Awais Anwar

Perspective on Recent Developments on Sulfur-Containing Agents and Hydrogen Sulfide Signaling

The last couple of years have witnessed the coming together of several initially unconnected lines of investigation which now link natural sulfur products to hydrogen sulfide release and wide ranging cardiovascular protection. It has become apparent that sulfur compounds contained within garlic, onions, mushrooms and various edible beans and fruits may be transformed chemically or enzymatically in the human body with subsequent formation of hydrogen sulfide. The latter has emerged during the last decade from a shadowy existence as toxic gas to be recognized as the third gaseous transmitter besides nitric oxide ( (.)NO) and carbon monoxide (CO). Hydrogen sulfide is formed endogenously in the human body by enzymes such as cystathionine beta-synthase (CBS) in the brain and cystathionine gamma-lyase (CSE) in liver, vascular and non-vascular smooth muscle. Although its exact chemical and biochemical modes of action are still not fully understood, levels of hydrogen sulfide in the brain and vasculature have unambiguously been associated with human health and disease. Not surprisingly, agents releasing hydrogen sulfide, as well as inhibitors of hydrogen sulfide synthesis (CBS and CSE inhibitors) have been investigated. Apart from linking our daily diet to a healthy brain and cardiovasculature, these findings may also provide new leads for drug design. Future studies will therefore need to focus on how such compounds are formed and transformed in the relevant plants, how food processing affects their chemical constitution, and how they release hydrogen sulfide (or control its levels) in the human body. Such multidisciplinary research should ultimately answer the all-important question if a hearty diet is also good for the heart.

The chemistry behind redox regulation with a focus on sulphur redox systems.

Sulphur metabolism in plants provides a wealth of natural products, including several chemically unusual substances, such as thiosulphinates, polysulphides and isothiocyanates. Many of these reactive sulphur species (RSS) exhibit a distinct redox behaviour in vitro, which translates into a rather interesting biological activity in vivo, such as antibiotic, fungicidal, pesticidal or anticancer activity. While the molecular basis for such activity has long remained obscure, research into sulphur-based redox systems during the past 5-10 years has achieved a better knowledge of the in vitro properties of RSS and has led to an improved understanding of their impact on intracellular redox signalling and control pathways in living cells. It has become apparent that the redox chameleon sulphur occurs in biological systems in about 10 different oxidation states, which give rise to an extensive and complicated network of sulphur-based redox events. Together, natural sulphur products from plants and their intracellular targets provide the basis for innovative design of novel antibiotics, fungicides, pesticides and anticancer agents.

Cell cycle arrest in early mitosis and induction of caspase-dependent apoptosis in U937 cells by diallyltetrasulfide (Al2S4)

Naturally occurring organic sulfur compounds (OSCs), such as linear allylsulfides from Allium species, are attracting attention in cancer research, since several OSCs were shown to act beneficially both in chemoprevention and in chemotherapy, while hardly exerting any harmful side effects. Hence, we investigated the possible role of different OSCs in the treatment of leukemia. Thereby, we found that the compounds tested in this study induced apoptosis in U937 cells, with an efficiency depending on the number of sulfides, and selected the most promising candidate, diallyltetrasulfide (Al2S4), for detailed mechanistic studies. Here we show that Al2S4 induced an accumulation of cells in early mitosis (G2/M phase), followed by the activation of caspase-dependent apoptosis. The compound counteracted different anti-apoptotic Bcl-2 family members (Bcl-xL, phospho-Bad and Bcl-2), promoted activation of Bax and Bak and induced the release of cytochrome c into the cytoplasm. Treatment by Al2S4 let to the identification of early apoptotic events including Bcl-xL degradation, Bak activation and release of cytochrome c followed by late events including Bcl-2 proteolysis, Bax activation, Bad dephosphorylation, caspase activation, nuclear fragmentation and phosphatidylserine exposure.

Allicin disrupts the cell's electrochemical potential and induces apoptosis in yeast

The volatile substance allicin gives crushed garlic (Allium sativum) its characteristic odor and is a pro-oxidant that undergoes thiol-disulfide exchange reactions with -SH groups in proteins and glutathione. The antimicrobial activity of allicin is suspected to be due to the oxidative inactivation of essential thiol-containing enzymes. We investigated the hypothesis that at threshold inhibitory levels allicin can shunt yeast cells into apoptosis by altering their overall redox status. Yeast cells were treated either with chemically synthesized, pure allicin or with allicin in garlic juice. Allicin-dependent cell oxidation was demonstrated with a redox-sensitive GFP construct and the shift in cellular electrochemical potential (E(hc)) from less than -215 to -181mV was calculated using the Nernst equation after the glutathione/glutathione disulfide couple (2GSH/GSSG) in the cell was quantified. Caspase activation occurred after allicin treatment, and yeast expressing a human antiapoptotic Bcl-XL construct was rendered more resistant to allicin. Also, a yeast apoptosis-inducing factor deletion mutant was more resistant to allicin than wild-type cells. We conclude that allicin in garlic juice can activate apoptosis in yeast cells through its oxidizing properties and that this presents an alternative cell-killing mechanism to the previously proposed specific oxidative inactivation of essential enzymes.

