Serum depletion induced cancer stem cell-like phenotype due to nitric oxide synthesis in oncogenic HRas transformed cells.

Cancer cells rewire their metabolism and mitochondrial oxidative phosphorylation (OXPHOS) to promote proliferation and maintenance. Cancer cells use multiple adaptive mechanisms in response to a hypo-nutrient environment. However, little is known about how cancer mitochondria are involved in the ability of these cells to adapt to a hypo-nutrient environment. Oncogenic HRas leads to suppression of the mitochondrial oxygen consumption rate (OCR), but oxygen consumption is essential for tumorigenesis. We found that in oncogenic HRas transformed cells, serum depletion reversibly increased the OCR and membrane potential. Serum depletion promoted a cancer stem cell (CSC)-like phenotype, indicated by an increase in CSC markers expression and resistance to anticancer agents. We also found that nitric oxide (NO) synthesis was significantly induced after serum depletion and that NO donors modified the OCR. An NOS inhibitor, SEITU, inhibited the OCR and CSC gene expression. It also reduced anchorage-independent growth by promoting apoptosis. In summary, our data provide new molecular findings that serum depletion induces NO synthesis and promotes mitochondrial OXPHOS, leading to tumor progression and a CSC phenotype. These results suggest that mitochondrial OCR inhibitors can be used as therapy against CSC.

[Analysis of the Neuraminidase Amino Acid Sequences of Influenza A/H1N1pdm09, A/H3N2, and B Viruses Isolated from Influenza Patients in the 2013/14 Japanese Influenza Season].

BACKGROUND: Neuraminidase (NA) is an essential surface protein for influenza virus replication. NA inhibitors are commonly used for the treatment of influenza patients in Japan. Several mutations that reduce the effect of NA inhibitors have been reported. We sequenced the whole NA segment of isolated virus from influenza patients and investigated the relation between the NA amino acid sequence and the 50% inhibitory concentration (IC50) of four NA inhibitors. MATERIALS AND METHODS: A total of 20 viruses that showed high or low IC50 of NA inhibitors were selected from A/H1N1pdm09, A/H3N2, and B isolates from the viruses isolated from patients in the 2013-14 influenza season. Viral RNA was extracted and RT-PCR was done. The amplified genome was sequenced using a next generation sequencer", and the deduced amino acid sequences were analyzed. RESULTS: Two A/H1N1pdm09 viruses that showed very high IC50 for oseltamivir (150 nM and 130 nM) contained the H275Y mutation. Otherwise, no significant relation was found between the NA amino acids and the IC50 of the four NA inhibitors. There was no significant relation between the NA amino acids and the IC50 of the four NA inhibitors for A/H3N2 viruses. The B viruses that showed a high IC50 for oseltamivir and laninamivir shared some amino acids. The B viruses that showed a high IC50 of zanamivir and peramivir also shared some amino acids. They were different from the shared amino acids found for oseltamivir and laninamivir. CONCLUSION: The previously reported H275Y mutation that causes oseltamivir resistance was found in the two A/H1N1pdm09 viruses that showed a very high IC50 for oseltamivir. No additional NA amino acid sequences related to the IC50 of the four NA inhibitors was found. The meaning of the shared amino acids among B viruses that showed a high IC50 would be an interesting target for further investigation.

[Analysis of influenza A/H3N2 neuraminidase genes obtained from influenza patients in the 2011/12 and 2012/13 seasons in Japan].

BACKGROUND: Influenza virus has neuraminidase (NA), a surface protein with enzymatic activity that is essential for virus replication. Mutation may affect the effectiveness of NA inhibitors that are used for the treatment of influenza patients. In this study, we determined the NA gene sequences from the clinical isolates of influenza patients to examine the chronological genetic changes and the relation to drug susceptibility. METHODS: For 96 A/H3N2 virus isolates the 50% inhibitory concentration (IC50) (48 each from the 2011-12 and 12-13 influenza seasons) was measured. RT-PCR was done with extracted viral RNA, followed by nucleotide sequencing. RESULTS: One putative amino acid mutation, D151N, was found in an NA activity-related cite in five of ninety-six tested isolate. The mutation did not affect the IC50 value. The mutations identified at amino acid positions 387 and 400 were statistically correlated with an increased IC50 value, although the change was less than ten times, suggesting no significant difference in the clinical effectiveness. A small number .of isolates showed mutation in the T and/or B cell epitope region of NA. CONCLUSION: No mutation that affected the IC50 value or effectiveness of NAIs was detected. Antigenic mutations of NA, which influence the selection of epidemic strains, were not determined. Continuous observation will be necessary to further clarify the genetic features of NA.

The functional role of arginine 901 at the C-terminus of the human anion transporter band 3 protein.

To determine which arginine residues are responsible for band 3-mediated anion transport, we analyzed hydroxyphenylglyoxal (HPG)-modified band 3 protein in native erythrocyte membranes. HPG-modification leads to inhibition of the transport of phosphoenolpyruvate, a substrate for band 3-mediated transport. We analyzed the HPG-modified membranes by reverse phase-HPLC, and determined that arginine 901 was modified by HPG. To determine the role of Arg 901 in the conformational change induced by anion exchange, we analyzed HPG-modification of the membranes when 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethypyrocarbonate (DEPC) was present. DNDS and DEPC fix band 3 in the outward and inward conformations, respectively. HPG-modification was unaffected in the presence of DEPC but decreased in the presence of DNDS. In addition to that, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which specifically reacts with the outward conformation of band 3, did not react with HPG-modified membranes. Furthermore, we expressed a band 3 mutant in which Arg 901 was replaced by alanine (R901A) on yeast membranes. The kinetic parameters indicated that the R901A mutation affected the rate of conformational change of the band 3 protein. From these results, we conclude that the most C-terminal arginine, Arg 901, has a functional role in the conformational change that is necessary for anion transport.