Glycyrrhizic Acid (from licorice roots) Inhibits Proliferation of Gastric Cancer Cells by Inducing Cell Cycle Arrest and Apoptosis

[ginfreely]

 

[ginfreely]

Abstract​

Purpose​

Glycyrrhizic acid (GA) is the main active ingredient extracted from Chinese herb licorice root, and it shows anti-tumor effects in many cancer types, while its role in gastric cancer (GC) is still unknown. In this study, we evaluated the effects of GA on GC cells and explored the underlying mechanisms.

Methods​

The anti-proliferation effect of GA on GC cells was assessed by CCK-8, colony formation, and EdU assay. The effects of GA on cell cycle and apoptosis were detected by flow cytometer. Western blotting was performed to explore the underlying mechanisms.

 

[ginfreely]

Results​

Our results showed that GA had a time- and dose-dependent inhibitory effect on proliferation of GC cells. Flow cytometer analysis demonstrated that GA would lead to G1/S-phase arrest and apoptosis. GA treatment down-regulated the levels of G1 phase-related proteins, including cyclin D1, D2, D3, E1, and E2. In terms of apoptosis, GA treatment up-regulated the levels of Bax, cleaved PARP, and pro-caspase-3, -8, -9, but did not influence their cleavage patterns. The expression of Bcl-2, survivin and p65 was attenuated after treatment. Besides, GA would down-regulate the phosphorylation of PI3K/AKT pathway.

Conclusion​

This study focused on inhibitory effect of GA on GC cells by inducing cell cycle arrest and apoptosis. Several important cyclins- and apoptosis-related proteins were involved in the regulation of GA to GC cells, and phosphorylated PI3K and AKT were attenuated. The results of this study indicated that GA is a potential and promising anti-cancer drug for GC.

 

[yubenOh]

Hmm...moi only know Glycyrrhetinic acid (GA) and glycyrrhizic acid and related salts and ester are well known for their skin-whitening effects and skin-conditioning activities.

 

[ginfreely]

Introduction​

Gastric cancer (GC) is the sixth most common malignancy following breast, prostate, lung, colorectal, and cervical cancers. It is the fifth leading cause of cancer-associated deaths following lung, breast, colorectal and liver cancers. Approximately half of the GC cases encountered occur in developing countries.1–4 Despite the improvement in diagnostic and treatment techniques, GC remains a major health issue.5 Current treatments for GC include surgery and chemotherapy, despite more and more drugs have been investigated for GC treatment,6–8 they still exhibit certain disadvantages. Therefore, it is urgent to develop molecular-targeted agents for the improvement in the treatment of this disease.

Glycyrrhizic acid (GA) is the main active ingredient of Chinese herb licorice root (Figure 1). Previous studies have shown that GA and its derivatives exhibit a variety of pharmacological effects, such as detoxification, anti-inflammatory, bronchodilatory, anti-tumor, anti-ulcer, and anti-viral functions.9 The anti-tumor effect of GA has been reported in various types of tumors, such as those of the lung, liver, breast and cervix as well as in hematological malignancies, such as leukemia.10–14 GA demonstrated low toxicity and its LD50 value was estimated to 2000 mg/kg in mice following a single oral dose.15 In the clinic, GA compounds are widely used in the treatment of viral hepatitis and hepatocellular carcinomas.16–18 Previous studies have shown that GA can regulate several important signaling proteins, including those that belong to cysteine-dependent aspartate-specific protease (caspase) and the Bcl-2 families, the nuclear factor-kappaB (NF-κB) protein, the high mobility group box-1 (HMGB1) protein, the extracellular regulated protein kinases (ERK), the phosphatidylinositol 3-kinase (PI3K)/AKT kinases and the c-Jun N-terminal kinase (JNK).19–22 However, a limited number of reports have investigated mechanism by which GA affects GC.

 

[ginfreely]

