion (both P < 0.05) which 

were promoted by PI3K/AKT pathway activation as shown 

in Figure 10f and g and Figure 10h and i. In conclusion, 

these results suggest that QFG and 5‑FU synergistically 

regulated the growth process of HCT-8 cells by regulating 

the PI3K/AKT pathway. 

DISCUSSION 
CRC ranks as the third most common malignant tumor in 

the world.[1] Current treatment choices for CRC primarily 

include surgery, radiotherapy and chemotherapy, with 

chemotherapy being the main approach for patients with 

inoperable or postoperative metastatic tumors.[2] 5‑FU, the 

first‑line chemotherapy regimen, is widely used in CRC. 

However, the side effects which can easily damage the 

normal organism, induce drug resistance and reduce patients’ 

quality of life limited the use of 5‑FU. Consequently, it is 

urgent to discover novel treatment strategies to overcome 

the limitations associated with chemotherapy. Combination 

therapy involving the simultaneous administration of 

multiple drugs has emerged as a promising approach. 

Traditional Chinese Medicine has better advantages in 

reducing toxicity and increasing efficacy, Combination 

therapy of TCM and 5‑FU not only increase the therapeutic 

effects, but alleviate the side effects at lower dose. This 

 a   c  

 b   d  

 f  
 e  



51 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

51  

  

  

 

 
Figure 5: Qingjie Fuzheng Granule combined with 5‑fluorouracil synergisticly promoted apoptosis in xenograft mice. (a and b) TUNEL staining 
assay (×400, bar = 50 m). (c and d) The expression of apoptosis‑related proteins in xenograft tissues. QFG: Qingjie Fuzheng Granule, 5‑FU: 
5‑Fluorouracil 

 

approach aims to achieve synergistic efficacy while reducing 

toxicity.[12] 

Patients' quality of life and chemotherapy effectiveness are 

both improved greatly by QFG, a clinical anti-tumor 

compound that is often utilized. To add to that, QFG may 

shield the gut mucosal from 5-FU. References [13,14]: It was 

proposed that there could be a synergistic impact when QFG 

and 5-FU are combined. Prior research has shown that QFG 

may control CRC development by inhibiting the PI3K/AKT 

pathway. [13] Multiple processes, including tumor growth, 

apoptosis, metastasis, and resistance to 5-FU treatment, are 

influenced by the tumor-related genes that are downstream 

targets of the PI3K/AKT signaling pathway. These genes 

include p21, Bcl-2, EMT, DPD, TS, and others. On the basis 

of these results, the purpose of this research is to investigate 

the combined effects of QFG and 5-FU on colorectal cancer. 

Furthermore, we endeavored to investigate how QFG and 

5-FU impacted the PI3K/AKT pathway and its subsequent 

processes, such as CRC cell proliferation, apoptosis, 

metastasis, and chemoresistance. Understanding how QFG 

and 5-FU work together to combat CRC is the objective.  

The unending advancement of tumors is mostly due to the 

unchecked growth of malignant cells. [16] As a crucial tumor 

suppressor and one of the major regulators of cell division, 

p53 is an important player in the field. One gene that 

suppresses cell cycle-dependent protein kinases is P21, 

which is a downstream gene of p53 [17]. Cyclin D1-CDK4 is 

unable to phosphorylate Rb (G1 phase cycle) because a 

complex formed by P21 and Cyclin D1-CDK4 suppresses 

protein kinase activity.  

protein that acts as an inhibitor. The G1 phase block and 

inhibition of cell proliferation are caused by Rb's inactive 

state binding to E2F, which inactivates E2F. pp. 18–21 The 

colony formation test was used to determine that a mixture of 

QFG and 5-FU may decrease cell growth in this investigation. 

We found that the combination of QFG and 5-FU decreased 

Ki67 positivity in tumor tissues by immunohistochemical 

assay. We also found that the combination of QFG and 5-FU 

increased p53 and p21 protein expression while inhibiting 

Cyclin D1 and CDK4 protein expression by Western bolt 

assay, compared to single drug. The results of the experiment 

show that it is possible to suppress the growth of colorectal 

cancer cells by combining QFG with 5-FU.  

