Autophagy regulates cisplatin‐induced stemness and chemoresistance via the upregulation of CD44, ABCB1 and ADAM17 in oral squamous cell carcinoma

Objective

We inspected the relevance of CD44, ABCB1 and ADAM17 in OSCC stemness and deciphered the role of autophagy/mitophagy in regulating stemness and chemoresistance.

Material and methods

A retrospective analysis of CD44, ABCB1 and ADAM17 with respect to the various clinico‐pathological factors and their correlation was analysed in sixty OSCC samples. Furthermore, the stemness and chemoresistance were studied in resistant oral cancer cells using sphere formation assay, flow cytometry and florescence microscopy. The role of autophagy/mitophagy was investigated by transient transfection of siATG14, GFP‐LC3, tF‐LC3, mKeima‐Red‐Mito7 and Western blot analysis of autophagic and mitochondrial proteins.

Results

In OSCC, high CD44, ABCB1 and ADAM17 expressions were correlated with higher tumour grades and poor differentiation and show significant correlation in their co‐expression. In vitro and OSCC tissue double labelling confirmed that CD44⁺ cells co‐expresses ABCB1 and ADAM17. Further, cisplatin (CDDP)‐resistant FaDu cells displayed stem‐like features and higher CD44, ABCB1 and ADAM17 expression. Higher autophagic flux and mitophagy were observed in resistant FaDu cells as compared to parental cells, and inhibition of autophagy led to the decrease in stemness, restoration of mitochondrial proteins and reduced expression of CD44, ABCB1 and ADAM17.

Conclusion

The CD44⁺/ABCB1⁺/ADAM17⁺ expression in OSCC is associated with stemness and chemoresistance. Further, this study highlights the involvement of mitophagy in chemoresistance and autophagic regulation of stemness in OSCC.     

Fractal fluctuations in human respiration.

The present study was designed to characterize respiratory fluctuations in awake, healthy adult humans under resting conditions. For this purpose, we recorded respiratory movements with a strain-gauge pneumograph in 20 subjects. We then used Allan factor, Fano factor, and dispersional analysis to test whether the fluctuations in the number of breaths, respiratory period, and breath amplitude were fractal (i.e., time-scale-invariant) or random in occurrence. Specifically, we measured the slopes of the power laws in the Allan factor, Fano factor, and dispersional analysis curves for original time series and compared these with the slopes of the curves for surrogates (randomized data sets). In addition, the Hurst exponent was calculated from the slope of the power law in the Allan factor curve to determine whether the long-range correlations among the fluctuations in breath number were positively or negatively correlated. The results can be summarized as follows. Fluctuations in all three parameters were fractal in nine subjects. There were four subjects in whom only the fluctuations in number of breaths and breath amplitude were fractal, three subjects in whom only the fluctuations in number of breaths were fractal, and two subjects in whom only fluctuations in breath number and respiratory period were fractal. Time-scale-invariant behavior was absent in the two remaining subjects. The results indicate that, in most cases, apparently random fluctuations in respiratory pattern are, in fact, correlated over more than one time scale. Moreover, the data suggest that fractal fluctuations in breath number, respiratory period, and breath amplitude are controlled by separate processes.     

Medullary lateral tegmental field neurons influence the timing and pattern of phrenic nerve activity in cats.

In an effort to characterize the role of the medullary lateral tegmental field (LTF) in regulating respiration, we tested the effects of selective blockade of excitatory (EAA) and inhibitory amino acid (IAA) receptors in this region on phrenic nerve activity (PNA) of vagus-intact and vagotomized cats anesthetized with dial-urethane. We found distinct patterns of changes in central respiratory rate, duration of inspiratory and expiratory phases of PNA (Ti and Te, respectively), and I-burst amplitude after selective blockade of EAA and IAA receptors in the LTF. First, blockade of N-methyl-D-aspartate (NMDA) receptors significantly (P < 0.05) decreased central respiratory rate primarily by increasing Ti but did not alter I-burst amplitude. Second, blockade of non-NMDA receptors significantly reduced I-burst amplitude without affecting central respiratory rate. Third, blockade of GABAA receptors significantly decreased central respiratory rate by increasing Te and significantly reduced I-burst amplitude. Fourth, blockade of glycine receptors significantly decreased central respiratory rate by causing proportional increases in Ti and Te and significantly reduced I-burst amplitude. These changes in PNA were markedly different from those produced by blockade of EAA or IAA receptors in the pre-B?tzinger complex. We propose that a proper balance of excitatory and inhibitory inputs to several functionally distinct pools of LTF neurons is essential for maintaining the normal pattern of PNA in anesthetized cats.     

Conformation and Trimer Association of the Transmembrane Domain of the Parainfluenza Virus Fusion Protein in Lipid Bilayers from Solid-State NMR: Insights into the Sequence Determinants of Trimer Structure and Fusion Activity

Enveloped viruses enter cells by using their fusion proteins to merge the virus lipid envelope and the cell membrane. While crystal structures of the water-soluble ectodomains of many viral fusion proteins have been determined, the structure and assembly of the C-terminal transmembrane domain (TMD) remains poorly understood. Here we use solid-state NMR to determine the backbone conformation and oligomeric structure of the TMD of the parainfluenza virus 5 fusion protein. ¹³C chemical shifts indicate that the central leucine-rich segment of the TMD is α-helical in POPC/cholesterol membranes and POPE membranes, while the Ile- and Val-rich termini shift to the β-strand conformation in the POPE membrane. Importantly, lipid mixing assays indicate that the TMD is more fusogenic in the POPE membrane than in the POPC/cholesterol membrane, indicating that the β-strand conformation is important for fusion by inducing membrane curvature. Incorporation of para-fluorinated Phe at three positions of the α-helical core allowed us to measure interhelical distances using ¹⁹F spin diffusion NMR. The data indicate that, at peptide:lipid molar ratios of ~ 1:15, the TMD forms a trimeric helical bundle with inter-helical distances of 8.2–8.4 Å for L493F and L504F and 10.5 Å for L500F. These data provide high-resolution evidence of trimer formation of a viral fusion protein TMD in phospholipid bilayers, and indicate that the parainfluenza virus 5 fusion protein TMD harbors two functions: the central α-helical core is the trimerization unit of the protein, while the two termini are responsible for inducing membrane curvature by transitioning to a β-sheet conformation.