Alexzander Asea

Heat Shock Proteins and Toll-Like Receptors

Researchers have only just begun to elucidate the relationship between heat shock proteins (HSP) and Toll-like receptors (TLR). HSP were originally described as an intracellular molecular chaperone of naïve, aberrantly folded, or mutated proteins and primarily implicated as a cytoprotective protein when cells are exposed to stressful stimuli. However, recent studies have ascribed novel functions to the Hsp70 protein depending on its localization: Surface-bound Hsp70 specifically activate natural killer (NK) cells, while Hsp70 released into the extracellular milieu specifically bind to Toll-like receptors (TLR) 2 and 4 on antigen-presenting cells (APC) and exerts immunoregulatory effects, including upregulation of adhesion molecules, co-stimulatory molecule expression, and cytokine and chemokine release-a process known as the chaperokine activity of Hsp70. This chapter discusses the most recent advances in the understanding of heat shock protein (HSP) and TLR interactions in general and highlights recent findings that demonstrate Hsp70 is a ligand for TLR and its biological significance.

Serum Heat Shock Protein 70 Level as a Biomarker of Exceptional Longevity

Heat shock proteins are highly conserved proteins that, when produced intracellularly, protect stress exposed cells. In contrast, extracellular heat shock protein 70 (Hsp70) has been shown to have both protective and deleterious effects. In this study, we assessed heat shock protein 70 for its potential role in human longevity. Because of the importance of HSP to disease processes, cellular protection, and inflammation, we hypothesized that: (1) Hsp70 levels in centenarians and centenarian offspring are different from controls and (2) alleles in genes associated with Hsp70 explain these differences. In this cross-sectional study, we assessed serum Hsp70 levels from participants enrolled in either the New England Centenarian Study (NECS) or the Longevity Genes Project (LGP): 87 centenarians (from LGP), 93 centenarian offspring (from NECS), and 126 controls (43 from NECS, 83 from LGP). We also examined genotypic and allelic frequencies of polymorphisms in HSP70-A1A and HSP70-A1B in 347 centenarians (266 from the NECS, 81 from the LGP), 260 NECS centenarian offspring, and 238 controls (NECS: 53 spousal controls and 106 septuagenarian offspring controls; LGP: 79 spousal controls). The adjusted mean serum Hsp70 levels (ng/mL) for the NECS centenarian offspring, LGP centenarians, LGP spousal controls, and NECS controls were 1.05, 1.13, 3.07, 6.93, respectively, suggesting that a low serum Hsp70 level is associated with longevity; however, no genetic associations were found with two SNPs within two hsp70 genes.

Petiveria alliacea extracts uses multiple mechanisms to inhibit growth of human and mouse tumoral cells

BackgroundThere is ethnopharmacological evidence that Petiveria alliacea can have antitumor activity; however, the mechanism of its cytotoxic activity is not well understood. We assessed multiple in vitro biological activities of an ethyl acetate soluble plant fraction over several tumor cell lines.MethodsTumor cell lines were evaluated using the following tests: trypan blue exclusion test, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide], flow cytometry, cytoskeleton organization analysis, cell cycle, mitochondria membrane depolarization, clonogenicity test, DNA fragmentation test and differential protein expression by HPLC-Chip/MS analysis. F4 fraction characterization was made by HPLC-MS.ResultsPetiveria alliacea fraction characterized by de-replication was found to alter actin cytoskeleton organization, induce G2 cell cycle arrest and cause apoptotic cell death in a mitochondria independent way. In addition, we found down regulation of cytoskeleton, chaperone, signal transduction proteins, and proteins involved in metabolic pathways. Finally up regulation of proteins involved in translation and intracellular degradation was also observed.ConclusionThe results of this study indicate that Petiveria alliacea exerts multiple biological activities in vitro consistent with cytotoxicity. Further studies in animal models are needed but Petiveria alliacea appears to be a good candidate to be used as an antitumor agent.

Combined lentiviral and RNAi technologies for the delivery and permanent silencing of the hsp25 gene.

Elevated heat shock protein 27 (Hsp27) expression has been found in a number of tumors, including breast, prostate, gastric, uterine, ovarian, head and neck, and tumor arising from the nervous system and urinary system, and determined to be a predictor of poor clinical outcome. Although the mechanism of action of Hsp27 has been well documented, there are currently no available inhibitors of Hsp27 in clinical trials. RNA interference (RNAi) has the potential to offer more specificity and flexibility than traditional drugs to silence gene expression. Not surprisingly, RNAi has become a major focus for biotechnology and pharmaceutical companies, which are now in the early stages of developing RNAi therapeutics, mostly based on short interfering RNA (siRNAs), to target viral infection, cancer, hypercholesterolemia, cardiovascular disease, macular degeneration, and neurodegenerative diseases. However, the critical issues associated with RNAi as a therapeutic are delivery, specificity, and stability of the RNAi reagents. To date, the delivery is currently considered the biggest hurdle, as the introduction of siRNAs systemically into body fluids can result in their degradation, off-target effects, and immune detection. In this chapter, we discuss a method of combined lentiviral and RNAi-based technology for the delivery and permanent silencing of the hsp25 gene.

