Nevertheless, there is likely considerable circulating anti-HIV serpin activity in plasma, such that the serpin anti-HIV activity in plasma may be saturated, and intravenous inoculation of hep-ATIII into the plasma has limited additive effect. In contrast subcutaneous administration of ET-ATIII targeting lymphoid organs maybe more effective because baseline serpin activity in lymphoid organs is minimal. We probed the underlying mechanism of hep-ATIII anti-HIV activity using software-supported interactome analysis, which allowed us to identify key host cell factors that are immediately downstream of drug treatment, and which in turn modulate the expression of overarching gene networks. We previously found that hep-ATIII activated two interactomes in HIV-1 infected PBMC: one interactome dependent on the NFkB transcription factor and a second interactome anchored by ERK1/2. These host factors are known to significantly impact HIV-1 replication. We now have expanded our analysis of transcriptional changes downstream of treatment with hep-ATIII, and studied these changes in the significantly more complex in vivo environment. Once again we found networks regulated by NFkB (Fig. 7) and ERK1/2-PTGS2 (Fig. 8) associated with hep-ATIII treatment confirming earlier in vitro results from PBMC acutely infected with HIV-1 [9]. There is a great need to counter HIV-induced inflammation and its consequences on the central nervous system (CNS) including HIV dementia. In the current studies we found that hep-ATIII treatment down-regulated NFkB after 7 days.
This is important since the NFkB dimer consisting of p50 and RelA isconsidered to be the largest contributor to activation of HIV transcription and inflammation [68]. Our second network was centered around ERK1/2 and seems to be dependent on PTGS2, an HIV inhibitory host cell factor described earlier [9]. Thus, another possible mechanism by which hep-ATIII might prevent HIV-induced dementia is through its anti-inflammatory effect since prostaglandins were found to block inflammation through inhibition of HIV-1 Tat-mediated ERK1/2 activation [69]. There are several limitations to these studies. (1) In our investigation of virus-induced cytotoxicity in the spleens of humanized mice, we have not characterized the specific cellular populations that are preserved in contrast to those that are lost, nor have we determined the mechanism by which hep-ATIII may prevent cytotoxicity. (2) We have not provided a mechanism for viral rebound after treatment with ET-ATIII. Our gene expression analyses suggest that the mechanism of ET-ATIII is through recruitment of innate antiviral mechanisms. Although it is unclear whether HIV can rapidly evolve resistance to host innate factors in such a rapid timeframe, we suspect that the rebound is most likely due to the clearance of ET-ATIII from the host, and hence loss of its suppressive activity. (3) Our non-human primate pilot studies are limited by the number of animals available, but we believe provide justification for a larger scale trial. Clearly testing a more prolonged administration regimen is needed to more fully evaluate the safety and efficacy of ET-ATIII as an anti-HIV therapeutic. In conclusion, our data suggest that activated ATIII targeted to lymph nodes may have substantial in vivo activity against HIV-1. Further understanding of the mechanisms by which hep-ATIII interferes with HIV replication in lymphoid tissues might have important implications for the design of therapeutic strategies that harness the innate immune system for both its direct antiretroviral potential and its ability to modulate the adaptive immune response.
Abstract
Background: Flavonoids are widely proposed as very interesting compounds with possible chemopreventive and therapeutic capacities. Methods & Results: In this study, we showed that in vitro treatment with the flavonoid Luteolin induced caspasedependent cell death in a model of human cutaneous squamous cell carcinoma (SCC) derived cells, representing a matched pair of primary tumor and its metastasis. Notably, no cytotoxic effects were observed in normal human keratinocytes when treated with similar doses of Luteolin. Luteolin-induced apoptosis was accompanied by inhibition of AKT signaling, and sensitivity decreased with tumor progression, as the primary MET1 SCC cells were considerably more sensitive to Luteolin than the isogenic metastatic MET4 cells. Extensive intracellular vacuolization was observed in Luteolin-treated MET4 cells, which were characterized as acidic lysosomal vacuoles, suggesting the involvement of autophagy. Transmission electron microscopy, mRFP-GFP-LC3 assay and p62 protein degradation, confirmed that Luteolin stimulated the autophagic process in the metastatic MET4 cells. Blocking autophagy using chloroquine magnified Luteolin-induced apoptosis in the metastatic SCC cells. Conclusion: Together, these results suggest that Luteolin has the capacity to induce selectively apoptotic cell death both in primary cutaneous SCC cells and in metastatic SCC cells in combination with chloroquine, an inhibitor of autophagosomal degradation. Hence, Luteolin might be a promising agent for the treatment of cutaneous SCC.
Citation: Verschooten L, Barrette K, Van Kelst S, Rubio Romero N, Proby C, et al. (2012) Autophagy Inhibitor Chloroquine Enhanced the Cell Death Inducing Effect of the Flavonoid Luteolin in Metastatic Squamous Cell Carcinoma Cells. PLoS ONE 7(10): e48264. doi:10.1371/journal.pone.0048264 Editor: Hari Koul, University of Colorado, United States of America Received April 15, 2012; Accepted September 21, 2012; Published October 26, 2012 Copyright: ?2012 Verschooten et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Funding was provided by the Instituut voor de Aanmoediging van Innovatie door Wetenschap en Technologie in Vlaanderen (personal PhD grant L.V.: SB/0661432), Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (grant G.0491.05), and Catholic University of Leuven (grant OT/04/42). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Introduction
Recent biochemical en preclinical studies provide evidence that flavonoids, bioactive compounds which can be derived from a variety of plants, possess multiple pharmacological activities, including antioxidant, anti-inflammatory and anticancer effects. Luteolin (LUT), one of the most common flavonoids, has the ability to induce apoptosis, to prevent carcinogenesis and to reduce tumorigenesis, which suggests its potential use as a therapeutic treatment [1], even in multidrug resistant cells [2]. Next to their role as conventional hydrogen-donation antioxidants [3,4], growing data have revealed that flavonoids exert their effects predominately through modulation of protein kinase signaling pathways [5,6]. LUT, amongst other flavonoids, acts as a competitive inhibitor of protein kinases (such as AKT, MEK1, PKC) [7], probably by direct binding to their ATP binding site, thereby altering the phosphorylation status and influencing multiple cell signaling pathways [5]. Since the inhibition of protein kinases appears to be an important strategy for cancer chemoprevention and cancer therapy [8], flavonoids have emerged as interesting biomolecules in that field [6]. Noteworthy, the activities of flavonoids appear to be very cell type dependent. Indeed, we recently discovered that LUT increased the resistance of normal human keratinocytes (NHK) to ultraviolet (UV) B-irradiation, a potent risk factor for skin carcinogenesis. However, LUT has no photoprotective effect on UVB-induced cell death of malignant keratinocytes derived from human cutaneous squamous cell carcinoma (SCC) [9]. SCC of the skin is a common cancer within the Caucasian population. The incidence of SCC is increasing worldwide, with epidemic proportions in Australia. Early primary SCC of the skin has a high curability and relatively low overall metastatic rate of 3 to 5%.