HIF-1α is a "master" gene controlling the hypoxic response in mammalian cells and thus plays an important role in tumor growth. In this study, we demonstrated that modulating the expression level of HIF-1α was able to increase or decrease apoptosis of a tumor cell line, A549, under hypoxic stress. Furthermore, this effect of HIF-1α was dependent upon the glycolysis pathway and pH changes of the culture medium, both of which appear to be regulated by HIF-1α during hypoxia.
During hypoxia, transportation of glucose into the cell and glycolytic enzyme activity are enhanced. Consistent with previous studies, we also found that hypoxia up-regulated expression of two key genes in the glycolysis pathway, HK1 and PGK1, as well as a glucose transportation gene, GLUT1 [3, 8, 9]. These genes were responsive to the level of HIF-1α, as down-regulation of HIF-1α with siRNA decreased their expression, while up-regulation of HIF-1α with an expression vector similarly increased expression of GLUT1, HK1, and PGK1. Taken together, our results indicate that the expression of key genes controlling glucose transportation and glycolysis are under the control of HIF-1α during hypoxia. Furthermore, over-expression HIF-1α in hypoxic A549 cells resulted in increased accumulation of lactate and decreased pH in the culture medium. When HIF-1α expression was inhibited with siRNA during hypoxia in the converse experiment, lactate levels instead decreased, accompanied by a restoration of normal pH in the culture medium. As lactate is the final product of glycolysis, increased glycolysis is expected to lead to acidosis of cell culture medium during hypoxia. Therefore, lactate accumulation and acidic pH may be an indicator of increased glycolytic activity. Thus, based on the result showing that HIF-1α regulates the expression of genes involved in both glucose transportation and glycolysis described above, we conclude that HIF-1α regulates the glycolysis pathway of A549 cells during hypoxia.
It is important to note that in another model, hypoxic rat cardiomyocytes, glucose-uptake and metabolism was found to be protective against hypoxia-induced apoptosis . In that study, glycolysis was shown to account for the protective effects, but other metabolic substrates provided no such protection from apoptosis . In contrast to this, the study presented here found that HIF-1α contributes to up-regulation of the glycolysis pathway of A549 cells during hypoxia. Furthermore, this increased metabolic activity leads to hypoxia-induced apoptosis in this lung cancer cell line. Although HIF-1α was found to be protective against hypoxia-induced apoptosis in cardiomyocytes, we instead found that increased HIF-1α expression further exacerbated apoptosis in A549 lung adenocarcinoma cells. We also found that inhibition of glycolysis with 2-DG attenuated HIF-1α-induced apoptosis in these cells during hypoxia. Both increased glycolysis and the resulting apoptosis could be inhibited by knocking-down HIF-1α expression with siRNA. Together, our results strongly implicate HIF-1α in the hypoxia-induced apoptosis of A549 cells, which depends on the glycolysis pathway and acidosis of the culture medium.
Despite examples to the contrary, this effect is not entirely unprecedented. In another relevant study, Schmaltz et al  reported that apoptosis in hypoxic cultures was primarily due to decreased pH of the culture medium, most likely caused by lactic acidosis. Here, we also observed that decreased pH triggered apoptosis in A549 cells. We were able to partially inhibit HIF-1α-induced apoptosis by increasing the buffering capacity of the medium to reduce acidosis. Further pointing to a role for HIF-1α in these processes, siRNA-knockdown of HIF-1α significantly blocked by the decrease of pH and apoptosis induced by hypoxia.
Consistent with our results, Krick et al  recently reported that hypoxia suppressed alveolar epithelial cell proliferation and enhanced alveolar type II cell apoptosis through activation of the HIF-1α/HRE axis and BNIP3, but they did not further investigate the role of acidosis in the action of BNIP3. Kubasiak et al , however, demonstrated that BNIP3 protein accumulated more rapidly under acidic pH and peaked at a significantly higher level than at neutral pH when cells were exposed to hypoxia. Acidosis was found to increase binding of BNIP3 to mitochondrial membranes, leading to apoptosis of hypoxic cells.
Taken together, when the cells are exposed to hypoxia, up-regulated expression of HIF-1α accelerates the glycolysis, which leads to the intracellular acidosis. Intracellular acidosis may activate the BNIP3 and promoted the hypoxic cell apoptosis. But this mechanism of HIF-1α on the hypoxia induced cell apoptosis needs further studies.