Background Information about the harmful effects of vaping is sparse and inconsistent, therefore, since the use of electronic cigarettes (e-CIGs) has become increasingly popular as a tool to limit tobacco smoking, it is urgent to establish the safety or the toxicity of the liquid vaporized by the atomizer of the commercial e-CIGs. Methods Skin (HaCaT) and lung (A549) cells, the main targets of cigarette smoke, were exposed to e-CIG vapor (e-CIG Mini Touch T-Fumo T-TEX) and cigarette smoke (UK research cigarette) in a smoke chamber in vitro. The cytotoxic effect of the exposure was analyzed in both cell types by ultrastructural morphology, Trypan Blue exclusion test and LDH assay. In addition, pro-inflammatory cytokines were measured in culture medium by the Bio-Plex cytokine assay kit. Results The cytotoxic components of e-CIG were restrained to the flavoring compound and, to a lesser extent, to nicotine and their effects were comparable to that of cigarette smoke. Humectants alone exhibited no cytotoxicity but induced the release of cytokines and pro-inflammatory mediators, mainly in keratinocytes. Conclusions Based on our results, we can state that e-CIG vapors exposure is not completely harmless, although far less toxic than CS. In fact, besides the deleterious effect of flavor and nicotine, even the humectants alone are able to evocate some adverse cellular events, such as enhanced cytokines release. This study will hopefully promote the development of truly innocuous e-CIGs to help people quit smoking.

Because cancers are caused by deregulation of hundreds of genes, an ideal anticancer agent should target multiple gene products or signaling pathways simultaneously. Recently, extensive research has addressed the chemotherapeutic potential of plant-derived compounds. Among the ever-increasing list of naturally occurring anticancer agents, Rottlerin appears to have great potentiality for being used in chemotherapy because it affects several cell machineries involved in survival, apoptosis, autophagy, and invasion. The underlying mechanisms that have been described are diverse, and the final, cell-specific, Rottlerin outcome appears to result from a combination of signaling pathways at multiple levels. This paper seeks to summarize the multifocal signal modulatory properties of Rottlerin, which merit to be further exploited for successful prevention and treatment of cancer.

Rottlerin is a natural product isolated from Mallotus philippinensis. This polyphenolic compound, originally described as a selective inhibitor of PKCδ, can inhibit many other PKC-unrelated kinases and has a number of biological actions, including mitochondrial uncoupling effects. We recently found that Rottlerin inhibits the transcription factor nuclear factor κB in different cell types, causing downregulation of cyclin D1 and growth arrest. The present study was carried out to clarify the surprising lack of effect of Rottlerin on MCF-7 cell viability, assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test. We found that Rottlerin causes overestimation of the MTT test, leading to inconsistent results between cell number and cell viability. Rottlerin, however, strongly differs from other antioxidant polyphenols, which directly reduce tetrazolium salts, since it does not exhibit any reactivity toward the tetrazolium salts in vitro nor does it modulate lactate dehydrogenase activity. The interference in the MTT assay occurred only in cultured cells, concomitantly with a decrease in the energy charge. Because the same MTT overestimation was observed in the presence of uncoupling agents, we conclude that the Rottlerin artifact is linked to its uncoupling action that, by accelerating oxidative chain, accidentally results in enhanced MTT reduction. These results suggest caution in the use of the MTT assay in the presence of Rottlerin and uncouplers in general.

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Since the ability of cancer cells to evade apoptosis often limits the efficacy of radiotherapy and chemotherapy, autophagy is emerging as an alternative target to promote cell death. Therefore, we wondered whether Rottlerin, a natural polyphenolic compound with antiproliferative effects in several cell types, can induce cell death in MCF-7 breast cancer cells. The MCF-7 cell line is a good model of chemo/radio resistance, being both apoptosis and autophagy resistant, due to deletion of caspase 3 gene, high expression of the antiapoptotic protein Bcl-2, and low expression of the autophagic Beclin-1 protein. The contribution of autophagy and apoptosis to the cytotoxic effects of Rottlerin was examined by light, fluorescence, and electron microscopic examination and by western blotting analysis of apoptotic and autophagic markers. By comparing caspases-3-deficient (MCF-73def) and caspases-3-transfected MCF-7 cells (MCF-73trans), we found that Rottlerin induced a noncanonical, Bcl-2-, Beclin 1-, Akt-, and ERK-independent autophagic death in the former- and the caspases-mediated apoptosis in the latter, in not starved conditions and in the absence of any other treatment. These findings suggest that Rottlerin could be cytotoxic for different cancer cell types, both apoptosis competent and apoptosis resistant.

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The parathyroid hormone-related protein (PTHrp), structurally similar to the parathyroid hormone (PTH) in its NH(2)-terminal part, was first identified as a tumour-derived peptide responsible for a paraneoplastic syndrome known as humoral hypercalcemia of malignancy. The PTHrp gene is expressed not only in cancer but also in normal tissues during adult and/or fetal life, where it plays predominantly paracrine and/or autocrine roles. In the skin PTHrp produced by keratinocytes acts on fibroblasts by complex cooperative circuits involving cytokines and growth factors. In this report, we studied the direct effects of synthetic PTHrp 1-40 on proliferation and collagen synthesis and matrix metalloproteinase-2 (MMP-2) activity in cultures of fibroblasts isolated from normal human skin. Fibroblasts exposure to varying doses of PTHrp for 48 h, significantly and dose-dependently inhibited proliferation evaluated by [(3)H]-thymidine incorporation into DNA. A dose-dependent stimulation of cAMP released into the medium was concomitantly observed. In contrast, PTHrp had no effect on collagen synthesis evaluated either by [(3)H]-proline incorporation or by radioimmunoassay (RIA) of the carboxyterminal fragment of type I procollagen (PICP). MMP-2 activity, evaluated by quantitative zymographic analysis, was significantly increased by PTHrp treatment at doses of 160 and 320 nM. These findings indicate that PTHrp may play a role in normal dermal physiology by controlling both fibroblast proliferation and extracellular matrix degradation.

Rottlerin, a natural product purified from Mallotus philippinensis, has a number of target molecules and biological effects. We recently found that Rottlerin caused growth arrest in MCF-7 breast cancer cells and human immortalized keratinocytes, through inhibition of NFκB and downregulation of cyclin D-1. To evaluate whether this effect could be generalized to primary cells, human microvascular endothelial cells were treated with Rottlerin. In this study, we demonstrated that Rottlerin prevents basal and TNFα-stimulated NFκB nuclear migration and DNA binding also in human microvascular endothelial cell, where NFκB inhibition was accompanied by the downregulation of NFκB target gene products, such as cyclin D-1 and endothelin-1, which are essential molecules for endothelial cell proliferation and survival. Rottlerin, indeed, inhibited human microvascular endothelial cells proliferation and tube formation on Matrigel. Rottlerin also increases cytoplasmic free calcium and nitric oxide levels and downregulates endothelin converting enzyme-1 expression, thus contributing to the drop in endothelin-1 and growth arrest. These results suggest that Rottlerin may prove useful in the development of therapeutic agents against angiogenesis.