The Role And Mechanism Of Lactate In The Tumor To Adapt To Glucose Starvation

The essential characteristic of tumors is rapid growth; therefore, tumors need to generate a large number of new blood vessels to provide nutrients. Nonetheless, compared with the normal tissue, tumor angiogenesis is disarranged and functions poorly. The rapid tumor growth and the physiological characteristics of the tumor vasculature mean that the rate of formation of blood vessels providing the energy substances can not catch up with the rate of increase in tumor volume, such that many tumors exist in a low glucose environment. The concentration of glucose in the colon and stomach tumor tissue is only 0.12 and 0.4mM, respectively. Tumors can survive in harsh environments, such as poor glucose and oxygen depletion. In the case of glucose deficiency, tumors in vivo adjust their own state to adapt to the environment and obtain more nutrition. By contrast, lack of glucose in cultured tumor cells in vitro does not support survival of tumor cells. Therefore, to explore how tumor survive glucose depletion in vivo is very important.Otto Warburg first observed that even in cases with good oxygen supply, tumors still preferred to utilize glucose via glycolysis. These changes in tumor metabolic patterns, increased glucose uptake and glycolysis as the main production method, eventually led to the accumulation of lactate, the end product of glycolysis, in the tumor. Some studies have reported increased lactate levels within tumors, which reflects the high metastasis rate and poor prognosis in human cervical cancers, human head-and-neck cancers, human rectal adenocarcinomas and non-small-cell lung cancers. Some studies have suggested that lactate could be used as an energy source by oxidative phosphorylation to generate ATP. Lactate could also be used as a signal molecule, such as promoting endothelial cells secrete VEGF, which can promote the migration of tumor cells and stimulate angiogenesis. Lactate could inhibit the activation of T cells, also has no effect on Treg cells, this function can help to complete the immune escape of tumor cells.In solid tumors, the growth of vascular development is far behind the rapid growth of the tumor, also led to the existence of the lack of glucose in tumor region. In blood rich regions, aerobic glycolysis consumes a large amount of glucose to produce lactate, whereas the lactate in the blood poor regions of tumor cells plays an important role. So we assumed that the lactate played an important role in the tumors lack of glucose.Part 1. Lactate maintained the tumor cells to survive in the glucose starvationTo determine the effect of lactate on glucose starvation, we exposed A549 cell lines to different culture conditions:complete medium containing different lactate concentrations and without glucose. We found that in glucose-free conditions, adding lactate into the medium could effectively prolong the survival times of the A549 cell line. The survival time of A549 cells in the presence of 20mM lactate was longer than that of any other concentration of lactate. We also confirmed the above phenomena in other types of tumor cell lines. The above results showed that lactate help tumor cells survive in the environment of the lack of glucose. Further we found that endogenous lactate also had the same effect, and the role of lactate needed hydrogen ions and lactate ions to participate. We determined whether under no glucose conditions, lactate could be used as an energy substrate. We found that the same concentration of pyruvate and lactate could not be the same as the extension of the tumor cells in glucose free environment in the survival time. Determination of dissolved oxygen in the culture of cells monitored in the Seahorse Bioscience XF24 instrument showed that, upon addition of lactate, oxidative phosphorylation levels did not change significantly. In addition, we examined the lactate concentrations in the cell culture:the extracellular lactate concentrations did not change significantly over the course of the experiment. Using RNA interference technology cut MCT1 and MCT4 expression level also did not significantly affect the role of lactate.Thus under glucose-free conditions, lactate is not an energy substrate. At the same time with the Real-time PCR detection under the regulation of lactate, the key enzyme in glycolysis, glucose metabolism, oxidative pentose phosphate pathway showed decreased expression. The use of glycolysis inhibitor 2-DG or aerobic respiration inhibitor rotenone compared with lactate they prolonged cell survival time in sugar free environment in a much smaller role. So the lactate in glucose-free environment indeed reduced the glucose