THP-1 Monocytic Cells Are Polarized to More Antitumorigenic Macrophages by Serial Treatment with Phorbol-12-Myristate-13-Acetate and PD98059.

Background and Objectives: As modulators of the tumor microenvironment, macrophages have been extensively studied for their potential in developing anticancer strategies, particularly in regulating macrophage polarization towards an antitumorigenic (M1) phenotype rather than a protumorigenic (M2) one in various experimental models. Here, we evaluated the effect of PD98059, a mitogen-activated protein kinase kinase MAPKK MEK1-linked pathway inhibitor, on the differentiation and polarization of THP-1 monocytes in response to phorbol-12-myristate-13-acetate (PMA) under various culture conditions for tumor microenvironmental application. Materials and Methods: Differentiation and polarization of THP-1 were analyzed by flow cytometry and RT-PCR. Polarized THP-1 subsets with different treatment were compared by motility, phagocytosis, and so on. Results: Clearly, PMA induced THP-1 differentiation occurs in adherent culture conditions more than nonadherent culture conditions by increasing CD11b expression up to 90%, which was not affected by PD98059 when cells were exposed to PMA first (post-PD) but inhibited when PD98059 was treated prior to PMA treatment (pre-PD). CD11bhigh THP-1 cells treated with PMA and PMA-post-PD were categorized into M0 (HLA-DRlow and CD206low), M1 (HLA-DRhigh and CD206low), and M2 (HLA-DRlow and CD206high), resulting in an increased population of M1 macrophages. The transcription levels of markers of macrophage differentiation and polarization confirmed the increased M1 polarization of THP-1 cells with post-PD treatment rather than with PMA-only treatment. The motility and cytotoxicity of THP-1 cells with post-PD treatment were higher than THP-1 cells with PMA, suggesting that post-PD treatment enhanced the anti-tumorigenicity of THP-1 cells. Confocal microscopy and flow cytometry showed the effect of post-PD treatment on phagocytosis by THP-1 cells. Conclusions: We have developed an experimental model of macrophage polarization with THP-1 cells which will be useful for further studies related to the tumor microenvironment.

Applications of Mesenchymal Stem Cells in Skin Regeneration and Rejuvenation.

Mesenchymal stem cells (MSCs) are multipotent stem cells derived from adult stem cells. Primary MSCs can be obtained from diverse sources, including bone marrow, adipose tissue, and umbilical cord blood. Recently, MSCs have been recognized as therapeutic agents for skin regeneration and rejuvenation. The skin can be damaged by wounds, caused by cutting or breaking of the tissue, and burns. Moreover, skin aging is a process that occurs naturally but can be worsened by environmental pollution, exposure to ultraviolet radiation, alcohol consumption, tobacco use, and undernourishment. MSCs have healing capacities that can be applied in damaged and aged skin. In skin regeneration, MSCs increase cell proliferation and neovascularization, and decrease inflammation in skin injury lesions. In skin rejuvenation, MSCs lead to production of collagen and elastic fibers, inhibition of metalloproteinase activation, and promote protection from ultraviolet radiation-induced senescence. In this review, we focus on how MSCs and MSC-derived molecules improve diseased and aged skin. Additionally, we emphasize that induced pluripotent stem cell (iPSC)-derived MSCs are potentially advanced MSCs, which are suitable for cell therapy.

α-Pinene Enhances the Anticancer Activity of Natural Killer Cells via ERK/AKT Pathway.

Natural killer (NK) cells are lymphocytes that can directly destroy cancer cells. When NK cells are activated, CD56 and CD107a markers are able to recognize cancer cells and release perforin and granzyme B proteins that induce apoptosis in the targeted cells. In this study, we focused on the role of phytoncides in activating NK cells and promoting anticancer effects. We tested the effects of several phytoncide compounds on NK-92mi cells and demonstrated that α-pinene treatment exhibited higher anticancer effects, as observed by the increased levels of perforin, granzyme B, CD56 and CD107a. Furthermore, α-pinene treatment in NK-92mi cells increased NK cell cytotoxicity in two different cell lines, and immunoblot assays revealed that the ERK/AKT pathway is involved in NK cell cytotoxicity in response to phytoncides. Furthermore, CT-26 colon cancer cells were allografted subcutaneously into BALB/c mice, and α-pinene treatment then inhibited allografted tumor growth. Our findings demonstrate that α-pinene activates NK cells and increases NK cell cytotoxicity, suggesting it is a potential compound for cancer immunotherapy.

Ginsenoside 20(S)-protopanaxadiol induces cell death in human endometrial cancer cells via apoptosis.

BACKGROUND: 20(S)-protopanaxadiol (20(S)-PPD), one of the aglycone derivatives of major ginsenosides, has been shown to have an anticancer activity toward a variety of cancers. This study was initiated with an attempt to evaluate its anti-cancer activity toward human endometrial cancer by cell and xenograft mouse models. METHODS: Human endometrial cancer (HEC)-1A cells were incubated with different 20(S)-PPD concentrations. 20(S)-PPD cytotoxicity was evaluated using MTT assay. Apoptosis was detected using the annexin V binding assay and cell cycle analysis. Cleaved poly (ADP-ribose) polymerase (PARP) and activated caspase-9 were assessed using western blotting. HEC-1A cell tumor xenografts in athymic mice were generated by inoculating HEC-1A cells into the flank of BALB/c female mice and explored to validate 20(S)-PPD anti-endometrial cancer toxicity. RESULTS: 20(S)-PPD inhibited HEC-1A cell proliferation in a dose-dependent manner with an IC50 value of 3.5 µM at 24 h. HEC-1A cells morphologically changed after 20(S)-PPD treatment, bearing resemblance to Taxol-treated cells. Annexin V-positive cell percentages were 0%, 10.8%, and 58.1% in HEC-1A cells when treated with 0, 2.5, and 5 µM of 20(S)-PPD, respectively, for 24 h. 20(S)-PPD subcutaneously injected into the HEC-1A cell xenograft-bearing mice three times a week for 17 days manifested tumor growth inhibition by as much as 18% at a dose of 80 mg/kg, which sharply contrasted to controls that showed an approximately 2.4-fold tumor volume increase. These events paralleled caspase-9 activation and PARP cleavage. CONCLUSION: 20(S)-PPD inhibits endometrial cancer cell proliferation by inducing cell death via a caspase-mediated apoptosis pathway. Therefore, the 20(S)-PPD-like ginsenosides are endowed with ample structural information that could be utilized to develop other ginsenoside-based anticancer agents.