Discussion​

The use of Chinese medicinal herbs has been widely applied in the clinic in China.23 Previous studies have shown that GA possesses anti-cancer activity against several types of cancers. In the present study, GA decreased proliferation of GC cells by inducing G1/S-phase arrest and apoptosis.
The inhibition of cellular proliferation can be caused by cell cycle arrest. Cyclins and cyclin-dependent kinases (CDKs) are critical proteins that regulate cell cycle progression. The results of the present study demonstrated that GA treatment attenuated the expression levels of G1 phase-related proteins. Activated D-type cyclins (cyclin D1, cyclin D2 and cyclin D3) bind to CDK4 or CDK6 to form the cyclin D-CDK4/CDK6 complexes, which result in phosphorylation of the retinoblastoma protein and subsequent dissociation of the E2F transcription factor. Once released, the E2F transcription factor promotes expression of downstream target genes and facilitates the S-phase entry. Deregulation of D-type cyclins, notably cyclin D1, can directly lead to proliferation and cell cycle progression from G1 to S phase.24 E-type cyclins (cyclin E1 and cyclin E2) are synthesized initially in the mid G1 phase. Their expression reaches its maximum levels at G1/S transition followed by their degradation at the end of S phase. They act as key regulators of the G1 to S phase transition. A similar action has been noted for the D-type cyclins. E-type cyclins bind to CDK2 and activate it, resulting in the S phase entry of the cells.25 The changes in the expression levels of cyclin proteins caused by GA treatment of GC cells were accompanied by G1/S-phase arrest of cell cycle.
The results of the present study demonstrated that both early and late apoptotic rates of MGC-803 cells were increased following GA treatment. Current studies have shown that GA exhibits different effects on the induction of apoptosis or necrosis under different conditions. In tumors, GA promotes apoptosis, while during liver, brain or kidney injury, it impedes apoptosis.26–28 It is well known that caspase family of proteins plays an important role in the progression of apoptosis. Caspase-dependent apoptosis is a “waterfall pattern” cascade reaction beginning with the activation of initiator-like caspase-2, −8, −9, and −10 enzymes and is continued with the increase in their expression levels. The self-activation activates endogenous and exogenous apoptotic pathways. Once activated, the initiators induce the expression of a series of downstream caspase proteins and ultimately activate caspase-3, −6, −7 and −14, which are the effectors of apoptotic process.29 However, the present study demonstrated that GA treatment did not change the levels of cleaved caspase proteins in MBC-803 cells, indicating GA mediated caspase-independent apoptosis in GC cells. Similar results were noted in breast cancer.10Moreover, the expression levels of other key apoptosis-associated proteins changed, including Bcl-2, Bax, cleaved PARP, survivin, and p65. Bcl-2 is an anti-apoptotic protein that prevents the release of apoptotic factors into cytoplasm, while Bax permeabilizes the outer mitochondrial membrane to promote the release of apoptotic factors and accelerate the apoptotic process.30The ratio of expression levels of Bcl-2 and Bax is used to reflect the apoptotic cascade.31 PARP is regarded as a DNA repair enzyme and a substrate of caspase family of enzymes. During induction of apoptosis, the full-length PARP protein is cleaved into a catalytic domain (89 kDa) and a binding domain (24 kDa), which is considered to be the hallmark of apoptosis.32 The survivin protein exhibits anti-apoptotic effects by inducing inhibition of caspase family proteins.33 Previous studies demonstrated that GA treatment attenuated survivin by blocking AKT/mTOR/STAT3 signaling pathway.11 P65 is an active subunit of NF-κB protein, which is responsible for regulation of cellular proliferation and apoptosis.34 It was reported that GA inhibited cell growth and induced apoptosis possibly by downregulating p65.35 The results presented in the current study revealed that GA acted via a caspase-independent apoptotic pathway.
The PI3K/AKT signaling pathway is involved in the regulation of several biological processes, including inhibition of apoptosis and cell proliferation. PI3K can be activated by tyrosine kinase receptors, G-protein-coupled receptors or Ras protein. This activation leads to the phosphorylation and subsequent activation of AKT at Ser473 and Thr308 residues.36 Phosphorylated AKT targets more than 100 proteins and leads to oncogenesis. It can protect cardiomyocytes from H2O2-induced injury by abating apoptosis.37 Low levels of phosphorylated AKT upregulated p21cip1 and p27kip1 leading to the induction of G1/S-phase arrest in thyroid carcinoma cells.38 Furthermore, downregulation of phosphorylated AKT inhibited proliferation and induced G1/S-phase arrest and apoptosis in glioma cells resulting to a significant reduction in the levels of Bcl-2 and cyclin D1.39 In the present study, GA attenuated phosphorylation of PI3K and AKT, which was probably responsible for cell cycle arrest and apoptosis in GC cells.
In conclusion, GA exhibited anti-tumor property in GC cells partly by inducing apoptosis and cell cycle arrest. These effects were associated with corresponding protein changes. In addition, the phosphorylation of PI3K/AKT pathway proteins was inhibited. The aforementioned data indicated that GA was a potential anti-tumor drug for GC treatment.
Go to:

Funding Statement​

This study was funded by Health Commission of Wuxi in Jiangsu Province (grant numbers: JCRCPY001 and Q201824).

 

[ginfreely]

Hmm...moi only know Glycyrrhetinic acid (GA) and glycyrrhizic acid and related salts and ester are well known for their skin-whitening effects and skin-conditioning activities.

Really? Guess what? I have never heard of GA until today. Obviously I have no need for skin whitening unlike your women.

