Biological features of tumor associated fibroblasts and effect of Transwell co-culture on proliferation and metastasis of gastric cancer cells / 中国药理学通报

; Aim To culture primary human gastric cancer associated fibroblasts (CAFs) and normal fibroblasts (NFs), and to explore the biological characteristics and their effects on gastric cancer cells. Methods After isolation and culture of CAFs and NFs, growth curve was drawn by MTT. The a-smooth muscle actin (ot-SMA) and Vimentin were detected by Immunofluorescence, Western blot and qRT-PCR. MGC-803 cells were co-cultured with CAFs and NFs in Transwell suspension mode. The migration and invasion ability of gastric cancer cells was detected by Transwell. The proliferation activity and AMD3100 on CAFs-gastric cancer co-culture system were compared by MTT. The acidic property, lactic acid and ROS contents of co-culture system were determined by PH meter, lactic acid and DCFH-DA method. Results The morphology of CAFs, NFs cells were in long spindle or flat star shape. The proliferation ability and overlapping growth phenomenon of CAFs were higher than those of NFs. The expression of ct-SMA and Vimentin cells was positive in CAFs, but low or negative in NFs cells. The activity of gastric cancer in low density co-culture group > medium density group > high density group, the PH value of CAFs co-culture system decreased, the content of lactic acid and ROS was high, and only CAFs low density co-culture group had significant effect on promoting cancer. Conclusions The co-culture of gastric cancer cells with CAFs and NFs is greatly affected by the proportion. Low density co-culture can significantly improve the proliferation and metastasis ability of gastric cancer cells. High density co-culture may in turn inhibit the growth and metastasis of cancer cells, which may be related to the content of lactic acid and ROS.

PKM2 promotes reductive glutamine metabolism.

OBJECTIVE: Pyruvate kinases M (PKM), including the PKM1 and PKM2 isoforms, are critical factors in glucose metabolism. PKM2 promotes aerobic glycolysis, a phenomenon known as " the Warburg effect". The purpose of this study was to identify the roles of PKM2 in regulating cellular metabolism. METHODS: The CRISPR/Cas9 system was used to generate the PKM-knockout cell model to evaluate the role of PKM in cellular metabolism. Lactate levels were measured by the Vitros LAC slide method on an autoanalyzer and glucose levels were measured by the autoanalyzer AU5800. The metabolism of 13C6-glucose or 13C5-glutamine was evaluated by liquid chromatography/mass spectrometry analyses. The effects of PKM on tumor growth were detected in vivo in a tumor-bearing mouse model. RESULTS: We found that both PKM1 and PKM2 enabled aerobic glycolysis, but PKM2 converted glucose to lactate much more efficiently than PKM1. As a result, PKM2 reduced glucose levels reserved for intracellular utilization, particularly for the production of citrate, and thus increased the α-ketoglutarate/citrate ratio to promote the generation of glutamine-derived acetyl-coenzyme A through the reductive pathway. Furthermore, reductive glutamine metabolism facilitated cell proliferation under hypoxia conditions, which supports in vivo tumor growth. In addition, PKM-deletion induced a reverse Warburg effect in tumor-associated stromal cells. CONCLUSIONS: PKM2 plays a critical role in promoting reductive glutamine metabolism and maintaining proton homeostasis. This study is helpful to increase the understanding of the physiological role of PKM2 in cancer cells.

The reverse Warburg effect is likely to be an Achilles' heel of cancer that can be exploited for cancer therapy.

Although survival outcomes of cancer patients have been improved dramatically via conventional chemotherapy and targeted therapy over the last decades, there are still some tough clinical challenges that badly needs to be overcome, such as anticancer drug resistance, inevitable recurrences, cancer progression and metastasis. Simultaneously, accumulated evidence demonstrates that aberrant glucose metabolism termed 'the Warburg effect' in cancer cell is closely associated with malignant phenotypes. In 2009, a novel 'two-compartment metabolic coupling' model, also named 'the reverse Warburg effect', was proposed and attracted lots of attention. Based on this new model, we consider whether this new viewpoint can be exploited for improving the existent anti-cancer therapeutic strategies. Our review focuses on the paradigm shift from 'the Warburg effect' to 'the reverse Warburg effect', the features and molecular mechanisms of 'the reverse Warburg effect', and then we discuss its significance in fundamental researches and clinical practice.