Released lipids regulate transient receptor potential channel (TRP)-dependent oral cancer pain.

BACKGROUND: Pain in the head neck area is an early symptom in oral cancer, supporting the hypothesis that cancer cells control the activities of surrounding nociceptors at the site of the tumor. Several reports implicate TRPV1 and TRPA1 in cancer pain, although there is a large gap in knowledge since the mechanisms for tumor-induced activation of these TRP receptors are unknown. Interestingly, TRP-active lipids such as linoleic acid, arachidonic acid, hydroxyoctadecadienoic acid and hydroxyeicosatetraenoic acid are significantly elevated in the saliva of oral cancer patients compared to normal patients, supporting a possible linkage between these lipids and oral cancer pain. We therefore hypothesize that oral squamous cell carcinomas release certain lipids that activate TRPV1 and/or TRPA1 on sensory neurons, contributing to the development of oral cancer pain. METHODS: Lipid extracts were made from conditioned media of three human oral squamous cell carcinoma (OSCC) cell lines as well as one normal human oral keratinocytes cell line. These were then injected intraplantarly into rat hindpaws to measure spontaneous nocifensive behavior, as well as thermal and mechanical allodynia. For interventional experiments, the animals were pretreated with AMG517 (TRPV1 antagonist) or HC030031 (TRPA1 antagonist) prior to extract injection. RESULTS: These studies demonstrate that lipids released from the three OSCC cell lines, but not the normal cell line, were capable of producing significant spontaneous nocifensive behaviors, as well as thermal and mechanical allodynia. Notably each of the cell lines produced a different magnitude of response for each of three behavioral assays. Importantly, pre-treatment with a TRPVI antagonist blocked lipid-mediated nocifensive and thermal hypersensitivity, but not mechanical hypersensitivity. In addition, pre-treatment with a TRPA1 antagonist only reversed thermal hypersensitivity without affecting lipid-induced nocifensive behavior or mechanical allodynia. CONCLUSIONS: These data reveal a novel mechanism for cancer pain and provide strong direction for future studies evaluating the cellular mechanism regulating the TRP-active lipids by OSCC tumors.

Probing peptide amphiphile self-assembly in blood serum.

There has been recent interest in designing smart diagnostic or therapeutic self-assembling peptide or polymeric materials that can selectively undergo morphological transitions to accumulate at a disease site in response to specific stimuli. Developing approaches to probe these self-assembly transitions in environments that accurately amalgamate the diverse plethora of proteins, biomolecules, and salts of blood is essential for creating systems that function in vivo. Here, we have developed a fluorescence anisotropy approach to probe the pH-dependent self-assembly transition of peptide amphiphile (PA) molecules that transform from spherical micelles at pH 7.4 to nanofibers under more acidic pH's in blood serum. By mixing small concentrations of a Ru(bipy)3(2+)-tagged PA with a Gd(DO3A)-tagged PA having the same lipid-peptide sequence, we showed that the pH dependence of self-assembly is minimally affected and can be monitored in mouse blood serum. These PA vehicles can be designed to transition from spherical micelles to nanofibers in the pH range 7.0-7.4 in pure serum. In contrast to the typical notion of serum albumin absorbing isolated surfactant molecules and disrupting self-assembly, our experiments showed that albumin does not bind these anionic PAs and instead promotes nanofibers due to a molecular crowding effect. Finally, we created a medium that replicates the transition pH in serum to within 0.08 pH units and allows probing self-assembly behavior using conventional spectroscopic techniques without conflicting protein signals, thus simplifying the development pathway from test tube to in vivo experimentation for stimuli-responsive materials.

A rationally designed pyrrolysyl-tRNA synthetase mutant with a broad substrate spectrum.

Together with tRNA(CUA)(Pyl), a rationally designed pyrrolysyl-tRNA synthetase mutant N346A/C348A has been successfully used for the genetic incorporation of a variety of phenylalanine derivatives with large para substituents into superfolder green fluorescent protein at an amber mutation site in Escherichia coli. This discovery greatly expands the genetically encoded noncanonical amino acid inventory and opens the gate for the genetic incorporation of other phenylalanine derivatives using engineered pyrrolysyl-tRNA synthetase-tRNA(CUA)(Pyl) pairs.