The process of programmed cell death, or apoptosis, is 

controlled by genes and results in the inevitable cessation of 

life. [8] In apoptosis, the Bcl-2 family is involved, and Bax 

dimers help open and increase the permeability of the 

mitochondrial membrane. A reduction in mitochondrial 

membrane permeability follows an increase in Bcl-2 

expression, and Bcl-2 may form heterodimers with Bax. As a 

result, cystathionase (Caspases) is activated when cellular 

mitochondrial membrane disruption triggers the release of 

cytochrome-c. Cell death is orchestrated by these cleaved 

caspases, one of which being Caspase 3. pages 22–24 This 

research used a battery of tests, including western blotting, 

flow cytometry, DAPI staining, and TUNEL 

immunofluorescence, to confirm elevated apoptosis. 

 a   b  

 c   d  



52 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

52  

  

  

 

 
Figure 6: Qingjie Fuzheng Granule (QFG) and 5‑fluorouracil (5‑FU) inhibited migration and invasion. (a and b) Migration assay and statistic 
analysis in HCT‑8 cells (×200, bar = 100 m). (c and d) Invasion assay and statistic analysis in HCT‑8 cells (×200, bar = 100 m). 
(e and f) EMT‑related proteins expression in HCT‑8 cells. (g and h) EMT‑related proteins expression in xenograft mice. *P < 0.05 compared 
to the control group; ^ P < 0.05 compared to the QFG group; #P < 0.05 compared to the 5‑FU group. QFG: Qingjie Fuzheng Granule, 5‑FU: 
5‑Fluorouracil 
 

EMT is a process in which epithelial cell phenotype 

transformed to mesenchymal cell phenotype under 

specific physiological or pathological conditions, which 

promotes the loss of intercellular adhesions, increases 

the metastatic capacity of cancer cells, inhibits apoptosis, 

and is an important factor affecting tumor recurrence and 

metastasis.[25-28] Cell-triggered EMT, which results in loss 

of epithelial cell integrity, and transcriptional repression 

of genes encoding epithelial cell-specific proteins such 

as E-Cadherin by transcription factors, contribute to the 

degradation of adherens junctions, and their adherens 

junctions are replaced by proteins capable of greater 

junctional flexibility (e.g., N‑Cadherin), leading to cell 

separation and enhanced cell motility.[29-34] Epithelial genes 

 a   c  

 b   d  

 e   f  

 g   h  



53 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

53  

  

  

 

 
Figure 7: Qingjie Fuzheng Granule (QFG) and 5‑fluorouracil (5‑FU) inhibited DPD and TS expression. (a and b) DPD and TS expression in HCT‑8 cells. 
(c and d) DPD and TS in tumor tissues. *P < 0.05 compared with the control group; ^ P < 0.05 compared with the QFG group; #P < 0.05 compared 
with the 5‑FU group. QFG: Qingjie Fuzheng Granule, 5‑FU: 5‑Fluorouracil 

 

are repressed by transcription factors while mesenchymal 

genes are activated, upregulate Vimentin (a waveform 

protein) expression, increase extracellular deposition of 

fibronectin, thereby facilitating metastasis.[35-37] In this study, 

migration and invasion assays revealed that QFG combined 

with 5‑FU significantly reduced the migrated and invased 

ability of CRC cells. In‑depth study, treatment with QFG 

and 5‑FU affected the expression of EMT‑related factor, 

inhibited EMT occurrence, and synergistically suppressed 

tumor cell metastasis. 

5-FU blocks the production of DNA in cells, which is how it 

fights tumors. Because it facilitates the conversion of dUMP 

to dTMP, the only thymidine nucleotide required for DNA 

synthesis and repair, TS is also recognized as the 5-FU target 

enzyme. Downregulation of DPD promotes 5-FU 

chemosensitivity, and DPD is a rate-limiting enzyme in 5-FU 

degradation. Sections 38–40 This research found that the 

combination of QFG and 5-FU substantially reduced DPD 

and TS expression compared to the single-drug therapy. This 

suggests that the combination of the two drugs may enhance 

the effectiveness of 5-FU by inhibiting 5-FU-related negative 

regulatory enzymes.  

