Mechanism Study on the Induction of Apoptosis in Nasopharyngeal Carcinoma 6-10B Cells by Grass Fruit Volatile Oil
Nasopharyngeal carcinoma (NPC) is one of the common malignant tumors. The International Agency for Research on Cancer (IARC) estimates that there will be 910677 new cases of NPC worldwide in 2020, and 763570000 patients may die from this cancer. According to WHO statistics, about 80% of NPC cases occur in China, seriously endangering people’s health and safety, and is one of the key malignant tumors for prevention and treatment in China. At present, radiation and chemotherapy are the main treatment methods, while local recurrence and distant metastasis are the main reasons for treatment failure; The main drugs used to treat nasopharyngeal carcinoma include paclitaxel, cisplatin, 5-fluorouracil, cetuximab, etc. However, these anti-tumor drugs are mostly non-specific drugs, accompanied by toxic side effects such as lymphocyte depletion, mucosal inflammation, throat/oral pain, anemia, and rash. Therefore, in order to more effectively treat nasopharyngeal carcinoma, scholars are committed to the development of new natural drugs with strong targeting and minimal toxic side effects for the treatment of nasopharyngeal carcinoma.

Amomum tsaoko Crevost et Lemaire is a dried and mature fruit of plants in the ginger family and the cardamom genus. It is mainly produced in Guangxi, Yunnan, Guizhou and other regions and is an important medicinal and edible plant in China; Traditional Chinese medicine believes that it has the effects of drying dampness, warming the middle, intercepting malaria, and removing phlegm. It is mainly used to treat symptoms such as cold dampness resistance, vomiting, malaria fever, plague fever, etc. It is widely used to treat hemorrhoids, throat infections, digestive system diseases, nausea, and abdominal pain. Modern research has shown that grass fruit has biological activities such as lipid-lowering, hypoglycemic, antioxidant, anti-tumor, anti mold, and anti-inflammatory and analgesic effects. As an important active ingredient in grass fruit, the volatile oil of grass fruit contains 1,8-eucalyptol, α – pinene, β – pinene, geranyl acetate, linalool, trans orange blossom tertiary alcohol, β – laurene, etc. It has biological activities such as antioxidant, gastrointestinal regulation, antibacterial, anti-tumor, and changes in drug permeability. However, there is relatively little research on its anti-tumor activity. Currently, only the research on the anti liver cancer effect of grass fruit volatile oil is available, and there are no reports on the related research on the inhibition of nasopharyngeal carcinoma by grass fruit volatile oil. Based on this, this study aims to explore the inhibitory effect of grass fruit volatile oil on the proliferation of nasopharyngeal carcinoma 6-10B cells, elucidate its mechanism of action, and provide scientific basis for the development and application of grass fruit volatile oil in the treatment of nasopharyngeal carcinoma and other tumors.

Nasopharyngeal carcinoma is a malignant tumor of the head and neck epithelial cells, and its main pathological type is non keratinized undifferentiated carcinoma. Early nasopharyngeal carcinoma is relatively better treated with radiotherapy alone, while the treatment effect for late stage lesions is unsatisfactory. Therefore, we are trying to combine radiotherapy and chemotherapy for the treatment of late stage nasopharyngeal carcinoma. However, the commonly used chemotherapy drugs currently have drawbacks such as drug resistance and high doses. Therefore, scholars are committed to exploring the effectiveness of natural medicine in treating nasopharyngeal carcinoma. Studies have shown that alkaloids such as ipecac, tomato glycosides, and camptothecin, as well as various natural products such as Sarcandra extract, have a certain inhibitory effect on the proliferation of nasopharyngeal carcinoma cells.

The volatile oil of grass fruit is the main active ingredient of grass fruit, which has the effects of antioxidant, regulating gastrointestinal function, antibacterial, anti-tumor, and changing drug permeability. There are few reports on the anti-tumor effects of grass fruit volatile oil, except for Zhang’s report that grass fruit volatile oil has an inhibitory effect on the proliferation of liver cancer cells. This study is the first to explore the inhibitory effect of grass fruit volatile oil on the proliferation of nasopharyngeal carcinoma cells. The results showed that grass fruit volatile oil has a dose-dependent inhibitory effect on the proliferation of 6-10B cells, with an IC50 of 0.14mg/mL at 24 hours; At the same time, morphological results showed that under the action of high concentrations (0.93 and 0.46mg/mL) of grass fruit volatile oil, tumor cells were completely fragmented and lysed, while low concentrations (0.23, 0.12, and 0.058mg/mL) of grass fruit volatile oil had a significant apoptosis inducing effect on nasopharyngeal carcinoma tumor cells 6-10B.

The mitochondrial pathway is one of the main pathways of cell apoptosis, and changes in mitochondrial membrane potential (∆ PSI m) are early markers of cell apoptosis. This study found that after 24 hours of treatment with grass fruit volatile oil at drug concentrations of 0.23, 0.12, and 0.058mg/mL, the mitochondrial membrane potential of 6-10B cells significantly changed, indicating that the mitochondrial apoptosis pathway is involved in the induction of nasopharyngeal carcinoma cell apoptosis by grass fruit volatile oil. The Bcl-2 family proteins play a crucial regulatory role in the mitochondrial apoptosis pathway. External apoptotic stimuli activate pro apoptotic factors (such as Bax, Bim, etc.), causing changes in mitochondrial membrane potential, resulting in mitochondrial swelling, increased outer membrane permeability, release of cytochrome C, activation of Caspase-9, and triggering the Caspase cascade reaction, leading to cell apoptosis. Western blot further analyzed the expression levels of mitochondrial pathway related proteins Bax, Bim, Bcl-2, Bcl xL, and apoptosis marker proteins Caspase-3/7/9, PARP, etc. The results further confirmed that the volatile oil of Caoguo induces apoptosis in nasopharyngeal carcinoma 6-10B cells through the mitochondrial apoptosis pathway.

In summary, the volatile oil from grass fruit has a significant inhibitory effect on the proliferation of nasopharyngeal carcinoma cells in vitro, and can induce apoptosis of 6-10B cells through the mitochondrial apoptosis pathway, thereby exerting anti-tumor biological effects; The research team will further explore the therapeutic effect and mechanism of Grass Fruit Volatile Oil in treating nasopharyngeal carcinoma in vivo, and analyze its inhibitory effect on the proliferation of other tumor cells. These studies will provide scientific basis for the development and application of Grass Fruit Volatile Oil in subsequent anti-tumor natural medicine, and also lay the foundation for the comprehensive development and utilization of medicinal and edible plant Grass Fruit.