Induction of cell cycle changes and modulation of apoptogenic/anti-apoptotic and extracellular signaling regulatory protein expression by water extracts of I'm-Yunity™ (PSP)

Throughout history, mushroom and mushroom products have always been revered as food delicacies and are also held in high esteem for their overall health benefits in many cultures, particularly the Orient [1‐4]. In East Asian societies, a variety of mushrooms are sold either fresh or as dietary supplements. These products are frequently consumed depending on season of the year as prophylactic measures for common ills and to improve the general well-being of individuals [5]. The notable regard mushrooms are given for promoting wellness of the public at large is perhaps in part attributed to the rather extensive anecdotal and scientific evidence reporting their disease preventive properties, focusing mostly on the potentiation of immune functions and regulation of biological responses [3, 4, 6]. Beginning in the 1990s, however, it has become increasingly clear that mushrooms, mushroom extracts, and indeed plant/botanical polysaccharides in general, have activities beyond that of the immune system, with suppression of tumorigenesis having the most medical relevance and significance [7‐10]. Thus, for instance, polysaccharides with 6-branched 1,3-β glucan structures isolated from the cultured fruit body of edible mushroom Sparassis crispa reportedly show antitumor activity when tested against Sarcoma 180 in the ICR strain mice [11, 12]. Antineoplastic activity has been demonstrated in polysaccharides isolated from Pleurotus tuber-regium[13], and from fruit body of cultivated Agricus blazei[14, 15]. Several polysaccharide-peptide, and polysaccharide-protein complexes with immunomodulatory and antitumor activities have been isolated and purified from mycelia cultures of Tricholoma Sp., an edible mushroom native to Hong Kong [16‐18]. Maitake, a mushroom indigenous to northeastern Japan, is recognized as a rich source of polysaccharides with a wide-range of biological and medicinal properties [19, 20]. Most notably, gel-purified D-fraction from Maitake characterized as heterogeneous β-(1→6)-branched β-(1→3)-linked alkali-soluble and acid-insoluble polysaccharides [21], show bioactivities spanning the control of immune response, suppression of tumor proliferation, induction of apoptosis, inhibition of metastasis, and regulation of angiogenesis [10, 21, 22]. Additionally, mushrooms reportedly also contain antitumor proteins capable of inducing apoptosis as well as cell cycle checkpoint arrest in cultured malignant cells [23].

Dietary supplements derived from edible mushroom known as Yunzhi, or Coriolus versicolor (Trametes versicolor, Fr.) – known as one of six Zhi's recorded in the "Shen Non Compendium Medica" some 2000 years ago – reportedly also show a number of medicinal properties [2, 24, 25]. Structural and functional analyses of Yunzhi have benefited from the discovery of the patented Cov-1 strain of Coriolus versicolor in 1984–1987 by Yang and coworkers, through an exhaustive screen of a large number of strains of Yunzhi [1, 4, 26]. Subsequently, an innovative industrial scale cultivation method using the mycelia of Cov-1 was developed, which led to the serendipitous discovery, isolation and purification of a family of polysaccharo-peptide, denoted I'm-Yunity™ (PSP) [4, 25, 27].

I'm-Yunity™ (PSP) has demonstrated potent immunomodulatory and antitumor activities in tissue culture studies, based principally on data using flow cytometry [27‐31]. In previous studies, we have observed that ethanol and water extracts of I'm-Yunity™ (PSP) exerted dose- and time-dependent anti-proliferative effects in human promyelocytic HL-60 leukemic cells. Flow cytometric analyses showed that low doses of ethanol and water extracts of I'm-Yunity™ (PSP) induced partial cell arrest in the G₁ phase, whereas high doses resulted in apoptotic cell death. Moreover, water extracts were found to have significantly more pronounced anti-cellular effects than ethanol extracts [28]. In addition, water and ethanol extracts of I'm-Yunity™ (PSP) markedly increased the secretion of IL-1β and IL-6 with concomitant reduction of IL-8 in HL-60 cells, while having no affect on growth and lymphokine expression in normal human lymphocytes [28]. The molecular details by which I'm-Yunity™ (PSP) exerts its biological effects remain incompletely understood.

To further investigate the mechanism of action of I'm-Yunity™ (PSP), we studied the effects of water extracts of I'm-Yunity™ (PSP) on growth, viability and cell cycle traverse using human HL-60 and U-937 leukemia cells. We found a similar degree of inhibition of cell growth and induction of apoptosis, accompanied by differential targeting of G₁/S and G₂/M cell cycle phase transition, respectively, in the HL-60 and U-937 cells. Furthermore, in HL-60 cells, we showed that growth suppression by water extracts of I'm-Yunity™ (PSP) was correlated with time- and dose-dependent inhibition of retinoblastoma protein Rb expression. Correspondingly, induction of apoptosis in I'm-Yunity™ (PSP)-treated cells – evident by cleavage of poly(ADP-ribose) polymerase (PARP) from its native 112-kDa form to the 89-kDa truncated product – was matched by a significant decrease in the expression of anti-apoptotic proteins bcl-2 and survivin, and an increase in apoptogenic proteins bax and cytochrome c. Moreover, HL-60 cells treated with I'm-Yunity™ (PSP) also showed a diminished p65 and p50 forms of transcription factor NF-κB, which paralleled the reduced expression of COX2. We further observed that suppression of HL-60 cell growth was positively correlated with an increase in signal transducer and activator family of transcription factors STAT1, and conversely, with reduction in the expression of activated form of ERK. We postulate that the observed cell cycle and apoptogenic/anti-apoptotic and extracellular signaling regulatory protein expression changes resulting from treatment by water extracts of I'm-Yunity™ (PSP) are mechanistically linked to its reported clinical attributes and general health beneficial effects.