Tumor growth may be accelerated by the PI3K/AKT 

signaling pathway. In order to partially activate AKT and 

then modulate its activity, PI3K activation may produce 

PIP3, which acts as a second messenger to bring PDK1 and 

AKT proteins to the plasma membrane.  

 

route that goes downstream. Tables 41–43 AKT produces 

several outcomes: 1) The phosphorylation of AKT hinders 

cell proliferation by affecting factors like p21; 2) The 

phosphorylation of AKT affects apoptosis by preventing the 

activity of BAD (Bcl-2-associated cell death agonist); 3) The 

phosphorylation of AKT positively controls the NF-B 

pathway, which regulates EMT and impacts cell metastasis. 

positions 11, 44, and 45 Treatment with QFG and 5-FU, as 

revealed by the results of the western blot experiment, 

disrupted the PI3K/AKT signaling pathway, according to this 

research. In this work, the PI3K/AKT signaling pathway was 

stimulated by 10% FBS, and the effects of the two medicines 

coupled with QFG were investigated. 10% FBS triggered the 

AKT signaling pathway. The combined treatment of QFG and 

5-FU controlled the proliferation, apoptosis, and metastasis of 

HCT-8 cells, which were followed by the activation of PI3K 

and AKT, according to functional tests. The findings show 

that the PI3K/AKT signaling system's downstream pathway 

may be regulated by combining QFG and 5-FU therapy, 

which in turn controls the proliferation, apoptosis, metastasis, 

and 5-FU chemoresistance of CRC. 

 

CONCLUSIONS 
In summary, we demonstrated for the first time that 

combined treatment QFG with 5‑FU has a synergistic 

ability against CRC, which inhibits cell proliferation, 

metastasis, induces apoptosis. The downstream of PI3K/ 

 a   b  

 c   d  



54 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

54  

  

  

 a   b  

 c   d  

 

 
Figure 8: Qingjie Fuzheng Granule (QFG) combined with 5‑fluorouracil (5‑FU) synergisticly inhibited the PI3K/AKT signaling pathway. (a and b) QFG 
and 5‑FU blocked phosphorylation of PI3K and AKT in HCT‑8 cells. (c and d) QFG and 5‑FU blocked phosphorylation of PI3K and AKT in tumor tissues. 
*P < 0.05 compared to the control group; ^ P < 0.05 compared to the QFG group; #P < 0.05 compared to the 5‑FU group. QFG: Qingjie Fuzheng 
Granule, 5‑FU: 5‑Fluorouracil 

 

Figure 9: Effect of Qingjie Fuzheng Granule (QFG) and 5‑fluorouracil (5‑FU) on the activation of the PI3K/AKT pathway by 10% FBS. (a and b) FBS 
induced AKT phosphonation in different time. (c and d) QFG and 5‑FU inhibited the AKT phosphonation. $P < 0.05 compared with the FBS Free group; 
*P < 0.05 compared with the FBS group. QFG: Qingjie Fuzheng Granule, 5‑FU: 5‑Fluorouracil 

 a   b  

 c   d  



55 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

55  

  

  

 
 
 
 
 
 
 
 
 
 
 

 
 c  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 e  

 
 a   b   d  

 
 
 
 
 
 
 
 
 
 
 
 
 

 
 g  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 f   h   i  

Figure 10: Qingjie Fuzheng Granule (QFG) combined with 5‑fluorouracil (5‑FU) regulated proliferation, apoptosis, migration and invasion via inhibiting 
PI3K/AKT pathway. (a) Morphology. (b and c) Colony formation and statistical analysis; (d and e) DAPI staining and statistical analysis (×200, 
ba r = 100 m). (f and g) Migration and statistical analysis (×200, bar = 100 m); (h and i) Invasion and statistical analysis (×200, bar = 100 m). 
$P < 0.05 compared with the FBS‑Free group; *P < 0.05 compared with the FBS group; ^ P < 0.05 compared with the FBS + QFG group; #P < 0.05 
compared with the FBS + 5‑FU group. QFG: Qingjie Fuzheng Granule, 5‑FU: 5‑Fluorouracil 