metabolism level of the tumor cells, but the extent of regulation was not enough to explain lactate for a long time to extend the tumor cell survival in glucose-free environment. We further by flow cytometry instrument to detect cell apoptosis, the results showed that under the participation of the lactate A549 cell apoptosis rate significantly decreased in the glucose-free environment, reducing the apparent apoptosis made long-term survival of tumor cells in sugar-free environments.Part 2. Lactate activates the Bcl-2 signaling pathway to modulate the apoptotic response of cancer cells to glucose starvationInterestingly, lactate had different effects on different cell lines, allowing cell lines to be divided into sensitive insensitive to lactate groups. We compared the genetic backgrounds of the five cell lines and found that the cell lines insensitive to lactate lacked PTEN function and increased AKT phosphorylation. Then we respectively used AKT inhibitors Perifosine and activators insulin-like growth factor 1, the results showed that lactate activated AKT to help reduce the apoptosis of tumor cells in the glucose-free environment. We further through the use of PI3K inhibitors and Western blot confirmed lactate activated AKT via the rapid phosphorylation at Thr-308 and Ser-473 mediated by PI3K. By Western blot test protein changed, RNA interference technology demonstration function and flow cytometry instrument detection of apoptosis, we found that the lactate raising the Bel-2 protein expression in reducing tumor cell apoptosis in glucose-free environment was very important, and AKT activation and mTOR upregulation by lactate in glucose free conditions led to the upregulation of Bcl-2. By detecting the mitochondrial cytochrome C release conditions and the activity of Caspase-9, we showed that lactate in glucose-free environment raised the Bcl-2 to inhibit cytochrome C/apoptotic protease activating factor-1/Caspase 9 apoptosis pathway to reduce the apoptosis of tumor cells.Part 3. The role of lactate in the tumor to adapt to glucose starvation in vivo studyThrough the establishment of xenograft model in nude mice by subcutaneous lung cancer, and treatment in tumor surrounding subcutaneous injection of lactate and normal saline, we found that lactate to promote the growth of subcutaneous human lung carcinoma, reduce the survival time of tumor bearing mice, reduce subcutaneous human lung carcinoma necrosis. By TUNEL method and detection the Caspase 3/7 activity we confirmed lactate reduced subcutaneous human lung carcinoma apoptosis. Immunohistochemical analysis for slice of transplantation tumor, the results pointed out that lactate group compared with normal saline group, Phospho-AKT (Thr308), Phospho-AKT (Ser) 473, the Bcl-2 and Phospho-mTOR (Ser) 2448 expressions were increasing. The above results showed that lactate promoted the activation of PI3K/AKT/mTOR/Bcl-2 signaling pathway subcutaneous human lung A549 carcinoma in nude mice.Conclusion1. Lactate helped tumor cells survive in the environment of the lack of glucose. The endogenous lactate also had the same effect, and the role of lactate needed hydrogen ions and lactate ions to participate. Lactate did not directly provide energy for the tumor cells, but lower levels of the glucose metabolism of tumor cells.2. Lactate rescues cancer cells from glucose starvation-induced cell apoptosis through regulation of the PI3K/AKT/mTOR/Bcl-2 signaling pathway. Lactate in glucose-free environment raised the Bcl-2 to inhibit cytochrome C/apoptotic protease activating factor-1/Caspase-9 apoptosis pathway to reduce the apoptosis of tumor cells.3. Lactate promoted the growth of subcutaneous human lung A549 carcinoma, reduced the survival time of tumor bearing mice, reduced subcutaneous human lung A549 carcinoma necrosis and apotosis. Lactate promoted the activation of PI3K/AKT/mTOR/Bcl-2 signaling pathway subcutaneous human lung A549 carcinoma in nude mice.Novelty1. Find and verify that lactate might take part in the development of tumor. Lactate might be a potential molecular target for prediction and treatment of tumor.2. Find and verify that lactate did not directly provide energy for the tumor cells, but lower levels of the glucose metabolism of tumor cells and reduce tumor cell apoptosis to sustain its survival.3. Preliminary reveal the molecular mechanism of lactate to promote survival tumor. Lactate rescues cancer cells from glucose starvation-induced cell apoptosis through regulation of the PI3K/AKT/mTOR/Bcl-2 signaling pathway. Lactate in glucose-free environment raised the Bcl-2 to inhibit cytochrome C/apoptotic protease activating factor-1/Caspase-9 apoptosis pathway to reduce the apoptosis of tumor cells.