A significant percentage of the estimated two million deaths recorded in the United States each year are attributable to cancer and its ensuing complications. Cancer presents clinically as a constellation of diseases and at all stages – from primary prevention to treatment – can be profoundly and significantly affected by nutrition and diets. The existence of such a link is well supported by decades of epidemiological studies showing that the consumption of plant-based diets is directly associated with reduced risk of cancer [64‐76]. The impact of nutrition and diets in prevention of carcinogenesis is formally conceptualized in chemoprevention by Sporn and others in the 1970s, which emphasizes the use of pharmacologic or natural agents to inhibit development or progression of invasive cancer [67‐69, 71, 72, 74, 77‐79]. Research in recent years has begun to focus on "bioactive compounds" in diets with chemopreventive attributes and delineation of their mechanism(s) [67, 70, 72].

Efficacy of chemopreventive agents may have multiple mechanistic bases: reversal of abnormal differentiation, suppression of cell replication/growth, and induction of apoptosis. Additionally, inhibition of carcinogen activation and increase in their removal by detoxification enzymes are also considered prudent cancer prevention strategies [71, 72, 74]. Since many of the very same chemopreventive attributes are found in I'm-Yunity™ (PSP), we propose that it, as an example of mushroom and mushroom products, may be included in this category of dietary agents. Indeed, based results from this communication and earlier studies, it may be suggested that I'm-Yunity™ (PSP) is an easily compliant adjunctive treatment particularly germane to human leukemia and other blood-borne hematologic malignancies. Since polysaccharide moieties predominate in I'm-Yunity™ (PSP), it is possible that less chemo- and radio-resistant clones will result from treatment using such an agent.

The cellular changes reported in this communication as a result of treatment with water extracts of I'm-Yunity™ (PSP) can be explained by the multiple effects it elicits, which include suppression of the retinoblastoma tumor suppressor protein Rb, increased release of cytochrome c from mitochondria to the cytosol, reduced expression of anti-apoptotic protein bcl-2 and survivin, and simultaneously, increase in bax, as well as a disruption in cell signaling events, as collectively depicted in the scheme in Figure 8.

A number of new molecular targets and mechanistic insights have surfaced from the present studies. First and foremost is the observation that I'm-Yunity™ (PSP) down-regulates the expression of NF-κB in HL-60 cells, thereby identifying a new, novel molecular target of this family of bioactive polysaccharo-peptides. It is noteworthy that the effect of I'm-Yunity™ (PSP) on NF-κB correlated well with the suppression of growth, suggesting a causal relationship. NF-κB is a transcription factor known to be functionally associated with cell survival; its constitutive activation in various human malignancies contributes to tumor transformation, development, and progression. These effects may relate to the ability of NF-κB to control the transcription of genes involved in cell growth and proliferation, suppression or escape of apoptosis, acquisition of new vasculature and blood supply, and dissemination to distant location for further growth and colony formation [52‐56]. Treatment of HL-60 cells with water extract of I'm-Yunity™ (PSP) caused a precipitous reduction in p65 and to a lesser degree also p50 subunits of NF-κB (Figure 5). This finding is significant since it proffers the notion that this family of polysaccharo-peptides acts in part by forestalling a pivotal factor required for the survival and propagation of tumor cells. The more pronounced decline in p65 relative to p50 may relate to the fact that these two subunits of NF-κB are known to be different gene products and not necessarily regulated in a coordinate fashion. Studies have shown, for example, that p50 is proteolytically derived from a p105 precursor and hence, in principle, could be subjected to more complex mechanism of control, compared to the p65 subunit. Taking these into consideration, it is entirely possible that the p65 and p50 subunits are differentially regulated depending on the dose of I'm-Yunity™ (PSP) used for treating the cells. Conceivably, therefore, low rather than high doses of I'm-Yunity™ (PSP) are effective in suppressing the expression of the p50 subunits. In contrast, a broader dose range of I'm-Yunity™ (PSP) affects the expression of the p65 subunit. It should also be pointed out that our findings do not necessarily contradict the role of I'm-Yunity™ (PSP) as an immune modulator, and similarly, other data pointing to NF-κB as a tumor suppressor rather than a promoter of tumorigenesis [52, 54]. It may well be that in normal immune cells, I'm-Yunity™ (PSP) at an appropriate dose range does indeed increases the expression of NF-κB and thereby promotes the increased transcription of genes in immune cells conducive to a heightened immune status. The same cell type consideration may equally apply to recent evidence showing that NF-κB can function as a tumor suppressor by actively repressing anti-apoptotic genes. The acquisition of a non-conventional role for NF-κB, i.e., functioning in tumor suppression rather than promotion, may also depend on stimuli impinging on cells, and just as plausible, the co-expression of genes acting in concert with NF-κB to facilitate transcriptional repressor of anti-apoptotic genes. It has been demonstrated, for example, that ARF (ADP-ribosylation factor) and p53 as well as certain inducers of NF-κB DNA-binding activity, such as ultraviolet light and some chemotherapeutic compounds, can induce the association of NF-κB subunits with transcriptional corepressor complexes, allowing them to function as repressors rather than activators of gene expression [80‐82]. Whether these considerations can apply to HL-60 cells, known to have p53-null status but do express the ARF [83‐89], in the context of cellular effects elicited by I'm-Yunity™ (PSP) remain to be studied. Further research will also focus on investigation of detailed changes in steady state levels and subcellular distribution of NF-κB, using control and treated cells harboring a stably transfected dominant-negative IκB gene in which amino acid serine at positions 32 and 34 of IκB is mutated to amino acid alanine. These changes should exclude its phosphorylation by IκB kinase, which is required for the degradation of IκB, coincident with the cytoplasm-to-nucleus translocation of NF-κB [51, 90, 91].

