Atypical protein kinase C isoforms differentially regulate directional keratinocyte migration during wound healing.

BACKGROUND: The epidermis possesses regenerative properties that become apparent only after wounding. Atypical protein kinase C (aPKC) isoforms aPKCζ and aPKCλ form a ternary complex with Par3 and Par6, and play crucial roles in establishing and maintaining epithelial cell polarity. The epidermal loss of aPKCλ results in progressive depletion of hair follicle stem cells. However, it is unclear whether aPKCs have equivalent activities in epidermal regeneration. OBJECTIVES: To clarify functional differences between aPKCζ and aPKCλ in cutaneous wound healing. METHODS: We compared cutaneous wound healing processes in vivo using mutant mice with genetic deletion of each aPKC isoform. We also analyzed functional differences between aPKCζ and aPKCλ in cell proliferation, directional cell migration, and formation of microtubules in vitro using primary keratinocytes established from each mutant mouse. RESULTS: Wound healing was significantly retarded in epidermis-specific aPKCλ knockout mice. In aPKCλ-deleted keratinocytes, the correct orientation of cell protrusions toward the wound was disrupted through the destabilization of Par6ß. The elongation of stabilized ß-tubulin was also deteriorated in aPKCλ-deleted keratinocytes, leading to defects in cell spreading. Conversely, wound healing and directional cell migration in aPKCζ-deleted mice were comparable to those in their control littermates. CONCLUSIONS: aPKCs are not functionally equivalent; aPKCλ, but not aPKCζ, plays a primary role in cutaneous wound healing.

Topical 3-bromopyruvate is a novel targeted therapy for melanoma in a preclinical model.

BACKGROUND: Targeting cancer metabolism is a promising strategy in improving cancer treatment. OBJECTIVE: To introduce a targeted therapy with topical 3-bromopyruvate (3BP), aglycolytic inhibitor, into the clinic in the near future. METHOD: We investigated the anti-tumor efficacy of 3BP on melanoma cells in vitro and in a preclinical model. RESULTS: Our cell-based study demonstrated that 3BP showed cytotoxicity for melanoma cells under anchorage-dependent or independent cell growth via a reactive oxygen species-mediated and caspase-independent cell death pathway. Moreover, 3BP inhibited both self-renewal potential and growth of slow-cycling phenotype in melanoma cells. Remarkably, the preclinical mouse xenograft model shed light on topical application of 3BP, showing significant anti-tumor effects with no apparent toxicity in surrounding normal tissues. CONCLUSION: We have now proposed that a targeted therapy with topical 3BP is an innovative strategy for adjuvant chemotherapy of technically or medically unresectable melanoma and possibly other skin cancers.

Rapid immunohistochemistry based on alternating current electric field for intraoperative diagnosis of brain tumors.

Rapid immunohistochemistry (R-IHC) can contribute to the intraoperative diagnosis of central nervous system (CNS) tumors. We have recently developed a new IHC method based on an alternating current electric field to facilitate the antigen-antibody reaction. To ensure the requirement of R-IHC for intraoperative diagnosis, 183 cases of CNS tumors were reviewed regarding the accuracy rate of diagnosis without R-IHC. The diagnostic accuracy was 90.7 % (166/183 cases) [corrected] in which definitive diagnoses were not provided in 17 cases because of the failure of glioma grading and differential diagnosis of lymphoma and glioma. To establish the clinicopathological application, R-IHC for frozen specimens was compared with standard IHC for permanent specimens. 33 gliomas were analyzed, and the Ki-67/MIB-1 indices of frozen specimens by R-IHC were consistent with the grade and statistically correlated with those of permanent specimens. Thus, R-IHC provided supportive information to determine the grade of glioma. For discrimination between glioma and lymphoma, R-IHC was able to provide clear results of CD20 and Ki-67/MIB-1 in four frozen specimens of CNS lymphoma as well as standard IHC. We conclude that the R-IHC for frozen specimens can provide important information for intraoperative diagnosis of CNS tumors.

A novel immunohistochemical staining method allows ultrarapid detection of lymph node micrometastases while conserving antibody.

We developed a novel ultrarapid immunohistochemical staining method in which an AC electric field is used to facilitate detection of tumor cells. Frozen sections of non-small cell lung cancer in lymph nodes were fixed in acetone for 2 min, after which they were incubated for 2 min with an anti-pancytokeratin antibody cocktail and then with EnVision(TM) complex under an alternating current (AC) electric field. The sections were then incubated with a chromogen (3,3'diaminobenzidine) for 3 min and counterstained with hematoxylin. This method enabled detection of tumor cells in frozen sections in less than 15 min. In addition, we were able to reduce the amount of antibody used by more than 90% when the sections were incubated under the AC electric field for a longer period. This method could be a useful tool for frozen section diagnosis and research. Furthermore, with this method the cost of immunohistochemical staining can be reduced.