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Kynurenine (KYN), the most abundant metabolite of tryptophan, is classically associated with immune tolerance and tumor immune escape. In the last years, KYN is in the spotlight in other biological processes. Here, we showed that KYN inhibited tyrosinase expression and melanin content in primary human melanocyte and keratinocyte co-cultures. Furthermore, KYN decreased melanosome content in a 3D human skin reconstruction model. In these experiments, we used tyrosine + NH4 Cl to induce pigmentation. We compared the inhibitory effect of KYN on melanogenesis with the already known inhibitory effect promoted by IFN-γ. Since increased KYN production depends on the IFN-γ-inducible enzyme indoleamine-2,3-dioxygenase (IDO), we propose that part of the effect of IFN-γ on melanogenesis involves KYN production. From that, we tested if, during melanogenesis, changes in tryptophan metabolism would occur. For this purpose, we measured tryptophan, KYN and downstream products along with pigmentation. There were no significant changes in Trp metabolism, except for the high consumption of kynurenic acid. Our data identify the skin as a potential target for the action of KYN relevant for skin physiology and pigmentation. The results are discussed concerning the high production of KYN in skin inflammatory disorders and cancer.

Background Human papillomavirus (HPV) infection is the central factor for cervical cancer, whereas epithelial immune mechanisms contribute to the progression of HPV infection and its associated lesions. The authors evaluated the expression of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) in cervicovaginal samples from women with normal cervical epithelium or with different degrees of squamous intraepithelial lesions (SILs) and cervical cancer. Methods IDO expression was analyzed by immunocytochemistry in liquid-based cytology samples from 165 women, of whom 42 had cervical changes subclassified as low-grade SIL (n = 6), high-grade SIL (n = 30), or squamous cell carcinoma (SCC) (n = 6), and 123 had negative Papanicolaou smears. IDO and TDO expression also were analyzed by immunohistochemistry, and HPV and other genital pathogens were evaluated by polymerase chain reaction analysis. Results Low IDO expression was observed in normal cervical epithelium irrespective of HPV status. Increased numbers of IDO-positive squamous cells and IDO-positive leukocytes were observed in women with SIL or SCC. TDO expression was detected in leukocytes infiltrating the stroma around intraepithelial or invasive cervical lesions. Higher IDO levels were detected in organotypic epithelial cultures established from keratinocytes transduced with the HPV16 E6/E7 oncoproteins. Conclusions The upregulation of IDO expression in leukocytes and squamous cells in HPV-associated SIL and SCC suggests that immunosuppressive mechanisms involving tryptophan metabolism may have a role in cervical carcinogenesis. Although previous studies have suggested the role of IDO in HPV pathogenesis, this is the first evidence of TDO involvement in the process. Furthermore, the current data emphasize the role of leukocytes, especially neutrophil-like cells, as an IDO source.

Studies about cervical carcinogenesis have demonstrated the increased expression of matrix-metalloproteinase (MMP) according to the grade of cervical intraepithelial lesions. Considering the importance of innovative techniques to introduce noninvasive and rapid diagnoses for patients, this study aimed to perform MMP-9 immunocytochemistry in cervical smears according to the cytopathological diagnoses, in order to monitor MMP activity in cervical smears. This cross-sectional study investigated the expression of MMP-9 in normal cervical smears, inflammatory cervical smears, squamous intraepithelial lesions, and cervical carcinoma. Cervical smears from 630 women were collected for cytopathological diagnoses and immunocytochemistry. Women with squamous intraepithelial lesions showed an increase in MMP-9 expression, with moderate to intense staining occurring with increasing cervical lesion grade. The prevalence of moderate to intense MMP-9 staining was 9% in normal cervical smears, 12% in cervical inflammation, 24% in low-grade squamous intraepithelial lesion (LSIL), 92% in high-grade squamous intraepithelial lesions (HSIL) and 100% in cervical carcinoma cases. In the specific case of LSIL, we found that association with MMP-9 is more evident when there is the simultaneous presence of an infectious agent. Thus, the expression of MMP-9 in cervical smears increases according to the grade of cervical lesion and LSIL in the presence of infectious agents showed higher MMP-9 expression than women with LSIL without infectious agents. Diagn. Cytopathol. 2014;42:827–833. © 2014 Wiley Periodicals, Inc.