 AKT signaling pathway involves tumor proliferation, 



56 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

56  

  

  

apoptosis, metastasis and other related processes. Our 

results demonstrate that the combined treatment of QFG 

with and 5‑FU exerts its inhibitory capability on CRC 

cells though regulating PI3K/AKT signaling pathway. This 

regulation leads to the modulation of gene expressions such 

as p53 and p21, resulting in a synergistic inhibition of CRC 

proliferation. Moreover, the combination therapy modulates 

the genes expression, such as Bax and Bcl-2, leading to 

a synergistic induction of CRC apoptosis. In addition, it 

regulates the EMT process, synergistically inhibiting CRC 

metastasis. Furthermore, the combination therapy inhibits 

the expression of DPD and TS, which enhances the efficacy 

of 5‑FU and further contributes to the inhibition of CRC 

progression. 

 

REFERENCES 
1. Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, 

et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence 

and mortality worldwide for 36 cancers in 185 countries. CA Cancer J 

Clin 2021;71:209-49. 

2. Kuipers EJ, Grady WM, Lieberman D, Seufferlein T, Sung JJ, 

Boelens PG, et al. Colorectal cancer. Nat Rev Dis Primers 2015;1:15065. 

3. Kim JH. Chemotherapy for colorectal cancer in the elderly. World J 

Gastroenterol 2015;21:5158‑66. 

4. Liao HF, Chen YJ, Yang YC. A novel polysaccharide of black 

soybean promotes myelopoiesis and reconstitutes bone marrow after 

5‑flurouracil‑ and irradiation‑induced myelosuppression. Life Sci 

2005;77:400-13. 

5. Lee JJ, Beumer JH, Chu E. Therapeutic drug monitoring of 5‑fluorouracil. 

Cancer Chemother Pharmacol 2016;78:447‑64. 

6. Paschke S, Hebart H, Goeb R, Staib L, Fleck U, Henne‑Bruns D, et al. 

Adjuvant chemotherapy of locally advanced colon cancer: Final results 

of a randomized trial comparing 5‑fluorouracil and folinic acid with 

folfiri. Visc Med 2019;35:124‑32. 

7. Zhang JH, Wang LH, Li XJ, Wang AP, Reng LQ, Xia FG, et al. 

Expression of Ang‑2/Tie‑2 and PI3K/AKT in colorectal cancer. Asian 

Pac J Cancer Prev 2014;15:8651‑6. 

8. Mondal P, Natesh J, Penta D, Meeran SM. Extract of Murraya koenigii 

selectively causes genomic instability by altering redox-status via 

targeting PI3K/AKT/Nrf2/caspase‑3 signaling pathway in human 

non‑small cell lung cancer. Phytomedicine 2022;104:154272. 

9. Wang Y, Zhang S, Liu J, Fang B, Yao J, Cheng B. Matrine inhibits the 

invasive and migratory properties of human hepatocellular carcinoma 

by regulating epithelial-mesenchymal transition. Mol Med Rep 

2018;18:911-9. 

10. Dong S, Liang S, Cheng Z, Zhang X, Luo L, Li L, et al. ROS/PI3K/ 

Akt and Wnt/β‑catenin signalings activate HIF‑1α‑induced metabolic 

reprogramming to impart 5‑fluorouracil resistance in colorectal cancer. 

J Exp Clin Cancer Res 2022;41:15. 

11. Ma Z, Lou S, Jiang Z. PHLDA2 regulates EMT and autophagy in 

colorectal cancer via the PI3K/AKT signaling pathway. Aging (Albany 

NY) 2020;12:7985‑8000. 

