Expression of Heat Shock Protein 70 in Oxidative Stress
In present research, the comparison between the negative control which represents the basal expression of HSP70 (31530597.5±923268; n=4), and the positive control that represents the expression of HSP70 as a response to the oxidant (4094970.25±10449032; n=4), has clearly indicated that treatment with 100 μM copper sulphates for 24 hours resulted in a significant up-regulation of HSP70 expression (p= 0.04), Figure 1 & Table 2). Overexpression of inducible HSP70 by oxidants has been reported by many other studies (6,7).
The mechanism by which this happens is still elusive, however it is suggested that HSP70 was induced secondary to the adverse effects of oxidative stress on protein conformation (8,9). The oxidative stress here is inducing as a result of increased copper sulphate level which is known to cause oxidative stress due to the overproduction of free radicals particularly hydroxyl radicals. These free radicals attack major cellular molecules and exhaust the antioxidant enzymes inside the cells (10). CuSO 4 can be reduced from Cu+2 to Cu+ by apoplastic ascorbate and/or other electron donors. Cu+ formed can participate in a Fenton reaction with apoplastic H2O2 to form the highly reactive hydroxyl radical (°OH) (11). Consequently, being attack by ROS, proteins may be oxidized, causing carbonylation and aromatic hydroxylation of some amino acids (12). Furthermore, it has been indicated by some studies that oxidation of protein thiols may result in formation of non-native intermolecular disulphide. These alterations in protein structure collectively, can cause proteins to destabilize, denature and unfold so hydrophobic domains are exposed. This would be probably initiating a signal for induction of the heat shock response (13). This has been shown in earlier studies that demonstrated a rapid induction of cytosolic HSP70 expression following the injection of denatured proteins into the cells (14).
Transcription of human HSP70 in response to accumulated denatured and abnormally folded proteins is mediated through the activation of heat shock transcription factor (HSF1). Biochemical and genetic studies have clearly demonstrated critical roles for mammalian heat shock factor 1 (HSF1) in stress-inducible HSP gene expression (15). Whether HSF1 directly senses stress or is regulated by an upstream signalling cascade is not yet fully understood. However, what is clear is that in non-stressed conditions –when HSP70 levels are in excess- HSF1 is found in the cytoplasm in a non-DNA binding monomeric form, by virtue of its being complexes with HSP70. In the case of oxidative stress, HSP70 recognizes the abnormally folded proteins, possibly through the recognition of the hydrophobic regions, forming longer lived complexes. As a result, HSF1 is liberated from HSP70; phosphorylated and converted to a trimer with the capacity to bind DNA. HSF then translocates from cytoplasm to nucleus where it binds to heat shock elements (HSE) in the promoter region of HSP70 coding genes, and subsequently activates HSPs transcription (16).
Consequently, it can be suggested that the significant induction of heat shock protein 70 mRNA observed in the present study, is possibly attributed to the accumulation of damaged proteins as a result of oxidative stress, and this was mediated, at the transcriptional level, by HSF1.
The induction of HSP70 following oxidative stress appears to be a part of cellular protective mechanisms to protect against subsequent stress, apparently by preventing or repairing stress-induced protein denaturation and promoting protein folding thus enhancing cells capability to survive (17,18). HSP70 was also shown to effectively inhibit the cellular death processes (19,20). The HSP70 seems to increase resistance against oxidative stress-mediated apoptosis. Reports revealed that HSP70 inhibits apoptosis induced by ethanol, hydrogen peroxide, TNF, UV radiation and several chemotherapeutic agents. All of these treatments have been found to produce ROS within cells during their apoptotic induction (21–23).
The mechanisms by which HSP70 confer protection from ROS and free radicals are not completely understood, but recent studies suggest several possible mechanisms such as : (1) inhibiting protein misfolding and aggregation (24); (2) refolding abnormally folded proteins (25); (3) directing damaged proteins to lysosomes (26), or to peroxisomes to be degraded (27); (4) increasing the expression or the activity of endogenous scavengers of ROS (28); (5) and it also seems to protect DNA, mitochondria from free radicals (29,30).
HSP70 Expression Level in Presence of Vitamin E
To assess whether the increase in HSP70 secondary to the stress caused by the oxidant could decrease in the presence of vitamin E; two groups of cells were incubated for 4 hours with α-tocopherol and palm tocotrienol-rich fraction respectively with concentration of 10, 20 and 30 μg/ml after the cells were pre-exposed to oxidative stress.
The present study has shown that the overall effect of vitamin E compounds seems to reduce the increase in HSP70 expression (Figure 1). α-Tocopherol tended to trigger a more constant rate of response to the dose given but unable to significantly induced the decrease of the HSP70 expression as compared to TRF (see Figures 1 and Table 3). This observation was supported by some other studies. Topbas and colleagues (31) demonstrated that in liver cells, HSP70 that was induced as a result of vitamin E deficiency, was significantly reduced following re-supplementation with vitamin E. Similarly, vitamin E, particularly α-tocopherol, has been shown to reduce HSP70 levels in human muscles and blood following exercise respectively (32,33), in leukocytes after treadmill running and in human skin fibroblasts after oxidative stress (35).