 

[ginfreely]

Discussion​

The use of Chinese medicinal herbs has been widely applied in the clinic in China.23 Previous studies have shown that GA possesses anti-cancer activity against several types of cancers. In the present study, GA decreased proliferation of GC cells by inducing G1/S-phase arrest and apoptosis.
The inhibition of cellular proliferation can be caused by cell cycle arrest. Cyclins and cyclin-dependent kinases (CDKs) are critical proteins that regulate cell cycle progression. The results of the present study demonstrated that GA treatment attenuated the expression levels of G1 phase-related proteins. Activated D-type cyclins (cyclin D1, cyclin D2 and cyclin D3) bind to CDK4 or CDK6 to form the cyclin D-CDK4/CDK6 complexes, which result in phosphorylation of the retinoblastoma protein and subsequent dissociation of the E2F transcription factor. Once released, the E2F transcription factor promotes expression of downstream target genes and facilitates the S-phase entry. Deregulation of D-type cyclins, notably cyclin D1, can directly lead to proliferation and cell cycle progression from G1 to S phase.24 E-type cyclins (cyclin E1 and cyclin E2) are synthesized initially in the mid G1 phase. Their expression reaches its maximum levels at G1/S transition followed by their degradation at the end of S phase. They act as key regulators of the G1 to S phase transition. A similar action has been noted for the D-type cyclins. E-type cyclins bind to CDK2 and activate it, resulting in the S phase entry of the cells.25 The changes in the expression levels of cyclin proteins caused by GA treatment of GC cells were accompanied by G1/S-phase arrest of cell cycle.
The results of the present study demonstrated that both early and late apoptotic rates of MGC-803 cells were increased following GA treatment. Current studies have shown that GA exhibits different effects on the induction of apoptosis or necrosis under different conditions. In tumors, GA promotes apoptosis, while during liver, brain or kidney injury, it impedes apoptosis.26–28 It is well known that caspase family of proteins plays an important role in the progression of apoptosis. Caspase-dependent apoptosis is a “waterfall pattern” cascade reaction beginning with the activation of initiator-like caspase-2, −8, −9, and −10 enzymes and is continued with the increase in their expression levels. The self-activation activates endogenous and exogenous apoptotic pathways. Once activated, the initiators induce the expression of a series of downstream caspase proteins and ultimately activate caspase-3, −6, −7 and −14, which are the effectors of apoptotic process.29 However, the present study demonstrated that GA treatment did not change the levels of cleaved caspase proteins in MBC-803 cells, indicating GA mediated caspase-independent apoptosis in GC cells. Similar results were noted in breast cancer.10Moreover, the expression levels of other key apoptosis-associated proteins changed, including Bcl-2, Bax, cleaved PARP, survivin, and p65. Bcl-2 is an anti-apoptotic protein that prevents the release of apoptotic factors into cytoplasm, while Bax permeabilizes the outer mitochondrial membrane to promote the release of apoptotic factors and accelerate the apoptotic process.30The ratio of expression levels of Bcl-2 and Bax is used to reflect the apoptotic cascade.31 PARP is regarded as a DNA repair enzyme and a substrate of caspase family of enzymes. During induction of apoptosis, the full-length PARP protein is cleaved into a catalytic domain (89 kDa) and a binding domain (24 kDa), which is considered to be the hallmark of apoptosis.32 The survivin protein exhibits anti-apoptotic effects by inducing inhibition of caspase family proteins.33 Previous studies demonstrated that GA treatment attenuated survivin by blocking AKT/mTOR/STAT3 signaling pathway.11 P65 is an active subunit of NF-κB protein, which is responsible for regulation of cellular proliferation and apoptosis.34 It was reported that GA inhibited cell growth and induced apoptosis possibly by downregulating p65.35 The results presented in the current study revealed that GA acted via a caspase-independent apoptotic pathway.
The PI3K/AKT signaling pathway is involved in the regulation of several biological processes, including inhibition of apoptosis and cell proliferation. PI3K can be activated by tyrosine kinase receptors, G-protein-coupled receptors or Ras protein. This activation leads to the phosphorylation and subsequent activation of AKT at Ser473 and Thr308 residues.36 Phosphorylated AKT targets more than 100 proteins and leads to oncogenesis. It can protect cardiomyocytes from H2O2-induced injury by abating apoptosis.37 Low levels of phosphorylated AKT upregulated p21cip1 and p27kip1 leading to the induction of G1/S-phase arrest in thyroid carcinoma cells.38 Furthermore, downregulation of phosphorylated AKT inhibited proliferation and induced G1/S-phase arrest and apoptosis in glioma cells resulting to a significant reduction in the levels of Bcl-2 and cyclin D1.39 In the present study, GA attenuated phosphorylation of PI3K and AKT, which was probably responsible for cell cycle arrest and apoptosis in GC cells.
In conclusion, GA exhibited anti-tumor property in GC cells partly by inducing apoptosis and cell cycle arrest. These effects were associated with corresponding protein changes. In addition, the phosphorylation of PI3K/AKT pathway proteins was inhibited. The aforementioned data indicated that GA was a potential anti-tumor drug for GC treatment.
Go to:

Funding Statement​

This study was funded by Health Commission of Wuxi in Jiangsu Province (grant numbers: JCRCPY001 and Q201824).

So when can China come out with a drug to treat stomach cancer? Everything seems to be potential to treat stomach cancer.