12. Tallarida RJ, Midic U, Lamarre NS, Obradovic Z. A search for interaction 

among combinations of drugs of abuse and the use of isobolographic 

analysis. J Clin Pharm Ther 2013;38:190‑5. 

13. Yang H, Liu JX, Shang HX, Lin S, Zhao JY, Lin JM. Qingjie Fuzheng 

granules inhibit colorectal cancer cell growth by the PI3K/AKT and 

ERK pathways. World J Gastrointest Oncol 2019;11:377‑92. 
14. Zhang L, Jin Y, Peng J, Chen W, Lisha L, Lin J. Qingjie Fuzheng granule 

attenuates 5‑fluorouracil‑induced intestinal mucosal damage. Biomed 

Pharmacother 2019;118:109223. 

15. Chou TC. Drug combination studies and their synergy quantification 

using the Chou-Talalay method. Cancer Res 2010;70:440-6. 

16. Fearon ER. Molecular genetics of colorectal cancer. Annu Rev Pathol 

2011;6:479-507. 

17. Liu X, Liu Y, Liu Z, Lin C, Meng F, Xu L, et al. CircMYH9 drives 

colorectal cancer growth by regulating serine metabolism and redox 

homeostasis in a p53-dependent manner. Mol Cancer 2021;20:114. 

18. Chen Z, Hou R, Gao S, Song D, Feng Y. Baicalein inhibits 

proliferation activity of human colorectal cancer cells HCT116 through 

downregulation of ezrin. Cell Physiol Biochem 2018;49:2035‑46. 

19. Bey C, Yong X, Eu V, Hong Z. Role of p53 and p21in senescence-like 

terminal proliferation arrest induced in human tumor cells by 

chemotherapeutic drugs. Oncogene 1999;18:3012-9. 

20. Schade AE, Oser MG, Nicholson HE, DeCaprio JA. Cyclin D‑CDK4 

relieves cooperative repression of proliferation and cell cycle gene 

expression by DREAM and RB. Oncogene 2019;38:4962-76. 

21. Xia Y, Deng Y, Zhou Y, Li D, Sun X, Gu L, et al. TSPAN31 suppresses 

cell proliferation in human cervical cancer through down-regulation 

of its antisense pairing with CDK4. Cell Biochem Funct 

2020;38:660-8. 

22. Zhang Y, Yang X, Ge X, Zhang F. Puerarin attenuates neurological 

deficits via Bcl‑2/Bax/cleaved caspase‑3 and Sirt3/SOD2 apoptotic 

pathways in subarachnoid hemorrhage mice. Biomed Pharmacother 

2019;109:726-33. 

23. Hassan M, Watari H, AbuAlmaaty A, Ohba Y, Sakuragi N. 

Apoptosis and molecular targeting therapy in cancer. Biomed Res Int 

2014;2014:150845. 

24. Li Y, Wang Y, Yu X, Yu T, Zheng X, Chu Q. Radix tetrastigma inhibits 

the non-small cell lung cancer via Bax/Bcl-2/caspase-9/caspase-3 

pathway. Nutr Cancer 2022;74:320‑32. 

25. Lamouille S, Xu J, Derynck R. Molecular mechanisms of 

epithelial‑mesenchymal transition. Nat Rev Mol Cell Biol 

2014;15:178-96. 

26. Dongre A, Weinberg RA. New insights into the mechanisms of 

epithelial‑mesenchymal transition and implications for cancer. Nat Rev 

Mol Cell Biol 2019;20:69-84. 

27. Zhang Y, Weinberg RA. Epithelial-to-mesenchymal transition in cancer: 

Complexity and opportunities. Front Med 2018;12:361-73. 

28. Mittal V. Epithelial mesenchymal transition in tumor metastasis. Annu 

Rev Pathol 2018;13:395‑412. 

29. van Roy F, Berx G. The cell‑cell adhesion molecule E‑cadherin. Cell 

Mol Life Sci 2008;65:3756-88. 

