The mechanistic coupling of mitochondrial ATP generation and oxPPP through proline metabolism that supports normal cells during stress and which appears to be dysfunctional in many tumorigenic cells provides a foundation for a possible model mechanism for the chemopreventive action of phenolic antioxidants, such as genistein, against certain cancers (100).
When soybean is consumed, its main phenolic phytochemical, the isoflavone genistein, is converted by intestinal flora bioprocessing and subsequent hepatic activities into a b-glucuronide conjugate. In an interesting twist of fate, the expression of b-glucuronidase is low or not detected in normal tissues, but is high in tumors (123). Therefore, in tissues that take up the genistein-b-glucuronide from the bloodstream, free and biologically active genistein is most likely to occur in a higher amount in tumor cells than in healthy cells due to the selective nature of b-glucuronidase expression in these tissues.
Once activated by glucuronidase, free genistein may stimulate proline metabolism through the activation of proline dehydrogenase (PDH) via p53. Genistein can induce p53 expression in colorectal cancer cells (124). The mechanism for stimulation of p53 by genistein is not clear, but other dietary antioxidants can activate p53 by a refl dependent redox mechanism (125,126). The transcription factor p53 can activate PDH, as well as ROS and apoptosis in cancer cells (127-129).
Stimulation of mitochondrial PDH in the tumor cell by phenolic antioxidants such as genistein should cause a demand for proline (117) that would have several major metabolic effects: (1) increased mitochondrial oxPHOS supported by proline oxidation would generate increased ROS that could leak into the cytosol and damage essential cellular components; (2) proline would be shunted to the mitochondria and away from collagen biosynthesis, potentially crippling tumor growth, expansion, and proliferation activities; and (3) energy metabolism would be redirected toward proline mediated mitochondrial ATP synthesis and away from TCA linked, NADH mediated oxPHOS via activity of the PL-PPP. An occurrence of these metabolic effects would be uniquely detrimental to the functioning of a tumor cell because, by all reported indications, normal cellular metabolism, including the antioxidant response system, in tumor cells is dysfunctional at mitochondrial and cytosolic levels.
First, increasing the activity of PDH would drive mitochondrial oxPHOS toward ATP synthesis, and produce increased ROS levels in the process. In a normal cell, increased ROS production by increased mitochondrial activity would likely be countered by activation of the cellular antioxidant enzyme response system, but in cancer cells antioxidant enzymes appear to be less active and may be less able to adequately protect tumor cells from oxidative damage, and potentially promote apoptosis (109,110,122,130). Because tumor cell mitochondria are already dysfunctional, increased respiration activities activity driven by genistein stimulated PDH activity should produce increased amounts of cytosolic ROS into the cytosol, endangering numerous essential cytosolic components such as proteins and organelles. MnSOD is expressed at low levels in normal cells, but at high levels in tumors, perhaps in response to high ROS levels (131). However, expression of catalase, glutathione peroxidase (GPx), and CuZnSOD is greatly diminished, which may facilitate accumulation of excessive oxygen radicals and oxidative damage in tumor cells (109,110). Although dietary antioxidants, such as curcumin, ascorbic acid, and flavanoids, have been shown to stimulate antioxidants and important phase II antioxidant enzymes such as SOD and catalase, genistein did not stimulate catalase or SOD in prostate cancer (132-135). In fact, genistein and soy isoflavone extracts have been shown to stimulate caspase-3 and apoptosis in cancer cells (58,62). Thus, phenolic antioxidants such as genistein may stimulate apoptosis linked activities that support the generation and accumulation of oxygen radicals and oxidative damage in tumor cells. The action of genistein could be further enhanced synergistically by other soluble phenolics from a food system. This is further supported by recent evidence that fermented soymilk and whole soy extracts inhibit tumor growth better than genistein alone (16,136,137).
Second, the stimulation of PDH activity by genistein and synergistic phenolic profiles would cause a metabolic demand for proline as a reductant to support mitochondrial oxPHOS (energy production) and may derail tumor growth supporting collagen biosynthesis by redirecting a necessary substrate (e.g., proline). Genistein has been shown to inhibit the growth and proliferation of cancer cells in vitro (32). If genistein activates of mitochondrial PDH and the proline cycle as part of a key stress response mechanism, proline cycle demand for proline may have higher cellular priority than collagen biosynthesis during times of stress (117).
