Re-oxygenation causes hypoxic tumor regression through restoration of p53 wild-type conformation and post-translational modifications

Abstract

Hypoxic tumors are resistant to conventional therapies through indirect mechanisms such as the selection of resistant phenotype under chronic hypoxia. Hyperbaric oxygen (HBO) therapy has been shown to increase oxygen level and induce apoptosis in hypoxic tumor. However, it could produce significant adverse effects including oxygen toxic seizures and severe radiation tissue injury due to high pressure. We have shown that repeated oxygenation at 30% O(2) (1 atmospheres absolute) results in significant regression of MCF-7 tumor xenografts without any adverse effect. In MCF-7 cells, re-oxygenation showed an eightfold increase in cellular apoptosis. Both in hypoxic tumor and in hypoxic cells, that exclusively favor p53 to exist in mutant conformation, re-oxygenation restores p53 wild-type conformation. The oxygen-mediated rescue of mutant p53 followed by its trans-activation is responsible for the induction of p53-downstream apoptotic, cell-cycle arrest and DNA-repair genes. Further, p53 trans-activation may thus be due to its post-translational modifications as a result of re-oxygenation. We have thus concluded that oxygen therapy without pressure, as opposed to HBO therapy, may be ideal for hypoxic tumor regression, which functions through oxygen-mediated rescue of mutant p53 followed by induction of apoptosis.

Figure 1

Re-oxygenation induces p53-dependent apoptosis in cancer cells. (a) The oxygen concentration in the peripheral and the core tissue regions of the MCF-7 tumors (approximately 2.2 cm³) were analyzed using EPR oximetry. The tumor core tissue shows 1.8 mm Hg pO₂, whereas the peripheral tumor tissue shows 14.2 mm Hg pO₂. The healthy non-tumor tissue shows 21.2 mm Hg pO₂ (n=5). ^*P<0.0148. (b) Cellular apoptosis is observed in hypoxic MCF-7 cells, which were re-oxygenated for 24 h at 10–100% oxygen concentration. Results show significant apoptotic fraction in hypoxic MCF-7 cells exposed to 30–100% oxygen (n=10, S.D., ANOVA). ^*P<0.042. (c) Annexin V staining of hypoxic MCF-7 cells shows significant increase in the apoptotic fraction of the hypoxic MCF-7 cells on re-oxygenation with 30% oxygen (n=10). (d) The effect of re-oxygenation (30% O₂) is observed on the cellular apoptotic fraction of various cancer cell lines with p53(+/+) and p53(−/−) status. The results show that re-oxygenation induces higher apoptosis in cancer cells with wild-type p53 (n=10, S.D., ANOVA). ^*P<0.022. (e) The role of p53 in re-oxygenation-induced apoptosis in hypoxic cancer cells using the MCF-7 p53(+/+), MCF-7 p53(−/−) and HCT p53(+/+), HCT p53(−/−) system. The results show that re-oxygenation induces 64 and 57% apoptosis in MCF-7 p53(+/+), HCT p53(+/+) cells. On the other hand a mere 17 and 21% apoptosis is observed in the MCF-7 p53(−/−), HCT p53(−/−) cells. ^*P<0.037

Figure 2

Re-oxygenation induces p53-dependent apoptosis in hypoxic tumors. (a) The volume of hypoxic MCF-7 p53(+/+), MCF-7 p53(−/−), HCT p53(+/+) and HCT p53(−/−) tumor xenografts is measured after treatment with cisplatin and re-oxygenation. Results show that cisplatin treatment does not cause any significant change in the tumor size. The re-oxygenation treatment induces >75% tumor size reduction in both MCF-7 p53(+/+) and HCT p53(+/+) tumors. On the other hand, re-oxygenation of MCF-7 p53(−/−) and HCT p53(−/−) tumors led to only 22 and 24% tumor regression (n=8, S.D., ANOVA). ^*P<0.034. (b) In-vivo apoptosis kit from FLIVO is used to analyze if the re-oxygenation-induced tumor regression is via apoptosis. The results show that re-oxygenation induces >75% apoptosis in both MCF-7 p53(+/+) and HCT p53(+/+) tumors and only 22 and 24% in MCF-7 p53(−/−) and HCT p53(−/−) tumors (n=8, S.D. ANOVA). ^*P<0.027

Figure 3

Re-oxygenation increases expression of p53 downstream genes. (a) Hypoxic and re-oxygenated cancer cells were transfected with luciferase cDNA constructs carrying p53 DNA-binding sites. Results show that p53 is unable to increase luciferase activity at the promoters of its downstream genes in hypoxic cancer cells (black bar). On re-oxygenation of the hypoxic cancer cells the luciferase activity of p53 downstream genes is significantly increased (blue bar) (n=11, S.D. ANOVA). (b) Real-time PCR using the apoptotic gene array kit from SA Biosciences (Valencia, CA, USA) shows that p53 downstream apoptotic genes were negatively regulated in hypoxic cancer cells (upper panel). Whereas the expression of these genes was significantly higher in re-oxygenated cancer cells (lower panel) (n=12). (c) Expression of proteins involved in p53 apoptotic pathway bax, apaf-1, puma, bad, bag-1, pig-3, bak-1, caspase-3, p53aip-1, pten-10 and tnfsf10 is analyzed in hypoxic (lane 2) and re-oxygenated (lane 3) cancer cells (n=10). Re-oxygenation-induced significant increase in the expression of these proteins

Figure 4

Re-oxygenation increases p53 post-translational modifications. Re-oxygenation converts mutant p53 to wild-type p53. (a) The p53 wild-type and mutant conformation were analyzed in hypoxic core tissue of control and treated MCF-7 tumor xenografts. Results show that p53 exists in a mutant conformation in hypoxic tumors. Cisplatin treatment is not effective in restoration of p53 (1620) conformation in the hypoxic zones of the solid tumors. On re-oxygenation of the tumors the wt p53 conformation showed a significant increase, and higher rate of the conversion of mutant p53 to wt p53 (n=7). (b) The ratio of p53 1620 and p53 240 form was analyzed in the hypoxic core tissue of MCF-7 p53(+/+) and HCT p53(+/+) tumors using in-vivo ELISA. The results show that re-oxygenation improves the p53-1620/p53-240 ratio and helps in synthesis of p53 1620 form. (c) The effect of re-oxygenation on the conversion of mt p53 to wt p53 is observed in hypoxic H1299 cells transfected with Wt p53 cDNA. IPP showed that at 0 h (i.e. normoxia) p53 1620 and p53 240 form are present in equal ration (lanes 3 and 4). After a brief hypoxic shock of 8 h, the p53 1620 form is stabilized (lanes 5 and 6). After a chronic hypoxia exposure of 54 h, the p53 1620 conformation is lost and p53 exists as 240-conformation (lanes 7 and 8). MCF-7 cells are re-oxygenated after 54 h of hypoxia and 6 h after re-oxygenation (60-h mark) p53 conformation is revived and IPP showed increase in 1620 form (lane 3). p53 wild-type conformation showed consistent rise with re-oxygenation and at 72 h mark p53 is mostly present in 1620 form (n=8). (d) The phosphorylation of p53 at Ser^{6, 9, 15, 20, 33, 37, 46, 315 and 392}, Thr^{18, 55, 81, 155 and 377} and acetylation of p53 at Lys^{120, 164, 305, 320, 373, 379 and 382} is analyzed using immunoprecipitation. Hypoxia abolishes p53 phosphorylation and acetylation whereas re-oxygenation induces phosphorylation and acetylation at p53 core domain along with p53 N and C termini (n=7)