30. Wong SH, Fang CM, Chuah LH, Leong CO, Ngai SC. E‑cadherin: 

Its dysregulation in carcinogenesis and clinical implications. Crit Rev 

Oncol Hematol 2018;121:11-22. 

31. Coopman P, Djiane A. Adherens junction and E‑cadherin complex 

regulation by epithelial polarity. Cell Mol Life Sci 2016;73:3535-53. 

32. Cao ZQ, Wang Z, Leng P. Aberrant N‑cadherin expression in cancer. 

Biomed Pharmacother 2019;118:109320. 

33. Radice GL. N‑cadherin‑mediated adhesion and signaling from 

development to disease: Lessons from mice. Prog Mol Biol Transl Sci 

2013;116:263-89. 

34. Mariotti A, Perotti A, Sessa C, Rüegg C. N‑cadherin as a therapeutic 

target in cancer. Expert Opin Investig Drugs 2007;16:451-65. 

35. Satelli A, Li S. Vimentin in cancer and its potential as a molecular target 

for cancer therapy. Cell Mol Life Sci 2011;68:3033-46. 

36. Battaglia RA, Delic S, Herrmann H, Snider NT. Vimentin on the move: 

New developments in cell migration. F1000Res 2018;7:v1000‑796. 

37. Ramos I, Stamatakis K, Oeste CL, Pérez‑Sala D. Vimentin as a 

multifaceted player and potential therapeutic target in viral infections. 

Int J Mol Sci 2020;21:4675. 

38. Omura K. Clinical implications of dihydropyrimidine 

dehydrogenase (DPD) activity in 5‑FU‑based chemotherapy: Mutations 

in the DPD gene, and DPD inhibitory fluoropyrimidines. Int J Clin 

Oncol 2003;8:132-8. 

39. Xie P, Mo JL, Liu JH, Li X, Tan LM, Zhang W, et al. Pharmacogenomics 



57 CTMJ | traditionalmedicinejournals.com Chinese Traditional Medicine Journal | 2025 | Vol 8 |Issue 1 

 

57  

  

  

 

of 5‑fluorouracil in colorectal cancer: Review and update. Cell 

Oncol (Dordr) 2020;43:989-1001. 

40. Akhter K, Enamur Rashid M. Study of thymidylate synthase (TS) 

and dihydropyrimidine dehydrogenase (DPD) expressions on 

5‑fluorouracil in oral squamous cell carcinoma. Asian Pac J Cancer 

Prev 2019;20:503‑8. 

41. Fan J, Bao Y, Meng X, Wang S, Li T, Chang X, et al. Mechanism of 

modulation through PI3K‑AKT pathway about Nepeta cataria L.’s 

extract in non-small cell lung cancer. Oncotarget 2017;8:31395-405. 

42. Chen H, Zhou L, Wu X, Li R, Wen J, Sha J, et al. The PI3K/AKT 

pathway in the pathogenesis of prostate cancer. Front Biosci (Landmark 

Ed) 2016;21:1084-91. 

43. Chen YH, Yang SF, Yang CK, Tsai HD, Chen TH, Chou MC, et al. 

Metformin induces apoptosis and inhibits migration by activating 

the AMPK/p53 axis and suppressing PI3K/AKT signaling in human 

cervical cancer cells. Mol Med Rep 2021;23:88. 

44. Wang R, Song F, Li S, Wu B, Gu Y, Yuan Y. Salvianolic acid A attenuates 

CCl(4)‑induced liver fibrosis by regulating the PI3K/AKT/mTOR, 

Bcl-2/Bax and caspase-3/cleaved caspase-3 signaling pathways. Drug 

Des Devel Ther 2019;13:1889-900. 

45. Ha SE, Kim SM, Vetrivel P, Kim HH, Bhosale PB, Heo JD, et al. 

Inhibition of Cell Proliferation and Metastasis by Scutellarein Regulating 

PI3K/Akt/NF‑κB Signaling through PTEN Activation in Hepatocellular 

Carcinoma. Int J Mol Sci 2021;22:8841. 