Antiproliferative effect of natural tetrasulfides in human breast cancer cells is mediated through the inhibition of the cell division cycle 25 phosphatases

For many years, in vitro and in vivo studies have reported that organosulfur compounds (OSCs), naturally found in Allium vegetables, are able to suppress the proliferation of various tumor cells. In spite of recent advances, the specific molecular mechanisms involved in OSC activity are still unclear. Considering the antiproliferative effects observed in cancer cells, we postulated that OSCs might target the cell division cycle (Cdc) 25 phosphatases which are crucial enzymes of the cell cycle. Our findings suggest phosphatases Cdc25 as possible targets of naturally occuring polysulfides contributing to their anticancer properties. We report on the inhibitory activity of tetrasulfides occurring naturally in garlic and onion towards the human Cdc25 phosphatases. Diallyl- and dipropyltetrasulfides have emerged as interesting irreversible inhibitors of the Cdc25 isoforms A and C in vitro. Furthermore, growth of both sensitive (MCF-7) and resistant (Vcr-R) human breast carcinoma cells was significantly decreased by these tetrasulfides. The observed antiproliferative effect appeared to be associated with a G2-M cell cycle arrest.

Naturally occurring reactive sulfur species, their activity against Caco-2 cells, and possible modes of biochemical action

Natural sulfur compounds from plants, bacteria, fungi, and animals frequently exhibit interesting biological activities, such as antioxidant, antimicrobial, and anticancer activity. Considering the recent developments in medicine (e.g. oxidative stress in aging, antibiotic resistant bacteria, selective anticancer agents) and agriculture (e.g. ‘green’ pesticides), several of these compounds have become the focus of interdisciplinary research. Among the various sulfur agents isolated to date, polysulfides, such as diallyltrisulfide and diallyltetrasulfide from garlic, are of particular interest, since they combine an unusual chemistry and biochemical mode(s) of action with a distinct biological activity, which includes antimicrobial activity and cytotoxicity against certain cancer cells. In many cases, the biological activity of these compounds is well established, but the underlying causes for this activity are hardly known. As part of our investigations, we have now confirmed the activity of diallyltrisulfide and diallyltetrasulfide against the fairly ‘robust’ Caco-2 colon cancer cell line. At the concentrations used, the activity observed for tri- and tetrasulfide is considerably higher than that of disulfide, while monosulfide is virtually inactive. Controls with the long chain carbon analog 1,9-decadiene count against solely lipophilic effects of diallyltetrasulfide, and together with the ‘ranking’ of activity, point toward a ‘special’ sulfur redox chemistry that emerges when shifting from di- to trisulfide. This special reactivity of polysulfides has previously been associated with certain oxidizing properties of the polysulfides. The electrochemical studies and thiol oxidation assays conducted as part of this study, however, count against the notion of diallyltrisulfide and diallyltetrasulfide as effective oxidants. On the contrary, the rather negative oxidation and reduction potentials associated with these agents point toward a reducing chemistry, which is confirmed in the nitrotetrazolium blue assay: the latter seems to indicate dioxygen reduction to the superoxide radical anion, although other reductive events or H2S release cannot be ruled out at this point. It is therefore likely that diallyltrisulfide and diallyltetrasulfide are reduced inside the cancer cells to perthiols and hydropolysulfides, which in turn trigger a lethal oxidative burst, for instance via superoxide radical anion formation. Further interdisciplinary studies are required to investigate in more detail the rather complicated chemical and biochemical processes, which ultimately may explain the biological activity that is clearly associated with many natural polysulfides.

How to Turn the Chemistry of Garlic into a ‘Botanical’ Pesticide

In the past, synthetic pesticides have played a major role in food crop protection. However, prompted by environmental and health concerns, many chemical pesticides have been banned or are under re-evaluation. Consequently, there is an urgent need for alternative “environmentally friendly” pest control methods. Garlic extracts display broad spectrum activity against numerous food crop pests offering a “botanical” alternative to many chemical pesticides. Underpinned by the molecular basis of redox active garlic polysulfides, this chapter describes the pathway taken to establish precedence for EU regulatory approval of a botanical pesticide.