Finally, and perhaps most importantly, induction of mitochondrial PDH activity and proline cycling by genistein would shift cellular energy metabolism in the tumor cell away from NADH linked mitochondrial oxPHOS to a proline linked mitochondrial oxPHOS system (100). Dysfunctional tumor cell mitochondria may produce increased ROS generation via increased mitochondrial respiration that may lead to mitochondrial membrane damage, caspase activation, and eventually apoptosis. In support of this idea, genistein has been shown previously to inhibit nonoxPPP ribose synthesis and cell proliferation in cancer cells (32,33). In healthy cells, stress induced stimulation of the proline cycle also drives the oxPPP via G6PDH recycling of NADPH, and G6PDH (and thereby the stress induced PL-PPP) can be inhibited by high NADPH levels. In contrast, in tumorigenic cells G6PDH is dysfunctional (with activity decreased by up to 90%) and is no longer inhibited by high NADPH (105,117,38). Therefore, while genistein stimulated PDH activity may be supported by NADPH cycling between proline biosynthesis and G6PDH activity, a dysfunctional G6PDH enzyme may not allow the tumor cell to disengage the stress response mechanism in the presence of high levels of NADPH. Because the dysfunctional enzyme operates at such a low efficiency (~10%) and likely does not produce high NADPH levels, there may not be enough NADPH produced to support the anabolic demands of the antioxidant enzyme response system which would further hinder the tumor cell to defend itself against oxPHOS derived ROS.
Further, genistein possesses other biological activities that may help to promote apoptosis in tumor cells. Aside from the stimulation of caspase-3 which could result from mitochondrial PDH activation, genistein can inhibit NF-kB (13), which can block apoptosis (139).
Starvation is an efficient way to kill a living organism, even a diseased cell. An effective strategy to starve a tumor cell could be to potentiate a switch in energy metabolism from a dysfunctional and inefficient TCA/NADH linked mechanism to an even more dysfunctional alternative pathway (100). In tumor cells, mitochondrial ATP-generating activities are known to be negatively altered and to function inefficiently (103,104,140). If the energy metabolism could somehow be forced to revert back to the dysfunctional mitochondria, the inefficiency of the system may starve the tumor cell of chemical energy (ATP) for cellular activities (100). For a tumor cell to knowingly switch a core metabolism to favor a pathway or mechanism that would be detrimental to its survival seems unlikely, but it is possible that just such an action may occur through the activation of an underlying key stress response mechanism that functions in normal, healthy cells, for which activation triggers may still remain even after the normal cell transitions into a tumorigenic cell (100).
Here we describe a potential mechanism by which phenolic antioxidants such as the isoflavone genistein may act to promote tumor cell death by inducing the diseased cell to switch its energy metabolism from growth-promoting TCA cycle/ NADH linked system to a dysfunctional proline linked system through the activation of a key stress response mechanism involving the PL-PPP, whereby the cell is essentially starved of chemical energy (100). As the PL-PPP in plants can be stimulated by dietary (89,92,93,41), the same may be true in animal (e.g., human) cells. We have described how p53 mediated activation of PDH by genistein may create a metabolic demand for proline, therein activating the PL-PPP and causing a shift in energy metabolism to facilitate mitochondrial ATP generation, even though components of the stress response mechanism in tumor cells may be dysfunctional. A mitochondrial demand for proline might divert proline away from collagen biosynthesis, which may explain the interrupted tumor growth and proliferation activities observed in cancer cells treated with phenolic compounds. It is unknown whether or not tumor cell PDH is dysfunctional (100).
ROS accumulation precipitated by increased activity of leaky mitochondria should stimulate activity of antioxidant enzymes and the oxPPP for NADPH to support reductant cycling systems (100). However, in tumor cells G6PDH is also dysfunctional and inefficient, such that the generation of adequate NADPH levels to support the demands of both proline synthesis and the antioxidant enzyme response system is unlikely. Further, as the dysfunctional G6PDH is no longer inactivated by high NADPH, it is likely that once the PL-PPP mediated stress response mechanism is activated, the tumor cell may not be able to disengage it, and may become locked in its fate and thus prevented from returning to TCA cycle/ NADH linked energy metabolism (100).
Further, our hypothetical model helps to explain how phenolic antioxidants such as genistein could have both detrimental effects against tumor cells and beneficial effects on healthy cells. In healthy cells, addition of a phenolic antioxidant that can stimulate mitochondrial activity and the antioxidant enzyme response system (i.e., SOD, catalase, GPx) may aid in protection against oxidative damage and thus promote healthy cellular conditions (102,142). Furthermore, as normal G6PDH can be inactivated by high NADPH levels, the PL-PPP-mediated stress response mechanism can be turned off as needed, something that a tumor cell may be unable to do and which may, ultimately, lead to the death of the diseased cell (100).
Numerous dietary phenolic antioxidants have been shown to inhibit cancer cell growth and proliferation, but until now no overall cellular mechanism that integrates the many observed phenolic linked activities has been put forth. We believe our hypothetical model for the chemopreventive actions of the soybean isoflavonoid genistein as described here offers a logical mode of action for dietary phenolic antioxidants based on key cellular metabolism linked to alternative energy and redox management. This model merits further experimental investigation in conjunction with genetic and signal transduction mechanisms associated with cancer biology (107). As various dietary phenolic compounds have been reported to possess activities that may facilitate apoptosis, our model provides a core mechanism by which many of these compounds could act, and a mechanism that could be supported by other such anticancer properties, such as NF-kB inhibition and related signal pathways.
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