The mechanism involved in this reduction is not well understood. However, this effect is possibly attributed to the potent antioxidant properties of vitamin E (32). It is likely that the effect of vitamin E on HSP70 was mediated through the scavenging of free radicals and ROS generated by the oxidant and its ability to break radical-propagated chain reactions. As HSF1 is sensitive to redox, it would be imperative to suggest that scavenging of free radicals and the subsequent reduction of the oxidative stress state of cells had possibly inhibited the activation of this factor, and consequently HSP70 (32,33).
Since the 1960s, vitamin E potent antioxidant function had been clearly recognized (36). The strong reducing capacity of vitamin E is related to its chemical structure. All vitamin E isomers have active hydroxyl groups attached to benzene ring-based structure and a hydrophobic side chain. It is the hydroxyl group that involves in reducing the free radicals since it can easily donate a hydrogen atom, leading to the formation of vitamin E radicals (chromanoxyl) (37). These chromanoxyl radicals are relatively stable and can be reduced back to vitamin E form by compounds naturally present in biological systems such as ascorbic acid and lipoic acid, in a process termed “recycling” of vitamin E (38). On the other hand, vitamin E side chains are important for the mobility and the incorporation of the vitamin molecules within the membrane phospholipids bilayer (39,40).
As a result of, the present study shows that vitamin E had maintained the reducing environment of the cell, hence prevented the activation of HSF1 and in turn HSP70 by ROS and other free radicals.
Comparative Effect of α-Tocopherol and Palm-Tocotrienol Rich Fraction on HSP70 Expression
With reference to the positive control (Table 3) HSP70 mRNA was significantly reduced by palm-TRF at 20μg and 30μg (p=0.028, 0.032 respectively; n=4), and this effect was more pronounced at 20μg palm-TRF (Table 3). In comparison with the negative control (Table 3), at these concentrations of palm-TRF, HSP70 expression was down-regulated to its basal levels (p=0.051, 0.224 respectively; n=4).
Palm-TRF also appeared to be more effective in reducing HSP70 expression than α-tocopherol (10, 20, 30μg). Although α-tocopherol (10, 20, 30μg) had caused a decline in HSP70 expression in a dose dependent manner (Figure 1), this decrease was not statistically significant compared to the positive control (p=0.814, 0.224, 0.052 respectively, see Table 3). However, at 30μg α-tocopherol, HSP70 expression was almost similar to the negative control (p=0.326).
The tocotrienol-rich-fraction (TRF) of palm oil has been anticipated as an efficient alternative for α-tocopherol due to its potent antioxidant properties in vitro (41–45). Although there was no report on TRF effect on HSP70, the present study findings are in agreement with a number of studies that suggest a higher antioxidant potency of tocotrienols compared to tocopherols in vitro; as HSP70 reduction has been earlier related in this discussion to vitamin E antioxidant function. It has been reported that α-tocotrienol exhibited significantly greater peroxyl radical scavenging potency than α-tocopherol in phosphatidylcholine liposomes (46). Furthermore, palm-TRF has been also shown to be a more effective inhibitor of LDL oxidation in endothelial cell lipid peroxidation than α-tocopherol in vitro (47). However, in contrast to these findings, a small number of studies suggested that tocopherols and tocotrienols exerted the same reactivity toward radicals and the same antioxidant activities against lipid peroxidation in solution and liposomal membranes. These conflicting findings may be due to the differences in the systems used to conduct these studies (47).
Several reasons have been suggested for the increased antioxidant activity of tocotrienols compared to tocopherols, mostly related to the difference in the hydrophobic side chain saturation level. The unsaturated side chain of tocotrienols has been proposed to allow for more efficient penetration into tissues and increase in the rate of transfer of tocotrienols between liposomal membranes than tocopherols (48). It has been suggested that the higher antioxidant potency of tocotrienols is due to a higher recycling efficiency of the chromanoxyl radicals of the tocotrienols (tocotrienoxyl radical) in membranes and lipoproteins, a more uniform distribution of tocotrienols in the cellular membranes, and stronger disordering effect on membranes than α-tocopherol. In addition, studies have indicated that α-tocotrienols located closer to the membrane surface, which may facilitate recycling (46). These properties are likely result in a more efficient interaction of the chromanol ring of tocotrienols with reactive oxygen species (46).
Despite the fact that the antioxidant efficiency of tocotrienols in vitro is higher than that of tocopherols, these compounds have shown to be less absorbed in vivo than α-tocopherol that is found to be the dominant vitamin E isomer in human plasma and tissue (49). Possible explanation for the reduced bioavailability includes (1) the presence of a transfer protein in the liver that specifically enriches VLDL with α-tocopherol leading to the secretion of these compounds from the liver in a manner that discriminates between tocopherols and tocotrienols (49), (2) less tissue retention and half-life (3) and the higher rate of tocotrienols metabolism compared to tocopherols (50).
Nevertheless, other studies have indicated that oral intake of tocotrienol makes it bioavailable to all vital organs; even though in significantly lower concentrations than α-tocopherol (51). Nanomolar tocotrienols has been demonstrated to be sufficient to exert antioxidant-independent protective effects in brain (48). The same study has suggested that at micromolar but not nanomolar α-tocotrienol can protect against chemically produced peroxyl radicals (48,52). This effect has been clearly shown in this study.