Oleanolic acid 3-acetate, a minor element of ginsenosides, induces apoptotic cell death in ovarian carcinoma and endometrial carcinoma cells via the involvement of a reactive oxygen species-independent mitochondrial pathway.

OBJECTIVES: Oleanolic acid, a minor element of ginsenosides, and its derivatives have been shown to have cytotoxicity against some tumor cells. The impact of cytotoxic effect of oleanolic acid 3-acetate on ovarian cancer SKOV3 cells and endometrial cancer HEC-1A cells were examined both in vivo and in vitro to explore the underlying mechanisms. METHODS: Cytotoxic effects of oleanolic acid 3-acetate were assessed by cell viability, phosphatidylserine exposure on the cell surface, mitochondrial release of cytochrome C, nuclear translocation of apoptosis-inducing factor, depolarization of mitochondrial transmembrane potential (ΔΨm), and generation of reactive oxygen species (ROS). In vivo inhibition of tumor growth was also assessed with xenografts in immunocompromised mice. RESULTS: Oleanolic acid 3-acetate exhibited potent cytotoxicity toward SKOV3 and HEC-1A cells by decreasing cell viability in a concentration-dependent manner. Importantly, oleanolic acid 3-acetate effectively suppressed the growth of SKOV3 cell tumor xenografts in immunocompromised mice. Furthermore, oleanolic acid 3-acetate induced apoptotic cell death as revealed by loss of ΔΨm, release of cytochrome c, and nuclear translocation of apoptosis-inducing factor with a concomitant activation of many proapoptotic cellular components including poly(ADP-ribose) polymerase, Bcl-2, and caspases-8, caspase-3, and caspase-7. Oleanolic acid 3-acetate, however, caused a decrease in ROS production, suggesting the involvement of an ROS-independent pathway in oleanolic acid 3-acetate-induced apoptosis in SKOV3 and HEC-1A cells. CONCLUSION: These findings support the notion that oleanolic acid 3-acetate could be used as a potent anticancer supplementary agent against ovarian and endometrial cancer. Oleanolic acid 3-acetate exerts its proapoptotic effects through a rather unique molecular mechanism that involves an unconventional ROS-independent but mitochondria-mediated pathway.

Loss of the dermis zinc transporter ZIP13 promotes the mildness of fibrosarcoma by inhibiting autophagy.

Fibrosarcoma is a skin tumor that is frequently observed in humans, dogs, and cats. Despite unsightly appearance, studies on fibrosarcoma have not significantly progressed, due to a relatively mild tumor severity and a lower incidence than that of other epithelial tumors. Here, we focused on the role of a recently-found dermis zinc transporter, ZIP13, in fibrosarcoma progression. We generated two transformed cell lines from wild-type and ZIP13-KO mice-derived dermal fibroblasts by stably expressing the Simian Virus (SV) 40-T antigen. The ZIP13-/- cell line exhibited an impairment in autophagy, followed by hypersensitivity to nutrient deficiency. The autophagy impairment in the ZIP13-/- cell line was due to the low expression of LC3 gene and protein, and was restored by the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza) treatment. Moreover, the DNA methyltransferase activity was significantly increased in the ZIP13-/- cell line, indicating the disturbance of epigenetic regulations. Autophagy inhibitors effectively inhibited the growth of fibrosarcoma with relatively minor damages to normal cells in xenograft assay. Our data show that proper control over autophagy and zinc homeostasis could allow for the development of a new therapeutic strategy to treat fibrosarcoma.

Endometrial cancer invasion depends on cancer-derived tumor necrosis factor-alpha and stromal derived hepatocyte growth factor.

Cancer invasion is an outcome of interactions of the cancer and the host cell. It is now becoming increasingly clear that ovarian hormones have a huge influence on such intercommunications in various types of cancers. Estrogen is known to aggravate the aggressiveness of the endometrial cancer whereas progesterone seems to act as a negative factor. Insight into the mode of ovarian hormonal actions could come from the studies of its regulation of the paracrine interactions between the endometrial cancer and the normal stromal cells during the cancer invasion. In this context, we report here that estrogen promotes the endometrial cancer invasion by inducing humoral interactions between the cancer and the stromal cells, i.e., estrogen stimulates tumor necrosis factor-alpha expression from the endometrial cancer cells, which, in turn, induces the stromal expression of hepatocyte growth factor (HGF), conferring the enhanced NK4 (HGF-antagonist/angiogenesis inhibitor)-sensitive invasion characteristic of the endometrial cancer cells. Additionally, we demonstrate a close correlation of the invasion of endometrial cancer cells with the expression and dimerization of integrin alpha(v)beta(5) as well as the activation of focal adhesion kinase as the consequences of paracrine interactions. Thus, understanding of paracrine interactions of cancer cells with host stromal cells can yield new insight into the architecture and function of cancer invasion and metastasis, leading to a development of a new cancer therapeutic intervention.