The effect of Shengcao phenol regulating the Akt/mTOR pathway on autophagy in glioma cells. Glioblastoma multimodal (GBM) is the most common malignant tumor in the central nervous system, with high malignancy and invasiveness. Even after comprehensive treatment with surgery, radiotherapy, and chemotherapy, it still faces the risk of recurrence and metastasis. Temozolomide (TMZ) is a first-line anti glioma drug in clinical practice. However, due to its serious adverse reactions such as blood toxicity, this greatly limits the clinical use of TMZ and seriously affects its efficacy. Therefore, seeking efficient and low toxicity new anti glioma drugs is of great significance.
Autophagy, as a programmed cell death mechanism, is a dynamic degradation and circulatory system. Autophagy is a dynamic process that involves three states: phagophores, autophagosomes, and autolysosomes. In recent years, research has found that abnormalities in cellular autophagy are closely related to the occurrence and development of tumors. In gliomas, abnormal regulation of autophagy can promote tumor growth, while intervening in autophagy can lead to glioma cell death and thus inhibit tumor progression. Therefore, exploring new glioma treatment methods targeting autophagy is a highly promising research direction.
The chemical structure of eriodictyol (ERD) is shown in Figure 1. It is a natural edible flavonoid widely found in vegetables and fruits, especially in citrus water where it is most abundant. Research has shown that resveratrol has multiple pharmacological effects, including antioxidant, anti-inflammatory, and anti-tumor effects. Our previous research found that resveratrol can induce apoptosis and inhibit metastasis of glioma cells, but its effect on autophagy has not been studied yet. The aim of this study is to explore the effects of resveratrol on the proliferation and autophagy of glioma cells through experiments, and further investigate the possible molecular mechanisms, providing a basis for elucidating the pharmacological mechanism of resveratrol against glioma.

 

Gliomas are central nervous system tumors with the highest incidence and mortality rates, characterized by strong invasiveness, rapid malignant progression, and extremely low cure rates, with a median survival period of less than one year. Due to the biological characteristics of highly invasive growth in GBM, it is almost impossible to guarantee complete tumor resection during surgery, and the postoperative recurrence rate is extremely high. Therefore, in clinical practice, a comprehensive treatment approach of surgery+radiotherapy and chemotherapy is usually adopted.

At present, there is no ideal therapeutic drug for glioma. The commonly used drugs in clinical practice mainly include biological alkylating agents, targeted anti-tumor drugs, and immune checkpoint inhibitors. TMZ is a first-line chemotherapy drug for treating GBM, but its selectivity is poor, it is prone to drug resistance, and it has strong side effects, often resulting in unsatisfactory treatment outcomes. Targeted anti-tumor drugs and immune checkpoint inhibitors have the characteristics of high selectivity and minimal side effects, and have gradually become mainstream anti-tumor drugs abroad. However, targeted drugs and immune checkpoint inhibitors are expensive, and long-term use imposes a heavy economic burden and pressure on patients. Therefore, the search for efficient, low toxicity, and affordable new anti glioma drugs is currently a hot topic in the field of glioma research. Traditional Chinese medicine and natural active products in our country have abundant sources, numerous types, relatively small toxic side effects, and have the characteristics of broad dimensions, multi-level, and multi-target comprehensive effects, which have unique advantages in anti-tumor treatment. So, exploring effective anti glioma ingredients from natural active products and traditional Chinese medicine may be an effective way to solve the current dilemma.

ERD, as a natural flavonoid, has attracted attention and research due to its wide range of biological activities. In the past, research on ERD mainly focused on anti-inflammatory and antioxidant effects, but our recent study found that resveratrol can significantly inhibit the metastasis of glioma cells and induce their apoptosis. In addition, Tang et al.’s study showed that in nasopharyngeal carcinoma CNE1 cells, ERD significantly inhibited cell proliferation through autophagy mediation. Wang et al. found that ERD plays a protective role in rat cerebral ischemia-reperfusion injury by regulating autophagy. The above research results suggest that ERD has a regulatory effect on cellular autophagy and may exert anti-cancer effects by affecting autophagy.

This study investigated the effect of ERD on autophagy in gliomas. Firstly, we confirmed through CCK-8 reagent and clone formation experiments that ERD has a good inhibitory effect on glioma cells, which is consistent with our previous research results. Next, to investigate whether ERD has an effect on autophagy in gliomas, we measured the expression levels of autophagy related proteins LC3, Beclin 1, and ATG5, and observed the number of autophagosomes through fluorescence. The results showed that the expression of LC3B, Beclin 1, and ATG5 was significantly upregulated in glioma cells treated with ERD, and the number of autophagosomes was also significantly increased. ERD induces autophagy in glioma cells. Autophagy is a dynamic process that includes three states: autophagosomes, autophagosomes, and autolysosomes. When cells undergo autophagy, autophagosomes are first formed, which then fuse with lysosomes to form autolysosomes, and subsequently degrade proteins and other substances enclosed within them. We transfected the cell line with GFP-mRFP-LC3B dual fluorescent plasmid to construct stable cells expressing red green fluorescent protein. We observed the autophagy flow by utilizing the characteristic of GFP fluorescent protein’s reduced fluorescence intensity under low pH conditions. When autophagosomes fuse with lysosomes to form autolysosomes, the pH value inside the vesicles decreases, and after the combination of red and green fluorescence, the cells show a reddish fluorescence. Otherwise, they tend to turn yellow. The results showed that after ERD treatment, glioma cells exhibited bright yellow fluorescence and increased expression of P62 protein, which is a substrate for autophagosomes. However, when ERD was co treated with the autophagy inducer RAP, the effect of ERD was partially reversed. The above experimental results suggest that ERD promotes the formation of autophagosomes in glioma cells, but inhibits autophagic flow.

The Akt/mTOR signaling pathway plays a crucial regulatory role in cellular autophagy and is a potential target for tumor therapy. Therefore, to further investigate the potential mechanism of ERD affecting autophagy in glioma cells, we examined the effect of ERD on the expression of Akt/mTOR pathway related proteins. The results showed that ERD could inhibit the expression of p-Ak and p-mTOR, and this effect was partially reversed by Akt agonist 740 Y-P. This suggests that ERD may affect the autophagy process of glioma cells by downregulating the Akt/mTOR pathway. Finally, we observed the effect of ERD on glioma autophagy in vivo using a nude mouse subcutaneous transplant tumor model, and the experimental results were consistent with those in vitro and in vivo.

In summary, this study preliminarily explored the effect of ERD on autophagy in glioma cells. It was found that ERD may partially inhibit the Akt/mTOR pathway, regulate the expression of autophagy related proteins ATG5, Beclin1, LC3, and P62, affect the autophagy process of glioma cells, thereby inhibiting glioma cell proliferation and ultimately exerting anti glioma effects. However, the molecular mechanism by which ERD regulates autophagic flow has not been fully elucidated in this study, which requires further investigation.