This study investigates the mechanism of action behind the long-term responses (12–16 months) of two BRAF WT melanoma patients to the AKT inhibitor MK-2206 in combination with paclitaxel and carboplatin. Although single agent MK-2206 inhibited phospho-AKT signaling, it did not impact in vitro melanoma growth or survival. The combination of MK-2206 with paclitaxel and carboplatin was cytotoxic in long-term colony formation and 3D spheroid assays, and induced autophagy. Autophagy was initially protective with autophagy inhibitors and deletion of ATG5 found to enhance cytotoxicity. Although prolonged autophagy induction (>6 days) led to caspase-dependent apoptosis, drug resistant clones still emerged. Autophagy inhibition enhanced the cell death response through reactive oxygen species and could be reversed by anti-oxidants. We demonstrate for the first time that AKT inhibition in combination with chemotherapy may have clinical activity in BRAF WT melanoma and show that an autophagy inhibitor may prevent resistance to these drugs.

Summary Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half-life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4-Nerolidylcatechol (4-NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4-NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl-1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4-NC is time-dependent upon the pro-apoptotic protein Noxa, which is able to bind and neutralize Mcl-1. We demonstrate the role of 4-NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4-NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.

Skin, the largest organ of the human body, is organized into an elaborate layered structure consisting mainly of the outermost epidermis and the underlying dermis. A subcutaneous adipose-storing hypodermis layer and various appendages such as hair follicles, sweat glands, sebaceous glands, nerves, lymphatics, and blood vessels are also present in the skin. These multiple components of the skin ensure survival by carrying out critical functions such as protection, thermoregulation, excretion, absorption, metabolic functions, sensation, evaporation management, and aesthetics. The study of how these biological functions are performed is critical to our understanding of basic skin biology such as regulation of pigmentation and wound repair. Impairment of any of these functions may lead to pathogenic alterations, including skin cancers. Therefore, the development of genetically controlled and well characterized skin models can have important implications, not only for scientists and physicians, but also for manufacturers, consumers, governing regulatory boards and animal welfare organizations. As cells making up human skin tissue grow within an organized three-dimensional (3D) matrix surrounded by neighboring cells, standard monolayer (2D) cell cultures do not recapitulate the physiological architecture of the skin. Several types of human skin recombinants, also called artificial skin, that provide this critical 3D structure have now been reconstructed in vitro. This review contemplates the use of these organotypic skin models in different applications, including substitutes to animal testing.

Invasive behavior is the pathological hallmark of malignant gliomas, being responsible for the failure of surgery, radiation, and chemotherapy. Matrix metalloproteinases (MMPs) are essential for proper ECM remodeling and invasion. The tumor and metastasis suppressor RECK protein regulates at least three members of the MMPs family: MMP-2, MMP-9, and MT1-MMP. In order to mimic the in vivo invasion process, A172 and T98G, respectively, non-invasive and invasive human glioblastoma cell lines, were cultured onto uncoated (control) or type I collagen gel-coated surface, and maintained for up to 7 days to allow establishment of the invasive process. We show that the collagen substrate causes decreased growth rates and morphological alterations correlated with the invasive phenotype. Electronic transmission microscopy of T98G cells revealed membrane invaginations resembling podosomes, which are typically found in cells in the process of crossing tissue boundaries, since they constitute sites of ECM degradation. Real time PCR revealed higher RECK mRNA expression in A172 cells, when compared to T98G cells and, also, in samples obtained from cultures where the invasive process was fully established. Interestingly, the collagen substrate increases RECK expression in A172 cells and the same tendency is displayed by T98G cells. MMPs-2 and -9 displayed higher levels of expression and activity in T98G cells, and their activities are also upregulated by collagen. Therefore, we suggest that: (1) RECK downregulation is critical for the invasiveness process displayed by T98G cells; (2) type 1 collagen could be employed to modulate RECK expression in glioblastoma cell lines. Since a positive correlation between RECK expression and patients survival has been noted in several types of tumors, our results may contribute to elucidate the complex mechanisms of malignant gliomas invasiveness. J. Cell. Biochem. © 2006 Wiley-Liss, Inc.