A second new finding revealed in the present studies is the ability of I'm-Yunity™ (PSP) to down regulate COX2 expression. Persistent chronic inflammation plays an important role in carcinogenesis [92‐94]. In the early phase of inflammation, COX2 facilitates the production of inflammatory prostaglandins. COX2 has been shown to be overexpressed in a variety of cancers [95‐97]. Further, there is compelling evidence from in vitro experimental studies that inhibition of COX2 decreases cellular proliferation, increases apoptosis, and modulates genes involved in cell cycle regulation [93, 94]. Therefore we measured COX2 expression in control and treated cells. Results in Figure 6 show that COX2 expression was significantly suppressed by treatment with water extracts of I'm-Yunity™ (PSP).

A third important observation relates to the modulation of potent signaling molecules by I'm-Yunity™ (PSP), as exemplified by an increase the expression of STAT1 in parallel with the suppression of the phosphorylated form of ERK (Figure 7). STATs and their protein partners JAKs (Janus kinases) are evolutionarily conserved signaling and cellular communication molecules that enable cytokines to mount a repertoire of host defenses and immunological responses. Similarly, ability of cells to respond to extracellular challenges also depend upon signaling pathways governed by the MAP kinase modules, with sequentially acting protein kinases including ERK as the archetypal form. In both examples, interaction of ligand or an external challenge with membrane-associated receptors are considered to be key initiation events. Because of the importance of the STAT and ERK signaling pathways in homeostatic control of cells, as discussed, it is of interest and pertinent to ask how they might be affected by I'm-Yunity™ (PSP). One possibility is that I'm-Yunity™ (PSP), having structural similarity to proteoglycans [4], act directly as agonists/antagonists of specific surface receptor ligands, and in turn, augment the trigger of the JAK/STAT signaling pathway while concurrently dampen the events directed to the ERK signaling cascade. A second possibility is that I'm-Yunity™ (PSP) functions as a growth factor/cytokine decoy to sequester/trap the relevant growth factor/cytokine required for HL-60 cellular function, or bind the pertinent receptor in ways that block further engagement and interaction with their physiological, cognate ligands. This possibility may be supported by structural resemblance existing between the bioactive constituents present in I'm-Yunity™ (PSP) with proteoglycans occurring in the extracellular matrix of cancer cells [98]. As mentioned in the Introduction, a broad spectrum of bioactive molecules, ranging from those with defined structures and functions, e.g., proteases, nucleases, to others with heterogeneous structures and ill-defined activities, e.g., polysaccharo-peptides and polysaccharides, have been identified and isolated from mushrooms [2, 4, 99, 100]. Finally, it is possible that I'm-Yunity™ (PSP) exerts its effect on STAT1 and ERK by disrupting intracellular signaling events, secondary to its internalization that may involve the formation of a complex between the polysaccharo-peptides with specific components in the serum to facilitate its uptake into cells. This mechanism may draw analogy from lipid-mediated delivery of highly charged and heterogeneous plasmid DNA into cultured mammalian cells and as vaccines [101], as well as the intestinal absorption of oral heparin (a heterogeneous, charged mucopolysaccharide) [102‐104] or heparan sulfate, e.g., chondroitin sulfate [105‐107]. Notably, some of these possibilities can be tested experimentally and are being considered for further investigation in our laboratory.

Treatment of human leukemia cells by water extracts of I'm-Yunity™ (PSP) causes cell cycle arrest and alterations in the expression of apoptogenic/anti-apoptotic and extracellular signaling regulatory proteins, the net result being a reduction in proliferation and an increase in apoptosis. These findings may be part of the mechanisms that underlie or contribute to its reported clinical attributes and overall health benefits.