In this study, the inhibitory effect of α-hederin on tumor cells was assessed, and the effects of glucose, lactate and intracellular ATP in cell culture medium on glycolysis in A549 cells were measured. Expression of GLUT1, HK2, PKM2, LDHA, MCT4 and glycolytic regulators c-Myc, HIF-1α, p53, Akt and SIRT6 were detected by WB assay to determine the inhibitory mechanism of α-hederin on non-small cell lung cancer A549 cells. We established a non-small cell lung cancer A549 allograft transplantation tumor model to investigate the effect of α-hederin on xenograft tumors in mice by evaluating tumor volume and weight, and immunohistochemistry was used to detect the expression of related protein in glycolysis. The results showed that α-hederin inhibited non-small cell lung cancer A549 in vivo and in vitro, significantly reduced non-small cell lung cancer A549 glucose uptake, reduced generation of lactate, and reduced ATP levels in cells. The mechanism of α-hederin occurred by activating SIRT6 and regulate c-Myc and HIF-1α, which reduced key glycolytic enzymes, GLUT1, HK2, PKM2, LDHA, and MCT4.
Otto Warburg discovered in the 1920s that glycolysis is the primary source of energy for cancer cells, even when oxygen levels are sufficient . Oxidative phosphorylation is a major way that normal cells produce energy; however, during the process of growth and proliferation, tumor cells need a large energy supply in a short period of time. Oxidative phosphorylation cannot provide energy for tumors very well, while the glycolysis pathway can quickly provide energy for tumors. Therefore, cancer cells primarily generate energy by glycolysis, and lactate produced by glycolysis provides an acidic environment for tumors to grow . The Warburg effect primarily manifests in glucose consumption, lactate production and ATP production in cancer cells. Therefore, by measuring the glucose consumption, lactate production and ATP levels, we can determine whether the drug has an impact on the glycolytic process. The results of this study demonstrated that α-hederin significantly reduced glucose uptake, lactate production and ATP levels in A549 cells of non-small cell lung cancer, indicating that glycolysis in A549 cells was significantly inhibited in response to α-hederin treatment.
During the process of aerobic glycolysis, glucose is transferred into the cytoplasm by initial glucose transporters (GLUTs). Then, glucose is catalyzed into glucose 6 phosphate by hexokinase (HK), followed by 6-phosphate glucose transfer into pyruvate by PKM2. Pyruvate is catalyzed to lactate by lactate dehydrogenase (LDH) and finally transported out of cell by MCTs. The results of this study indicate that α-hederin significantly reduces expression of GLUT1, HK2, PKM2, LDHA, and MCT4 among the glycolytic proteins of A549 cells of NSCLC both in vitro and in vivo.
GLUT1, HK2, PKM2, LDHA, MCT4 and other relevant proteins involved in glycolysis play a role in directly affecting the glycolytic process. However, these key enzymes are also regulated by relevant regulatory factors. Akt play a very important role in cell energy metabolism, and Akt may enhance aerobic glycolysis of tumor cells . p53 is a tumor suppressor gene, and p53 reprograms energy metabolism to negatively regulate cell glycolysis by promoting mitochondrial oxidative phosphorylation and inhibiting glycolysis [18, 19]. GLUTs and glycolytic related catalytic enzymes are also regulated by p53 to inhibit glycolysis of cancer cells. Expression of GLUT1 and GLUT4 can be directly inhibited and are indirectly adjusted by GLUT3 . p53 also regulates the PI3K/Akt/mTOR pathway to regulate glucose metabolism . Hypoxia inducible factor-1 (HIF-1α) is in a class of transcription factors that are heterodimers composed of subunits to adapt to the response of tumor cells to environmental changes in the hypoxic environment. HIF-1α can regulate glucose uptake, glycolytic enzymes, and expression of single carboxylic acid transporters, such as GLUT1, GLUT3, HK2, fructose phosphate kinase 2 (PFK2), aldolase A (ALDOA), enolization enzyme (ENO), pyruvate kinase M (PKM), and expression of LDHA and MCT 4 [22–24]. c-Myc is a member of the Myc gene family of oncogenes and is associated with a wide variety of tumor development, and c-Myc promotes glucose absorption by up-regulating GLUT1 [25–26], enhancing transcription of glycolytic enzymes HK2, PFK, and LDHA [27–28]. c-Myc also up-regulates expression of MCT and PKM2 [29–31]. In this study, we found that α-hederin significantly reduces expression of glycolytic regulators HIF-1α and c-Myc in non-small cell lung cancer A549 cells but had no significant effect on expression of p53 or Akt.
SIRT6 plays a key regulatory role in gene transcription, metabolism, maintenance of genomic stability and the integrity of telomeres, thus regulating the occurrence and development of diabetes, obesity, heart disease, cancer and other diseases, SIRT6 regulate the metabolism of fat and glucose, which is a key regulator of energy stress and closely related to the process of tumors , SIRT6 inhibited the activity of transcription factor HIF-1α and inhibited glucose oxidation and glycolysis through the citric acid cycle , SIRT6 was also found to co-inhibit the transcription activity of the central oncogene MYC of the ribosome gene , we found that α-hederin significantly increase expression of SIRT6, we used a SIRT6 inhibitor, OSS_128167, for verification. The results showed that expression of HIF-1α and c-Myc were decreased in OSS_128167 treatment in combination with α-hederin compared to OSS_128167 alone. Combined with the results of previous experiments, our results demonstrate that α-hederin activates expression of SIRT6 then regulate tumour glycolysis.