Release of Heat Shock Proteins: Passive Versus Active Release Mechanisms

There is now no doubt that heat shock proteins have a profound immunoregulatory effect on the host’s immune system. This knowledge has successfully been harnessed to generate a number of important clinical trails. However, one intriguing question that remains to be answered is how heat shock proteins (HSP) which do not have peptide leader sequence targeting secretion can gain access to the extracellular milieu. This chapter will discuss the most recent findings in the area of HSP release and attempts to broadly categorize these findings into two basic mechanisms; the passive and active mechanisms

Heat Shock Protein-Based Therapies

Heat shock proteins (HSP) are a family of evolutionary conserved proteins induced by cellular stressors. HSP are essential players in the development and progression of cancer inducing resistance to conventional treatment in a wide range of human tumors. Overexpression of HSP27, a member of HSP family, is associated with apoptosis inhibition, enhanced migration and metastasis and several clinical features of cancer including drug resistance promotion. At present, HSP27 could be a promising strategy to enhance sensitivity of tumors to cancer treatment. A plethora of novel compounds present in the diet, including flavonoids, can efficiently inhibit the growth of tumor cells by acting as natural “chemopreventers”. Many works reported the efficient targeting of HSP27 by using small inhibitors and dietary natural compounds in order to enhance the effectiveness of cancer therapies. In this chapter, we reviewed the current status of treatments based in dietary components targeting HSP27 as a novel strategy to circumvent chemotherapy cytotoxicity in cancer patients. Moreover we addressed the current status of HSP27 overexpression in many types of human cancers and highlighted the prominent role of targeting HSP27 as a novel therapeutic strategy in cancer.

Internalization of exogenous ADP-ribosylation factor 6 (Arf6) proteins into cells.

Endogenous Arf6 is a myristoylated protein mainly involved in endosomal membrane traffic and structural organization at the plasma membrane. It has been shown that Arf6 mediates cancer cell invasion and shedding of plasma membrane microvesicles derived from tumor cells. In this article, we determined that Arf6 proteins both in the GDP and GTPγS bound forms can enter cells when simply added in the cell culture medium without requiring the myristoyl group. The GTPγS bound can enter cells at a faster rate than the GDP-bound Arf6. Despite the role of the endogenous Arf6 in endocytosis and membrane trafficking, the internalization of exogenous Arf6 may involve non-endocytic processes. As protein therapeutics is becoming important in medicine, we examined the effect of the uptake of Arf6 proteins on cellular functions and determined that exogenous Arf6 inhibits proliferation, invasion, and migration of cells. Future studies of the internalization of Arf6 mutants will reveal key residues that play a role in the internalization of Arf6 and its interaction and possible structural conformations bound to the plasma membrane.

Heat Shock Proteins and Plants

Small heat shock proteins are one of the fi ve classes of heat shock proteins, a family named after their expression in response to heat shock. Despite their name some members of this family have been shown to express during a gamut of non-stress conditions in a variety of plant species. Small HSPs have been known to accumulate during plant developmental stages like pollen development, seed maturation stages, early seed germination and also in storage organs. Interestingly, aging induced accumulation of small HSPs has also been observed in a few species. The spatial and temporal accumulation pattern of small HSPs also correlates well with other seed abundant proteins like late embryogenesis abundant (LEA) proteins. Regulation of these developmental stages responsive and non-stress induced small HSPs is also distinct from the heat stress regulated transcript induction in terms of involvement of some novel and exclusive transcription activators like ABI3 and HsfA9. Small HSPs are known to function as molecular chaperone and thus their role in plant development especially during seed development has been discussed in the light of their functional implication during these stages.

Heat Shock Proteins and Cancer

Heat shock proteins (HSP) play multiple roles in cellular physiology and pathology depending on a wide variety of factors including its relative location within the cell (intracellular, plasma membrane or extracellular milieu), the age of the cell or whether it has undergone neoplastic transformation. In normal non-transformed cells, HSP play a cytoprotective role and protect cells from adverse stressful stimuli via chaperoning naive, misfolded and/or denatured proteins by a process known as the stress response. However, cancer cells have commandeered this function and the result is increased resistance to a number of anti-cancer therapies including hyperthermia, radiation and a wide range of chemotherapeutic agents. Recent advances in our understanding of this dual role of HSP have led to the development of pharmacological and molecular tool to target HSP for therapeutic gain. In this chapter, we highlight evidence for the involvement of HSP in the pathology of various cancers and discuss their proposed mechanism of action and therapeutic potential