Study on the involvement of glycolysis in regulating the inhibition of proliferation of chronic myeloid leukemia cells by ceramide
Chronic myelogenous leukemia (CML) is a clonal proliferative leukemia of bone marrow cells, accounting for about 15% of adult leukemia cases. Its annual incidence rate is 2/100000, high in people aged 50~55 years. Chronic myeloid leukemia is a tumor associated with the formation of BCR-ABL1 fusion protein through Philadelphia chromosome translocation. At present, tyrosine kinase inhibitor (TKI) targeted drugs are mainly used for treatment, which can significantly improve the prognosis of CML patients. However, there is still a recurrence problem of TKI resistance caused by the instability of the tyrosine kinase gene of BCR-ABL1 oncogene. Traditional Chinese medicine has therapeutic effects on CML, such as enhancing chemotherapy efficacy and improving the body’s immune function. Discovering anti CML lead compounds in traditional Chinese medicine and analyzing their mechanisms of action can provide new ideas and experimental basis for the treatment of CML.

Rocaglamide (RocA), derived from the branches and leaves of the traditional Chinese medicine plant Spartina alterniflora, is a natural product containing a cyclopentane benzofuran skeleton. It has functions such as preventing and treating respiratory diseases such as cough and asthma, inflammatory skin diseases, anti-inflammatory effects, reducing drug damage, neuroprotection, and anti-tumor effects. Previous studies have found that ceramide inhibits the growth and proliferation of tumor cells at nanomolar concentrations. Its anti-tumor mechanism mainly involves inhibiting the translation initiation factors eIF4E and eIF4A of tumor cells, blocking the cell cycle by activating the ATM/ATR-Chk1/CHK2 checkpoint pathway, and activating tumor cells without affecting the MAPK p38 and JNK pathways of normal cells. Recent studies have found that sulfonamide can improve chemotherapy efficacy and enhance the sensitivity of leukemia cells. Multiple studies have shown that sulfonamide has great potential as a candidate small molecule compound for the development of anti-tumor drugs.

Some tumor cells undergo metabolic changes to meet their energy and biosynthetic needs for survival and rapid proliferation, and the increased levels of aerobic glycolysis (i.e., the Wolberg effect) are one of the metabolic markers of tumor cells. Aerobic glycolysis has become an important target for selective intervention in leukemia. It is not clear whether sulfonamide inhibits CML proliferation by regulating CML glycolysis. This paper uses CML cell line K562 as a cell model to investigate the effects of ceramide on CML cell proliferation and glycolysis, and explore the regulatory role of aerobic glycolysis in the inhibition of CML cell proliferation by ceramide.
Melitamide is an active substance extracted from the rice orchid that has anti-tumor effects. This article explores whether sulfonamide inhibits the proliferation of K562 cells through aerobic glycolysis. The results of MTT assay showed that the growth and proliferation of K562 cells were significantly inhibited by Melitamide in a time – and concentration dependent manner, and its IC50 for inhibiting the proliferation of K562 cells reached the nanomolar level. The use of flow cytometry technology has demonstrated that azepam inhibits the proliferation and induces apoptosis of K562 cells, blocking the G2/M phase of the cell cycle.
Tumor cells still exhibit high levels of glucose uptake, glycolysis, and lactate levels under aerobic conditions, known as the Warburg effect. The transcription factor c-Myc is widely involved in regulating cellular functions such as cell proliferation, cell survival, tissue remodeling, and angiogenesis, and also initiates metabolic reprogramming of tumor cells. c-Myc upregulates glucose transporters and almost all glycolytic enzymes, thereby promoting tumor cell glucose metabolism. 13C labeled glucose showed that c-Myc promoted glucose consumption in Burkitt lymphoma and c-Myc driven liver cancer. Research has found that c-Myc promotes the secretion of lactate, which is not only a metabolic substance involved in glycolysis but also participates in promoting angiogenesis, immune escape, cell migration, metabolic demand, and other carcinogenic effects. We found through glucose starvation treatment of K562 cells that the removal of glucose reduced the lactate production level and protein expression level of c-Myc, and was not conducive to the proliferation of K562 cells. This confirms that the growth and proliferation of K562 cells depend on the supply of glucose, reflecting the correlation between c-Myc levels and glucose levels in K562 cells. Under starvation conditions, it was found that the relative inhibition rate of formamide on K562 cells after glucose removal was lower than that of the glucose added group. Therefore, it is speculated that formamide’s inhibition of K562 cell growth is partially dependent on glucose, meaning that formamide cannot further induce growth inhibition in K562 cells without glucose supply. Melitamide also significantly reduced the glucose consumption and lactate production levels in the supernatant of K562 cells, indicating that Melitamide inhibited aerobic glycolysis in K562 cells.
Hexokinase 2 (HK2) is a specific key enzyme in tumor cell glycolysis, which can bind to the outer mitochondrial membrane to maintain high-speed aerobic glycolysis of tumor cells and provide energy for rapid proliferation of tumor cells. The researchers confirmed that HK2 plays an important role in the occurrence and development of breast cancer, lung cancer and other tumors by using mouse models; Hexokinase 1 (HK1) is widely expressed in adult tissues and is considered a housekeeping subtype of hexokinase; Phosphofructokinase P (PFKP) plays a crucial role in the initiation and metastasis of cancer. Some studies have shown that PFKP has the potential to be a diagnostic marker or drug target for breast cancer and lung cancer; Pyruvate kinase 2 (PKM2) is expressed in all cells with high nucleic acid synthesis rates, such as normal proliferating cells, embryonic cells, and especially tumor cells; Lactate dehydrogenase A (LDHA) catalyzes the mutual transformation of pyruvate and L-lactic acid. The LDHA level of many human tumor cells is higher than that of normal cells. Inhibiting LDHA can reduce the proliferation of tumor cells such as head and neck cancer, pancreatic cancer, cervical cancer and prostate cancer. We analyzed the effects of ceramide on the protein expression levels of HK2, HK1, PFKP, PKM2, LDHA, and oncogenic transcription factor c-Myc in K562 cells and found that ceramide hardly regulated the protein expression levels of HK1, PFKP, PKM2, and LDHA, but significantly downregulated the protein expression levels of c-Myc and HK2. We speculate that Melinamide exerts its anti-K562 cell activity by downregulating c-Myc and HK2 and inhibiting aerobic glycolysis pathways. The regulatory relationship between c-Myc and HK2 requires further exploration.