Supplementary MaterialsSupplementary Info. a profound influence on SP cells as evidenced by its capability to induce a substantial upsurge in the percentage of SP cells in the overall cancer cell population, and glucose starvation causes a rapid depletion of SP cells. Mechanistically, glucose upregulates the SP fraction through ATP-mediated suppression of AMPK and activation of the Akt pathway, leading to elevated expression of the ATP-dependent efflux pump ABCG2. Importantly, inhibition of glycolysis by 3-BrOP significantly reduces SP cells and impairs their ability to form tumors models to test the effect of glucose on the SP subpopulation. Flow cytometry sorting method was employed to separate SP cells from the non-SP cells, which were then compared for their metabolic properties and for the expression of relevant genes. We found that SP cells are more active in glycolysis when compared to the non-SP cells. Addition of glucose to the culture medium induced a significant increase in SP subpopulation in culture. We also revealed that several key genes involved in glucose metabolism were differentially expressed in SP and non-SP cells, and that the Akt pathway seemed to play a key role in mediating glucose-induced increase in SP cells. Finally, we investigated the potential therapeutic effect of glycolytic inhibition on the viability of SP cells and their ability to form tumor (known to affect HK-2 and PDK-1 expression) and c-Myc (known to affect HK-2 expression) appeared similar in SP and non-SP cells (Figures 2c and d), suggesting that the high expression of YH239-EE PDK1 and low expression of HK2 in SLC2A3 SP cells are unlikely due to differential expression of HIF-1or c-Myc in SP and non-SP cells. Glucose induces a reversible increase of SP cells in the cancer cell population Based on the observation that SP cells were highly glycolytic (Figure 2a), we postulated that glucose in the tissue environment might have a significant impact on SP cells. To test this possibility, we first cultured A549 cells in medium containing various concentrations of glucose and analyzed the percent of SP cells. As shown in Figure 3a, A549 cells in their routine culture medium (F12K) with 1260?mg/l glucose contained 5.04% SP cells. When the cells were switched to a medium containing a higher level of glucose (2000?mg/l, RPMI1640), there was a time-dependent increase in SP cells, which reached 26.48% YH239-EE at 72?h. In contrast, when the cells were switched to glucose-free RPMI1640 medium, the SP population dramatically decreased to 0.86% in 24?h and to less than 0.1% in 72?h (Figure 3a). Interestingly, A549 cells continuing to proliferate YH239-EE through the 1st 24?h within the glucose-free moderate, as the % of SP cells decreased substantially during this time period period (Supplementary Shape S2). Cell proliferation ceased once the cells had been cultured within the absence of blood sugar for an extended time frame (48C72?h, Supplementary Shape S2). Open up in another window Shape 3 Aftereffect of blood sugar on SP cell small fraction in lung tumor and cancer of the colon cell lines. (a) The lung tumor A549 cells had been maintained in regular F12K moderate including 1260?mg/l blood sugar. A portion from the cells was turned to RPMI 1640 moderate containing higher blood sugar (2000?mg/l) and another part of the cells was switched to YH239-EE glucose-free RPMI 1640 moderate. The cells cultured under three different circumstances had been analyzed for % of SP cells at 24, 48, and 72?h. (b) LoVo cells (human being cancer of the colon) had been taken care of in F12K moderate including 1260?mg/l blood sugar, switched to glucose-free RPMI 1640 moderate for 24?h, and changed with fresh medium including 2000 then?mg/l blood sugar for 24 and 48?h. The cells cultured under each one of these conditions had been gathered for SP evaluation. (c) Lung tumor cells (NCI-H460) had been maintained in RPMI 1640 medium containing 2000?mg/l glucose, switched to glucose-free RPMI 1640.